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Jingmei

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Pandemic_PMC

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Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2139893/

Inhibition of S. aureus α-Hemolysin and B. anthracis Lethal Toxin by β-Cyclodextrin Derivatives

Many pathogens utilize the formation of trans-membrane pores in target cells in the process of infection. A great number of pore-forming proteins, both bacterial and viral, are considered to be important virulence factors, which makes them attractive targets for the discovery of new therapeutic agents. Our research is based on the idea that compounds designed to block the pores can inhibit the action of virulence factors, and that the chances to find high affinity blocking agents increases if they have the same symmetry as the target pore. Recently, we demonstrated that derivatives of β-cyclodextrin inhibited anthrax lethal toxin (LeTx) action by blocking the trans-membrane pore formed by the protective antigen (PA) subunit of the toxin. To test the broader applicability of this approach, we sought β-cyclodextrin derivatives capable of inhibiting the activity of Staphylococcas aureus α-hemolysin (α-HL), which is regarded as a major virulence factor playing an important role in staphylococcal infection. We identified several amino acid derivatives of β-cyclodextrin that inhibited the activity of α-HL and LeTx in cell-based assays at low micromolar concentrations. One of the compounds was tested for the ability to block ion conductance through the pores formed by α-HL and PA in artificial lipid membranes. We anticipate that this approach can serve as the basis for a structure-directed drug discovery program to find new and effective therapeutics against various pathogens that utilize pore-forming proteins as virulence factors.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8402886/

Isolation and Characterisation of the Bundooravirus Genus and Phylogenetic Investigation of the Salasmaviridae Bacteriophages

Bacillus is a highly diverse genus containing over 200 species that can be problematic in both industrial and medical settings. This is mainly attributed to Bacillus sp. being intrinsically resistant to an array of antimicrobial compounds, hence alternative treatment options are needed. In this study, two bacteriophages, PumA1 and PumA2 were isolated and characterized. Genome nucleotide analysis identified the two phages as novel at the DNA sequence level but contained proteins similar to phi29 and other related phages. Whole genome phylogenetic investigation of 34 phi29-like phages resulted in the formation of seven clusters that aligned with recent ICTV classifications. PumA1 and PumA2 share high genetic mosaicism and form a genus with another phage named WhyPhy, more recently isolated from the United States of America. The three phages within this cluster are the only candidates to infect B. pumilus . Sequence analysis of B. pumilus phage resistant mutants revealed that PumA1 and PumA2 require polymerized and peptidoglycan bound wall teichoic acid (WTA) for their infection. Bacteriophage classification is continuously evolving with the increasing phages' sequences in public databases. Understanding phage evolution by utilizing a combination of phylogenetic approaches provides invaluable information as phages become legitimate alternatives in both human health and industrial processes. 1. Introduction Organisms that belong to the Bacillus genus are Gram-positive, aerobic, endospore forming rods [ 1 ]. They are diverse and important environmental microbes; however, members of this genus have been implicated with human disease [ 2 , 3 ]. These organisms most commonly include Bacillus cereus and Bacillus anthracis , which are capable of causing severe foodborne illnesses and anthrax, respectively [ 4 ]. Bacillus pumilus has also been known to intermittently cause food borne illness and contamination in assumed sterile areas [ 5 ]. This is largely due to the ability of B. pumilus endospores to survive extreme environments, including hydrogen peroxide treatment, which is a common method of equipment sterilization [ 6 ]. B. pumilus genomes also contain an arsenal of genes able to survive oxidative stress and antimicrobial compounds [ 7 ]. This alarming robustness, along with the looming threat of antibiotic resistance, suggests that alternative treatments to chemical sterilization and antibiotics are required. Bacteriophages (or phages) are viruses that propagate by infecting and lysing bacterial cells. Phages are predicted to be the most diverse and abundant biological entities on the planet [ 8 , 9 ]. Due to their ability to alter bacterial genomes through horizontal gene transfer and impact the population dynamics within microbial communities, they play a major role in microbial ecology and evolution [ 10 , 11 ]. Phages have also gained great interest as potential therapeutic agents to be used as an alternative to antibiotics [ 12 , 13 ]. With the emergence of next generation sequencing, an abundance of phage sequences are continuously deposited into public databases, providing comprehensive information on phage genetic diversity and taxonomy [ 14 , 15 , 16 , 17 ]. The abundance of sequence data has led to the creation of the Mycobacteriophage databases that has subsequently become the Actinobacteriophage database (phagesDB.org) [ 18 ]. This website has expanded to also include a Bacillus phage database with over 1400 Bacillus phage sequences uploaded. This useful resource has provided us with information on the genetic diversity and evolution of Bacillus phages. It is becoming increasingly important that the more Bacillus phage genome sequences we have, the better we understand their evolution and interactions with the environment and their hosts [ 19 ]. Grose et al. [ 20 ] conducted a thorough analysis of 93 Bacillus phage sequences and generated clusters based on genomic similarities. The groupings were based on that described previously for the Mycobacterium phages [ 21 ] and the phi29-like phages [ 22 ]. Cluster B was noted for phages relating to phi29, now classified in the Salasvirus genus. At the time, a small subset of phage sequences was available and was classified into three sub-clusters, including phi29 and PZA (grouped in sub-cluster B1), B103 and Nf (sub-cluster B2), and GA-1 (sub-cluster B3) [ 20 ]. In 2018, Schilling, Hoppert, and Hertel [ 9 ] discussed 21 phi29-like phage sequences available in GenBank, providing preliminary insights into this group's genetic relatedness [ 9 ]. However, due to advances in viral classification techniques and the addition of other phi29-like phages in Genbank, this work is outdated. Recently, Li, et al. [ 23 ] isolated one of the newest members of this group, DLc1, and concluded that the now present 30 phi29-like phages should be included within the Salasvirus genus. However, this study failed to use multiple reticulate phylogeny methods for this classification and relied only on basic genome comparison methods. As of earlier this year, the International Committee on Virus Taxonomy (ICTV) reviewed the phi29-like phages and classified them in the new Salasmaviridae family [ 24 ]. However, within this report there were no detailed conclusions about these new taxonomic rankings, therefore, validation of this work is needed. In this study we isolated two novel B. pumilus phages, PumA1 and PumA2, from Australian soil samples. These phages were characterized based on morphology, host range, and genome sequence. Annotation and sequence analysis revealed that both phages are novel at the DNA sequence but share conserved protein families, genome structure, and phenotypic characteristics similar to phi29 and other Salasmaviridae phages. This study also provides an in-depth genomic analysis of the newly classified Salasmaviridae phages and insights into their evolution and diversity. 2. Materials and Methods 2.1. Bacterial Strains and Media In this study, Bacillus pumilus LTU1 strain was used, isolated by Dr Darryl Reanney, from a soil sample collected from Victoria, Australia. Bacterial cultures were grown on LB (1% tryptone (Oxoid, Adelaide, Australia), 0.5% yeast extract (Oxoid), and 1% sodium chloride (Sigma)) broth or agar (LB plus 1.4% agar (Oxoid)) at 28 °C. All chemicals were obtained from Sigma, Sydney, Australia) unless otherwise stated. 2.2. Isolation and Purification of Phages and DNA Extraction Bacteriophages were isolated from soil samples taken from multiple points within 30 km of Darwin, Australia. The soil samples (1 g) were suspended in 2 mL of sterile water. The mixture was vortexed for 60 s and then centrifuged for 5 min before being filtered through a cellulose acetate membrane filter (0.2-µm pore size) to remove cells and other solid matter. Following incubations, the remaining bacterial cells were removed by centrifugation and filtration through a 0.2-µm cellulose acetate membrane filter. Lawn plates of B. pumilus were prepared and 20 µL aliquots of enriched supernatants were applied onto the lawn plates and allowed to dry. Plates were incubated overnight and visually inspected for the presence of the plaques the following day. Single plaques were observed and isolated from two different soil samples in the Northern Territory in Australia. Plaques were purified through six rounds of dilution and re-isolation to ensure their purity. 2.3. Phage DNA Isolation, Genome Sequencing, and Annotation Purified phage particles were polyethylene glycol (PEG) precipitated followed by a proteinase K treatment to extract DNA as described previously [ 25 ]. Isolated phage DNA (100 ng) were prepared using the NEBNext ® Ultra™ II DNA Library Prep Kit (NEB) (Australia) followed by whole genome sequencing on an Illumina MiSeq v3 600-cycle kit with 300 bp paired-end reads. Raw data were filtered using Trim Galore v0.6.4 with the default settings (Q scores of ≥20, with automatic adapter detection) [ 26 ]. Phage genomes were assembled with SPAdes v3.12.0 with default settings. The genome termini were corrected upon manual inspection of raw sequencing reads using CLC Genomics Workbench v9.5.4 (Qiagen, Melbourne, Victoria, Australia). Putative open reading frames (ORFs) were predicted using Glimmer v3 and manually confirmed [ 27 ]. Sequence similarity searches were conducted using the predicted amino acid sequences against the GenBank database and the BLASTP algorithm was used with an E-value significance cut off of 10 −4 [ 28 , 29 ]. Conserved domains and motifs were identified using the conserved domain database (CDD) ( http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml ) (accessed on 7 March 2019) and the Pfam database ( http://pfam.sanger.ac.uk ) (accessed on 7 March 2019) [ 30 ]. The presence of genes encoding tRNAs was screened for using ARAGORN ( http://130.235.244.92/ARAGORN/ ) (accessed on 7 March 2019) [ 31 ]. 2.4. Electron Microscopy Copper grids (ProSciTech, Townsville, Queensland, Australia) coated with carbon and formvar were subjected to a glow discharge treatment for 60 s. A total of 20 µL of high titer (>10 9 pfu/mL) phage filtrates were placed onto the grids, incubated for 10 min, and followed by removal of excess residue with filter paper. Grids were washed twice with 5 µL MilliQ, each wash being absorbed with filter paper. The grids were then negatively stained with 3 µL of 2% ( w / v ) uranyl acetate, followed by immediate removal with filter paper and one final MilliQ wash as outlined previously. The grids were then left to dry in a laminar flow for 30 min before imaging. The grids were examined under a JEOL JEM02010HC electron microscope. 2.5. Nucleotide Sequence The nucleotide sequences for phages vB_BpuP_PumA1 and vB_BpuP_PumA2 have been deposited in GenBank under the accession numbers MN524844 and MN524845, respectively. 2.6. Identification of Phage Resistant B. pumilus Mutants Lawn plates of B. pumilus and either PumA1 or PumA2 flooded in high titer were grown for 24 h. Colonies that emerged in the clearings were picked, streaked out for a total of three times, and re-spotted with phage to test that their resistance was stable. DNA extractions of the wild type B. pumilus and the strains showing phage resistance were prepared using the Wizard Genomic DNA Purification Kit (Promega, Sydney, Australia) as per manufacturer's instructions. The DNA samples were then prepared for next generation sequencing as outlined above. For the wild type B. pumilus , the raw sequencing data was trimmed using Trim Galore v0.6.4 and the genome assembled using Unicycler v0.4.8. For SNP analysis, Snippy (Galaxy V.4.5.0) was used. The assembled wild type B. pumilus was the reference genome and each mutant strain was compared for differences. 2.7. Whole Genome Analysis and Clustering The NCBI and Bacillus Phage (phagesdb.org) Databases were examined for related phi29-like phages. Thirty-four complete phage sequences were found on either database and used for this study ( Table 1 ). A dot plot using the Genome Pair Rapid Dotter program (GEPARD) was chosen as a preliminary guide for clustering [ 32 ]. The genome sequences were collated into one FASTA file in the order of nucleotide identity found through BLASTN results. Clusters were validated using VIRIDIC v1.0 software [ 33 ]. Whole genome alignments of all phages and their clusters were undertaken using BLASTn comparisons and visualized with Easyfig [ 34 ]. For gene content analysis, a Clustal Omega alignment was performed and visualized in Splitstree4 [ 35 , 36 ]. Using the Neighbor-Joining method, a consensus network tree was developed. To create the reticulate gene sharing network, vConTACT v2.0 0.9.17 [ 37 ], a gene mapping program, was used. Gene2Genome was first used to assign each protein coding sequence of each phage and map it to its contig/genome ID. This output file was then combined with the collated FASTA file previously used and ran in vConTACT v.2.0. The network output file of vConTACT 2v.2.0 was then visualized in Cytoscape v3.8.1 [ 38 ]. Finally, a core protein phylogeny tree was created using both the DNA polymerase and DNA encapsidation ATPase in VICTOR [ 39 ]. All pairwise comparisons of the nucleotide sequences were conducted using the Genome-BLAST Distance Phylogeny (GBDP) method and branch support was inferred from 100 pseudo-bootstrap replicates each. The tree was then visualized using iTOL v6.2 [ 40 ]. The tree was rooted at midpoint and meta-data overlayed. 2.1. Bacterial Strains and Media In this study, Bacillus pumilus LTU1 strain was used, isolated by Dr Darryl Reanney, from a soil sample collected from Victoria, Australia. Bacterial cultures were grown on LB (1% tryptone (Oxoid, Adelaide, Australia), 0.5% yeast extract (Oxoid), and 1% sodium chloride (Sigma)) broth or agar (LB plus 1.4% agar (Oxoid)) at 28 °C. All chemicals were obtained from Sigma, Sydney, Australia) unless otherwise stated. 2.2. Isolation and Purification of Phages and DNA Extraction Bacteriophages were isolated from soil samples taken from multiple points within 30 km of Darwin, Australia. The soil samples (1 g) were suspended in 2 mL of sterile water. The mixture was vortexed for 60 s and then centrifuged for 5 min before being filtered through a cellulose acetate membrane filter (0.2-µm pore size) to remove cells and other solid matter. Following incubations, the remaining bacterial cells were removed by centrifugation and filtration through a 0.2-µm cellulose acetate membrane filter. Lawn plates of B. pumilus were prepared and 20 µL aliquots of enriched supernatants were applied onto the lawn plates and allowed to dry. Plates were incubated overnight and visually inspected for the presence of the plaques the following day. Single plaques were observed and isolated from two different soil samples in the Northern Territory in Australia. Plaques were purified through six rounds of dilution and re-isolation to ensure their purity. 2.3. Phage DNA Isolation, Genome Sequencing, and Annotation Purified phage particles were polyethylene glycol (PEG) precipitated followed by a proteinase K treatment to extract DNA as described previously [ 25 ]. Isolated phage DNA (100 ng) were prepared using the NEBNext ® Ultra™ II DNA Library Prep Kit (NEB) (Australia) followed by whole genome sequencing on an Illumina MiSeq v3 600-cycle kit with 300 bp paired-end reads. Raw data were filtered using Trim Galore v0.6.4 with the default settings (Q scores of ≥20, with automatic adapter detection) [ 26 ]. Phage genomes were assembled with SPAdes v3.12.0 with default settings. The genome termini were corrected upon manual inspection of raw sequencing reads using CLC Genomics Workbench v9.5.4 (Qiagen, Melbourne, Victoria, Australia). Putative open reading frames (ORFs) were predicted using Glimmer v3 and manually confirmed [ 27 ]. Sequence similarity searches were conducted using the predicted amino acid sequences against the GenBank database and the BLASTP algorithm was used with an E-value significance cut off of 10 −4 [ 28 , 29 ]. Conserved domains and motifs were identified using the conserved domain database (CDD) ( http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml ) (accessed on 7 March 2019) and the Pfam database ( http://pfam.sanger.ac.uk ) (accessed on 7 March 2019) [ 30 ]. The presence of genes encoding tRNAs was screened for using ARAGORN ( http://130.235.244.92/ARAGORN/ ) (accessed on 7 March 2019) [ 31 ]. 2.4. Electron Microscopy Copper grids (ProSciTech, Townsville, Queensland, Australia) coated with carbon and formvar were subjected to a glow discharge treatment for 60 s. A total of 20 µL of high titer (>10 9 pfu/mL) phage filtrates were placed onto the grids, incubated for 10 min, and followed by removal of excess residue with filter paper. Grids were washed twice with 5 µL MilliQ, each wash being absorbed with filter paper. The grids were then negatively stained with 3 µL of 2% ( w / v ) uranyl acetate, followed by immediate removal with filter paper and one final MilliQ wash as outlined previously. The grids were then left to dry in a laminar flow for 30 min before imaging. The grids were examined under a JEOL JEM02010HC electron microscope. 2.5. Nucleotide Sequence The nucleotide sequences for phages vB_BpuP_PumA1 and vB_BpuP_PumA2 have been deposited in GenBank under the accession numbers MN524844 and MN524845, respectively. 2.6. Identification of Phage Resistant B. pumilus Mutants Lawn plates of B. pumilus and either PumA1 or PumA2 flooded in high titer were grown for 24 h. Colonies that emerged in the clearings were picked, streaked out for a total of three times, and re-spotted with phage to test that their resistance was stable. DNA extractions of the wild type B. pumilus and the strains showing phage resistance were prepared using the Wizard Genomic DNA Purification Kit (Promega, Sydney, Australia) as per manufacturer's instructions. The DNA samples were then prepared for next generation sequencing as outlined above. For the wild type B. pumilus , the raw sequencing data was trimmed using Trim Galore v0.6.4 and the genome assembled using Unicycler v0.4.8. For SNP analysis, Snippy (Galaxy V.4.5.0) was used. The assembled wild type B. pumilus was the reference genome and each mutant strain was compared for differences. 2.7. Whole Genome Analysis and Clustering The NCBI and Bacillus Phage (phagesdb.org) Databases were examined for related phi29-like phages. Thirty-four complete phage sequences were found on either database and used for this study ( Table 1 ). A dot plot using the Genome Pair Rapid Dotter program (GEPARD) was chosen as a preliminary guide for clustering [ 32 ]. The genome sequences were collated into one FASTA file in the order of nucleotide identity found through BLASTN results. Clusters were validated using VIRIDIC v1.0 software [ 33 ]. Whole genome alignments of all phages and their clusters were undertaken using BLASTn comparisons and visualized with Easyfig [ 34 ]. For gene content analysis, a Clustal Omega alignment was performed and visualized in Splitstree4 [ 35 , 36 ]. Using the Neighbor-Joining method, a consensus network tree was developed. To create the reticulate gene sharing network, vConTACT v2.0 0.9.17 [ 37 ], a gene mapping program, was used. Gene2Genome was first used to assign each protein coding sequence of each phage and map it to its contig/genome ID. This output file was then combined with the collated FASTA file previously used and ran in vConTACT v.2.0. The network output file of vConTACT 2v.2.0 was then visualized in Cytoscape v3.8.1 [ 38 ]. Finally, a core protein phylogeny tree was created using both the DNA polymerase and DNA encapsidation ATPase in VICTOR [ 39 ]. All pairwise comparisons of the nucleotide sequences were conducted using the Genome-BLAST Distance Phylogeny (GBDP) method and branch support was inferred from 100 pseudo-bootstrap replicates each. The tree was then visualized using iTOL v6.2 [ 40 ]. The tree was rooted at midpoint and meta-data overlayed. 3. Results 3.1. Isolation of Bacteriophages and Their Morphological Features After screening multiple soil samples collected in Darwin (Northern Territory, Australia), two samples from different locations produced plaques on lawn plates of B. pumilus (LTU1). They were isolated, purified, and named vB_BpuP_PumA1 (PumA1) and vB_BpuP_PumA2 (PumA2). For host range analysis, the phages were tested against other Bacillus species in our culture collection including B. anthracis , B. subtilis, B. mycoides , and B. cereus . Both phages exclusively lysed B. pumilus, suggesting a narrow host range (regarding available strains). Transmission electron microscopy imaging of negatively stained phages demonstrated that PumA1 and PumA2 displayed short tails and small, elongated capsids of 52 ± 5 nm (length) × 29 ± 6 nm (width) ( Figure 1 ). 3.2. Sequencing and Genomic Features of PumA1 and PumA2 PumA1 and PumA2 were sequenced using Illumina sequencing technology. Genome assembly revealed both phages had linear genomes of 18,466 bp and 18,932 bp, respectively. Annotation of the PumA1 and PumA2 genomes revealed that they contained 26 and 28 putative open reading frames, respectively, and no tRNA genes ( Figure 2 ). The genomes of both PumA1 and PumA2 share 82% similarity over 98% of the genomes. When compared to DNA sequences in the GenBank database, both phages are unique, with only 4% to 20% of the genomes sharing between 65% and 73% sequence identities with other phi29-like phage genomes. The phage genomes were flanked by 11 bp inverted repeat sequences (5′-AATGTAAAGGT-3′) consistent with phi29-like phages that all contain variations of inverted repeats [ 53 ]. The predicted amino acid sequence of each open reading frame was used in a BLASTp analysis to determine the closest homologue. Predicted functions can be assigned to sixteen gene products ( Table S1 ). The genes were numbered consecutively with the exceptions of orf2.1 and orf2.2 , which are present in PumA2 but not in PumA1. The genome organization and structure are similar to that of phage phi29 and its relatives, and the gene products share conserved similarities at the amino acid level [ 54 ]. The genomes of both PumA1 and PumA2 can be separated into three different modules based on the direction the genes are transcribed (Regions I, II, and III) ( Figure 2 ). Regions I and III (also known as the early genes) contain genes that are transcribed in the same direction and are located at the 5′ and 3′ ends of the genomes. Region II (also referred to as the late genes) is located in the center of the genome and is transcribed in the opposite direction to the other genes [ 9 ]. Region I of the genomes encompasses eleven genes for PumA1 and thirteen genes for PumA2. The proteins encoded by the open reading frames orf1-orf6 in both phage genomes have no known predicted function and are noted as hypothetical proteins. However, orf7 to orf11 are described and associated with the phi29-like phages [ 9 , 44 , 45 ]. The genes encoding the DNA polymerase ( orf7 ) are highly conserved between the phi29-like phages, and when compared using BLASTn, most of this gene (83% coverage) is 65% homologous to the DNA polymerase of phage phi29. The terminal binding protein in PumA1 and PumA2 is encoded by orf8 , adjacent to the DNA polymerase [ 55 ]. The remaining genes within the first region encode a DNA transcriptional activator for the late genes ( orf9 ), containing a characteristic conserved motif pfam05464 similar to that observed in phi29 [ 56 , 57 ], followed by a gene encoding single stranded binding protein ( orf10 ) and double stranded binding protein ( orf11 ). Region III contains four genes ( orf23-orf26 ) transcribed in the same direction as the genes in region I. Three of the genes have unknown functions, and orf24 encodes a DNA replication organizer with a pfam06720 motif. Region II contains genes orf12-orf22 that encode structural and morphogenesis genes. The head morphogenesis protein, orf12 , contains a pfam11418 motif, similar to phi29 scaffolding protein [ 58 ]. This is followed by orf13 , a putative major head protein containing a bacterial Ig-like domain (pfam02368) and orf14 , a head fiber protein and motif (pfam11133). The major tail protein ( orf15 ) contains a pfam16838 motif, conserved across groups of podoviruses [ 50 , 59 ]. The proteins that connect the phage head and tail are encoded by orf16 containing a pfam05352 [ 60 ], orf17 encoding a lower collar protein with the PHA00148 motif [ 61 , 62 ], and orf18 encoding a minor structural protein that is suspected to form the pre-neck appendage protein. Orf18 's closest homologues are found in other phi29-like phages WhyPhy and SRT01hs and a Staphylococcus phage ST134 with a conserved motif TIGR04523. This is followed by another morphogenesis protein, orf19 , with the characteristic motif pfam01551. A putative holin is encoded by orf20 with the pfam05105 motif conserved in bacteriophage holin proteins. Orf21 encodes an endolysin with two motifs, pfam01520 and pfam01476 [ 63 ]. The last gene in the module orf22 encodes a protein that is predicted to encode a podovirus DNA encapsidation protein with the characteristic motif pfam05894 [ 64 ]. 3.3. Whole Genome Comparison and Clustering of phi29-Like Phages Since the genome organization and protein homologies of PumA1 and PumA2 were reminiscent of phi29, we next investigated the evolutionary relationship shared between PumA1, PumA2, and other phi29-like phages. The GenBank and Bacillus phage databases were searched for complete phage genomes that shared genomic similarities to phi29. This led to the identification of 34 phages, including the two isolated in this study ( Table 1 ). Hatfull, et al. [ 65 ] previously described a classification system using a four-method approach to cluster 60 mycobacteriophage genomes. We employed their approach as a guide and included whole genome comparisons, network and clustering analysis, and candidate gene phylogenetics to organize the phi29-like phages into respective clusters ( Figure S1 ). The clusters were expanded from the ones previously described by Grose, Jensen, Burnett, and Breakwell [ 20 ] and employ the same naming style of B and subclusters numbered (e.g., B1). The number of clusters were expanded from three [ 20 ] to seven and three singleton phages. New members were added to existing clusters and align with the ICTV classifications. While no new clusters or potential genera were formed from this analysis, this study provides a justification for the ICTV taxonomic rankings and framework for future clustering and classification of phages. 3.4. Dot Plot Analysis and Genomic Identities Dot plot analysis of the 34 genomes revealed six clusters and two phages not pertaining to a cluster, referred to as singletons ( Figure S2 ). To further define the clusters and provide a numerical value to their similarities, we used the Virus Intergenomic Distance Calculator (VIRIDIC) to calculate intergenomic similarities between each phage. VIRIDIC combines several similarity algorithms with genome alignment and length ratios to capture overall relatedness of prokaryotic viruses [ 33 ]. In correlation with the dot plot analysis, the heatmap presents the 34 phages in the same clustering pattern ( Figure 3 ). Each cluster shares similarities of between 65.65–99.87% ( Table S2 ). The clusters are now referred to as from B1 to B7, expanding from previous clusters [ 20 ]. 3.5. Genome Map Alignments Each cluster was aligned to show the dissimilarities between genomes ( Figure 4 ). PumA1 and PumA2 contain an extra 796 bp and 1240 bp respectively compared to the ancestral phage phi29 in the 5′ early gene region. These hyperplastic regions are common throughout the phi29-like phages, where large insertions of up to 8256 bp are seen in the largest phi29-like phage, DLc1. Each cluster also shares the same pattern of insertions between them. The additional genes have no known functions but are presumed to be involved in the infection or replication processes since they are located within that region. Interestingly, the phages with genome sizes over 20 kb no longer contain the head fiber gene, a characteristic feature of phi29. The singletons show the least similarity to phi29 and other phages in this group, with small regions of similarity to their closest related phage. 3.6. Gene Sharing Networks Reticulate networks have been recently shown to provide an accurate representation of phage relatedness versus traditional rooted phylogenetics, since phages undergo many recombination and horizontal gene transfer events [ 66 ]. To test if these methods aligned with the comparison methods previously mentioned, two reticulate methods were used. Firstly, an unrooted phylogenetic network was created in Splitstree4 using whole genome CLUSTAL Omega alignment ( Figure 5 ). This network showed a consistent pattern of clustering in agreement with the other techniques. vConTACT v.2.0 was then used in conjunction with the Splitstree network. vConTACT v.2.0 is a newly developed software for virus classification that extracts, aligns, and clusters all predicted input proteins [ 37 ]. The protein clusters are then used to calculate viral clusters (VC) by am "edge" weight or statistical confidence due to the amount of protein clusters that each phage shares. This is compared to a global network of phages in the GenBank database. Figure 6 depicts the global network produced from vConTACT v.2.0 with various phages color coded by their host genera. The phi29-like phages are not connected to the main network, showing little gene-sharing outside of this group of phages. When the phi29-like network is expanded, there is a clear differentiation of each phage cluster, denoted by the individual cluster colors. While these phages do not connect to the main network, they share proteins with two other phages that are not infective for Bacillus , Lactococcus phage Asccphi28 [ 67 ] and Weisella phage phiYS61 [ 68 ], which are genotypically similar to the phi29-like phages. This form of reticulate phylogenetics helped corroborate the established clusters previously outlined and is an accurate tool for investigating phage gene-sharing and evolution. 3.7. Candidate Gene Analysis Finally, a phylogenetic tree was created combining two conserved and integral genes, DNA polymerase and DNA encapsidation ATPase ( Figure 7 ). The tree correlates with the whole genome comparison approaches as the phages are seen to group with their clusters. The tree splits into two distinctive branches with singletons DLc1 and MG-B1 and clusters B6 and B7 diverging from the rest of the clusters. This correlates with the genome sizes of these phages, as they are the largest genomes of the phi29-like group. The branch distances are also short within the clusters, particularly B6 members, signifying small base pair substitutions. The other factors that are seen to contribute to the phages' evolution are host species and country or region they were isolated. These clusters also agree with the ICTV taxonomic rankings as outlined by metadata on the tree. 3.8. PumA1 and PumA2 Host Receptor Site PumA1 and PumA2 displayed a narrow host range, only able to infect the B. pumilus strain in our collection. This appears to be a characteristic of the B4 cluster or Bundooravirus genus. It has been shown that phi29 requires polymerized teichoic acid for its attachment to B. subtilis [ 69 ]. Given the observed host range differences between phi29 and the two phages isolated in this study, we pursued an investigation into the host receptor of PumA1 and PumA2. B. pumilus colonies that developed stable resistance to PumA1 and PumA2 were isolated, and whole genome sequenced to determine which genomic modifications were causing resistance to phages. Mutations were found in either the tagF or tagT genes, which are a part of the teichoic acid synthesis operon [ 70 , 71 , 72 ]. Four of the seven variant strains isolated had modifications to the tagF gene, and the other three had mutations in the tagT gene ( Table S3 ). The majority of the mutants (A2M1, A2M11, A1M3, A2M14, and A1M5) contained frameshift mutations resulting in early termination of protein translation. Most of these frameshifts occurred near the N-terminus of the respective proteins likely resulting in non-functional TagT or TagF proteins. The remaining mutant had a single amino acid substitution in TagF (G688S). It is unclear how this mutation affects protein structure and function. 3.1. Isolation of Bacteriophages and Their Morphological Features After screening multiple soil samples collected in Darwin (Northern Territory, Australia), two samples from different locations produced plaques on lawn plates of B. pumilus (LTU1). They were isolated, purified, and named vB_BpuP_PumA1 (PumA1) and vB_BpuP_PumA2 (PumA2). For host range analysis, the phages were tested against other Bacillus species in our culture collection including B. anthracis , B. subtilis, B. mycoides , and B. cereus . Both phages exclusively lysed B. pumilus, suggesting a narrow host range (regarding available strains). Transmission electron microscopy imaging of negatively stained phages demonstrated that PumA1 and PumA2 displayed short tails and small, elongated capsids of 52 ± 5 nm (length) × 29 ± 6 nm (width) ( Figure 1 ). 3.2. Sequencing and Genomic Features of PumA1 and PumA2 PumA1 and PumA2 were sequenced using Illumina sequencing technology. Genome assembly revealed both phages had linear genomes of 18,466 bp and 18,932 bp, respectively. Annotation of the PumA1 and PumA2 genomes revealed that they contained 26 and 28 putative open reading frames, respectively, and no tRNA genes ( Figure 2 ). The genomes of both PumA1 and PumA2 share 82% similarity over 98% of the genomes. When compared to DNA sequences in the GenBank database, both phages are unique, with only 4% to 20% of the genomes sharing between 65% and 73% sequence identities with other phi29-like phage genomes. The phage genomes were flanked by 11 bp inverted repeat sequences (5′-AATGTAAAGGT-3′) consistent with phi29-like phages that all contain variations of inverted repeats [ 53 ]. The predicted amino acid sequence of each open reading frame was used in a BLASTp analysis to determine the closest homologue. Predicted functions can be assigned to sixteen gene products ( Table S1 ). The genes were numbered consecutively with the exceptions of orf2.1 and orf2.2 , which are present in PumA2 but not in PumA1. The genome organization and structure are similar to that of phage phi29 and its relatives, and the gene products share conserved similarities at the amino acid level [ 54 ]. The genomes of both PumA1 and PumA2 can be separated into three different modules based on the direction the genes are transcribed (Regions I, II, and III) ( Figure 2 ). Regions I and III (also known as the early genes) contain genes that are transcribed in the same direction and are located at the 5′ and 3′ ends of the genomes. Region II (also referred to as the late genes) is located in the center of the genome and is transcribed in the opposite direction to the other genes [ 9 ]. Region I of the genomes encompasses eleven genes for PumA1 and thirteen genes for PumA2. The proteins encoded by the open reading frames orf1-orf6 in both phage genomes have no known predicted function and are noted as hypothetical proteins. However, orf7 to orf11 are described and associated with the phi29-like phages [ 9 , 44 , 45 ]. The genes encoding the DNA polymerase ( orf7 ) are highly conserved between the phi29-like phages, and when compared using BLASTn, most of this gene (83% coverage) is 65% homologous to the DNA polymerase of phage phi29. The terminal binding protein in PumA1 and PumA2 is encoded by orf8 , adjacent to the DNA polymerase [ 55 ]. The remaining genes within the first region encode a DNA transcriptional activator for the late genes ( orf9 ), containing a characteristic conserved motif pfam05464 similar to that observed in phi29 [ 56 , 57 ], followed by a gene encoding single stranded binding protein ( orf10 ) and double stranded binding protein ( orf11 ). Region III contains four genes ( orf23-orf26 ) transcribed in the same direction as the genes in region I. Three of the genes have unknown functions, and orf24 encodes a DNA replication organizer with a pfam06720 motif. Region II contains genes orf12-orf22 that encode structural and morphogenesis genes. The head morphogenesis protein, orf12 , contains a pfam11418 motif, similar to phi29 scaffolding protein [ 58 ]. This is followed by orf13 , a putative major head protein containing a bacterial Ig-like domain (pfam02368) and orf14 , a head fiber protein and motif (pfam11133). The major tail protein ( orf15 ) contains a pfam16838 motif, conserved across groups of podoviruses [ 50 , 59 ]. The proteins that connect the phage head and tail are encoded by orf16 containing a pfam05352 [ 60 ], orf17 encoding a lower collar protein with the PHA00148 motif [ 61 , 62 ], and orf18 encoding a minor structural protein that is suspected to form the pre-neck appendage protein. Orf18 's closest homologues are found in other phi29-like phages WhyPhy and SRT01hs and a Staphylococcus phage ST134 with a conserved motif TIGR04523. This is followed by another morphogenesis protein, orf19 , with the characteristic motif pfam01551. A putative holin is encoded by orf20 with the pfam05105 motif conserved in bacteriophage holin proteins. Orf21 encodes an endolysin with two motifs, pfam01520 and pfam01476 [ 63 ]. The last gene in the module orf22 encodes a protein that is predicted to encode a podovirus DNA encapsidation protein with the characteristic motif pfam05894 [ 64 ]. 3.3. Whole Genome Comparison and Clustering of phi29-Like Phages Since the genome organization and protein homologies of PumA1 and PumA2 were reminiscent of phi29, we next investigated the evolutionary relationship shared between PumA1, PumA2, and other phi29-like phages. The GenBank and Bacillus phage databases were searched for complete phage genomes that shared genomic similarities to phi29. This led to the identification of 34 phages, including the two isolated in this study ( Table 1 ). Hatfull, et al. [ 65 ] previously described a classification system using a four-method approach to cluster 60 mycobacteriophage genomes. We employed their approach as a guide and included whole genome comparisons, network and clustering analysis, and candidate gene phylogenetics to organize the phi29-like phages into respective clusters ( Figure S1 ). The clusters were expanded from the ones previously described by Grose, Jensen, Burnett, and Breakwell [ 20 ] and employ the same naming style of B and subclusters numbered (e.g., B1). The number of clusters were expanded from three [ 20 ] to seven and three singleton phages. New members were added to existing clusters and align with the ICTV classifications. While no new clusters or potential genera were formed from this analysis, this study provides a justification for the ICTV taxonomic rankings and framework for future clustering and classification of phages. 3.4. Dot Plot Analysis and Genomic Identities Dot plot analysis of the 34 genomes revealed six clusters and two phages not pertaining to a cluster, referred to as singletons ( Figure S2 ). To further define the clusters and provide a numerical value to their similarities, we used the Virus Intergenomic Distance Calculator (VIRIDIC) to calculate intergenomic similarities between each phage. VIRIDIC combines several similarity algorithms with genome alignment and length ratios to capture overall relatedness of prokaryotic viruses [ 33 ]. In correlation with the dot plot analysis, the heatmap presents the 34 phages in the same clustering pattern ( Figure 3 ). Each cluster shares similarities of between 65.65–99.87% ( Table S2 ). The clusters are now referred to as from B1 to B7, expanding from previous clusters [ 20 ]. 3.5. Genome Map Alignments Each cluster was aligned to show the dissimilarities between genomes ( Figure 4 ). PumA1 and PumA2 contain an extra 796 bp and 1240 bp respectively compared to the ancestral phage phi29 in the 5′ early gene region. These hyperplastic regions are common throughout the phi29-like phages, where large insertions of up to 8256 bp are seen in the largest phi29-like phage, DLc1. Each cluster also shares the same pattern of insertions between them. The additional genes have no known functions but are presumed to be involved in the infection or replication processes since they are located within that region. Interestingly, the phages with genome sizes over 20 kb no longer contain the head fiber gene, a characteristic feature of phi29. The singletons show the least similarity to phi29 and other phages in this group, with small regions of similarity to their closest related phage. 3.6. Gene Sharing Networks Reticulate networks have been recently shown to provide an accurate representation of phage relatedness versus traditional rooted phylogenetics, since phages undergo many recombination and horizontal gene transfer events [ 66 ]. To test if these methods aligned with the comparison methods previously mentioned, two reticulate methods were used. Firstly, an unrooted phylogenetic network was created in Splitstree4 using whole genome CLUSTAL Omega alignment ( Figure 5 ). This network showed a consistent pattern of clustering in agreement with the other techniques. vConTACT v.2.0 was then used in conjunction with the Splitstree network. vConTACT v.2.0 is a newly developed software for virus classification that extracts, aligns, and clusters all predicted input proteins [ 37 ]. The protein clusters are then used to calculate viral clusters (VC) by am "edge" weight or statistical confidence due to the amount of protein clusters that each phage shares. This is compared to a global network of phages in the GenBank database. Figure 6 depicts the global network produced from vConTACT v.2.0 with various phages color coded by their host genera. The phi29-like phages are not connected to the main network, showing little gene-sharing outside of this group of phages. When the phi29-like network is expanded, there is a clear differentiation of each phage cluster, denoted by the individual cluster colors. While these phages do not connect to the main network, they share proteins with two other phages that are not infective for Bacillus , Lactococcus phage Asccphi28 [ 67 ] and Weisella phage phiYS61 [ 68 ], which are genotypically similar to the phi29-like phages. This form of reticulate phylogenetics helped corroborate the established clusters previously outlined and is an accurate tool for investigating phage gene-sharing and evolution. 3.7. Candidate Gene Analysis Finally, a phylogenetic tree was created combining two conserved and integral genes, DNA polymerase and DNA encapsidation ATPase ( Figure 7 ). The tree correlates with the whole genome comparison approaches as the phages are seen to group with their clusters. The tree splits into two distinctive branches with singletons DLc1 and MG-B1 and clusters B6 and B7 diverging from the rest of the clusters. This correlates with the genome sizes of these phages, as they are the largest genomes of the phi29-like group. The branch distances are also short within the clusters, particularly B6 members, signifying small base pair substitutions. The other factors that are seen to contribute to the phages' evolution are host species and country or region they were isolated. These clusters also agree with the ICTV taxonomic rankings as outlined by metadata on the tree. 3.8. PumA1 and PumA2 Host Receptor Site PumA1 and PumA2 displayed a narrow host range, only able to infect the B. pumilus strain in our collection. This appears to be a characteristic of the B4 cluster or Bundooravirus genus. It has been shown that phi29 requires polymerized teichoic acid for its attachment to B. subtilis [ 69 ]. Given the observed host range differences between phi29 and the two phages isolated in this study, we pursued an investigation into the host receptor of PumA1 and PumA2. B. pumilus colonies that developed stable resistance to PumA1 and PumA2 were isolated, and whole genome sequenced to determine which genomic modifications were causing resistance to phages. Mutations were found in either the tagF or tagT genes, which are a part of the teichoic acid synthesis operon [ 70 , 71 , 72 ]. Four of the seven variant strains isolated had modifications to the tagF gene, and the other three had mutations in the tagT gene ( Table S3 ). The majority of the mutants (A2M1, A2M11, A1M3, A2M14, and A1M5) contained frameshift mutations resulting in early termination of protein translation. Most of these frameshifts occurred near the N-terminus of the respective proteins likely resulting in non-functional TagT or TagF proteins. The remaining mutant had a single amino acid substitution in TagF (G688S). It is unclear how this mutation affects protein structure and function. 4. Discussion Advances in whole genome sequencing techniques and the rise of antibiotic resistance has resulted in an abundance of publicly available sequencing data and reinvigoration of phage-based studies. In this study, we isolated two novel, narrow host range Bacillus pumilus phages, PumA1 and PumA2. Both phages were unable to form plaques on B. pumilus strains that had mutations in the tagT or tagF genes. Wall teichoic acid (WTA) is a major component of Gram-positive cell walls, with the tag operons encoding the necessary machine for its synthesis [ 70 , 71 ]. TagF is a poly(glycerol phosphate) polymerase that plays a key role in the formation of the glycerol phosphate chains in WTA. Single nucleotide polymorphisms have been shown to significantly alter the function of TagF, resulting in decreased polymerase activity [ 71 ]. Whereas tagT , which encodes a LytR-CspA-Psr (LCP) family protein, is implicated in the final stage of WTA synthesis, in which it catalyzes the attachment between the teichoic acid polymers and peptidoglycan. In B. subtilis mutants in which the tagTUV operon was knocked out, cells appear more rounded and lack the typical rod shape and teichoic acid present in growth cultures but are detached from peptidoglycan [ 70 ]. Given that both phages studied here and phi29 appear to share the same surface receptor, we hypothesize that the Bundooravirus phages have differentiated tail structures to specifically detect B. pumilus teichoic acid. The Salasmaviridae (or phi29-like) group contain the smallest known genome sizes of phages that infect Bacillus sp. The highly conserved phages of this family are distributed throughout several continents, with PumA1 and PumA2 currently the only phages isolated from Australia. The multi-approach clustering indicated a correlation of Salasmaviridae phages by host range and genome size. This was aptly demonstrated with the Bundooravirus phages isolated in this study, which appeared to have a narrow host range against B. pumilus . We also noticed a correlation of cluster formation with the geographical location of phage isolation. This pattern is likely driven by the host bacterium's biogeography and population; this may become clearer as more Salasmaviridae phages are isolated and sequenced [ 72 , 73 ]. Since the Salasmaviridae phages follow a strict lytic lifecycle with no evidence of lysogenic activity, there may be low gene-content flux and recombination event rates between themselves, their hosts, and other phages [ 74 ]. However, environmental and host pressures can naturally result in genomic mutations, and as a phage gains new adaptations such as expanded host range, this can result in enough genetic variance to be included as a new species and genus [ 19 , 66 , 75 ]. While the phage genomes remain well conserved in their arrangement and modules of essential genes, there appears to be three main regions where insertions are present, all in the "early" replication gene modules. These additional genes correlate to increased genome size and how the phages cluster, reminiscent of hyperplastic regions seen in other groups of phages [ 76 ]. While most of these early genes have unknown functions, it is hypothesized that early proteins are associated with phage-host interactions. This includes genes that encode for protection from host degradation and restriction, anti-CRISPR, and inhibition of host transcription [ 77 ]. Though they are not essential for phage function, and can be lost and gained readily, they have the potential to be advantageous for adaption to their host [ 78 ]. Mutations leading to increased genome and capsid sizes tend to be more favorable and have been conserved throughout the Northopvirinae phages [ 79 ]. In contrast, the structural or "late" gene modules remain almost identical in arrangement and constitution throughout the Salasmaviridae phages. One notable difference seen in the "late genes" region is that any phage with a genome larger than 20 kb had lost the "head fiber" gene. The head fibers are involved in sensing and interact with the bacteria cell wall but are not essential for phage viability [ 80 , 81 ]. Phages that have acquired new genes are seen to have an enlarged capsid and altered capsid architecture in response [ 79 ]. It is hypothesized that since the fibers have no essential functions and the addition of newer genes has either forced or allowed these phages to expand their capsids, the head fibers are no longer able to attach to this new capsid structure. Nevertheless, this needs to be investigated further. 5. Conclusions PumA1 and PumA2 represent a novel genus in the newly formed Salasmaviridae family, Bundooravirus . Multiple clustering approaches, including reticulate networks, resulted in the clustering of all current phi29-like phages, including those recently classified by the ICTV. This clustering agrees with taxonomic rankings and allows for the addition of several phages into this family. Thornton and Baseball_field should be included in the Claudivirus genus, BSTP4 should be classified in the Salasvirus genus, and DLc1 should be classified into the Northopvirinae sub-family but in its own genus. WhyPhy should be classified into the Bundooravirus genus with PumA1 and PumA2. Current data suggests the Bundooravirus phages are unique in their host range and require B. pumilus specific teichoic acid residues for infection. The Salasmaviridae (or phi29-like) phages are globally distributed but remain well conserved in genome organization and protein domains. However, there are three patterns of clustering that contribute to their evolution and classification, including geographical location, host range, and genome size. This study demonstrated that a combination of whole genome comparisons and rooted and reticulate phylogenetic models can be used to our advantage to order and classify phages. Understanding phage evolution and their relationships with other phages and the environment will provide us with invaluable information into phage phylogenetics as their usage in medical and industrial processes continues.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5799738/

Catalogue of Tenebrionidae ( Coleoptera ) of North America

Abstract This catalogue includes all valid family-group (8 subfamilies, 52 tribes, 14 subtribes), genus-group (349 genera, 86 subgenera), and species-group names (2825 species, 215 subspecies) of darkling beetles ( Coleoptera : Tenebrionidae ) known to occur in North America 1 and their available synonyms. Data on extant, subfossil and fossil taxa are given. For each name the author and year and page number of the description are provided, with additional information (e.g., type species for genus-group names, author of synonymies for invalid taxa) depending on the taxon rank. Several new nomenclatural acts are included. One new genus, Lepidocnemeplatia Bousquet and Bouchard, is described. Spelaebiosis Bousquet and Bouchard [for Ardoinia Özdikmen, 2004], Blapstinus marcuzzii Aalbu [for Blapstinus kulzeri Marcuzzi, 1977], and Hymenorus campbelli Bouchard [for Hymenorus oculatus Doyen and Poinar, 1994] are proposed as new replacement names. Supporting evidence is provided for the conservation of usage of Tarpela micans (Fabricius, 1798) nomen protectum over Tarpela vittata (Olivier, 1793) nomen oblitum . The generic names Psilomera Motschulsky, 1870 [= Stenomorpha Solier, 1836], Steneleodes Blaisdell, 1909 [= Xysta Eschscholtz, 1829], Ooconibius Casey, 1895 and Euconibius Casey, 1895 [= Conibius LeConte, 1851] are new synonyms (valid names in square brackets). The following 127 new synonymies of species-group names, listed in their original combination, are proposed (valid names, in their current combination, placed in square brackets): Bothrasida mucorea Wilke, 1922 [= Pelecyphorus guanajuatensis (Champion, 1884)]; Parasida zacualpanicola Wilke, 1922 [= Pelecyphorus asidoides Solier, 1836]; Stenosides kulzeri Pallister, 1954, Stenosides bisinuatus Pallister, 1954, and Parasida trisinuata Pallister, 1954 [= Pelecyphorus dispar (Champion, 1892)]; Asida favosa Champion, 1884 and Asida similata Champion, 1884 [= Pelecyphorus fallax (Champion, 1884)]; Ologlyptus bicarinatus Champion, 1884 [= Pelecyphorus indutus (Champion, 1884)]; Parasida laciniata Casey, 1912 and Parasida cristata Pallister, 1954 [= Pelecyphorus liratus (LeConte, 1854)]; Parasida esperanzae Wilke, 1922 and Parasida mixtecae Wilke, 1922 [= Pelecyphorus longipennis (Champion, 1884)]; Parasida tolucana Casey, 1912 [= Pelecyphorus scutellaris (Champion, 1884)]; Parasida purpusi Wilke, 1922 [= Pelecyphorus tristis (Champion, 1884)]; Astrotus nosodermoides Champion, 1892 [= Pelecyphorus erosus (Champion, 1892)]; Astrotus seticornis var. humeralis Champion, 1884 [= Pelecyphorus seticornis (Champion, 1884)]; Pactostoma breviuscula Casey, 1912, Pactostoma exoleta Casey, 1912, Pactostoma luteotecta Casey, 1912, Pactostoma monticola Casey, 1912, Pactostoma obtecta Casey, 1912, and Pactostoma sigillata Casey, 1912 [= Pelecyphorus anastomosis (Say, 1824)]; Ologlyptus canus Champion, 1884 and Ologlyptus sinuaticollis Champion, 1884 [= Pelecyphorus graciliformis (Solier, 1836)]; Gonasida elata reducta Casey, 1912, Gonasida elata prolixa Casey, 1912, and Gonasida aucta Casey, 1912 [= Philolithus elatus compar (Casey, 1912)]; Gonasida alaticollis Casey, 1912 [= Philolithus elatus difformis (LeConte, 1854)]; Gonasida gravida Casey, 1912 [= Philolithus elatus elatus (LeConte, 1853)]; Pelecyphorus aegrotus limbatus Casey, 1912 [= Philolithus aegrotus aegrotus (LeConte, 1861)]; Pelecyphorus corporalis Casey, 1912, Pelecyphorus reptans Casey, 1912, Pelecyphorus socer Casey, 1912, Pelecyphorus abscissus Casey, 1912, Pelecyphorus fumosus Casey, 1912, Pelecyphorus parvus Casey, 1912, Pelecyphorus morbillosus pacatus Casey, 1912, Pelecyphorus morbillosus sobrius Casey, 1912, Pelecyphorus piceus Casey, 1912, Pelecyphorus piceus crudelis Casey, 1912, Pelecyphorus snowi Casey, 1912, and Pelecyphorus subtenuis Casey, 1912 [= Philolithus morbillosus (LeConte, 1858)]; Bothrasida sanctae-agnae Wilke, 1922 [= Stenomorpha funesta (Champion, 1884)]; Asida flaccida Horn, 1896 [= Stenomorpha embaphionides (Horn, 1894)]; Asida angustula Casey, 1890, Stethasida stricta Casey, 1912, Stethasida muricatula languida Casey, 1912, Stethasida pertinax Casey, 1912, Stethasida socors Casey, 1912, Stethasida angustula inepta Casey, 1912, Stethasida tenax Casey, 1912, and Stethasida vegrandis Casey, 1912 [= Stenomorpha muricatula (LeConte, 1851)]; Stethasida obsoleta expansa Casey, 1912, Stethasida obsoleta opacella Casey, 1912, Stethasida brevipes Casey, 1912, Stethasida torpida Casey, 1912, Stethasida convergens Casey, 1912, Stethasida discreta Casey, 1912, Stethasida longula Casey, 1912, Stethasida adumbrata Casey, 1912, Stethasida occulta Casey, 1912, Stethasida tarsalis Casey, 1912, Stethasida unica Casey, 1912, and Pelecyphorus laevigatus Papp, 1961 [= Stenomorpha obsoleta (LeConte, 1851)]; Trichiasida eremica Wilke, 1922 [= Stenomorpha difficilis (Champion, 1884)]; Trichiasida lineatopilosa Casey, 1912 [= Stenomorpha hirsuta (LeConte, 1851)]; Trichiasida tenella Casey, 1912 [= Stenomorpha hispidula (LeConte, 1851)]; Trichiasida duplex Casey, 1912 [= Stenomorpha villosa (Champion, 1884)]; Alaudes squamosa Blaisdell, 1919, Alaudes testacea Blaisdell, 1919, and Alaudes fallax Fall, 1928 [= Alaudes singularis Horn, 1870]; Edrotes barrowsi Dajoz, 1999 [= Edrotes ventricosus LeConte, 1851]; Nyctoporis tetrica Casey, 1907 and Nyctoporis maura Casey, 1907 [= Nyctoporis aequicollis Eschscholtz, 1831]; Nyctoporis pullata Casey, 1907 [= Nyctoporis sponsa Casey, 1907]; Eleodes tibialis forma oblonga Blaisdell, 1909 [= Eleodes tibialis Blaisdell, 1909]; Eleodes ( manni var.) variolosa Blaisdell, 1917 [= Eleodes constrictus LeConte, 1858]; Eleodes cordata forma sublaevis Blaisdell, 1909, Eleodes cordata forma intermedia Blaisdell, 1909, Eleodes cordata forma oblonga Blaisdell, 1909, Eleodes cordata forma elongata Blaisdell, 1909, and Eleodes ( cordata var.) adulterina Blaisdell, 1917 [= Eleodes cordata Eschscholtz, 1829]; Eleodes hornii var. monticula Blaisdell, 1918 and Eleodes manni sierra Blaisdell, 1925 [= Eleodes fuchsii Blaisdell, 1909]; Eleodes parvicollis var. squalida Blaisdell, 1918 [= Eleodes parvicollis Eschscholtz, 1829]; Eleodes reflexicollis Mannerheim, 1843 and Eleodes parvicollis forma farallonica Blaisdell, 1909 [= Eleodes planata Eschscholtz, 1829]; Eleodes indentata Blaisdell, 1935 [= Eleodes rotundipennis LeConte, 1857]; Eleodes intricata Mannerheim, 1843 [= Eleodes scabrosa Eschscholtz, 1829]; Eleodes horni fenyesi Blaisdell, 1925 [= Eleodes tenebrosa Horn, 1870]; Eleodes cordata var. horrida Blaisdell, 1918 [= Eleodes tuberculata Eschscholtz, 1829]; Eleodes oblonga Blaisdell, 1933 [= Eleodes versatilis Blaisdell, 1921]; Eleodes dentipes marinae Blaisdell, 1921 [= Eleodes dentipes Eschscholtz, 1829]; Eleodes carbonaria forma glabra Blaisdell, 1909 [= Eleodes carbonaria carbonaria (Say, 1824)]; Eleodes granosa forma fortis Blaisdell, 1909 [= Eleodes granosa LeConte, 1866]; Eleodes pilosa forma ordinata Blaisdell, 1909 [= Eleodes pilosa Horn, 1870]; Trogloderus costatus pappi Kulzer, 1960 [= Trogloderus tuberculatus Blaisdell, 1909]; Trogloderus costatus mayhewi Papp, 1961 [= Trogloderus vandykei La Rivers, 1946]; Bolitophagus cristatus Gosse, 1840 [= Bolitotherus cornutus (Fabricius, 1801)]; Eleates explanatus Casey, 1890 [= Eleates depressus (Randall, 1838)]; Blapstinus sonorae Casey, 1890 [= Blapstinus brevicollis LeConte, 1851]; Blapstinus falli Blaisdell, 1929 [= Blapstinus castaneus Casey, 1890]; Blapstinus brunneus Casey, 1890 and Blapstinus coronadensis Blaisdell, 1892 [= Blapstinus histricus Casey, 1890]; Blapstinus hesperius Casey, 1890 [= Blapstinus intermixtus Casey, 1890]; Blapstinus cinerascens Fall, 1929 [= Blapstinus lecontei Mulsant and Rey, 1859]; Blapstinus niger Casey, 1890 and Blapstinus cribricollis Casey, 1890 [= Blapstinus pimalis Casey, 1885]; Blapstinus arenarius Casey, 1890 [= Blapstinus pratensis LeConte, 1859]; Blapstinus gregalis Casey, 1890 [= Blapstinus substriatus Champion, 1885]; Blapstinus hydropicus Casey, 1890 [= Blapstinus sulcatus LeConte, 1851]; Blapstinus hospes Casey, 1890 [= Blapstinus vestitus LeConte, 1859]; Notibius reflexus Horn, 1894 [= Conibius opacus (LeConte, 1866)]; Notibius affinis Champion, 1885 [= Conibius rugipes (Champion, 1885)]; Conibius parallelus LeConte, 1851 [= Conibius seriatus LeConte, 1851]; Nocibiotes rubripes Casey, 1895 [= Nocibiotes caudatus Casey, 1895]; Nocibiotes gracilis Casey, 1895 and Nocibiotes acutus Casey, 1895 [= Nocibiotes granulatus (LeConte, 1851)]; Conibius alternatus Casey, 1890 [= Tonibius sulcatus (LeConte, 1851)]; Pedinus suturalis Say, 1824 [= Alaetrinus minimus (Palisot de Beauvois, 1817)]; Menedrio longipennis Motschulsky, 1872 [= Tenebrio obscurus Fabricius, 1792]; Hymenophorus megops Hatch, 1965 and Telesicles magnus Hatch, 1965 [= Hymenorus sinuatus Fall, 1931]; Andrimus concolor Casey, 1891 and Andrimus convergens Casey, 1891 [= Andrimus murrayi (LeConte, 1866)]; Mycetochara marshalli Campbell, 1978 [= Mycetochara perplexata Marshall, 1970]; Phaleria globosa LeConte, 1857 [= Phaleria picta Mannerheim, 1843]. The following subspecies of Trogloderus costatus LeConte, 1879 are given species rank: Trogloderus nevadus La Rivers, 1943, Trogloderus tuberculatus Blaisdell, 1909, and Trogloderus vandykei La Rivers, 1946. The following taxa, previously thought to be junior synonyms, are considered valid: Amphidora Eschscholtz, 1829; Xysta Eschscholtz, 1829; Helops confluens (Casey, 1924). Two new combinations are proposed: Stenomorpha spinimana (Champion, 1892) and Stenomorpha tenebrosa (Champion, 1892) [from the genus Parasida Casey, 1912]. The type species [placed in square brackets] of the following 12 genus-group taxa are designated for the first time: Lagriola Kirsch, 1874 [ Lagriola operosa Kirsch, 1874]; Locrodes Casey, 1907 [ Emmenastus piceus Casey, 1890]; Falacer Laporte, 1840 [ Acanthopus cupreus Laporte, 1840 (= Helops contractus Palisot de Beauvois, 1812)]; Blapylis Horn, 1870 [ Eleodes cordata Eschscholtz, 1829]; Discogenia LeConte, 1866 [ Eleodes scabricula LeConte, 1858]; Metablapylis Blaisdell, 1909 [ Eleodes nigrina LeConte, 1858]; Steneleodes Blaisdell, 1909 [ Eleodes longicollis LeConte, 1851]; Scaptes Champion, 1886 [ Scaptes squamulatus Champion, 1886 (= Asida tropica Kirsch, 1866)]; Aspidius Mulsant and Rey, 1859 [ Blaps punctata Fabricius, 1792]; Cryptozoon Schaufuss, 1882 [ Cryptozoon civile Schaufuss, 1882]; Halophalerus Crotch, 1874 [ Phaleria rotundata LeConte, 1851]; Dignamptus LeConte, 1878 [ Dignamptus stenochinus LeConte, 1878]. Two species previously known from South America [ Nilio lebasi J. Thomson and Platydema erotyloides Chevrolat] are reported for the first time from North America. Citation Bousquet Y, Thomas DB, Bouchard P, Smith AD, Aalbu RL, Johnston AM, Steiner WE Jr. (2018) Catalogue of Tenebrionidae (Coleoptera) of North America. ZooKeys 728: 1–455. https://doi.org/10.3897/zookeys.728.20602 Introduction Darkling beetles ( Coleoptera : Tenebrionidae ) form a species-rich and morphologically diverse family with approximately 2300 genera and 20000 species worldwide ( Matthews et al. 2010 ), and many more taxa to be described. The first thorough classification of the Tenebrionidae was provided by Lacordaire (1859) and was based entirely on the external morphology of adults. With relatively few exceptions, his classification schema was followed by subsequent workers for approximately 100 years ( Watt 1967 ). The family classification was eventually reviewed using morphological characters of immature stages ( Watt 1975 ) although significant changes did not appear until the first comprehensive investigation of adult internal structures including defense glands, female ovipositor, and female genital tube ( Tschinkel and Doyen 1980 , Doyen and Tschinkel 1982 ). The first higher-level phylogeny of the family based on molecular data was published only recently ( Kergoat et al. 2014 ). As a result of these comparative and phylogenetic studies, several taxa previously treated as separate families (e.g., Lagriidae , Alleculidae , Nilionidae ) are now included within Tenebrionidae . Additionally, many taxa previously included in Tenebrionidae are now classified in other families (see Table 1 for North American genera, Aalbu (2006) for worldwide taxa). Table 1. List of North American genera previously included in Tenebrionidae but currently classified in another family. Genus Current placement Boros Herbst, 1797 Boridae Dacoderus LeConte, 1858 Salpingidae Megazopherus Casey, 1907 Zopheridae Meralius Casey, 1907 Zopheridae Noserodes Casey, 1907 Zopheridae Noserus LeConte, 1862 Zopheridae Nosoderma Solier, 1841 Zopheridae Phellopsis LeConte, 1862 Zopheridae Phloeodes LeConte, 1862 Zopheridae Sesaspis Casey, 1907 Zopheridae Usechus Motschulsky, 1845 Zopheridae Verodes Casey, 1907 Zopheridae Zopherinus Casey, 1907 Zopheridae Zopherodes Casey, 1907 Zopheridae Zopherus Laporte, 1840 Zopheridae The aim of this work is to synthesize available taxonomic, nomenclatural, and distributional information for all darkling beetles known from North America. Methods Nomenclatural data All nomenclaturally available family-, genus- and species-group names are included. Extant taxa and subfossils from the Pleistocene (see Doyen and Miller 1980 , Doyen and Poinar 1994 ) are given in the main catalogue. Impression fossils from major North American deposits are listed in Appendix 1, although the taxonomic assignment of these often-fragmentary fossils needs to be confirmed. Fossil species described from amber are listed in Appendix 2. Taxa incorrectly recorded from North America are given in Appendix 3. Subfamilies are listed in a phylogenetic framework but valid tribal, generic, and specific names are given in alphabetic order; listings of all invalid names are chronological. The author and year and page number of the original description are provided for each scientific name. The type genus for each family-group name and the type species and type fixation for each genus-group name are included. The reference in which a given generic or specific name is first placed in synonymy with the current valid name is listed. Type-species designations in Lucas (1920) were accepted when a single species was listed under a particular genus-group name (see Alonso-Zarazaga and Lyal 2009 , Bousquet et al. 2015 ). For genera with valid subgenera, synonyms are given under the nominotypical subgenus when relevant. Every species-group name are listed in its original combination, as given in the publication even if the agreement in gender with the generic name is incorrect. In cases where a species-group name older than the one currently recognized as valid is available but of doubtful application (e.g., Latridius pubescens Say, 1826), we have retained usage of the younger, accepted name as valid and treated the older name as nomen dubium . The synonymy list for adventive species focuses primarily on names used in the North American literature, other sources (e.g., Löbl et al. 2008a ) can be consulted for data on all invalid names. The classification used follows Bouchard et al. (2011) but also includes corrections and subsequent additions. The gender of all valid genera listed in the catalogue has been determined following the provision of Article 30 ( ICZN 1999 ) and indicated after the name using the initials M [Masculine], F [Feminine], and N [Neuter]. Therefore, the gender of the generic names Liodema , Platydema , and Scaphidema is herein treated as feminine following Article 30.1.3 ( ICZN 1999 ; see Kerzhner (2003) and Löbl and Smetana (2010 : 34) for further comments); the ending is derived from the Greek " demas (body silhouette)." The gender of Alaudes is treated as masculine following Article 30.2.4 ( ICZN 1999 ). The gender of Eleodes , originally treated as feminine by Eschscholtz (1829) , was changed to masculine by Somerby and Doyen (1976) and has been followed subsequently in the literature. However, Eleodes is feminine following Article 30.1.4.4 ( ICZN 1999 ) which says that "a compound genus-group name ending in the suffix ... - odes is to be treated as masculine unless its author, when establishing the name, stated that it had another gender or treated it as such by combining it with an adjectival species-group name in another gender form." If necessary, the ending of all valid species-group names has been modified according to the gender of the generic name with which the species is currently combined. All specific names that are nouns in apposition need not agree in gender with the generic name and retain their original endings. The author(s) of every new nomenclatural act proposed in the catalogue is given in square brackets (e.g., "[ADS]") except for the first typification of genus-group names. One of these new acts needs further development. The genus-group taxon Lepidocnemeplatia was proposed by Kaszab (1938 : 80) as a subgenus of Cnemeplatia Costa, 1847 to include two species, Cnemeplatia laticollis Champion, 1885 and C. sericea Horn, 1870. Unfortunately, Kaszab did not designate a type species for his new genus and therefore the name is unavailable from that date ( ICZN 1999 : Article 13.3). Löbl and Merkl (2003 : 245) designated C. sericea Horn, 1870 as type species of Lepidocnemeplatia , the first typification for the taxon, and subsequently Löbl et al. (2008a : 140) credited authorship of the name to Löbl and Merkl (2003) . However, since Löbl and Merkl (2003 : 245) failed to indicate that they were establishing a new nominal taxon, a mandatory requirement for all new names published after 1999 ( ICZN 1999 : Article 16.1), the name cannot be attributed to them and is still a nomen nudum . In order to make the name available we here proposed the generic name Lepidocnemeplatia 2 Bousquet and Bouchard, new genus; type species (here designated): Cnemeplatia sericea Horn, 1870. The reader is referred to Kaszab (1938 : 79–80, couplet 1" of his key) for a description of the character states that differentiate the taxon ( ICZN 1999 : Article 13.1.2). A summary of all new nomenclatural acts is available in the Abstract. Distributional data This catalogue documents all species of Tenebrionidae from Greenland, Alaska, and Canada south to Panama, and also includes islands of the West Indies. Records from the Netherlands Antilles (consisting of several islands in the West Indies), the Venezuelan island Margarita, and Trinidad and Tobago off the northeast coast of Venezuela are not included since the fauna of these islands is more closely affiliated with the South American fauna. When known, the states (for Mexico and continental United States) and the provinces and territories (for Canada) are listed in parentheses for each species. For West Indian records, we give the political unit names for species found in the Lucayan Archipelago and Greater Antilles, occasionally with specific islands in parenthesis; political unit names are not provided for species occurring in the Lesser Antilles ( LAN ) and Virgin Islands ( VIS ), though sometimes we include specific islands in parentheses, especially when the species is known from only one or two islands. Further details regarding the West Indies geographical units can be found in Ivie and Hart (2016 : Table 1 ). Many Mexican state records in this catalogue came from localities listed by Champion in volume IV, parts 1 and 2, of the Biologia Centrali-Americana [1884–1893] and the gazetteer of Selander and Vaurie (1962) was used consistently to associate localities with states (including the federal district). Amongst the geographical units used (see list below), HIS (for Hispaniola) is used when only the island record is known and LC (for Lower California) when only the peninsula record is known. South America ( SA ) is placed at the end of a species record list to indicate that the species extends into South America. Distributional records listed in square brackets (e.g., "[NM]") are considered doubtful. South American species introduced accidentally into North America are indicated with a subscript " i " beside the country record (i.e., " USA i "). Bibliographic data References are provided for all scientific names included if a page number (related to the description of the taxon) is provided after the year of publication. Based on evidence previously published (see Bousquet 2016a : 211, 265), the dates of publication of Germar's Coleopterorum species novae dated 1824 on its title page, and of Hope's The Coleopterist's manual, part the third , dated 1840 on its title page, are given as 1823 and 1841 respectively. As discussed by Bousquet (2016b) , Mäklin's "Monographie der Gattung Strongylium " was published in 1867, not 1864 as given by several authors. List of acronyms used for geographic units 3 BAH Bahamas BEL Belize BER Bermuda CAN Canada [AB: Alberta; BC: British Columbia; MB: Manitoba; NB: New Brunswick; NF: Newfoundland and Labrador; NS: Nova Scotia; NT: Northwest Territories; NU: Nunavut; ON: Ontario; PE: Prince Edward Island; QC: Quebec; SK: Saskatchewan; YT: Yukon Territory] CAY Cayman Islands CRI Costa Rica CUB Cuba DOM Dominican Republic GRE Greenland GUA Guatemala HAI Haiti HIS Hispaniola HON Honduras JAM Jamaica LAN Lesser Antilles (including among others Anguilla, Antigua and Barbuda, Montserrat, Guadeloupe, Dominica, Martinique, Saint Lucia, Grenada, Barbados) LC Lower California MEX Mexico [AG: Aguascalientes; BC: Baja California; BS: Baja California Sur; CA: Campeche; CH: Chihuahua; CI: Chiapas; CL: Colima; CO: Coahuila; DU: Durango; FD: Federal District; GE: Guerrero; GU: Guanajuato; HI: Hidalgo; JA: Jalisco; ME: México; MI: Michoacán; MO: Morelos; NA: Nayarit; NL: Nuevo León; OA: Oaxaca; PU: Puebla; QR: Quintana Roo; QU: Querétaro; SI: Sinaloa; SL: San Luis Potosí; SO: Sonora; TA: Tamaulipas; TB: Tabasco; TL: Tlaxcala; VE: Veracruz; YU: Yucatán; ZA: Zacatecas] NIC Nicaragua PAN Panama PRI Puerto Rico [includes Vieques] SAL El Salvador TUR Turks and Caicos Islands USA United States of America [AK: Alaska; AL: Alabama; AR: Arkansas; AZ: Arizona; CA: California; CO: Colorado; CT: Connecticut; DC: District of Columbia; DE: Delaware; FL: Florida; GA: Georgia; IA: Iowa; ID: Idaho; IL: Illinois; IN: Indiana; KS: Kansas; KY: Kentucky; LA: Louisiana; MA: Massachusetts; MD: Maryland; ME: Maine; MI: Michigan; MN: Minnesota; MO: Missouri; MS: Mississippi; MT: Montana; NC: North Carolina; ND: North Dakota; NE: Nebraska; NH: New Hampshire; NJ: New Jersey; NM: New Mexico; NV: Nevada; NY: New York; OH: Ohio; OK: Oklahoma; OR: Oregon; PA: Pennsylvania; RI: Rhode Island; SC: South Carolina; SD: South Dakota; TN: Tennessee; TX: Texas; UT: Utah; VA: Virginia; VT: Vermont; WA: Washington; WI: Wisconsin; WV: West Virginia; WY: Wyoming] VIS Virgin Islands [includes US Virgin Islands and British Virgin Islands: Saint Thomas, Saint Croix] SA South America Nomenclatural data All nomenclaturally available family-, genus- and species-group names are included. Extant taxa and subfossils from the Pleistocene (see Doyen and Miller 1980 , Doyen and Poinar 1994 ) are given in the main catalogue. Impression fossils from major North American deposits are listed in Appendix 1, although the taxonomic assignment of these often-fragmentary fossils needs to be confirmed. Fossil species described from amber are listed in Appendix 2. Taxa incorrectly recorded from North America are given in Appendix 3. Subfamilies are listed in a phylogenetic framework but valid tribal, generic, and specific names are given in alphabetic order; listings of all invalid names are chronological. The author and year and page number of the original description are provided for each scientific name. The type genus for each family-group name and the type species and type fixation for each genus-group name are included. The reference in which a given generic or specific name is first placed in synonymy with the current valid name is listed. Type-species designations in Lucas (1920) were accepted when a single species was listed under a particular genus-group name (see Alonso-Zarazaga and Lyal 2009 , Bousquet et al. 2015 ). For genera with valid subgenera, synonyms are given under the nominotypical subgenus when relevant. Every species-group name are listed in its original combination, as given in the publication even if the agreement in gender with the generic name is incorrect. In cases where a species-group name older than the one currently recognized as valid is available but of doubtful application (e.g., Latridius pubescens Say, 1826), we have retained usage of the younger, accepted name as valid and treated the older name as nomen dubium . The synonymy list for adventive species focuses primarily on names used in the North American literature, other sources (e.g., Löbl et al. 2008a ) can be consulted for data on all invalid names. The classification used follows Bouchard et al. (2011) but also includes corrections and subsequent additions. The gender of all valid genera listed in the catalogue has been determined following the provision of Article 30 ( ICZN 1999 ) and indicated after the name using the initials M [Masculine], F [Feminine], and N [Neuter]. Therefore, the gender of the generic names Liodema , Platydema , and Scaphidema is herein treated as feminine following Article 30.1.3 ( ICZN 1999 ; see Kerzhner (2003) and Löbl and Smetana (2010 : 34) for further comments); the ending is derived from the Greek " demas (body silhouette)." The gender of Alaudes is treated as masculine following Article 30.2.4 ( ICZN 1999 ). The gender of Eleodes , originally treated as feminine by Eschscholtz (1829) , was changed to masculine by Somerby and Doyen (1976) and has been followed subsequently in the literature. However, Eleodes is feminine following Article 30.1.4.4 ( ICZN 1999 ) which says that "a compound genus-group name ending in the suffix ... - odes is to be treated as masculine unless its author, when establishing the name, stated that it had another gender or treated it as such by combining it with an adjectival species-group name in another gender form." If necessary, the ending of all valid species-group names has been modified according to the gender of the generic name with which the species is currently combined. All specific names that are nouns in apposition need not agree in gender with the generic name and retain their original endings. The author(s) of every new nomenclatural act proposed in the catalogue is given in square brackets (e.g., "[ADS]") except for the first typification of genus-group names. One of these new acts needs further development. The genus-group taxon Lepidocnemeplatia was proposed by Kaszab (1938 : 80) as a subgenus of Cnemeplatia Costa, 1847 to include two species, Cnemeplatia laticollis Champion, 1885 and C. sericea Horn, 1870. Unfortunately, Kaszab did not designate a type species for his new genus and therefore the name is unavailable from that date ( ICZN 1999 : Article 13.3). Löbl and Merkl (2003 : 245) designated C. sericea Horn, 1870 as type species of Lepidocnemeplatia , the first typification for the taxon, and subsequently Löbl et al. (2008a : 140) credited authorship of the name to Löbl and Merkl (2003) . However, since Löbl and Merkl (2003 : 245) failed to indicate that they were establishing a new nominal taxon, a mandatory requirement for all new names published after 1999 ( ICZN 1999 : Article 16.1), the name cannot be attributed to them and is still a nomen nudum . In order to make the name available we here proposed the generic name Lepidocnemeplatia 2 Bousquet and Bouchard, new genus; type species (here designated): Cnemeplatia sericea Horn, 1870. The reader is referred to Kaszab (1938 : 79–80, couplet 1" of his key) for a description of the character states that differentiate the taxon ( ICZN 1999 : Article 13.1.2). A summary of all new nomenclatural acts is available in the Abstract. Distributional data This catalogue documents all species of Tenebrionidae from Greenland, Alaska, and Canada south to Panama, and also includes islands of the West Indies. Records from the Netherlands Antilles (consisting of several islands in the West Indies), the Venezuelan island Margarita, and Trinidad and Tobago off the northeast coast of Venezuela are not included since the fauna of these islands is more closely affiliated with the South American fauna. When known, the states (for Mexico and continental United States) and the provinces and territories (for Canada) are listed in parentheses for each species. For West Indian records, we give the political unit names for species found in the Lucayan Archipelago and Greater Antilles, occasionally with specific islands in parenthesis; political unit names are not provided for species occurring in the Lesser Antilles ( LAN ) and Virgin Islands ( VIS ), though sometimes we include specific islands in parentheses, especially when the species is known from only one or two islands. Further details regarding the West Indies geographical units can be found in Ivie and Hart (2016 : Table 1 ). Many Mexican state records in this catalogue came from localities listed by Champion in volume IV, parts 1 and 2, of the Biologia Centrali-Americana [1884–1893] and the gazetteer of Selander and Vaurie (1962) was used consistently to associate localities with states (including the federal district). Amongst the geographical units used (see list below), HIS (for Hispaniola) is used when only the island record is known and LC (for Lower California) when only the peninsula record is known. South America ( SA ) is placed at the end of a species record list to indicate that the species extends into South America. Distributional records listed in square brackets (e.g., "[NM]") are considered doubtful. South American species introduced accidentally into North America are indicated with a subscript " i " beside the country record (i.e., " USA i "). Bibliographic data References are provided for all scientific names included if a page number (related to the description of the taxon) is provided after the year of publication. Based on evidence previously published (see Bousquet 2016a : 211, 265), the dates of publication of Germar's Coleopterorum species novae dated 1824 on its title page, and of Hope's The Coleopterist's manual, part the third , dated 1840 on its title page, are given as 1823 and 1841 respectively. As discussed by Bousquet (2016b) , Mäklin's "Monographie der Gattung Strongylium " was published in 1867, not 1864 as given by several authors. List of acronyms used for geographic units 3 BAH Bahamas BEL Belize BER Bermuda CAN Canada [AB: Alberta; BC: British Columbia; MB: Manitoba; NB: New Brunswick; NF: Newfoundland and Labrador; NS: Nova Scotia; NT: Northwest Territories; NU: Nunavut; ON: Ontario; PE: Prince Edward Island; QC: Quebec; SK: Saskatchewan; YT: Yukon Territory] CAY Cayman Islands CRI Costa Rica CUB Cuba DOM Dominican Republic GRE Greenland GUA Guatemala HAI Haiti HIS Hispaniola HON Honduras JAM Jamaica LAN Lesser Antilles (including among others Anguilla, Antigua and Barbuda, Montserrat, Guadeloupe, Dominica, Martinique, Saint Lucia, Grenada, Barbados) LC Lower California MEX Mexico [AG: Aguascalientes; BC: Baja California; BS: Baja California Sur; CA: Campeche; CH: Chihuahua; CI: Chiapas; CL: Colima; CO: Coahuila; DU: Durango; FD: Federal District; GE: Guerrero; GU: Guanajuato; HI: Hidalgo; JA: Jalisco; ME: México; MI: Michoacán; MO: Morelos; NA: Nayarit; NL: Nuevo León; OA: Oaxaca; PU: Puebla; QR: Quintana Roo; QU: Querétaro; SI: Sinaloa; SL: San Luis Potosí; SO: Sonora; TA: Tamaulipas; TB: Tabasco; TL: Tlaxcala; VE: Veracruz; YU: Yucatán; ZA: Zacatecas] NIC Nicaragua PAN Panama PRI Puerto Rico [includes Vieques] SAL El Salvador TUR Turks and Caicos Islands USA United States of America [AK: Alaska; AL: Alabama; AR: Arkansas; AZ: Arizona; CA: California; CO: Colorado; CT: Connecticut; DC: District of Columbia; DE: Delaware; FL: Florida; GA: Georgia; IA: Iowa; ID: Idaho; IL: Illinois; IN: Indiana; KS: Kansas; KY: Kentucky; LA: Louisiana; MA: Massachusetts; MD: Maryland; ME: Maine; MI: Michigan; MN: Minnesota; MO: Missouri; MS: Mississippi; MT: Montana; NC: North Carolina; ND: North Dakota; NE: Nebraska; NH: New Hampshire; NJ: New Jersey; NM: New Mexico; NV: Nevada; NY: New York; OH: Ohio; OK: Oklahoma; OR: Oregon; PA: Pennsylvania; RI: Rhode Island; SC: South Carolina; SD: South Dakota; TN: Tennessee; TX: Texas; UT: Utah; VA: Virginia; VT: Vermont; WA: Washington; WI: Wisconsin; WV: West Virginia; WY: Wyoming] VIS Virgin Islands [includes US Virgin Islands and British Virgin Islands: Saint Thomas, Saint Croix] SA South America Results Overall diversity A total of 128 valid and invalid family-group, 612 genus-group, and 4065 species-group taxa (excluding fossils listed in Appendices 1, 2) are listed in this catalogue. The subfamily Pimeliinae is the most diverse with 908 valid species-group taxa, followed by the Tenebrioninae (808), Alleculinae (418), Stenochiinae (361), Diaperinae (260), Lagriinae (256), Phrenapatinae (22), and Nilioninae (6). Thirty-seven species in three subfamilies are adventive (Table 2 ), several of which are pests of stored grain products. Mexico and the continental United States of America are by far the most diverse political regions with 1224 and 1230 valid species-group taxa respectively, while at the other extreme Greenland and Bermuda only have two each (Table 3 ). Table 2. List of adventive species documented in North America. Data on the origin, date of first detection in North America, and microhabitat associations in nature are given for each species as far as known. * = pest of stored grain products. Species Origin Date of detection Microhabitat Placement Alphitobius diaperinus (Panzer, 1797) Africa <1866 *Animal nests, caves, guano Tenebrioninae : Alphitobiini Alphitobius laevigatus (Fabricius, 1781) Africa <1866 *Animal nests, caves, guano Tenebrioninae : Alphitobiini Alphitophagus bifasciatus (Say, 1824) Europe, probably <1824 *Animal nests, caves, guano Diaperinae : Diaperini : Adelinina Anchophthalmops menouxi (Mulsant and Rey, 1853) Africa; probably not established <1870 Leaf litter on sandy soil, probably Tenebrioninae : Pedinini : Platynotina Blaps ( Blaps ) lethifera lethifera Marsham, 1802 Europe <1889 Under rocks, wood, in caves Tenebrioninae : Blaptini : Blaptina Blaps ( Blaps ) mucronata Latreille, 1804 Europe <1889 Under rocks, wood, in caves Tenebrioninae : Blaptini : Blaptina Ceropria induta (Wiedemann, 1819) Asia 1998 Polypore fungi, rotten wood Diaperinae : Diaperini : Diaperina Ellipsodes ( Anthrenopsis ) ziczac (Motschulsky, 1873) Asia, probably via Antilles 1891 Under leaf litter on sandy soil Diaperinae : Crypticini Gnatocerus ( Echocerus ) maxillosus (Fabricius, 1801) Asia, probably via Europe <1866 *Animal nests, caves, under bark Diaperinae : Diaperini : Adelinina Gnatocerus ( Gnatocerus ) cornutus (Fabricius, 1798) Asia, probably via Europe <1866 *Animal nests, caves, under bark Diaperinae : Diaperini : Adelinina Gondwanocrypticus pictus (Gebien, 1928) South America 1954 Under leaf litter near ant nests Diaperinae : Crypticini Gondwanocrypticus platensis (Fairmaire, 1884) South America 1929 Under leaf litter near ant nests Diaperinae : Crypticini Gonocephalum ( Gonocephalum ) sericeum (Baudi di Selve, 1875) Northwest Africa + Arabian Peninsula 1980 Under leaf litter, wood, rocks Tenebrioninae : Opatrini : Opatrina Latheticus oryzae Waterhouse, 1880 Old World 1908 *Animal nests, caves, under bark Tenebrioninae : Triboliini Leichenum canaliculatum variegatum (Klug, 1833) Madagascar 1906 Under leaf litter on sandy soil Tenebrioninae : Pedinini : Leichenina Lyphia tetraphylla (Fairmaire, 1857) Europe <1902 In dead wood, other insect burrows Tenebrioninae : Triboliini Myrmechixenus lathridioides Crotch, 1873 Europe <1883 Under leaf litter, in soil Diaperina : Myrmechixenini Opatroides punctulatus Brulle, 1832 Middle East 2003 Under leaf litter, wood, rocks Tenebrioninae : Opatrini : Opatrina Palorus cerylonoides (Pascoe, 1863) Indo-Malayan, probably 2004 Under bark dry wood, plant debris Tenebrioninae : Palorini Palorus genalis Blair, 1930 Old World 1937 Under bark dry wood, plant debris Tenebrioninae : Palorini Palorus ratzeburgii (Wissmann, 1848) North Africa, probably <1897 *Under bark dry wood, plant debris Tenebrioninae : Palorini Palorus subdepressus (Wollaston, 1864) Africa, probably <1882 *Under bark dry wood, plant debris Tenebrioninae : Palorini Pentaphyllus testaceus (Hellwig, 1792) Europe 2005 Polypore fungi, under bark Diaperinae : Diaperini : Diaperina Platydema woldai Triplehorn and Phillips, 1998 Central America, probably 1964 With orchid plants; at lights in forest Diaperinae : Diaperini : Diaperina Plesiophthalmus spectabilis Harold, 1875 Asia; probably not established 2013 Rotten wood Tenebrioninae : Amarygmini Poecilocrypticus formicophilus Gebien, 1928 South America 1978 Under leaf litter near ant nests Diaperinae : Crypticini Strongylium cultellatum Maklin, 1867 Asia 2010 Dead standing wood Stenochiinae : Stenochiini Tenebrio molitor Linnaeus, 1758 Africa, probably <1837 *Animal nests, caves Tenebrioninae : Tenebrionini Tenebrio obscurus Fabricius, 1792 Africa, probably <1869 *Animal nests, caves Tenebrioninae : Tenebrionini Trachyscelis aphodioides Latreille, 1809 Europe <1846 Under plant debris on beach sand Diaperinae : Trachyscelini Tribolium ( Tribolium ) castaneum (Herbst, 1797) Africa, probably <1866 *Under bark dry wood, plant debris Tenebrioninae : Triboliini Tribolium ( Tribolium ) confusum Jacquelin du Val, 1862 Africa, probably <1893 *Under bark dry wood, plant debris Tenebrioninae : Triboliini Tribolium ( Tribolium ) destructor Uyttenboogaart, 1934 Africa <1948 *Under bark dry wood, plant debris Tenebrioninae : Triboliini Tribolium ( Tribolium ) madens (Charpentier, 1825) Africa, probably <1866 *Under bark dry wood, plant debris Tenebrioninae : Triboliini Tyrtaeus dobsoni Hinton, 1947 Unknown; probably via Europe 2002 Under bark and in dead wood Diaperinae : Gnathidiini : Anopidiina Ulomina carinata Baudi di Selve, 1876 Asia 1952 Under bark dry wood, plant debris Tenebrioninae : Palorini Ulomoides ocularis (Casey, 1891) Asia <1891 Dry pods of Tamarindus L. Diaperinae : Diaperini : Diaperina Table 3. Number of valid species-group taxa by political region. Data excludes impression and amber fossils. Political region Lagriinae Nilioninae Phrenapatinae Pimeliinae Tenebrioninae Alleculinae Diaperinae Stenochiinae Total BAH : Bahamas 0 0 0 8 22 14 25 1 70 BEL : Belize 20 0 3 2 20 5 32 28 110 BER : Bermuda 0 0 0 0 1 0 1 0 2 CAN : Canada 7 0 2 8 58 28 24 10 137 CAY : Cayman Islands 1 0 0 1 14 4 16 0 36 CRI : Costa Rica 44 0 6 12 27 23 40 44 196 CUB : Cuba 2 0 0 23 57 25 37 31 175 DOM : Dominican Republic 0 0 0 2 18 13 19 19 71 GRE : Greenland 0 0 0 0 2 0 0 0 2 GUA : Guatemala 60 2 4 20 70 57 66 71 350 HAI : Haiti 2 0 0 3 10 8 13 11 47 HON : Honduras 1 0 0 8 10 5 16 3 43 JAM : Jamaica 2 0 1 3 19 11 17 0 53 LAN : Lesser Antilles 11 0 3 6 50 11 31 18 130 MEX : Mexico 101 1 4 392 348 135 129 115 1225 NIC : Nicaragua 41 1 3 16 49 19 36 62 227 PAN : Panama 70 5 8 13 51 41 60 87 335 PRI : Puerto Rico 3 0 1 2 22 4 19 10 61 SAL : El Salvador 0 0 0 2 4 4 8 0 18 TUR : Turks and Caicos Islands 0 0 0 0 1 2 2 0 5 USA : United States of America 38 0 2 546 362 154 79 46 1227 VIS : Virgin Islands 0 0 0 0 3 2 1 1 7 A significant proportion of new species-group taxa (41%) were described between the years 1880 and 1910 (Fig. 1 ). A noticeable decrease in the number of new species-group taxa proposed occurred between 1940–1960, with a small resurgence since then (at a rate of approximately 100 new taxa per decade, see Figs 1 , 2 ). Over 3000 North American species-group taxa are currently recognized as valid (Fig. 2 ), approximately 15% of the world fauna. Figure 1. Number of North American species-group taxa described over time, by decade. Data excludes adventive species as well as impression and amber fossils. Figure 2. Cumulative number of North American species-group taxa described over time, by decade. Data excludes adventive species as well as impression and amber fossils. Line with square marker = total available taxa, line with circular marker = currently valid taxa. Significant contributions (see Table 4 ) The British entomologist George Charles Champion [b. 1851, d. 1927], working on the fauna of Mexico and Central America, proposed the highest number of new tenebrionid taxa found on the continent (83 genus-group, 906 species-group taxa) followed by the American Thomas Lincoln Casey [b. 1857, d. 1925], working mainly on the fauna of the United States of America (80 genus-group, 792 species-group taxa). Frank Ellsworth Blaisdell [b. 1862, d. 1947] (338 species-group taxa), John Lawrence LeConte [b. 1825, d. 1883] (270 species-group taxa), George Henry Horn [b. 1840, d. 1897] (133 species-group taxa), and John Milton Campbell [b. 1935] (103 species-group taxa) also contributed significantly to describing the North American darkling beetle fauna. Table 4. Significant contributions to the description of new North American Tenebrionidae genus-group and species-group taxa (list includes the top ten contributors in each category). Data excludes adventive taxa as well as impression and amber fossils. * = given in parentheses is the country where the person produced taxonomic works, when different from the country of origin. Author Country of origin* New genus-group names New species-group names Blaisdell, Frank Ellsworth USA 20 338 Campbell, John Milton USA (Canada) 10 103 Casey, Thomas Lincoln USA 80 792 Champion, George Charles United Kingdom 83 906 Dejean, Pierre Francois Marie Auguste France 16 0 Doyen, John Thomas USA 5 79 Fall, Henry Clinton USA 1 66 Horn, George Henry USA 20 133 Laporte, Francois Louis (Comte de Castelnau) United Kingdom (France) 12 40 LeConte, John Lawrence USA 56 277 Maklin, Friedrich [Fredrik] Wilhelm Finland 3 66 Marcuzzi, Giorgio Italy 7 84 Motschulsky, Victor de Russia 12 24 Mulsant, Martial Etienne France 13 18 Pascoe, Francis Polkinghorne United Kingdom 12 4 Solier, Antoine Joseph Jean France 17 35 Triplehorn, Charles Albert USA 4 86 Overall diversity A total of 128 valid and invalid family-group, 612 genus-group, and 4065 species-group taxa (excluding fossils listed in Appendices 1, 2) are listed in this catalogue. The subfamily Pimeliinae is the most diverse with 908 valid species-group taxa, followed by the Tenebrioninae (808), Alleculinae (418), Stenochiinae (361), Diaperinae (260), Lagriinae (256), Phrenapatinae (22), and Nilioninae (6). Thirty-seven species in three subfamilies are adventive (Table 2 ), several of which are pests of stored grain products. Mexico and the continental United States of America are by far the most diverse political regions with 1224 and 1230 valid species-group taxa respectively, while at the other extreme Greenland and Bermuda only have two each (Table 3 ). Table 2. List of adventive species documented in North America. Data on the origin, date of first detection in North America, and microhabitat associations in nature are given for each species as far as known. * = pest of stored grain products. Species Origin Date of detection Microhabitat Placement Alphitobius diaperinus (Panzer, 1797) Africa <1866 *Animal nests, caves, guano Tenebrioninae : Alphitobiini Alphitobius laevigatus (Fabricius, 1781) Africa <1866 *Animal nests, caves, guano Tenebrioninae : Alphitobiini Alphitophagus bifasciatus (Say, 1824) Europe, probably <1824 *Animal nests, caves, guano Diaperinae : Diaperini : Adelinina Anchophthalmops menouxi (Mulsant and Rey, 1853) Africa; probably not established <1870 Leaf litter on sandy soil, probably Tenebrioninae : Pedinini : Platynotina Blaps ( Blaps ) lethifera lethifera Marsham, 1802 Europe <1889 Under rocks, wood, in caves Tenebrioninae : Blaptini : Blaptina Blaps ( Blaps ) mucronata Latreille, 1804 Europe <1889 Under rocks, wood, in caves Tenebrioninae : Blaptini : Blaptina Ceropria induta (Wiedemann, 1819) Asia 1998 Polypore fungi, rotten wood Diaperinae : Diaperini : Diaperina Ellipsodes ( Anthrenopsis ) ziczac (Motschulsky, 1873) Asia, probably via Antilles 1891 Under leaf litter on sandy soil Diaperinae : Crypticini Gnatocerus ( Echocerus ) maxillosus (Fabricius, 1801) Asia, probably via Europe <1866 *Animal nests, caves, under bark Diaperinae : Diaperini : Adelinina Gnatocerus ( Gnatocerus ) cornutus (Fabricius, 1798) Asia, probably via Europe <1866 *Animal nests, caves, under bark Diaperinae : Diaperini : Adelinina Gondwanocrypticus pictus (Gebien, 1928) South America 1954 Under leaf litter near ant nests Diaperinae : Crypticini Gondwanocrypticus platensis (Fairmaire, 1884) South America 1929 Under leaf litter near ant nests Diaperinae : Crypticini Gonocephalum ( Gonocephalum ) sericeum (Baudi di Selve, 1875) Northwest Africa + Arabian Peninsula 1980 Under leaf litter, wood, rocks Tenebrioninae : Opatrini : Opatrina Latheticus oryzae Waterhouse, 1880 Old World 1908 *Animal nests, caves, under bark Tenebrioninae : Triboliini Leichenum canaliculatum variegatum (Klug, 1833) Madagascar 1906 Under leaf litter on sandy soil Tenebrioninae : Pedinini : Leichenina Lyphia tetraphylla (Fairmaire, 1857) Europe <1902 In dead wood, other insect burrows Tenebrioninae : Triboliini Myrmechixenus lathridioides Crotch, 1873 Europe <1883 Under leaf litter, in soil Diaperina : Myrmechixenini Opatroides punctulatus Brulle, 1832 Middle East 2003 Under leaf litter, wood, rocks Tenebrioninae : Opatrini : Opatrina Palorus cerylonoides (Pascoe, 1863) Indo-Malayan, probably 2004 Under bark dry wood, plant debris Tenebrioninae : Palorini Palorus genalis Blair, 1930 Old World 1937 Under bark dry wood, plant debris Tenebrioninae : Palorini Palorus ratzeburgii (Wissmann, 1848) North Africa, probably <1897 *Under bark dry wood, plant debris Tenebrioninae : Palorini Palorus subdepressus (Wollaston, 1864) Africa, probably <1882 *Under bark dry wood, plant debris Tenebrioninae : Palorini Pentaphyllus testaceus (Hellwig, 1792) Europe 2005 Polypore fungi, under bark Diaperinae : Diaperini : Diaperina Platydema woldai Triplehorn and Phillips, 1998 Central America, probably 1964 With orchid plants; at lights in forest Diaperinae : Diaperini : Diaperina Plesiophthalmus spectabilis Harold, 1875 Asia; probably not established 2013 Rotten wood Tenebrioninae : Amarygmini Poecilocrypticus formicophilus Gebien, 1928 South America 1978 Under leaf litter near ant nests Diaperinae : Crypticini Strongylium cultellatum Maklin, 1867 Asia 2010 Dead standing wood Stenochiinae : Stenochiini Tenebrio molitor Linnaeus, 1758 Africa, probably <1837 *Animal nests, caves Tenebrioninae : Tenebrionini Tenebrio obscurus Fabricius, 1792 Africa, probably <1869 *Animal nests, caves Tenebrioninae : Tenebrionini Trachyscelis aphodioides Latreille, 1809 Europe <1846 Under plant debris on beach sand Diaperinae : Trachyscelini Tribolium ( Tribolium ) castaneum (Herbst, 1797) Africa, probably <1866 *Under bark dry wood, plant debris Tenebrioninae : Triboliini Tribolium ( Tribolium ) confusum Jacquelin du Val, 1862 Africa, probably <1893 *Under bark dry wood, plant debris Tenebrioninae : Triboliini Tribolium ( Tribolium ) destructor Uyttenboogaart, 1934 Africa <1948 *Under bark dry wood, plant debris Tenebrioninae : Triboliini Tribolium ( Tribolium ) madens (Charpentier, 1825) Africa, probably <1866 *Under bark dry wood, plant debris Tenebrioninae : Triboliini Tyrtaeus dobsoni Hinton, 1947 Unknown; probably via Europe 2002 Under bark and in dead wood Diaperinae : Gnathidiini : Anopidiina Ulomina carinata Baudi di Selve, 1876 Asia 1952 Under bark dry wood, plant debris Tenebrioninae : Palorini Ulomoides ocularis (Casey, 1891) Asia <1891 Dry pods of Tamarindus L. Diaperinae : Diaperini : Diaperina Table 3. Number of valid species-group taxa by political region. Data excludes impression and amber fossils. Political region Lagriinae Nilioninae Phrenapatinae Pimeliinae Tenebrioninae Alleculinae Diaperinae Stenochiinae Total BAH : Bahamas 0 0 0 8 22 14 25 1 70 BEL : Belize 20 0 3 2 20 5 32 28 110 BER : Bermuda 0 0 0 0 1 0 1 0 2 CAN : Canada 7 0 2 8 58 28 24 10 137 CAY : Cayman Islands 1 0 0 1 14 4 16 0 36 CRI : Costa Rica 44 0 6 12 27 23 40 44 196 CUB : Cuba 2 0 0 23 57 25 37 31 175 DOM : Dominican Republic 0 0 0 2 18 13 19 19 71 GRE : Greenland 0 0 0 0 2 0 0 0 2 GUA : Guatemala 60 2 4 20 70 57 66 71 350 HAI : Haiti 2 0 0 3 10 8 13 11 47 HON : Honduras 1 0 0 8 10 5 16 3 43 JAM : Jamaica 2 0 1 3 19 11 17 0 53 LAN : Lesser Antilles 11 0 3 6 50 11 31 18 130 MEX : Mexico 101 1 4 392 348 135 129 115 1225 NIC : Nicaragua 41 1 3 16 49 19 36 62 227 PAN : Panama 70 5 8 13 51 41 60 87 335 PRI : Puerto Rico 3 0 1 2 22 4 19 10 61 SAL : El Salvador 0 0 0 2 4 4 8 0 18 TUR : Turks and Caicos Islands 0 0 0 0 1 2 2 0 5 USA : United States of America 38 0 2 546 362 154 79 46 1227 VIS : Virgin Islands 0 0 0 0 3 2 1 1 7 A significant proportion of new species-group taxa (41%) were described between the years 1880 and 1910 (Fig. 1 ). A noticeable decrease in the number of new species-group taxa proposed occurred between 1940–1960, with a small resurgence since then (at a rate of approximately 100 new taxa per decade, see Figs 1 , 2 ). Over 3000 North American species-group taxa are currently recognized as valid (Fig. 2 ), approximately 15% of the world fauna. Figure 1. Number of North American species-group taxa described over time, by decade. Data excludes adventive species as well as impression and amber fossils. Figure 2. Cumulative number of North American species-group taxa described over time, by decade. Data excludes adventive species as well as impression and amber fossils. Line with square marker = total available taxa, line with circular marker = currently valid taxa. Significant contributions (see Table 4 ) The British entomologist George Charles Champion [b. 1851, d. 1927], working on the fauna of Mexico and Central America, proposed the highest number of new tenebrionid taxa found on the continent (83 genus-group, 906 species-group taxa) followed by the American Thomas Lincoln Casey [b. 1857, d. 1925], working mainly on the fauna of the United States of America (80 genus-group, 792 species-group taxa). Frank Ellsworth Blaisdell [b. 1862, d. 1947] (338 species-group taxa), John Lawrence LeConte [b. 1825, d. 1883] (270 species-group taxa), George Henry Horn [b. 1840, d. 1897] (133 species-group taxa), and John Milton Campbell [b. 1935] (103 species-group taxa) also contributed significantly to describing the North American darkling beetle fauna. Table 4. Significant contributions to the description of new North American Tenebrionidae genus-group and species-group taxa (list includes the top ten contributors in each category). Data excludes adventive taxa as well as impression and amber fossils. * = given in parentheses is the country where the person produced taxonomic works, when different from the country of origin. Author Country of origin* New genus-group names New species-group names Blaisdell, Frank Ellsworth USA 20 338 Campbell, John Milton USA (Canada) 10 103 Casey, Thomas Lincoln USA 80 792 Champion, George Charles United Kingdom 83 906 Dejean, Pierre Francois Marie Auguste France 16 0 Doyen, John Thomas USA 5 79 Fall, Henry Clinton USA 1 66 Horn, George Henry USA 20 133 Laporte, Francois Louis (Comte de Castelnau) United Kingdom (France) 12 40 LeConte, John Lawrence USA 56 277 Maklin, Friedrich [Fredrik] Wilhelm Finland 3 66 Marcuzzi, Giorgio Italy 7 84 Motschulsky, Victor de Russia 12 24 Mulsant, Martial Etienne France 13 18 Pascoe, Francis Polkinghorne United Kingdom 12 4 Solier, Antoine Joseph Jean France 17 35 Triplehorn, Charles Albert USA 4 86 Catalogue of Tenebrionidae ( Coleoptera ) of North America Family TENEBRIONIDAE Latreille, 1802 Tenebrionites Latreille, 1802: 165. Type genus: Tenebrio Linnaeus, 1758. Subfamily LAGRIINAE Latreille, 1825 Lachnaedes Billberg, 1820a: 34. Type genus: Lachna Billberg, 1820 (= Lagria Fabricius, 1775). Nomen oblitum (see Favret and Bouchard 2016 ). Lagriariae Latreille, 1825: 381. Type genus: Lagria Fabricius, 1775. Note. Use of younger family-group name conserved over Lachnina Billberg, 1820 ( ICZN 1999 : Article 40.2) (see Bouchard et al. 2005 ). Tribe Belopini Reitter, 1917 Belopinae Reitter, 1917: 59. Type genus: Belopus Gebien, 1911. Genus Adelonia Laporte, 1840 [F] Adelonia Laporte, 1840: 221. Type species: Uloma filiformis Laporte, 1840, monotypy. Merotemnus Horn, 1870: 367. Type species: Merotemnus elongatus Horn, 1870 (= Uloma filiformis Laporte, 1840), monotypy. Synonymy: Spilman (1961a : 49). Rhacius Champion, 1885: 120. Type species: Rhacius sulcatulus Champion, 1885, subsequent designation ( Gebien 1941 : 805). Synonymy: Spilman (1961a : 50). Adelonia filiformis (Laporte, 1840) MEX (BC BS) Uloma filiformis Laporte, 1840: 221. Merotemnus elongatus Horn, 1870: 367. Synonymy: Gebien (1908b : 160). Adelonia insularis Doyen, 1983 MEX (NA [Islas Marías]) Adelonia insularis Doyen, 1983: 85. Adelonia quadricollis (Champion, 1885) GUA BEL PAN / SA Rhacius quadricollis Champion, 1885: 121. Adelonia sulcatula (Champion, 1885) 4 USA (TX) MEX (GE JA MO OA PU SO VE YU) GUA HON NIC CRI PAN / CUB CAY JAM / SA Rhacius sulcatulus Champion, 1885: 121. Genus Rhypasma Pascoe, 1862 [N] Rhypasma Pascoe, 1862: 325. Type species: Rhypasma pusillum Pascoe, 1862, monotypy. Derosimus Fairmaire, 1904: 62. Type species: Derosimus quadricollis Fairmaire, 1904, monotypy. Synonymy: Blair (1935 : 104). Rhypasma costaricense Marcuzzi, 1976 CRI Rhypasma costaricense Marcuzzi, 1976: 119. Rhypasma haitianum Marcuzzi, 1954 CUB HAI Rhypasma haitianum Marcuzzi, 1954a: 82. Rhypasma livae Ferrer and Ødegaard, 2005 NIC PAN Rhypasma livae Ferrer and Ødegaard, 2005: 635. Tribe Eschatoporiini Blaisdell, 1906 Eschatoporini Blaisdell, 1906: 78. Type genus: Eschatoporis Blaisdell, 1906. Genus Eschatoporis Blaisdell, 1906 [M] Eschatoporis Blaisdell, 1906: 76. Type species: Eschatoporis nunenmacheri Blaisdell, 1906, monotypy. Eschatoporis nunenmacheri Blaisdell, 1906 USA (CA) Eschatoporis nunenmacheri Blaisdell, 1906: 78. Eschatoporis styx Aalbu, Kanda and Smith, 2017 USA (CA) Eschatoporis styx Aalbu, Kanda and Smith, 2017: 140. Tribe Goniaderini Lacordaire, 1859 Goniadérides Lacordaire, 1859: 390. Type genus: Goniadera Perty, 1832. Phobeliina Ardoin, 1961: 33. Type genus: Phobelius Blanchard, 1845. Genus Anaedus Blanchard, 1842 [M] Aspisoma Duponchel and Chevrolat, 1841: 210 [junior homonym of Aspisoma Laporte, 1833]. Type species: Aspisoma fulvipenne Duponchel and Chevrolat, 1841, original designation. Anaedus Blanchard, 1842: pl. 14. Type species: Anaedus punctatissimus Blanchard, 1842, monotypy. Synonymy: Lacordaire (1859 : 396). Anaedes Agassiz, 1846: 36. Unjustified emendation of Anaedus Blanchard, 1842, not in prevailing usage. Aspidosoma Agassiz, 1846: 36. Unjustified emendation of Aspisoma Duponchel and Chevrolat, 1841, not in prevailing usage. Anaedus aeneotinctus Champion, 1893 MEX (GE) Anaedus aeneotinctus Champion, 1893a: 543. Anaedus apicicornis Champion, 1886 MEX (JA) PAN Anaedus apicicornis Champion, 1886: 236. Anaedus brevicollis Champion, 1886 GUA Anaedus brevicollis Champion, 1886: 236. Anaedus brunneus (Ziegler, 1844) [Fig. 3 ] CAN (ON) USA (AL AR DC FL IN KS KY LA MA MD MO MS NC NJ OH PA RI SC TN VA WI) Figure 3. Anaedus brunneus (Ziegler, 1844). Scale bar = 1 mm. Pandarus brunneus Ziegler, 1844: 45. Anaedus impressicollis Pic, 1917 MEX Anaedus impressicollis Pic, 1917: 19. Anaedus inangulatus (Pic, 1934) NIC Aspisoma inangulata Pic, 1934: 35. Anaedus longicornis Champion, 1886 USA (TX) MEX (GU OA) GUA Anaedus longicornis Champion, 1886: 235. Anaedus maculatus Champion, 1886 NIC PAN Anaedus maculatus Champion, 1886: 235. Anaedus marginatus Champion, 1886 NIC PAN Anaedus marginatus Champion, 1886: 236. Anaedus mexicanus Champion, 1886 MEX (VE) Anaedus mexicanus Champion, 1886: 234. Anaedus nitidissimus Pic, 1917 CRI Anaedus nitidissimus Pic, 1917: 20. Anaedus pallidus Schaeffer, 1915 USA (TX) Anaedus pallidus Schaeffer, 1915: 238. Anaedus punctatissimus Blanchard, 1842 MEX (DU JA OA PU SI VE) GUA NIC CRI PAN / SA Anaedus punctatissimus Blanchard, 1842: pl. 14. Anaedus quadrinotatus Champion, 1896 LAN Anaedus quadrinotatus Champion, 1896: 26. Anaedus rotundicollis LeConte, 1851 USA (AZ) MEX (BS) Anoedus rotundicollis LeConte, 1851: 150. Anaedus setulosus Champion, 1886 MEX (TB) NIC PAN / SA Anaedus setulosus Champion, 1886: 237. Anaedus similis Champion, 1886 MEX (VE) GUA NIC Anaedus similis Champion, 1886: 234. Anaedus texanus Linell, 1899 USA (TX) Anoedus texanus Linell, 1899: 182. Anaedus villosus Champion, 1893 GUA CRI PAN Anaedus villosus Champion, 1893a: 543. Genus Goniadera Perty, 1832 [F] Goniadera Perty, 1832: 62 5 . Type species: Goniadera crenata Perty, 1832, monotypy. Goniodera Agassiz, 1846: 165. Unjustified emendation of Goniadera Perty, 1832, not in prevailing usage. Subgenus Aemymone Bates, 1868 Aemymone Bates, 1868: 314. Type species: Aemymone cariosa Bates, 1868, original designation. Goniadera championi Ferrer and Delatour, 2007 MEX (VE) PAN Aemymone crenata Champion, 1893a: 542 [junior secondary homonym of Goniadera crenata Perty, 1832]. Goniadera championi Ferrer and Delatour, 2007: 286. Replacement name for Goniadera crenata (Champion, 1893). Subgenus Goniadera Perty, 1832 Goniadera Perty, 1832: 62. Type species: Goniadera crenata Perty, 1832, monotypy. Goniadera alternata Champion, 1886 MEX (VE) GUA BEL PAN Goniadera alternata Champion, 1886: 231. Goniadera dissipata Kirsch, 1866 PAN / JAM LAN / SA Goniadera dissipata Kirsch, 1866: 197. Goniadera nicaraguensis Champion, 1886 NIC Goniadera nicaraguensis Champion, 1886: 230. Goniadera obscuriceps Pic, 1913 NIC / SA Goniadera obscuriceps Pic, 1913a: 125. Goniadera oculata oculata Champion, 1886 MEX (GE OA VE YU) BEL NIC CRI PAN Goniadera oculata Champion, 1886: 230. Goniadera pilosa Champion, 1886 NIC CRI PAN Goniadera pilosa Champion, 1886: 230. Goniadera pseudorepanda Ferrer and Delatour, 2007 MEX (GE VE YU) GUA NIC CRI / SA Goniadera pseudorepanda Ferrer and Delatour, 2007: 296. Goniadera repanda (Fabricius, 1801) MEX (JA VE) GUA BEL NIC CRI / SA Melandrya repanda Fabricius, 1801a: 165. Subgenus Opatresthes Gebien, 1928 Opatresthes Gebien, 1928b: 192. Type species: Opatresthes binodosus Gebien, 1928, subsequent designation ( Gebien 1941 : 817). Goniadera maesi Ferrer and Delatour, 2007 NIC Goniadera maesi Ferrer and Delatour, 2007: 287. Goniadera quadrinodosa (Gebien, 1928) 6 CRI / SA Opatresthes quadrinodosus Gebien, 1928b: 193. Genus Paratenetus Spinola, 1844 [M] Paratenetus Spinola, 1844: 116. Type species: Paratenetus punctatus Spinola, 1844, subsequent designation ( Lucas 1920 : 483). Lagriola Kirsch, 1874: 409. Type species: Lagriola operosa Kirsch, 1874, present designation . Synonymy: Matthews and Lawrence (2015 : 289). Storthephora Mäklin, 1875a: 658. Type species: Storthephora denticollis Mäklin, 1875, subsequent designation ( Bousquet and Bouchard 2014 : 26). Synonymy: Champion (1893c : 47). Paratenetus brevipennis Champion, 1886 PAN Paratenetus brevipennis Champion, 1886: 242. Paratenetus championi Matthews and Lawrence, 2015 PAN Paratenetus denticulatus Champion, 1886: 243 [secondary homonym of Paratenetus denticulatus (Kirsch, 1874)]. Paratenetus championi Matthews and Lawrence, 2015: 311. Replacement name for Paratenetus denticulatus Champion, 1886. Paratenetus constrictus Champion, 1893 MEX (CI TB VE) GUA BEL CRI PAN Paratenetus constrictus Champion, 1893a: 546. Paratenetus corticarioides Champion, 1886 MEX (OA) GUA Paratenetus corticarioides Champion, 1886: 241. Paratenetus crenulatus Champion, 1886 PAN Paratenetus crenulatus Champion, 1886: 242. Paratenetus exutus Bousquet and Bouchard, 2014 CAN (AB MB NB NS ON QC SK) USA (AL AR CT DC FL IA IL IN KS KY LA MD ME MI MN MO MS NC ND NJ NY OH OK PA TN TX VA WI WV) Paratenetus exutus Bousquet and Bouchard, 2014: 39. Paratenetus foveithorax Ferrer and Ødegaard, 2005 PAN Paratenetus foveithorax Ferrer and Ødegaard, 2005: 635. Paratenetus fuscus LeConte, 1850 [Fig. 4 ] CAN (AB BC MB NT ON QC SK) USA (CO CT DC IA KS MA MD MI MT ND NE NM NY OH RI SD TN VT WI WY) Figure 4. Paratenetus fuscus LeConte, 1850. Scale bar = 1 mm. Paratenetus fuscus LeConte, 1850: 223. Paratenetus crinitus Fall, 1907a: 253. Synonymy: Bousquet and Bouchard (2014 : 31). Paratenetus gibbipennis Motschulsky, 1869 CAN (MB ON QC) USA (AL CT GA IL MA ME MI MN MO NC ND NE NH NJ NY OH PA RI SC TN TX VA WI) Paratenetus gibbipennis Motschulsky, 1869: 193. Paratenetus cribratus Motschulsky, 1869: 193. Synonymy: Bousquet and Bouchard (2014 : 29). Paratenetus grandicornis Motschulsky, 1869 NIC PAN Paratenetus grandicornis Motschulsky, 1869: 193. Paratenetus inermis Champion, 1893 GUA Paratenetus inermis Champion, 1893a: 545. Paratenetus koltzei Pic, 1939 MEX Paratenetus koltzei Pic, 1939: 9. Paratenetus longicornis Pic, 1925 LAN (Guadeloupe) Paratenetus longicornis Pic, 1925a: 6. Paratenetus mexicanus Pic, 1925 MEX (SI) Paratenetus mexicanus Pic, 1925a: 6. Paratenetus nigricornis Champion, 1893 MEX (TB VE) GUA BEL PAN Paratenetus nigricornis Champion, 1893a: 544. Paratenetus obovatus Champion, 1886 BEL Paratenetus obovatus Champion, 1886: 241. Paratenetus punctatus Spinola, 1844 CAN (MB NB ON QC) USA (AR CT DC GA IA IL IN KS KY LA MA MD ME MI MN MO MS NC NH NJ NY OH OK PA RI SC TN TX VA VT WI WV WY) Latridius pubescens Say, 1826: 265 [ nomen dubium , see Bousquet and Bouchard (2014 : 33)]. Paratenetus punctatus Spinola, 1844: 118. Paratenetus punctulatus Champion, 1893 MEX (TB VE) BEL Paratenetus punctulatus Champion, 1893a: 545. Paratenetus ruficornis Champion, 1886 PAN Paratenetus ruficornis Champion, 1886: 239. Paratenetus sexdentatus Champion, 1893 GUA BEL PAN Paratenetus sexdentatus Champion, 1893a: 546. Paratenetus testaceus Pic, 1920 MEX (PU) CRI Paratenetus testaceus Pic, 1920: 2. Paratenetus texanus Bousquet and Bouchard, 2014 USA (FL LA TX) MEX (CI NA TA) Paratenetus texanus Bousquet and Bouchard, 2014: 45. Paratenetus tibialis Champion, 1886 MEX (GE TB VE) GUA BEL Paratenetus tibialis Champion, 1886: 239. Paratenetus tropicalis Motschulsky, 1869 MEX (TB VE) GUA BEL PAN Paratenetus tropicalis Motschulsky, 1869: 193. Paratenetus tuberculatus Champion, 1886 PAN Paratenetus tuberculatus Champion, 1886: 242. Paratenetus villosus Champion, 1886 MEX (VE) GUA PAN Paratenetus villosus Champion, 1886: 240. Genus Phobelius Blanchard, 1842 [M] Phobelius Blanchard, 1842: pl. 14. Type species: Phobelius crenatus Blanchard, 1842, monotypy. Phobelius mexicanus Doyen, 1990 MEX (JA) GUA Phobelius mexicanus Doyen, 1990: 217. Phobelius nevermanni Kulzer, 1961 CRI Phobelius nevermanni Kulzer, 1961a: 226. Genus Phymatestes Pascoe, 1867 [M] Phymatodes Dejean, 1834: 203. Type species: Lagria tuberculata Fabricius, 1792, monotypy. Note. Dejean's name has been suppressed by the Commission in Opinion 1525 ( ICZN 1989 ). Phymatestes Pascoe, 1867: 142. Replacement name for Phymatodes Dejean, 1834 [as Phymatodes Blanchard, 1845]. Note. This name has been placed on the Official List of Generic Names in Zoology in Opinion 1525 ( ICZN 1989 ). Phymatestes agnei Ferrer and Ødegaard, 2005 PAN Phymatestes agnei Ferrer and Ødegaard, 2005: 634. Phymatestes charbonnelae Ferrer and Moraguès, 2003 LAN (Grenada) Phymatestes charbonnelae Ferrer and Moraguès, 2003: 161. Genus Prateus LeConte, 1862 [M] Prateus LeConte, 1862a: 238. Type species: Prateus fusculus LeConte, 1862, original designation. Prateus fusculus LeConte, 1862 USA (AL AR DC FL MD MS NC NY OH OK SC TN TX VA WV) MEX (TA) Prateus fusculus LeConte, 1862a: 238. Genus Xanthicles Champion, 1886 [M] Xanthicles Champion, 1886: 231. Type species: Xanthicles caraboides Champion, 1886, subsequent designation ( Gebien 1941 : 815). Xanthicles caraboides Champion, 1886 CRI Xanthicles caraboides Champion, 1886: 232. Xanthicles hirsutus Champion, 1886 CRI Xanthicles hirsutus Champion, 1886: 232. Tribe Lagriini Latreille, 1825 Lagriariae Latreille, 1825: 381. Type genus: Lagria Fabricius, 1775. Subtribe Statirina Blanchard, 1845 Statyrites Blanchard, 1845: 39. Type genus: Statira Lepeletier and Audinet-Serville, 1828. Genus Arthromacra Kirby, 1837 [F] Arthromacra Kirby, 1837: 238. Type species: Arthromacra donacioides Kirby, 1837 (= Lagria aenea Say, 1824), monotypy. Macrarthra Agassiz, 1846: 219. Unjustified emendation of Arthromacra Kirby, 1837, not in prevailing usage. Arthromacra aenea aenea (Say, 1824) CAN (MB NB NS ON PE QC) USA (CT DC DE MA MD ME MI NC NH NJ NY OH PA TN VA VT WI WV) Lagria aenea Say, 1824b: 287 [junior primary homonym of Lagria aenea Fabricius, 1775]. Arthromacra donacioides Kirby, 1837: 239. Synonymy: LeConte (1859d : 191). Lagria viridis Melsheimer, 1845: 311. Synonymy: Parsons (1976 : 222). Arthromacra aenea glabricollis Blatchley, 1910 USA (IL IN KY MO OH PA VA WI) Arthromacra glabricollis Blatchley, 1910: 1285. Arthromacra aenea lengi Parsons, 1976 USA (GA NC PA SC TN WV) Arthromacra aenea lengi Parsons, 1976: 224. Arthromacra aenea rugosecollis Leng, 1914 USA (GA NC TN) Arthromacra aenea var. rugosecollis Leng, 1914: 287. Arthromacra appalachiana Leng, 1917 USA (NC SC TN VA) Arthromacra appalachiana Leng, 1917: 18. Arthromacra pilosella Leng, 1917 USA (KY NC SC TN) Arthromacra pilosella Leng, 1917: 18. Arthromacra robinsoni Leng, 1914 USA (NC SC VA) Arthromacra robinsoni Leng, 1914: 286. Genus Colparthrum Kirsch, 1866 [N] Colparthrum Kirsch, 1866: 204. Type species: Colparthrum gerstaeckeri Kirsch, 1866, monotypy. Subgenus Colparthrum Kirsch, 1866 Colparthrum Kirsch, 1866: 204. Type species: Colparthrum gerstaeckeri Kirsch, 1866, monotypy. Colparthrum aenescens Borchmann, 1936 CRI Colparthrum aenescens Borchmann, 1936: 444. Colparthrum decoratum bilunulatum (Pic, 1912) PAN Statira bilunulata Pic, 1912b: 76. Colparthrum decoratum decoratum (Mäklin, 1863) MEX (VE) GUA NIC PAN Statira decorata Mäklin, 1863: 588. Colparthrum decoratum maklini Borchmann, 1936 7 [No region originally mentioned but presumably from Mexico and/or Central America] Colparthrum decoratum var. mäklini Borchmann, 1936: 447. Colparthrum foveiceps Champion, 1889 PAN Colparthrum foveiceps Champion, 1889: 68. Colparthrum grande Borchmann, 1936 CRI Colparthrum grandis Borchmann, 1936: 439. Colparthrum majus Borchmann, 1916 MEX Colparthrum decoratum var. major Borchmann, 1916: 233. Subgenus Pseudocolparthrum Borchmann, 1916 Pseudocolparthrum Borchmann, 1916: 236. Type species: Colparthrum calcaratum Champion, 1889, subsequent designation ( Borchmann 1936 : 452). Colparthrum calcaratum Champion, 1889 NIC CRI PAN Colparthrum calcaratum Champion, 1889: 71. Colparthrum sulcicolle Champion, 1889 NIC PAN Colparthrum sulcicolle Champion, 1889: 69. Colparthrum vitticolle Champion, 1889 NIC Colparthrum vitticolle Champion, 1889: 70. Genus Disema Mäklin, 1875 [F] Disema Mäklin, 1875a: 646. Type species: Disema bimaculata Mäklin, 1875, subsequent designation ( Lucas 1920 : 244). Disema singularis (Champion, 1889) PAN Sphragidophorus singularis Champion, 1889: 64. Genus Epicydes Champion, 1889 [M] Epicydes Champion, 1889: 60. Type species: Epicydes oculatus Champion, 1889, subsequent designation ( Borchmann 1936 : 429). Subgenus Cybostira Borchmann, 1936 Cybostira Borchmann, 1936: 430. Type species: Cybostira caligata Borchmann, 1936, original designation. Epicydes caligatus (Borchmann, 1936) CRI Cybostira caligata Borchmann, 1936: 430. Subgenus Epicydes Champion, 1889 Epicydes Champion, 1889: 60. Type species: Epicydes oculatus Champion, 1889, subsequent designation ( Borchmann 1936 : 429). Epicydes oculatus Champion, 1889 MEX (OA VE) GUA Epicydes oculatus Champion, 1889: 61. Epicydes vicinus Champion, 1889 GUA NIC Epicydes vicinus Champion, 1889: 61. Genus Meniscophorus Champion, 1889 [M] Meniscophorus Champion, 1889: 64. Type species: Meniscophorus amazonicus Champion, 1889, subsequent designation ( Lucas 1920 : 404). Meniscophorus costatus Champion, 1889 PAN Meniscophorus costatus Champion, 1889: 65. Genus Meropria Borchmann, 1921 [F] Meropria Borchmann, 1921: 228. Type species: Statira glabrata Mäklin, 1863, original designation. Meropria chiriquina (Champion, 1889) PAN Statira chiriquina Champion, 1889: 9. Meropria denticulata (Champion, 1889) PAN / SA Statira denticulata Champion, 1889: 7. Meropria glabrata (Mäklin, 1863) MEX (GE MO OA VE) GUA BEL CRI Statira glabrata Mäklin, 1863: 587. Meropria interrupta (Champion, 1889) GUA NIC PAN Statira interrupta Champion, 1889: 8. Meropria unidentata (Champion, 1889) MEX (CI VE) GUA BEL Statira unidentata Champion, 1889: 8. Genus Nevermanniella Borchmann, 1936 [F] Nevermanniella Borchmann, 1936: 332. Type species: Statira albolineata Champion, 1889, original designation. Nevermanniella albolineata (Champion, 1889) MEX (VE) BEL NIC Statira albolineata Champion, 1889: 36. Genus Othryades Champion, 1889 [M] Othryades Champion, 1889: 72. Type species: Othryades fragilicornis Champion, 1889, monotypy. Othryades fragilicornis Champion, 1889 PAN Othryades fragilicornis Champion, 1889: 72. Genus Rhaibodera Borchmann, 1921 [F] Rhaibodera Borchmann, 1921: 219. Type species: Rhaibodera pachycera Borchmann, 1921, original designation. Rhaibodera crassicornis (Champion, 1889) MEX (TB) Statira crassicornis Champion, 1889: 18. Genus Rhosaces Champion, 1889 [M] Rhosaces Champion, 1889: 73. Type species: Rhosaces clavipes Champion, 1889, monotypy. Rhosaces clavipes Champion, 1889 PAN Rhosaces clavipes Champion, 1889: 73. Genus Sphragidophorus Champion, 1889 [M] Sphragidophorus Champion, 1889: 61. Type species: Statira cyanipennis Mäklin, 1863, subsequent designation ( Lucas 1920 : 603). Sphragidophorus cyanipennis (Mäklin, 1863) MEX (VE) GUA PAN Statira cyanipennis Mäklin, 1863: 591. Sphragidophorus ocularis Borchmann, 1936 CRI Sphragidophorus ocularis Borchmann, 1936: 507. Sphragidophorus violaceus Champion, 1889 PAN Sphragidophorus violaceus Champion, 1889: 63. Genus Statira Lepeletier and Audinet-Serville, 1828 [F] Statira Lepeletier and Audinet-Serville, 1828: 479. Type species: Statira agroides Lepeletier and Audinet-Serville, 1828, subsequent designation ( Blanchard 1844 : pl. 53bis). Subgenus Spinostatira Pic, 1918 Spinostatira Pic, 1918a: 22. Type species: Statira spinipes Pic, 1918, subsequent designation ( Borchmann 1936 : 247). Statira costaricensis Champion, 1889 CRI / SA Statira costaricensis Champion, 1889: 36. Subgenus Statira Lepeletier and Audinet-Serville, 1828 Statira Lepeletier and Audinet-Serville, 1828: 479. Type species: Statira agroides Lepeletier and Audinet-Serville, 1828, subsequent designation ( Blanchard 1844 : pl. 53bis). Statira aeneipennis Champion, 1889 GUA Statira aeneipennis Champion, 1889: 25. Statira aeneotincta Champion, 1889 MEX (VE) GUA Statira aeneotincta Champion, 1889: 27. Statira aerata Champion, 1889 MEX (VE) GUA Statira aerata Champion, 1889: 26. Statira agraeformis Champion, 1889 PAN Statira agraeformis Champion, 1889: 12. Statira albofasciata Champion, 1889 MEX (VE) GUA PAN Statira albofasciata Champion, 1889: 44. Statira alternans Champion, 1889 MEX (OA) Statira alternans Champion, 1889: 30. Statira amicta Borchmann, 1936 CRI Statira amicta Borchmann, 1936: 294. Statira analis Borchmann, 1921 MEX Statira analis Borchmann, 1921: 273. Statira angustula Champion, 1889 GUA Statira angustula Champion, 1889: 15. Statira antennalis Borchmann, 1936 CRI Statira antennalis Borchmann, 1936: 313. Statira asperata Champion, 1889 PAN / LAN / SA Statira asperata Champion, 1889: 49. Statira antillarum Champion, 1896: 36. Synonymy: Champion (1917 : 230). Statira basalis Horn, 1888 USA (AL AR FL GA LA MD MO MS NC SC TX VA) Statira basalis Horn, 1888: 31. Statira bicolor Champion, 1889 PAN Statira bicolor Champion, 1889: 47. Statira biseriata Borchmann, 1921 MEX Statira biseriata Borchmann, 1921: 263. Statira borchmanni Nevermann, 1926 CRI Statira borchmanni Nevermann, 1926: 113. Statira brevipilis Champion, 1889 MEX (VE) Statira brevipilis Champion, 1889: 39. Statira caeruleipennis Champion, 1889 MEX (MI) Statira caeruleipennis Champion, 1889: 14. Statira ciliata Champion, 1889 GUA Statira ciliata Champion, 1889: 42. Statira collaris Champion, 1889 MEX (VE) Statira collaris Champion, 1889: 13. Statira colorata Fall, 1909 MEX (BC BS) Statira colorata Fall, 1909: 165. Statira conspicillata conspicillata Mäklin, 1863 MEX (CI TB VE) GUA BEL NIC PAN Statira conspicillata Mäklin, 1863: 589. Statira conspicillata lateannulata Borchmann, 1936 GUA Statira conspicillata var. lateannulata Borchmann, 1936: 291. Statira corrosa Champion, 1889 MEX GUA / SA Statira corrosa Champion, 1889: 37. Statira cribrata Champion, 1889 GUA Statira cribrata Champion, 1889: 42. Statira croceicollis Mäklin, 1863 USA (AL FL GA MS) Statira croceicollis Mäklin, 1863: 594. Statira cruciata Champion, 1917 NIC Statira cruciata Champion, 1917: 254. Statira cupreotincta Champion, 1889 NIC PAN Statira cupreotincta Champion, 1889: 23. Statira curticollis Champion, 1889 MEX (GE ME MO OA) GUA Statira curticollis Champion, 1889: 24. Statira defecta Schaeffer, 1905 USA (AZ NM) Statira defecta Schaeffer, 1905b: 175. Statira dolera Parsons, 1966 USA (FL GA) Statira dolera Parsons, 1966: 249. Statira dumalis Parsons, 1973 USA (CA) Statira dumalis Parsons, 1973: 1. Statira erina Parsons, 1976 USA (TX) Statira erina Parsons, 1976: 219. Statira evanescens evanescens Champion, 1889 MEX (DU GE JA MO OA VE) NIC Statira evanescens Champion, 1889: 34. Statira evanescens obscuripennis Borchmann, 1921 MEX Statira evanescens var. obscuripennis Borchmann, 1921: 260. Statira flohri Champion, 1893 MEX (OA) Statira flohri Champion, 1893b: 452. Statira foveicollis Champion, 1889 BEL NIC PAN Statira foveicollis Champion, 1889: 18. Statira fulva Fleutiaux and Sallé, 1890 LAN Statira fulva Fleutiaux and Sallé, 1890: 431. Statira gagatina Melsheimer, 1845 [Fig. 5 ] CAN (QC) USA (AL AR CT DC DE IA IL IN KS KY MA MD MI MS NC NJ NY OH PA SC TN VT WI) Figure 5. Statira gagatina Melsheimer, 1845. Scale bar = 1 mm. Statyra gagatina Melsheimer, 1845: 311. Statyra resplendens Melsheimer, 1845: 311. Synonymy: Parsons (1976 : 220). Lagria fusca Melsheimer, 1845: 311. Synonymy: Parsons (1966 : 252). Statira glabricollis Borchmann, 1936 CRI Statira glabricollis Borchmann, 1936: 315. Statira guatemalensis Champion, 1889 GUA Statira guatemalensis Champion, 1889: 48. Statira guttata Borchmann, 1921 MEX ("Tenancingo") Statira guttata Borchmann, 1921: 250. Statira haitiensis Champion, 1917 HAI Statira haitiensis Champion, 1917: 255. Statira heliconiae Borchmann, 1936 CRI Statira heliconiae Borchmann, 1936: 292. Statira heliophila Borchmann, 1936 CRI Statira heliophila Borchmann, 1936: 276. Statira hirsuta Champion, 1889 USA (TX) MEX (CI GE JA VE) NIC Statira hirsuta Champion, 1889: 50. Statira simulans Schaeffer, 1905a: 180. Synonymy: Parsons (1966 : 246). Statira horrida Champion, 1889 GUA Statira horrida Champion, 1889: 38. Statira huachucae Schaeffer, 1905 USA (AZ NM) Statira huachucae Schaeffer, 1905b: 176. Statira ignita Champion, 1889 MEX (VE) Statira ignita Champion, 1889: 23. Statira inaequicollis Borchmann, 1936 CRI Statira inaequicollis Borchmann, 1936: 287. Statira inconstans Champion, 1889 GUA NIC Statira inconstans Champion, 1889: 16. Statira ingae Borchmann, 1936 CRI Statira ingae Borchmann, 1936: 295. Statira ingens Champion, 1889 NIC CRI PAN Statira ingens Champion, 1889: 12. Statira irazuensis Champion, 1889 CRI Statira irazuensis Champion, 1889: 22. Statira irregularis Champion, 1889 GUA Statira irregularis Champion, 1889: 45. Statira isthmiaca Champion, 1889 PAN Statira isthmiaca Champion, 1889: 19. Statira laevicollis Champion, 1889 MEX (CL GE) Statira laevicollis Champion, 1889: 46. Statira latitator Parsons, 1973 USA (CA) MEX (BC) Statira latitator Parsons, 1973: 3. Statira leptotracheloides Champion, 1889 MEX (DU) Statira leptotracheloides Champion, 1889: 52. Statira liebecki Leng, 1924 USA (AL FL SC) Statira liebecki Leng, 1924: 187. Statira limbata Champion, 1889 MEX (TB VE YU) BEL Statira limbata Champion, 1889: 14. Statira limonis Borchmann, 1936 CRI Statira limonis Borchmann, 1936: 315. Statira marmorata Champion, 1889 MEX (VE) GUA Statira marmorata Champion, 1889: 43. Statira mediosignata Borchmann, 1921 MEX ("Santiago Ixcuintla") Statira mediosignata Borchmann, 1921: 278. Statira melanocephala Mäklin, 1863 MEX (VE) Statira melanocephala Mäklin, 1863: 593. Statira metallica Champion, 1889 NIC CRI PAN Statira metallica Champion, 1889: 16. Statira mexicana Champion, 1889 MEX (PU VE) Statira mexicana Champion, 1889: 26. Statira microps Champion, 1889 MEX (CI TB) GUA Statira microps Champion, 1889: 44. Statira minima minima Champion, 1889 NIC PAN Statira minima Champion, 1889: 47. Statira minima subatra Borchmann, 1936 "Mittelamerika" Statira minima var. subatra Borchmann, 1936: 276. Statira multiformis Champion, 1889 MEX (VE) GUA NIC PAN Statira multiformis Champion, 1889: 19. Statira multipunctata Champion, 1889 MEX (MO) Statira multipunctata Champion, 1889: 49. Statira nevermanni Borchmann, 1936 CRI Statira nevermanni Borchmann, 1936: 255. Statira nigripennis affinis Mäklin, 1875 MEX Statira affinis Mäklin, 1875a: 642. Statira nigripennis championi Pic, 1912 MEX Statira nigripennis var. championi Pic, 1912a: 20. Statira nigripennis humeralis Mäklin, 1875 MEX Statira humeralis Mäklin, 1875a: 642. Statira nigripennis nigripennis Mäklin, 1875 MEX (JA MO) Statira nigripennis Mäklin, 1875a: 641. Statira nigroaenea Champion, 1889 MEX (DU) Statira nigroaenea Champion, 1889: 40. Statira nigrofasciata Borchmann, 1921 MEX ("Navarrete") Statira nigrofasciata Borchmann, 1921: 257. Statira nigromaculata Champion, 1889 USA (AZ TX) MEX (OA VE) GUA Statira nigromaculata Champion, 1889: 33. Statira nigrosparsa Mäklin, 1863 MEX (GE VE) GUA NIC Statira nigrosparsa Mäklin, 1863: 590. Statira nodulosa Champion, 1889 GUA Statira nodulosa Champion, 1889: 31. Statira opaca Borchmann, 1936 CRI Statira opaca Borchmann, 1936: 283. Statira opacicollis Horn, 1888 USA (AZ) Statira opacicollis Horn, 1888: 30. Statira paradoxa Borchmann, 1936 CRI Statira paradoxa Borchmann, 1936: 306. Statira patricia Borchmann, 1921 CRI Statira patricia Borchmann, 1921: 269. Statira penicillata Champion, 1889 MEX (VE) Statira penicillata Champion, 1889: 30. Statira perforata Champion, 1917 MEX Statira perforata Champion, 1917: 262. Statira pici Blackwelder, 1945 CRI Statira bimaculata Borchmann, 1936: 291 [junior primary homonym of Statira bimaculata Pic, 1912]. Statira pici Blackwelder, 1945: 500. Replacement name for Statira bimaculata Borchmann, 1936. Statira picta Champion, 1889 NIC PAN Statira picta Champion, 1889: 36. Statira pilifera Champion, 1893 MEX (VE) Statira pilifera Champion, 1893b: 451. Statira pilipes Champion, 1889 MEX (CI) Statira pilipes Champion, 1889: 43. Statira pluripunctata Horn, 1888 USA (AZ NM TX UT) MEX (GE) Statira pluripunctata Horn, 1888: 29. Statira sulcicrus Champion, 1889: 51. Synonymy: Parsons (1966 : 245). Statira pueblensis Champion, 1889 MEX (PU) Statira pueblensis Champion, 1889: 51. Statira pulchella Mäklin, 1863 USA (TX) MEX (SL TA VE) NIC Statira pulchella Mäklin, 1863: 589. Statira punctatissima Champion, 1889 MEX (CI) Statira punctatissima Champion, 1889: 38. Statira punctipennis Champion, 1889 GUA Statira punctipennis Champion, 1889: 28. Statira reticulaticollis Borchmann, 1936 NIC CRI Statira reticulaticollis Borchmann, 1936: 269. Statira robusta Schaeffer, 1905 USA (AZ CO NM TX) Statira robusta Schaeffer, 1905a: 180. Statira rugicollis Champion, 1889 MEX (VE) Statira rugicollis Champion, 1889: 48. Statira rugipes Champion, 1889 MEX (DU) Statira rugipes Champion, 1889: 52. Statira schmidti Borchmann, 1936 CRI Statira schmidti Borchmann, 1936: 304. Statira scitula Champion, 1889 MEX (VE) GUA Statira scitula Champion, 1889: 10. Statira setigera Champion, 1889 MEX (GU) Statira setigera Champion, 1889: 41. Statira simplex Borchmann, 1936 CRI Statira simplex Borchmann, 1936: 284. Statira sobrina Champion, 1889 MEX Statira sobrina Champion, 1889: 22. Statira spiculifera Champion, 1893 MEX (VE) Statira spiculifera Champion, 1893b: 451. Statira suavis Champion, 1889 MEX (DU) Statira suavis Champion, 1889: 15. Statira subnitida LeConte, 1866 MEX (BC BS) Statira subnitida LeConte, 1866b: 141. Statira testacea Champion, 1889 MEX (VE) Statira testacea Champion, 1889: 28. Statira tolensis Champion, 1889 PAN Statira tolensis Champion, 1889: 20. Statira triangulifer Champion, 1889 MEX (CI VE) GUA BEL Statira triangulifer Champion, 1889: 34. Statira tristis Mäklin, 1875 MEX Statira tristis Mäklin, 1875a: 639. Statira tropicalis Champion, 1889 MEX (VE) GUA BEL NIC Statira tropicalis Champion, 1889: 10. Statira tuberculifera Champion, 1889 GUA Statira tuberculifera Champion, 1889: 32. Statira tuberosa Champion, 1889 MEX (VE) Statira tuberosa Champion, 1889: 31. Statira variabilis inexpecta Borchmann, 1936 PAN Statira variabilis var. inexpecta Borchmann, 1936: 282. Statira variabilis variabilis Champion, 1889 GUA PAN Statira variabilis Champion, 1889: 11. Statira veraecrucis Champion, 1889 MEX (VE) Statira veraecrucis Champion, 1889: 35. Statira veraepacis Champion, 1889 GUA Statira veraepacis Champion, 1889: 24. Statira vilis Mäklin, 1863 MEX (CI TB VE) GUA BEL PAN Statira vilis Mäklin, 1863: 592. Statira villosa Champion, 1889 MEX (GE) Statira villosa Champion, 1889: 39. Statira viridicollis Champion, 1889 PAN Statira viridicollis Champion, 1889: 17. Statira vittata Champion, 1896 LAN Statira vittata Champion, 1896: 37. Genus Uroplatopsis Champion, 1889 [F] Uroplatopsis Champion, 1889: 53. Type species: Uroplatopsis imitator Champion, 1889, subsequent designation ( Lucas 1920 : 666). Uroplatopsis appendiculata Champion, 1889 PAN Uroplatopsis appendiculata Champion, 1889: 59. Uroplatopsis dilaticornis Champion, 1889 PAN Uroplatopsis dilaticornis Champion, 1889: 58. Uroplatopsis excavata Champion, 1889 PAN Uroplatopsis excavata Champion, 1889: 59. Uroplatopsis imitator Champion, 1889 NIC Uroplatopsis imitator Champion, 1889: 54. Uroplatopsis mimica Champion, 1889 PAN Uroplatopsis mimica Champion, 1889: 57. Uroplatopsis planicollis Champion, 1889 PAN Uroplatopsis planicollis Champion, 1889: 56. Uroplatopsis reducta Pic, 1931 CRI Uroplatopsis reducta Pic, 1931: 33. Uroplatopsis reticulata Champion, 1889 PAN Uroplatopsis reticulata Champion, 1889: 56. Uroplatopsis simulans nevermanni Borchmann, 1936 CRI Uroplatopsis simulans var. nevermanni Borchmann, 1936: 482. Uroplatopsis simulans simulans Champion, 1889 PAN Uroplatopsis simulans Champion, 1889: 58. Uroplatopsis vermiculata Champion, 1889 NIC Uroplatopsis vermiculata Champion, 1889: 55. Tribe Lupropini Lesne, 1926 Lypropsini Lesne, 1926: 68. Type genus: Luprops Hope, 1833 [as Lyprops , incorrect subsequent spelling of the type genus name, not in prevailing usage]. Genus Lorelus Sharp, 1876 [M] Lorelus Sharp, 1876: 76. Type species: Lorelus priscus Sharp, 1876, monotypy. Lorelopsis Champion, 1896: 15. Type species: Lorelopsis pilosus Champion, 1896, monotypy. Synonymy: Doyen (1993 : 295). Lorelus angustulus Champion, 1913 GUA Lorelus angustulus Champion, 1913: 165. Lorelus bicolor Doyen, 1993 PRI Lorelus bicolor Doyen, 1993: 296. Lorelus brevicornis Champion, 1896 LAN Lorelus brevicornis Champion, 1896: 14. Lorelus breviusculus Champion, 1913 PAN Lorelus breviusculus Champion, 1913: 165. Lorelus cribricollis Kaszab, 1940 LAN (Guadeloupe) Lorelus cribricollis Kaszab, 1940: 156. Lorelus curticollis Champion, 1913 MEX (VE) GUA PAN Lorelus curticollis Champion, 1913: 164. Lorelus curvipes Champion, 1913 GUA Lorelus curvipes Champion, 1913: 163. Lorelus exilis Champion, 1913 GUA Lorelus exilis Champion, 1913: 166. Lorelus glabratus Doyen, 1993 PRI Lorelus glabratus Doyen, 1993: 297. Lorelus guadeloupensis Kaszab, 1940 LAN (Guadeloupe) Lorelus guadeloupensis Kaszab, 1940: 155. Lorelus pilosus (Champion, 1896) LAN Lorelopsis pilosus Champion, 1896: 16. Lorelus trapeziderus Champion, 1913 GUA PAN Lorelus trapeziderus Champion, 1913: 167. Lorelus wolcotti Doyen, 1993 PRI Lorelus wolcotti Doyen, 1993: 295. Incertae sedis: Lagriinae Genus Pseudesarcus Champion, 1913 8 [M] Pseudesarcus Champion, 1913: 115. Type species: Pseudesarcus villosus Champion, 1913, original designation. Pseudesarcus villosus Champion, 1913 PAN Pseudesarcus villosus Champion, 1913: 116. Subfamily NILIONINAE Oken, 1843 Nilioniden Oken, 1843: 484. Type genus: Nilio Latreille, 1802. Genus Nilio Latreille, 1802 [M] Nilio Latreille, 1802: 179 [as Nilion ] 9 . Type species: Coccinella villosa Fabricius, 1787, monotypy. Subgenus Nilio Latreille, 1802 Nilio Latreille, 1802: 179 [as Nilion ]. Type species: Coccinella villosa Fabricius, 1787, monotypy. Nilio chiriquensis Champion, 1888 PAN Nilio chiriquensis Champion, 1888: 471. Nilio fulvopilosus Champion, 1888 PAN Nilio fulvo-pilosus Champion, 1888: 471. Nilio lebasi J. Thomson, 1860 9 PAN / SA New North American record Nilio lebasei J. Thomson, 1860: 10. Nilio sallei J. Thomson, 1860 MEX (VE) GUA Nilio sallei J. Thomson, 1860: 10. Nilio sallaei Champion, 1888: 470. Unjustified emendation of Nilio sallei J. Thomson, 1860, not in prevailing usage. Nilio thomsoni Champion, 1888 GUA NIC PAN Nilio thomsoni Champion, 1888: 471. Nilio villosus (Fabricius, 1787) PAN / SA Coccinella villosa Fabricius, 1787: 379 [junior primary homonym of Coccinella villosa Geoffroy, 1785]. Subfamily PHRENAPATINAE Solier, 1834 Phrépatides Solier, 1834: 488. Type genus: Phrenapates Gray, 1832. Tribe Archaeoglenini Watt, 1975 Archaeoglenini Watt, 1975: 412. Type genus: Archaeoglenes Broun, 1893. Genus Archaeoglenes Broun, 1893 [M] Archaeoglenes Broun, 1893: 188. Type species: Archaeoglenes costipennis Broun, 1893, monotypy. Archaeoglenes bollensis Watrous, 1982 PAN Archaeoglenes bollensis Watrous, 1982: 140. Archaeoglenes occidentalis Lawrence, 1979 MEX (CI) BEL PAN / SA Archaeoglenes occidentalis Lawrence [in Doyen and Lawrence], 1979: 358. Archaeoglenes pecki Lawrence, 1979 JAM Archaeoglenes pecki Lawrence [in Doyen and Lawrence], 1979: 358. Archaeoglenes puntaensis Watrous, 1982 PAN Archaeoglenes puntaensis Watrous, 1982: 141. Tribe Penetini Lacordaire, 1859 Pénétides Lacordaire, 1859: 318. Type genus: Peneta Lacordaire, 1859. Phthorini Boddy, 1965: 144. Type genus: Phtora Mulsant, 1854 [as Phthora , incorrect subsequent spelling, not in prevailing usage]. Genus Clamoris des Gozis, 1886 [F] Phtora Mulsant, 1854: 228 [junior homonym of Phtora Germar, 1835]. Type species: Phtora crenata Mulsant, 1854, monotypy. Clamoris des Gozis, 1886: 25. Replacement name for Phtora Mulsant, 1854. Phthora Champion, 1893a: 531. Unjustified emendation of Phtora Mulsant, 1854, not in prevailing usage. Clamoris americana (Horn, 1874) [Fig. 6 ] CAN (BC) USA (CA OR WA) Figure 6. Clamoris americana (Horn, 1874). Scale bar = 1 mm. Phthora americana Horn, 1874a: 35. Clamoris armata (Champion, 1893) GUA Phthora armata Champion, 1893a: 532. Clamoris elongata (Champion, 1893) MEX (VE) NIC Phthora elongata Champion, 1893a: 532. Genus Cleolaus Champion, 1886 [M] Cleolaus Champion, 1886: 142. Type species: Peneta sommeri Lacordaire, 1859, original designation. Cleolaus sommeri (Lacordaire, 1859) MEX (OA) Peneta sommeri Lacordaire, 1859: 320. Genus Daochus Champion, 1886 [M] Daochus Champion, 1886: 139. Type species: Daochus mandibularis Champion, 1886, monotypy. Daochus mandibularis Champion, 1886 GUA BEL Daochus mandibularis Champion, 1886: 140. Genus Dioedus LeConte, 1862 [M] Dioedus LeConte, 1862a: 238. Type species: Dioedus punctatus LeConte, 1862, monotypy. Arrhabaeus Champion, 1886: 144. Type species: Arrhabaeus convexus Champion, 1886, monotypy. Synonymy: Kaszab (1977b : 314). Dioedus convexus (Champion, 1886) CRI PAN Arrhabaeus convexus Champion, 1886: 145. Dioedus debilis (Champion, 1896) LAN Arrhabaeus debilis Champion, 1896: 20. Dioedus guadeloupensis (Fleutiaux and Sallé, 1890) LAN Arrhabaeus guadeloupensis Fleutiaux and Sallé, 1890: 424. Dioedus minor (Fleutiaux and Sallé, 1890) LAN Arrhabaeus guadeloupensis minor Fleutiaux and Sallé, 1890: 425. Dioedus punctatus LeConte, 1862 [Fig. 7 ] CAN (ON) USA (AL CT DC FL IA IL IN KS MD MI MO NC NJ NY OH SC VA WI WV) / PRI Figure 7. Dioedus punctatus LeConte, 1862. Scale bar = 1 mm. Dioedus punctatus LeConte, 1862a: 238. Genus Peneta Lacordaire, 1859 [F] Peneta Lacordaire, 1859: 319. Type species: Peneta lebasii Lacordaire, 1859, subsequent designation ( Lucas 1920 : 492). Peneta costaricensis Gebien, 1928 CRI Peneta costaricensis Gebien, 1928a: 147. Peneta nevermanni Gebien, 1928 CRI Peneta nevermanni Gebien, 1928a: 148. Peneta nuchicornis Gebien, 1928 CRI PAN Peneta nuchicornis Gebien, 1928a: 146. Peneta obtusicornis Kirsch, 1866 PAN / SA Peneta obtusicornis Kirsch, 1866: 191. Peneta panamensis Champion, 1886: 142. Synonymy: Champion (1893a : 531). Genus Telchis Champion, 1886 [M] Telchis Champion, 1886: 142. Type species: Telchis clavicornis Champion, 1886, monotypy. Telchis clavicornis Champion, 1886 PAN Telchis clavicornis Champion, 1886: 143. Genus Zypoetes Champion, 1893 [M] Zypoetes Champion, 1893a: 532. Type species: Zypoetes epieroides Champion, 1893, monotypy. Zypoetes epieroides Champion, 1893 MEX (VE) GUA BEL NIC Zypoetes epieroides Champion, 1893a: 533. Tribe Phrenapatini Solier, 1834 Phrépatides Solier, 1834: 488. Type genus: Phrenapates Gray, 1832. Genus Delognatha Lacordaire, 1859 [F] Delognatha Lacordaire, 1859: 315. Type species: Delognatha lacordairei Lacordaire, 1859, subsequent designation ( Gebien 1940 : 756). Note. The name Delognatha Agassiz, 1846 has been suppressed for the purposes of both the Principle of Priority and the Principle of Homonymy in Opinion 2250 ( ICZN 2010 ). Delognatha persimilis Gebien, 1928 CRI Delognatha persimilis Gebien, 1928a: 142. Genus Phrenapates Gray, 1832 [M] Phrenapates Gray [in Griffith and Pidgeon], 1832: 91. Type species: Phrenapates bennettii Gray, 1832, monotypy. Phrenapates bennettii Gray, 1832 GUA NIC CRI PAN / SA Phrenapates bennettii Gray [in Griffith and Pidgeon], 1832: 91. Subfamily PIMELIINAE Latreille, 1802 Pimeliariae Latreille, 1802: 166. Type genus: Pimelia Fabricius, 1775. Tribe Anepsiini LeConte, 1862 Anepsiini LeConte, 1862a: 215. Type genus: Anepsius LeConte, 1851. Batuliini Horn, 1870: 270. Type genus: Batulius LeConte, 1851. Anchommini Horn, 1878b: 558. Type genus: Anchomma LeConte, 1858. Genus Anchomma LeConte, 1858 [N] Anchomma LeConte, 1858b: 63. Type species: Anchomma costatum LeConte, 1858, monotypy. Anchomma costatum LeConte, 1858 USA (CA) Anchomma costatum LeConte, 1858b: 63. Genus Anepsius LeConte, 1851 [M] Anepsius LeConte, 1851: 147. Type species: Anepsius delicatulus LeConte, 1851, monotypy. Anepsius delicatulus LeConte, 1851 USA (AZ CA NV UT) MEX (SO) Anepsius delicatulus LeConte, 1851: 148. Anepsius catenulosus Casey, 1907: 505. Synonymy (with A. atratus Casey): Casey (1911 : 254). Anepsius atratus Casey, 1907: 506. Synonymy: Doyen (1987 : 351). Anepsius brunneus Casey, 1907: 506. Synonymy: Doyen (1987 : 351). Anepsius nebulosus Casey, 1907: 507. Synonymy: Doyen (1987 : 351). Anepsius bicolor Casey, 1907: 507. Synonymy: Doyen (1987 : 351). Anepsius deficiens Casey, 1907: 507. Synonymy: Doyen (1987 : 351). Anepsius minutus Doyen, 1987 USA (TX) MEX (NL) Anepsius minutus Doyen, 1987: 352. Anepsius montanus Casey, 1891 [Fig. 8 ] CAN (AB) USA (CO ND NE NM WY) Figure 8. Anepsius montanus Casey, 1891. Scale bar = 1 mm. Anepsius montanus Casey, 1891: 55. Anepsius valens Casey, 1907 USA (AZ) Anepsius valens Casey, 1907: 504. Genus Batuliodes Casey, 1907 [M] Batuliodes Casey, 1907: 499. Type species: Batulius rotundicollis LeConte, 1851, original designation. Batuliodes confluens (Blaisdell, 1923) MEX (BC BS) Anepsius confluens Blaisdell, 1923: 243. Anepsius angulatus Blaisdell, 1923: 244. Synonymy: Doyen (1987 : 366). Batuliodes obesus Doyen, 1987 USA (CA) Batuliodes obesus Doyen, 1987: 369. Batuliodes rotundicollis (LeConte, 1851) USA (AZ CA NV) Batulius rotundicollis LeConte, 1851: 148. Batuliodes spatulatus Doyen, 1987 USA (AZ CA UT) MEX (SO) Batuliodes spatulatus Doyen, 1987: 368. Batuliodes wasbaueri Doyen, 1987 USA (CA) MEX (BC) Batuliodes wasbaueri Doyen, 1987: 367. Genus Batuliomorpha Doyen, 1987 [F] Batuliomorpha Doyen, 1987: 359. Type species: Batuliomorpha comata Doyen, 1987, original designation. Batuliomorpha comata Doyen, 1987 USA (AZ CA) Batuliomorpha comata Doyen, 1987: 361. Batuliomorpha imperialis Doyen, 1987 USA (CA) Batuliomorpha imperialis Doyen, 1987: 359. Batuliomorpha tibiodentata Doyen, 1987 MEX (BS) Batuliomorpha tibiodentata Doyen, 1987: 362. Genus Batulius LeConte, 1851 [M] Batulius LeConte, 1851: 148. Type species: Batulius setosus LeConte, 1851, subsequent designation ( Casey 1907 : 497). Batulius setosus LeConte, 1851 USA (AZ CA) MEX (BC) Batulius setosus LeConte, 1851: 148. Tribe Asidini Fleming, 1821 Asidadae Fleming, 1821: 51. Type genus: Asida Latreille, 1802. Astroti Horn, 1870: 289. Type genus: Astrotus J.L. LeConte, 1858. Craniotini LeConte and Horn, 1883: 361. Type genus: Craniotus LeConte, 1851. Genus Ardamimicus Smith, 2013 [M] Ardamimicus Smith, 2013: 601. Type species: Ardamimicus cognatoi Smith, 2013, original designation. Ardamimicus cognatoi Smith, 2013 USA (TX) MEX (CH DU) Ardamimicus cognatoi Smith, 2013: 602. Genus Craniotus LeConte, 1851 [M] Craniotus LeConte, 1851: 142. Type species: Craniotus pubescens LeConte, 1851, monotypy. Craniotus mardecortesi Aalbu, Smith and Sánchez Piñero, 2015 MEX (BC) Craniotus mardecortesi Aalbu, Smith and Sánchez Piñero, 2015: 94. Craniotus pubescens LeConte, 1851 USA (AZ CA NV) MEX (BC) Craniotus pubescens LeConte, 1851: 142. Craniotus blaisdelli Tanner, 1963: 169. Synonymy: Aalbu et al. (2015 : 96). Craniotus triplehorni Aalbu, Smith and Sánchez Piñero, 2015 MEX (BC) Craniotus triplehorni Aalbu, Smith and Sánchez Piñero, 2015: 95. Genus Ferveoventer Smith, 2013 [M] Ferveoventer Smith, 2013: 604. Type species: Ferveoventer browni Smith, 2013, original designation. Ferveoventer browni Smith, 2013 USA (NM TX) Ferveoventer browni Smith, 2013: 605. Ferveoventer planatus (Champion, 1884) MEX (MO) Ologlyptus planatus Champion, 1884: 69. Genus Heterasida Casey, 1912 [F] Heterasida Casey, 1912: 76, 165. Type species: Pelecyphorus bifurcus LeConte, 1861, original designation. Heterasida bifurcus (LeConte, 1861) 10 MEX (BS) Pelecyphorus bifurcus LeConte, 1861a: 337. Heterasida connivens (LeConte, 1866) MEX (BS) Pelecyphorus connivens LeConte, 1866b: 110. Heterasida tantilla Casey, 1912: 167. Synonymy: Smith (2013 : 607). Heterasida exilis Casey, 1912: 168. Synonymy: Smith (2013 : 607). Genus Litasida Casey, 1912 [F] Litasida Casey, 1912: 77, 184. Type species: Litasida townsendi Casey, 1912, original designation. Litasida townsendi Casey, 1912 USA (AZ) MEX (CH) Litasida townsendi Casey, 1912: 185. Genus Micrasida Smith, 2013 [F] Micrasida Smith, 2013: 608. Type species: Micrasida obrienorum Smith, 2013, original designation. Micrasida obrienorum Smith, 2013 MEX (NL) Micrasida obrienorum Smith, 2013: 608. Genus Microschatia Solier, 1836 [F] Microschatia Solier, 1836: 474. Type species: Microschatia punctata Solier, 1836, monotypy. Pycnonotida Casey, 1912: 75, 89. Type species: Microschatia inaequalis LeConte, 1851, original designation. Synonymy: Brown and Doyen (1992 : 546). Acroschatia Wilke, 1922: 269. Type species: Microschatia robusta Horn, 1893, original designation. Synonymy: Brown and Doyen (1992 : 546). Microschatia cedrosensis Brown and Doyen, 1992 MEX (BS) Microschatia cedrosensis Brown and Doyen, 1992: 568. Microschatia championi Horn, 1893 USA (AZ CA) MEX (BC BS) Microschatia championi Horn, 1893: 140. Microschatia costulata Brown and Doyen, 1992 USA (CA) MEX (BC) Microschatia costulata Brown and Doyen, 1992: 570. Microschatia inaequalis LeConte, 1851 USA (CA) MEX (BC) Microschatia inaequalis LeConte, 1851: 129. Microschatia puncticollis LeConte, 1851: 129. Synonymy: Horn (1870 : 282). Pycnonotida laxicollis Casey, 1912: 91. Synonymy: Brown and Doyen (1992 : 572). Pycnonotida araneoides Casey, 1912: 92. Synonymy: Brown and Doyen (1992 : 572). Pycnonotida inaequalis diversa Casey, 1912: 92. Synonymy: Brown and Doyen (1992 : 572). Pycnonotida impar Casey, 1912: 93. Synonymy: Brown and Doyen (1992 : 572). Microschatia morata Horn, 1878 USA (AZ NM) MEX (CH DU SO) Microschatia morata Horn, 1878a: 56. Microschatia planata Doyen and Brown, 1992 MEX (BC BS) Microschatia planata Doyen and Brown [in Brown and Doyen], 1992: 576. Microschatia polita Horn, 1893 USA (AZ) Microschatia polita Horn, 1893: 141. Microschatia punctata Solier, 1836 MEX (HI QU) Microschatia punctata Solier, 1836: 475. Microschatia robusta Horn, 1893 USA (TX) MEX (CH CO NL TA) Microschatia robusta Horn, 1893: 142. Microschatia rockefelleri Pallister, 1954 MEX (CH DU) Microschatia rockefelleri Pallister, 1954: 15. Microschatia solieri Brown and Doyen, 1992 MEX (HI) Microschatia solieri Brown and Doyen, 1992: 552. Microschatia sulcipennis LeConte, 1858 USA (TX) Microschatia sulcipennis LeConte, 1858a: 18. Genus Pelecyphorus Solier, 1836 [M] Pelecyphorus Solier, 1836: 467. Type species: Pelecyphorus mexicanus Solier, 1836, subsequent designation ( Hope 1841 : 110). Subgenus Astrotus LeConte, 1858 Astrotus LeConte, 1858a: 19. Type species: Microschatia contorta LeConte, 1853, original designation. Pelecyphorus alveolatus (Casey, 1912) USA (TX) Astrotus alveolatus Casey, 1912: 83. Pelecyphorus contortus (LeConte, 1853) USA (TX) Microschatia contorta LeConte, 1853: 446. Pelecyphorus fasciculatus (Champion, 1892) MEX (MO) Asida fasciculata Champion, 1892: 495. Pelecyphorus guanajuatensis (Champion, 1884) MEX (CH DU GU) Asida guanajuatensis Champion, 1884: 56. Bothrasida mucorea Wilke, 1922: 270. New synonymy [ADS]. Pelecyphorus hebes (Champion, 1892) MEX (DU) Ologlyptus hebes Champion, 1892: 506. Pelecyphorus regularis (Horn, 1870) USA (TX) MEX (TA) Astrotus regularis Horn, 1870: 290. Subgenus Pelecyphorus Solier, 1836 Pelecyphorus Solier, 1836: 467. Type species: Pelecyphorus mexicanus Solier, 1836, subsequent designation ( Hope 1841 : 110). Pelecyphorus mexicanus Solier, 1836 MEX (PU QU VE) Pelecyphorus mexicanus Solier, 1836: 469. Subgenus Pleisiasida Smith, 2013 Parasida Casey, 1912: 76, 126 [junior homonym of Parasida Daday, 1904]. Type species: Parasida laciniata Casey, 1912, original designation. Pleisiasida Smith, 2013: 610. Replacement name for Parasida Casey, 1912. Pelecyphorus asidoides Solier, 1836 MEX (HI ME) 11 Pelecyphorus asidoides Solier, 1836: 471. Parasida zacualpanicola Wilke, 1922: 272. New synonymy [ADS]. Pelecyphorus bibasalis (Casey, 1912) MEX (DU) Parasida bibasalis Casey, 1912: 128. Pelecyphorus dispar (Champion, 1892) MEX (CH DU) Asida dissimilis Champion, 1884: 59 [junior primary homonym of Asida dissimilis Allard, 1869]. Asida dispar Champion, 1892: 496. Replacement name for Asida dissimilis Champion, 1884. Stenosides kulzeri Pallister, 1954: 12. New synonymy [ADS]. Stenosides bisinuatus Pallister, 1954: 13. New synonymy [ADS]. Parasida trisinuata Pallister, 1954: 22. New synonymy [ADS]. Pelecyphorus fallax (Champion, 1884) MEX (DU FD GU HI ME PU) Asida fallax Champion, 1884: 57. Asida favosa Champion, 1884: 58. New synonymy [ADS]. Asida similata Champion, 1884: 58. New synonymy [ADS]. Pelecyphorus foveolatus Solier, 1836 MEX (OA VE) Pelecyphorus foveolatus Solier, 1836: 472. Pelecyphorus indutus (Champion, 1884) MEX (OA) Asida induta Champion, 1884: 56. Ologlyptus bicarinatus Champion, 1884: 69. New synonymy [ADS]. Pelecyphorus intricatus (Champion, 1892) MEX (CH JA) Asida intricata Champion, 1892: 493. Pelecyphorus laticollis (Champion, 1884) MEX (DU GU) Asida laticollis Champion, 1884: 58. Pelecyphorus liratus (LeConte, 1854) USA (AZ NM) MEX (CH DU) Euschides liratus LeConte, 1854c: 223. Parasida laciniata Casey, 1912: 128. New synonymy [ADS]. Parasida cristata Pallister, 1954: 24. New synonymy [ADS]. Pelecyphorus longipennis (Champion, 1884) MEX (OA PU VE) Asida longipennis Champion, 1884: 56. Parasida esperanzae Wilke, 1922: 271. New synonymy [ADS]. Parasida mixtecae Wilke, 1922: 271. New synonymy [ADS]. Pelecyphorus obliviosus (Wilke, 1922) MEX (CH DU) Parasida obliviosa Wilke, 1922: 270. Pelecyphorus planatulus (Casey, 1912) MEX (DU) Parasida planatula Casey, 1912: 129. Pelecyphorus scutellaris (Champion, 1884) MEX (DU FD ME OA PU VE) Asida scutellaris Champion, 1884: 56. Parasida tolucana Casey, 1912: 130. New synonymy [ADS]. Pelecyphorus sexcostatus LeConte, 1861 MEX (BS) Pelecyphorus sexcostatus LeConte, 1861a: 337. Pelecyphorus tristis (Champion, 1884) MEX (PU VE) Asida tristis Champion, 1884: 55. Parasida purpusi Wilke, 1922: 271. New synonymy [ADS]. Subgenus Poliorcetes Champion, 1884 Poliorcetes Champion, 1884: 70. Type species: Poliorcetes platesthoides Champion, 1884, monotypy. Pelecyphorus platesthoides (Champion, 1884) MEX (OA) Poliorcetes platesthoides Champion, 1884: 71. Subgenus Sicharbas Champion, 1884 Sicharbas Champion, 1884: 67. Type species: Sicharbas lobatus Champion, 1884, monotypy. Pelecyphorus debilis (Champion, 1884) MEX (PU) Astrotus debilis Champion, 1884: 66. Pelecyphorus erosus (Champion, 1892) MEX (HI) Astrotus erosus Champion, 1892: 504. Astrotus nosodermoides Champion, 1892: 505. New synonymy [ADS]. Pelecyphorus lobatus (Champion, 1884) MEX (GE MO) Sicharbas lobatus Champion, 1884: 67. Pelecyphorus seticornis (Champion, 1884) MEX (ME) Astrotus seticornis Champion, 1884: 67. Astrotus seticornis var. humeralis Champion, 1884: 67. New synonymy [ADS]. Pelecyphorus undatus (Champion, 1892) MEX (DU) Astrotus undatus Champion, 1892: 504. Subgenus Stenosides Solier, 1836 Stenosides Solier, 1836: 484. Type species: Stenosides graciliformis Solier, 1836, monotypy. Pactostoma LeConte, 1858a: 19. Type species: Asida anastomosis Say, 1824, original designation. Synonymy (with Ologlyptus Lacordaire): LeConte (1862a : 222). Ologlyptus Lacordaire, 1859: 158. Unnecessary replacement name for Stenosides Solier, 1836. Pelecyphorus anastomosis (Say, 1824) USA (AR AZ CO KS NM TX) MEX (CH DU) Asida anastomosis Say, 1824a: 256. Pactostoma anastomosis salebrosa Casey, 1912: 87. Synonymy: Pallister (1954 : 12). Pactostoma breviuscula Casey, 1912: 87. New synonymy [ADS]. Pactostoma exoleta Casey, 1912: 87. New synonymy [ADS]. Pactostoma luteotecta Casey, 1912: 88. New synonymy [ADS]. Pactostoma monticola Casey, 1912: 88. New synonymy [ADS]. Pactostoma obtecta Casey, 1912: 89. New synonymy [ADS]. Pactostoma sigillata Casey, 1912: 89. New synonymy [ADS]. Pelecyphorus graciliformis (Solier, 1836) MEX (FD HI OA PU SL) Stenosides graciliformis Solier, 1836: 486. Ologlyptus canus Champion, 1884: 68. New synonymy [ADS]. Ologlyptus sinuaticollis Champion, 1884: 69. New synonymy [ADS]. Pelecyphorus limosus (Champion, 1884) MEX Astrotus limosus Champion, 1884: 66. Pelecyphorus texanus (Wickham, 1903) USA (TX) Ologlyptus texanus Wickham, 1903: 72. Subgenus Ucalegon Champion, 1884 Ucalegon Champion, 1884: 65. Type species: Ucalegon pulchellus Champion, 1884, monotypy. Pelecyphorus pulchellus (Champion, 1884) MEX (GE OA) Ucalegon pulchellus Champion, 1884: 65. Subgenus Zaleucus Champion, 1892 Zamolxis Champion, 1884: 70 [junior homonym of Zamolxis Stål, 1865]. Type species: Zamolxis dilatatus Champion, 1884, monotypy. Zaleucus Champion, 1892: 491. Replacement name for Zamolxis Champion, 1884. Pelecyphorus dilatatus (Champion, 1884) MEX (PU) Zamolxis dilatatus Champion, 1884: 70. Genus Philolithus Lacordaire, 1858 [M] Philolithus Lacordaire [in LeConte], 1858a: 18. Type species: Pelecyphorus carinatus LeConte, 1851, subsequent designation ( Casey 1912 : 79). Subgenus Glyptasida Casey, 1912 Glyptasida Casey, 1912: 75, 95. Type species: Pelecyphorus sordidus LeConte, 1853, original designation. Philolithus aeger (LeConte, 1858) USA (NM TX) Pelecyphorus aeger LeConte, 1858a: 19. Glyptasida sycophanta Casey, 1912: 104. Synonymy: Lockwood and Pollock (2009 : 21). Philolithus rugosissimus (Champion, 1884) USA (AZ NM) MEX (CH CO SL) Asida rugosissima Champion, 1884: 53. Philolithus sordidus (LeConte, 1853) [Fig. 9 ] CAN (AB SK) USA (AZ CO KS MT ND NE NM OK SD TX UT WY) MEX (CH CO DU ZA) Figure 9. Philolithus ( Glyptasida ) sordidus (LeConte, 1853). Scale bar = 1 mm. Pelecyphorus sordidus LeConte, 1853: 445. Pelecyphorus subcostatus LeConte, 1853: 446. Synonymy: Henshaw (1882 : 255). Pelecyphorus irregularis LeConte, 1858a: 19. Synonymy: Champion (1892 : 492). Pelecyphorus costipennis LeConte, 1858a: 20. Synonymy: Champion (1892 : 492). Asida interrupta Champion, 1884: 53. Synonymy: Champion (1892 : 492). Glyptasida parvicollis Casey, 1912: 97. Synonymy: Lockwood and Pollock (2009 : 8). Glyptasida sordida porcatula Casey, 1912: 97. Synonymy: Lockwood and Pollock (2009 : 8). Glyptasida subpubescens Casey, 1912: 98. Synonymy: Lockwood and Pollock (2009 : 8). Glyptasida turgescens Casey, 1912: 98. Synonymy: Lockwood and Pollock (2009 : 8). Glyptasida turgescens furtiva Casey, 1912: 99. Synonymy: Lockwood and Pollock (2009 : 8). Glyptasida turgescens obesa Casey, 1912: 99. Synonymy: Lockwood and Pollock (2009 : 8). Glyptasida procrustes Casey, 1912: 99. Synonymy: Lockwood and Pollock (2009 : 8). Glyptasida costipennis fulvisetis Casey, 1912: 100. Synonymy: Lockwood and Pollock (2009 : 8). Glyptasida strigipennis Casey, 1912: 100. Synonymy: Lockwood and Pollock (2009 : 8). Glyptasida turbulenta Casey, 1912: 101. Synonymy: Lockwood and Pollock (2009 : 8). Glyptasida aegra imperfecta Casey, 1912: 102. Synonymy: Lockwood and Pollock (2009 : 7). Glyptasida aegra pigra Casey, 1912: 102. Synonymy: Lockwood and Pollock (2009 : 8). Glyptasida aegra plena Casey, 1912: 102. Synonymy: Lockwood and Pollock (2009 : 8). Glyptasida heres Casey, 1912: 103. Synonymy: Lockwood and Pollock (2009 : 8). Glyptasida crenicollis Casey, 1912: 103. Synonymy: Lockwood and Pollock (2009 : 8). Subgenus Gonasida Casey, 1912 Gonasida Casey, 1912: 75, 117. Type species: Pelecyphorus elatus LeConte, 1853, original designation. Philolithus elatus compar (Casey, 1912) USA (CO ID KS NE NM NV OR UT WY) Gonasida compar Casey, 1912: 120. Gonasida elata reducta Casey, 1912: 121. New synonymy [based on Brown (1971 : 262) unpublished thesis]. Gonasida elata prolixa Casey, 1912: 121. New synonymy [based on Brown (1971 : 262) unpublished thesis]. Gonasida aucta Casey, 1912: 122. New synonymy [based on Brown (1971 : 262) unpublished thesis]. Philolithus elatus difformis (LeConte, 1854) USA (AZ NM UT) MEX (CH) Pelecyphorus difformis LeConte, 1854c: 223. Gonasida alaticollis Casey, 1912: 122. New synonymy [based on Brown (1971 : 255) unpublished thesis]. Philolithus elatus elatus (LeConte, 1853) USA (AZ NM TX) MEX (CH) Pelecyphorus elatus LeConte, 1853: 445. Gonasida gravida Casey, 1912: 119. New synonymy [based on Brown (1971 : 251) unpublished thesis]. Philolithus elatus infernus (Casey, 1912) USA (AZ NM UT) Gonasida inferna Casey, 1912: 119. Subgenus Herthasida Wilke, 1922 Herthasida Wilke, 1922: 269. Type species: Asida ingens Champion, 1892, monotypy. Philolithus ingens (Champion, 1892) MEX (CO) Asida ingens Champion, 1892: 503. Subgenus Philolithus Lacordaire, 1858 Philolithus Lacordaire [in LeConte], 1858a: 18. Type species: Pelecyphorus carinatus LeConte, 1851, subsequent designation ( Casey 1912 : 79). Philolithus actuosus (Horn, 1870) USA (CA) Asida actuosa Horn, 1870: 284. Philolithus adversus (Casey, 1912) USA (AZ) Pelecyphorus adversus Casey, 1912: 114. Philolithus aegrotus aegrotus (LeConte, 1861) MEX (BC) Pelecyphorus aegrotus LeConte, 1861a: 337. Pelecyphorus aegrotus limbatus Casey, 1912: 107. New synonymy [ADS]. Philolithus carinatus (LeConte, 1851) USA (CA) Pelecyphorus carinatus LeConte, 1851: 128 [junior secondary homonym of Asida carinata Solier, 1836]. Asida carinifera Gebien, 1910a: 128. Replacement name for Asida carinata (LeConte, 1851) 12 . Philolithus densicollis (Horn, 1894) [Fig. 10 ] CAN (BC) USA (OR WA) Figure 10. Philolithus ( Philolithus ) densicollis (Horn, 1894). Scale bar = 1 mm. Asida densicollis Horn, 1894b: 417. Pelecyphorus corrosus Casey, 1912: 117. Synonymy: Boddy (1965 : 137). Philolithus haruspex ellipsipennis (Casey, 1912) USA (UT) Pelecyphorus haruspex ellipsipennis Casey, 1912: 116. Philolithus haruspex haruspex (Casey, 1912) USA (AZ ID OR NV UT) Pelecyphorus haruspex Casey, 1912: 115. Philolithus jaegeri (Papp, 1961) USA (CA) Pelecyphorus jaegeri Papp, 1961b: 107. Philolithus morbillosus (LeConte, 1858) USA (AZ) MEX (SO) Pelecyphorus morbillosus LeConte, 1858b: 74. Pelecyphorus corporalis Casey, 1912: 107. New synonymy [ADS]. Pelecyphorus reptans Casey, 1912: 108. New synonymy [ADS]. Pelecyphorus socer Casey, 1912: 108. New synonymy [ADS]. Pelecyphorus abscissus Casey, 1912: 109. New synonymy [ADS]. Pelecyphorus fumosus Casey, 1912: 109. New synonymy [ADS]. Pelecyphorus parvus Casey, 1912: 110. New synonymy [ADS]. Pelecyphorus morbillosus pacatus Casey, 1912: 110. New synonymy [ADS]. Pelecyphorus morbillosus sobrius Casey, 1912: 110. New synonymy [ADS]. Pelecyphorus piceus Casey, 1912: 111. New synonymy [ADS]. Pelecyphorus piceus crudelis Casey, 1912: 111. New synonymy [ADS]. Pelecyphorus snowi Casey, 1912: 111. New synonymy [ADS]. Pelecyphorus subtenuis Casey, 1912: 112. New synonymy [ADS]. Philolithus opimus (Casey, 1912) USA (CA) Pelecyphorus opimus Casey, 1912: 115. Philolithus pantex (Casey, 1912) USA (NV UT) Pelecyphorus pantex Casey, 1912: 116. Philolithus porcatus (Papp, 1961) USA (CA) Pelecyphorus porcatus Papp, 1961b: 109. Philolithus quadripennis (Casey, 1912) MEX ( LC ) Pelecyphorus quadripennis Casey, 1912: 113. Philolithus reflexus (Casey, 1912) USA (CA) Pelecyphorus reflexus Casey, 1912: 114. Philolithus rugosus (Papp, 1961) USA (CA) Pelecyphorus rugosus Papp, 1961c: 157. Philolithus sophistes (Casey, 1912) USA (CA) Pelecyphorus sophistes Casey, 1912: 113. Philolithus uteanus (Casey, 1924) USA (UT) Pelecyphorus uteanus Casey, 1924: 308. Subgenus Tisamenes Champion, 1884 Tisamenes Champion, 1884: 64. Type species: Tisamenes truquii Champion, 1884, monotypy. Philolithus truquii (Champion, 1884) MEX (FD HI) Tisamenes truquii Champion, 1884: 64. Genus Stenomorpha Solier, 1836 [F] Stenomorpha Solier, 1836: 487. Type species: Stenomorpha blapsoides Solier, 1836, subsequent designation ( Desmarest 1860 : 150). Euschides LeConte, 1851: 127. Unnecessary replacement name for Stenomorpha Solier, 1836. Subgenus Asidina Casey, 1912 Asidina Casey, 1912: 169. Type species: Pelecyphorus parallelus LeConte, 1851, original designation. Stenomorpha confluens (LeConte, 1851) USA (AZ CA) MEX (BC SO) Pelecyphorus confluens LeConte, 1851: 128. Stenomorpha parallela (LeConte, 1851) USA (AZ CA) MEX (SO) Pelecyphorus parallelus LeConte, 1851: 128 [junior secondary homonym of Asida parallela Solier, 1836]. Asida neglecta Gebien, 1910a: 134. Replacement name for Asida parallela (LeConte, 1851) 13 . Asidina teres Casey, 1912: 171. Synonymy: Triplehorn and Brown (1971 : 84). Asida parallela terricola Blaisdell, 1923: 254. Synonymy: Triplehorn and Brown (1971 : 84). Stenomorpha rugicollis (Triplehorn and Brown, 1971) USA (AZ) Asidina rugicollis Triplehorn and Brown, 1971: 76. Stenomorpha semilaevis (Horn, 1870) USA (AZ CA NV) Asida semilaevis Horn, 1870: 284. Subgenus Asidopsis Casey, 1912 Asidopsis Casey, 1912: 77, 185. Type species: Asida opaca Say, 1824, original designation. Stenomorpha abbreviata (Casey, 1912) USA (NM) Asidopsis abbreviata Casey, 1912: 198. Stenomorpha cochisensis (Casey, 1912) USA (AZ NM) Asidopsis cochisensis Casey, 1912: 189. Stenomorpha coenosa (Casey, 1912) USA (NM) Asidopsis coenosa Casey, 1912: 200. Stenomorpha collaris (Champion, 1892) MEX (AG GU JA) Asida marginicollis Champion, 1884: 60 [junior primary homonym of Asida marginicollis Rosenhauer, 1856]. Asida collaris Champion, 1892: 499. Replacement name for Asida marginicollis Champion, 1884. Stenomorpha collega (Casey, 1912) USA (KS) Asidopsis collega Casey, 1912: 198. Stenomorpha consentanea (Casey, 1912) USA (CA) Asidopsis consentanea Casey, 1912: 192. Stenomorpha divaricata (Blaisdell, 1923) MEX (BS) Asida divaricata Blaisdell, 1923: 255. Stenomorpha dolosa (Casey, 1912) USA (MT SD WY) Asidopsis dolosa Casey, 1912: 194. Stenomorpha durangoensis (Casey, 1912) MEX (DU) Asidopsis durangoensis Casey, 1912: 201. Stenomorpha eximia (Casey, 1912) USA (TX) Asidopsis eximia Casey, 1912: 188. Stenomorpha forreri (Champion, 1884) MEX (DU) Asida forreri Champion, 1884: 55. Stenomorpha gracilipes (Casey, 1912) USA (AZ) Asidopsis gracilipes Casey, 1912: 189. Stenomorpha humeralis (Triplehorn and Flores, 2002) MEX (CH) Asidopsis humeralis Triplehorn and Flores, 2002: 288. Stenomorpha immunda (Casey, 1912) USA (NM) MEX (DU) Asidopsis immunda Casey, 1912: 199. Stenomorpha macra (Horn, 1883) USA (AZ NM) Asidopsis macra Horn, 1883: 304. Stenomorpha mancipata (Horn, 1878) USA (NM) MEX (CH) Asida mancipata Horn, 1878a: 56. Asidopsis woodgatei Casey, 1912: 197. Synonymy: Triplehorn and Flores (2002 : 285). Stenomorpha nitidula (Casey, 1912) USA (NM) Asidopsis nitidula Casey, 1912: 196. Stenomorpha obsidiana (Casey, 1912) USA (CO) Asidopsis obsidiana Casey, 1912: 193. Stenomorpha olsoni (Triplehorn and Flores, 2002) USA (AZ) Asidopsis olsoni Triplehorn and Flores, 2002: 286. Stenomorpha opaca (Say, 1824) [Fig. 11 ] CAN (AB SK) USA (CO KS MT ND NE NM SD TX) Figure 11. Stenomorpha ( Asidopsis ) opaca (Say, 1824). Scale bar = 1 mm. Asida opaca Say, 1824a: 254. Stenomorpha pinalica (Casey, 1912) USA (AZ) Asidopsis pinalica Casey, 1912: 190. Stenomorpha planata (Horn, 1894) USA (CA) MEX Asida planata Horn, 1894b: 415. Stenomorpha polita futilis (Casey, 1912) USA (KS) Asidopsis polita futilis Casey, 1912: 194. Stenomorpha polita polita (Say, 1824) CAN (AB SK) USA (CO ID KS MT NE NM OK SD TX WY) Asida polita Say, 1824a: 255. Stenomorpha polita subopaca (Casey, 1912) USA (KS) Asidopsis polita subopaca Casey, 1912: 193. Stenomorpha quadricollis (Horn, 1880) USA (AZ NM) Asidopsis quadricollis Horn, 1880: 151. Stenomorpha servilis (Casey, 1912) USA (CO) Asidopsis servilis Casey, 1912: 199. Stenomorpha suavis (Casey, 1912) USA (AZ) Asidopsis suavis Casey, 1912: 190. Stenomorpha tensa (Casey, 1912) USA (CO) Asidopsis tensa Casey, 1912: 197. Subgenus Bothrasida Casey, 1912 Bothrasida Casey, 1912: 76, 122. Type species: Asida clathrata Champion, 1884, original designation. Stenomorpha baroni (Casey, 1912) MEX (GE) Bothrasida baroni Casey, 1912: 124. Stenomorpha clathrata (Champion, 1884) MEX (GE ME MO OA PU) Asida clathrata Champion, 1884: 54. Stenomorpha funesta (Champion, 1884) MEX (PU) Asida funesta Champion, 1884: 53. Bothrasida sanctae-agnae Wilke, 1922: 270. New synonymy [ADS]. Subgenus Megasida Casey, 1912 Megasida Casey, 1912: 77, 202. Type species: Asida obliterata Champion, 1892, original designation. Stenomorpha foeda (Champion, 1892) MEX (CO DU) Asida foeda Champion, 1892: 498. Stenomorpha latissima (Champion, 1892) MEX (DU) Asida latissima Champion, 1892: 500. Stenomorpha magnifica (Pallister, 1954) MEX (DU) Megasida magnifica Pallister, 1954: 30. Stenomorpha moricoides (Champion, 1892) MEX (CO DU) Asida moricoides Champion, 1892: 497. Stenomorpha obliterata (Champion, 1892) USA (NM TX) MEX (CH) Asida obliterata Champion, 1892: 497. Stenomorpha rufipes (Champion, 1884) MEX (SL) Asida rufipes Champion, 1884: 62. Stenomorpha segregata (Champion, 1892) MEX (CH CO DU) Asida segregata Champion, 1892: 497. Stenomorpha tarda (Champion, 1892) MEX (CO) Asida tarda Champion, 1892: 498. Stenomorpha tenuicollis (Triplehorn, 1967) USA (AZ NM TX) Megasida tenuicollis Triplehorn, 1967: 40. Stenomorpha zacatecensis (Pallister, 1954) MEX (ZA) Megasida zacatecensis Pallister, 1954: 32. Subgenus Notiasida Casey, 1912 Notiasida Casey, 1912: 76, 124. Type species: Notiasida abstrusa Casey, 1912, original designation. Stenomorpha abstrusa (Casey, 1912) MEX (FD) Notiasida abstrusa Casey, 1912: 125. Stenomorpha evertissima (Casey, 1912) MEX (CH DU) Notiasida evertissima Casey, 1912: 126. Stenomorpha geminata (Champion, 1892) MEX (CH DU) Asida geminata Champion, 1892: 492. Stenomorpha lata (Champion, 1884) MEX (SL) Asida lata Champion, 1884: 60. Stenomorpha lugubris (Wilke, 1922) MEX (SL) Pelecyphorus lugubris Wilke, 1922: 269. Stenomorpha suturalis (Champion, 1884) MEX (FD OA VE) Asida suturalis Champion, 1884: 55. Subgenus Platasida Casey, 1912 Platasida Casey, 1912: 77, 182. Type species: Asida embaphionides Horn, 1894, original designation. Stenomorpha embaphionides (Horn, 1894) MEX (BS) Asida embaphionides Horn, 1894b: 419. Asida flaccida Horn, 1896: 379. New synonymy [ADS]. Subgenus Pycnomorpha Motschulsky, 1870 Pycnomorpha Motschulsky, 1870: 398. Type species: Pycnomorpha californica Motschulsky, 1870, monotypy. Stenomorpha californica (Motschulsky, 1870) MEX (BC) Pycnomorpha californica Motschulsky, 1870: 399. Stenomorpha gabbii (Horn, 1880) USA (CA) MEX (BS) Asida gibbicollis Horn, 1870: 288 [junior primary homonym of Asida gibbicollis Pérez Arcas, 1865]. Asida gabbii Horn, 1880: 152. Replacement name for Asida gibbicollis Horn, 1870. Stenomorpha tumidicollis Blaisdell, 1943 MEX (BC) Stenomorpha tumidicollis Blaisdell, 1943: 226. Subgenus Stenomorpha Solier, 1836 Stenomorpha Solier, 1836: 487. Type species: Stenomorpha blapsoides Solier, 1836, subsequent designation ( Desmarest 1860 : 150). Psilomera Motschulsky, 1870: 400. Type species: Pelecyphorus angulatus LeConte, 1851, monotypy. New synonymy [YB]. Stenomorpha advena (Casey, 1912) USA (CO) Euschides advena Casey, 1912: 143. Stenomorpha amplicollis (Casey, 1912) USA (CA) Euschides amplicollis Casey, 1912: 153. Stenomorpha angulata (LeConte, 1851) USA (CA) Pelecyphorus angulatus LeConte, 1851: 127. Stenomorpha blanda (Champion, 1884) MEX (AG GU JA) Asida blanda Champion, 1884: 63. Stenomorpha blapsoides alutacea Wilke, 1922 MEX (ME) Stenomorpha blapsoides alutacea Wilke, 1922: 272. Stenomorpha blapsoides blapsoides Solier, 1836 MEX (CO FD GU JA ME OA PU SI VE) Stenomorpha blapsoides Solier, 1836: 491. Stenomorpha brevimargo (Casey, 1912) USA (AZ) Euschides brevimargo Casey, 1912: 134. Stenomorpha caliginosa (Casey, 1912) USA (AZ) Euschides caliginosus Casey, 1912: 137. Stenomorpha captiosa (Horn, 1870) USA (CA) Asida captiosa Horn, 1870: 287. Stenomorpha clarissae Wilke, 1922 MEX (ME) Stenomorpha clarissae Wilke, 1922: 273. Stenomorpha compressa (Horn, 1870) USA (CA) Asida lecontei var. compressa Horn, 1870: 287. Stenomorpha congruens congruens (Casey, 1912) USA (NM) Euschides congruens Casey, 1912: 164. Stenomorpha congruens lubrica (Casey, 1912) USA (AZ) Euschides congruens lubricus Casey, 1912: 164. Stenomorpha consobrina (Horn, 1870) USA (ID OR) Asida consobrina Horn, 1870: 287. Stenomorpha consors (Casey, 1912) USA (NM) Euschides consors Casey, 1912: 162. Stenomorpha consueta (Casey, 1912) USA (AZ) Euschides consuetus Casey, 1912: 161. Stenomorpha convexa (LeConte, 1859) USA (CA KS) Euschides convexa LeConte, 1859a: 14. Stenomorpha convexicollis (LeConte, 1854) USA (CA CO KS NM TX) MEX (CH DU) Euschides convexicollis LeConte, 1854c: 224. Stenomorpha corrugans (Casey, 1912) USA (AZ) Euschides corrugans Casey, 1912: 143. Stenomorpha costata Solier, 1836 MEX (ME VE) Stenomorpha costata Solier, 1836: 490. Stenomorpha crassa (Casey, 1912) USA (CA) Euschides crassus Casey, 1912: 154. Stenomorpha cressoni (Blaisdell, 1933) USA (CA) Euschides cressoni Blaisdell, 1933b: 191. Stenomorpha cribrata (Casey, 1912) USA (NM) Euschides cribratus Casey, 1912: 140. Stenomorpha crinita (Casey, 1912) USA (OR) Euschides crinitus Casey, 1912: 148. Stenomorpha deceptor (Casey, 1912) USA (CA) Euschides deceptor Casey, 1912: 154. Stenomorpha directa (Casey, 1912) USA (AZ) Euschides directa Casey, 1912: 141. Stenomorpha evanescens (Casey, 1912) USA (CA) Euschides evanescens Casey, 1912: 146. Stenomorpha facilis (Casey, 1912) USA (KS) Euschides facilis Casey, 1912: 165. Stenomorpha fastigiosa (Casey, 1912) USA (NM) Euschides fastigiosus Casey, 1912: 157. Stenomorpha globicollis (Casey, 1912) USA (NE) Euschides globicollis Casey, 1912: 158. Stenomorpha gracilior (Casey, 1912) USA (KS) Euschides gracilior Casey, 1912: 160. Stenomorpha gravidipes (Casey, 1912) USA (CA) Euschides gravidipes Casey, 1912: 155. Stenomorpha huachucae (Casey, 1912) USA (AZ) Euschides huachucae Casey, 1912: 162. Stenomorpha implicans (Casey, 1912) USA (AZ) Euschides implicans Casey, 1912: 136. Stenomorpha inhabilis inhabilis (Casey, 1912) USA (NM) Euschides inhabilis Casey, 1912: 156. Stenomorpha inhabilis retusa (Casey, 1912) USA (KS) Euschides inhabilis retusus Casey, 1912: 157. Stenomorpha integer (Casey, 1912) USA (CA) Euschides integer Casey, 1912: 153. Stenomorpha lecontei lecontei (Horn, 1870) USA (CA) Pelecyphorus costipennis LeConte, 1859b: 76 [junior primary homonym of Pelecyphorus costipennis LeConte, 1858]. Asida lecontei Horn, 1870: 286. Replacement name for Asida costipennis (LeConte, 1859). Stenomorpha lecontei gigantea (Blaisdell, 1921) USA (CA) Euschides lecontei gigantea Blaisdell, 1921b: 209. Stenomorpha lecontella lecontella (Blaisdell, 1936) USA (CA) Euschides lecontella Blaisdell, 1936b: 227. Stenomorpha lecontella tempestalis (Blaisdell, 1936) USA (CA) Euschides lecontella tempestalis Blaisdell, 1936b: 229. Stenomorpha luctata (Horn, 1870) USA (CA NV) Asida luctata Horn, 1870: 286. Stenomorpha marginata duplicans (Casey, 1912) USA [AZ] Euschides marginatus duplicans Casey, 1912: 136. Stenomorpha marginata esuriens (Casey, 1912) USA (CA) Euschides marginatus esuriens Casey, 1912: 137. Stenomorpha marginata marginata (LeConte, 1851) USA (AZ CA NM TX) MEX (CH SO) Pelecyphorus marginatus LeConte, 1851: 128. Stenomorpha maritima imula (Casey, 1912) USA (CA) Euschides maritimus imulus Casey, 1912: 151. Stenomorpha maritima maritima (Casey, 1912) USA (CA) Euschides maritimus Casey, 1912: 151. Stenomorpha mckittricki (Pierce, 1954) USA (CA) 14 Parasida mckittricki Pierce, 1954a: 43. Stenomorpha montezuma Wilke, 1922 MEX (DU) Stenomorpha montezuma Wilke, 1922: 272. Stenomorpha musiva Wilke, 1922 MEX (ME) Stenomorpha musiva Wilke, 1922: 273. Stenomorpha neutralis (Casey, 1912) USA (CA) Euschides neutralis Casey, 1912: 146. Stenomorpha oblonga (Casey, 1924) USA (NM) Euschides oblongus Casey, 1924: 309. Stenomorpha obovata gliscans (Casey, 1912) USA (AZ) Euschides obovatus gliscans Casey, 1912: 161. Stenomorpha obovata nitidipennis (Casey, 1912) USA (AZ) Euschides obovatus nitidipennis Casey, 1912: 160. Stenomorpha obovata obovata (LeConte, 1851) USA (AZ CA TX) MEX (CH) Euschides obovata LeConte, 1851: 127. Stenomorpha oregonensis (Casey, 1924) USA (OR) Euschides oregonensis Casey, 1924: 309. Stenomorpha orizabae Wilke, 1922 MEX (VE) Stenomorpha orizabae Wilke, 1922: 273. Stenomorpha papagoana (Casey, 1912) USA (AZ) Euschides papagoanus Casey, 1912: 163. Stenomorpha pollens pollens (Casey, 1912) USA (AZ) Euschides pollens Casey, 1912: 134. Stenomorpha pollens proxima (Casey, 1912) USA (AZ) Euschides pollens proximus Casey, 1912: 134. Stenomorpha procurrens (Casey, 1912) USA (AZ) Euschides procurrens Casey, 1912: 137. Stenomorpha puncticollis (LeConte, 1866) USA (OR WA) Euschides puncticollis LeConte, 1866b: 111 [junior secondary homonym of Asida puncticollis Solier, 1836]. Asida robusta Gebien, 1910a: 135. Replacement name for Asida puncticollis (LeConte, 1866) 15 . Stenomorpha rimata rimata (LeConte, 1854) USA (TX) Pelecyphorus rimatus LeConte, 1854c: 223. Stenomorpha rimata subplanata (Casey, 1912) USA (AZ) Euschides rimatus subplanatus Casey, 1912: 139. Stenomorpha rudis (Casey, 1912) USA (AZ) Euschides rudis Casey, 1912: 139. Stenomorpha rugata (Casey, 1912) USA (AZ) Euschides rugatus Casey, 1912: 138. Stenomorpha rustica (Casey, 1912) USA (AZ) Euschides rusticus Casey, 1912: 135. Stenomorpha satiata (Casey, 1912) USA (AZ) Euschides satiatus Casey, 1912: 138. Stenomorpha semirufa (Casey, 1912) USA (AZ) Euschides semirufus Casey, 1912: 140. Stenomorpha severa (Casey, 1912) USA (NM) Euschides severus Casey, 1912: 144. Stenomorpha socialis (Casey, 1912) USA (NM) Euschides socialis Casey, 1912: 162. Stenomorpha speculata (Blaisdell, 1936) USA (CA) Euschides speculatus Blaisdell, 1936b: 225. Stenomorpha sphaericollis (Champion, 1884) MEX (AG SL) Asida sphaericollis Champion, 1884: 64. Stenomorpha sponsor (Casey, 1912) USA (AZ) Euschides sponsor Casey, 1912: 135. Stenomorpha spurcans (Casey, 1912) USA (CA) Euschides spurcans Casey, 1912: 145. Stenomorpha strigosula (Casey, 1912) USA (AZ) Euschides strigosulus Casey, 1912: 163. Stenomorpha subcruenta (Casey, 1912) USA (NM) Euschides subcruentus Casey, 1912: 140. Stenomorpha subcylindrica (Horn, 1870) USA (AZ) Asida marginata var. subcylindrica Horn, 1870: 288. Stenomorpha subelegans (Casey, 1912) USA (CA) Euschides subelegans Casey, 1912: 152. Stenomorpha tetrica (Casey, 1912) USA (UT) Euschides tetricus Casey, 1912: 149. Stenomorpha tularensis (Casey, 1912) USA (CA) Euschides tularensis Casey, 1912: 152. Stenomorpha uhdei Wilke, 1922 MEX (ME) Stenomorpha uhdei Wilke, 1922: 273. Stenomorpha umbrosa (Champion, 1884) MEX (GU) Asida umbrosa Champion, 1884: 62. Stenomorpha vigens (Casey, 1912) USA (AZ) Euschides vigens Casey, 1912: 159. Subgenus Stethasida Casey, 1912 Stethasida Casey, 1912: 78, 203. Type species: Pelecyphorus muricatulus LeConte, 1851, original designation. Stenomorpha flohri (Champion, 1892) MEX (JA) Asida flohri Champion, 1892: 496. Stenomorpha muricatula (LeConte, 1851) USA (CA) Pelecyphorus muricatulus LeConte, 1851: 128. Asida angustula Casey, 1890b: 370. New synonymy [ADS]. Stethasida stricta Casey, 1912: 210. New synonymy [ADS]. Stethasida muricatula languida Casey, 1912: 211. New synonymy [ADS]. Stethasida pertinax Casey, 1912: 211. New synonymy [ADS]. Stethasida socors Casey, 1912: 212. New synonymy [ADS]. Stethasida angustula inepta Casey, 1912: 213. New synonymy [ADS]. Stethasida tenax Casey, 1912: 213. New synonymy [ADS]. Stethasida vegrandis Casey, 1912: 214. New synonymy [ADS]. Stenomorpha obsoleta (LeConte, 1851) USA (CA) Pelecyphorus obsoletus LeConte, 1851: 128. Stethasida obsoleta expansa Casey, 1912: 205. New synonymy [ADS]. Stethasida obsoleta opacella Casey, 1912: 205. New synonymy [ADS]. Stethasida brevipes Casey, 1912: 206. New synonymy [ADS]. Stethasida torpida Casey, 1912: 206. New synonymy [ADS]. Stethasida convergens Casey, 1912: 207. New synonymy [ADS]. Stethasida discreta Casey, 1912: 207. New synonymy [ADS]. Stethasida longula Casey, 1912: 207. New synonymy [ADS]. Stethasida adumbrata Casey, 1912: 208. New synonymy [ADS]. Stethasida occulta Casey, 1912: 208. New synonymy [ADS]. Stethasida tarsalis Casey, 1912: 208. New synonymy [ADS]. Stethasida unica Casey, 1912: 209. New synonymy [ADS]. Pelecyphorus laevigatus Papp, 1961c: 159. New synonymy [ADS]. Subgenus Trichiasida Casey, 1912 Trichiasida Casey, 1912: 77, 172. Type species: Pelecyphorus hirsutus LeConte, 1851, original designation. Stenomorpha acerba (Horn, 1878) USA (AZ NV UT) Asida acerba Horn, 1878a: 56. Stenomorpha difficilis (Champion, 1884) MEX (HI ME SL) Asida difficilis Champion, 1884: 61. Trichiasida eremica Wilke, 1922: 274. New synonymy [ADS]. Stenomorpha hirsuta (LeConte, 1851) USA (CA) Pelecyphorus hirsutus LeConte, 1851: 127. Trichiasida lineatopilosa Casey, 1912: 175. New synonymy [ADS]. Stenomorpha hispidula (LeConte, 1851) USA (AZ CA) Pelecyphorus hispidulus LeConte, 1851: 127. Trichiasida tenella Casey, 1912: 177. New synonymy [ADS]. Stenomorpha horrida (Champion, 1892) USA (TX) MEX (TA) Asida horrida Champion, 1892: 500. Stenomorpha idahoensis (Boddy, 1957) USA (ID) Trichiasida idahoensis Boddy, 1957: 187. Stenomorpha ignava (Casey, 1912) USA (AZ) Trichiasida ignava Casey, 1912: 180. Stenomorpha impetrata (Horn, 1894) USA (CA) Asida impetrata Horn, 1894b: 418. Stenomorpha impotens (Casey, 1912) USA (AZ) Trichiasida impotens Casey, 1912: 180. Stenomorpha lutulenta (Doyen, 1990) MEX (JA) Trichiasida lutulenta Doyen, 1990: 225. Stenomorpha palmeri (Champion, 1884) MEX (SL) Asida palmeri Champion, 1884: 59. Stenomorpha pubescens (Champion, 1884) MEX Asida pubescens Champion, 1884: 61. Stenomorpha subpilosa Solier, 1836 MEX (PU) Stenomorpha subpilosa Solier, 1836: 490. Stenomorpha thoracica (Champion, 1884) MEX Asida thoracica Champion, 1884: 62. Stenomorpha unicostata (Champion, 1892) MEX (GE) Asida unicostata Champion, 1892: 501. Stenomorpha villosa (Champion, 1884) MEX (PU) Asida villosa Champion, 1884: 60. Trichiasida duplex Casey, 1912: 178 16 . New synonymy [ADS]. [incertae sedis] Stenomorpha catalinae (Blaisdell, 1923) MEX (BS) Asida catalinae Blaisdell, 1923: 256. Stenomorpha furcata (Champion, 1892) USA (TX) MEX (CO DU) Asida furcata Champion, 1892: 499. Stenomorpha granicollis (Blaisdell, 1923) MEX (SO) Asida granicollis Blaisdell, 1923: 256. Stenomorpha roosevelti Smith, Miller and Wheeler, 2011 MEX (CO) Stenomorpha roosevelti Smith, Miller and Wheeler, 2011: 30. Stenomorpha spinimana (Champion, 1892) MEX (DU) New combination [ADS]. Asida spinimanus Champion, 1892: 494. Stenomorpha subvittata (Horn, 1894) MEX (BS) Asida subvittata Horn, 1894b: 416. Stenomorpha tenebrosa (Champion, 1892) MEX (CO) New combination [ADS]. Asida tenebrosa Champion, 1892: 495. Stenomorpha wickhami (Horn, 1894) USA (AZ CA) Asida wickhami Horn, 1894b: 420. Asidina liberta Casey, 1912: 171. Synonymy: Triplehorn and Brown (1971 : 84). Tribe Branchini LeConte, 1862 Branchini LeConte, 1862a: 222. Type genus: Branchus LeConte, 1862 Genus Anectus Horn, 1866 [M] Anectus Horn, 1866: 399. Type species: Anectus vestitus Horn, 1866, monotypy. Anectus vestitus Horn, 1866 HON Anectus vestitus Horn, 1866: 399. Genus Branchus LeConte, 1862 17 [M] Branchus LeConte, 1862a: 222. Type species: Branchus floridanus LeConte, 1862, monotypy. Branchus floridanus LeConte, 1862 USA (FL) Branchus floridanus LeConte, 1862a: 223. Branchus geraceorum Steiner, 2006 BAH Branchus geraceorum Steiner, 2006: 10. Branchus jamaicensis Marcuzzi, 1977 JAM Branchus jamaicensis Marcuzzi, 1977: 10. Branchus obscurus Horn, 1866 MEX (GE) GUA NIC Branchus obscurus Horn, 1866: 398. Branchus opatroides Champion, 1892 MEX (JA VE) Branchus opatroides Champion, 1892: 507. Branchus saxatilis Steiner, 2005 BAH Branchus saxatilis Steiner, 2005: 443. Branchus whiteheadi Steiner, 1991 USA (TX) Branchus whiteheadi Steiner, 1991: 426. Branchus woodii LeConte, 1866 BAH CUB Branchus woodii LeConte, 1866b: 111. Genus Oxinthas Champion, 1884 [M] Oxinthas Champion, 1884: 72. Type species: Oxinthas praocioides Champion, 1884, monotypy. Oxinthas nicaraguensis Merkl, 1992 NIC Oxinthas nicaraguensis Merkl, 1992: 89. Oxinthas praocioides Champion, 1884 MEX (OA) Oxinthas praocioides Champion, 1884: 72. Tribe Cnemeplatiini Jacquelin du Val, 1861 Cnéméplatiites Jacquelin du Val, 1861: 286. Type genus: Cnemeplatia Costa, 1847. Subtribe Cnemeplatiina Jacquelin du Val, 1861 Cnéméplatiites Jacquelin du Val, 1861: 286. Type genus: Cnemeplatia Costa, 1847. Genus Alaudes Horn, 1870 [M] Alaudes Horn, 1870: 361. Type species: Alaudes singularis Horn, 1870, monotypy. Alaudes alternatus Fall, 1928 USA (CA) Alaudes alternata Fall, 1928: 148. Alaudes setigerus Blaisdell, 1919 USA (CA) Alaudes setigera Blaisdell, 1919a: 310. Alaudes singularis Horn, 1870 USA (CA ID NV OR) Alaudes singularis Horn, 1870: 362. Alaudes squamosa Blaisdell, 1919a: 309. New synonymy [RLA]. Alaudes testacea Blaisdell, 1919a: 311. New synonymy [RLA]. Alaudes fallax Fall, 1928: 150. New synonymy [RLA]. Genus Lepidocnemeplatia Bousquet and Bouchard, new genus [F] Lepidocnemeplatia Bousquet and Bouchard, new genus. Type species: Cnemeplatia sericea Horn, 1870. Lepidocnemeplatia laticollis (Champion, 1885) MEX (ME) NIC CRI PAN / SA Cnemeplatia laticollis Champion, 1885: 136. Lepidocnemeplatia sericea (Horn, 1870) USA (AZ CA NV OR TX WA) MEX (BS CH DU MO NL PU SO VE) NIC Cnemeplatia sericea Horn, 1870: 360. Tribe Cnemodinini Gebien, 1910 Cnemodini Horn, 1870: 266. Type genus: Cnemodus Horn, 1870 (= Cnemodinus Cockerell, 1906). Cnemodininae Gebien, 1910a: 4. Type genus: Cnemodinus Cockerell, 1906. Genus Cnemodinus Cockerell, 1906 [M] Cnemodus Horn, 1870: 266 [junior homonym of Cnemodus Herrich-Schaeffer, 1850]. Type species: Cnemodus testaceus Horn, 1870, monotypy. Cnemodinus Cockerell, 1906: 242. Replacement name for Cnemodus Horn, 1870. Cnemodinus angustus (Casey, 1907) USA (AZ) Cnemodus angustus Casey, 1907: 284. Cnemodinus subhyalinus (Casey, 1907) USA (UT) Cnemodus subhyalinus Casey, 1907: 285. Cnemodinus testaceus (Horn, 1870) USA (AZ CA) Cnemodus testaceus Horn, 1870: 266. Tribe Coniontini G.R. Waterhouse, 1858 Coniontidae G.R. Waterhouse, 1858: 59. Type genus: Coniontis Eschscholtz, 1829. Coelini Casey, 1907: 500. Type genus: Coelus Eschscholtz, 1829. Eusatti Doyen, 1984b: 11. Type genus: Eusattus LeConte, 1851. Genus Coelus Eschscholtz, 1829 [M] Coelus Eschscholtz, 1829: 5. Type species: Coelus ciliatus Eschscholtz, 1829, monotypy. Coelomorpha Casey, 1890a: 182. Type species: Coelomorpha maritima Casey, 1890, monotypy. Synonymy: Doyen (1972 : 371). Pseudocoelus Casey, 1908: 152. Type species: Coelus pacificus Fall, 1897, subsequent designation ( Doyen 1976 : 608). Synonymy: Blaisdell (1919b : 322). Coelus ciliatus Eschscholtz, 1829 [Fig. 12 ] CAN (BC) USA (CA OR WA) MEX (BC) Figure 12. Coelus ciliatus Eschscholtz, 1829. Scale bar = 1 mm. Coelus ciliatus Eschscholtz, 1829: 5. Coelus arenarius Casey, 1890a: 179. Synonymy: Doyen (1976 : 614). Coelus latus Casey, 1895: 612. Synonymy (with C. arenarius Casey): Fall (1901 : 166). Coelus curtulus Casey, 1895: 612. Synonymy: Fall (1901 : 166). Coelus ciliatus longulus Casey, 1908: 154. Synonymy: Blaisdell (1919b : 333). Coelus debilis Casey, 1908: 155. Synonymy: Doyen (1976 : 614). Coelus sternalis Casey, 1908: 156. Synonymy: Doyen (1976 : 614). Coelus obscurus Casey, 1908: 156. Synonymy (with C. arenarius Casey): Blaisdell (1919b : 334). Coelus scolopax Casey, 1908: 157. Synonymy (with C. arenarius Casey): Blaisdell (1919b : 334). Coelus amplicollis Casey, 1908: 157. Synonymy (with C. latus Casey): Blaisdell (1919b : 334). Coelus ciliatus var. sparsus Blaisdell, 1919b: 325. Synonymy (with C. ciliatus debilis Casey): Gebien (1938 : 409). Coelus globosus LeConte, 1851 USA (CA) MEX (BC) Coelus globosus LeConte, 1851: 133. Coelus grossus Casey, 1890a: 178. Synonymy: Doyen (1976 : 617). Coelus solidus Casey, 1908: 153. Synonymy (with C. globosus grossus Casey): Blaisdell (1919b : 333). Coelus saginatus Casey, 1908: 154. Synonymy: Doyen (1976 : 617). Coelus gracilis Blaisdell, 1939 USA (CA) Coelus gracilis Blaisdell, 1939a: 16. Coelus maritimus (Casey, 1889) MEX (BC) Coelomorpha maritima Casey, 1890a: 183. Coelomorpha pallens Casey, 1908: 160. Synonymy: Doyen (1976 : 620). Coelus pacificus Fall, 1897 USA (CA) Coelus pacificus Fall, 1897: 241. Coelus remotus Fall, 1897: 241. Synonymy: Doyen (1976 : 618). Genus Coniontis Eschscholtz, 1829 [F] Coniontis Eschscholtz, 1829: 7. Type species: Coniontis viatica Eschscholtz, 1829, subsequent designation ( Casey 1908 : 57). Coelotaxis Horn, 1876a: 200. Type species: Coelotaxis punctulata Horn, 1876, subsequent designation ( Gebien 1938 : 289). Synonymy: Doyen (1972 : 373). Coniontellus Casey, 1890b: 388. Type species: Coniontis obesa LeConte, 1851, subsequent designation ( Casey 1908 : 57). Synonymy: Doyen (1972 : 373). Coniontides Casey, 1908: 57, 78. Type species: Coniontis lata LeConte, 1866, original designation. Synonymy: Doyen (1972 : 373). Crypticomorpha Casey, 1908: 81, 140. Type species: Coniontis tenuis Casey, 1908, monotypy. Synonymy: Aalbu et al. (2002 : 487). Brachyontis Casey, 1908: 82, 141. Type species: Coniontis globulina Casey, 1895, monotypy. Synonymy: Aalbu et al. (2002 : 487). Coniontis abdominalis LeConte, 1859 USA (CA) Coniontis abdominalis LeConte, 1859b: 77. Coniontis strenua Casey, 1908: 84. Synonymy: Doyen (1977 : 2). Coniontis tristis Casey, 1908: 84. Synonymy: Doyen (1977 : 2). Coniontis gravis Casey, 1908: 85. Synonymy: Doyen (1977 : 2). Coniontis rugosa Casey, 1908: 85. Synonymy: Doyen (1977 : 2). Coniontis tenebrosa Casey, 1908: 86. Synonymy: Doyen (1977 : 2). Coniontis abdominalis caseyi Pierce, 1954c: 145. Synonymy: Doyen and Miller (1980 : 3). Coniontis abdominalis labreae Pierce, 1954c: 146. Synonymy: Doyen and Miller (1980 : 3). Coniontis abdominalis fragmans Pierce, 1954c: 148. Synonymy: Doyen and Miller (1980 : 3). Coniontis tristis alpha Pierce, 1954c: 148. Synonymy: Doyen and Miller (1980 : 3). Coniontis tristis asphalti Pierce, 1954c: 149. Synonymy: Doyen and Miller (1980 : 3). Coniontis tristis latigula Pierce, 1954c: 149. Synonymy: Doyen and Miller (1980 : 3). Coniontis blissi Pierce, 1954c: 149. Synonymy: Doyen and Miller (1980 : 3). Coniontis pectoralis paraelliptica Pierce, 1954c: 153. Synonymy: Doyen and Miller (1980 : 3). Coniontis pectoralis interrupta Pierce, 1954c: 154. Synonymy: Doyen and Miller (1980 : 3). Coniontis affinis LeConte, 1851 USA (CA OR) Coniontis affinis LeConte, 1851: 130. Coniontis expansa Casey, 1908: 120. Synonymy: Doyen (1977 : 2). Coniontis franciscana Casey, 1908: 120. Synonymy: Doyen (1977 : 2). Coniontis truncata Casey, 1908: 120. Synonymy: Doyen (1977 : 2). Coniontis suturalis Casey, 1908: 121. Synonymy: Doyen (1977 : 2). Coniontis audax Casey, 1908: 121. Synonymy: Doyen (1977 : 2). Coniontis symmetrica Casey, 1908: 122. Synonymy: Doyen (1977 : 2). Coniontis convergens Casey, 1908: 122. Synonymy: Doyen (1977 : 2). Coniontis anxia Casey, 1908: 123. Synonymy: Doyen (1977 : 2). Coniontis affinis patruelis Casey, 1908: 123. Synonymy: Doyen (1977 : 2). Coniontis oregona Casey, 1908: 124. Synonymy: Doyen (1977 : 2). Coniontis pagana Casey, 1908: 130. Synonymy: Doyen (1977 : 2). Coniontis atronitens Casey, 1908 USA (CA) Coniontis atronitens Casey, 1908: 110. Coniontis blaisdelli Casey, 1908 USA (CA) Coniontis blaisdelli Casey, 1908: 97. Coniontis callida Casey, 1908 USA (CA) Coniontis callida Casey, 1908: 128. Coniontis shastanica Casey, 1908: 128. Synonymy: Doyen (1977 : 2). Coniontis conferta Casey, 1908: 129. Synonymy: Doyen (1977 : 2). Coniontis agrestis Casey, 1908: 131. Synonymy: Doyen (1977 : 2). Coniontis congesta Casey, 1908: 131. Synonymy: Doyen (1977 : 2). Coniontis costulata Casey, 1908 USA (CA) Coniontis costulata Casey, 1908: 89. Coniontis elliptica Casey, 1884 USA (CA) Coniontis elliptica Casey, 1884: 46. Coniontis laevigata Casey, 1908: 88. Synonymy: Doyen (1977 : 2). Coniontis elliptica catalinae Casey, 1908: 88. Synonymy: Doyen (1977 : 2). Coniontis cuneata Casey, 1908: 111. Synonymy: Gebien (1938 : 286). Coniontis elongata Casey, 1890 USA (CA) Coniontis elongata Casey, 1890b: 380. Coniontis rotundicollis Casey, 1908: 97. Synonymy: Doyen (1977 : 3). Coniontis innocua Casey, 1908: 99. Synonymy: Doyen (1977 : 3). Coniontis elongata limatula Casey, 1908: 99. Synonymy: Doyen (1977 : 3). Coniontis cylindrica Casey, 1908: 100. Synonymy: Doyen (1977 : 3). Coniontis obsidiana Casey, 1908: 100. Synonymy: Blaisdell (1935b : 120). Coniontis longicollis Casey, 1908: 101. Synonymy: Doyen (1977 : 3). Coniontis eschscholtzii Mannerheim, 1840 USA (CA) Coniontis eschscholtzii Mannerheim, 1840: 138. Coniontis extricata Casey, 1908 USA (CA OR) Coniontis extricata Casey, 1908: 124. Coniontis marginata Casey, 1908: 125. Synonymy: Doyen (1977 : 3). Coniontis minuta Casey, 1908: 126. Synonymy: Doyen (1977 : 3). Coniontis parva Casey, 1908: 126. Synonymy: Doyen (1977 : 3). Coniontis perpolita Casey, 1908: 127. Synonymy: Doyen (1977 : 3). Coniontis pudica Casey, 1908: 127. Synonymy: Doyen (1977 : 3). Coniontis nemoralis slevini Blaisdell, 1924a: 86. Synonymy: Doyen (1977 : 3). Coniontis farallonica Casey, 1895 USA (CA) Coniontis farallonica Casey, 1895: 610. Coniontis genitiva Casey, 1890 USA (CA) Coniontis genitiva Casey, 1890b: 385. Coniontis verna Casey, 1908: 94. Synonymy: Doyen (1977 : 3). Coniontis opacicollis Casey, 1908: 101. Synonymy: Doyen (1977 : 3). Coniontis globulina Casey, 1895 USA (CA) Coniontis globulina Casey, 1895: 610. Coniontis histrio Casey, 1908 USA (AZ) Coniontis histrio Casey, 1908: 91. Coniontis hoppingi Blaisdell, 1918 USA (CA) Coniontis hoppingi Blaisdell, 1918a: 7. Coniontis inaequalis Casey, 1890 USA (CA) Coniontis inaequalis Casey, 1890b: 375. Coniontis inornata Casey, 1908 USA (CA) Coniontis inornata Casey, 1908: 130. Coniontis integer Casey, 1908 USA (CA) Coniontis integer Casey, 1908: 87. Coniontis keiferi (Blaisdell, 1943) MEX (BC) Coniontides keiferi Blaisdell, 1943: 189. Coniontis lamentabilis Blaisdell, 1924 USA (CA) Coniontis lamentabilis Blaisdell, 1924a: 85. Coniontis lanei Boddy, 1957 USA (ORWA) Coniontis lanei Boddy, 1957: 191. Coniontis lariversi Blaisdell, 1941 USA (NV) Coniontis lariversi Blaisdell, 1941c: 131. Coniontis lassenica Casey, 1908 USA (CA NV) Coniontis lassenica Casey, 1908: 95. Coniontis nevadensis Casey, 1908: 95. Synonymy: Doyen (1977 : 3). Coniontis nevadensis carsonica Casey, 1908: 95. Synonymy: Doyen (1977 : 3). Coniontis lata LeConte, 1866 USA (CA) Coniontis lata LeConte, 1866b: 113. Coniontis lata var. insularis Casey, 1890b: 377. Synonymy: Doyen (1977 : 3). Coniontides finitimus Casey, 1908: 79. Synonymy: Doyen (1977 : 3). Coniontides clementinus Casey, 1908: 80. Synonymy: Blaisdell (1921b : 212). Coniontis malkini (Boddy, 1957) USA (CA) Coniontellus malkini Boddy, 1957: 188. Coniontis microsticta Casey, 1908 USA (CA) Coniontis microsticta Casey, 1908: 107. Coniontis inconspicua Casey, 1908: 108. Synonymy: Blaisdell (1924a : 84). Coniontis muscula Blaisdell, 1918 USA (CA) Coniontis globulina var. muscula Blaisdell, 1918a: 9. Coniontis nemoralis borealis Boddy, 1957 USA (CA OR) Coniontis nemoralis borealis Boddy, 1957: 192. Coniontis nemoralis nemoralis Eschscholtz, 1829 USA (CA) Coniontis nemoralis Eschscholtz, 1829: 8. Coniontis obesa LeConte, 1851 USA (CA CO ID MT NV OR WY) Coniontis obesus LeConte, 1851: 131. Coniontellus inflatus Casey, 1890b: 389. Synonymy: Doyen (1977 : 3). Coniontellus subglaber Casey, 1890b: 389. Synonymy: Doyen (1977 : 3). Coniontellus hystrix Casey, 1908: 142. Synonymy (with C. inflatus Casey): La Rivers (1947b : 214). Coniontellus longipennis Casey, 1908: 143. Synonymy (with C. inflatus Casey): La Rivers (1947b : 214). Coniontellus ampliatus Casey, 1908: 144. Synonymy (with C. inflatus Casey): La Rivers (1947b : 214). Coniontellus argutus Casey, 1908: 145. Synonymy: Doyen (1977 : 3). Coniontellus micans Casey, 1908: 145. Synonymy: Doyen (1977 : 3). Coniontis oblonga Casey, 1908 USA (CA) Coniontis oblonga Casey, 1908: 92. Coniontis opaca Horn, 1870 USA (CA) Coniontis opaca Horn, 1870: 296. Coniontis ancilla Casey, 1908: 91. Synonymy: Doyen (1977 : 3). Coniontis degener Casey, 1908: 93. Synonymy: Doyen (1977 : 3). Coniontis ovalis LeConte, 1851 [Fig. 13 ] CAN (BC) USA (AK CA CO ID MT NV OR UT WA) Figure 13. Coniontis ovalis LeConte, 1851. Scale bar = 1 mm. Coniontis ovalis LeConte, 1851: 131. Coniontis alutacea Casey, 1890b: 383. Synonymy: Doyen (1977 : 4). Coniontis breviuscula Casey, 1908: 133. Synonymy: Boddy (1957 : 190). Coniontis sculptipennis Casey, 1908: 134. Synonymy: Boddy (1957 : 190). Coniontis regularis Casey, 1908: 134. Synonymy: Doyen (1977 : 4). Coniontis punctata Casey, 1908: 135. Synonymy (with C. regularis Casey): Boddy (1965 : 141). Coniontis parilis Casey, 1908: 135. Synonymy: Boddy (1957 : 190). Coniontis vancouveri Casey, 1908: 136. Synonymy: Boddy (1957 : 190). Coniontis uteana Casey, 1908: 136. Synonymy: Doyen (1977 : 4). Coniontis inepta Casey, 1908: 137. Synonymy: Doyen (1977 : 4). Coniontis oblita Casey, 1908: 137. Synonymy: Doyen (1977 : 4). Coniontis arida Casey, 1908: 138. Synonymy: Doyen (1977 : 4). Coniontis weidti Casey, 1908: 138. Synonymy: Doyen (1977 : 4). Coniontis acerba Casey, 1908: 139. Synonymy: Doyen (1977 : 4). Coniontis anita Casey, 1908: 139. Synonymy: Doyen (1977 : 4). Coniontis corvina Casey, 1908: 140. Synonymy: Doyen (1977 : 4). Coniontis ovalis okanagani Boddy, 1957: 190. Synonymy: Doyen (1977 : 4). Coniontis pallidicornis Casey, 1890 USA (CA) Coniontis pallidicornis Casey, 1890b: 385. Coniontis obsolescens Casey, 1908: 92. Synonymy: Doyen (1977 : 4). Coniontis parallela Casey, 1890 USA (CA) Coniontis parallela Casey, 1890b: 386. Coniontis parviceps Casey, 1890 USA (CA) MEX (BC) Coniontis parviceps Casey, 1890b: 387. Coniontis filiola Casey, 1908: 115. Synonymy: Doyen (1977 : 4). Coniontis pectoralis Casey, 1908 USA (CA) Coniontis pectoralis Casey, 1908: 86. Coniontis levettei Casey, 1908: 87. Synonymy: Doyen (1977 : 4). Coniontis picescens Casey, 1908: 87. Synonymy: Doyen (1977 : 4). Coniontis perspicua Casey, 1908 USA (CA) Coniontis perspicua Casey, 1908: 114. Coniontis proba Casey, 1908 USA (ID OR) Coniontis proba Casey, 1908: 105. Coniontis puncticollis LeConte, 1851 USA (CA) Coniontis puncticollis LeConte, 1851: 131. Coniontis exigua Casey, 1908: 106. Synonymy: Doyen (1977 : 4). Coniontis paupercula Casey, 1908: 106. Synonymy: Doyen (1977 : 4). Coniontis inflexula Casey, 1908: 107. Synonymy (with C. exigua Casey): Casey (1911 : 253). Coniontis picipes Casey, 1908: 107. Synonymy: Doyen (1977 : 4). Coniontis punctipes Casey, 1890 USA (CA) Coniontis punctipes Casey, 1890b: 380. Coniontis punctulata (Horn, 1876) USA (CA) MEX Coelotaxis punctulata Horn, 1876a: 201. Coelotaxis muricata Horn, 1876a: 201. Synonymy: Doyen (1977 : 4). Coelotaxis angustula Casey, 1890a: 177. Synonymy: Doyen (1977 : 4). Coelotaxis densa Casey, 1908: 149. Synonymy: Doyen (1977 : 4). Coelotaxis frontalis Casey, 1908: 149. Synonymy: Doyen (1977 : 4). Coniontis rainieri Boddy, 1957 USA (WA) Coniontis rainieri Boddy, 1957: 191. Coniontis remnans Pierce, 1954 USA (CA) 18 Coniontis remnans Pierce, 1954c: 155. Coniontis robusta Horn, 1870 USA (CA) Coniontis robusta Horn, 1870: 296. Coniontis luctuosa Casey, 1908: 89. Synonymy: Doyen (1977 : 5). Coniontis sanfordii Blaisdell, 1895 USA (CA) Coniontis sanfordii Blaisdell, 1895: 235. Coniontis santarosae Blaisdell, 1921 USA (CA) Coniontis santarosae Blaisdell, 1921b: 209. Coniontis setosa Casey, 1890 USA (ID NV OR UT WA) Coniontis setosus Casey, 1890b: 387. Coniontis obtusa Casey, 1908: 116. Synonymy: Doyen (1977 : 5). Coniontis wickhami Casey, 1908: 117. Synonymy: Doyen (1977 : 5). Coniontis lanuginosa Casey, 1908: 117. Synonymy: Doyen (1977 : 5). Coniontis pubifera Casey, 1908: 118. Synonymy: Boddy (1965 : 140). Coniontis sparsa Casey, 1908 USA (CA) Coniontis sparsa Casey, 1908: 110. Coniontis subpubescens LeConte, 1851 USA (CA OR) Coniontis subpubescens LeConte, 1851: 131. Coniontis montana Casey, 1890b: 384. Synonymy: Doyen (1977 : 5). Coniontis canonica Casey, 1908: 114. Synonymy: Blaisdell (1918a : 13). Coniontis tenuis Casey, 1908 USA (CA) Coniontis tenuis Casey, 1908: 141. Coniontis thoracica Casey, 1908 USA (CA) Coniontis thoracica Casey, 1908: 104. Coniontis timida Casey, 1908 USA (CA) Coniontis timida Casey, 1908: 102. Coniontis conicicollis Casey, 1908: 102. Synonymy: Doyen (1977 : 5). Coniontis lucidula Casey, 1908: 103. Synonymy: Doyen (1977 : 5). Coniontis protensa Casey, 1908: 104. Synonymy: Doyen (1977 : 5). Coniontis vandykei Blaisdell, 1921 USA (CA) Coniontides vandykei Blaisdell, 1921b: 212. Coniontis ventura Blaisdell, 1924 USA (CA) Coniontis globulina ventura Blaisdell, 1924a: 83. Coniontis viatica Eschscholtz, 1829 USA (CA) Coniontis viatica Eschscholtz, 1829: 7. Coniontis wadei Casey, 1924 USA (WA) Coniontis wadei Casey, 1924: 313. Genus Conisattus Casey, 1895 [M] Conisattus Casey, 1895: 614. Type species: Conisattus rectus Casey, 1895, monotypy. Conisattus rectus Casey, 1895 USA (OR WA) Conisattus rectus Casey, 1895: 614. Conisattus nelsoni Boddy, 1957: 188. Synonymy: Doyen (1984b : 26). Genus Eusattus LeConte, 1851 [M] Eusattus LeConte, 1851: 131. Type species: Eusattus difficilis LeConte, 1851, subsequent designation ( Casey 1908 : 56). Discodemus LeConte, 1862a: 223. Type species: Zophosis reticulata Say, 1824, monotypy. Synonymy: LeConte (1866a : 60). Conipinus LeConte, 1862a: 223. Type species: Eusattus dubius LeConte, 1851, subsequent designation ( Gebien 1938 : 284). Synonymy: LeConte (1866a : 60). Nesostes Casey, 1908: 56, 58. Type species: Eusattus robustus LeConte, 1866, original designation. Synonymy: Triplehorn (1968b : 379). Megasattus Casey, 1908: 56. Type species: Eusattus erosus Horn, 1870, original designation. Synonymy: Triplehorn (1968b : 379). Eusattodes Casey, 1908: 56. Type species: Eusattus laevis LeConte, 1866, original designation. Synonymy: Triplehorn (1968b : 379). Sphaeriontis Casey, 1908: 56, 75. Type species: Eusattus muricatus LeConte, 1851, original designation. Synonymy: La Rivers (1949 : 180). Coelosattus Blaisdell, 1927: 166. Type species: Coelosattus fortineri Blaisdell, 1927 (= Eusattus dilatatus LeConte, 1851), monotypy. Synonymy: Doyen (1972 : 373). Eusattus araneosus (Blaisdell, 1923) MEX (BS) Megasattus araneosus Blaisdell, 1923: 266. Eusattus arenarius Doyen, 1984 MEX (BC BS) Eusattus arenarius Doyen, 1984b: 75. Eusattus aridus Doyen, 1984 MEX (BC BS) Eusattus aridus Doyen, 1984b: 43. Eusattus catalinensis Doyen, 1984 MEX (BS) Eusattus catalinensis Doyen, 1984b: 45. Eusattus catavinus Doyen, 1984 MEX (BC) Eusattus catavinus Doyen, 1984b: 46. Eusattus cedrosensis Doyen, 1984 MEX (BC) Eusattus cedrosensis Doyen, 1984b: 47. Eusattus ceralboensis Doyen, 1984 MEX (BS) Eusattus ceralboensis Doyen, 1984b: 48. Eusattus cienegus Doyen, 1984 MEX (CO) Eusattus cienegus Doyen, 1984b: 49. Eusattus ciliatoides Doyen, 1984 MEX (BC) Eusattus ciliatoides Doyen, 1984b: 75. Eusattus ciliatus Horn, 1894 MEX (BC) Eusattus ciliatus Horn, 1894b: 422. Eusattus convexus LeConte, 1851 USA (AZ CO KS MO NM OK TX UT WY) MEX (CH DU SO) Eusattus convexus LeConte, 1851: 132. Eusattus sculptus Champion, 1892: 510. Synonymy: Doyen (1984b : 35). Eusattus rotundus Casey, 1908: 72. Synonymy: Doyen (1977 : 5). Eusattus turgidus Casey, 1908: 73. Synonymy: Doyen (1977 : 5). Eusattus subnitens Casey, 1908: 73. Synonymy: Doyen (1984b : 35). Eusattus peropacus Casey, 1908: 74. Synonymy: Doyen (1977 : 5). Eusattus acutus Casey, 1908: 74. Synonymy: Doyen (1977 : 5). Eusattus quadratus Casey, 1924: 311. Synonymy: Doyen (1977 : 5). Eusattus subvelutinus Casey, 1924: 312. Synonymy: Doyen (1977 : 5). Eusattus woodgatei Casey, 1924: 312. Synonymy: Doyen (1977 : 5). Eusattus costatus Horn, 1870 USA (CA) MEX (BC BS) Eusattus costatus Horn, 1870: 293. Megasattus sternalis Blaisdell, 1923: 268. Synonymy: Doyen (1984b : 50). Eusattus crypticus Doyen, 1984 MEX (BS) Eusattus crypticus Doyen, 1984b: 90. Eusattus depressus Champion, 1884 MEX (CH NA SI SO) Eusattus depressus Champion, 1884: 75. Eusattus puncticeps Blaisdell, 1923: 269. Synonymy: Doyen (1984b : 87). Eusattus difficilis LeConte, 1851 USA (CA NV) MEX (BC SO) Eusattus difficilis LeConte, 1851: 132. Eusattus coquilletti Linell, 1899: 180. Synonymy: Doyen (1977 : 5). Eusattus agnatus Casey, 1908: 70. Synonymy: Doyen (1977 : 5). Eusattus compositus Casey, 1908: 71. Synonymy: Doyen (1977 : 5). Eusattus congener Casey, 1908: 71. Synonymy: Doyen (1977 : 5). Eusattus acutangulus Casey, 1908: 72. Synonymy: Doyen (1977 : 5). Eusattus dilatatus LeConte, 1851 USA (AZ CA) MEX (SO) Eusattus dilatatus LeConte, 1851: 132. Coelosattus fortineri Blaisdell, 1927: 167. Synonymy: Doyen (1977 : 5). Eusattus dubius abditus Doyen, 1984 MEX (BC) Eusattus dubius abditus Doyen, 1984b: 82. Eusattus dubius arizonensis Doyen, 1984 USA (AZ CA NV) Eusattus dubius arizonensis Doyen, 1984b: 81. Eusattus dubius dubius LeConte, 1851 USA (AZ CA NV UT) Eusattus dubius LeConte, 1851: 132. Eusattus nanus Casey, 1895: 613. Synonymy: Doyen (1977 : 5). Eusattus oblongulus Casey, 1908: 67. Synonymy: Doyen (1977 : 5). Conipinus spaldingi Casey, 1924: 313. Synonymy: Doyen (1977 : 5). Eusattus dubius setosus Doyen, 1984 MEX (BS) Eusattus dubius setosus Doyen, 1984b: 82. Eusattus erosus erosus Horn, 1870 MEX (BS) Eusattus erosus Horn, 1870: 294. Eusattus erosus laeviventris (Blaisdell, 1923) MEX (BS) Megasattus laeviventris Blaisdell, 1923: 267. Eusattus erosus manuelis (Blaisdell, 1923) MEX (BS) Megasattus erosus manuelis Blaisdell, 1923: 266. Eusattus franciscanus Doyen, 1984 MEX (BS) Eusattus franciscanus Doyen, 1984b: 54. Eusattus hirsutus Doyen, 1984 USA (NV) Eusattus hirsutus Doyen, 1984b: 66. Eusattus laevis LeConte, 1866 MEX (BS) Eusattus laevis LeConte, 1866b: 113. Eusattus mexicanus Champion, 1892 MEX (CO GE JA) Eusattus mexicanus Champion, 1892: 510. Eusattus minimus Doyen, 1984 MEX (NL) Eusattus minimus Doyen, 1984b: 38. Eusattus muricatus diabloensis Doyen, 1984 MEX (BC) Eusattus muricatus diabloensis Doyen, 1984b: 71. Eusattus muricatus muricatus LeConte, 1851 USA (AZ CA CO ID NM NV OR TX UT WA) Eusattus muricatus LeConte, 1851: 132. Sphaeriontis acomana Casey, 1908: 76. Synonymy: La Rivers (1949 : 180). Sphaeriontis latissima Casey, 1924: 310. Synonymy: La Rivers (1949 : 180). Sphaeriontis fulvescens Casey, 1924: 310. Synonymy: La Rivers (1949 : 180). Eusattus nitidipennis LeConte, 1851 MEX (CO GU NL PU VE ZA) Eusattus nitidipennis LeConte, 1851: 133. Eusattus brevis Champion, 1884: 75. Synonymy: Doyen (1977 : 6). Eusattus obliteratus Champion, 1892 MEX (CO DU) Eusattus obliteratus Champion, 1892: 510 19 . Eusattus pallidus adustus Doyen, 1984 MEX (BS) Eusattus pallidus adustus Doyen, 1984b: 84. Eusattus pallidus immaculatus Doyen, 1984 MEX (BS) Eusattus pallidus immaculatus Doyen, 1984b: 85. Eusattus pallidus pallidus Doyen, 1984 MEX (BS) Eusattus pallidus pallidus Doyen, 1984b: 84. Eusattus phreatophilus Doyen, 1984 USA (CA NV) Eusattus phreatophilus Doyen, 1984b: 71. Eusattus planulus Doyen, 1984 MEX (BS) Eusattus planulus Doyen, 1984b: 57. Eusattus politus cruzensis Doyen, 1984 USA (CA) Eusattus politus cruzensis Doyen, 1984b: 93. Eusattus politus politus Horn, 1883 USA (CA) Eusattus politus Horn, 1883: 304. Eusattus vanduzeei Blaisdell, 1921b: 214. Synonymy: Doyen (1984b : 93). Eusattus pons Triplehorn, 1968 USA (TX) MEX (CH CO DU) Eusattus pons Triplehorn, 1968b: 376. Eusattus productus LeConte, 1858 USA (AZ CA) MEX (BC SO) Eusattus productus LeConte, 1858a: 20. Eusattus explanatus Casey, 1908: 68. Synonymy: Doyen (1977 : 6). Eusattus vicinus Casey, 1908: 68. Synonymy: Doyen (1977 : 6). Eusattus lobatus Casey, 1908: 68. Synonymy: Doyen (1977 : 6). Eusattus puberulus LeConte, 1854 USA (TX) Eusattus puberulus LeConte, 1854a: 84. Eusattus reticulatus (Say, 1824) USA (AZ CO NM OK TX UT) MEX (CH SO) Tenebrio gibbus DeGeer, 1778: 652 [junior primary homonym of Tenebrio gibbus Linnaeus, 1767]. Tenebrio striatus Retzius, 1783: 134 [junior primary homonym of Tenebrio striatus Müller, 1776]. Replacement name for Tenebrio gibbus DeGeer, 1778. Zophosis reticulata Say, 1824a: 250. Synonymy: Ferrer and Holston (2011 : 252). Discodemus corrosus Casey, 1908: 61. Synonymy: Doyen (1977 : 6). Discodemus brevipennis Casey, 1908: 61. Synonymy: Doyen (1977 : 6). Discodemus elongatulus Casey, 1908: 61. Synonymy: Doyen (1977 : 6). Discodemus depressulus Casey, 1908: 62. Synonymy: Doyen (1977 : 6). Discodemus subsericeus Casey, 1908: 62. Synonymy: Doyen (1977 : 6). Discodemus knausi Casey, 1908: 62. Synonymy: Doyen (1977 : 6). Eusattus robustus LeConte, 1866 USA (CA) Eusattus robustus LeConte, 1866b: 112. Eusattus robustus postremus Casey, 1908: 59. Synonymy: Doyen (1977 : 6). Eusattus rudei Doyen, 1984 MEX (BS) Eusattus rudei Doyen, 1984b: 91. Eusattus secutus Horn, 1894 MEX (BS) Eusattus secutus Horn, 1894b: 421. Eusattus venosus Champion, 1892 MEX (CO JA NA) Eusattus venosus Champion, 1892: 509. Eusattus vizcainensis Doyen, 1984 MEX (BC BS) Eusattus vizcainensis Doyen, 1984b: 85. Tribe Cryptoglossini LeConte, 1862 Centrioptérides Lacordaire, 1859: 134 [ nomen oblitum , see Aalbu 2006 : 57]. Type genus: Centrioptera Mannerheim, 1843. Cryptoglossini LeConte, 1862a: 220 [ nomen protectum ]. Type genus: Cryptoglossa Solier, 1837. Genus Asbolus LeConte, 1851 [M] Asbolus LeConte, 1851: 129. Type species: Asbolus verrucosus LeConte, 1851, subsequent designation ( Aalbu 2005 : 721). Asbolus laevis LeConte, 1851 USA (AZ CA) MEX (BC SO) Asbolus laevis LeConte, 1851: 130. Cryptoglossa laevis subsimilis Casey, 1924: 308. Synonymy: Aalbu (2005 : 730). Asbolus mexicanus angularis (Horn, 1894) USA (AZ CA) MEX (BC BS) Centrioptera angularis Horn, 1894b: 414. Asbolus mexicanus mexicanus (Champion, 1884) USA (NM TX) MEX (CH CO DU NL) Cryptoglossa mexicana Champion, 1884: 73. Cryptoglossa granulifera Champion, 1892: 508. Synonymy: Aalbu (2005 : 722). Asbolus papillosus (Triplehorn, 1964) USA (CA) MEX (SO) Cryptoglossa laevis papillosa Triplehorn, 1964a: 48. Asbolus verrucosus LeConte, 1851 USA (AZ CA NM NV UT) MEX (BC SO) Asbolus verrucosus LeConte, 1851: 129. Cryptoglossa verrucosa carinulatus Blaisdell, 1945: 25. Synonymy: Aalbu (2005 : 724). Genus Cryptoglossa Solier, 1837 [F] Cryptoglossa Solier, 1837: 680. Type species: Cryptoglossa bicostata Solier, 1837, monotypy. Centrioptera Mannerheim, 1843: 279. Type species: Centrioptera caraboides Mannerheim, 1843, monotypy. Synonymy: Aalbu et al. (2002 : 486). Oochila LeConte, 1862a: 220. Type species: Asbolus infaustus LeConte, 1854, original designation. Synonymy (with Centrioptera Mannerheim): Horn (1870 : 278). Amblycyphus Motschulsky, 1870: 401. Type species: Amblycyphus asperatus Motschulsky, 1870 (= Centrioptera pectoralis Blaisdell, 1921), monotypy. Synonymy: Aalbu et al. (1995 : 483). Cryptoglossa asperata (Horn, 1870) MEX (BS) Centrioptera asperata Horn, 1870: 279. Centrioptera asperata discreta Blaisdell, 1923: 249. Synonymy: Aalbu (2005 : 709). Centrioptera asperata subornata Blaisdell, 1923: 249. Synonymy: Aalbu (2005 : 709). Centrioptera asperata planata Blaisdell, 1923: 250. Synonymy: Aalbu (2005 : 709). Cryptoglossa bicostata Solier, 1837 MEX (OA PU) Cryptoglossa bicostata Solier, 1837: 681. Cryptoglossa caraboides (Mannerheim, 1843) MEX (GE MO PU) Centrioptera caraboides Mannerheim, 1843: 280. Cryptoglossa infausta (LeConte, 1854) USA (TX) MEX (CO DU TA) Asbolus infaustus LeConte, 1854a: 84. Centrioptera spiculosa Champion, 1892: 508. Synonymy: Champion (1893a : 572). Centrioptera texana Blaisdell, 1924b: 88. Synonymy: Aalbu (2005 : 705). Cryptoglossa michelbacheri (Blaisdell, 1943) MEX (BS) Centrioptera michelbacheri Blaisdell, 1943: 222. Cryptoglossa muricata (LeConte, 1851) USA (AZ CA NV UT) MEX (BC SO) Centrioptera muricata LeConte, 1851: 142. Centrioptera utensis Casey, 1907: 513. Synonymy: Aalbu (2005 : 712). Centrioptera sculptiventris Blaisdell, 1923: 247. Synonymy: Aalbu (2005 : 712). Centrioptera serrata Casey, 1924: 306. Synonymy: Aalbu (2005 : 712). Centrioptera elongata Casey, 1924: 306. Synonymy: Aalbu (2005 : 712). Cryptoglossa seriata cerralvoensis Aalbu, 2005 MEX (BS) Cryptoglossa seriata cerralvoensis Aalbu, 2005: 721. Cryptoglossa seriata seriata LeConte, 1861 USA (AZ CA) MEX (BS) Cryptoglossa seriata LeConte, 1861a: 337. Cryptoglossa spiculifera pectoralis (Blaisdell, 1921) USA (CA) MEX (BC BS) Amblycyphus asperatus Motschulsky, 1870: 404 [junior secondary homonym of Cryptoglossa asperata (Horn, 1870) 20 ]. Centrioptera pectoralis Blaisdell, 1921b: 198. Synonymy: Aalbu et al. (1995 : 483). Centrioptera dulzurae Blaisdell, 1921b: 199. Synonymy: Aalbu et al. (1995 : 483). Centrioptera chamberlini Blaisdell, 1923: 246. Synonymy: Aalbu (2005 : 717). Cryptoglossa spiculifera spiculifera (LeConte, 1861) MEX (BS) Centrioptera spiculifera LeConte, 1861a: 337. Cryptoglossa variolosa (Horn, 1870) USA (AZ CA NM) MEX (SI SO) Centrioptera variolosa Horn, 1870: 280. Genus Schizillus Horn, 1874 [M] Schizillus Horn, 1874a: 33. Type species: Schizillus laticeps Horn, 1874, monotypy. Schizillus laticeps Horn, 1874 USA (AZ CA NV UT) MEX (BC) Schizillus laticeps Horn, 1874a: 33. Schizillus convexus Blaisdell, 1921b: 203. Synonymy: Aalbu (2005 : 732). Schizillus lomae Blaisdell, 1921b: 206. Synonymy: Aalbu (2005 : 732). Schizillus opacus Casey, 1924: 307. Synonymy: Aalbu (2005 : 732). Schizillus nunenmacheri Blaisdell, 1921 USA (AZ CA NV UT) Schizillus nunenmacheri Blaisdell, 1921b: 204. Schizillus beali Parker, 1955: 148. Synonymy: Aalbu (2005 : 734). Tribe Edrotini Lacordaire, 1859 Édrotides Lacordaire, 1859: 31. Type genus: Edrotes LeConte, 1851. Triorophi LeConte and Horn, 1883: 362. Type genus: Triorophus LeConte, 1851. Auchmobii LeConte and Horn, 1883: 362. Type genus: Auchmobius LeConte, 1851. Trimytini Casey, 1907: 278. Type genus: Trimytis Leconte, 1851. Eurymetoponini Casey, 1907: 278. Type genus: Eurymetopon Eschscholtz, 1831. Trientomini Casey, 1907: 278. Type genus: Trientoma Solier, 1835. Genus Armalia Casey, 1907 [F] Armalia Casey, 1907: 289, 330. Type species: Emmenastus texanus LeConte, 1866, original designation. Armalia alata (Champion, 1884) GUA NIC Emmenastus alatus Champion, 1884: 13. Emmenastus salvini Champion, 1884: 13. Synonymy: Champion (1892 : 482). Armalia angularis Casey, 1907 USA (TX) Armalia angularis Casey, 1907: 331. Armalia belti (Champion, 1884) MEX (YU) GUA HON NIC Emmenastus belti Champion, 1884: 11. Emmenastus rotundicollis Champion, 1884: 11. Synonymy: Champion (1892 : 480). Emmenastus intermedius Champion, 1884: 12. Synonymy: Champion (1892 : 480). Armalia brevipennis (Champion, 1884) MEX (NA) Emmenastus brevipennis Champion, 1884: 10. Armalia canaliculata (Champion, 1884) MEX (SI) Emmenastus canaliculatus Champion, 1884: 10. Armalia chiriquensis (Champion, 1884) PAN / SA Emmenastus chiriquensis Champion, 1884: 9. Armalia longicornis (Champion, 1884) GUA Emmenastus longicornis Champion, 1884: 9. Armalia solitaria (Champion, 1884) MEX (OA) Emmenastus solitarius Champion, 1884: 11. Armalia texana (LeConte, 1866) USA (TX) Emmenastus texanus LeConte, 1866b: 108. Armalia variabilis (Champion, 1884) MEX (VE) HON Emmenastus variabilis Champion, 1884: 10. Genus Auchmobius LeConte, 1851 [M] Auchmobius LeConte, 1851: 139. Type species: Auchmobius sublaevis LeConte, 1851, monotypy. Auchmobius angelicus Blaisdell, 1934 USA (CA) Auchmobius angelicus Blaisdell, 1934b: 249. Auchmobius parvicollis Blaisdell, 1934 USA (CA) Auchmobius parvicollis Blaisdell, 1934b: 246. Auchmobius picipes Blaisdell, 1934 USA (CA) Auchmobius picipes Blaisdell, 1934b: 252. Auchmobius sanfordi Blaisdell, 1934 USA (CA) Auchmobius sanfordi Blaisdell, 1934b: 257. Auchmobius slevini Blaisdell, 1934 USA (CA) Auchmobius slevini Blaisdell, 1934b: 243. Auchmobius subboreus Blaisdell, 1934 USA (CA NV) Auchmobius subboreus Blaisdell, 1934b: 254. Auchmobius sublaevis LeConte, 1851 USA (CA) Auchmobius sublaevis LeConte, 1851: 140. Auchmobius subovalis Blaisdell, 1934 USA (CA) Auchmobius subovalis Blaisdell, 1934b: 238. Genus Chilometopon Horn, 1874 [N] Chilometopon Horn, 1874a: 31. Type species: Trimytis abnormis Horn, 1870, subsequent designation ( Casey 1907 : 367). Prometopion Casey, 1907: 366, 370. Type species: Prometopion amplipenne Casey, 1907 (= Chilometopon helopioides Horn, 1874), original designation. Synonymy: MacLachlan and Olson (1990 : 72). Chilometopon abnorme (Horn, 1870) USA (AZ CA NV OR UT) MEX (BC SO) Trimytis abnormis Horn, 1870: 261. Chilometopon castaneum Casey, 1907: 373. Synonymy: MacLachlan and Olson (1990 : 74). Chilometopon brevipenne Casey, 1907: 374. Synonymy: MacLachlan and Olson (1990 : 74). Chilometopon ensifer Casey, 1907: 374. Synonymy: MacLachlan and Olson (1990 : 74). Chilometopon brachystomum Doyen, 1983 USA (AZ CA NV) MEX (BC) Chilometopon brachystomum Doyen, 1983: 81. Chilometopon cribricolle Blaisdell, 1923 MEX (BS) Chilometopon cribricolle Blaisdell, 1923: 230. Chilometopon helopioides Horn, 1874 USA (AZ CA ID NM NV UT) MEX (BC) Chilometopon helopioides Horn, 1874a: 31. Prometopion amplipenne Casey, 1907: 372. Synonymy: MacLachlan and Olson (1990 : 78). Chilometopon microps MacLachlan and Olson, 1990 USA (CA) Chilometopon microps MacLachlan and Olson, 1990: 76. Chilometopon pallidum Casey, 1890 USA (AZ CA NM NV TX UT) MEX (BC CH) Chilometopon pallidum Casey, 1890b: 367. Chilometopon rugiceps Blaisdell, 1923 MEX (BC) Chilometopon rugiceps Blaisdell, 1923: 229. Genus Cryptadius LeConte, 1851 [M] Cryptadius LeConte, 1851: 140. Type species: Cryptadius inflatus LeConte, 1851, monotypy. Cryptadius inflatus blaisdelli Thomas, 1985 MEX (BS) Cryptadius inflatus blaisdelli Thomas, 1985: 197. Cryptadius inflatus inflatus LeConte, 1851 USA (CA) MEX (BC) Cryptadius inflatus LeConte, 1851: 140. Cryptadius oviformis Casey, 1907: 328. Synonymy: Thomas (1985 : 196). Cryptadius punctipennis Casey, 1907: 328. Synonymy: Thomas (1985 : 196). Cryptadius curvipes Casey, 1907: 329. Synonymy: Thomas (1985 : 196). Cryptadius sonorae Berry, 1974 MEX (BS SO) Cryptadius sonorae Berry, 1974: 175. Cryptadius tarsalis Blaisdell, 1923 MEX (BC BS SO) Cryptadius tarsalis Blaisdell, 1923: 212. Cryptadius angulatus Blaisdell, 1923: 210. Synonymy: Thomas (1985 : 198). Cryptadius sinuatus Blaisdell, 1923: 211. Synonymy: Thomas (1985 : 198). Cryptadius andrewsi Berry, 1977: 561. Synonymy: Thomas (1985 : 198). Genus Ditaphronotus Casey, 1907 [M] Ditaphronotus Casey, 1907: 341. Type species: Emmenastus foveicollis Champion, 1884, original designation. Ditaphronotus championi Casey, 1907 NIC Ditaphronotus championi Casey, 1907: 342. Ditaphronotus confusus (Champion, 1884) MEX (CI) GUA Emmenastus confusus Champion, 1884: 15. Ditaphronotus foveicollis (Champion, 1884) GUA NIC CRI Emmenastus foveicollis Champion, 1884: 14. Ditaphronotus laevicollis (Champion, 1884) PAN Emmenastus laevicollis Champion, 1884: 15. Genus Edrotes LeConte, 1851 [M] Edrotes LeConte, 1851: 140. Type species: Edrotes ventricosus LeConte, 1851, monotypy. Hedrotes Gemminger [in Gemminger and Harold], 1870: 1816. Unjustified emendation of Edrotes LeConte, 1851, not in prevailing usage. Subgenus Edrotes LeConte, 1851 Edrotes LeConte, 1851: 140. Type species: Edrotes ventricosus LeConte, 1851, monotypy. Edrotes fossor Triplehorn, 1972 MEX (BS) Edrotes fossor Triplehorn, 1972: 27. Edrotes leechi Doyen, 1968 USA (AZ CO UT) Edrotes leechi Doyen, 1968: 218. Edrotes rotundus (Say, 1824) USA (AZ CO NM TX UT) MEX (BC CH) Pimelia rotunda Say, 1824a: 251. Edrotes globosus Casey, 1890a: 175. Synonymy: La Rivers (1947a : 325). Edrotes inflatus Casey, 1907: 454. Synonymy: La Rivers (1947a : 325). Edrotes puncticeps Casey, 1907: 454. Synonymy: La Rivers (1947a : 325). Edrotes intermixtus Casey, 1907: 455. Synonymy: La Rivers (1947a : 325). Edrotes oblongulus Casey, 1907: 455. Synonymy: La Rivers (1947a : 325). Edrotes lineatus Casey, 1907: 456. Synonymy: La Rivers (1947a : 325). Edrotes subaequalis Casey, 1907: 456. Synonymy: La Rivers (1947a : 325). Edrotes angustulus Casey, 1907: 456. Synonymy: La Rivers (1947a : 325). Edrotes desertus Blaisdell, 1943: 212. Synonymy: La Rivers (1947a : 325). Edrotes ventricosus LeConte, 1851 USA (AZ CA ID NV OR) MEX (BC BS SO) Edrotes ventricosus LeConte, 1851: 141. Edrotes nitidus Casey, 1890a: 175. Synonymy: La Rivers (1947a : 321). Edrotes orbus Casey, 1907: 452. Synonymy: La Rivers (1947a : 321). Edrotes angusticollis Casey, 1907: 452. Synonymy: La Rivers (1947a : 321). Edrotes longipennis Casey, 1907: 453. Synonymy: La Rivers (1947a : 321). Edrotes mexicanus Blaisdell, 1923: 241. Synonymy: La Rivers (1947a : 321). Edrotes asperatus Blaisdell, 1923: 241. Synonymy: La Rivers (1947a : 321). Edrotes laticollis Casey, 1924: 300. Synonymy: La Rivers (1947a : 321). Edrotes longicornis Casey, 1924: 300. Synonymy: La Rivers (1947a : 321). Edrotes variipilis Casey, 1924: 301. Synonymy: La Rivers (1947a : 321). Edrotes barrowsi Dajoz, 1999: 320. New synonymy [RLA]. Subgenus Odrotes La Rivers, 1947 Odrotes La Rivers, 1947a: 320. Type species: Edrotes arens La Rivers, 1947, monotypy. Edrotes arens La Rivers, 1947 USA (AZ CA) MEX (BC) Edrotes arens La Rivers, 1947a: 320. Genus Emmenastrichus Horn, 1894 [M] Emmenastrichus Horn, 1894b: 413. Type species: Emmenastrichus cribratus Horn, 1894, subsequent designation ( Casey 1907 : 289). Emmenastrichus cribratus Horn, 1894 MEX (BS) Emmenastrichus cribratus Horn, 1894b: 413. Emmenastrichus erosus Horn, 1894 MEX (BS) Emmenastrichus erosus Horn, 1894b: 414. Genus Emmenides Casey, 1907 [M] Emmenides Casey, 1907: 329. Type species: Emmenastus punctatus LeConte, 1866, original designation. Emmenides apicalis Blaisdell, 1923 MEX (BS) Emmenides apicalis Blaisdell, 1923: 215. Emmenides catalinae Blaisdell, 1923 MEX (BS) Emmenides catalinae Blaisdell, 1923: 216. Emmenides igualensis (Champion, 1892) MEX (GE) Emmenastus igualensis Champion, 1892: 484. Emmenides obsoletus Blaisdell, 1923 MEX (BS) Emmenides obsoletus Blaisdell, 1923: 216. Emmenides punctatus (LeConte, 1866) USA (AZ TX) MEX (BS) Emmenastus punctatus LeConte, 1866b: 106. Emmenides subdescalceatus Blaisdell, 1923 MEX (BS) Emmenides subdescalceatus Blaisdell, 1923: 213. Genus Eremocantor Smith and Wirth, 2016 [M] Eremocantor Smith and Wirth, 2016: 582. Type species: Eremocantor marioni Smith and Wirth, 2016, original designation. Eremocantor marioni Smith and Wirth, 2016 USA (TX) Eremocantor marioni Smith and Wirth, 2016: 583. Genus Eschatomoxys Blaisdell, 1935 [M] Eschatomoxys Blaisdell, 1935d: 125. Type species: Eschatomoxys wagneri Blaisdell, 1935, original designation. Eschatomoxys andrewsi Aalbu and Thomas, 2008 USA (CA) Eschatomoxys andrewsi Aalbu and Thomas [in Pape et al.], 2008 : 529. Eschatomoxys paco Aalbu and Thomas, 2008 MEX (BC) Eschatomoxys paco Aalbu and Thomas [in Pape et al.], 2008 : 527. Eschatomoxys pholeter Thomas and Pape, 2008 USA (AZ) Eschatomoxys pholeter Thomas and Pape [in Pape et al.], 2008 : 525. Eschatomoxys rosei Aalbu and Thomas, 2008 MEX (BC) Eschatomoxys rosei Aalbu and Thomas [in Pape et al.], 2008 : 530. Eschatomoxys tanneri Sorenson and Stones, 1959 USA (AZ UT) Eschatomoxys tanneri Sorenson and Stones, 1959: 63. Eschatomoxys wagneri Blaisdell, 1935 USA (AZ CA) Eschatomoxys wagneri Blaisdell, 1935d: 125. Genus Eurymetopon Eschscholtz, 1831 [N] Eurymetopon Eschscholtz, 1831: 5, 8. Type species: Eurymetopon rufipes Eschscholtz, 1831, subsequent designation ( Casey 1907 : 288). Eurymetopon ochraceum Eschscholtz, 1831 USA (CA) Eurymetopon ochraceum Eschscholtz, 1831: 8. Eurymetopon rufipes Eschscholtz, 1831 USA (AZ CA) MEX (BS SO) Eurymetopon rufipes Eschscholtz, 1831: 8. Genus Garridoa Marcuzzi, 1985 [F] Garridoa Marcuzzi, 1985: 180. Type species: Garridoa kaszabi Marcuzzi, 1985, monotypy. Garridoa kaszabi Marcuzzi, 1985 CUB Garridoa kaszabi Marcuzzi, 1985: 180. Genus Hylocrinus Casey, 1907 [M] Hylocrinus Casey, 1907: 289, 331. Type species: Eurymetopon longulum LeConte, 1851, original designation. Subgenus Hylocrinus Casey, 1907 Hylocrinus Casey, 1907: 289, 331. Type species: Eurymetopon longulum LeConte, 1851, original designation. Hylocrinus ambiguus (Champion, 1884) PAN Emmenastus ambiguus Champion, 1884: 13. Hylocrinus angustus (Casey, 1890) USA (AZ) Emmenastus angustus Casey, 1890b: 352. Hylocrinus blaisdelli Casey, 1907 USA (CA) Hylocrinus blaisdelli Casey, 1907: 336. Hylocrinus breviusculus Casey, 1907 USA (TX) Hylocrinus breviusculus Casey, 1907: 334. Hylocrinus cunctans Casey, 1907 USA (TX) Hylocrinus cunctans Casey, 1907: 336. Hylocrinus delicatulus Casey, 1907 USA (AZ NV UT) Hylocrinus delicatulus Casey, 1907: 334. Hylocrinus depressulus Casey, 1907 USA (CA) Hylocrinus depressulus Casey, 1907: 335. Hylocrinus filitarsis Casey, 1907 USA (CA) Hylocrinus filitarsis Casey, 1907: 333. Hylocrinus guatemalensis (Champion, 1884) GUA Emmenastus guatemalensis Champion, 1884: 14. Hylocrinus longulus (LeConte, 1851) USA (AZ CA) MEX (BC SO) Eurymetopon longulum LeConte, 1851: 139. Hylocrinus magnus Blaisdell, 1923 MEX (SO) Hylocrinus magnus Blaisdell, 1923: 219. Hylocrinus tenuis Casey, 1907 USA (AZ) Hylocrinus tenuis Casey, 1907: 333. Subgenus Locrodes Casey, 1907 Locrodes Casey, 1907: 332. Type species: Emmenastus piceus Casey, 1890, present designation . Hylocrinus brunnescens Casey, 1907 USA (UT) Hylocrinus brunnescens Casey, 1907: 338. Hylocrinus fraternus Casey, 1907 USA (ID UT) Hylocrinus fraternus Casey, 1907: 338. Hylocrinus insularis Blaisdell, 1923 MEX (BS) Hylocrinus insularis Blaisdell, 1923: 218. Hylocrinus laborans Casey, 1907 USA (NV UT) Hylocrinus laborans Casey, 1907: 337. Hylocrinus mexicanus (Champion, 1892) MEX (FD) Emmenastus mexicanus Champion, 1892: 481. Hylocrinus oblongulus Casey, 1907 USA (CA) MEX (BC BS) Hylocrinus oblongulus Casey, 1907: 337. Hylocrinus parallelus (Champion, 1884) MEX (JA ME MO OA PU SI) Emmenastus parallelus Champion, 1884: 12. Hylocrinus piceus (Casey, 1890) USA (CA) Emmenastus piceus Casey, 1890b: 353. Hylocrinus seriatus (Champion, 1892) MEX (OA) Emmenastus seriatus Champion, 1892: 482. Hylocrinus subapterus (Champion, 1892) MEX (DU) Emmenastus subapterus Champion, 1892: 481. Hylocrinus tenebrosus (Champion, 1884) MEX (AG FD GU) Emmenastus tenebrosus Champion, 1884: 12. Hylocrinus umbrosus Casey, 1907 USA (UT) Hylocrinus umbrosus Casey, 1907: 338. Subgenus Paravius Casey, 1907 Paravius Casey, 1907: 332. Type species: Emmenastus marginatus Casey, 1890, monotypy. Hylocrinus marginatus (Casey, 1890) MEX (BC) Emmenastus marginatus Casey, 1890b: 351. Hylocrinus vicinus (Champion, 1884) USA (CA) Emmenastus vicinus Champion, 1884: 8. Genus Melanastus Casey, 1907 [M] Melanastus Casey, 1907: 289. Type species: Eurymetopon atrum LeConte, 1851, original designation. Melanastus acuminatus Casey, 1907 USA (CO) Melanastus acuminatus Casey, 1907: 362. Melanastus acutus (Horn, 1870) [Fig. 14 ] CAN (AB SK) USA (NE) Figure 14. Melanastus acutus (Horn, 1870). Scale bar = 1 mm. Emmenastus acutus Horn, 1870: 270. Melanastus aequicollis Casey, 1907 USA (CA) Melanastus aequicollis Casey, 1907: 360. Melanastus ater (LeConte, 1851) USA (CA ID OR) Eurymetopon atrum LeConte, 1851: 139. Melanastus coarcticollis (Casey, 1890) USA (NM) Emmenastus coarcticollis Casey, 1890b: 364. Melanastus crassicornis (Casey, 1890) USA (CA) Emmenastus crassicornis Casey, 1890b: 363. Melanastus exiguus Casey, 1907 USA (CO) Melanastus exiguus Casey, 1907: 363. Melanastus exoletus Casey, 1907 USA (CA) Melanastus exoletus Casey, 1907: 357. Melanastus fallax (Casey, 1890) USA (NM) Emmenastus fallax Casey, 1890b: 361. Melanastus finitimus Casey, 1907 USA (CO) Melanastus finitimus Casey, 1907: 359. Melanastus implicans Casey, 1907 USA (CO) Melanastus implicans Casey, 1907: 358. Melanastus lucidulus Casey, 1907 USA (CA) Melanastus lucidulus Casey, 1907: 358. Melanastus ludius Casey, 1907 USA (UT) Melanastus ludius Casey, 1907: 361. Melanastus moestus Casey, 1907 USA (CA) Melanastus moestus Casey, 1907: 355. Melanastus nitidus (Casey, 1890) USA (AZ) Emmenastus nitidus Casey, 1890b: 362. Melanastus nuperus Casey, 1907 USA (AZ) Melanastus nuperus Casey, 1907: 364. Melanastus obesus (LeConte, 1851) USA (CA) MEX (BC BS) Eurymetopon obesum LeConte, 1851: 139. Emmenastus nanulus Casey, 1884: 45. Synonymy: Casey (1907 b: 363). Melanastus obscurus Blaisdell, 1923 MEX (SO) Melanastus obscurus Blaisdell, 1923: 226. Melanastus obtusus (LeConte, 1866) USA (CA) Emmenastus obtusus LeConte, 1866b: 107. Melanastus otiosus Casey, 1907 USA (CA) Melanastus otiosus Casey, 1907: 358. Melanastus parvus Casey, 1907 USA (CO) Melanastus parvus Casey, 1907: 362. Melanastus sonoricus Casey, 1907 MEX (CH MI) Melanastus sonoricus Casey, 1907: 364. Melanastus sterilis Casey, 1907 USA (CA) Melanastus sterilis Casey, 1907: 357. Melanastus texanus Blaisdell, 1926 USA (TX) Melanastus texanus Blaisdell, 1926b: 22. Melanastus thoracicus (Casey, 1890) USA (CA) Emmenastus thoracicus Casey, 1890b: 362. Melanastus vegrandis Casey, 1907 USA (CA) Melanastus vegrandis Casey, 1907: 360. Genus Mencheres Champion, 1884 [M] Mencheres Champion, 1884: 5. Type species: Mencheres nicaraguensis Champion, 1884, subsequent designation ( Lucas 1920 : 403). Mencheres elongatus Champion, 1884 GUA Mencheres elongatus Champion, 1884: 6. Mencheres nicaraguensis Champion, 1884 NIC Mencheres nicaraguensis Champion, 1884: 5. Genus Mesabates Champion, 1884 [M] Mesabates Champion, 1884: 3. Type species: Mesabates latifrons Champion, 1884, monotypy. Mesabates latifrons Champion, 1884 MEX (OA PU) Mesabates latifrons Champion, 1884: 3. Mesabates spissicornis Champion, 1892 MEX (SI) Mesabates spissicornis Champion, 1892: 479. Genus Mesabatodes Casey, 1907 [M] Mesabatodes Casey, 1907: 517. Type species: Mesabates inaequalis Champion, 1892, original designation. Mesabatodes inaequalis (Champion, 1892) MEX (AG CH DU) Mesabates inaequalis Champion, 1892: 480. Genus Metoponium Casey, 1907 [N] Metoponium Casey, 1907: 288. Type species: Eurymetopon abnorme LeConte, 1851, original designation. Subgenus Metoponiopsis Casey, 1907 Metoponiopsis Casey, 1907: 290. Type species: Eurymetopon bicolor Horn, 1870, monotypy. Metoponium bicolor (Horn, 1870) USA (AZ CA) MEX (BC) Eurymetopon bicolor Horn, 1870: 268. Subgenus Metoponium Casey, 1907 Metoponium Casey, 1907: 288. Type species: Eurymetopon abnorme LeConte, 1851, original designation. Metoponium abnorme abnorme (LeConte, 1851) USA (CA) Eurymetopon abnorme LeConte, 1851: 138. Metoponium abnorme faustum Casey, 1907 USA (CA) Metoponium faustum Casey, 1907: 292. Metoponium abnorme laticolle Casey, 1907 USA (AZ) MEX (BC BS) Metoponium laticolle Casey, 1907: 291. Metoponium angelicum Blaisdell, 1923 MEX (BC) Metoponium angelicum Blaisdell, 1923: 203. Metoponium arizonicum Casey, 1907 USA (AZ) Metoponium arizonicum Casey, 1907: 294. Metoponium candidum Casey, 1907 USA (AZ) MEX (SO) Metoponium candidum Casey, 1907: 292. Metoponium cognitum Casey, 1907 USA (TX) Metoponium cognitum Casey, 1907: 305. Metoponium concors Casey, 1907 USA (CA) Metoponium concors Casey, 1907: 305. Metoponium congener (Casey, 1890) USA (TX) Eurymetopon congener Casey, 1890b: 333. Metoponium convexicolle (LeConte, 1851) USA (AZ CA NV) MEX (BC BS) Eurymetopon convexicolle LeConte, 1851: 139. Metoponium crassum Casey, 1907 USA (AZ) Metoponium crassum Casey, 1907: 299. Metoponium cribriceps Casey, 1907 USA (NM TX) Metoponium cribriceps Casey, 1907: 305. Metoponium cylindricum (Casey, 1890) USA (CA) Eurymetopon cylindricum Casey, 1890b: 337. Metoponium dubium (Casey, 1884) USA (AZ) Eurymetopon dubium Casey, 1884: 44. Eurymetopon carbonatum Casey, 1884: 43. Synonymy: Horn (1885b : 110). Metoponium edax Casey, 1907 USA (CA) Metoponium edax Casey, 1907: 309. Metoponium egregium Casey, 1907 USA (CA) Metoponium egregium Casey, 1907: 300. Metoponium emarginatum (Casey, 1884) USA (AZ) Eurymetopon emarginatum Casey, 1884: 41. Eurymetopon piceum Casey, 1884: 40. Synonymy: Horn (1885b : 110). Eurymetopon papagonum Casey, 1884: 42. Synonymy: Horn (1885b : 110). Eurymetopon sculptile Casey, 1884: 44. Synonymy: Horn (1885b : 110). Metoponium erosum Blaisdell, 1943 MEX (BS) Metoponium erosum Blaisdell, 1943: 175. Metoponium extensum Casey, 1907 USA (AZ) Metoponium extensum Casey, 1907: 300. Metoponium fatigans Casey, 1907 USA (AZ) Metoponium fatigans Casey, 1907: 304. Metoponium fusculum (Casey, 1890) USA (AZ CA) Eurymetopon fusculum Casey, 1890b: 335. Metoponium gravidum Casey, 1907 USA (CA) Metoponium gravidum Casey, 1907: 308. Metoponium gulosum Casey, 1907 USA (CA) Metoponium gulosum Casey, 1907: 307. Metoponium hebes Casey, 1907 USA (AZ) Metoponium hebes Casey, 1907: 301. Metoponium insulare Casey, 1907 USA (CA) Metoponium insulare Casey, 1907: 308. Metoponium integer Casey, 1907 USA (CA) Metoponium integer Casey, 1907: 310. Metoponium ludificans Casey, 1907 USA (TX) Metoponium ludificans Casey, 1907: 303. Metoponium molestum Casey, 1907 USA (CA) Metoponium molestum Casey, 1907: 309. Metoponium nevadense Casey, 1907 USA (NV) Metoponium nevadense Casey, 1907: 307. Metoponium opacipenne Casey, 1907 USA (CA) Metoponium opacipenne Casey, 1907: 309. Metoponium pacificum Blaisdell, 1923 MEX (BS) Metoponium pacificum Blaisdell, 1923: 202. Metoponium pallescens Casey, 1907 USA (AZ) Metoponium pallescens Casey, 1907: 293. Metoponium parvuliceps Casey, 1907 USA (AZ) Metoponium parvuliceps Casey, 1907: 296. Metoponium perforatum anceps Casey, 1907 USA (NM) Metoponium anceps Casey, 1907: 294. Metoponium perforatum congruens Casey, 1907 USA (NM) Metoponium congruens Casey, 1907: 293. Metoponium perforatum perforatum (Casey, 1890) USA (AZ) Eurymetopon perforatum Casey, 1890b: 334. Metoponium phoenicis Casey, 1907 USA (AZ) Metoponium phoenicis Casey, 1907: 301. Metoponium politum (Casey, 1890) USA (TX) Eurymetopon politum Casey, 1890b: 338. Metoponium probatum Casey, 1907 USA (CA) Metoponium probatum Casey, 1907: 310. Metoponium procerum Casey, 1907 USA (AZ) Metoponium procerum Casey, 1907: 297. Metoponium prolixum Casey, 1907 USA (AZ) Metoponium prolixum Casey, 1907: 298. Metoponium rufescens Casey, 1907 USA (AZ) Metoponium rufescens Casey, 1907: 302. Metoponium rufopiceum Casey, 1907 USA (AZ) Metoponium rufopiceum Casey, 1907: 296. Metoponium saginatum Casey, 1907 USA (TX) Metoponium saginatum Casey, 1907: 296. Metoponium socium socium Casey, 1907 USA (AZ) Metoponium socium Casey, 1907: 295. Metoponium socium subsimile Casey, 1907 USA (AZ) Metoponium subsimile Casey, 1907: 295. Metoponium subovale Casey, 1907 USA (UT) Metoponium subovale Casey, 1907: 307. Metoponium tersum Casey, 1907 USA (CA) Metoponium tersum Casey, 1907: 306. Metoponium testaceum Casey, 1907 USA (CA) Metoponium testaceum Casey, 1907: 303. Metoponium transversum Blaisdell, 1943 MEX (BS) Metoponium transversum Blaisdell, 1943: 174. Metoponium truncaticeps Casey, 1907 USA (AZ) Metoponium truncaticeps Casey, 1907: 299. Genus Micrarmalia Casey, 1907 [F] Micrarmalia Casey, 1907: 516. Type species: Emmenastus constrictus Champion, 1892, monotypy. Micrarmalia constricta (Champion, 1892) MEX (GE MO) Emmenastus constrictus Champion, 1892: 482. Genus Micromes Casey, 1907 [M] Micromes Casey, 1907: 432, 441. Type species: Stibia ovipennis Horn, 1874, original designation. Micromes maritimus (Casey, 1892) USA (CA) Stibia maritima Casey, 1891: 52. Micromes ovipennis (Horn, 1874) USA (CA) Stibia ovipennis Horn, 1874a: 28. Genus Orthostibia Blaisdell, 1923 [F] Orthostibia Blaisdell, 1923: 235. Type species: Orthostibia frontalis Blaisdell, 1923, original designation. Orthostibia fraterna Blaisdell, 1943 MEX (BS) Orthostibia fraterna Blaisdell, 1943: 211. Orthostibia frontalis Blaisdell, 1923 MEX (BS) Orthostibia frontalis Blaisdell, 1923: 236. Orthostibia muricata Blaisdell, 1943 MEX (BS) Orthostibia muricata Blaisdell, 1943: 210. Genus Oxygonodera Casey, 1907 [F] Oxygonodera Casey, 1907: 433, 444. Type species: Oxygonodera villosa Casey, 1907, original designation. Oxygonodera grandiceps Casey, 1907 USA (UT) Oxygonodera grandiceps Casey, 1907: 446. Oxygonodera hispidula (Horn, 1874) USA (ID OR UT WA) Stibia hispidula Horn, 1874a: 29. Oxygonodera villosa Casey, 1907 USA (UT) Oxygonodera villosa Casey, 1907: 445. Genus Pescennius Champion, 1884 [M] Pescennius Champion, 1884: 3. Type species: Pescennius villosus Champion, 1884, monotypy. Pescennius villosus Champion, 1884 MEX (PU) Pescennius villosus Champion, 1884: 4. Genus Pimeliopsis Champion, 1892 [F] Pimeliopsis Champion, 1892: 477. Type species: Pimeliopsis granulata Champion, 1892, monotypy. Pimeliopsis granulata Champion, 1892 MEX (GE) Pimeliopsis granulata Champion, 1892: 477. Genus Posides Champion, 1884 [M] Posides Champion, 1884: 6. Type species: Posides dissidens Champion, 1884, monotypy. Posides dissidens Champion, 1884 MEX (PU) Posides dissidens Champion, 1884: 6. Genus Soemias Champion, 1884 [F] Soemias Champion, 1884: 4. Type species: Soemias minuta Champion, 1884, monotypy. Soemias minuta Champion, 1884 MEX (VE) Soemias minuta Champion, 1884: 5. Genus Steriphanides Casey, 1907 [M] Steriphanides Casey, 1907: 515. Type species: Emmenastus stolidus Champion, 1892, monotypy. Steriphanides stolidus (Champion, 1892) MEX (OA) Emmenastus stolidus Champion, 1892: 483. Genus Steriphanus Casey, 1907 [M] Steriphanus Casey, 1907: 289. Type species: Emmenastus conicicollis Casey, 1890, original designation. Steriphanus aridus Casey, 1907 USA (AZ) Steriphanus aridus Casey, 1907: 347. Steriphanus conicicollis (Casey, 1890) USA (AZ) Emmenastus conicicollis Casey, 1890b: 355. Steriphanus convexus convexus (LeConte, 1866) USA (AZ NM TX) Emmenastus convexus LeConte, 1866b: 107. Steriphanus convexus unicolor Casey, 1907 USA (NM) Steriphanus unicolor Casey, 1907: 346. Steriphanus curtus (Champion, 1884) MEX (DU PU) Emmenastus curtus Champion, 1884: 16. Steriphanus discrepans Casey, 1907 USA (AZ) Steriphanus discrepans Casey, 1907: 343. Steriphanus discretus (Casey, 1890) USA (AZ) Emmenastus discretus Casey, 1890b: 354. Steriphanus durus Blaisdell, 1923 MEX (BC) Steriphanus durus Blaisdell, 1923: 224. Steriphanus ellipticus (Champion, 1884) MEX ("Pensacola") Emmenastus ellipticus Champion, 1884: 8. Steriphanus estebani Blaisdell, 1923 MEX (SO) Steriphanus estebani Blaisdell, 1923: 225. Steriphanus glabratus (Champion, 1884) MEX (JA OA PU) Emmenastus glabratus Champion, 1884: 16. Steriphanus hilaris Casey, 1907 USA (AZ UT) Steriphanus hilaris Casey, 1907: 345. Steriphanus lentus (Champion, 1884) MEX (CH CO DU) Emmenastus lentus Champion, 1884: 16. Steriphanus libertus Casey, 1907 USA (AZ) Steriphanus libertus Casey, 1907: 350. Steriphanus lubricans Casey, 1907 USA (AZ NV) Steriphanus lubricans Casey, 1907: 345. Steriphanus lustrans Casey, 1907 USA (AZ) Steriphanus lustrans Casey, 1907: 344. Steriphanus mancus (Champion, 1884) MEX (GE PU) Emmenastus mancus Champion, 1884: 15. Steriphanus nigrans Casey, 1907 USA (AZ) Steriphanus nigrans Casey, 1907: 347. Steriphanus nitescens Casey, 1907 USA (TX) Steriphanus nitescens Casey, 1907: 344. Steriphanus perovatus Casey, 1907 USA (TX) Steriphanus perovatus Casey, 1907: 351. Steriphanus picipes (Champion, 1884) MEX (OA) Emmenastus picipes Champion, 1884: 17. Steriphanus placidus Casey, 1907 MEX (FD) Steriphanus placidus Casey, 1907: 347. Steriphanus proprius Casey, 1907 USA (AZ) Steriphanus proprius Casey, 1907: 347. Steriphanus pulvinatus (Champion, 1884) MEX (FD HI OA) Emmenastus pulvinatus Champion, 1884: 17. Steriphanus rugicollis (Champion, 1884) MEX (SL) Emmenastus rugicollis Champion, 1884: 17. Steriphanus rutilans Casey, 1907 USA (TX) Steriphanus rutilans Casey, 1907: 346. Steriphanus subopacus alutaceus Casey, 1907 USA (AZ) MEX (BC SO) Steriphanus alutaceus Casey, 1907: 347. Steriphanus subopacus peropacus Casey, 1907 USA (AZ) Steriphanus peropacus Casey, 1907: 349. Steriphanus subopacus subopacus (Horn, 1870) USA (AZ TX) MEX (BC BS SO) Emmenastus subopacus Horn, 1870: 269. Steriphanus torpidus Blaisdell, 1923: 221. Synonymy: Sánchez Piñero and Aalbu (2002 : 132). Steriphanus mucronatus Blaisdell, 1923: 223. Synonymy: Sánchez Piñero and Aalbu (2002 : 132). Steriphanus tardus Blaisdell, 1923 MEX (SO) Steriphanus tardus Blaisdell, 1923: 222. Genus Stibia Horn, 1870 [F] Stibia Horn, 1870: 260. Type species: Stibia puncticollis Horn, 1870, monotypy. Eutriorophus Casey, 1924: 296. Type species: Eutriorophus tuckeri Casey, 1924, original designation. Synonymy: Blaisdell (1933b : 210). Stibia blairi Blaisdell, 1936 USA (AZ CA) MEX (BC) Stibia blairi Blaisdell, 1936a: 88. Stibia cribrata Blaisdell, 1923 MEX (BS) Stibia cribrata Blaisdell, 1923: 239 21 . Stibia fallaciosa fallaciosa Blaisdell, 1936 MEX (BS) Stibia fallaciosa Blaisdell, 1936a: 70. Stibia fallaciosa interstitialis Blaisdell, 1936 MEX (BS) Stibia fallaciosa interstitialis Blaisdell, 1936a: 73. Stibia ferruginea Blaisdell, 1943 MEX (BS) Stibia ferruginea Blaisdell, 1943: 208. Stibia freyi Kulzer, 1959 MEX (SI) Stibia freyi Kulzer, 1959: 614. Stibia granulata Blaisdell, 1923 MEX (BS) Stibia granulata Blaisdell, 1923: 238. Stibia imperialis Blaisdell, 1936 USA (AZ CA) Stibia imperialis Blaisdell, 1936a: 94. Stibia puncticollis martinensis Blaisdell, 1936 MEX (BC) Stibia puncticollis martinensis Blaisdell, 1936a: 83. Stibia puncticollis puncticollis Horn, 1870 USA (CA) MEX (BC BS SO) Stibia puncticollis Horn, 1870: 260. Stibia hannai Blaisdell, 1925b: 329. Synonymy: Blaisdell (1936a : 81). Stibia sparsa Blaisdell, 1923 MEX (BC BS) Stibia sparsa Blaisdell, 1923: 237. Stibia tortugensis Blaisdell, 1936a: 100. Synonymy: Sánchez Piñero and Aalbu (2002 : 132). Stibia tanneri Blaisdell, 1936 USA (CA) Stibia tanneri Blaisdell, 1936a: 97. Stibia tuckeri (Casey, 1924) USA (AZ) Eutriorophus tuckeri Casey, 1924: 297. Stibia williamsi Blaisdell, 1925 MEX (BC) Stibia williamsi Blaisdell, 1925b: 328. Genus Stictodera Casey, 1907 [F] Stictodera Casey, 1907: 289, 352. Type species: Emmenastus pinguis LeConte, 1866, original designation. Stictodera pinguis (LeConte, 1866) MEX (BS) Emmenastus pinguis LeConte, 1866b: 107. Genus Telabis Casey, 1890 [M] 22 Telabis Casey, 1890b: 331. Type species: Eurymetopon longipenne Casey, 1890, subsequent designation ( Casey 1907 : 288). Telabis alienus Casey, 1907 USA (AZ) Telabis aliena Casey, 1907: 325. Telabis amicus Casey, 1907 USA (UT) Telabis amica Casey, 1907: 318. Telabis asperus Casey, 1907 USA (CO) Telabis aspera Casey, 1907: 322. Telabis blandus Casey, 1907 USA (TX) Telabis blanda Casey, 1907: 326. Telabis brevicollis (Champion, 1884) MEX (CO) Eurymetopon brevicolle Champion, 1884: 7. Telabis compar Casey, 1907 USA (AZ) Telabis compar Casey, 1907: 321. Telabis crassulus (Casey, 1890) USA (AZ TX) Eurymetopon crassulum Casey, 1890b: 344. Telabis curticollis Casey, 1907 USA (AZ) Telabis curticollis Casey, 1907: 321. Telabis debilis (Casey, 1890) USA (AZ) Eurymetopon debile Casey, 1890b: 343. Telabis discors (Casey, 1890) USA (TX) Eurymetopon discors Casey, 1890b: 342. Telabis famelicus Casey, 1907 USA (NM) Telabis famelica Casey, 1907: 323. Telabis fidelis Casey, 1907 USA (CA) Telabis fidelis Casey, 1907: 320. Telabis hirtipes Blaisdell, 1923 MEX (BC BS) Telabis hirtipes Blaisdell, 1923: 205. Telabis histricus (Casey, 1890) USA (AZ) Eurymetopon histricum Casey, 1890b: 340. Telabis incisus Casey, 1907 USA (CA) Telabis incisa Casey, 1907: 322. Telabis inops Casey, 1907 USA (AZ) Telabis inops Casey, 1907: 325. Telabis latipennis Blaisdell, 1923 MEX (BS) Telabis latipennis Blaisdell, 1923: 207. Telabis lobifrons Casey, 1907 USA (AZ) Telabis lobifrons Casey, 1907: 318. Telabis longipennis (Casey, 1890) USA (NM) Eurymetopon longipenne Casey, 1890b: 339. Telabis lunulatus Blaisdell, 1923 MEX (BC BS) Telabis lunulata Blaisdell, 1923: 206. Telabis lustrellus Casey, 1907 USA (NM) Telabis lustrella Casey, 1907: 323. Telabis mimeticus Casey, 1907 USA (TX) Telabis mimetica Casey, 1907: 319. Telabis muricatulus (Casey, 1890) USA (AZ TX) Eurymetopon muricatulum Casey, 1890b: 341. Telabis nevadensis Blaisdell, 1925 USA (NV) Telabis nevadensis Blaisdell, 1925c: 372. Telabis obtusus Casey, 1907 USA (AZ) Telabis obtusa Casey, 1907: 317. Telabis opacellus Casey, 1907 USA (CA) Telabis opacella Casey, 1907: 316. Telabis ovalis Casey, 1907 USA (AZ) Telabis ovalis Casey, 1907: 324. Telabis pavidus Casey, 1907 USA (NM) Telabis pavida Casey, 1907: 324. Telabis prominens Casey, 1907 USA (TX) Telabis prominens Casey, 1907: 314. Telabis proxima Casey, 1907: 315. Synonymy: Casey (1911 : 253). Telabis punctulatus (LeConte, 1866) MEX (BC BS SO) Eurymetopon punctulatum LeConte, 1866b: 105. Telabis rubidus Casey, 1907 USA (TX) Telabis rubida Casey, 1907: 315. Telabis serratus (LeConte, 1866) USA (AZ CA ID NM NV OR TX) MEX (BC BS SO) Eurymetopon serratum LeConte, 1866b: 106. Telabis sodalis (Horn, 1870) USA (AZ CA) MEX (BS) Eurymetopon sodalis Horn, 1870: 268. Telabis timidus Casey, 1907 USA (AZ) Telabis timida Casey, 1907: 320. Telabis uteanus Casey, 1907 USA (UT) Telabis uteana Casey, 1907: 317. Telabis vafer Casey, 1907 USA (AZ) Telabis vafra Casey, 1907: 315. Telabis vapidus Casey, 1907 USA (TX) Telabis vapida Casey, 1907: 322. Genus Telaponium Blaisdell, 1923 [N] Telaponium Blaisdell, 1923: 209. Type species: Telaponium castaneum Blaisdell, 1923, original designation. Telaponium castaneum Blaisdell, 1923 MEX (BS) Telaponium castaneum Blaisdell, 1923: 209. Telaponium pingue Blaisdell, 1943 MEX (BS) Telaponium pingue Blaisdell, 1943: 179. Genus Texaponium Thomas, 1984 [N] Texaponium Thomas, 1984: 658. Type species: Cryptadius triplehorni Berry, 1974, original designation. Texaponium triplehorni (Berry, 1974) USA (TX) Cryptadius triplehorni Berry, 1974: 172. Genus Tlascalinus Casey, 1907 [M] Tlascalinus Casey, 1907: 370. Type species: Trimytis flohri Champion, 1892, monotypy. Tlascalinus flohri (Champion, 1892) MEX (FD) Trimytis flohri Champion, 1892: 478. Genus Trichiotes Casey, 1907 [M] Trichiotes Casey, 1907: 432, 443. Type species: Trichiotes seriatus Casey, 1907, original designation. Trichiotes lightfooti Wirth and Smith, 2017 MEX (CO) Trichiotes lightfooti Wirth and Smith, 2017: 535. Trichiotes seriatus Casey, 1907 USA (NM TX) MEX (CO NL) Trichiotes seriatus Casey, 1907: 444. Genus Trientoma Solier, 1835 [F] Trientoma Solier, 1835b: 256. Type species: Trientoma varvasi Solier, 1835, monotypy. Trientoma cayensis Garrido and Gutiérrez, 1995 CUB Trientoma cayensis Garrido and Gutiérrez, 1995b: 48. Trientoma convexipennis Allard, 1883 CUB Trientoma convexipennis Allard, 1883: 14. Trientoma garridoi Marcuzzi, 1988 CUB Trientoma garridoi Marcuzzi, 1988: 69. Trientoma guadeloupensis Fleutiaux and Sallé, 1890 LAN Trientoma guadeloupensis Fleutiaux and Sallé, 1890: 421. Trientoma jilae Steiner, 2006 BAH Trientoma jilae Steiner, 2006: 3. Trientoma kaszabi Marcuzzi, 1985 CUB Trientoma kaszabi Marcuzzi, 1985: 181. Trientoma kochi Marcuzzi, 1977 CAY Trientoma kochi Marcuzzi, 1977: 6. Trientoma laevis Allard, 1883 HAI Trientoma laevis Allard, 1883: 14. Trientoma maisiensis Marcuzzi, 1988 CUB Trientoma maisiensis Marcuzzi, 1988: 67. Trientoma zayasi Marcuzzi, 1988: 70. Synonymy: Garrido and Gutiérrez (1995b : 48). Trientoma martinicensis Allard, 1883 LAN (Martinique) Trientoma martinicensis Allard, 1883: 14. Trientoma puertoricensis Marcuzzi, 1977 PRI Trientoma puertoricensis Marcuzzi, 1977: 7. Trientoma rugifrons Champion, 1884 MEX / HIS Trientoma rugifrons Champion, 1884: 2. Trientoma ryticephala Allard, 1883 HAI Trientoma ryticephala Allard, 1883: 14. Trientoma sallei Kraatz, 1865 MEX / HAI DOM Trientoma sallei Kraatz, 1865: 74. Trientoma mexicana Champion, 1884: 2. Synonymy: Champion (1892 : 479). Trientoma siboneyensis Marcuzzi, 1988 CUB Trientoma siboneyensis Marcuzzi, 1988: 71. Trientoma varvasi Solier, 1835 CUB Trientoma varvasi Solier, 1835b: 257. Trientoma voegeliorum Steiner, 2006 BAH Trientoma voegeliorum Steiner, 2006: 8. Trientoma wickhami Casey, 1907 BAH Trientoma wickhami Casey, 1907: 377. Genus Trimytantron Ardoin, 1977 [N] Trimytantron Ardoin, 1977b: 381. Type species: Trimytantron decui Ardoin, 1977, original designation. Bielawskia Marcuzzi, 1985: 179. Type species: Bielawskia cubana Marcuzzi, 1985 (= Trimytantron decui Ardoin, 1977), monotypy. Synonymy: Marcuzzi (1998a : 153). Trimytantron armasi Garrido and Gutiérrez, 1997 CUB Trimytantron armasi Garrido and Gutiérrez, 1997: 32. Trimytantron cavernicolous Garrido and Gutiérrez, 1997 CUB Trimytantron cavernicolous Garrido and Gutiérrez, 1997: 34. Trimytantron cubanum Ardoin, 1977 CUB Trimytantron cubanum Ardoin, 1977c: 388. Trimytantron decui Ardoin, 1977 CUB Trimytantron decui Ardoin, 1977b: 382. Bielawskia cubana Marcuzzi, 1985: 179 [junior secondary homonym of Trimytantron cubanum Ardoin, 1977]. Synonymy: Garrido and Gutiérrez (1997 : 30). Trimytantron garridoi Marcuzzi, 1998a: 153. Replacement name for Trimytantron cubanum (Marcuzzi, 1985). Trimytantron escambrayense Garrido and Gutiérrez, 1997 CUB Trimytantron escambrayensis Garrido and Gutiérrez, 1997: 33. Trimytantron litorale Garrido and Gutiérrez, 1997 CUB Trimytantron litoralis Garrido and Gutiérrez, 1997: 30. Trimytantron minus 23 Garrido and Gutiérrez, 1997 CUB Trimytantron minor Garrido and Gutiérrez, 1997: 36. Trimytantron negreai Ardoin, 1977 CUB Trimytantron negreai Ardoin, 1977c: 387. Trimytantron poeyi Ardoin, 1977 CUB Trimytantron poeyi Ardoin, 1977b: 383. Trimytantron pumilum Garrido and Gutiérrez, 1997 CUB Trimytantron pumilus Garrido and Gutiérrez, 1997: 35. Trimytantron punctulaticeps Garrido and Gutiérrez, 1997 CUB Trimytantron punctulaticeps Garrido and Gutiérrez, 1997: 34. Trimytantron sierrae Garrido and Gutiérrez, 1997 CUB Trimytantron sierrae Garrido and Gutiérrez, 1997: 31. Trimytantron vinai Ardoin, 1977 CUB Trimytantron viñai Ardoin, 1977c: 388. Genus Trimytis LeConte, 1851 [F] Trimytis LeConte, 1851: 141. Type species: Trimytis pruinosa LeConte, 1851, monotypy. Pimalius Casey, 1907: 367. Type species: Trimytis pulverea Horn, 1870, original designation. Synonymy: MacLachlan and Olson (1990 : 79). Trimytis ceralboensis Blaisdell, 1943 MEX (BS) Trimytis ceralboensis Blaisdell, 1943: 196. Trimytis obovata Champion, 1892: 478 MEX (CH) Trimytis obovata Champion, 1892: 478. Trimytis obtusa Horn, 1894 MEX (BS) Trimytis obtusa Horn, 1894b: 412. Trimytis pruinosa LeConte, 1851 USA (AZ CO KS MT NE NM SD TX WY) Trimytis pruinosa LeConte, 1851: 141. Trimytis nympha Casey, 1907: 368. Synonymy: MacLachlan and Olson (1990 : 80). Trimytis tonsa Casey, 1907: 369. Synonymy: MacLachlan and Olson (1990 : 80). Trimytis ignava Casey, 1907: 369. Synonymy: MacLachlan and Olson (1990 : 81). Trimytis trapezifera Casey, 1924: 299. Synonymy: MacLachlan and Olson (1990 : 81). Trimytis pulverea Horn, 1870 USA (AZ) Trimytis pulverea Horn, 1870: 261. Trimytis subsenilis Blaisdell, 1923 MEX (SO) Trimytis subsenilis Blaisdell, 1923: 227. Genus Triorophus LeConte, 1851 [M] Triorophus LeConte, 1851: 141. Type species: Triorophus laevis LeConte, 1851, subsequent designation ( Casey 1907 : 432). Triorophus basalis Casey, 1907 USA (AZ) Triorophus basalis Casey, 1907: 437. Triorophus brevis Casey, 1907 USA (TX) Triorophus brevis Casey, 1907: 439. Triorophus gracilicornis Casey, 1907 USA (CA) Triorophus gracilicornis Casey, 1907: 437. Triorophus gravidulus Casey, 1907 USA (AZ) Triorophus gravidulus Casey, 1907: 437. Triorophus histrio Casey, 1907 USA (AZ) Triorophus histrio Casey, 1907: 437. Triorophus laevis laevis LeConte, 1851 USA (AZ CA NV) MEX (SO) Triorophus laevis LeConte, 1851: 141. Triorophus laevis politus Casey, 1907 USA (CA NV) Triorophus politus Casey, 1907: 435. Triorophus lariversi Blaisdell, 1942 USA (NV) Triorophus lariversi Blaisdell, 1942: 132. Triorophus laticeps Casey, 1924 USA (TX) Triorophus laticeps Casey, 1924: 297. Triorophus lecontei Casey, 1890 USA (TX) MEX (CH DU) Triorophus lecontei Casey, 1890b: 327. Triorophus longicornis Casey, 1907 USA (AZ) Triorophus longicornis Casey, 1907: 438. Triorophus mixtus Casey, 1907 USA (TX) Triorophus mixtus Casey, 1907: 440. Triorophus mundulus Casey, 1907 USA (AZ) Triorophus mundulus Casey, 1907: 436. Triorophus nevadensis Casey, 1924 USA (NV) Triorophus nevadensis Casey, 1924: 298. Triorophus nodiceps LeConte, 1853 USA (TX) MEX (CO) Triorophus nodiceps LeConte, 1853: 446. Triorophus puberulus Casey, 1924 USA (CA) Triorophus puberulus Casey, 1924: 298. Triorophus punctatus LeConte, 1851 USA (CA) Triorophus punctatus LeConte, 1851: 142. Triorophus rugiceps LeConte, 1851 USA (CA ID) Triorophus rugiceps LeConte, 1851: 142. 24 Triorophus simplex Casey, 1907 USA (AZ) Triorophus simplex Casey, 1907: 436. Triorophus subpubescens Horn, 1870 USA (CA) Triorophus subpubescens Horn, 1870: 259. Triorophus terebratulus Casey, 1907 USA (AZ) Triorophus terebratulus Casey, 1907: 436. Triorophus thoracicus Casey, 1924 USA (AZ) Triorophus thoracicus Casey, 1924: 298. Genus Triphalopsis Blaisdell, 1923 [F] Triphalopsis Blaisdell, 1923: 232. Type species: Triphalopsis partida Blaisdell, 1923, original designation. Triphalopsis californica Doyen, 1983 USA (CA) MEX (BC) Triphalopsis californicus Doyen, 1983: 87. Triphalopsis impressicollis Blaisdell, 1943 MEX (BC) Triphalopsis impressicollis Blaisdell, 1943: 203. Triphalopsis partida Blaisdell, 1923 MEX (BC BS SO) Triphalopsis partida Blaisdell, 1923: 232. Triphalopsis minor Blaisdell, 1923: 233. Synonymy: Sánchez Piñero and Aalbu (2002 : 132). Genus Triphalopsoides Doyen, 1990 [M] Triphalopsoides Doyen, 1990: 222. Type species: Triphalopsoides lasiodorsa Doyen, 1990, monotypy. Triphalopsoides lasiodorsa Doyen, 1990 MEX (JA) Triphalopsoides lasiodorsa Doyen, 1990: 224. Genus Triphalus LeConte, 1866 [M] Triphalus LeConte, 1866b: 104. Type species: Triphalus perforatus LeConte, 1866, monotypy. Triphalus cribricollis Horn, 1895 MEX (BS) Triphalus cribricollis Horn, 1895: 251. Triphalus impressifrons Blaisdell, 1943 MEX (BS) Triphalus impressifrons Blaisdell, 1943: 202. Triphalus perforatus LeConte, 1866 MEX (BS) Triphalus perforatus LeConte, 1866b: 104. Triphalus subcylindricus Blaisdell, 1923 MEX (BS) Triphalus subcylindricus Blaisdell, 1923: 234. Genus Troglogeneion Aalbu, 1985 [N] Troglogeneion Aalbu, 1985: 541. Type species: Troglogeneion zapoteca Aalbu, 1985, monotypy. Troglogeneion zapoteca Aalbu, 1985 MEX (OA) Troglogeneion zapoteca Aalbu, 1985: 542. Tribe Epitragini Blanchard, 1845 Lygophila Rafinesque, 1815: 113 [ nomen oblitum , see Bouchard et al. (2007 : 386)]. Type genus: Lygophilus Rafinesque, 1815 (= Epitragus Latreille, 1802). Épitragites Blanchard, 1845: 16 [ nomen protectum ]. Type genus: Epitragus Latreille, 1802. Genus Bothrotes Casey, 1907 [M] Bothrotes Casey, 1907: 379, 398. Type species: Epitragus canaliculatus Say, 1824, original designation. Bothrotes angusticollis (Champion, 1884) MEX (GE JA SI) Epitragus angusticollis Champion, 1884: 26. Bothrotes bicarinatus (Champion, 1884) MEX (CL VE) Epitragus bicarinatus Champion, 1884: 25. Bothrotes canaliculatus acutus (LeConte, 1866) USA (FL KS NM OK TX) MEX (CO) Epitragus acutus LeConte, 1866b: 108. Bothrotes fortis Casey, 1907: 399. Synonymy: Freude (1967 : 283). Bothrotes subrudis Casey, 1907: 400. Synonymy: Freude (1967 : 283). Bothrotes pensus Casey, 1907: 400. Synonymy: Freude (1967 : 283). Bothrotes knausi Casey, 1907: 401. Synonymy: Freude (1967 : 283). Bothrotes canaliculatus arundinis (LeConte, 1866) USA (DE GA MD NC NJ NY SC VA) Epitragus arundinis LeConte, 1866b: 108. Bothrotes pinorum Casey, 1924: 304. Synonymy: Freude (1967 : 281). Bothrotes canaliculatus canaliculatus (Say, 1824) USA (AZ CO IL KS MO NM OH SD TX WI) MEX (CH DU SO ZA) Epitragus canaliculatus Say, 1824b: 281. Bothrotes canaliculatus mexicanus Freude, 1967 MEX (DU NL TA) Bothrotes canaliculatus mexicanus Freude, 1967: 285. Bothrotes canus (Champion, 1884) MEX (GE) Epitragus canus Champion, 1884: 34. Bothrotes cristatus (Champion, 1892) MEX (CO GE) Epitragus cristatus Champion, 1892: 485. Bothrotes foveatus (Champion, 1884) MEX (OA VE) Epitragus foveatus Champion, 1884: 29. Bothrotes hoegei (Champion, 1884) MEX (MI VE) Epitragus högei Champion, 1884: 26. Bothrotes inaequalis (Champion, 1884) MEX (OA PU VE) Epitragus inaequalis Champion, 1884: 32. Bothrotes incisus (Champion, 1884) MEX (NA [Islas Marías]) Epitragus incisus Champion, 1884: 28. Bothrotes littoralis (Champion, 1884) MEX (GE JA MO NA OA SI) Epitragus littoralis Champion, 1884: 27. Bothrotes ornatus (Champion, 1884) MEX (DU GU PU VE) Epitragus ornatus Champion, 1884: 26. Bothrotes plumbeus plumbeus (LeConte, 1866) USA (AZ CA CO KS NE NM SD TX) Epitragus plumbeus LeConte, 1866b: 109. Bothrotes aeneicollis Casey, 1907: 401. Synonymy: Freude (1967 : 287). Bothrotes chalceus Casey, 1907: 402. Synonymy: Freude (1967 : 287). Bothrotes affinis Casey, 1907: 405. Synonymy: Freude (1967 : 287). Bothrotes pertinax Casey, 1907: 405. Synonymy: Freude (1967 : 287). Bothrotes picipennis Casey, 1907: 406. Synonymy: Freude (1967 : 287). Bothrotes secutor Casey, 1907: 406. Synonymy: Freude (1967 : 287). Bothrotes secutor var. apertus Casey, 1907: 406. Synonymy: Freude (1967 : 287). Bothrotes acomanus Casey, 1907: 407. Synonymy: Freude (1967 : 287). Bothrotes neglectus Casey, 1907: 407. Synonymy: Freude (1967 : 287). Bothrotes insitus Casey, 1907: 408. Synonymy: Freude (1967 : 287). Bothrotes funebris Casey, 1907: 409. Synonymy: Freude (1967 : 287). Bothrotes plumbeus rorulentus (Champion, 1884) MEX (CO GU SI) Epitragus rorulentus Champion, 1884: 27. Bothrotes plumbeus tenebrosus Casey, 1907 USA (AZ) MEX (SO) Bothrotes tenebrosus Casey, 1907: 403. Bothrotes occipitalis Casey, 1907: 403. Synonymy: Freude (1967 : 296). Bothrotes confertus Casey, 1907: 404. Synonymy: Freude (1967 : 296). Bothrotes eversus Casey, 1907: 404. Synonymy: Freude (1967 : 296). Bothrotes perditus Casey, 1907: 409. Synonymy: Freude (1967 : 296). Bothrotes amplificans Casey, 1907: 410. Synonymy: Freude (1967 : 296). Bothrotes obsolescens Casey, 1907: 411. Synonymy: Freude (1967 : 296). Bothrotes scutatus occidentalis Freude, 1967 MEX (CH CL JA NA OA) Bothrotes scutatus occidentalis Freude, 1967: 304. Bothrotes scutatus scutatus (Champion, 1884) MEX (GU) Epitragus scutatus Champion, 1884: 28. Genus Conoecus Horn, 1885 [M] Conoecus Horn, 1885c: 159. Type species: Conoecus ovipennis Horn, 1885, monotypy. Conoecus ovipennis estriatus Casey, 1907 USA (LA TX) Conoecus estriatus Casey, 1907: 431. Conoecus ovipennis ovipennis Horn, 1885 USA (TX) Conoecus ovipennis Horn, 1885c: 159. Genus Cyrtomius Casey, 1907 [M] Cyrtomius Casey, 1907: 379. Type species: Cyrtomius cavicauda Casey, 1907(= Epitragus plicatus Champion, 1884), original designation. Subgenus Cyrtomius Casey, 1907 Cyrtomius Casey, 1907: 379. Type species: Cyrtomius cavicauda Casey, 1907(= Epitragus plicatus Champion, 1884), original designation. Cyrtomius chevrolati (Champion, 1884) MEX (DU GU MO PU VE) GUA NIC Epitragus chevrolati Champion, 1884: 30. Cyrtomius freyi Freude, 1967 MEX (GE JA MI PU) Cyrtomius freyi Freude, 1967: 229. Cyrtomius plicatus (Champion, 1884) MEX (FD OA VE) Epitragus plicatus Champion, 1884: 31. Cyrtomius cavicauda Casey, 1907: 384. Synonymy: Freude (1967 : 231). Subgenus Grandicyrtomius Freude, 1967 Grandicyrtomius Freude, 1967: 225. Type species: Epitragus grandis Champion, 1884, original designation. Cyrtomius grandis (Champion, 1884) MEX (CI DU GE JA MO OA PU SI SO VE) Epitragus grandis Champion, 1884: 31. [incertae sedis] Cyrtomius gaigli Freude, 1986 GUA Cyrtomius gaigli Freude, 1986: 27. Cyrtomius polli Freude, 1986 GUA Cyrtomius polli Freude, 1986: 26. Genus Epitragodes Casey, 1890 [M] Epitragodes Casey, 1890b: 365. Type species: Epitragus tomentosus LeConte, 1866, monotypy. Epitragodes tomentosus macilentus Casey, 1907 USA (AL FL GA NC SC VA) / BAH Epitragodes tomentosus macilentus Casey, 1907: 425. Epitragodes debilicollis Casey, 1907: 423. Synonymy: Freude (1968 : 86). Epitragodes pardalis Casey, 1907: 423. Synonymy: Freude (1968 : 86). Epitragodes cuprascens Casey, 1907: 424. Synonymy: Freude (1968 : 86). Epitragodes tomentosus tomentosus (LeConte, 1866) USA (FL GA) / BAH Epitragus tomentosus LeConte, 1866b: 109. Epitragodes floridanus Casey, 1907: 424. Synonymy: Freude (1968 : 86). Epitragodes obesulus Casey, 1907: 425. Synonymy: Freude (1968 : 86). Genus Epitragopsis Casey, 1907 [F] Epitragopsis Casey, 1907: 386. Type species: Epitragus godmani Champion, 1884, original designation. Epitragopsis communis (Champion, 1884) MEX (OA VE) GUA BEL HON Epitragus communis Champion, 1884: 36. Epitragopsis godmani (Champion, 1884) PAN / SA Epitragus godmani Champion, 1884: 36. Epitragopsis auratus Marcuzzi, 1961: 8. Synonymy: Freude (1968 : 71). Epitragopsis ruatanensis (Champion, 1892) HON NIC Epitragus ruatanensis Champion, 1892: 488. Genus Epitragosoma Brown and Triplehorn, 2002 [N] Epitragosoma Brown and Triplehorn, 2002: 515. Type species: Epitragosoma arenaria Brown and Triplehorn, 2002, original designation. Epitragosoma arenarium Brown and Triplehorn, 2002 USA (NM TX) Epitragosoma arenaria Brown and Triplehorn, 2002: 519. Genus Epitragus Latreille, 1802 [M] Epitragus Latreille, 1802: 165. Type species: Epitragus fuscus Latreille, 1804, subsequent monotypy in Latreille (1804 : 322). Subgenus Epitragus Latreille, 1802 Epitragus Latreille, 1802: 165. Type species: Epitragus fuscus Latreille, 1804, subsequent monotypy in Latreille (1804 : 322). Epitragus antillensis Marcuzzi, 1961 JAM Epitragus antillensis Marcuzzi, 1961: 28. Epitragus aurulentus aurulentus Kirsch, 1866 NIC CRI PAN / CUB JAM HIS PRI LAN / SA Epitragus aurulentus Kirsch, 1866: 189. Epitragus jamaicensis Champion, 1896: 3. Synonymy: Freude (1967 : 158). Epitragus emarginatus Champion, 1884 PAN Epitragus emarginatus Champion, 1884: 24. Epitragus consimilis Marcuzzi, 1961: 34. Synonymy: Freude (1967 : 164). Epitragus gaigli Freude, 1986 GUA Epitragus gaigli Freude, 1986: 25. Epitragus mexicanus Marcuzzi, 1961 MEX (OA) Epitragus mexicanus Marcuzzi, 1961: 29. Epitragus nigricans Champion, 1884 PAN / SA Epitragus nigricans Champion, 1884: 24. Epitragus puberulus Kirsch, 1886: 332. Synonymy: Freude (1967 : 162). Epitragus roscidus Erichson, 1849 LAN / SA Epitragus roscidus Erichson, 1849: 565. Epitragus exaratus Champion, 1896: 2. Synonymy: Freude (1967 : 166). Epitragus sallei Champion, 1884 25 MEX (CI VE YU) GUA HON NIC CRI / SA Epitragus sallaei Champion, 1884: 24. Epitragus rigens Casey, 1907: 381. Synonymy: Freude (1967 : 155). Genus Hemasodes Casey, 1907 [M] Hemasodes Casey, 1907: 378. Type species: Schoenicus vestitus Champion, 1884, original designation. Hemasodes vestitus (Champion, 1884) MEX (GE JA OA VE) Schoenicus vestitus Champion, 1884: 22. Schoenicus yucatanensis Champion, 1884: 22. Synonymy: Freude (1967 : 182). Genus Lobometopon Casey, 1907 [N] Lobometopon Casey, 1907: 379, 385. Type species: Epitragus fusiformis Casey, 1890, original designation. Lobometopon acutangulum (Champion, 1884) MEX (CI OA) GUA Epitragus acutangulus Champion, 1884: 31. Lobometopon aeratum (Champion, 1884) MEX (CL GE JA NA OA VE) Epitragus aeratus Champion, 1884: 33. Lobometopon aurichalceum (Champion, 1884) USA (AZ) MEX (GU OA SI) Epitragus aurichalceus Champion, 1884: 33. Lobometopon tuckeri Casey, 1924: 301. Synonymy: Freude (1968 : 44). Lobometopon cupreum (Champion, 1884) GUA NIC CRI PAN Epitragus cupreus Champion, 1884: 34. Lobometopon bicaviceps Casey, 1907: 394. Synonymy: Freude (1968 : 36). Lobometopon alveolatum Casey, 1907: 394. Synonymy: Freude (1968 : 36). Lobometopon fusiforme cribricolle Casey, 1907 USA (KS NE NM SD TX) MEX (NL) Lobometopon cribricolle Casey, 1907: 391. Lobometopon jucundum Casey, 1907: 392. Synonymy: Freude (1968 : 50). Lobometopon obscurum Casey, 1907: 395. Synonymy: Freude (1968 : 50). Lobometopon fusiforme fusiforme (Casey, 1890) USA (AZ) MEX (SO) Epitragus fusiformis Casey, 1890b: 365. Lobometopon symmetricum Casey, 1907: 389. Synonymy: Freude (1968 : 50). Lobometopon pimalicum Casey, 1907: 389. Synonymy: Freude (1968 : 50). Lobometopon aeneopiceum Casey, 1907: 390. Synonymy: Freude (1968 : 50). Lobometopon docile Casey, 1907: 389. Synonymy: Freude (1968 : 50). Lobometopon propinquum Casey, 1907: 391. Synonymy: Freude (1968 : 50). Lobometopon aequipenne Casey, 1907: 393. Synonymy: Freude (1968 : 50). Lobometopon morrisoni Casey, 1907: 393. Synonymy: Freude (1968 : 50). Lobometopon fusiforme uintanum Casey, 1907 USA (AZ NM NV UT) Lobometopon uintanum Casey, 1907: 388. Lobometopon parvicolle Casey, 1907: 392. Synonymy: Freude (1968 : 50). Lobometopon alticola Casey, 1924: 302. Synonymy: Freude (1968 : 50). Lobometopon woodgatei Casey, 1924: 302. Synonymy: Freude (1968 : 50). Lobometopon provoanum Casey, 1924: 303. Synonymy: Freude (1968 : 50). Lobometopon guatemalense (Champion, 1884) GUA BEL SAL HON NIC CRI Epitragus guatemalensis Champion, 1884: 32. Lobometopon lucidum (Champion, 1884) MEX (DU NA PU SI SO) Epitragus lucidus Champion, 1884: 34. Lobometopon metallicum (Champion, 1884) MEX (CH CI CL DU FD GE GU ME MO OA PU QU SL VE) GUA CRI Epitragus metallicus Champion, 1884: 29. Epitragus gracilis Casey, 1890b: 366. Synonymy: Freude (1968 : 40). Lobometopon aberrans Casey, 1907: 387. Synonymy: Freude (1968 : 40). Lobometopon micans (Champion, 1884) MEX (CI FD OA) Epitragus micans Champion, 1884: 32. Lobometopon obovatum (Champion, 1884) MEX (GE OA) Epitragus obovatus Champion, 1884: 35. Lobometopon ovale (Casey, 1885) USA (TX) Epitragus ovalis Casey, 1885: 184. Lobometopon parviceps (Champion, 1884) MEX (CL OA) Epitragus parviceps Champion, 1884: 34. Genus Metopoloba Casey, 1907 [F] Metopoloba Casey, 1907: 379. Type species: Epitragus pruinosus Horn, 1870, original designation. Metopoloba pruinosa mexicana Freude, 1967 MEX (SO) Metopoloba pruinosa mexicana Freude, 1967: 259. Metopoloba pruinosa pruinosa (Horn, 1870) USA (AZ CA NV UT) Epitragus pruinosus Horn, 1870: 264. Metopoloba bifossiceps Casey, 1907: 413. Synonymy: Freude (1967 : 248). Metopoloba proba Casey, 1907: 414. Synonymy: Freude (1967 : 248). Metopoloba punctiventris Casey, 1907: 414. Synonymy: Freude (1967 : 248). Metopoloba perpolita Casey, 1907: 415. Synonymy: Freude (1967 : 248). Metopoloba californica Casey, 1907: 419. Synonymy: Freude (1967 : 248). Lobometopon juabense Casey, 1924: 303. Synonymy: Freude (1967 : 248). Metopoloba pruinosa subpilosa Blaisdell, 1943 MEX (BS) Metopoloba subpilosa Blaisdell, 1943: 199. Metopoloba pruinosa subseriata Casey, 1907 USA (AZ NM TX) Metopoloba subseriata Casey, 1907: 415. Metopoloba snowi Casey, 1907: 416. Synonymy: Freude (1967 : 253). Metopoloba densiventris Casey, 1907: 417. Synonymy: Freude (1967 : 253). Metopoloba contaminans Casey, 1907: 418. Synonymy: Freude (1967 : 253). Metopoloba amplexa Casey, 1907: 418. Synonymy: Freude (1967 : 253). Metopoloba sublaeviceps Casey, 1907: 418. Synonymy: Freude (1967 : 253). Metopoloba angulata Casey, 1907: 419. Synonymy: Freude (1967 : 253). Metopoloba pruinosa werneri Freude, 1967 MEX (BS) Metopoloba pruinosa werneri Freude, 1967: 259. Genus Ortheolus Casey, 1907 [M] Ortheolus Casey, 1907: 380. Type species: Schoenicus oculatus Champion, 1884, original designation. Ortheolus antillarum (Champion, 1896) LAN Schoenicus antillarum Champion, 1896: 5. Schoenicus brunneus Champion, 1896: 4. Synonymy: Freude (1968 : 106). Ortheolus caraibicus caraibicus Marcuzzi, 1961 LAN / SA Ortheolus caraibicus Marcuzzi, 1961: 38. Ortheolus oculatus oculatus (Champion, 1884) PAN Schoenicus oculatus Champion, 1884: 18. Ortheolus panamensis (Champion, 1884) CRI PAN Schoenicus panamensis Champion, 1884: 18. Genus Pechalius Casey, 1907 [M] Pechalius Casey, 1907: 379, 420. Type species: Pechalius subvittatus Casey, 1907, original designation. Epitragoma Casey, 1907: 386. Type species: Epitragus vestitus Casey, 1891, monotypy. Synonymy: Freude (1968 : 61). Pechalius bradleyi Triplehorn, 1974 USA (NM) Pechalius bradleyi Triplehorn, 1974: 73. Pechalius dentiger (Horn, 1870) USA (AZ) MEX (SO) Epitragus dentiger Horn, 1870: 265. Pechalius pilosus (Champion, 1884) MEX (CH TA VE) Epitragus pilosus Champion, 1884: 34. Pechalius subvittatus Casey, 1907 USA (TX) MEX (DU) Pechalius subvittatus Casey, 1907: 421. Pechalius vestitus (Casey, 1891) USA (AZ) Epitragus vestitus Casey, 1891: 53. Genus Phegoneus Casey, 1907 [M] Phegoneus Casey, 1907: 380, 426. Type species: Epitragodes julichi Casey, 1891, original designation. Subgenus Pectphegoneus Freude, 1968 Pectphegoneus Freude, 1968: 90. Type species: Schoenicus pectoralis Champion, 1884, monotypy. Phegoneus pectoralis (Champion, 1884) MEX (CL GE JA MI MO PU) Schoenicus pectoralis Champion, 1884: 21. Subgenus Phegoneus Casey, 1907 Phegoneus Casey, 1907: 380, 426. Type species: Epitragodes julichi Casey, 1891, original designation. Phegoneus basalis (Champion, 1884) MEX (OA VE) Schoenicus basalis Champion, 1884: 21. Phegoneus chalybeus (Champion, 1884) MEX (MI NA OA PU SI SL VE) Schoenicus chalybeus Champion, 1884: 20. Phegoneus difficilis (Champion, 1884) MEX (DU GE JA MI OA SI VE) Schoenicus difficilis Champion, 1884: 20. Phegoneus julichi (Casey, 1891) USA (TX) Epitragodes jülichi Casey, 1891: 55. Phegoneus rufipes impressus (Champion, 1884) CRI Schoenicus impressus Champion, 1884: 20. Phegoneus rufipes rufipes (Champion, 1884) MEX (YU) NIC Schoenicus rufipes Champion, 1884: 19. Phegoneus salvini salvini (Champion, 1884) GUA SAL CRI Schoenicus salvini Champion, 1884: 19. Schoenicus niger Champion, 1884: 20. Synonymy: Freude (1968 : 96). Phegoneus salvini subaeneus Casey, 1907 PAN Phegoneus subaeneus Casey, 1907: 428. Phegoneus viridis (Champion, 1884) MEX (CI CL DU GE JA OA PU SI) GUA CRI Schoenicus viridis Champion, 1884: 19. Genus Polemiotus Casey, 1907 [M] Polemiotus Casey, 1907: 379, 381. Type species: Epitragus submetallicus LeConte, 1854, original designation. Polemiotus submetallicus (LeConte, 1854) USA (AZ) Epitragus submetallicus LeConte, 1854c: 224. Polemiotus humeralis Casey, 1907: 382. Synonymy: Freude (1967 : 223). Polemiotus humeralis var. acuticauda Casey, 1907: 383. Synonymy: Freude (1967 : 223). Genus Schoenicus LeConte, 1866 [M] Schoenicus LeConte, 1866b: 109. Type species: Schoenicus puberulus LeConte, 1866, monotypy. Schoenicus puberulus LeConte, 1866 USA (FL GA MD MS NC NJ NY SC) Schoenicus puberulus LeConte, 1866b: 110. Genus Tydeolus Champion, 1884 [M] Tydeolus Champion, 1884: 37. Type species: Tydeolus atratus Champion, 1884, subsequent designation ( Kirby 1885 : 80). Tydeolus atratus Champion, 1884 MEX (PU) Tydeolus atratus Champion, 1884: 37. Tydeolus tibialis Champion, 1884: 37. Synonymy: Freude (1968 : 122). Tydeolus singularis Champion, 1884: 37. Synonymy: Freude (1968 : 122). Tribe Nyctoporini Lacordaire, 1859 Nyctoporides Lacordaire, 1859: 130. Type genus: Nyctoporis Eschscholtz, 1831. Genus Nyctoporis Eschscholtz, 1831 [F] Nyctoporis Eschscholtz, 1831: 10, 11. Type species: Nyctoporis cristata Eschscholtz, 1831, subsequent designation ( Hope 1841 : 124). Emeax Pascoe, 1866: 450. Type species: Emeax sculpturatus Pascoe, 1866 (= Nyctoporis cristata Eschscholtz, 1831), monotypy. Synonymy: LeConte (1873 : 334). Enneacoides Fairmaire, 1881: 277. Type species: Enneacoides vinculiger Fairmaire, 1881 (= Nyctoporis carinata LeConte, 1851), monotypy. Synonymy: Gebien (1908a : 287). Nyctoporis aequicollis Eschscholtz, 1831 USA (CA) Nyctoporis aequicollis Eschscholtz, 1831: 12. Nyctoporis tetrica Casey, 1907: 510. New synonymy [RLA]. Nyctoporis maura Casey, 1907: 512. New synonymy [RLA]. Nyctoporis carinata LeConte, 1851 USA (CA) Nyctoporis carinata LeConte, 1851: 138. Nyctoporis segnis Casey, 1907: 511. Synonymy: Blaisdell (1931 : 43). Enneacoides vinculiger Fairmaire, 1881: 277. Synonymy: Gebien (1910: 118) 26 . Nyctoporis cristata Eschscholtz, 1831 USA (CA) Nyctoporis cristata Eschscholtz, 1831: 11. Nyctoporis galeata LeConte, 1857: 49. Synonymy: Casey (1907 b: 510). Emeax sculpturatus Pascoe, 1866: 450. Synonymy: Carter (1914 : 406). Nyctoporis sponsa Casey, 1907 USA (CA) Nyctoporis sponsa Casey, 1907: 510. Nyctoporis pullata Casey, 1907: 510. New synonymy [RLA]. Nyctoporis vandykei Blaisdell, 1931 USA (CA) Nyctoporus [sic!] vandykei Blaisdell, 1931: 41. Tribe Stenosini Schaum, 1859 Tagénites Solier, 1834: 503. Type genus: Tagenia Latreille, 1802 (= Stenosis Herbst, 1799). Note. Use of younger name Stenosini conserved (Art. 40.2) (see Bouchard et al. 2005 : 523). Stenosidae Schaum, 1859: 66. Type genus: Stenosis Herbst, 1799. Typhlusechini Casey, 1907: 281. Type genus: Typhlusechus Linell, 1897. Araeoschizini Casey, 1907: 484. Type genus: Araeoschizus LeConte, 1851. Genus Araeoschizus LeConte, 1851 [M] Araeoschizus LeConte, 1851: 138. Type species: Araeoschizus costipennis LeConte, 1851, monotypy. Araeoschizus aalbui Papp, 1981 MEX (BS) Araeoschizus aalbui Papp, 1981: 316. Araeoschizus agustinus Papp, 1998 MEX (BC) Araeoschizus agustinus Papp, 1998: 90. Araeoschizus airmeti Tanner, 1945 USA (ID NV OR) Araeoschizus airmeti Tanner, 1945: 125. Araeoschizus alinae Dajoz, 1984 USA (UT) Araeoschizus alinae Dajoz, 1984: 246. Araeoschizus andrewsi Papp, 1981 USA (CA) Araeoschizus andrewsi Papp, 1981: 318. Araeoschizus antennatus antennatus Blaisdell, 1943 MEX (BC) Araeoschizus antennatus Blaisdell, 1943: 215. Araeoschizus antennatus blaisdelli Papp, 1989 MEX (BC) Araeoschizus antennatus blaisdelli Papp, 1989: 338. Araeoschizus antennatus clarki Papp, 1989 MEX (BC) Araeoschizus antennatus clarki Papp, 1989: 335. Araeoschizus apachensis Papp, 1981 USA (AZ) Araeoschizus apachensis Papp, 1981: 367. Araeoschizus arizonicus Dajoz, 1989 USA (AZ NM) Araeoschizus arizonicus Dajoz, 1989b: 33. Araeoschizus armatus Horn, 1870 USA (CA NV) Araeoschizus armatus Horn, 1870: 275. Araeoschizus blomi Papp, 1998 MEX (BC) Araeoschizus blomi Papp, 1998: 93. Araeoschizus colossalis Papp, 1981 USA (AZ) Araeoschizus colossalis Papp, 1981: 346. Araeoschizus costipennis LeConte, 1851 USA (CA) Araeoschizus costipennis LeConte, 1851: 138. Araeoschizus decipiens Horn, 1890 USA (AZ CO NM TX UT) MEX (CH DU SO) Araeoschizus decipiens Horn, 1890: 342. Araeoschizus dolenterus Papp, 1981 MEX (PU) Araeoschizus dolenterus Papp, 1981: 349. Araeoschizus doyeni Papp, 1981 USA (CA) Araeoschizus doyeni Papp, 1981: 375. Araeoschizus duplicatus Casey, 1907 USA (WY) Araeoschizus duplicatus Casey, 1907: 491. Araeoschizus elegantulus Papp, 1981 MEX (BS) Araeoschizus elegantulus Papp, 1981: 325. Araeoschizus exiguus Casey, 1907 USA (CA) Araeoschizus exiguus Casey, 1907: 487. Araeoschizus expeditionis Papp, 1981 MEX (DU) Araeoschizus expeditionis Papp, 1981: 351. Araeoschizus fimbriatus Casey, 1890 USA (AZ) Araeoschizus fimbriatus Casey, 1890b: 369. Araeoschizus giulianii Papp, 1981 MEX (SO) Araeoschizus giulianii Papp, 1981: 393. Araeoschizus hardyi Papp, 1981 USA (CA) Araeoschizus hardyi Papp, 1981: 308. Araeoschizus hardyorum Papp, 1981 USA (UT) Araeoschizus hardyorum Papp, 1981: 395. Araeoschizus hystrix Papp, 1981 USA (CA) Araeoschizus hystrix Papp, 1981: 330. Araeoschizus interjectus Papp, 1981 MEX (BS) Araeoschizus interjectus Papp, 1981: 332. Araeoschizus kaszabi Papp, 1981 USA (CA) Araeoschizus kaszabi Papp, 1981: 397. Araeoschizus kubai Papp, 1981 USA (AZ) Araeoschizus kubai Papp, 1981: 400. Araeoschizus lariversi Papp, 1981 USA (CA) Araeoschizus lariversi Papp, 1981: 306. Araeoschizus lecontei Papp, 1981 USA (AZ) Araeoschizus lecontei Papp, 1981: 335. Araeoschizus limbatus Blaisdell, 1943 MEX (BS) Araeoschizus limbatus Blaisdell, 1943: 214. Araeoschizus magdae Papp, 1989 MEX (GE) Araeoschizus magdae Papp, 1989: 338. Araeoschizus mexicanus Champion, 1892 MEX (DU GE OA) Araeoschizus mexicanus Champion, 1892: 491. Araeoschizus microcephalus Papp, 1981 MEX (CH) Araeoschizus microcephalus Papp, 1981: 379. Araeoschizus muthi Dajoz, 1998 USA (CA) Araeoschizus muthi Dajoz, 1998: 87. Araeoschizus orientalis Dajoz, 1991 USA (TX) Araeoschizus orientalis Dajoz, 1991: 172. Araeoschizus percellosus Papp, 1981 MEX (BC) Araeoschizus percellosus Papp, 1981: 312. Araeoschizus problematicus Papp, 1981 MEX (ZA) Araeoschizus problematicus Papp, 1981: 402. Araeoschizus regularis Horn, 1870 USA (AZ UT) MEX (SO) Araeoschizus regularis Horn, 1870: 274. Araeoschizus rufus Dajoz, 1991 USA (CA) Araeoschizus rufus Dajoz, 1991: 165. Araeoschizus setosiformis Papp, 1981 USA (UT) Araeoschizus setosiformis Papp, 1981: 382. Araeoschizus similaris Papp, 1981 USA (NM) Araeoschizus similaris Papp, 1981: 384. Araeoschizus simplex Casey, 1890 USA (AZ NM TX) MEX (CH) Araeoschizus simplex Casey, 1890b: 369. Araeoschizus simulans Casey, 1907 USA (CA) Araeoschizus simulans Casey, 1907: 488. Araeoschizus squamulissimus Papp, 1981 MEX (BC) Araeoschizus squamulissimus Papp, 1981: 340. Araeoschizus sulcicollis disjunctus Papp, 1981 USA (CA) Araeoschizus sulcicollis disjunctus Papp, 1981: 364. Araeoschizus sulcicollis sulcicollis Horn, 1870 USA (CA NV) Araeoschizus sulcicollis Horn, 1870: 274. Araeoschizus tenuis Casey, 1907 USA (AZ) Araeoschizus tenuis Casey, 1907: 486. Araeoschizus texanus Dajoz, 1989 USA (TX) Araeoschizus texanus Dajoz, 1989a: 149. Araeoschizus utahensis Papp, 1981 USA (UT) Araeoschizus utahensis Papp, 1981: 389. Araeoschizus wasbauerorum Papp, 1981 MEX (SO) Araeoschizus wasbauerorum Papp, 1981: 342. Genus Caribanosis Nabozhenko, Kirejtshuk, Merkl, Varela, Aalbu and Smith, 2016 [M] Caribanosis Nabozhenko, Kirejtshuk, Merkl, Varela, Aalbu and Smith, 2016: 568 27 . Type species: Rhypasma quisqueyanus Garrido and Varela, 2011, original designation. Caribanosis quisqueyanus (Garrido and Varela, 2011) DOM Rhypasma quisqueyanus Garrido and Varela, 2011: 32. Genus Discopleurus Lacordaire, 1859 [M] Pleurophorus Solier, 1851: 162 [junior homonym of Pleurophorus Mulsant, 1842]. Type species: Pleurophorus quadricollis Solier, 1851, monotypy. Discopleurus Lacordaire, 1859: 105. Replacement name for Pleurophorus Solier, 1851. Discopleurus mesoamericanus Aalbu and Andrews, 1996 HON CRI PAN Discopleurus mesoamericanus Aalbu and Andrews, 1996: 27. Genus Typhlusechus Linell, 1897 [M] Typhlusechus Linell, 1897: 154. Type species: Typhlusechus singularis Linell, 1897, original designation. Typhlusechus balsasensis Aalbu and Andrews, 1985 MEX (MI) Typhlusechus balsasensis Aalbu and Andrews, 1985: 4. Typhlusechus chemehuevii Aalbu and Andrews, 1985 USA (CA) Typhlusechus chemehuevii Aalbu and Andrews, 1985: 3. Typhlusechus ignotus Doyen, 1990 MEX (JA) Typhlusechus ignotus Doyen, 1990: 227. Typhlusechus peninsularis Aalbu and Andrews, 1985 MEX (BS) Typhlusechus peninsularis Aalbu and Andrews, 1985: 5. Typhlusechus singularis Linell, 1897 USA (CA) Typhlusechus singularis Linell, 1897: 155. Typhlusechus spilmani Aalbu and Andrews, 1985 USA (TX) MEX (DU) Typhlusechus spilmani Aalbu and Andrews, 1985: 6. Tribe Vacronini Gebien, 1910 Vacroninae Gebien, 1910a: 118. Type genus: Vacronus Casey, 1907 (= Alaephus Horn, 1870). Genus Alaephus Horn, 1870 [M] Alaephus Horn, 1870: 346. Type species: Alaephus pallidus Horn, 1870, monotypy. Vacronus Casey, 1907: 501, 508. Type species: Vacronus tenuicornis Casey, 1907, original designation. Synonymy: Doyen and Lawrence (1979 : 350). Alaephus convergens Casey, 1924 USA (UT) Alaephus convergens Casey, 1924: 324. Alaephus gracilicornis Casey, 1924 USA (NM) Alaephus gracilicornis Casey, 1924: 325. Alaephus gracilis Fall, 1905 USA (AZ) Alaephus gracilis Fall, 1905: 276. Alaephus longicornis Casey, 1924 USA (CA) Alaephus longicornis Casey, 1924: 325. Alaephus macilentus Casey, 1891 USA (AZ CA NM) Alaephus macilentus Casey, 1891: 61. Alaephus nitidipennis Fall, 1905: 275. Synonymy: Fall (1907b : 175). Alaephus madarensis Casey, 1924 USA (CA) Alaephus madarensis Casey, 1924: 324. Alaephus nevadensis Tanner, 1965 USA (NV) Alaephus nevadensis Tanner [in Tanner and Packtam], 1965: 39. Alaephus pallidus Horn, 1870 USA (CA) Alaephus pallidus Horn, 1870: 346. Alaephus puberulus Fall, 1907 USA (AZ UT) Alaephus puberulus Fall, 1907b: 175. Alaephus quadricollis Casey, 1924 USA (UT) Alaephus quadricollis Casey, 1924: 326. Alaephus tenuicornis (Casey, 1907) USA (CA) Vacronus tenuicornis Casey, 1907: 508. Genus Eupsophulus Cockerell, 1906 [M] Eupsophus Horn, 1870: 347 [junior homonym of Eupsophus Fitzinger, 1843]. Type species: Eupsophus castaneus Horn, 1870, monotypy. Eupsophulus Cockerell, 1906: 242. Replacement name for Eupsophus Horn, 1870. Eupsophulus brevipennis Casey, 1924 USA (AZ) Eupsophulus brevipennis Casey, 1924: 323. Eupsophulus castaneus (Horn, 1870) USA (AZ CA NV TX) MEX (BC CO SO) Eupsophus castaneus Horn, 1870: 347. Eupsophulus horni (Champion, 1885) MEX (BS) Eupsophus horni Champion, 1885: 122. Subfamily TENEBRIONINAE Latreille, 1802 Tenebrionites Latreille, 1802: 165. Type genus: Tenebrio Linnaeus, 1758. Tribe Acropteronini Doyen, 1989 Acropteronini Doyen, 1989: 288. Type genus: Acropteron Perty, 1832. Genus Acropteron Perty, 1832 [N] Acropteron Perty, 1832: 64. Type species: Acropteron rufipes Perty, 1832, subsequent designation ( Hope 1841 : 133). Arthroplatus Solier, 1851: 246. Type species: Arthroplatus pallipes Solier, 1851, monotypy. Synonymy: Mäklin (1862 : 1). Acropteron agriloides Mäklin, 1862 MEX (CI GE OA TB VE) GUA Acropteron agriloides Mäklin, 1862: 17. Acropteron angulicolle Champion, 1886 NIC Acropteron angulicolle Champion, 1886: 255. Acropteron belti Champion, 1886 NIC PAN Acropteron belti Champion, 1886: 253. Acropteron brunneum Mäklin, 1862 CRI / SA Acropteron brunneum Mäklin, 1862: 15. Acropteron calcaratum Champion, 1886 GUA Acropteron calcaratum Champion, 1886: 255. Acropteron chabrieri Fleutiaux and Sallé, 1890 LAN Acropteron chabrieri Fleutiaux and Sallé, 1890: 429. Acropteron laevipes Champion, 1886 NIC Acropteron laevipes Champion, 1886: 257. Acropteron langurioides Champion, 1886 PAN Acropteron langurioides Champion, 1886: 254. Acropteron longipenne Champion, 1886 GUA PAN Acropteron longipenne Champion, 1886: 256. Acropteron maklini Champion, 1886 PAN Acropteron mäklini Champion, 1886: 254. Acropteron mexicanum Champion, 1886 MEX (VE) Acropteron mexicanum Champion, 1886: 256. Acropteron puncticolle Champion, 1886 PAN Acropteron puncticolle Champion, 1886: 256. Acropteron quadraticolle Champion, 1896 LAN Acropteron quadraticolle Champion, 1896: 29. Acropteron rugipes Champion, 1886 NIC Acropteron rugipes Champion, 1886: 257. Tribe Alphitobiini Reitter, 1917 Alphitobiini Reitter, 1917: 58. Type genus: Alphitobius Stephens, 1829. Genus Alphitobius Stephens, 1829 [M] Alphitobius Stephens, 1829: 19. Type species: Helops picipes Panzer, 1794 (= Opatrum laevigatum Fabricius, 1781), monotypy. Heterophaga Dejean, 1834: 199. Type species: Tenebrio mauritanicus Fabricius, 1792 (= Opatrum laevigatum Fabricius, 1781), subsequent designation ( Duponchel 1845 : 601). Synonymy: Wollaston (1854 : 498). Cryptops Solier, 1851: 235. Type species: Cryptops ulomoides Solier, 1851 (= Tenebrio diaperinus Panzer, 1797), monotypy. Synonymy: Philippi (1887 : 735). Microphyes MacLeay, 1873: 286. Type species: Microphyes rufipes MacLeay, 1873 (= Opatrum laevigatum Fabricius, 1781), monotypy. Synonymy: Blair (1914 : 486). Alphitobius diaperinus (Panzer, 1797) [Fig. 15 ] CAN (AB BC MB NB NF NS ON PE QC SK) USA (FL GA IN MD MI NC NY OH SC SD VA WA WI) MEX (CH DU GU PU QR VE) NIC / BAH CUB CAY JAM HAI DOM PRI LAN / SA – Adventive Figure 15. Alphitobius diaperinus (Panzer, 1797). Scale bar = 1 mm. Tenebrio diaperinus Panzer, 1797: 16. Cryptops ulomoides Solier, 1851: 236. Synonymy: Schawaller and Grimm (2014 : 174). Alphitobius laevigatus (Fabricius, 1781) CAN (BC NB ON QC) USA (CT FL GA ID IN NY OH OR PA SC SD WA WI) MEX (BS CH CO DU NL TA VE YU) BEL NIC CRI PAN / CUB CAY JAM PRI LAN / SA – Adventive Opatrum laevigatum Fabricius, 1781: 90. Helops piceus Olivier, 1793: 50. Synonymy: Blair (1914 : 486). Helops picipes Panzer, 1794: 4. Synonymy: Spilman (1966 : 7). Alphitobius piceus var. ruficolor Pic, 1925b: 11. Synonymy: Gebien (1940 : 779). Tribe Amarygmini Gistel, 1848 Amarygmiidae Gistel, 1848: [10]. Type genus: Amarygmus Dalman, 1823. Mégacanthides Lacordaire, 1859: 467. Type genus: Megacantha Westwood, 1843. Méracanthides Lacordaire, 1859: 464. Type genus: Meracantha Kirby, 1837. Genus Cymatothes Dejean, 1834 [M] Cymatothes Dejean, 1834 [30 June]: 208. Type species: Helops undatus Fabricius 1792 (= Erotylus nebulosus Fabricius, 1781), monotypy. Physignathus Gistel, 1834 [23 September]: 22 [junior homonym of Physignathus Cuvier, 1829]. Type species: Helops undatus Fabricius, 1792 (= Erotylus nebulosus Fabricius, 1781), monotypy. Synonymy: Bousquet and Bouchard (2017a : 132). Pyanisia Laporte, 1840: 235. Type species: Helops undatus Fabricius, 1792 (= Erotylus nebulosus Fabricius, 1781), subsequent designation ( Lacordaire 1859 : 476). Synonymy: Chevrolat (1847 : 643). Pyganisia Hope, 1841: 133. Type species: Helops undatus Fabricius, 1792 (= Erotylus nebulosus Fabricius, 1781), original designation. Synonymy: Chevrolat (1847 : 643). Cymatothes fumosus (Champion, 1887) GUA Pyanisia fumosa Champion, 1887: 331. Cymatothes laevis (Champion, 1893) MEX (GE) Pyanisia laevis Champion, 1893a: 561. Cymatothes longicollis (Champion, 1887) GUA Pyanisia longicollis Champion, 1887: 331. Cymatothes nebulosus nebulosus (Fabricius, 1781) MEX GUA BEL NIC CRI PAN / CUB HAI LAN / SA Erotylus nebulosus Fabricius, 1781: 158. Helops undatus Fabricius, 1792a: 122. Synonymy: Blair (1935 : 103). Cymatothes opacus Solier, 1848 USA (FL) MEX (CH CI DU GE OA PU VE) Cymatothes opacus Solier, 1848: 180. Cymatothes coarctatus Solier, 1848: 181. Synonymy: Champion (1887 : 330). Cymatothes unicolor Solier, 1848 USA (AL FL TX) MEX (CI DU JA NA VE) GUA BEL NIC CRI PAN / BAH CUB PRI LAN Helops tristis Laporte, 1840: 236 [junior primary homonym of Helops tristis Rossi, 1790]. Cymatothes unicolor Solier, 1848: 182. Synonymy: Champion (1887 : 330). Cymatothes uniformis (C.O. Waterhouse, 1878) JAM Hoplonyx uniformis C.O. Waterhouse, 1878: 306. Genus Meracantha Kirby, 1837 [F] Meracantha Kirby, 1837: 237. Type species: Meracantha canadensis Kirby, 1837 (= Helops contractus Palisot de Beauvois, 1812), monotypy. Falacer Laporte, 1840: 233. Type species: Acanthopus cupreus Laporte, 1840 (= Helops contractus Palisot de Beauvois, 1812), present designation . Synonymy: Lacordaire (1859 : 466). Physocoelus Haldeman, 1850: 5. Type species: Helops contractus Palisot de Beauvois, 1812, monotypy. Synonymy: Lacordaire (1859 : 466). Meracantha contracta (Palisot de Beauvois, 1812) CAN (ON) USA (AL CT FL GA IA IN LA MD MI NC NY OH PA SC TN TX VA WI) Helops contractus Palisot de Beauvois, 1812: 121. Meracantha canadensis Kirby, 1837: 238. Synonymy: Melsheimer (1853 : 140). Acanthopus cupreus Laporte, 1840: 233. Synonymy: Lacordaire (1859 : 466). Acanthopus rugosus Laporte, 1840: 233. Synonym (in doubt): Papp (1961d : 135). Helops tumidus Melsheimer, 1846: 61. Synonymy: Melsheimer (1853 : 140). Psorodes inflata Solier, 1848: 167. Synonymy (with M. canadensis Kirby): Schaum (1850 : 181). Genus Plesiophthalmus Motschulsky, 1857 [M] Plesiophthalmus Motschulsky, 1857: 34. Type species: Plesiophthalmus nigrocyaneus Motschulsky, 1857, monotypy. Cyriogeton Pascoe, 1871: 356. Type species: Cyriogeton insignis Pascoe, 1871, monotypy. Synonymy: Masumoto (1989a : 536). Plesiophthalmus spectabilis Harold, 1875 USA (MD) 28 – Adventive Plesiophthalmus spectabilis Harold, 1875: 293. Plesiophthalmus obesus Marseul, 1876: 319. Synonymy: Harold (1876 : 85). Plesiophthalmus arciferens Fairmaire [in Deyrolle and Fairmaire], 1878: 120. Synonymy: Masumoto (1989b : 743). Plesiophthalmus subparallelus Pic, 1916: 11. Synonymy: Masumoto (1989b : 743). Tribe Amphidorini LeConte, 1862 Amphidorae LeConte, 1862a: 239. Type genus: Amphidora Eschscholtz, 1829. Eleodiini Blaisdell, 1909: 27. Type genus: Eleodes Eschscholtz, 1829. Eleodopsinae Blaisdell, 1939b: 51. Type genus: Eleodopsis Blaisdell, 1939 (= Eleodes Eschscholtz, 1829). Lariversiina La Rivers, 1948: 98. Type genus: Lariversius Blaisdell, 1947. Trogloderina La Rivers, 1948: 98. Type genus: Trogloderus LeConte, 1879. Genus Eleodes Eschscholtz, 1829 29 [F] Eleodes Eschscholtz, 1829: 8. Type species: Eleodes dentipes Eschscholtz, 1829, subsequent designation ( Hope 1841 : 124). Elaeodes Gemminger [in Gemminger and Harold], 1870: 1868. Unjustified emendation of Eleodes Eschscholtz, 1829, not in prevailing usage. Subgenus Amphidora Eschscholtz, 1829 Amphidora Eschscholtz, 1829: 9. Type species: Amphidora littoralis Eschscholtz, 1829, monotypy. Status revised [ADS & MAJ]. Eleodes littoralis (Eschscholtz, 1829) USA (CA) Amphidora littoralis Eschscholtz, 1829: 9 30 . Eleodes nigropilosa (LeConte, 1851) USA (CA) MEX (BC) Amphidora nigropilosa LeConte, 1851: 136. Amphidora tenebrosa Horn, 1870: 329. Synonymy: Triplehorn (1996 : 13). Eleodes subdeplanata (Blaisdell, 1943) MEX (BC BS) Amphidora subdeplanata Blaisdell, 1943: 252. Subgenus Ardeleodes Blaisdell, 1937 Ardeleodes Blaisdell, 1937b: 128 31 . Type species: Eleodes tibialis Blaisdell, 1909, original designation. Eleodes tibialis Blaisdell, 1909 MEX (BS) Eleodes tibialis Blaisdell, 1909: 313. Eleodes tibialis forma oblonga Blaisdell, 1909: 315. New synonymy [YB]. Subgenus Blapylis Horn, 1870 Blapylis Horn, 1870: 315. Type species: Eleodes cordata Eschscholtz, 1829, present designation . Synonymy: Doyen and Lawrence (1979 : 367). Eleodopsis Blaisdell, 1939b: 52. Type species: Eleodopsis subvestita Blaisdell, 1939, original designation. Synonymy: Spilman (1962b : 59). Eleodes alticola Blaisdell, 1925 USA (CA) Eleodes parvicollis alticola Blaisdell, 1925c: 387. Eleodes aristata Somerby, 1977 USA (CA) Eleodes aristata Somerby, 1977: 22. Eleodes bishopensis Somerby and Doyen, 1976 USA (CA) Eleodes bishopensis Somerby and Doyen, 1976: 257. Eleodes blanchardii Blaisdell, 1909 USA (CA) Eleodes blanchardii Blaisdell, 1909: 339. Eleodes brunnipes Casey, 1890 USA (CA CO ID NV WY) Eleodes brunnipes Casey, 1890b: 402. Eleodes pimelioides var. brevisetosa Blaisdell, 1918b: 162. Synonymy: Blaisdell (1925a : 80). Eleodes caseyi Blaisdell, 1909 USA (CA NV) Eleodes caseyi Blaisdell, 1909: 388. Eleodes clavicornis Eschscholtz, 1829 USA (CA) Eleodes clavicornis Eschscholtz, 1829: 11. Eleodes impressicollis Boheman, 1858: 90. Synonymy: LeConte (1866a : 60). Eleodes consobrina LeConte, 1851 USA (CA) MEX (BC) Eleodes consobrina LeConte, 1851: 135. Eleodes veseyi LeConte, 1858c: 187. Synonymy: LeConte (1866a : 60). Eleodes constricta LeConte, 1858 CAN (BC) USA (CA UT) Eleodes constrictus LeConte, 1858c: 187. Eleodes [ manni var.] variolosa Blaisdell, 1917: 223. New synonymy [based on Somerby (1972 : 161) unpublished thesis]. Eleodes cooperi Somerby and Doyen, 1976 USA (CA) Eleodes cooperi Somerby and Doyen, 1976: 253. Eleodes cordata Eschscholtz, 1829 USA (CA) Eleodes cordata Eschscholtz, 1829: 12. Eleodes cordata forma sublaevis Blaisdell, 1909: 381. New synonymy [based on Somerby (1972 : 254) unpublished thesis]. Eleodes cordata forma intermedia Blaisdell, 1909: 381. New synonymy [YB]. Eleodes cordata forma oblonga Blaisdell, 1909: 383. New synonymy [YB]. Eleodes cordata forma elongata Blaisdell, 1909: 383. New synonymy [YB]. Eleodes [ cordata var.] adulterina Blaisdell, 1917: 224. New synonymy [based on Somerby (1972 : 254) unpublished thesis]. Eleodes fuchsii Blaisdell, 1909 USA (CA) Eleodes fuchsii Blaisdell, 1909: 343. Eleodes hornii var. monticula Blaisdell, 1918c: 385. New synonymy [based on Somerby (1972 : 199) unpublished thesis]. Eleodes manni sierra Blaisdell, 1926a: 78. New synonymy [based on Somerby (1972 : 199) unpublished thesis]. Eleodes hoppingii Blaisdell, 1909 USA (CA) Eleodes hoppingii Blaisdell, 1909: 368. Eleodes hornii hornii Blaisdell, 1909 USA (CA) Eleodes hornii Blaisdell, 1909: 350. Eleodes hybrida Blaisdell, 1917 USA (CA) Eleodes [ cordata var.] hybrida Blaisdell, 1917: 225. Eleodes inculta LeConte, 1861 USA (CA) Eleodes inculta LeConte, 1861b: 352. Eleodes inculta var. affinis Blaisdell, 1918c: 384. Synonymy: Miller (1985 : 21). Eleodes kaweana Blaisdell, 1933 USA (CA) Eleodes kaweana Blaisdell, 1933b: 203. Eleodes lariversi Somerby and Doyen, 1976 USA (CA) Eleodes lariversi Somerby and Doyen, 1976: 256. Eleodes lecontei Horn, 1870 USA (CO) Eleodes subaspera LeConte, 1866b: 115 [junior primary homonym of Eleodes subaspera Solier, 1848]. Eleodes lecontei Horn, 1870: 316. Replacement name for Eleodes subaspera LeConte, 1866. Eleodes manni Blaisdell, 1917 USA (WA) Eleodes manni Blaisdell, 1917: 221. Eleodes nana Blaisdell, 1909 USA (CA NV) Eleodes tenebrosa var. nana Blaisdell, 1909: 328. Eleodes neotomae Blaisdell, 1909 USA (CA) Eleodes neotomae Blaisdell, 1909: 347. Eleodes novoverrucula Boddy, 1957 CAN (AB BC) USA (ID MT OR WA) Eleodes novoverrucula Boddy, 1957: 195. Eleodes nunenmacheri Blaisdell, 1918 USA (CA OR) Eleodes nunenmacheri Blaisdell, 1918b: 163. Eleodes oregona Blaisdell, 1941 USA (OR) Eleodes oregona Blaisdell, 1941b: 157. Eleodes orophila Somerby, 1977 USA (AZ NM) Eleodes orophilus Somerby, 1977: 24. Eleodes panamintensis Somerby, 1977 USA (CA) Eleodes panamintensis Somerby, 1977: 20. Eleodes parvicollis Eschscholtz, 1829 USA (CA) Eleodes parvicollis Eschscholtz, 1829: 11. Eleodes parvicollis var. squalida Blaisdell, 1918c: 380. New synonymy [based on Somerby (1972 : 188) unpublished thesis]. Eleodes patulicollis Blaisdell, 1932 USA (UT) Eleodes manni dilaticollis Blaisdell, 1925c: 388 [junior primary homonym of Eleodes dilaticollis Champion, 1884]. Eleodes patulicollis Blaisdell, 1932a: 78. Replacement name for Eleodes dilaticollis Blaisdell, 1925. Eleodes pimelioides Mannerheim, 1843 [Fig. 16 ] CAN (BC) USA (CA OR UT WA) Figure 16. Eleodes ( Blapylis ) pimelioides Mannerheim, 1843. Scale bar = 1 mm. Eleodes pimelioides Mannerheim, 1843: 274. Eleodes viator LeConte, 1858c: 188. Synonymy: Horn (1870 : 318). Eleodes nunenmacheri var. verrucula Blaisdell, 1918b: 164. Synonymy: Bousquet and Campbell (1991 : 257) [based on Somerby (1972 : 238) unpublished thesis]. Eleodes pimelioides var. patruelis Blaisdell, 1918c: 382. Synonymy (with E. nunenmacheri verrucula Blaisdell): Boddy (1957 : 197). Eleodes planata Eschscholtz, 1829 USA (CA) Eleodes planata Eschscholtz, 1829: 12. Eleodes reflexicollis Mannerheim, 1843: 270. New synonymy [based on Somerby (1972 : 187) unpublished thesis]. Eleodes parvicollis forma farallonica Blaisdell, 1909: 356. New synonymy [based on Somerby (1972 : 187) unpublished thesis]. Eleodes producta Mannerheim, 1843 USA (CA) Eleodes producta Mannerheim, 1843: 271. Eleodes propinqua Blaisdell, 1918 USA (CA) Eleodes propinqua Blaisdell, 1918b: 165. Eleodes robinetti Boddy, 1957 USA (OR WA) Eleodes robinetti Boddy, 1957: 194. Eleodes rotundipennis LeConte, 1857 CAN (BC) USA (OR WA) Eleodes rotundipennis LeConte, 1857: 50. Eleodes stricta LeConte, 1857: 50. Synonymy: Boddy (1965 : 158). Eleodes subligata LeConte, 1857: 50. Synonymy: Boddy (1965 : 158). Eleodes indentata Blaisdell, 1935a: 28. New synonymy [based on Somerby (1972 : 237) unpublished thesis]. Eleodes scabripennis LeConte, 1859 USA (CA) Eleodes scabripennis LeConte, 1859b: 77. Eleodes scabriventris Blaisdell, 1933 USA (CA) Eleodes scabriventris Blaisdell, 1933b: 202. Eleodes scabrosa Eschscholtz, 1829 USA (CA OR) Eleodes scabrosa Eschscholtz, 1829: 11. Eleodes intricata Mannerheim, 1843: 273. New synonymy [based on Somerby (1972 : 163) unpublished thesis]. Eleodes schlingeri Somerby and Doyen, 1976 USA (CA) Eleodes schlingeri Somerby and Doyen, 1976: 254. Eleodes schwarzii Blaisdell, 1909 USA (ID OR WA) Eleodes schwarzii Blaisdell, 1909: 406. Eleodes snowii Blaisdell, 1909 USA (AZ CO NM NV) Eleodes snowii Blaisdell, 1909: 317. Eleodes spilmani Somerby and Doyen, 1976 USA (CA) Eleodes spilmani Somerby and Doyen, 1976: 258. Eleodes strumosa Blaisdell, 1932 USA (UT NV) Eleodes strumosa Blaisdell, 1932a: 76. Eleodes subvestita (Blaisdell, 1939) USA (CA) Eleodopsis subvestita Blaisdell, 1939b: 53. Eleodes tenebrosa Horn, 1870 CAN (BC) USA (CA NV UT) Eleodes tenebrosa Horn, 1870: 316. Eleodes horni fenyesi Blaisdell, 1926a: 77. New synonymy [based on Somerby (1972 : 161) unpublished thesis]. Eleodes triplehorni Somerby and Doyen, 1976 MEX (BC) Eleodes triplehorni Somerby and Doyen, 1976: 252. Eleodes trita Blaisdell, 1917 USA (CA OR) Eleodes [ parvicollis var.] trita Blaisdell, 1917: 225. Eleodes tuberculata Eschscholtz, 1829 USA (CA) Eleodes tuberculata Eschscholtz, 1829: 12. Eleodes cordata var. horrida Blaisdell, 1918c: 383. New synonymy [based on Somerby (1972 : 255) unpublished thesis]. Eleodes versatilis Blaisdell, 1921 USA (OR WA) Eleodes rotundipennis versatilis Blaisdell, 1921b: 217. Eleodes oblonga Blaisdell, 1933b: 206 [junior primary homonym of Eleodes tibialis oblonga Blaisdell, 1909]. New synonymy [based on Somerby (1972 : 190) unpublished thesis]. Eleodes formosus Thomas, 2005: 551. Replacement name for Eleodes oblonga Blaisdell, 1933. Eleodes volcanensis Somerby, 1977 USA (CA OR) Eleodes volcanensis Somerby, 1977: 23. Eleodes wakelandi Somerby, 1977 USA (ID OR UT) Eleodes wakelandi Somerby, 1977: 19. Subgenus Caverneleodes Triplehorn, 1975 Caverneleodes Triplehorn, 1975: 39. Type species: Eleodes easterlai Triplehorn, 1975, original designation. Eleodes easterlai Triplehorn, 1975 USA (TX) MEX (CO) Eleodes easterlai Triplehorn, 1975: 39. Eleodes grutus Aalbu, Smith and Triplehorn, 2012 32 MEX (NL) Eleodes grutus Aalbu, Smith and Triplehorn, 2012: 206. Eleodes guadalupensis Aalbu, Smith and Triplehorn, 2012 USA (NM) Eleodes guadalupensis Aalbu, Smith and Triplehorn, 2012: 208. Eleodes labialis Triplehorn, 1975 USA (TX) MEX (CH) Eleodes labialis Triplehorn, 1975: 42. Eleodes leptoscelis Triplehorn, 1975 USA (AZ) Eleodes leptoscelis Triplehorn, 1975: 42. Eleodes microps Aalbu, Smith and Triplehorn, 2012 USA (CA) Eleodes microps Aalbu, Smith and Triplehorn, 2012: 200. Eleodes reddelli Triplehorn, 2007 MEX (NL) Eleodes reddelli Triplehorn, 2007: 639. Eleodes rugosifrons Triplehorn and Reddell, 1991 MEX (CO NL) Eleodes rugosifrons Triplehorn and Reddell, 1991: 527. Eleodes sprousei Triplehorn and Reddell, 1991 MEX (NL TA) Eleodes sprousei Triplehorn and Reddell, 1991: 525. Eleodes thomasi Aalbu, Smith and Triplehorn, 2012 MEX (CO NL) Eleodes thomasi Aalbu, Smith and Triplehorn, 2012: 204. Eleodes wheeleri Aalbu, Smith and Triplehorn, 2012 USA (AZ) Eleodes wheeleri Aalbu, Smith and Triplehorn, 2012: 208. Eleodes wynnei Aalbu, Smith and Triplehorn, 2012 USA (AZ UT) Eleodes wynnei Aalbu, Smith and Triplehorn, 2012: 201. Subgenus Chaseleodes Thomas, 2015 Chaseleodes Thomas, 2015: 122. Type species: Elaeodes curta Champion, 1884, original designation. Eleodes connata Solier, 1848 MEX (CH DU FD ME MI MO PU TL VE) Eleodes connata Solier, 1848: 243. Eleodes curta Champion, 1884 MEX (JA ME MI) Elaeodes curta Champion, 1884: 82. Subgenus Cratidus LeConte, 1862 Cratidus LeConte, 1862a: 239. Type species: Amphidora osculans LeConte, 1851, monotypy. Eleodes osculans (LeConte, 1851) USA (CA) MEX (BC) Amphidora osculans LeConte, 1851: 136. Cratidus fuscipilosus Casey, 1890b: 407. Synonymy: Triplehorn (1996 : 13). Cratidus ovipennis Casey, 1924: 328. Synonymy: Triplehorn (1996 : 13). Eleodes behrii Grinnell, 1908: 213. Synonymy: Doyen and Miller (1980 : 3). Eleodes intermedia Grinnell, 1908: 215. Synonymy: Doyen and Miller (1980 : 3). Eleodes ursus Triplehorn, 1996 MEX (BC BS) Cratidus rotundicollis Horn, 1870: 328 [junior secondary homonym of Eleodes rotundicollis Eschscholtz, 1829]. Eleodes ursus Triplehorn, 1996: 14. Replacement name for Eleodes rotundicollis (Horn, 1870). Subgenus Discogenia LeConte, 1866 Discogenia LeConte, 1866b: 117. Type species: Eleodes scabricula LeConte, 1858, present designation . Eleodes acutangula Blaisdell, 1921 USA (CA) Eleodes acutangula Blaisdell, 1921b: 225. Eleodes marginata Eschscholtz, 1829 USA (CA) Eleodes marginata Eschscholtz, 1829: 10. Eleodes fischerii Mannerheim, 1840: 137. Synonymy: Horn (1870 : 320). Eleodes scabricula deplanata Blaisdell, 1909 USA (CA) Eleodes scabricula forma deplanata Blaisdell, 1909: 444. Eleodes scabricula scabricula LeConte, 1858 USA (CA) Eleodes scabricula LeConte, 1858c: 187. Subgenus Eleodes Eschscholtz, 1829 Eleodes Eschscholtz, 1829: 8. Type species: Eleodes dentipes Eschscholtz, 1829, subsequent designation ( Hope 1841 : 124). Eleodes acuticauda LeConte, 1851 USA (CA) MEX (BC) Eleodes acuticauda LeConte, 1851: 135. Eleodes laticollis LeConte, 1851: 135. Synonymy: Horn (1870 : 314). Eleodes acuticauda forma punctata Blaisdell, 1909: 278. Synonymy: Triplehorn et al. (2015 : 161). Eleodes laticollis apprima Blaisdell, 1921b: 219. Synonymy: Triplehorn (1996 : 9). Eleodes acuta (Say, 1824) USA (CO KS NM SD TX) Blaps acuta Say, 1824a: 258. Eleodes acuta pernigra Blaisdell, 1937b: 128. Synonymy: Triplehorn et al. (2015 : 161). Eleodes adumbrata Blaisdell, 1925 MEX (BC) Eleodes adumbrata Blaisdell, 1925b: 332. Eleodes armata LeConte, 1851 USA (AZ CA ID NV OR UT) MEX (BC SO) Eleodes armata LeConte, 1851: 134. Eleodes armata impotens Blaisdell, 1895: 236. Synonymy: Triplehorn et al. (2015 : 163). Eleodes armata forma sinuata Blaisdell, 1909: 266. Synonymy: Triplehorn et al. (2015 : 163). Eleodes armata var. pumila Blaisdell, 1933b: 197. Synonymy: Triplehorn et al. (2015 : 163). Eleodes amedeensis Blaisdell, 1933b: 199. Synonymy: Triplehorn et al. (2015 : 163). Eleodes striatipennis Blaisdell, 1942: 134. Synonymy: Triplehorn et al. (2015 : 163). Eleodes curvidens Triplehorn and Cifuentes-Ruiz, 2011 MEX (GU MI MO PU) Eleodes curvidens Triplehorn and Cifuentes-Ruiz, 2011: 66. Eleodes dentipes Eschscholtz, 1829 USA (CA) Eleodes dentipes Eschscholtz, 1829: 10. Eleodes elegans Casey, 1890b: 401. Synonymy: Blaisdell (1909 : 251). Eleodes prominens Casey, 1890b: 401. Synonymy: Blaisdell (1909 : 251). Eleodes confinis Blaisdell, 1895: 237. Synonymy: Blaisdell (1909 : 251). Eleodes dentipes forma pertenuis Blaisdell, 1909: 253. Synonymy: Triplehorn et al. (2015 : 164). Eleodes dentipes forma elongata Blaisdell, 1909: 254. Synonymy: Gebien (1938 : 57). Eleodes dentipes forma robusta Blaisdell, 1909: 255. Synonymy: Triplehorn et al. (2015 : 164). Eleodes dentipes var. perpunctata Blaisdell, 1918c: 386. Synonymy: Gebien (1938 : 57). Eleodes dentipes marinae Blaisdell, 1921b: 218. New synonymy [ADS & MAJ]. Eleodes dentipes montana Blaisdell, 1925c: 385 [junior primary homonym of Eleodes montana Champion, 1884]. Synonymy: Triplehorn et al. (2015 : 164). Eleodes dentipes tularensis Blaisdell, 1925c: 386. Synonymy: Triplehorn et al. (2015 : 164). Eleodes paradoxa Blaisdell, 1932a: 78. Replacement name for Eleodes montana Blaisdell, 1925. Eleodes dentipes sordida Blaisdell, 1935a: 30. Synonymy: Triplehorn et al. (2015 : 164). Eleodes discincta Blaisdell, 1925 MEX (BC BS) Eleodes discincta Blaisdell, 1925b: 333. Eleodes eschscholtzii Solier, 1848 USA (AZ NM) MEX (BS CO DU SI SO) Eleodes eschscholtzii Solier, 1848: 254. Eleodes lucae LeConte, 1866b: 114. Synonymy: Triplehorn (1996 : 10). Eleodes wickhami Horn, 1891: 41. Synonymy: Triplehorn et al. (2015 : 167). Eleodes femorata LeConte, 1851 USA (CA) MEX (BC BS) Eleodes femorata LeConte, 1851: 134. Eleodes militaris Horn, 1870: 310. Synonymy: Triplehorn (1996 : 7). Eleodes militaris forma subedentata Blaisdell, 1909: 270. Synonymy: Triplehorn et al. (2015 : 167). Eleodes inepta Blaisdell, 1925b: 334. Synonymy: Triplehorn (1996 : 7). Eleodes marthae Blaisdell, 1943: 243. Synonymy: Triplehorn (1996 : 7). Eleodes fiski Triplehorn, 2015 MEX (TA) Eleodes fiski Triplehorn [in Triplehorn, Thomas and Smith], 2015: 170. Eleodes gracilis distans Blaisdell, 1909 USA (CA) Eleodes gracilis var. distans Blaisdell, 1909: 242. Eleodes gracilis gracilis LeConte, 1858 USA (AZ NM TX) MEX (CH CO DU SI SO ZA) Eleodes gracilis LeConte, 1858c: 184. Eleodes grandicollis grandicollis Mannerheim, 1843 USA (AZ CA NV) MEX (BC) Eleodes grandicollis Mannerheim, 1843: 266. Eleodes elongata Grinnell, 1908: 215. Synonymy: Doyen and Miller (1980 : 3). Eleodes grandicollis valida Boheman, 1858 USA (CA) Eleodes valida Boheman, 1858: 90. Eleodes hispilabris (Say, 1824) CAN (AB MB SK) USA (AZ CA CO ID KS MT ND NE NM NV OK OR SD TX UT WA WY) MEX (CH CO NL SO TA) Blaps hispilabris Say, 1824a: 259. Eleodes sulcata LeConte, 1852: 67 [junior primary homonym of Eleodes sulcatus Eschscholtz, 1829]. Synonymy: Horn (1870 : 313). Eleodes connexa LeConte, 1857: 49. Synonymy: Triplehorn et al. (2015 : 174). Eleodes nupta LeConte, 1858c: 183. Synonymy: Triplehorn et al. (2015 : 174). Eleodes binotata Walker, 1866: 329. Synonymy: Triplehorn et al. (2015 : 174). Elaeodes lecontei Gemminger, 1870: 122. Replacement name for Elaeodes sulcata LeConte, 1852. Eleodes hispilabris forma sculptilis Blaisdell, 1909: 220. Synonymy: Triplehorn et al. (2015 : 174). Eleodes hispilabris forma elongata Blaisdell, 1909: 220 [primary homonym of Eleodes dentipes forma elongata Blaisdell, 1909]. Synonymy: Triplehorn et al. (2015 : 174). Eleodes hispilabris forma laevis Blaisdell, 1909: 220. Synonymy (with E. binotata Walker): Blair (1921 : 283). Eleodes subpinguis Blaisdell, 1909: 247. Synonymy: Triplehorn et al. (2015 : 174). Eleodes hispilabris var. imitabilis Blaisdell, 1918b: 167. Synonymy: Triplehorn et al. (2015 : 174). Eleodes hispilabris var. attenuata Blaisdell, 1918b: 168 [junior secondary homonym of Eleodes attenuatus (LeConte, 1851)]. Synonymy: Triplehorn et al. (2015 : 174). Eleodes hispilabris immundus Blaisdell, 1925a: 79. Replacement name for Eleodes hispilabris elongata Blaisdell, 1909. Eleodes loretensis Blaisdell, 1923 MEX (BC BS) Eleodes loretensis Blaisdell, 1923: 262. Eleodes mexicana Blaisdell, 1943 MEX (BC BS) Eleodes mexicana Blaisdell, 1943: 246. Eleodes simondsi Blaisdell, 1943: 247. Synonymy: Triplehorn (1996 : 11). Eleodes blaisdelli Blackwelder, 1945: 521. Unnecessary replacement name for Eleodes mexicana Blaisdell, 1943. Eleodes mirabilis Triplehorn, 2007 USA (TX) MEX (NL SL TA) Eleodes mirabilis Triplehorn, 2007: 634. Eleodes moesta Blaisdell, 1921 MEX (BC BS) Eleodes sanmartinensis moesta Blaisdell, 1921b: 221. Eleodes morbosa Blaisdell, 1925b: 335. Synonymy: Triplehorn (1996 : 11). Eleodes muricatula Triplehorn, 2007 MEX (CO SL ZA) Eleodes muricatulus Triplehorn, 2007: 637. Eleodes obscura dispersa LeConte, 1858 USA (AZ CO NM UT) Eleodes dispersa LeConte, 1858c: 182. Eleodes deleta LeConte, 1858c: 182. Synonymy: Horn (1870 : 305). Eleodes obscura glabriuscula Blaisdell, 1925 USA (AZ NM TX) MEX (CH) Eleodes obscura glabriuscula Blaisdell, 1925c: 383. Eleodes obscura obscura (Say, 1824) USA (CO MT NE NM TX WY) Blaps obscura Say, 1824a: 259. Eleodes obscura sulcipennis Mannerheim, 1843 [Fig. 17 ] CAN (BC) USA (AZ CA ID MT NM NV OR UT WA) MEX (CH DU SO) Figure 17. Eleodes ( Eleodes ) obscura sulcipennis Mannerheim, 1843. Scale bar = 1 mm. Eleodes sulcipennis Mannerheim, 1843: 266. Eleodes arata LeConte, 1858c: 182. Synonymy: Horn (1870 : 306). Eleodes conjuncta Walker, 1866: 329. Synonymy: Leng (1920 : 227). Eleodes convexicollis Walker, 1866: 328. Synonymy: Leng (1920 : 227). Eleodes rossi Blaisdell, 1943 MEX (BS) Eleodes rossi Blaisdell, 1943: 241. Eleodes rugosa Perbosc, 1839 MEX (SL TA VE) Eleodes rugosa Perbosc, 1839: 263. Eleodes caudata Solier, 1848: 255. Synonymy: Champion (1884 : 77). Eleodes samalayucae Triplehorn, 2007 MEX (CH) Eleodes samalayucae Triplehorn, 2007: 641. Eleodes sanmartinensis Blaisdell, 1921 MEX (BC) Eleodes sanmartinensis Blaisdell, 1921b: 220. Eleodes scyroptera Triplehorn, 2007 MEX (AG DU GU HI NL QU ZA) Eleodes scyropterus Triplehorn, 2007: 635. Eleodes spinipes macrura Champion, 1892 USA (AZ NM TX) MEX (AG CH CO DU JA NA SO ZA) Elaeodes macrura Champion, 1892: 511. Eleodes ventricosa var. falli Blaisdell, 1909: 305. Synonymy: Triplehorn (2010 : 377). Eleodes spinipes spinipes Solier, 1848 MEX (GU HI NL QU SL TA) Eleodes spinipes Solier, 1848: 253. Eleodes spinipes ventricosa LeConte, 1858 USA (TX) MEX (CO NL TA) Eleodes ventricosa LeConte, 1858c: 186. Eleodes sponsa LeConte, 1858 USA (AZ CO NM TX UT) Eleodes sponsa LeConte, 1858c: 184. Eleodes sponsa forma convexa Blaisdell, 1909: 215. Synonymy: Triplehorn et al. (2015 : 187). Eleodes subcylindrica Casey, 1890 USA (AZ CA NV) MEX (BC) Eleodes subcylindricus Casey, 1890b: 400. Eleodes armata forma subedentata Blaisdell, 1909: 262. Synonymy: Blaisdell (1910 : 62). Eleodes suturalis (Say, 1824) USA (AZ CO KS MN ND NE NM OK SD TX UT WY) Blaps suturalis Say, 1824a: 257. Eleodes texana LeConte, 1858c: 182. Synonymy: Triplehorn et al. (2009: 432). Eleodes tenuipes Casey, 1890 USA (NM TX) MEX (CH) Eleodes tenuipes Casey, 1890b: 399. Eleodes vanduzeei Blaisdell, 1923 MEX (BS) Eleodes vanduzeei Blaisdell, 1923: 264. Subgenus Heteropromus Blaisdell, 1909 Heteropromus Blaisdell, 1909: 179. Type species: Eleodes veterator Horn, 1874, monotypy. Eleodes veterator Horn, 1874 33 USA (LA TX) Eleodes vetorator Horn, 1874a: 33. Subgenus Litheleodes Blaisdell, 1909 Litheleodes Blaisdell, 1909: 114. Type species: Blaps extricata Say, 1824, subsequent designation ( Triplehorn and Thomas 2015 : 11). Eleodes arcuata Casey, 1884 USA (AZ NM TX) MEX (CH CO SO) Eleodes arcuata Casey, 1884 [August]: 47. Elaeodes sonorae Champion, 1884 [December]: 85. Synonymy: Gebien (1938 : 53). Eleodes aspera LeConte, 1866 USA (AZ CO UT) Eleodes aspera LeConte, 1866b: 115. Eleodes corvina Blaisdell, 1921 USA (CA OR) Eleodes corvina Blaisdell, 1921b: 224. Eleodes extricata (Say, 1824) [Fig. 18 ] CAN (AB BC SK) USA (AZ CO ID KS MT ND NE NM NV OK OR SD TX UT WY) MEX (CH CO SO) Figure 18. Eleodes ( Litheleodes ) extricata (Say, 1824). Scale bar = 1 mm. Blaps extricata Say, 1824a: 261. Eleodes cognata Haldeman, 1852: 376. Synonymy: Triplehorn and Thomas (2015 : 16). Eleodes extricata var. arizonensis Blaisdell, 1909: 125. Synonymy: Triplehorn and Thomas (2015 : 16). Eleodes extricata forma elongata Blaisdell, 1909: 123. Synonymy: Triplehorn and Thomas (2015 : 16). Eleodes extricata forma convexicollis Blaisdell, 1909: 123 [junior primary homonym of Eleodes convexicollis Walker, 1866]. Synonymy: Triplehorn and Thomas (2015 : 16). Eleodes extricata var. utahensis Blaisdell, 1921a: 131. Synonymy: Triplehorn and Thomas (2015 : 16). Eleodes extricata frigida La Rivers, 1943b: 54. Synonymy: Triplehorn and Thomas (2015 : 16). Eleodes extricatus convexinotus Thomas, 2005: 551. Replacement name for Eleodes extricatus convexicollis Blaisdell, 1909. Eleodes granulata LeConte, 1857 CAN (BC) USA (CA CO ID NM OR WA) Eleodes subaspera Solier, 1848: 246 [ nomen dubium , see Horn (1870 : 309)]. Eleodes granulata LeConte, 1857: 50. Synonymy (in doubt): Horn (1870 : 309). Eleodes obtusa LeConte, 1861b: 352. Synonymy: Horn (1870 : 309). Eleodes letcheri var. vandykei Blaisdell, 1909: 136. Synonymy: Triplehorn and Thomas (2015 : 18). Eleodes vandykei var. modificata Blaisdell, 1921a: 131. Synonymy: Triplehorn and Thomas (2015 : 18). Eleodes vandykei similis Blaisdell, 1942: 142. Synonymy: Triplehorn and Thomas (2015 : 18). Eleodes hirtipennis Triplehorn, 1964 USA (CO) Eleodes hirtipennis Triplehorn, 1964b: 60. Eleodes letcheri Blaisdell, 1909 USA (ID NV OR UT) Eleodes letcheri Blaisdell, 1909: 133. Eleodes papillosa Blaisdell, 1917 USA (CA) Eleodes granulata forma tuberculata Blaisdell, 1909: 131 [junior primary homonym of Eleodes tuberculata Eschscholtz, 1829]. Eleodes papillosa Blaisdell, 1917: 226. Replacement name for Eleodes tuberculata Blaisdell, 1909. Eleodes subtuberculata Walker, 1866 CAN (BC) USA (CA ID MT OR WA) Eleodes subtuberculata Walker, 1866: 328. Subgenus Melaneleodes Blaisdell, 1909 Melaneleodes Blaisdell, 1909: 36. Type species: Blaps carbonaria Say, 1824, subsequent designation ( Triplehorn and Thomas 2012 : 254). Eleodes anthracina anthracina Blaisdell, 1909 USA (AZ NM) MEX (CH SO) Eleodes quadricollis var. anthracina Blaisdell, 1909: 87. Eleodes anthracina lustrans Blaisdell, 1909 USA (AZ) Eleodes quadricollis var. lustrans Blaisdell, 1909: 89. Eleodes carbonaria carbonaria (Say, 1824) USA (AZ CO NM NV TX UT WY) MEX (CH CO DU JA NL SL SO TA ZA) Blaps carbonaria Say, 1824a: 260. Eleodes vicina LeConte, 1851: 133. Synonymy: Triplehorn and Thomas (2012 : 256). Eleodes immunis LeConte, 1858c: 186. Synonymy: Horn (1870 : 308). Eleodes porcatus Casey, 1890b: 396. Synonymy: Triplehorn and Thomas (2012 : 257). Eleodes carbonaria forma interstitialis Blaisdell, 1909: 47. Synonymy: Triplehorn and Thomas (2012 : 257). Eleodes carbonaria forma glabra Blaisdell, 1909: 47. New synonymy [ADS & MAJ]. Eleodes mazatzalensis Blaisdell, 1925c: 379. Synonymy: Triplehorn and Thomas (2012 : 257). Eleodes carbonaria chihuahuensis Champion, 1884 USA (AZ NM) MEX (CH CO DU SO) Elaeodes chihuahuensis Champion, 1884: 86. Eleodes nitidus Casey, 1891: 58. Synonymy: Triplehorn and Thomas (2012 : 270). Eleodes ampla Blaisdell, 1909: 53. Synonymy: Triplehorn and Thomas (2012 : 270). Eleodes ampla var. dolosa Blaisdell, 1909: 57. Synonymy (with E. nitidus Casey): Blaisdell (1910 : 65). Eleodes lineata Blaisdell, 1939b: 55. Synonymy: Triplehorn and Thomas (2012 : 270). Eleodes carbonaria disjuncta Triplehorn and Thomas, 2012 MEX (GU HI ME ZA) Eleodes carbonarius disjunctus Triplehorn and Thomas, 2012: 269. Eleodes carbonaria knausii Blaisdell, 1909 USA (CO NM) Eleodes knausii Blaisdell, 1909: 67. Eleodes carbonaria nuevoleonensis Triplehorn and Thomas, 2012 MEX (CO NL) Eleodes carbonarius nuevoleonensis Triplehorn and Thomas, 2012: 265. Eleodes carbonaria obsoleta (Say, 1824) CAN (AB MB SK) USA (AZ CO KS MT ND NE NM OK SD TX UT WY) MEX (SO) Blaps obsoleta Say, 1824a: 261. Eleodes obsoleta forma glabra Blaisdell, 1909: 60. Synonymy: Triplehorn and Thomas (2012 : 272). Eleodes obsoleta forma annectans Blaisdell, 1909: 60. Synonymy: Triplehorn and Thomas (2012 : 272). Eleodes obsoleta forma punctata Blaisdell, 1909: 60. Synonymy: Triplehorn and Thomas (2012 : 272). Eleodes carbonaria omissoides Blaisdell, 1935 MEX (DU NA NL SI SO ZA) Eleodes omissoides Blaisdell, 1935c: 157. Eleodes carbonaria omissa LeConte, 1858 USA (CA NV) MEX (BC) Eleodes omissa LeConte, 1858c: 186. Eleodes interrupta Blaisdell, 1892: 241. Synonymy: Blaisdell (1909 : 72). Eleodes omissa forma catalinae Blaisdell, 1909: 73. Synonymy: Triplehorn and Thomas (2012 : 258). Eleodes omissa forma communis Blaisdell, 1909: 73. Synonymy: Triplehorn and Thomas (2012 : 258). Eleodes omissa forma emarginata Blaisdell, 1909: 74. Synonymy: Triplehorn and Thomas (2012 : 258). Eleodes omissa var. pygmaea Blaisdell, 1909: 77. Synonymy: Triplehorn (1996 : 5). Eleodes omissa var. peninsularis Blaisdell, 1909: 79. Synonymy: Triplehorn (1996 : 5). Eleodes omissa tumida Blaisdell, 1933b: 194. Synonymy: Triplehorn (1996 : 5). Eleodes carbonaria soror LeConte, 1858 USA (TX) MEX (CO NL TA) Eleodes soror LeConte, 1858c: 185. Eleodes halli Blaisdell, 1941 USA (AZ UT) Eleodes fuscipilosa Blaisdell, 1925c: 376 [junior secondary homonym of Eleodes fuscipilosus (Casey, 1890)]. Eleodes halli Blaisdell, 1941a: 37. Synonymy: Triplehorn and Thomas (2012 : 275). Eleodes humeralis LeConte, 1857 CAN (BC) USA (CA ID OR UT WA) Eleodes humeralis LeConte, 1857: 50. Eleodes latiuscula Walker, 1866: 329. Synonymy: LeConte (1873 : 334). Eleodes neomexicana Blaisdell, 1909 USA (NM TX) Eleodes pedinoides var. neomexicana Blaisdell, 1909: 113. Eleodes parowana Blaisdell, 1925 USA (UT) Eleodes parowana Blaisdell, 1925c: 374. Eleodes parowana mimica Blaisdell, 1925c: 375. Synonymy: Triplehorn and Thomas (2012 : 276). Eleodes pedinoides LeConte, 1858 USA (NM TX) MEX (CO NL TA) Eleodes pedinoides LeConte, 1858c: 183. Eleodes asperata LeConte, 1858c: 183. Synonymy: Horn (1870 : 307). Eleodes quadricollis Eschscholtz, 1829 USA (CA) Eleodes quadricollis Eschscholtz, 1829: 12. Eleodes tarsalis Casey, 1890b: 399. Synonymy: Casey (1893 : 597). Eleodes cuneaticollis Casey, 1890b: 397. Synonymy: Triplehorn and Thomas (2012 : 272). Eleodes rileyi reducta Blaisdell, 1925 USA (UT) Eleodes reducta Blaisdell, 1925c: 377. Eleodes rileyi rileyi Casey, 1891 USA (AZ CA CO ID MT NM NV UT WY) Eleodes rileyi Casey, 1891: 57. Eleodes humeralis forma tuberculo-muricata Blaisdell, 1909: 97. Synonymy: Triplehorn and Thomas (2012 : 274). Eleodes humeralis forma granulato-muricata Blaisdell, 1909: 97. Synonymy: Triplehorn and Thomas (2012 : 274). Eleodes quadricollis lassenica Blaisdell, 1925c: 373. Synonymy: Triplehorn and Thomas (2012 : 274). Eleodes coloradensis Blaisdell, 1925c: 380. Synonymy: Triplehorn and Thomas (2012 : 274). Eleodes concinna Blaisdell, 1925c: 381. Synonymy: Triplehorn and Thomas (2012 : 274). Eleodes tanneri Blaisdell, 1932a: 74. Synonymy: Triplehorn and Thomas (2012 : 274). Eleodes rufipes rufipes Pierre, 1976 MEX (PU/VE [Pico de Orizaba]) Eleodes alticola rufipes Pierre, 1976: 708. Eleodes rufipes transvolcanensis Thomas, 2005 MEX (ME/MO/PU [Popocatépetl]) Eleodes alticola Pierre, 1976: 706 [junior primary homonym of Eleodes alticola Blaisdell, 1925]. Eleodes transvolcanensis Thomas, 2005: 553 34 . Replacement name for Eleodes alticola Pierre, 1976. Eleodes tricostata (Say, 1824) [Fig. 19 ] CAN (AB MB SK) USA (AZ CO IA KS MN MO MT ND NE NM OK SD TX WI WY) MEX (CO TA) Figure 19. Eleodes ( Melaneleodes ) tricostata (Say, 1824). Scale bar = 1 mm. Blaps tricostata Say, 1824a: 262. Pimelia alternata Kirby, 1837: 232. Synonymy: LeConte (1851 : 133). Eleodes planata Solier, 1848: 366 [junior primary homonym of Eleodes planata Eschscholtz, 1829]. Synonymy: LeConte (1866a : 60). Eleodes robusta LeConte, 1858c: 183. Synonymy: Horn (1870 : 307). Eleodes tricostata forma ovalis Blaisdell, 1909: 106. Synonymy: Triplehorn and Thomas (2012 : 274). Eleodes tricostata forma costata Blaisdell, 1909: 106. Synonymy: Triplehorn and Thomas (2012 : 274). Eleodes wenzeli speculicollis Blaisdell, 1925 USA (TX) Eleodes speculicollis Blaisdell, 1925c: 382. Eleodes wenzeli wenzeli Blaisdell, 1925 USA (NM TX) Eleodes wenzeli Blaisdell, 1925c: 381. Subgenus Metablapylis Blaisdell, 1909 Metablapylis Blaisdell, 1909: 391. Type species: Eleodes nigrina LeConte, 1858, present designation . Eleodes aalbui Triplehorn, 2007 USA (CA) Eleodes aalbui Triplehorn, 2007: 628. Eleodes californica Blaisdell, 1929 USA (CA) Eleodes californica Blaisdell, 1929a: 165. Eleodes delicata Blaisdell, 1929 USA (AZ TX UT) MEX (BC) Eleodes delicata Blaisdell, 1929a: 164. Eleodes dissimilis Blaisdell, 1909 USA (AZ CA NM NV TX UT) MEX (SO) Eleodes dissimilis Blaisdell, 1909: 398. Eleodes nevadensis Blaisdell, 1909 USA (AZ CA NV UT) Eleodes dissimilis var. nevadensis Blaisdell, 1909: 402. Eleodes nigrina difformis Blaisdell, 1925 USA (ID OR WA) Eleodes nigrina difformis Blaisdell, 1925c: 389. Eleodes nigrina maclayi Boddy, 1957 USA (OR) Eleodes nigrina maclayi Boddy, 1957: 197. Eleodes nigrina nigrina LeConte, 1858 [Fig. 20 ] CAN (BC) USA (AZ CA CO ID KS ND NE NM NV OR TX UT) Figure 20. Eleodes ( Metablapylis ) nigrina nigrina LeConte, 1858. Scale bar = 1 mm. Eleodes nigrina LeConte, 1858c: 186. Eleodes nigrina perlonga Blaisdell, 1909 USA (ID WY) Eleodes nigrina var. perlonga Blaisdell, 1909: 398. Subgenus Omegeleodes Triplehorn and Thomas, 2012 Omegeleodes Triplehorn and Thomas, 2012: 253. Type species: Eleodes debilis LeConte, 1858, original designation. Eleodes debilis LeC4onte, 1858 USA (AZ NM TX) MEX (AG CH CO DU MI NL QU SI SL SO ZA) Eleodes debilis LeConte, 1858c: 185. Subgenus Promus LeConte, 1862 Promus LeConte, 1862a: 226. Type species: Blaps opaca Say, 1824, original designation. Eleodes anachronus Triplehorn, 2010 35 MEX (HI JA OA QU SL TA VE) Eleodes anachronus Triplehorn, 2010: 373. Eleodes bidens Triplehorn, 2007 MEX (DU) Eleodes bidens Triplehorn, 2007: 641. Eleodes brucei Triplehorn, 2007 MEX (DU ZA) Eleodes brucei Triplehorn, 2007: 638. Eleodes calcarata Champion, 1884 MEX (GU) Elaeodes calcarata Champion, 1884: 86. Eleodes composita Casey, 1891 USA (TX) Eleodes compositus Casey, 1891: 58. Eleodes erratica Champion, 1884 MEX (NA SI) Elaeodes erratica Champion, 1884: 87. Eleodes exarata Champion, 1884 MEX (SL) Elaeodes exarata Champion, 1884: 78. Eleodes fusiformis LeConte, 1858 USA (CO KS NE NM TX WY) Eleodes fusiformis LeConte, 1858c: 184. Eleodes goryi Solier, 1848 USA (NM TX) MEX (PU TA VE) Eleodes goryi Solier, 1848: 251. Eleodes seriata LeConte, 1858c: 185. Synonymy: Champion (1885 : 93). Eleodes hoegei Champion, 1885 MEX (PU VE) Elaeodes högei Champion, 1885: 91. Eleodes insularis Linell, 1899 MEX (BC BS) Eleodes insularis Linell, 1899: 181. Eleodes terricola Blaisdell, 1910: 61. 36 Synonymy: Triplehorn (1971 : 58). Eleodes knullorum Triplehorn, 1971 USA (AZ NM TX) MEX (CO HI) Eleodes knullorum Triplehorn, 1971: 56. Eleodes longicornis Champion, 1884 MEX (DU) Elaeodes longicornis Champion, 1884: 87. Eleodes madrensis Johnston, 2015 USA (AZ NM) MEX (SO) Eleodes madrensis Johnston, 2015: 14. Eleodes montana Champion, 1884 MEX (SL) Elaeodes montana Champion, 1884: 86. Eleodes opaca (Say, 1824) [Fig. 21 ] CAN (AB MB SK) USA (CO KS ND NE OK SD TX) Figure 21. Eleodes ( Promus ) opaca (Say, 1824). Scale bar = 1 mm. Blaps opaca Say, 1824a: 263. Eleodes spiculifera Triplehorn, 2007 USA (TX) Eleodes spiculiferus Triplehorn, 2007: 632. Eleodes spinolae Solier, 1848 MEX (CO FD GE GU HI ME MO OA PU VE) Eleodes spinolae Solier, 1848: 253. Eleodes striolata LeConte, 1858 USA (TX) MEX (CO) Eleodes striolata LeConte, 1858c: 185. Eleodes subnitens LeConte, 1851 USA (AZ) MEX (SO) Eleodes subnitens LeConte, 1851: 134. Eleodes subnitens forma sinuata Blaisdell, 1909: 163. Synonymy: Johnston (2015 : 14). Eleodes watrousi Triplehorn, 2007 MEX (DU) Eleodes watrousi Triplehorn, 2007: 640. Subgenus Pseudeleodes Blaisdell, 1909 Pseudeleodes Blaisdell, 1909: 146. Type species: Eleodes granosa LeConte, 1866, monotypy. Trichoderulus Blaisdell, 1923: 281. Type species: Trichoderulus longipilosus Blaisdell, 1923 (= Eleodes tribulus Thomas, 2005), original designation. Synonymy: Johnston (2016 : 672). Eleodes caudifera LeConte, 1858 USA (AZ CO NM TX UT) MEX (CH) Eleodes caudifera LeConte, 1858c: 184. Eleodes caudifera forma glabra Blaisdell, 1909: 228. Synonymy: Johnston (2016 : 671). Eleodes caudifera forma scabra Blaisdell, 1909: 228. Synonymy: Johnston (2016 : 671). Eleodes caudifera forma sublaevis Blaisdell, 1909: 228. Synonymy: Johnston (2016 : 671). Eleodes granosa LeConte, 1866 USA (CA NV OR) Eleodes granosa LeConte, 1866b: 116. Eleodes granosa forma fortis Blaisdell, 1909: 150. New synonymy [YB]. Eleodes granosa var. pilifera Boddy, 1957: 193. Synonymy: Johnston (2016 : 671). Eleodes inyoensis Tanner, 1961: 68. Synonymy: Johnston (2016 : 671). Eleodes inornata Johnston, 2016 USA (NV) Eleodes inornatus Johnston, 2016: 669. Eleodes leechi Tanner, 1961 USA (CO UT) Eleodes leechi Tanner, 1961: 63. Eleodes longipilosa Horn, 1891 USA (CA ID NV OR) Eleodes longipilosa Horn, 1891: 42. Eleodes pilosa Horn, 1870 USA (CA ID NM NV OR UT WA WY) Eleodes pilosa Horn, 1870: 314. Eleodes pilosa forma ordinata Blaisdell, 1909: 143. New synonymy [YB]. Eleodes obesus Doyen, 1985b: 232. Synonymy: Johnston (2016 : 673). Eleodes spoliata Blaisdell, 1933 USA (OR) Eleodes spoliata Blaisdell, 1933b: 196. Eleodes tribulus Thomas, 2005 USA (AZ) MEX (SO) Amphidora caudata Horn, 1870: 330 [junior secondary homonym of Eleodes caudata Solier, 1848]. Trichoderulus longipilosus Blaisdell, 1923: 281 [junior secondary homonym of Eleodes longipilosus Horn, 1891]. Synonymy: Triplehorn and Aalbu (1987 : 371). Eleodes blaisdelli Doyen [in Doyen and Lawrence], 1979: 367 [junior primary homonym of Eleodes blaisdelli Blackwelder, 1945]. Replacement name for Eleodes longipilosus Blaisdell, 1923. Eleodes tribulus Thomas, 2005: 552. Replacement name for Eleodes caudatus (Horn, 1870). Subgenus Tricheleodes Blaisdell, 1909 Tricheleodes Blaisdell, 1909: 138. Type species: Eleodes hirsuta LeConte, 1861, by subsequent designation ( Johnston 2016 : 666). Eleodes hirsuta LeConte, 1861 USA (CA NV UT) Eleodes hirsuta LeConte, 1861b: 352. Subgenus Xysta Eschscholtz, 1829 Xysta Eschscholtz, 1829: 9. Type species: Eleodes gravida Eschscholtz, 1829, subsequent designation ( Hope 1841 : 124). Status revised [ADS & MAJ]. Steneleodes Blaisdell, 1909: 409. Type species: Eleodes longicollis LeConte, 1851, present designation . New synonymy [ADS & MAJ]. Holeleodes Blaisdell, 1937b: 132. Type species: Eleodes beameri Blaisdell, 1937 (= Elaeodes hepburni Champion, 1884), original designation. Synonymy (with Steneleodes Blaisdell): Johnston (2015 : 12). Eleodes angulata (Eschscholtz, 1829) MEX (FD ME) Xysta angulata Eschscholtz, 1829: 9. Eleodes angusta Eschscholtz, 1829 MEX (DU FD GU HI JA ME MI OA PU VE) Eleodes angusta Eschscholtz, 1829: 13. Eleodes blapoides Eschscholtz, 1829 MEX (OA) Eleodes blapoides Eschscholtz, 1829: 12. Elaeodes blaptoides Champion, 1884: 78. Unjustified emendation of Eleodes blapoides Eschscholtz, 1829, not in prevailing usage. Eleodes coarctata Champion, 1885 MEX (ME PU TA) Elaeodes coarctata Champion, 1885: 91. Eleodes corrugans Triplehorn, 2007 MEX (MI) Eleodes corrugans Triplehorn, 2007: 630. Eleodes distincta Solier, 1848 MEX (HI OA PU QU SL TA VE) Eleodes distincta Solier, 1848: 239. Eleodes forreri Champion, 1884 MEX (CH DU) Elaeodes forreri Champion, 1884: 88. Eleodes gigantea Mannerheim, 1843 USA (CA) MEX (BC) Eleodes gigantea Mannerheim, 1843: 267. Eleodes gentilis LeConte, 1858c: 187. Synonymy: Triplehorn (1996 : 15). Eleodes estriatus Casey, 1890b: 398. Synonymy: Triplehorn (1996 : 15). Eleodes gigantea var. meridionalis Blaisdell, 1918c: 387. Synonymy: Triplehorn (1996 : 15). Eleodes glabricollis Champion, 1884 MEX (AG GU NL SL) Elaeodes glabricollis Champion, 1884: 85. Eleodes gravida (Eschscholtz, 1829) MEX (OA) Xysta gravida Eschscholtz, 1829: 9. Eleodes hepburni Champion, 1884 USA (AZ NM) MEX (CH CO DU JA SI SO) Elaeodes hepburni Champion, 1884: 88. Eleodes compressitarsis Blaisdell, 1935c: 158. Synonymy: Triplehorn (2010 : 376). Eleodes beameri Blaisdell, 1937b: 132. Synonymy: Triplehorn (2010 : 376). Eleodes bryanti Blaisdell, 1937b: 134. Synonymy (with E. beameri Blaisdell): Triplehorn and Doyen (1972 : 79). Eleodes palmerleensis Blaisdell, 1937b: 136. Synonymy (with E. beameri Blaisdell): Triplehorn and Doyen (1972 : 79). Eleodes innocens LeConte, 1866 MEX (BS) Eleodes innocens LeConte, 1866b: 114. Eleodes laevigata blapsoides Solier, 1848 MEX Eleodes laevigata var. blapsoïdes Solier, 1848: 244. Eleodes laevigata laevigata Solier, 1848 MEX (ME OA PU VE) GUA Eleodes laevigata Solier, 1848: 244. Eleodes longicollis LeConte, 1851 USA (AZ CO KS NM NV OR TX UT WY) MEX (AG CH CO DU MI NL SL SO ZA) Eleodes longicollis LeConte, 1851: 134. Eleodes haydenii LeConte, 1858c: 186. Synonymy: Horn (1870 : 311). Eleodes mutilata Blaisdell, 1921 MEX (BS) Eleodes mutilata Blaisdell, 1921b: 222. Eleodes olida Champion, 1892 MEX (GE) Elaeodes olida Champion, 1892: 516. Eleodes ornatipennis Blaisdell, 1937 USA (NM) MEX (CH) Eleodes ornatipennis Blaisdell, 1937b: 129. Eleodes peropaca Champion, 1892 MEX (DU) Elaeodes peropaca Champion, 1892: 517. Eleodes platypennis Triplehorn, 2007 MEX (JA) Eleodes platypennis Triplehorn, 2007: 637. Eleodes ponderosa Champion, 1884 MEX (OA PU) Elaeodes ponderosa Champion, 1884: 84. Eleodes punctigera Blaisdell, 1935 MEX (DU) Eleodes punctigera Blaisdell, 1935c: 157. Eleodes ruida (Say, 1835) MEX (MO PU VE) Blaps ruida Say, 1835: 183. Eleodes coriacea Solier, 1848: 249. Synonymy: Champion (1884 : 84). Eleodes sallaei Champion, 1885 MEX (GU JA OA PU QU SL VE) Elaeodes sallaei Champion, 1885: 89. Eleodes solieri Champion, 1885 MEX (CH CO GU OA PU SL VE) Blaps celsa Say, 1835: 185 [ nomen dubium ]. Elaeodes solieri Champion, 1885: 89. Synonymy (in doubt): Champion (1885 : 89). Eleodes stolida Champion, 1885 MEX Elaeodes stolida Champion, 1885: 92. Eleodes sulcatula Champion, 1884 MEX (ME) Elaeodes sulcatula Champion, 1884: 83. Eleodes tenebricosa Gemminger, 1870 MEX (ME OA) Eleodes obscura Solier, 1848: 245 [junior secondary homonym of Eleodes obscurus (Say, 1824)]. Elaeodes tenebricosa Gemminger, 1870: 122. Replacement name for Elaeodes obscura Solier, 1848. Eleodes tessellata Champion, 1892 MEX (MI) Elaeodes tessellata Champion, 1892: 517. [incertae sedis] Eleodes aequalis (Say, 1835) MEX (DU ME OA PU) Blaps aequalis Say, 1835: 185. Eleodes alutacea Solier, 1848: 240. Synonymy (in doubt): Champion (1884 : 80). Eleodes maillei Solier, 1848: 247. Synonymy (with E. alutacea Solier): Champion (1884 : 80). Eleodes amaura Champion, 1892 MEX (GU HI OA PU TA) Elaeodes amaura Champion, 1892: 514. 37 Eleodes barbata Wickham, 1918 USA (AZ CO NM UT) Eleodes barbata Wickham, 1918: 256. Eleodes brevicollis Gemminger, 1870 MEX Eleodes obsoleta Solier, 1848: 238 [junior secondary homonym of Eleodes obsoleta (Say, 1823)]. Eleodes brevicollis Gemminger [in Gemminger and Harold], 1870: 1868. Replacement name for Eleodes obsoleta Solier, 1848. Eleodes cylindrica (Herbst, 1799) "Nordamerika" Blaps cylindrica Herbst, 1799: 185. Eleodes dilaticollis Champion, 1884 MEX (GU HI ME MI PU) Elaeodes dilaticollis Champion, 1884: 83. Eleodes ebenina (Solier, 1848) MEX Nycterinus ebeninus Solier, 1848: 269. Eleodes elongatula Eschscholtz, 1829 MEX Eleodes elongatula Eschscholtz, 1829: 13. Eleodes impolita (Say, 1835) MEX (ME OA PU VE) Blaps impolita Say, 1835: 183. Eleodes aubei Solier, 1848: 245. Synonymy: Champion (1885 : 90). Eleodes maura (Say, 1835) MEX (GU OA PU) Blaps maura Say, 1835: 184. Eleodes melanaria Eschscholtz, 1829 MEX Eleodes melanaria Eschscholtz, 1829: 13. Eleodes obliterata (Say, 1835) MEX Blaps obliterata Say, 1835: 184. Eleodes polita Champion, 1892 MEX (ME MO) Elaeodes polita Champion, 1892: 513. Eleodes rotundicollis (Eschscholtz, 1829) MEX (PU SL VE) Xysta rotundicollis Eschscholtz, 1829: 9. Blaps parva Say, 1835: 186. Synonymy (in doubt): Champion (1884 : 82). Eleodes scapularis Champion, 1884 MEX (GU ME) Elaeodes scapularis Champion, 1884: 81. Eleodes segregata Champion, 1892 MEX (CH DU GE MI) Elaeodes segregata Champion, 1892: 513. Eleodes striata (Guérin-Méneville, 1834) MEX (TA) Xysta striata Guérin-Méneville, 1834: 30. Eleodes sulcata (Eschscholtz, 1829) MEX (MO) Xysta sulcata Eschscholtz, 1829: 9. Genus Eleodimorpha Blaisdell, 1909 [F] Eleodimorpha Blaisdell, 1909: 477. Type species: Eleodimorpha bolcan Blaisdell, 1909, original designation. Eleodimorpha bolcan Blaisdell, 1909 USA (CA) Eleodimorpha bolcan Blaisdell, 1909: 479. Genus Embaphion Say, 1824 [N] Embaphion Say, 1824a: 254. Type species: Akis muricata Say, 1824, monotypy. Embaphion contractum blaisdelli Benedict, 1927 USA (NM) Embaphion contractum blaisdelli Benedict, 1927: 46. Embaphion contractum contractum Blaisdell, 1909 USA (NM) Embaphion contractum Blaisdell, 1909: 460. Embaphion contusum contusum LeConte, 1858 USA (AZ CO KS NM WY) Embaphion contusum LeConte, 1858a: 20. Embaphion contusum grande Blaisdell, 1909 USA (NM) Embaphion contusum forma grandis Blaisdell, 1909: 471. Embaphion contusum laminatum Casey, 1890 USA (TX) Embaphion laminatum Casey, 1890b: 403. Embaphion depressum (LeConte, 1851) USA (CA) Eleodes depressa LeConte, 1851: 136. Embaphion elongatum Horn, 1870 USA (CA ID NV OR UT) Embaphion elongatum Horn, 1870: 321. Embaphion glabrum Blaisdell, 1909 USA (AZ NM UT) Embaphion glabrum Blaisdell, 1909: 457. Embaphion mexicanum Blaisdell, 1935 MEX (CH) Embaphion mexicanum Blaisdell, 1935c: 160. Embaphion muricatum (Say, 1824) [Fig. 22 ] CAN (AB SK) USA (CO KS NE SD TX) MEX (TA) Figure 22. Embaphion muricatum (Say, 1824). Scale bar = 1 mm. Akis muricata Say, 1824a: 253. Embaphion concavum LeConte, 1853: 446. Synonymy: Horn (1870 : 320). Embaphion planum Horn, 1870 USA (CO KS NM UT WY) Embaphion planum Horn, 1870: 321. Genus Lariversius Blaisdell, 1947 [M] Lariversius Blaisdell, 1947: 59. Type species: Lariversius tibialis Blaisdell, 1947, original designation. Lariversius tibialis Blaisdell, 1947 USA (NV) Lariversius tibialis Blaisdell, 1947: 61. Genus Neobaphion Blaisdell, 1925 [N] Neobaphion Blaisdell, 1925c: 390. Type species: Eleodes planipennis LeConte, 1866, monotypy. Neobaphion alleni Triplehorn, 1989 USA (ID OR) Neobaphion alleni Triplehorn, 1989: 458. Neobaphion elongatum Blaisdell, 1933 USA (CA NV) Neobaphion elongatum Blaisdell, 1933b: 208. Neobaphion papula Triplehorn and Aalbu, 1985 USA (NV) Neobaphion papula Triplehorn and Aalbu, 1985 [11 July]: 588. Eleodes insolitus Doyen, 1985b [11 July]: 230. Synonymy: Triplehorn (1989 : 460). Neobaphion planipenne (LeConte, 1866) USA (AZ CO NM UT) Eleodes planipennis LeConte, 1866b: 116. Genus Trogloderus LeConte, 1879 [M] Trogloderus LeConte, 1879a: 2. Type species: Trogloderus costatus LeConte, 1879, monotypy. Trogloderus costatus LeConte, 1879 USA (CA ID NV) Trogloderus costatus LeConte, 1879a: 3. Trogloderus nevadus La Rivers, 1943 USA (ID NV) Status revised [MAJ] Trogloderus nevadus La Rivers, 1943a: 437. Trogloderus tuberculatus Blaisdell, 1909 USA (CA) Status revised [MAJ] Trogloderus tuberculatus Blaisdell, 1909: 490. Trogloderus costatus pappi Kulzer, 1960: 310. New synonymy [MAJ] Trogloderus vandykei La Rivers, 1946 USA (AZ CA) Status revised [MAJ] Trogloderus costatus vandykei La Rivers, 1946: 41. Trogloderus costatus mayhewi Papp, 1961a: 33. New synonymy [MAJ] Tribe Apocryphini Lacordaire, 1859 Apocryphides Lacordaire, 1859: 432. Type genus: Apocrypha Eschscholtz, 1831. Genus Apocrypha Eschscholtz, 1831 [F] Apocrypha Eschscholtz, 1831: 13. Type species: Apocrypha anthicoides Eschscholtz, 1831, monotypy. Compsomorphus Solier, 1851: 208. Type species: Compsomorphus elegans Solier, 1851, monotypy. Synonymy: Lacordaire (1859 : 433). Apocrypha anthicoides Eschscholtz, 1831 USA (CA) Apocrypha anthicoides Eschscholtz, 1831: 13. Apocrypha dyschirioides LeConte, 1851: 137. Synonymy: Doyen and Kitayama (1980 : 122). Apocrypha clivinoides Horn, 1870 USA (CA) Apocrypha clivinoides Horn, 1870: 391. Apocrypha setosa Doyen and Kitayama, 1980 USA (CA) Apocrypha setosa Doyen and Kitayama, 1980: 126. Genus Pseudapocrypha Champion, 1886 [F] Pseudapocrypha Champion, 1886: 260. Type species: Pseudapocrypha lacordairii Champion, 1886, monotypy. Pseudapocrypha lacordairii Champion, 1886 MEX (CI) GUA Pseudapocrypha lacordairii Champion, 1886: 260. Tribe Blaptini Leach, 1815 Blapsida Leach, 1815: 101. Type genus: Blaps Fabricius, 1775. Subtribe Blaptina Leach, 1815 Blapsida Leach, 1815: 101. Type genus: Blaps Fabricius, 1775. Genus Blaps Fabricius, 1775 [F] Blaps Fabricius, 1775: 254. Type species: Tenebrio mortisagus Linnaeus, 1758, subsequent designation ( Latreille 1810 : 429). Subgenus Blaps Fabricius, 1775 Blaps Fabricius, 1775: 254. Type species: Tenebrio mortisagus Linnaeus, 1758, subsequent designation ( Latreille 1810 : 429). Blaps lethifera lethifera Marsham, 1802 CAN (QC) USA (IN MD NJ NY OH VA) – Adventive Blaps lethifera Marsham, 1802: 479. Blaps similis Latreille, 1804: 279. Synonymy: Seidlitz (1893 : 317). Blaps mucronata Latreille, 1804 USA (MD NY OH) – Adventive Blaps mucronata Latreille, 1804: 278. Tribe Bolitophagini Kirby, 1837 Eledonaedes Billberg, 1820b: 392 [ nomen oblitum , see Bouchard et al. 2011 ]. Type genus: Eledona Latreille, 1797. Bolitophagidae Kirby, 1837: 236 [ nomen protectum ]. Type genus: Bolitophagus Illiger, 1798. Rhipidandri LeConte, 1862a: 236. Type genus: Rhipidandrus LeConte, 1862. Eutomides Lacordaire, 1865: 369. Type genus: Eutomus Lacordaire, 1865 (= Rhipidandrus LeConte, 1862). Genus Bolitophagus Illiger, 1798 [M] Bolitophagus Illiger, 1798: 100. Type species: Silpha reticulata Linnaeus, 1767, subsequent designation (C.G. Thomson 1859 : 115). Boletophagus Agassiz, 1846: 48. Unjustified emendation of Bolitophagus Illiger, 1798, not in prevailing usage. Bolitophagus corticola Say, 1826 CAN (NB NS ON PE QC) USA (CT DC FL GA IN MA MD ME MI MO NC NH NJ NY OH PA SC TN TX VA WI) Boletophagus corticola Say, 1826: 238. Genus Bolitotherus Candèze, 1861 [M] Bolitotherus Candèze, 1861: 367. Type species: Bolitophagus cornutus Fabricius, 1801, subsequent designation ( LeConte 1862b : 236). Phellidius LeConte, 1862a: 236. Type species: Bolitophagus cornutus Fabricius, 1801, original designation. Synonymy: LeConte (1866a : 62). Bolitotherus cornutus (Fabricius, 1801) 38 [Fig. 23 ] CAN (AB MB NB NS ON PE QC SK) USA (CT FL GA IA IL IN KY LA MA MD ME MI MN MS NC NE NH NJ NY OH PA RI SC TN TX VAVT WI) Figure 23. Bolitotherus cornutus (Fabricius, 1801). Scale bar = 1 mm. Opatrum bifurcum Fabricius, 1798: 40. Bolitophagus cornutus Fabricius, 1801a: 112. Synonymy: Fabricius (1801a : 113). Bolitophagus cristatus Gosse, 1840: 251. New synonymy [YB]. Genus Eleates Casey, 1886 [M] Eleates Casey, 1886: 253. Type species: Eleates occidentalis Casey, 1886, monotypy. Eleates depressus (Randall, 1838) CAN (BC MB NB NT ON QC SK) USA (AR GA MD ME MI NH NYOH OR PA TN VAVT WA WI) Eledona depressa Randall, 1838 [February]: 21. Bolitophagus tetraopes Newman, 1838 [April]: 378. Synonymy: LeConte (1854b : 219). Eleates explanatus Casey, 1890b: 486. New synonymy [YB]. Eleates occidentalis Casey, 1886 USA (CA) Eleates occidentalis Casey, 1886: 254. Genus Megeleates Casey, 1895 [M] Megeleates Casey, 1895: 623. Type species: Megeleates sequoiarum Casey, 1895, monotypy. Megeleates sequoiarum Casey, 1895 USA (CA OR WA) Megeleates sequoiarum Casey, 1895: 624. Genus Rhipidandrus LeConte, 1862 [M] Rhipidandrus LeConte, 1862a: 236. Type species: Xyletinus flabellicornis Sturm, 1826 (= Melolontha paradoxa Palisot de Beauvois, 1818), monotypy. Eutomus Lacordaire, 1865: 369. Type species: Eutomus micrographus Lacordaire, 1865, subsequent designation ( Barber 1914 : 191). Synonymy: LeConte and Horn (1883 : 232). Heptaphylla Friedenreich, 1883: 375. Type species: Heptaphylla fungicola Friedenreich, 1883, monotypy. Synonymy: Arrow (1904 : 31). Cherostus C.O. Waterhouse, 1894: 68. Type species: Cherostus walkeri Waterhouse, 1894, subsequent designation ( Merkl and Kompantzeva 1996 : 91). Synonymy: Gebien (1939 : 762). Rhipidandrus championi Sharp, 1905 GUA PAN Rhipidandrus championi Sharp, 1905: 691. Rhipidandrus cornutus (Arrow, 1904) MEX (DU OA) / HIS PRI LAN / SA Cherostus cornutus Arrow, 1904: 31. Rhipidandrus fulvomaculatus Dury, 1914 USA (FL) / BAH Rhipidandrus fulvomaculata Dury, 1914: 168. Rhipidandrus jamaicensis (Arrow, 1904) JAM Cherostus jamaicensis Arrow, 1904: 32. Rhipidandrus mexicanus Sharp, 1905 MEX (VE) GUA BEL Rhipidandrus mexicanus Sharp, 1905: 691. Rhipidandrus micrographus (Lacordaire, 1865) PRI LAN / SA Eutomus micrographus Lacordaire, 1865: 370. Rhipidandrus panamaensis (Barber, 1914) PAN Eutomus panamaensis Barber, 1914: 193. Rhipidandrus paradoxus (Palisot de Beauvois, 1818) [Fig. 24 ] CAN (ON QC) USA (DC FL GA IN KS KY LA MD MI NC NY OH SC TX VA WI) Figure 24. Rhipidandrus paradoxus (Palisot de Beauvois, 1818). Scale bar = 1 mm. Melolontha paradoxa Palisot de Beauvois, 1818: 173. Xylotinus flabellicornis Sturm, 1826: 59. Synonymy: LeConte (1873 : 329, 335). Rhipidandrus peninsularis Horn, 1894 USA (AZ TX) MEX (BS) Rhipidandrus peninsularis Horn, 1894b: 392. Rhipidandrus sulcatus (Gorham, 1898) BAH CUB CAY HIS LAN Eutomus sulcatus Gorham, 1898: 333. Tribe Centronopini Doyen, 1989 Centronopini Doyen, 1989: 284. Type genus: Centronopus Solier, 1848. Genus Centronopus Solier, 1848 [M] Centronopus Solier, 1848: 258. Type species: Centronopus extensicollis Solier, 1848 (= Tenebrio suppressus Say, 1835), original designation. Subgenus Centronopus Solier, 1848 Centronopus Solier, 1848: 258. Type species: Centronopus extensicollis Solier, 1848 (= Tenebrio suppressus Say, 1835), original designation. Centronopus grandicollis Champion, 1885 MEX (DU FD HI ME MO SL TA VE) Centronopus grandicollis Champion, 1885: 100. Centronopus suppressus (Say, 1835) MEX (FD HI ME MO PU SL TA VE) Tenebrio suppressus Say, 1835: 187. Centronopus extensicollis Solier, 1848: 260. Synonymy: Lacordaire (1859 : 361). Subgenus Menechides Motschulsky, 1872 Menechides Motschulsky, 1872: 26. Type species: Helops calcaratus Fabricius, 1798, original designation. Scotobates Rye, 1877: 341. Type species: Helops calcaratus Fabricius, 1798, subsequent designation ( Lucas 1920 : 587). Synonymy: Lucas (1920 : 403). Pyres Champion, 1885: 100. Type species: Pyres metallicus Champion, 1885 (= Centronopus speciosus Pascoe, 1883), subsequent designation ( Gebien 1941 : 336). Synonymy: Spilman (1962a : 3). Centronopus batesi (Champion, 1885) PAN / SA Pyres batesi Champion, 1885: 101. Centronopus beardsleyi Spilman, 1962 MEX (CL JA) Centronopus beardsleyi Spilman, 1962a: 11. Centronopus bimaculatus Champion, 1892 MEX (VE YU) BEL Centronopus bimaculatus Champion, 1892: 521. Centronopus calcaratus (Fabricius, 1798) CAN (NS ON QC) USA (AL CT DC FL GA IA IL IN KS LA MA MD ME MI MO MS NC NE NH NJ NY OH PA SC TN VA VT WI WV) Tenebrio aeneus DeGeer, 1775: 53 [junior primary homonym of Tenebrio aeneus Scopoli, 1763] [ nomen dubium ]. Helops calcaratus Fabricius, 1798: 52. Synonymy (in doubt): LeConte (1866a : 61). Tenebrio coracinus Knoch, 1801: 172. Synonymy: LeConte (1866a : 61). Helops caroliniensis Palisot de Beauvois, 1817: 162. Synonymy (in doubt): LeConte (1866a : 61). Tenebrio reflexus Say, 1825: 203. Synonymy: Melsheimer (1853 : 139). Centronopus nigrofasciatus (Gebien, 1928) CRI Pyres nigrofasciatus Gebien, 1928b: 182. Centronopus opacus LeConte, 1859 USA (AR KS MT OK SD TX) Centronopus opacus LeConte, 1859a: 15. Centronopus speciosus Pascoe, 1883 NIC CRI Centronopus speciosus Pascoe, 1883: 439. Pyres metallicus Champion, 1885: 101. Synonymy: Champion (1892 : 521). Genus Scotobaenus LeConte, 1859 [M] Scotobaenus LeConte, 1859b: 87. Type species: Scotobaenus parallelus LeConte, 1859, monotypy. Scotobaenus parallelus LeConte, 1859 USA (CA OR WA) MEX Scotobaenus parallelus LeConte, 1859b: 88. Scotobaenus punctatus (Blaisdell, 1933) USA (CA) Centronopus punctatus Blaisdell, 1933c: 220. Scotobaenus simplex (Blaisdell, 1937) USA (CA) Centronopus simplex Blaisdell, 1937a: 95. Scotobaenus wagneri (Blaisdell, 1933) USA (CA) Centronopus wagneri Blaisdell, 1933c: 218. Genus Tauroceras Hope, 1841 [N] Tauroceras Hope, 1841: 130. Type species: Tenebrio cornutus Fabricius, 1775, original designation. Tauroceropedus Pic, 1913b: 4. Type species: Tauroceropedus difformipes Pic, 1913, subsequent designation ( Gebien 1941 : 344). Synonymy: Ferrer et al. (2005 : 272). Tauroceras barclayi Ferrer, Soldati and Delatour, 2005 JAM Tauroceras barclayi Ferrer, Soldati and Delatour, 2005: 284. Tauroceras cornutum (Fabricius, 1775) CUB JAM Tenebrio cornutus Fabricius, 1775: 256. Tauroceras girardi Ferrer, Soldati and Delatour, 2005 MEX (QR) GUA HON NIC CRI Tauroceras girardi Ferrer, Soldati and Delatour, 2005: 288. Tauroceras mulata Zayas, 1988 CUB Tauroceras mulata Zayas, 1988: 94. Tribe Cerenopini Horn, 1870 Cerenopi Horn, 1870: 323. Type genus: Cerenopus LeConte, 1851. Genus Argoporis Horn, 1870 [F] Argoporis Horn, 1870: 325. Type species: Cerenopus costipennis LeConte, 1851, subsequent designation ( Gebien 1937 : 797). Threnus Motschulsky, 1870: 404. Type species: Threnus niger Motschulsky, 1870, original designation. Synonymy: Aalbu et al. (1995 : 483) 39 . Argoporis aequalis Blaisdell, 1923 MEX (SO) Argoporis aequalis Blaisdell, 1923: 259. Argoporis alutacea Casey, 1890 USA (AZ) MEX (SO) Argoporis alutacea Casey, 1890b: 406. Argoporis labialis Blaisdell, 1923: 258. Synonymy: Berry (1980 : 18). Argoporis angusta Casey, 1924: 331. Synonymy: Berry (1980 : 18). Argoporis hebes Casey, 1924: 332. Synonymy: Berry (1980 : 18). Argoporis tibialis Casey, 1924: 332. Synonymy: Berry (1980 : 18). Argoporis apicalis apicalis Blaisdell, 1943 MEX (BC BS) Argoporis apicalis Blaisdell, 1943: 234. Argoporis insularis Berry, 1980: 54. Synonymy: Sánchez Piñero and Aalbu (2002 : 132). Argoporis apicalis californica Berry, 1980 USA (AZ) MEX (BC) Argoporis apicalis californica Berry, 1980: 22. Argoporis atripes Horn, 1870 MEX (AG DU GU HI JA MI SI SL SO) Argoporis atripes Horn, 1870: 325. Argoporis bicolor (LeConte, 1851) USA (AZ CA) MEX (SO) Cerenopus bicolor LeConte, 1851: 143. Argoporis tuckeri Casey, 1924: 332. Synonymy: Berry (1980 : 27). Argoporis brevicollis Champion, 1885 MEX (DU SI) Argoporis brevicollis Champion, 1885: 94. Argoporis carinata Berry, 1980 USA (AZ) MEX (CH NA SI SO) Argoporis carinata Berry, 1980: 30. Argoporis cavifrons Champion, 1885 MEX (DU SI) Argoporis cavifrons Champion, 1885: 95. Argoporis colimensis Berry, 1980 MEX (CL) Argoporis colimensis Berry, 1980: 34. Argoporis costipennis (LeConte, 1851) USA (AZ NM) MEX (SO) Cerenopus costipennis LeConte, 1851: 143. 40 Argoporis lateralis Casey, 1924: 331. Synonymy: Berry (1980 : 38). Argoporis costulata (Horn, 1870) MEX (BC BS) Cerenopus costulatus Horn, 1870: 326. Argoporis craigi Berry, 1980 MEX (CH DU) Argoporis craigi Berry, 1980: 42. Argoporis crassicornis Champion, 1885 MEX (DU NA SI) Argoporis crassicornis Champion, 1885: 94. Argoporis cribrata (LeConte, 1861) MEX (BS) Cerenopus cribratus LeConte, 1861a: 337. Argoporis deltodonta Berry, 1980 MEX (Tres Marias Islands) Argoporis deltodonta Berry, 1980: 46. Argoporis durangoensis Berry, 1980 MEX (CH DU) Argoporis durangoensis Berry, 1980: 47. Argoporis ebenina Horn, 1894 MEX (BS) Argoporis ebenina Horn, 1894b: 424. Argoporis estebanensis Berry, 1980 MEX (BS) Argoporis estebanensis Berry, 1980: 50. Argoporis impressa Blaisdell, 1925 MEX (BC BS) Argoporis impressa Blaisdell, 1925b: 330. Argoporis inconstans Horn, 1894 MEX (BC BS) Argoporis inconstans Horn, 1894b: 425. Argoporis laevicollis Champion, 1892 MEX (DU SI) Argoporis laevicollis Champion, 1892: 520. Argoporis longipes Blaisdell, 1923 MEX (BS) Argoporis longipes Blaisdell, 1923: 260. Argoporis nigra inflata Berry, 1980 MEX (BS) Argoporis constanzae inflata Berry, 1980: 37. Argoporis nigra nigra (Motschulsky, 1870) MEX (BS) Threnus niger Motschulsky, 1870: 406 41 . Argoporis constanzae constanzae Berry, 1980: 35. Synonymy: Aalbu et al. (1995 : 483). Argoporis obregonensis Berry, 1980 MEX (SO) Argoporis obregonensis Berry, 1980: 58. Argoporis regalis Berry, 1980 MEX (BS) Argoporis regalis Berry, 1980: 59. Argoporis rufipes femorata Berry, 1980 MEX (CH DU GU SI SL ZA) Argoporis rufipes femorata Berry, 1980: 63. Argoporis rufipes nitida Casey, 1890 USA (AZ NM TX) MEX (CH CO) Argoporis nitida Casey, 1890b: 406. Argoporis rufipes rufipes Champion, 1885 MEX (AG CH CO SL SO ZA) Argoporis rufipes Champion, 1885: 94. Argoporis tridentata Champion, 1892 MEX (CL GE JA MI) Argoporis tridentata Champion, 1892: 519. Argoporis unicalcarata Champion, 1892 MEX (AG JA NA SO) Argoporis unicalcarata Champion, 1892: 519. Genus Cerenopus LeConte, 1851 [M] Cerenopus LeConte, 1851: 143 42 . Type species: Cerenopus concolor LeConte, 1851, subsequent designation ( Lucas 1920 : 173). Cerenopus angustatus Horn, 1894 MEX (BS) Cerenopus angustatus Horn, 1894b: 426. Cerenopus aterrimus Horn, 1894 MEX (BS) Cerenopus aterrimus Horn, 1894b: 425. Cerenopus concolor LeConte, 1851 USA (AZ CA NV) MEX (BC BS) Cerenopus concolor LeConte, 1851: 143. Cerenopus hermanus Berry, 1975 MEX (BS) Cerenopus hermanus Berry, 1975: 931. Cerenopus punctatus Berry, 1975 MEX (BS) Cerenopus punctatus Berry, 1975: 932. Tribe Eulabini Horn, 1870 Eulabes Horn, 1870: 323. Type genus: Eulabis Eschscholtz, 1829. Genus Apsena LeConte, 1862 [F] Apsena LeConte, 1862a: 228. Type species: Eulabis pubescens LeConte, 1851, original designation. Apsena barbarae Blaisdell, 1932 USA (CA) Apsena barbarae Blaisdell, 1932b: 61. Apsena grossa (LeConte, 1866) USA (CA) Eulabis grossa LeConte, 1866b: 118. Apsena insularis Blaisdell, 1932 MEX (BC) Apsena insularis Blaisdell, 1932b: 58. Apsena laticornis laticornis (Casey, 1891) USA (CA) Eulabis laticornis Casey, 1891: 60. Apsena labreae Pierce, 1954b: 98. Synonymy: Doyen and Miller (1980 : 2). Apsena laticornis subvestita Blaisdell, 1932 USA (CA) Apsena laticornis var. subvestita Blaisdell, 1932b: 68. Apsena leachi Blaisdell, 1932 USA (CA) Apsena leachi Blaisdell, 1932b: 70. Apsena pubescens pubescens (LeConte, 1851) USA (CA) Eulabis pubescens LeConte, 1851: 143. Eulabis crassicornis Casey, 1890b: 404. Synonymy: Blaisdell (1925a : 83). Apsena pubescens rufescens Blaisdell, 1932 MEX (BC) Apsena pubescens rufescens Blaisdell, 1932b: 56. Apsena rufipes opaca Blaisdell, 1932 USA (CA) Apsena rufipes var. opaca Blaisdell, 1932b: 81. Apsena rufipes rufipes (Eschscholtz, 1829) USA (CA) Eulabis rufipes Eschscholtz, 1829: 15. Eulabis montana Casey, 1924: 330. Synonymy: Blaisdell (1925a : 84). Apsena rufipes simplex Blaisdell, 1932 USA (CA) Apsena rufipes simplex Blaisdell, 1932b: 84. Genus Epantius LeConte, 1851 [M] Epantius LeConte, 1851: 144. Type species: Epantius obscurus LeConte, 1851, monotypy. Epantius obscurus LeConte, 1851 USA (CA) MEX (BC) Epantius obscurus LeConte, 1851: 144. Genus Eulabis Eschscholtz, 1829 [F] Eulabis Eschscholtz, 1829: 14. Type species: Eulabis bicarinata Eschscholtz, 1829, subsequent designation ( Blaisdell 1932b : 44). Eulabis bicarinata Eschscholtz, 1829 USA (CA) Eulabis bicarinata Eschscholtz, 1829: 15. Tribe Helopini Latreille, 1802 Helopii Latreille, 1802: 176. Type genus: Helops Fabricius, 1775. Genus Helops Fabricius, 1775 43 [M] Helops Fabricius, 1775: 257. Type species: Tenebrio caeruleus Linnaeus, 1758, subsequent designation ( Hope 1841 : 133) (see ICZN 2009 ) 44 . Stenotrichus LeConte, 1862a: 239. Type species: Amphidora rufipes LeConte, 1851, original designation. Synonymy: Aalbu et al. (2002 : 496). Biomorphus Motschulsky, 1872: 38. Type species: Biomorphus tuberculatus Motschulsky, 1872 (= Amphidora attenuata LeConte, 1851), original designation. Synonymy: Aalbu et al. (1995 : 485). Coscinoptilix Allard, 1876: 15 [as Coscinopter ] 45 . Type species: Coscinoptilix gracilicornis Allard, 1876, monotypy. Synonymy: Champion (1887 : 312). Helops angustus LeConte, 1859 USA (CA) Helops angustus LeConte, 1859b: 77. Helops arizonensis Horn, 1874 USA (AZ NM) Helops arizonensis Horn, 1874a: 36. Helops attenuatus (LeConte, 1851) USA (CA NV) Amphidora attenuata LeConte, 1851: 136. Biomorphus tuberculatus Motschulsky, 1872: 40. Synonymy: Aalbu et al. (1995 : 485). Helops bachei LeConte, 1861 USA (CA) Helops bachei LeConte, 1861b: 353. Helops benitensis Blaisdell, 1925 MEX (BC) Helops benitensis Blaisdell, 1925b: 339. Helops blaisdelli Casey, 1891 USA (CA) Helops blaisdelli Casey, 1891: 66. Helops blandi Bousquet and Bouchard, 2012 CAN (NB) USA (MD NJ NY SC VA) Helops gracilis Bland, 1864: 319 [junior primary homonym of Helops gracilis Fischer von Waldheim, 1823]. Helops blandi Bousquet and Bouchard [in Nabozhenko et al.], 2012 : 729. Replacement name for Helops gracilis Bland, 1864. Helops callosus Casey, 1890 USA (NM) Helops callosa Casey, 1890b: 489. Helops cavifrons Champion, 1887 GUA Helops cavifrons Champion, 1887: 313. Helops cisteloides Germar, 1823 USA (AR FL GA LA MD MO NC NJ OH SC TX VA) Helops cisteloides Germar, 1823: 159. Helops confluens (Casey, 1924) USA (CA) Status revised [RLA] Stenotrichus confluens Casey, 1924: 329. Helops coxalis Champion, 1887 MEX (MI) Helops coxalis Champion, 1887: 317. Helops crockeri Blaisdell, 1933 MEX (BC [Guadalupe Is.]) Helops crockeri Blaisdell, 1933a: 89. Helops cupripennis Champion, 1887 MEX (OA) Helops cupripennis Champion, 1887: 319. Helops cylindriformis Casey, 1891 USA (NM) Helops cylindriformis Casey, 1891: 68. Helops difficilis Horn, 1878 USA (CO WY) Helops difficilis Horn, 1878a: 57. Helops discipulus Casey, 1891 USA (CA) Helops discipula Casey, 1891: 67. Helops discretus LeConte, 1866 USA (TX) Helops discretus LeConte, 1866b: 134. Helops edwardsii Horn, 1870 USA (CA OR WA) Helops edwardsii Horn, 1870: 395. Helops enitescens Champion, 1893 GUA Helops enitescens Champion, 1893a: 557. Helops exsculptus Champion, 1887 GUA Helops exsculptus Champion, 1887: 314. Helops farctus LeConte, 1858 USA (TX) Helops farcta LeConte, 1858b: 74. Helops fresnoensis Blaisdell, 1931 USA (CA) Helops fresnoënsis Blaisdell, 1931: 44. Helops gracilicornis (Allard, 1876) MEX (VE) Coscinoptilix gracilicornis Allard, 1876: 52. Helops guadalupensis Casey, 1890 MEX (BC [Guadalupe Is.]) Helops guadalupensis Casey, 1890b: 488. Helops impolitus LeConte, 1866 USA (TX) Helops impolitus LeConte, 1866b: 132. Helops inanis (Allard, 1877) MEX (MO PU) Tarpela inanis Allard, 1877b: 262. Helops funebris Champion, 1887: 316. Synonymy: Champion (1893a : 556). Helops laetus LeConte, 1857 CAN (BC) USA (CA OR WA) Helops laetus LeConte, 1857: 50. Helops longicornis Champion, 1887 MEX (DU) Helops longicornis Champion, 1887: 314. Helops noguerai Doyen, 1990 MEX (JA) Helops noguerai Doyen, 1990: 236. Helops obtusangulus Blaisdell, 1921 USA (CA) Helops obtusangula Blaisdell, 1921b: 228. Helops opacus LeConte, 1859 USA (CA ID NV OR UT) Helops opacus LeConte, 1859c: 284. Helops panamensis Champion, 1887 PAN Helops panamensis Champion, 1887: 319. Helops perforatus Horn, 1880 USA (TX) Helops perforatus Horn, 1880: 153. Helops pernitens LeConte, 1861 [Fig. 25 ] CAN (BC) USA (CA OR WA) Figure 25. Helops pernitens LeConte, 1861. Scale bar = 1 mm. Helops pernitens LeConte, 1861b: 353. Helops pinguis Horn, 1894 MEX (BS) Helops pinguis Horn, 1894b: 430. Helops politus Say, 1826 USA (FL) Helops politus Say, 1826: 240. Helops pueblensis Champion, 1887 MEX (GE PU) Helops pueblensis Champion, 1887: 317. Helops punctatostriatus Champion, 1887 MEX Helops punctato-striatus Champion, 1887: 316. Helops punctatus Gemminger, 1870 USA (CA) Helops punctipennis LeConte, 1866b: 133 [junior primary homonym of Helops punctipennis Lucas, 1846]. Helops punctatus Gemminger, 1870: 123 [junior primary homonym of Helops punctatus Fabricius, 1801] 46 . Replacement name for Helops punctipennis LeConte, 1866. Helops punctiventris Champion, 1887 MEX (GU) Helops punctiventris Champion, 1887: 320. Helops rastratus Champion, 1893 MEX (CH) Helops rastratus Champion, 1893a: 557. Helops rufipes (LeConte, 1851) USA (CA) Amphidora rufipes LeConte, 1851: 136. Amphidora parallela Casey, 1924: 328. Synonymy: Blaisdell (1933b : 210). Helops rugiceps Champion, 1887 GUA Helops rugiceps Champion, 1887: 315. Helops rugicollis LeConte, 1866 USA (CA) Helops rugicollis LeConte, 1866b: 133. Helops rugulosus LeConte, 1851 USA (CA) Helops rugulosus LeConte, 1851: 151. Helops scintillatus Doyen, 1990 MEX (JA NA) Helops scintillatus Doyen, 1990: 239. Helops seriatoporus Champion, 1893 MEX (CH) Helops seriatoporus Champion, 1893a: 558. Helops seriatus (Allard, 1877) USA (CA) Catomus seriatus Allard, 1877b: 46. Helops simulator Blaisdell, 1921 USA (CA OR) Helops simulator Blaisdell, 1921b: 226. Helops sparsus Blaisdell, 1943 MEX (BC) Helops sparsus Blaisdell, 1943: 274. Helops spiethi Pallister, 1954 MEX (DU) Helops spiethi Pallister, 1954: 50. Helops spilmani Pallister, 1954 MEX (CH DU) Helops spilmani Pallister, 1954: 49. Helops spissicornis Champion, 1893 MEX (DU) Helops spissicornis Champion, 1893a: 558. Helops spretus Horn, 1880 USA (NV) Helops spretus Horn, 1880: 153. Helops stenotrichoides Blaisdell, 1895 USA (CA) Helops stenotrichoides Blaisdell, 1895: 238. Helops strigicollis Horn, 1885 USA (CA) Helops strigicollis Horn, 1885c: 161. Helops suavis Champion, 1887 MEX (OA) GUA Helops suavis Champion, 1887: 318. Helops sulcipennis LeConte, 1866 USA (AL GA NC SC TN VA) Helops sulcipennis LeConte, 1866b: 133. Helops sumptuosus (Allard, 1876) MEX Diastixus sumptuosus Allard, 1876: 57. Helops tristis Palisot de Beauvois, 1817 USA (SC) Helops tristis Palisot de Beauvois, 1817: 138 [junior primary homonym of Helops tristis Rossi, 1790]. 47 Helops tumescens LeConte, 1866 USA (AZ CA) Helops tumescens LeConte, 1866b: 134. Genus Nalassus Mulsant, 1854 [M] Nalassus Mulsant, 1854: 323. Type species: Helops dryadophilus Mulsant, 1854, subsequent designation ( Nabozhenko 2001 : 630). Nalassus aereus (Germar, 1823) USA (AL CT DC DE GA IL IN KS KY MD MO MS NC NJ NY OH PA SC TN VA WV) Helops aereus Germar, 1823: 160. Helops pullus Say, 1826: 240. Synonymy: LeConte (1866a : 63). Helops aratus Say, 1826: 241. Synonymy: LeConte (1866a : 63). Helops carolina Manee, 1924: 40. Synonymy: Steiner (2009 : 332). Nalassus californicus (Mannerheim, 1843) USA (CA ID NV OR WA) MEX Helops californicus Mannerheim, 1843: 287. Nalassus convexulus (LeConte, 1861) CAN (AB BC) USA (CA CO ID MT NE NV OR UT WA WY) Helops convexulus LeConte, 1861b: 353. Helops inclusus Walker, 1866: 330. Synonymy: Blair (1921 : 283). Helops montanus LeConte, 1879b: 518. Synonymy: Bousquet and Campbell (1991 : 258). Helops regulus Blaisdell, 1921b: 227. Synonymy: Boddy (1965 : 177). Genus Nautes Pascoe, 1866 [M] Nautes Pascoe, 1866: 475. Type species: Nautes fervidus Pascoe, 1866, monotypy. Nautes alternans Champion, 1893 GUA Nautes alternans Champion, 1893a: 550. Nautes antennatus Champion, 1887 PAN Nautes antennatus Champion, 1887: 281. Nautes asperipennis Allard, 1894 CUB Nautes asperipennis Allard, 1894: 259. Nautes azurescens (Jacquelin du Val, 1857) USA (FL) / BAH CUB Helops azurescens Jacquelin du Val, 1857: 153. Helops viridimicans Horn, 1878a: 57. Synonymy: Steiner (2005 : 454). Nautes belti Allard, 1877 NIC PAN Nautes belti Allard, 1877b: 59. Nautes breviceps Champion, 1887 PAN Nautes breviceps Champion, 1887: 282. Nautes chrysomeloides Champion, 1887 BEL Nautes chrysomeloides Champion, 1887: 284. Nautes enoplopoides Champion, 1887 GUA Nautes enoplopoides Champion, 1887: 287. Nautes fervidus Pascoe, 1866 MEX (VE) GUA NIC Nautes fervidus Pascoe, 1866: 476. Nautes aeneus Bates, 1870: 270. Synonymy: Champion (1887 : 278). Nautes glabratus Champion, 1887 MEX (VE) Nautes glabratus Champion, 1887: 278. Nautes guanahani Steiner, 2006 BAH Nautes guanahani Steiner, 2006: 29. Nautes hilaris Champion, 1887 GUA Nautes hilaris Champion, 1887: 286. Nautes laeviventris Champion, 1887 GUA Nautes laeviventris Champion, 1887: 285. Nautes magnificus Champion, 1887 GUA Nautes magnificus Champion, 1887: 284. Nautes nitidissimus Champion, 1887 MEX (VE) Nautes nitidissimus Champion, 1887: 286. Nautes nodulosus Champion, 1887 GUA Nautes nodulosus Champion, 1887: 287. Nautes rufipes Allard, 1876 CUB Nautes rufipes Allard, 1876: 45. Nautes splendens Champion, 1887 PAN Nautes splendens Champion, 1887: 280. Nautes stabilis Champion, 1893 MEX (OA VE) Nautes stabilis Champion, 1893a: 550. Nautes striatipennis Champion, 1887 MEX (OA) Nautes striatipennis Champion, 1887: 283. Nautes tinctus Champion, 1887 GUA Nautes tinctus Champion, 1887: 279. Nautes tricolor Champion, 1893 MEX (PU) Nautes tricolor Champion, 1893a: 551. Nautes varians Champion, 1887 MEX (OA VE) Nautes varians Champion, 1887: 281. Nautes versicolor Champion, 1887 GUA Nautes versicolor Champion, 1887: 284. Genus Neohelops Dajoz, 2001 [M] Neohelops Dajoz, 2001: 356. Type species: Neohelops texanus Dajoz, 2001, original designation. Neohelops texanus Dajoz, 2001 USA (TX) Neohelops texanus Dajoz, 2001: 357. Genus Tarpela Bates, 1870 [F] Tarpela Bates, 1870: 272. Type species: Tarpela brownii Bates, 1870, subsequent designation ( Gebien 1943 : 407). Lamperos Allard, 1876: 4. Type species: Helops micans Fabricius, 1798, subsequent designation ( Nabozhenko and Löbl 2008 : 256). Synonymy: Champion (1887 : 288). Tarpela aerifera Allard, 1876 MEX (PU VE) PAN Tarpela aerifera Allard, 1876: 47. Tarpela allardi Champion, 1887 MEX (VE) Tarpela allardi Champion, 1887: 307. Tarpela amabilis Champion, 1887 GUA Tarpela amabilis Champion, 1887: 308. Tarpela atra Allard, 1876 MEX (DU JA MI PU) Tarpela atra Allard, 1876: 46. Tarpela azteca Champion, 1887 MEX (GU) Tarpela azteca Champion, 1887: 300. Tarpela brownii Bates, 1870 NIC PAN Tarpela brownii Bates, 1870: 272. Tarpela cactivora Zayas, 1988 CUB Tarpela cactivora Zayas, 1988: 105. Tarpela catenata Champion, 1895 MEX (YU) Tarpela catenulata Champion, 1893a: 552 [junior primary homonym of Tarpela catenulata Allard, 1877]. Tarpela catenata Champion, 1895: 215. Unjustified emendation of Tarpela catenulata Champion, 1893. Tarpela cisteliformis Allard, 1877 MEX GUA Tarpela cisteliformis Allard, 1877b: 57. Tarpela contigua Champion, 1887 MEX (MI) Tarpela contigua Champion, 1887: 298. Tarpela corpulenta Champion, 1887 MEX (DU) Tarpela corpulenta Champion, 1887: 292. Tarpela costata Champion, 1887 MEX (GE JA SI) Tarpela costata Champion, 1887: 293. Tarpela crassipes Champion, 1887 MEX (OA) Tarpela crassipes Champion, 1887: 306. Tarpela cupreoviridis Allard, 1877 MEX (YU) GUA NIC Tarpela cupreo-viridis Allard, 1877b: 57. Tarpela cuprosa Zayas, 1988: 106 CUB Tarpela cuprosa Zayas, 1988: 106. Tarpela depressa Champion, 1887 MEX (JA YU) Tarpela depressa Champion, 1887: 306. Tarpela docilis Champion, 1887 MEX (VE) Tarpela docilis Champion, 1887: 312. Tarpela durangoensis Champion, 1887 MEX (DU) Tarpela durangoensis Champion, 1887: 292. Tarpela eximia (Bates, 1870) NIC Nautes eximius Bates, 1870: 271. Tarpela fallax Champion, 1887 MEX (TA VE) Tarpela fallax Champion, 1887: 301. Tarpela flohri Champion, 1893 MEX (MO) Tarpela flohri Champion, 1893a: 553. Tarpela foveipennis Champion, 1887 MEX (CI) Tarpela foveipennis Champion, 1887: 294. Tarpela foveolata Champion, 1893 MEX (TA) Tarpela foveolata Champion, 1893a: 554. Tarpela fragilicornis Champion, 1887 MEX (OA) Tarpela fragilicornis Champion, 1887: 309. Tarpela granulipennis (Jacquelin du Val, 1857) CUB Helops granulipennis Jacquelin du Val, 1857: 154. Tarpela guerreroensis Champion, 1893 MEX (GE) Tarpela guerreroensis Champion, 1893a: 555. Tarpela hispidula Allard, 1876 MEX Tarpela hispidula Allard, 1876: 47. Tarpela hoegei Champion, 1887 MEX (DU) Tarpela högei Champion, 1887: 297. Tarpela inaequalis Champion, 1887 PAN Tarpela inaequalis Champion, 1887: 290. Tarpela incilis Champion, 1893 MEX (JA) Tarpela incilis Champion, 1893a: 553. Tarpela jalapensis Champion, 1887 MEX (GE VE) Tarpela jalapensis Champion, 1887: 296. Tarpela marginicollis Champion, 1887 GUA Tarpela marginicollis Champion, 1887: 302. Tarpela micans (Fabricius, 1798) CAN (ON QC) USA (AL CT GA IL IN MA MD NC NY OH SC TN VA) Helops vittatus Olivier, 1793: 45 [ nomen oblitum : see Appendix 4 for supporting references]. Helops micans Fabricius, 1798: 51 [ nomen protectum ]. Synonymy: Illiger (1802 : 343). Helops taeniatus Palisot de Beauvois, 1812: 121. Synonymy: Dejean (1821 : 70). Tarpela nigerrima Champion, 1893 MEX (GE) Tarpela nigerrima Champion, 1893a: 555. Tarpela oblonga Champion, 1887 MEX (VE) Tarpela oblonga Champion, 1887: 298. Tarpela oblongopunctata Bates, 1870 MEX Tarpela oblongopunctata Bates, 1870: 273. Tarpela occidentalis (Allard, 1877) JAM Nesotes occidentalis Allard, 1877b: 40. Helops mutabilis C.O. Waterhouse, 1878: 304. Synonymy: Champion (1894 : lxxxv). Tarpela propinqua (C.O. Waterhouse, 1878) JAM Helops propinquus C.O. Waterhouse, 1878: 305. Tarpela pulchra Champion, 1893 MEX (VE) Tarpela pulchra Champion, 1893a: 551. Tarpela puncticeps Champion, 1887 GUA Tarpela puncticeps Champion, 1887: 303. Tarpela reticulata Champion, 1887 HON Tarpela reticulata Champion, 1887: 293. Tarpela sculptilis Champion, 1887 MEX (VE) Tarpela sculptilis Champion, 1887: 295. Tarpela setigera Champion, 1887 MEX (VE) Tarpela setigera Champion, 1887: 297. Tarpela silvicola Champion, 1887 GUA Tarpela silvicola Champion, 1887: 309. Tarpela sinuaticollis Champion, 1887 PAN Tarpela sinuaticollis Champion, 1887: 303. Tarpela socia Champion, 1887 MEX (GE JA SI) Tarpela socia Champion, 1887: 299. Tarpela subparallela Champion, 1887 MEX (SL) Tarpela subparallela Champion, 1887: 300. Tarpela subvittata Champion, 1887 GUA Tarpela subvittata Champion, 1887: 305. Tarpela suturalis Champion, 1887 GUA Tarpela suturalis Champion, 1887: 310. Tarpela teapensis Champion, 1893 MEX (TB) Tarpela teapensis Champion, 1893a: 556. Tarpela tenuicornis Champion, 1887 GUA Tarpela tenuicornis Champion, 1887: 289. Tarpela thoracica Champion, 1887 NIC Tarpela thoracica Champion, 1887: 293. Tarpela torrida Champion, 1887 MEX (DU YU) Tarpela torrida Champion, 1887: 291. Tarpela totonicapamensis Champion, 1887 GUA Tarpela totonicapamensis Champion, 1887: 311. Tarpela tropicalis Champion, 1887 GUA Tarpela tropicalis Champion, 1887: 304. Tarpela undulata (LeConte, 1866) USA (FL GA IN MD NC OH PA SC TN VA) Helops americanus Palisot de Beauvois, 1812: 122 [ nomen dubium ] 48 . Helops undulatus LeConte, 1866b: 132. Synonymy: Horn (1885a : 89). Tarpela venusta (Say, 1824) USA (AL GA MD MO NC NY OH PA SC TN VA) Helops venustus Say, 1824b: 284. Tarpela veraepacis Champion, 1887 GUA Tarpela veraepacis Champion, 1887: 295. Tarpela virescens (Laporte, 1840) "Amérique du Nord" Helops virescens Laporte, 1840: 235. Tribe Melanimonini Seidlitz, 1894 Microzoumates Mulsant, 1854: 176. Type genus: Microzoum Dejean, 1834 (= Melanimon Steven 1829). Melanimonina Seidlitz, 1894: 449. Type genus: Melanimon Steven, 1829. Note.Use of younger family-group name conserved ( ICZN 1999 : Art. 40.2) (see Bouchard et al. 2005 ). Genus Cheirodes Gené, 1839 [M] Cheirodes Gené, 1839: 73. Type species: Cheirodes sardous Gené, 1839, monotypy. Anemia Laporte, 1840: 218. Type species: Anemia granulata Laporte, 1840, monotypy. Synonymy: Spilman (1973 : 41). Chirodes Agassiz, 1846: 81. Unjustified emendation of Cheirodes Gené, 1839, not in prevailing usage. Cheirodes californicus (Horn, 1870) USA (CA NV OR WA) Anaemia californica Horn, 1870: 378. Tribe Metaclisini Steiner, 2016 Metaclisini Steiner, 2016: 542. Type genus: Metaclisa Jacquelin du Val, 1861. Genus Metaclisa Jacquelin du Val, 1861 [F] Amarantha Motschulsky, 1859: 141 [ nomen oblitum , see Bouchard et al. (2007 : 393)]. Type species: Amarantha viridis Motschulsky, 1859, monotypy. Metaclisa Jacquelin du Val, 1861: 296 [ nomen protectum ]. Type species: Platydema parallela Fairmaire, 1855 (= Diaperis azurea Waltl, 1838), original designation. Synonymy: Lewis (1891 : 70). Tharsus LeConte, 1862a: 233. Type species: Tharsus seditiosus LeConte, 1862, monotypy. Synonymy: Steiner (2016 : 538). Metaclisa atra LeConte, 1866 USA (AL DC FL GA LA MD MO MS NC PA SC TX VA) Metaclisa atra LeConte, 1866b: 127. Haplandrus collaris Casey, 1924: 320. Synonymy: Steiner (2016 : 537). Haplandrus subangusta Casey, 1924: 320. Synonymy: Steiner (2016 : 537). Metaclisa marginalis Horn, 1870 CAN (BC) USA (CA OR WA) Metaclisa marginalis Horn, 1870: 369. Metaclisa seditiosa (LeConte, 1862) USA (AL FL GA KY MD NC OH SC TN TX VA WV) / BAH Tharsus seditiosus LeConte, 1862a: 233. Tribe Opatrini Brullé, 1832 Opatrites Brullé, 1832: 213. Type genus: Opatrum Fabricius, 1775. Subtribe Opatrina Brullé, 1832 Opatrites Brullé, 1832: 213. Type genus: Opatrum Fabricius, 1775. Blapstinites Mulsant and Rey, 1853: 258. Type genus: Blapstinus Dejean, 1821. Genus Aconobius Casey, 1895 [M] Aconobius Casey, 1895: 617. Type species: Conibiosoma laciniata Casey, 1891, original designation. Aconobius densus Casey, 1914 USA (NM) Aconobius densus Casey, 1914: 377. Aconobius laciniatus (Casey, 1891) USA (AZ) Conibiosoma laciniata Casey, 1891: 64. Aconobius nigripes Casey, 1914 USA (TX) Aconobius nigripes Casey, 1914: 378. Genus Ammodonus Mulsant and Rey, 1859 [M] Ammodonus Mulsant and Rey, 1859: 143. Type species: Opatrum fossor LeConte, 1847, monotypy. Pseudonomus Fairmaire, 1884: 510. Type species: Pseudonomus dermestiformis Fairmaire, 1884, monotypy. Synonymy: Gebien (1939 : 470). Scaptes Champion, 1886: 222. Type species: Scaptes squamulatus Champion, 1886 (= Asida tropica Kirsch, 1866), present designation . Synonymy: Fall (1912 : 48). Trichotoides Marcuzzi, 1954b: 23. Type species: Scaptes hintoni Kaszab, 1949, monotypy. Synonymy: Ferrer and Moraguès (2001 : 499). Ammodonus ciliatus (Champion, 1896) LAN / SA Scaptes ciliatus Champion, 1896: 9. Ammodonus fossor (LeConte, 1847) CAN (ON) USA (AL AR DE IL IN KS MD MN NC NE NJ NY OH OK SC TX WI WV) Opatrum fossor LeConte, 1847: 92. Ammodonus granosus Fall, 1912 USA (AZ) MEX (BS) Ammodonus granosus Fall, 1912: 47. Ammodonus tropicus (Kirsch, 1866) USA (AZ CA) MEX (AG CH CI JA MI NA NL OA PU SI SO VE) GUA BEL SAL HON NIC CRI PAN / CUB JAM / SA Asida tropica Kirsch, 1866: 190. Scaptes squamulatus Champion, 1886: 223. Synonymy: Champion (1893a : 542). Genus Blapstinus Dejean, 1821 [M] Blapstinus Dejean, 1821: 66. Type species: Blaps punctata Fabricius, 1792, monotypy. Heteropus Laporte, 1840: 221 [junior homonym of Heteropus Palisot de Beauvois, 1820]. Type species: Heteropus holosericeus Laporte, 1840, monotypy. Synonymy: Lacordaire (1859 : 250). Pedonoeces G.R. Waterhouse, 1845: 32. Type species: Pedonoeces galapagoensis G.R. Waterhouse, 1845, subsequent designation ( Aalbu and Triplehorn 1991 : 170). Synonymy: Aalbu and Triplehorn (1991 : 170). Tessaromma Boheman, 1858: 91 [junior homonym of Tessaromma Newman, 1840]. Type species: Tessaromma lugubris Boheman, 1858, subsequent designation ( Aalbu and Triplehorn 1991 : 170). Synonymy: Aalbu and Triplehorn (1991 : 170). Lachnoderes Mulsant and Rey, 1859: 166. Type species: Pedonoeces pubescens G.R. Waterhouse, 1845, monotypy. Synonymy: Aalbu and Triplehorn (1991 : 170). Aspidius Mulsant and Rey, 1859: 187. Type species: Blaps punctata Fabricius, 1792, present designation . Synonymy: Champion (1885 : 124). Lodinus Mulsant and Rey, 1859: 195. Type species: Lodinus nigroaeneus Mulsant and Rey, 1859 (= Blapstinus punctulatus Solier, 1851), monotypy. Synonymy: Gemminger [in Gemminger and Harold] (1870 : 1923). Blapstinus aciculus Blatchley, 1917 USA (FL) / BAH Blapstinus aciculus Blatchley, 1917: 275. Blapstinus amnosus Blaisdell, 1923 MEX (BC) Blapstinus amnosus Blaisdell, 1923: 272. Blapstinus angustatus Champion, 1893 MEX (OA) Blapstinus angustatus Champion, 1893a: 528. Blapstinus aridus Blaisdell, 1923 MEX (SO) Blapstinus aridus Blaisdell, 1923: 270. Blapstinus atratus Champion, 1885 MEX (GE PU YU) GUA NIC PAN Blapstinus atratus Champion, 1885: 131. Blapstinus auripilis Horn, 1870 USA (AZ) Blapstinus auripilis Horn, 1870: 353. Blapstinus barri Boddy, 1957 USA (ID OR) Blapstinus barri Boddy, 1957: 198. Blapstinus brevicollis LeConte, 1851 USA (AZ CA) MEX (SO) Blapstinus brevicollis LeConte, 1851: 147. Blapstinus sonorae Casey, 1890b: 431. New synonymy [based on Davis (1970 : 131) unpublished thesis]. Blapstinus buqueti Champion, 1885 CRI PAN / LAN / SA Blapstinus buqueti Champion, 1885: 128. Blapstinus piliferus Fairmaire, 1892: 82. Synonymy: Marcuzzi (1951 : 75). Blapstinus castaneus Casey, 1890 USA (AZ CA TX) Blapstinus castaneus Casey, 1890b: 432. Blapstinus falli Blaisdell, 1929b: 21. New synonymy [based on Davis (1970 : 257) unpublished thesis]. Blapstinus cubanus cubanus Marcuzzi, 1962 CUB CAY Blapstinus cubanus Marcuzzi, 1962: 33. Blapstinus cubanus grandturki Marcuzzi, 1965 BAH Blapstinus cubanus grandturki Marcuzzi, 1965: 130. Blapstinus cylindriformis Doyen, 1990 MEX (GE JA) Blapstinus cylindriformis Doyen, 1990: 232. Blapstinus decui Ardoin, 1977 CUB Blapstinus decui Ardoin, 1977c: 390. Blapstinus debilis Casey, 1890 USA (FL TX) Blapstinus debilis Casey, 1890b: 458. Blapstinus densipunctatus Blaisdell, 1943 MEX (BS) Blapstinus densipunctatus Blaisdell, 1943: 256. Blapstinus dilatatus LeConte, 1851 USA (AZ CA CO SD UT) MEX (CH SO) Opatrum pullum Say, 1826: 237 [ nomen dubium ]. Blapstinus dilatatus LeConte, 1851: 146. Synonymy (in doubt): LeConte (1866a : 61). Blapstinus discolor Horn, 1870 CAN (BC) USA (CA ID NV OR UT WA) MEX (BS) Blapstinus discolor Horn, 1870: 354. Blapstinus oregonensis Casey, 1890b: 435. Synonymy: Davis (1982 : 254). Blapstinus fuliginosus Casey, 1890b: 438. Synonymy: Davis (1982 : 254). Blapstinus rufipes Casey, 1890b: 439. Synonymy: Davis (1982 : 254). Blapstinus crassicornis Casey, 1890b: 440. Synonymy: Davis (1982 : 254). Blapstinus elongatus Casey, 1890b: 441. Synonymy: Horn (1894b : 351). Blapstinus lepidus Casey, 1890b: 444. Synonymy: Davis (1982 : 254). Blapstinus aequalis Casey, 1890b: 445. Synonymy: Davis (1982 : 254). Blapstinus funebris Casey, 1890b: 446. Synonymy: Davis (1982 : 254). Blapstinus parallelus Casey, 1890b: 448. Synonymy: Davis (1982 : 254). Blapstinus inquisitus Casey, 1890b: 449. Synonymy: Davis (1982 : 254). Blapstinus domingoensis (Marcuzzi, 1998) DOM Diastolinus domingoensis Marcuzzi, 1998b: 222. Blapstinus dominicus Marcuzzi, 1962 JAM HAI PRI LAN Blapstinus dominicus Marcuzzi, 1962: 34. Blapstinus egenus Champion, 1885 MEX (JA SI VE) GUA NIC PAN / SA Blapstinus egenus Champion, 1885: 129. Blapstinus emmenastoides Champion, 1885 MEX (OA VE) GUA Blapstinus emmenastoides Champion, 1885: 131. Blapstinus errabundus Champion, 1885 MEX (OA VE YU) NIC PAN Blapstinus errabundus Champion, 1885: 127. Blapstinus exiguus Champion, 1893 MEX (OA YU) Blapstinus exiguus Champion, 1893a: 529. Blapstinus faulkneri Aalbu and Triplehorn, 1991 MEX ("Revillagigedo Is.") Blapstinus faulkneri Aalbu and Triplehorn, 1991: 173. Blapstinus fortis LeConte, 1878 USA (AZ CO FL GA KS LA MD MO NC NM OK SC SD TX) MEX (CO JA MO OA SL VE YU) GUA BEL NIC CRI PAN / BAH CUB CAY Opatrinus punctulatus Jacquelin du Val, 1857: 141 [junior secondary homonym of Blapstinus punctulatus Solier, 1851]. Blapstinus fortis LeConte, 1878a: 420. Synonymy: Casey (1890b : 429). Blapstinus interstitialis Champion, 1885: 125. Replacement name for Blapstinus punctulatus (Jacquelin du Val, 1857). Blapstinus fuscus Casey, 1890 USA (FL LA OK TX [NM]) MEX Blapstinus fuscus Casey, 1890b: 427. Blapstinus genaroi (Garrido, 2004) DOM Diastolinus genaroi Garrido, 2004b: 41. Blapstinus grandis Champion, 1885 MEX (JA SI) NIC CRI Blapstinus grandis Champion, 1885: 125. Blapstinus haitensis Marcuzzi, 1962 HAI DOM Blapstinus haitensis Marcuzzi, 1962: 34. Blapstinus hispaniolensis (Marcuzzi, 1998) DOM Diastolinus hispaniolensis Marcuzzi, 1998b: 220. Blapstinus histricus Casey, 1890 USA (AZ CA FL NV NM TX) Blapstinus histricus Casey, 1890b: 433. Blapstinus brunneus Casey, 1890b: 453. New synonymy [based on Davis (1970 : 220) unpublished thesis]. Blapstinus coronadensis Blaisdell, 1892: 242. New synonymy [based on Davis (1970 : 220) unpublished thesis]. Blapstinus humilis Casey, 1890 USA (FL) / BAH Blapstinus humilis Casey, 1890b: 459. 49 Blapstinus inflatitibia (Marcuzzi, 1977) CAY Diastolinus inflatitibia Marcuzzi, 1977: 17. Blapstinus insularis Champion, 1885 PAN Blapstinus insularis Champion, 1885: 127. Blapstinus intermedius Champion, 1885 MEX GUA NIC Blapstinus intermedius Champion, 1885: 129. Blapstinus intermixtus Casey, 1890 CAN (BC) USA (AZ CA ID OR UT WA) Blapstinus intermixtus Casey, 1890b: 451. Blapstinus hesperius Casey, 1890b: 454. New synonymy [based on Davis (1970 : 232) unpublished thesis]. Blapstinus jamaicensis Marcuzzi, 1962 JAM Blapstinus jamaicensis Marcuzzi, 1962: 35. Blapstinus kalik Steiner, 2006 BAH Blapstinus kalik Steiner, 2006: 18. Blapstinus kaszabi Marcuzzi, 1985 "Ciudad, Central America" Blapstinus kaszabi Marcuzzi, 1985: 183. Blapstinus klapperichi (Marcuzzi, 1998) DOM Diastolinus klapperichi Marcuzzi, 1998b: 219. Blapstinus lecontei Mulsant and Rey, 1859 USA (CA) Blapstinus pubescens LeConte, 1851: 147 [junior secondary homonym of Blapstinus pubescens (G.R. Waterhouse, 1845)]. Blapstinus lecontii Mulsant and Rey, 1859: 192. Replacement name for Blapstinus pubescens LeConte, 1851. Blapstinus cinerascens Fall, 1929: 58. New synonymy [based on Davis (1970 : 100) unpublished thesis]. Blapstinus californicus Aalbu and Triplehorn, 1991: 170 [junior primary homonym of Blapstinus californicus Motschulsky, 1845]. Replacement name for Blapstinus pubescens LeConte, 1851. Blapstinus longicollis Champion, 1885 GUA NIC Blapstinus longicollis Champion, 1885: 126. Blapstinus longipennis Champion, 1885 MEX (SI) Blapstinus longipennis Champion, 1885: 130. Blapstinus longulus LeConte, 1851 USA (AZ) Blapstinus longulus LeConte, 1851: 147. Blapstinus marcuzzii Aalbu, new replacement name JAM Blapstinus kulzeri Marcuzzi, 1977: 28 [junior primary homonym of Blapstinus kulzeri Kaszab, 1969] 50 . Blapstinus marcuzzii Aalbu, new replacement name for Blapstinus kulzeri Marcuzzi, 1977. Blapstinus metallicus (Fabricius, 1801) CAN (AB MB NB NS ON PE QC SK) USA (CO CT DE FL GA IA IL IN KS LA MA MD MI MN MO MS NC ND NE NH NJ NY OH OR PA RI SC SD VA WI WV WY) Blaps metallica Fabricius, 1801a: 143. Opatrum interruptum Say, 1824a: 264. Synonymy: LeConte (1866a : 61). Blapstinus aeneolus Melsheimer, 1846: 66. Synonymy: LeConte (1866a : 61). Blapstinus luridus Mulsant and Rey, 1859: 193. Synonymy: LeConte (1866a : 61). Blapstinus mexicanus Champion, 1885 MEX (CI YU) Blapstinus mexicanus Champion, 1885: 124. Blapstinus moestus Melsheimer, 1846 CAN (ON) USA (CT DC DE GA IL IN MA MD MI NC NH NJ NY OH RI SC VA WI) Blapstinus moestus Melsheimer, 1846: 65. Blapstinus nitidus Champion, 1885 MEX (VE) Blapstinus nitidus Champion, 1885: 130. Blapstinus obliteratus Champion, 1885 PAN Blapstinus obliteratus Champion, 1885: 132. Blapstinus opacus Mulsant and Rey, 1859 51 LAN Blapstinus opacus Mulsant and Rey, 1859: 186. Blapstinus opacus martinensis Marcuzzi, 1977: 29. Synonymy: Ivie and Hart (2016 : 466). Blapstinus orlandoi Ivie and Hart, 2016 JAM Diastolinus jamaicensis Garrido, 2004a: 37 [junior secondary homonym of Blapstinus jamaicensis Marcuzzi, 1962]. Blapstinus orlandoi Ivie and Hart, 2016: 466. Replacement name for Blapstinus jamaicensis (Garrido, 2004). Blapstinus pacificus Aalbu and Triplehorn, 1991 MEX ("Revillagigedo Is.") Blapstinus pacificus Aalbu and Triplehorn, 1991: 171. Blapstinus palmeri Champion, 1885 MEX (CH CO NL) Blapstinus palmeri Champion, 1885: 128. Blapstinus paradoxus Blaisdell, 1923 MEX (SO) Blapstinus paradoxus Blaisdell, 1923: 271. Blapstinus pimalis Casey, 1885 USA (AZ CA CO NM NV TX UT) MEX (SO) Blapstinus pimalis Casey, 1885 [January]: 185. Blapstinus umbrosus Champion, 1885 [October]: 127. Synonymy: Champion (1893a : 527). Blapstinus niger Casey, 1890b: 436. New synonymy [based on Davis (1970 : 306) unpublished thesis]. Blapstinus cribricollis Casey, 1890b: 437. New synonymy [based on Davis (1970 : 306) unpublished thesis]. Blapstinus pinorum Casey, 1914 USA (GA NC SC) Blapstinus pinorum Casey, 1914: 377. Blapstinus pratensis LeConte, 1859 CAN (AB) USA (CO KS MT NE NM OK SD TX) MEX (CH DU SO TA) Blapstinus pratensis LeConte, 1859a: 15. Blapstinus arenarius Casey, 1890b: 457. New synonymy [based on Davis (1970 : 177) unpublished thesis]. Blapstinus puertoricensis (Marcuzzi, 1977) PRI LAN Diastolinus puertoricensis Marcuzzi, 1977: 20. Blapstinus pulverulentus Mannerheim, 1843 USA (CA OR WA) Blapstinus pulverulentus Mannerheim, 1843: 276. Blapstinus californicus Motschulsky, 1845a: 77. Synonymy: Horn (1870 : 355). Blapstinus punctatus anxius Mulsant and Rey, 1859 [no locality given originally but probably from the Antilles] Blapstinus punctatus var. anxius Mulsant and Rey, 1859: 190. Blapstinus punctatus punctatus (Fabricius, 1792) PRI LAN Blaps punctata Fabricius, 1792a: 109. Diastolinus fuscicornis Chevrolat, 1877e: viii. Synonymy: Ivie and Hart (2016 : 467). Blapstinus puncticollis Champion, 1893 MEX (GE) Blapstinus puncticollis Champion, 1893a: 529. Blapstinus simulans barbadensis Marcuzzi, 1962 LAN Blapstinus simulans barbadensis Marcuzzi, 1962: 36. Blapstinus simulans simulans Marcuzzi, 1954 LAN / SA Blapstinus simulans Marcuzzi, 1954b: 15. Blapstinus striatulus Mulsant and Rey, 1859 PRI LAN (St. Barthélemy) Blapstinus striatulus Mulsant and Rey, 1859: 183. Blapstinus striatus Guérin-Méneville, 1831 CUB Blapstinus striatus Guérin-Méneville, 1831a: pl. 4. Blapstinus substriatus Champion, 1885 [Fig. 26 ] CAN (AB BC MB SK YT) USA (AZ CA CO ID KS MN MT ND NM NV OR SD TX UT WA WY) MEX (CH CO DU FD GU ME PU SL VE) NIC Figure 26. Blapstinus substriatus Champion, 1885. Scale bar = 1 mm. Blapstinus substriatus Champion, 1885: 128. Blapstinus gregalis Casey, 1890b: 442. New synonymy [based on Davis (1970 : 232) unpublished thesis]. Blapstinus sulcatus LeConte, 1851 USA (AZ CA CO KS NV UT) Blapstinus sulcatus LeConte, 1851: 147. Blapstinus hydropicus Casey, 1890b: 461. New synonymy [based on Davis (1970 : 106) unpublished thesis]. Blapstinus sulcipennis Champion, 1885 MEX (YU) GUA / CUB CAY Blapstinus sulcipennis Champion, 1885: 129. Blapstinus tibialis Champion, 1885 GUA NIC CRI Blapstinus tibialis Champion, 1885: 125. Blapstinus validus Casey, 1890 USA (AZ CA) Blapstinus validus Casey, 1890b: 429. Blapstinus vandykei Blaisdell, 1942 USA (AZ CA NM NV TX) Blapstinus vandykei Blaisdell, 1942: 136. Blapstinus vestitus LeConte, 1859 USA (CO KS SD TX UT WY) Blapstinus vestitus LeConte, 1859a: 15. Blapstinus hospes Casey, 1890b: 455. New synonymy [based on Davis (1970 : 187) unpublished thesis]. Blapstinus yucatanus Champion, 1893 MEX (YU) Blapstinus yucatanus Champion, 1893a: 526. Genus Bycrea Pascoe, 1868 [F] Bycrea Pascoe, 1868: xii. Type species: Bycrea villosa Pascoe, 1868, monotypy. Bycrea villosa Pascoe, 1868 USA (AZ) MEX (DU GE GU JA MI MO NL OA PU SI SO TA YU) GUA SAL CRI / SA Bycrea villosa Pascoe, 1868: xii. Genus Cenophorus Mulsant and Rey, 1859 [M] Cenophorus Mulsant and Rey, 1859: 177. Type species: Cenophorus viduus Mulsant and Rey, 1859, monotypy. Cenophorus viduus Mulsant and Rey, 1859 HAI Cenophorus viduus Mulsant and Rey, 1859: 177. Genus Conibiosoma Casey, 1890 [N] Conibiosoma Casey, 1890b: 476. Type species: Conibius elongatus Horn, 1870, monotypy. Conibiosoma elongatum (Horn, 1870) USA (CA NV) Conibius elongatus Horn, 1870: 351. Genus Conibius LeConte, 1851 [M] Conibius LeConte, 1851: 145. Type species: Conibius seriatus LeConte, 1851, subsequent designation ( Lucas 1920 : 199). Ooconibius Casey, 1895: 618. Type species: Notibius opacus LeConte, 1866, monotypy. New synonymy [RLA]. Euconibius Casey, 1895: 618. Type species: Notibius gagates Horn, 1870, monotypy. New synonymy [RLA]. Conibius brunnipes Champion, 1885 MEX (HI JA ME MI MO NA OA PU QU SI TL) GUA Conibius brunnipes Champion, 1885: 133. Conibius gagates (Horn, 1870) USA (AZ) MEX (CH SO) Notibius gagates Horn, 1870: 357. Conibius guadalupensis Casey, 1890 MEX (BC [Guadalupe Is.]) Conibius guadalupensis Casey, 1890b: 470. Conibius oblongus Blaisdell, 1943 MEX (BC) Conibius oblongus Blaisdell, 1943: 257. Conibius opacus (LeConte, 1866) USA (AZ) MEX (BC BS SO) Notibius opacus LeConte, 1866b: 118. Notibius reflexus Horn, 1894b: 429. New synonymy [RLA]. Conibius rotundicollis Linell, 1899 USA (TX) MEX (HI NL) Conibius rotundicollis Linell, 1899: 182. Conibius rugipes (Champion, 1885) MEX (OA PU) Notibius rugipes Champion, 1885: 132. Notibius affinis Champion, 1885: 132. New synonymy [RLA]. Conibius seriatus LeConte, 1851 USA (CA OR) MEX (BC) Conibius seriatus LeConte, 1851: 146. Conibius parallelus LeConte, 1851: 146. New synonymy [RLA]. Conibius troglodytes Champion, 1893 MEX (GE MO PU) Conibius troglodytes Champion, 1893a: 530. Conibius uniformis Casey, 1890 USA (AZ NM TX) MEX (CH CO DU ZA) Conibius uniformis Casey, 1890b: 471. Conibius ventralis Blaisdell, 1923 MEX (BS) Conibius ventralis Blaisdell, 1923: 274. Genus Cybotus Casey, 1890 [M] Cybotus Casey, 1890b: 482. Type species: Blapstinus estriatus LeConte, 1878, monotypy. Cybotus estriatus (LeConte, 1878) USA (FL) MEX (QR) HON Blapstinus estriatus LeConte, 1878a: 420. Genus Diastolinus Mulsant and Rey, 1859 [M] Diastolinus Mulsant and Rey, 1859: 138. Type species: Blaps clathrata Fabricius, 1792, subsequent designation ( Lucas 1920 : 236). Sellio Mulsant and Rey, 1859: 169. Type species: Blaps tibidens Quensel, 1806, subsequent designation ( Gebien 1938 : 407). Synonymy: Ivie and Hart (2016 : 468). Ctesicles Champion, 1896: 7. Type species: Ctesicles insularis Champion, 1896, subsequent designation ( Lucas 1920 : 214). Synonymy: Ivie and Hart (2016 : 468). Diastolinus azuaensis Hart and Ivie, 2016 DOM Diastolinus azuaensis Hart and Ivie, 2016a: 506. Diastolinus clathratus (Fabricius, 1792) VIS (St. Croix) Blaps clathrata Fabricius, 1792a: 109. Diastolinus chalumeaui Hart and Ivie, 2016 LAN Diastolinus chalumeaui Hart and Ivie, 2016a: 494. Diastolinus clavatus Mulsant and Rey, 1859 PRI LAN Diastolinus clavatus Mulsant and Rey, 1859: 155. Diastolinus hummelincki Marcuzzi, 1962: 28 [junior primary homonym of Diastolinus hummelincki Marcuzzi, 1949]. Synonymy: Hart and Ivie (2016a : 497). Diastolinus mulsanti Marcuzzi and d'Aguilar, 1971: 79. Replacement name for Diastolinus hummelincki Marcuzzi, 1962. Diastolinus coarctatus (Mulsant and Rey, 1859) DOM Sellio coarctatus Mulsant and Rey, 1859: 170. Diastolinus estebani Garrido, 2004b: 42. Synonymy: Hart and Ivie (2016a : 507). Diastolinus desecheo Hart and Ivie, 2016 PRI (Desecheo Island) Diastolinus desecheo Hart and Ivie, 2016a: 509. Diastolinus doyeni Hart and Ivie, 2016 PRI Diastolinus doyeni Hart and Ivie, 2016a: 511. Diastolinus espoloni Garrido, 2007 DOM Diastolinus espoloni Garrido, 2007: 46. Diastolinus gladiator (Garrido, 2005) DOM Sellio gladiator Garrido, 2005: 120. Diastolinus hoppae Hart and Ivie, 2016 LAN Diastolinus hoppae Hart and Ivie, 2016a: 522. Diastolinus insularis (Champion, 1896) LAN (St. Vincent) Ctesicles insularis Champion, 1896: 7. Diastolinus leewardensis Hart and Ivie, 2016 LAN Diastolinus leewardensis Hart and Ivie, 2016a: 498. Diastolinus maritimus (Champion, 1896) LAN Ctesicles maritimus Champion, 1896: 8. Diastolinus perforatus (Schönherr, 1806) LAN Opatrum perforatum Schönherr, 1806: 146. Diastolinus realinoi Marcuzzi, 2002 [ CUB ] 52 Diastolinus realinoi Marcuzzi, 2002: 398. Diastolinus shieli Hart and Ivie, 2016 LAN (Redonda) Diastolinus shieli Hart and Ivie, 2016a: 504. Diastolinus tibidens (Quensel, 1806) PRI LAN Blaps tibidens Quensel [in Schönherr], 1806: 147. Diastolinus vaderi Hart and Ivie, 2016 HAI Diastolinus vaderi Hart and Ivie, 2016a: 518. Diastolinus victori Garrido, 2002 PRI Diastolinus elongatus Marcuzzi, 1977: 15 [junior primary homonym of Diastolinus elongatus Marcuzzi, 1976]. Diastolinus victori Garrido, 2002: 39. Replacement name for Diastolinus elongatus Marcuzzi, 1977. Genus Ephalus LeConte, 1862 [M] Ephalus LeConte, 1862a: 228. Type species: Heliopates latimanus LeConte, 1847, monotypy. Ephalus latimanus (LeConte, 1847) CAN (NS) USA (CT MA ME NH NJ NY RI) Heliopates latimanus LeConte, 1847: 92. Genus Gonocephalum Solier, 1834 [N] Gonocephalum Solier, 1834: 498. Type species: Opatrum fuscum Herbst, 1793 (= Opatrum rusticum Olivier, 1811), subsequent designation ( Gebien 1939 : 443). Subgenus Gonocephalum Solier, 1834 Gonocephalum Solier, 1834: 498. Type species: Opatrum fuscum Herbst, 1793 (= Opatrum rusticum Olivier, 1811), subsequent designation ( Gebien 1939 : 443). Gonocephalum sericeum (Baudi di Selve, 1875) USA (CA) – Adventive Opatrum sericeum Baudi di Selve, 1875: 701. Genus Hummelinckia Marcuzzi, 1954 [F] Hummelinckia Marcuzzi, 1954b: 19. Type species: Hummelinckia caraibica Marcuzzi, 1954, monotypy. Hummelinckia caraibica Marcuzzi, 1954 LAN Hummelinckia caraibica Marcuzzi, 1954b: 19. Genus Mecysmus Horn, 1870 [M] Mecysmus Horn, 1870: 349. Type species: Blapstinus angustus LeConte, 1851, monotypy. Mecysmus advena Casey, 1890 USA (SD TX) Mecysmus advena Casey, 1890b: 466. Mecysmus angustus (LeConte, 1851) USA (AZ CA) Blapstinus angustus LeConte, 1851: 147. Mecysmus laticollis Casey, 1890 CAN (AB) USA (TX) Mecysmus laticollis Casey, 1890b: 463. Mecysmus parvulus Casey, 1890 USA (NM TX) Mecysmus parvulus Casey, 1890b: 466. Mecysmus tenuis Casey, 1890 USA (CA) Mecysmus tenuis Casey, 1890b: 465. Genus Nevisia Marcuzzi, 1986 [F] Nevisia Marcuzzi, 1985: 179. Type species: Diastolinus barbudensis Marcuzzi, 1962, monotypy. Nevisia barbudensis (Marcuzzi, 1962) LAN Diastolinus barbudensis Marcuzzi, 1962: 29. Diastolinus barbudensis antiguanus Marcuzzi, 1962: 30. Synonymy: Ivie and Hart (2016 : 469). Genus Nocibiotes Casey, 1895 [M] Nocibiotes Casey, 1895: 617. Type species: Notibius granulatus LeConte, 1851, subsequent designation ( Gebien 1938 : 407). Nocibiotes caudatus Casey, 1895 USA (AZ) MEX (BC) Nocibiotes caudatus Casey, 1895: 619, 621. Nocibiotes rubripes Casey, 1895: 619. New synonymy [RLA]. Nocibiotes crassipes (Casey, 1890) USA (CA) Conibius crassipes Casey, 1890b: 475. Nocibiotes granulatus (LeConte, 1851) USA (AZ CA) MEX (BS SO) Notibius granulatus LeConte, 1851: 145. Nocibiotes gracilis Casey, 1895: 619. New synonymy [RLA]. Nocibiotes acutus Casey, 1895: 619, 620. New synonymy [RLA]. Nocibiotes rossi (Blaisdell, 1943) MEX (BS) Tonibius rossi Blaisdell, 1943: 260. Genus Notibius LeConte, 1851 [M] Notibius LeConte, 1851: 144. Type species: Notibius puberulus LeConte, 1851, subsequent designation ( Gebien 1938 : 406). Notibius puberulus LeConte, 1851 USA (AZ CA NV OR UT) MEX (BC) Notibius puberulus LeConte, 1851: 145. Notibius substriatus Casey, 1890b: 479. Synonymy: Horn (1894a : 41). Notibius laticeps Casey, 1890b: 480. Synonymy: Horn (1894a : 41). Notibius puncticollis LeConte, 1851 USA (CA NV UT) Notibius puncticollis LeConte, 1851: 145. Genus Opatroides Brullé, 1832 [M] Opatroides Brullé, 1832: 219. Type species: Opatroides punctulatus Brullé, 1832, monotypy. Opatroides punctulatus Brullé, 1832 USA (CA NV) – Adventive Opatroides punctulatus Brullé, 1832: 220. Genus Penichrus Champion, 1885 [M] Penichrus Champion, 1885: 134. Type species: Penichrus blapstinoides Champion, 1885, monotypy. Penichrus blapstinoides Champion, 1885 PAN Penichrus blapstinoides Champion, 1885: 135. Genus Platylus Mulsant and Rey, 1859 [M] Platylus Mulsant and Rey, 1859: 134. Type species: Blaps dilatata Fabricius, 1798, monotypy. Platylus dilatatus (Fabricius, 1798) PRI (Vieques) VIS (St. Thomas) Blaps dilatata Fabricius, 1798: 47. Genus Pseudephalus Casey, 1924 [M] Pseudephalus Casey, 1924: 333. Type species: Pseudephalus brevicornis Casey, 1924, original designation. Pseudephalus brevicornis Casey, 1924 USA (AL FL) HON CRI Pseudephalus brevicornis Casey, 1924: 333. Genus Tonibiastes Casey, 1895 [M] Tonibiastes Casey, 1895: 617. Type species: Notibius costipennis Horn, 1894, original designation. Tonibiastes costipennis (Horn, 1894) MEX (BC BS) Notibius costipennis Horn, 1894: 430. Genus Tonibius Casey, 1895 [M] Tonibius Casey, 1895: 617. Type species: Notibius sulcatus LeConte, 1851, subsequent designation ( Lucas 1920 : 644). Tonibius sulcatus (LeConte, 1851) USA (CA) MEX (BC BS SO) Notibius sulcatus LeConte, 1851: 145. Conibius alternatus Casey, 1890b: 473. New synonymy [RLA]. Genus Trichoton Hope, 1841 [N] Trichoton Hope, 1841: 111. Type species: Trichoton cayennense Hope, 1841, original designation. Subgenus Trichoton Hope, 1841 Trichoton Hope, 1841: 111. Type species: Trichoton cayennense Hope, 1841, original designation. Epilasium Curtis, 1844: 222. Type species: Epilasium rotundatum Curtis, 1844, monotypy. Synonymy: Mulsant and Rey (1853 : 245). Trichoton curvipes Champion, 1885 PAN / SA Trichoton curvipes Champion, 1885: 136. Trichoton lapidicola Champion, 1885 NIC / SA Trichoton lapidicola Champion, 1885: 136. Trichoton marcuzzi Kulzer, 1961 LAN / SA Trichoton marcuzzi Kulzer, 1961a: 212. Trichoton mexicanum Kulzer, 1961 MEX (CL) Trichoton mexicanum Kulzer, 1961a: 211. Trichoton sordidum (LeConte, 1851) USA (AZ CA NV) MEX (BC JA SO) NIC Blapstinus sordidus LeConte, 1851: 146. Genus Ulus Horn, 1870 [M] Ulus Horn, 1870: 358. Type species: Blapstinus crassus LeConte, 1851, subsequent designation ( Lucas 1920 : 665). Ulus comatus Champion, 1893 MEX (VE) Ulus comatus Champion, 1893a: 530. Ulus crassus (LeConte, 1851) USA (AZ CA UT) MEX (BS YU) Blapstinus crassus LeConte, 1851: 146. Ulus elongatulus Casey, 1890 USA (AZ TX) Ulus elongatulus Casey, 1890b: 414. Ulus fimbriatus Casey, 1890 USA (TX) MEX (CH) Ulus fimbriatus Casey, 1890b: 413. Ulus hirsutus Champion, 1885 MEX (CH DU JA PU SI VE YU) GUA BEL NIC CRI PAN / CAY JAM Ulus hirsutus Champion, 1885: 133. Ulus latus Blaisdell, 1892 USA (CA) Ulus latus Blaisdell, 1892: 243. Ulus lineatulus Champion, 1885 MEX (JA) GUA NIC Ulus lineatulus Champion, 1885: 134. Ulus maritimus Casey, 1890 USA (AL FL MS TX) Ulus maritimus Casey, 1890b: 414. Ulus obliquus (LeConte, 1866) MEX (BC BS) Blapstinus obliquus LeConte, 1866b: 117. Genus Xerolinus Ivie and Hart, 2016 [M] Xerolinus Ivie and Hart, 2016: 470. Type species: Diastolinus sallei Mulsant and Rey, 1859, original designation. Xerolinus alfaroi (Garrido and Gutiérrez, 1996) CUB Diastolinus alfaroi Garrido and Gutiérrez, 1996a: 228. Xerolinus alutaceus (Casey, 1890) USA (FL) / CUB Blapstinus opacus LeConte, 1878a: 420 [junior primary homonym of Blapstinus opacus Mulsant, 1859]. Blapstinus alutaceus Casey, 1890b: 423. Synonymy: Casey (1890b : 423). Diastolinus trinitatis Marcuzzi, 1976: 127. Synonymy: Ivie and Hart (2016 : 470). Xerolinus armasi (Marcuzzi, 1988) CUB Diastolinus armasi Marcuzzi, 1988: 72. Xerolinus bahamae (Marcuzzi, 1965) BAH Diastolinus bahamae Marcuzzi, 1965: 125. Xerolinus bielawskii (Marcuzzi, 1985) CUB Diastolinus bielawskii Marcuzzi, 1985: 182. Xerolinus burtoni (Garrido and Gutiérrez, 1996) CAY Diastolinus burtoni Garrido and Gutiérrez, 1996b: 232. Xerolinus caguamensis (Marcuzzi, 1988) CUB Diastolinus caguamensis Marcuzzi, 1988: 74. Xerolinus camanoensis Hart and Ivie, 2016 VIS Xerolinus camanoensis Hart and Ivie, 2016b: 885. Xerolinus caymanensis (Marcuzzi, 1977) CAY Diastolinus caymanensis Marcuzzi, 1977: 12. Xerolinus cubanus (Marcuzzi, 1962) CUB Diastolinus cubanus Marcuzzi, 1962: 30. Xerolinus dentipes (Marcuzzi, 1977) CUB CAY Diastolinus dentipes Marcuzzi, 1977: 13. Diastolinus diformis Marcuzzi, 1977: 14. Synonymy: Garrido and Gutiérrez (1996b : 232). Xerolinus difficilis (Marcuzzi, 1976) CUB Diastolinus difficilis Marcuzzi, 1976: 126. Xerolinus dispar (Casey, 1890) USA (FL) Blapstinus dispar Casey, 1890b: 424. Xerolinus dozieri (Marcuzzi, 1965) TUR Diastolinus dozieri Marcuzzi, 1965: 128. Xerolinus elongatus (Marcuzzi, 1976) CUB Diastolinus elongatus Marcuzzi, 1976: 126. Xerolinus garridoi (Marcuzzi, 1988) CUB Diastolinus garridoi Marcuzzi, 1988: 78. Xerolinus hernandezi (Marcuzzi, 1988) CUB (Isla de Juventud) Diastolinus hernandezi Marcuzzi, 1988: 72. Xerolinus juraguensis (Marcuzzi, 1988) CUB Diastolinus juraguensis Marcuzzi, 1988: 75. Xerolinus kulzeri (Marcuzzi, 1965) BAH (Mayaguana) Diastolinus kulzeri Marcuzzi, 1965: 127. Xerolinus macamboensis (Marcuzzi, 1988) CUB Diastolinus macamboensis Marcuzzi, 1988: 77. Diastolinus garciai Marcuzzi, 1988: 79. Synonymy: Garrido and Gutiérrez (1996a : 226). Xerolinus minor (Marcuzzi, 1977) CAY Diastolinus minor Marcuzzi, 1977: 18. Xerolinus orientalis (Garrido and Gutiérrez, 1996) CUB Diastolinus orientalis Garrido and Gutiérrez, 1996a: 226. Xerolinus puncticeps (Mulsant and Rey, 1859) CUB Blapstinus puncticeps Mulsant and Rey, 1859: 181. Xerolinus rufoclavatus (Zayas, 1988) CUB Blapstinus rufoclavatus Zayas, 1988: 91. Xerolinus sallei (Mulsant and Rey, 1859) HAI DOM Diastolinus sallei Mulsant and Rey, 1859: 144. Diastolinus costipennis Mulsant and Rey, 1859: 149. Synonymy: Ivie and Hart (2016 : 473). Diastolinus puncticollis Mulsant and Rey, 1859: 147. Synonymy: Ivie and Hart (2016 : 473). Diastolinus assoi Garrido, 2004b: 40. Synonymy: Ivie and Hart (2016 : 473). Xerolinus smalli (Garrido, 2004) CUB Diastolinus smalli Garrido, 2004c: 46. Xerolinus swearingenae Hart and Ivie, 2016 JAM Xerolinus swearingenae Hart and Ivie, 2016b: 889. Xerolinus that (Steiner, 2006) BAH Diastolinus that Steiner, 2006: 25. Xerolinus this (Steiner, 2006) BAH Diastolinus this Steiner, 2006: 21. Xerolinus waterhousii (Mulsant and Rey, 1859) CUB Diastolinus waterhousii Mulsant and Rey, 1859: 152. Diastolinus kaszabi Marcuzzi, 1976: 125. Synonymy: Ivie and Hart (2016 : 474). Xerolinus zayasi (Marcuzzi, 1988) CUB Diastolinus zayasi Marcuzzi, 1988: 75. Tribe Palorini Matthews, 2003 Palorinae Matthews, 2003: 50. Type genus: Palorus Mulsant, 1854. Genus Palorus Mulsant, 1854 [M] Palorus Mulsant, 1854: 250. Type species: Hypophloeus depressus Fabricius, 1790, monotypy. Caenocorse C.G. Thomson, 1859: 117. Type species: Hypophloeus depressus Fabricius, 1790, original designation. Synonymy: Bedel (1906 : 92). Eba Pascoe, 1863: 129. Type species: Eba cerylonoides Pascoe, 1863, monotypy. Synonymy: Carter and Zeck (1937 : 194). Circomus Fleischer, 1900: 236. Type species: Hypophloeus subdepressus Wollaston, 1864, monotypy. Synonymy: Löbl et al. (2008a : 276). Palorus cerylonoides (Pascoe, 1863) USA (FL) / LAN – Adventive Eba cerylonoides Pascoe, 1863: 129. Palorus genalis Blair, 1930 BEL / LAN – Adventive Palorus genalis Blair, 1930: 140. Palorus ratzeburgii (Wissmann, 1848) [Fig. 27 ] CAN (BC NS ON QC) USA (DC FL GA IN MI NY OH OR SC WA WI) / CUB – Adventive Figure 27. Palorus ratzeburgii (Wissmann, 1848). Scale bar = 1 mm. Hypophloeus ratzeburgii Wissmann, 1848: 77. Palorus subdepressus (Wollaston, 1864) CAN (MB ON) USA (CT DC FL GA ID IN MI OH OR PA SC SD WI) MEX / LAN – Adventive Hypophloeus subdepressus Wollaston, 1864: 499. Genus Ulomina Baudi di Selve, 1876 [F] Ulomina Baudi di Selve, 1876: 235. Type species: Ulomina carinata Baudi di Selve, 1876, monotypy. Coelopalorus Blair, 1930: 135. Type species: Palorus foveicollis Blair, 1930 (= Ulomina carinata Baudi di Selve, 1876), monotypy. Synonymy: Scupola (2002 : 186). Ulomina carinata Baudi di Selve, 1876 USA (AL FL) / CUB – Adventive Ulomina carinata Baudi di Selve, 1876: 236. Palorus foveicollis Blair, 1930: 136. Synonymy: Scupola (2002 : 186). Tribe Pedinini Eschscholtz, 1829 Pediniden Eschscholtz, 1829: 4. Type genus: Pedinus Latreille, 1797. Subtribe Leichenina Mulsant, 1854 Leichenaires Mulsant, 1854: 179. Type genus: Leichenum Dejean, 1834. Genus Leichenum Dejean, 1834 [N] Leichenum Dejean, 1834: 194. Type species: Opatrum pictum Fabricius, 1801, monotypy. Lichenum Agassiz, 1846: 209. Unjustified emendation of Leichenum Dejean, 1834, not in prevailing usage. Leichenum canaliculatum variegatum (Klug, 1833) USA (AL FL GA MS NC SC) / BAH CUB LAN – Adventive Opatrum variegatum Klug, 1833: 88. Subtribe Platynotina Mulsant and Rey, 1853 Platynotaires Mulsant and Rey, 1853: 263. Type genus: Platynotus Fabricius, 1801. Genus Alaetrinus Iwan, 1995 [M] Alaetrinus Iwan, 1995: 24. Type species: Tenebrio pullus Sahlberg, 1823, original designation. Alaetrinus aciculatus (LeConte, 1858) USA (CA KS NM OK TX) MEX (NL TA) Opatrinus aciculatus LeConte, 1858b: 75. Alaetrinus minimus (Palisot de Beauvois, 1817) USA (AL AR CA CT DC DE FL GA IL IN KS KY LA MA MD MO MS NC NJ NY OH OK PA SC TN TX VA WV) / BAH CUB Tenebrio minimus Palisot de Beauvois, 1817: 164. Pedinus suturalis Say, 1824a: 263 [ nomen dubium ]. New synonymy [PB] 53 . Opatrum notum Say, 1826: 237. Synonymy: Mulsant and Rey (1853 : 309). Alaetrinus moestus (Mulsant and Rey, 1853) MEX (VE) BEL / SA Opatrinus moestus Mulsant and Rey, 1853: 307. Opatrinus lüderwaldti Gebien, 1928a: 112. Synonymy: Iwan (1995 : 47). Alaetrinus pullus (Sahlberg, 1823) USA (FL) MEX (CI QR TB VE YU) GUA BEL HON / BER BAH CUB JAM DOM PRI LAN / SA Tenebrio pullus C.R. Sahlberg, 1823: 16. Opatrinus anthracinus Mulsant and Rey, 1853: 304. Synonymy: Champion (1885 : 123). Opatrinus puertoricensis Marcuzzi, 1977: 23. Synonymy: Iwan (1995 : 38). Genus Anchophthalmops Koch, 1956 [M] Anchophthalmops Koch, 1956: 173. Type species: Anchophthalmops brevipleurum Koch, 1956, original designation. Anchophthalmops menouxi (Mulsant and Rey, 1853) USA (KS) – Adventive Selinus menouxii Mulsant and Rey, 1853: 322. Opatrinus sayi Horn, 1870: 349. Synonymy: Iwan (1995 : 52). Genus Opatrinus Dejean, 1821 [M] Opatrinus Dejean, 1821: 66. Type species: Opatrum clathratum Fabricius, 1787, monotypy. Hopatrinus Agassiz, 1846: 185. Unjustified emendation of Opatrinus Dejean, 1821, not in prevailing usage. Opatrinus clathratus (Fabricius, 1787) PAN / LAN / SA Opathrum clathratum Fabricius, 1787: 379. Blaps gemellata Olivier, 1795: [60] 9. Synonymy: Iwan (1995 : 16). Helops aethiops Fabricius, 1801a: 162. Synonymy: Gebien (1906 : 212). Opatrinus geminatus Erichson, 1849: 565. Synonymy: Gebien (1906 : 212). Opatrinus gridellii Marcuzzi, 1949: 342. Synonymy: Iwan (1995 : 16). Opatrinus armasi Garrido and Gutiérrez, 1994a: 121. Synonymy: Iwan (2002 : 282). Opatrinus gibbicollis Mulsant and Rey, 1853 PAN / SA Opatrinus gibbicollis Mulsant and Rey, 1853: 303. Tribe Tenebrionini Latreille, 1802 Tenebrionites Latreille, 1802: 165. Type genus: Tenebrio Linnaeus, 1758. Biuini Skopin, 1978: 224. Type genus: Bius Dejean, 1834. Genus Bius Dejean, 1834 [M] Bius Dejean, 1834: 205. Type species: Trogosita thoracicus Fabricius, 1792, monotypy. Bius estriatus (LeConte, 1851) CAN (AB BC YT) USA (CA ID NM MI OR WA) Tenebrio estriatus LeConte, 1851: 149. Genus Bouchardandrus Steiner, 2016 [M] Bouchardandrus Steiner, 2016: 543. Type species: Haplandrus concolor LeConte, 1866, original designation. Bouchardandrus concolor (LeConte, 1866) CAN (MB ON QC) USA (MI MN OH WI) Haplandrus concolor LeConte, 1866b: 121. Genus Idiobates Casey, 1891 [M] Idiobates Casey, 1891: 62. Type species: Tenebrio castaneus Knoch, 1801, monotypy. Idiobates castaneus (Knoch, 1801) CAN (ON QC) USA (AL AR DE FL GA IL IN KY MD MI NC NH NJ NY OH OK PA SC TN VA WI WV) Tenebrio castaneus Knoch, 1801: 171. Tenebrio interstitialis Say, 1824a: 266. Synonymy: Melsheimer (1853 : 139). Genus Neatus LeConte, 1862 [M] Neatus LeConte, 1862a: 233. Type species: Helops tenebrioides Palisot de Beauvois, 1812, monotypy. Neatus tenebrioides (Palisot de Beauvois, 1812) CAN (AB MB NB ON QC SK) USA (AL AR AZ CA CO CT DE FL GA IA IL IN KS KY LA MD MI MN MO MS MT NC ND NE NH NJ NM OH OK PA SC SD TN TX UT VA VT WI WV) Helops tenebrioïdes Palisot de Beauvois, 1812: 121. Tenebrio badius Say, 1824a: 265. Synonymy: LeConte (1859d : 156). Tenebrio rufinasus Say, 1831: 8 [ nomen dubium ]. Synonymy (in doubt with Tenebrio picipes Herbst sensu North American authors = Neatus tenebrioides ): Papp (1961d : 130, as rufimanus ). Genus Rhinandrus LeConte, 1866 [M] Rhinandrus LeConte, 1866b: 119. Type species: Rhinandrus gracilis LeConte, 1866, monotypy. Exerestus Bates, 1870: 268. Type species: Exerestus jansonii Bates, 1870 (= Rhinandrus elongatus Horn, 1866), monotypy. Synonymy: Bates (1872 : 98). Proderops Fairmaire, 1873: 393. Type species: Proderops foraminosus Fairmaire, 1873 (= Rhinandrus elongatus Horn, 1866), monotypy. Synonymy (with Exerestus Bates): Kraatz (1880 : 133). Rhinandrus elongatus Horn, 1866 MEX (YU) NIC CRI Rhinandrus elongatus Horn, 1866: 400. Exerestus jansonii Bates, 1870: 269. Synonymy: LeConte (1873 : 334). Proderops foraminosus Fairmaire, 1873: 394. Synonymy: Champion (1885 : 102). Rhinandrus foveolatus (Kraatz, 1880) MEX (OA) Proderops foveolatus Kraatz, 1880: 133. Rhinandrus gracilis LeConte, 1866 MEX (BS) Rhinandrus gracilis LeConte, 1866b: 120. Rhinandrus helopioides (Kraatz, 1880) MEX (OA) Exerestus helopioides Kraatz, 1880: 135. Rhinandrus obsoletus Champion, 1885 MEX (DU SI) Rhinandrus obsoletus Champion, 1885: 102. Genus Tenebrio Linnaeus, 1758 [M] Tenebrio Linnaeus, 1758: 417. Type species: Tenebrio molitor Linnaeus, 1758, subsequent designation ( Latreille 1810 : 429). Menedrio Motschulsky, 1872: 27. Type species: Tenebrio obscurus Fabricius, 1792, original designation. Synonymy: Heyden et al. (1883 : 134). Tenebrionellus Crotch, 1874: 105. Unnecessary replacement name for Tenebrio Linnaeus, 1758. Tenebrio molitor Linnaeus, 1758 [Fig. 28 ] CAN (AB BC MB NB NF NS ON PE QC SK) USA (AK FL GA ID IN MA MD MI NC OH OR SC SD WA WI) CRI / CUB PRI – Adventive Figure 28. Tenebrio molitor Linnaeus, 1758. Scale bar = 1 mm. Tenebrio molitor Linnaeus, 1758: 417. Tenebrio obscurus Fabricius, 1792 GRE CAN (AB BC NS ON QC SK) USA (FL GA ID IN MA MD MI NC OH OR SC SD WA WI) – Adventive Tenebrio obscurus Fabricius, 1792a: 111. Menedrio longipennis Motschulsky, 1872: 37. New synonymy [YB]. Tenebrio obscurus pollens Casey, 1924: 321. Synonymy: Bousquet and Campbell (1991 : 259). Genus Zophobas Dejean, 1834 [M] Zophobas Dejean, 1834 [30 June]: 204. Type species: Helops morio Fabricius, 1777 (= Tenebrio atratus Fabricius, 1775), subsequent designation ( Motschulsky 1872 : 26). Subgenus Macrozophobas Pic, 1913 Macrozophobas Pic, 1913b: 6. Type species: Macrozophobas gracilicornis Pic, 1913 (= Zophobas maculicollis Kirsch, 1866), monotypy. Zophobas klingelhoefferi Kraatz, 1880 MEX (VE) Zophobas klingelhöfferi Kraatz, 1880: 126. Zophobas maculicollis Kirsch, 1866 PAN / SA Zophobas maculicollis Kirsch, 1866: 196. Macrozophobas gracilicornis Pic, 1913b: 6. Synonymy: Gebien (1941 : 335). Zophobas signatus Champion, 1885 MEX GUA BEL HON NIC CRI PAN Zophobas signatus Champion, 1885: 104. Subgenus Zophobas Dejean, 1834 Zophobas Dejean, 1834 [30 June]: 204. Type species: Helops morio Fabricius, 1777 (= Tenebrio atratus Fabricius, 1775), subsequent designation ( Motschulsky 1872 : 26). Pythonissus Gistel, 1834 [23 September]: 21. Type species: Helops morio Fabricius, 1777 (= Tenebrio atratus Fabricius, 1775), subsequent designation ( Bousquet and Bouchard 2017a : 132). Synonymy: Bousquet and Bouchard (2017a : 132). Zophobas atratus (Fabricius, 1775) USA (CA FL) MEX (GE JA OA SL TB VE YU) GUA NIC CRI PAN / BAH CUB JAM HAI DOM PRI LAN / SA Tenebrio atratus Fabricius, 1775: 256. Helops morio Fabricius, 1777: 241. Synonymy: Marcuzzi and d'Aguilar (1971 : 90) 54 . Tenebrio elongatus Palisot de Beauvois, 1817: 164 [junior primary homonym of Tenebrio elongatus Herbst, 1797]. Synonymy (with H. morio Fabricius): Chevrolat (1853 : 638). Zophobas rugipes Kirsch, 1866: 197 55 . Synonymy: Tschinkel (1984 : 332). Zophobas concolor Wollaston, 1870: 33. Synonymy (with H. morio Fabricius): Champion (1896 : 26). Zophobas alternans Kraatz, 1880: 131. Synonymy: Ferrer (2011 : 298). Zophobas batavorum Marcuzzi, 1959: 88. Synonymy: Ferrer (2011 : 297). Zophobas costatus Pic, 1921 DOM Zophobas costatus Pic, 1921a: 10. Zophobas diversicolor Pic, 1921 HON Zophobas rugipes var. diversicolor Pic, 1921a: 9. Zophobas macretus Kraatz, 1880 MEX (CI ME OA TA VE YU) GUA NIC CRI Zophobas macretus Kraatz, 1880: 130. Zophobas opacus (Sahlberg, 1823) USA (NM) MEX (GE VE) GUA SAL NIC CRI PAN / PRI LAN / SA Helops opacus C.R. Sahlberg, 1823: 17. Zophabas [sic!] subnitidus Motschulsky, 1872: 35. Synonymy: Ferrer (2011 : 301). Zophabas [sic!] laticollis Motschulsky, 1872: 36. Synonymy: Ferrer (2011 : 301). Zophobas ambiguus Kraatz, 1880: 124. Synonymy: Ferrer (2011 : 301). Zophobas kraatzi Champion, 1885: 105. Synonymy: Ferrer (2011 : 301). Zophobas diversipes Pic, 1921a: 8. Synonymy: Ferrer (2011 : 301). Zophobas cubanus Marcuzzi, 1976: 128. Synonymy: Ferrer (2011 : 301). Zophobas tridentatus Kraatz, 1880 NIC PAN / SA Zophobas tridentatus Kraatz, 1880: 124. Zophobas kirschi Kraatz, 1880: 127. Synonymy: Ferrer (2011 : 299). Zophobas pedestris Champion, 1885: 103. Synonymy: Ferrer (2011 : 299). Zophobas elongatior Pic, 1921a: 9. Synonymy: Ferrer (2011 : 299). [incertae sedis] Zophobas subnitens (Horn, 1874) USA (AZ) MEX (SO) Nyctobates subnitens Horn, 1874a: 35. Rhinandrus sublaevis Horn, 1885c: 160. Synonymy: Spilman (1962b : 60). Tribe Toxicini Oken, 1843 Toxiciden Oken, 1843: 484. Type genus: Toxicum Latreille, 1802. Subtribe Dysantina Gebien, 1922 Dysantinae Gebien, 1922: 289. Type genus: Dysantes Pascoe, 1869. Eudysantina Bouchard, Lawrence, Davies and Newton, 2005: 508. Type genus: Eudysantes Bouchard et al., 2005 (= Dysantes Pascoe, 1869). Note. Dysantes Pascoe was first made available in 1869 [on 1 January], not in 1871 as previously noted. The name is a senior homonym of the ichneumonid Dysantes Förster, 1869 [May], which was incorrectly dated 1868. The replacement name of the family-group name proposed by Bouchard et al. (2005) was thus unnecessary. A full explanation about this case will be issued in a forthcoming publication by YB and PB. Genus Diceroderes Solier, 1841 [M] Diceroderes Solier, 1841: 30, 46. Type species: Diceroderes mexicanus Solier, 1841, original designation. Prosomenes Blanchard, 1845: 10. Type species: Diceroderes mexicanus Solier, 1841, subsequent monotypy ( Chevrolat 1847 : 562). Note. The generic name Prosomenes was listed as a junior synonym of Dicérodères by Blanchard (1845 : 10). However, because the name was treated before 1961 as an available name and adopted as the name of a taxon (e.g., Chevrolat 1847 : 562), it is made available thereby but dates from its first publication as a synonym ( ICZN 1999 : Article 11.6.1). Diceroderes cusucoensis Smith, 2015 GUA HON Diceroderes cusucoensis Smith [in Smith and Cifuentes-Ruiz], 2015: 62. Diceroderes mexicanus Solier, 1841 MEX (HI PU VE) Diceroderes mexicanus Solier, 1841: 49. Diceroderes ocozocoautlaensis Smith, 2015 MEX (CI) Diceroderes ocozocoautlaensis Smith [in Smith and Cifuentes-Ruiz], 2015: 63. Diceroderes skelleyi Smith, 2015 GUA Diceroderes skelleyi Smith [in Smith and Cifuentes-Ruiz], 2015: 65. Diceroderes subtriplehorni Smith and Cifuentes-Ruiz, 2015 MEX (OA PU VE) Diceroderes subtriplehorni Smith and Cifuentes-Ruiz, 2015: 67. Genus Ozolais Pascoe, 1866 [F] Ozolais Pascoe, 1866: 457. Type species: Ozolais scruposa Pascoe, 1866, monotypy. Ozolais elongata Champion, 1886 NIC PAN Ozolais elongata Champion, 1886: 228. Ozolais lutosa Champion, 1886 CRI Ozolais lutosa Champion, 1886: 227. Ozolais nodosa Champion, 1886 NIC Ozolais nodosa Champion, 1886: 228. Ozolais tuberculifera Champion, 1896 LAN Ozolais tuberculifera Champion, 1896: 10. Ozolais verrucosa Champion, 1886 PAN Ozolais verrucosa Champion, 1886: 226. Genus Wattius Kaszab, 1982 56 [M] Wattius Kaszab, 1982: 50. Type species: Calymmus cucullatus Pascoe, 1871, original designation. Wattius andersoni Smith and Sanchez, 2015 CUB Wattius andersoni Smith and Sanchez, 2015: 118. Wattius emmabaconae Smith and Sanchez, 2015 DOM Wattius emmabaconae Smith and Sanchez, 2015: 121. Wattius variegatus (Champion, 1886) NIC Calymmus variegatus Champion, 1886: 225. Wattius viatorus Smith and Sanchez, 2015 BAH CUB Wattius viatorus Smith and Sanchez, 2015: 125. Tribe Triboliini Gistel, 1848 Triboliidae Gistel, 1848: [4]. Type genus: Tribolium MacLeay, 1825. Genus Aesymnus Champion, 1886 [M] Aesymnus Champion, 1886: 168. Type species: Aesymnus nitidus Champion, 1886, monotypy. Aesymnus nitidus Champion, 1886 MEX (VE) PAN Aesymnus nitidus Champion, 1886: 168. Genus Hypogena Dejean, 1834 [F] Hypogena Dejean, 1834: 199. Type species: Tenebrio biimpressus Latreille, 1813, monotypy. Ulosonia Laporte, 1840: 220. Type species: Uloma tricornis Laporte, 1840 (= Phaleria tricornis Dalman, 1823), subsequent designation ( Gebien 1940 : 786). Synonymy: Jacquelin du Val (1857 : 148). Hypogena biimpressa (Latreille, 1813) MEX (CI DU JA OA PU SI TA VE YU) GUA BEL NIC PAN / CUB DOM LAN / SA Tenebrio biimpressus Latreille, 1813: 17. Hypogena canaliculata (Champion, 1886) NIC CRI PAN Ulosonia canaliculata Champion, 1886: 164. Hypogena dejeani (Champion, 1886) GUA / SA Ulosonia dejeani Champion, 1886: 165. Hypogena depressa (Champion, 1886) MEX (MO) Ulosonia depressa Champion, 1886: 164. Hypogena marginata (LeConte, 1851) USA (AZ CA) MEX (BS) Uloma marginata LeConte, 1851: 149. Hypogena tricornis (Dalman, 1823) USA (FL TX) MEX (BS JA OA PU VE YU) GUA BEL NIC CRI / BAH CUB CAY / SA Phaleria tricornis Dalman, 1823: 59. Ulosonia tricornis Laporte, 1840: 220. Synonymy: Spilman (1973 : 42) 57 . Genus Latheticus C.O. Waterhouse, 1880 [M] Latheticus C.O. Waterhouse, 1880: 147. Type species: Latheticus oryzae C.O. Waterhouse, 1880, monotypy. Latheticus oryzae C.O. Waterhouse, 1880 [Fig. 29 ] CAN (AB MB NB QC SK) USA (FL GA MD MI NC OH SC TX) / CUB HIS – Adventive Figure 29. Latheticus oryzae C.O. Waterhouse, 1880. Scale bar = 1 mm. Latheticus oryzae C.O. Waterhouse, 1880: 148. Latheticus prosopis Chittenden, 1904 USA (AZ CA FL) MEX (BS) Latheticus prosopis Chittenden, 1904: 167. Genus Lyphia Mulsant and Rey, 1859 [F] Lyphia Mulsant and Rey, 1859: 166. Type species: Lyphia ficicola Mulsant and Rey, 1859 (= Bius tetraphyllus Fairmaire, 1857), monotypy. Lyphia tetraphylla (Fairmaire, 1857) USA (DC FL GA MD OH) – Adventive Bius tetraphyllus Fairmaire, 1857: 534. Lyphia ficicola Mulsant and Rey, 1859: 166. Synonymy: Marseul (1876 : 113). Hypophloeus rugosus Dury, 1902: 171. Synonymy (with L. ficicola Mulsant and Rey): Schwarz [in Dury] (1902 : [198]). Genus Metulosonia Bates, 1873 [F] Metulosonia Bates, 1873d: 261. Type species: Metulosonia horni Bates, 1873, subsequent designation ( Gebien 1940 : 1061). Metulosonia horni Bates, 1873 PAN Metulosonia horni Bates, 1873d: 262. Metulosonia reflexa (Chevrolat, 1878) MEX (VE) GUA BEL NIC Peltoides reflexus Chevrolat, 1878c: 237. Genus Mycotrogus Horn, 1870 [M] Mycotrogus Horn, 1870: 367. Type species: Mycotrogus piceus Horn, 1870, subsequent designation ( Lucas 1920 : 427). Mycotrogus angustus Horn, 1870 USA (AZ CA) Mycotrogus angustus Horn, 1870: 368. Mycotrogus mentalis Blaisdell, 1923 USA (AZ) MEX (BC BS) Mycotrogus mentalis Blaisdell, 1923: 279. Mycotrogus paripunctatus Spilman, 1963 CUB Mycotrogus paripunctatus Spilman, 1963: 23. Mycotrogus piceus Horn, 1870 USA (CA) Mycotrogus piceus Horn, 1870: 367. Genus Spelaebiosis 58 Bousquet and Bouchard, new replacement name [F] Orghidania Ardoin, 1977b: 383 [junior homonym of Orghidania Capuse, 1971]. Type species: Orghidania torrei Ardoin, 1977, monotypy. Ardoinia Özdikmen, 2004: 202 [junior homonym of Ardoinia Kaszab, 1969]. Replacement name for Orghidania Ardoin, 1977. Spelaebiosis Bousquet and Bouchard, new replacement name for Ardoinia Özdikmen, 2004. Spelaebiosis torrei (Ardoin, 1977) CUB Orghidania torrei Ardoin, 1977b: 384. Genus Tribolium MacLeay, 1825 [N] Tribolium MacLeay, 1825: 47. Type species: Colydium castaneum Herbst, 1797, monotypy. Subgenus Aphanotus LeConte, 1862 Aphanotus LeConte, 1862a: 233. Type species: Eulabis brevicornis LeConte, 1859, original designation. Tribolium brevicorne (LeConte, 1859) CAN (BC) USA (CA OR WA) Eulabis brevicornis LeConte, 1859b: 78. Tribolium parallelum (Casey, 1890) USA (AZ) MEX (CL) Aphanotus parallelus Casey, 1890b: 483. Tribolium setosum Triplehorn, 1978 USA (AZ) Tribolium setosum Triplehorn, 1978: 73. Subgenus Tribolium MacLeay, 1825 59 Tribolium MacLeay, 1825: 47. Type species: Colydium castaneum Herbst, 1797, monotypy. Stene Stephens, 1829: 19. Type species: Tenebrio ferrugineus Fabricius sensu auctorum (= Colydium castaneum Herbst, 1797), monotypy. Synonymy: Shuckard (1840 : vii). Margus Dejean, 1834: 200. Type species: Tenebrio ferrugineus Fabricius sensu auctorum (= Colydium castaneum Herbst, 1797), monotypy. Synonymy: Guérin-Méneville (1846 : cxvii). Tribolium audax Halstead, 1969 CAN (AB BC MB ON QC SK) USA (FL MI MN OH PA SD UT VA) Tribolium audax Halstead, 1969: 296. Tribolium castaneum (Herbst, 1797) CAN (AB BC MB NB NS ON PE QC SK) USA (AL CA FL GA ID IL IN MA MD MI MS NC NY OH OR PA SC SD TN VA WA WI) MEX (BS CO GE NL OA) GUA NIC PAN / CUB CAY JAM HAI DOM PRI LAN / SA – Adventive Dermestes navalis Fabricius, 1775: 56. Note. This name was suppressed for the purposes of the Principle of Priority ( ICZN 1987 ). Colydium castaneum Herbst, 1797: 282. Synonymy: Schönherr (1806 : 153). Tribolium confusum Jacquelin du Val, 1862 [Fig. 30 ] CAN (AB BC MB NB NF NS ON PE QC SK) USA (AK CT FL GA ID IL IN MD MI NC NJ NY OH OR PA SC SD VA WA WI) MEX (CO GU NL) PAN / CUB JAM HAI DOM PRI / SA – Adventive Figure 30. Tribolium ( Tribolium ) confusum Jacquelin du Val, 1862. Scale bar = 1 mm. Tribolium confusum Jacquelin du Val, 1862: 181. Tribolium destructor Uyttenboogaart, 1934 GRE CAN (AB BC MB NB NF NS ON PE QC SK YT) USA (CA WA WI) – Adventive Tribolium destructor Uyttenboogaart, 1934: 21. Tribolium madens (Charpentier, 1825) CAN (MB NB NS ON QC) USA (KY MD MI NM PA) – Adventive Tenebrio madens Charpentier, 1825: 218. Tribolium linsleyi Hinton, 1948 MEX (CL) Tribolium linsleyi Hinton, 1948: 32. Tribe Ulomini Blanchard, 1845 Ulomites Blanchard, 1845: 16. Type genus: Uloma Dejean, 1821. Alégoriides Lacordaire, 1859: 325. Type genus: Alegoria Laporte, 1840. Genus Alegoria Laporte, 1840 [F] Alegoria Laporte, 1840: 221. Type species: Alegoria dilatata Laporte, 1840, monotypy. Alegoria castelnaui Fleutiaux and Sallé, 1890 60 LAN Allegoria [sic!] castelnaui Fleutiaux and Sallé, 1890: 425. Alegoria dilatata Laporte, 1840 MEX GUA HON NIC PAN / LAN / SA Alegoria dilatata Laporte, 1840: 221. Alegoria sallei Bates, 1873 MEX (OA VE) Alegoria sallei Bates, 1873a: 181. Alegoria sallaei Champion, 1886: 149. Unjustified emendation of Alegoria sallei Bates, 1873, not in prevailing usage. Genus Antimachus Gistel, 1829 [M] Antimachus Gistel, 1829: 1055. Type species: Phaleria furcifera Dalman, 1821, monotypy. Ceratupis Perty, 1830: 57. Type species: Ceratupis nigerrima Perty, 1830, monotypy. Synonymy: Lacordaire (1859 : 330). Antimachus ardoini Chalumeau, 1982 LAN (Martinique) Antimachus ardoini Chalumeau, 1982: 188. Antimachus coriaceus Lacordaire, 1859 NIC PAN / SA Antimachus coriacea Lacordaire, 1859: 331. Antimachus roudeni Fleutiaux and Sallé, 1890 LAN Antimachus roudeni Fleutiaux and Sallé, 1890: 426. Genus Eutochia LeConte, 1862 [F] Eutochia LeConte, 1862a: 238. Replacement name for Aniara Lacordaire, 1859. Subgenus Eutochia LeConte, 1862 Aniara Lacordaire, 1859: 336 [junior homonym of Aniara Hope, 1838]. Type species: Uloma picea Melsheimer, 1846, monotypy. Eutochia LeConte, 1862a: 238. Replacement name for Aniara Lacordaire, 1859. Delopygus LeConte, 1866b: 129. Type species: Delopygus crenatus LeConte, 1866, monotypy. Synonymy: Horn (1870 : 372). Aniarus Gemminger [in Gemminger and Harold], 1870: 1964. Unjustified emendation of Aniara Lacordaire, 1859, not in prevailing usage. Eutochia crenata (LeConte, 1866) USA (TX) Delopygus crenatus LeConte, 1866b: 130. Eutochia picea (Melsheimer, 1846) [Fig. 31 ] USA (AL AR DC FL GA IL IN KY MD MO NC NJ NY OH OK PA SC TN TX VA WV) Figure 31. Eutochia ( Eutochia ) picea (Melsheimer, 1846). Scale bar = 1 mm. Uloma picea Melsheimer, 1846: 64. Genus Pheres Champion, 1886 [M] Pheres Champion, 1886: 150. Type species: Pheres batesi Champion, 1886, monotypy. Pheres batesi Champion, 1886 PAN Pheres batesi Champion, 1886: 150. Genus Uleda Laporte, 1840 [F] Uleda Laporte, 1840: 220. Type species: Uloma diaperoides Laporte, 1840, monotypy. Uleda tarsalis (Perroud and Mulsant, 1856) MEX / SA 61 Melasia tarsalis Perroud and Mulsant, 1856: 163. Uleda grossa Champion, 1886: 151. Synonymy: Gebien (1940 : 771). Genus Uloma Dejean, 1821 [F] Uloma Dejean, 1821: 67. Type species: Tenebrio culinaris Linnaeus, 1758 (see ICZN 1975). Uloma antillarum Champion, 1896 LAN Uloma antillarum Champion, 1896: 22. Uloma armata Champion, 1886 GUA BEL Uloma armata Champion, 1886: 154. Uloma carolynae Doyen, 1985 MEX (CI) Uloma carolynae Doyen, 1985a: 518. Uloma divergens Champion, 1886 GUA Uloma divergens Champion, 1886: 155. Uloma extraordinaria Spilman, 1961 CUB Uloma extraordinaria Spilman, 1961b: 113. Uloma fossulata Champion, 1886 MEX (VE) GUA BEL Uloma fossulata Champion, 1886: 153. Uloma grenadensis Champion, 1896 LAN Uloma grenadensis Champion, 1896: 23. Uloma imberbis LeConte, 1866 USA (AL AR DE FL GA IA IL IN KS KY LA MD MI MO NC NJ NY OH OK SC TN TX VA) Uloma imberbis LeConte, 1866b: 123. Uloma impressa Melsheimer, 1846 CAN (ON) USA (AL FL GA IA IL IN LA MA MD MI NC NE NH NJ NY OH PA RI SC VA WI) Uloma impressa Melsheimer, 1846: 64. Uloma laevicollis Champion, 1886 GUA NIC CRI PAN Uloma laevicollis Champion, 1886: 153. Uloma longula LeConte, 1861 CAN (BC) USA (CA OR WA) Uloma longula LeConte, 1861b: 353. Uloma mentalis Horn, 1870 CAN (ON) USA (AL AR FL GA IN KS MD MI MS NC NY OH SC TN TX WI) Uloma mentalis Horn, 1870: 371. Uloma mexicana (Lacordaire, 1859) MEX (VE) GUA BEL SAL NIC CRI Antimachus mexicana Lacordaire, 1859: 331. Uloma moensis Marcuzzi, 2000 CUB Uloma moensis Marcuzzi, 2000: 286. Uloma parvula Champion, 1896 LAN Uloma parvula Champion, 1896: 23. Uloma punctulata LeConte, 1866 USA (FL GA IN LA MD MI MS NC NY OH SC TN TX VA WI) Uloma punctulata LeConte, 1866b: 124. 62 Uloma cava LeConte, 1866b: 124. Synonymy: Horn (1870 : 372). Uloma retusa ephippigera (Guérin-Méneville, 1831) MEX (VE) BEL NIC CRI PAN / LAN / SA Phaleria ephippiger Guérin-Méneville, 1831b: pl. 2. Uloma bicolor Kirsch, 1874: 403. Synonymy: Gebien (1928a : 160). Uloma retusa var. dimidiata Champion, 1886: 154. Synonymy (with U. bicolor Kirsch): Gebien (1911a : 403). Uloma retusa retusa (Fabricius, 1801) MEX (JA QR SI VE YU) / PRI LAN / SA Tenebrio retusus Fabricius, 1801a: 149. Uloma rubens Laporte, 1840 "Amérique du Nord" Uloma rubens Laporte, 1840: 220 63 . Uloma spinipes Champion, 1886 GUA Uloma spinipes Champion, 1886: 155. Uloma sulcata Champion, 1896 LAN Uloma sulcata Champion, 1896: 21. Subfamily ALLECULINAE Laporte, 1840 Alléculites Laporte, 1840: 242. Type genus: Allecula Fabricius, 1801. Tribe Alleculini Laporte, 1840 Alléculites Laporte, 1840: 242. Type genus: Allecula Fabricius, 1801. Subtribe Alleculina Laporte, 1840 Alléculites Laporte, 1840: 242. Type genus: Allecula Fabricius, 1801. Upinellae LeConte, 1866b: 137. Type genus: Upinella Mulsant, 1856. Genus Aeanes Champion, 1893 [M] Aeanes Champion, 1893a: 566. Type species: Aeanes angusticollis Champion, 1893, monotypy. Aeanes angusticollis Champion, 1893 MEX (GE) Aeanes angusticollis Champion, 1893a: 567. Genus Alethia Champion, 1888 [F] Alethia Champion, 1888: 417. Type species: Alethia sallaei Champion, 1888, original designation. Alethia azteca Champion, 1888 MEX (GU) Alethia azteca Champion, 1888: 418. Alethia carbonaria Schaeffer, 1905 USA (AZ) Alethia carbonaria Schaeffer, 1905b: 176. Hymenorus liebecki Fall, 1931b: 245. Synonymy: Marshall (1970b : 1). Alethia funerea Champion, 1888 MEX (GE) Alethia funerea Champion, 1888: 419. Alethia hoegei Champion, 1888 MEX (CH) Alethia högei Champion, 1888: 420. Alethia lepturoides Champion, 1888 MEX Alethia lepturoides Champion, 1888: 419. Alethia longipennis Champion, 1888 MEX (AG) Alethia longipennis Champion, 1888: 418. Alethia nitidipennis Champion, 1893 MEX (GE) Alethia nitidipennis Champion, 1893a: 565. Alethia quadricollis (Fall, 1931) USA (TX) Hymenorus quadricollis Fall, 1931b: 246. Alethia sallaei Champion, 1888 MEX (GU) Alethia sallaei Champion, 1888: 417. Alethia subnitida Champion, 1888 MEX (GE JA) Alethia subnitida Champion, 1888: 418. Genus Allecula Fabricius, 1801 [F] Allecula Fabricius, 1801b: 21. Type species: Allecula morio Fabricius, 1801, subsequent designation ( Duponchel 1840 : 283). Allecula angustata Champion, 1888 MEX (HI MO) Allecula angustata Champion, 1888: 416. Allecula belti Champion, 1888 NIC Allecula belti Champion, 1888: 414. Allecula brachyptera Doyen, 1990 MEX (JA) Allecula brachyptera Doyen, 1990: 241. Allecula caribea Campbell, 1971 PRI Allecula caribea Campbell, 1971: 67. Allecula castaneipennis Champion, 1888 CRI PAN / SA Allecula castaneipennis Champion, 1888: 412. Allecula depressa Champion, 1888 MEX (OA) Allecula depressa Champion, 1888: 415. Allecula ferox Champion, 1888 GUA Allecula ferox Champion, 1888: 413. Allecula gaumeri Champion, 1888 MEX (YU) Allecula gaumeri Champion, 1888: 414. Allecula inconspicua Borchmann, 1937 MEX (VE) Allecula inconspicua Borchmann, 1937: 212. Allecula laticeps Champion, 1888 MEX (OA) Allecula laticeps Champion, 1888: 416. Allecula opacipennis Champion, 1888 MEX (OA) Allecula opacipennis Champion, 1888: 415. Allecula pilipes Champion, 1888 MEX (VE) Allecula pilipes Champion, 1888: 414. Allecula ramosi Campbell, 1971 DOM PRI Allecula ramosi Campbell, 1971: 66. Allecula rugicollis Champion, 1888 MEX (GE JA) Allecula rugicollis Champion, 1888: 412. Allecula veraepacis Champion, 1888 GUA Allecula veraepacis Champion, 1888: 413. Genus Amaropsis Champion, 1893 [F] Amaropsis Champion, 1893a: 567. Type species: Amaropsis annulicornis Champion, 1893, monotypy. Amaropsis annulicornis Champion, 1893 MEX (VE) Amaropsis annulicornis Champion, 1893a: 568. Genus Charisius Champion, 1888 [M] Charisius Champion, 1888: 421. Type species: Charisius fasciatus Champion, 1888, subsequent designation ( Lucas 1920 : 178). Narses Champion, 1888: 423. Type species: Narses subalatus Champion, 1888, monotypy. Synonymy: Campbell (2014a : 271). Charisius apterus Campbell, 2014 MEX (OA) Charisius apterus Campbell, 2014a: 278. Charisius fasciatus Champion, 1888 MEX (CI) GUA SAL HON Charisius fasciatus Champion, 1888: 421. Charisius granulatus Campbell, 2014 GUA Charisius granulatus Campbell, 2014a: 277. Charisius howdenorum Campbell, 2014 MEX (CI) Charisius howdenorum Campbell, 2014a: 287. Charisius mexicanus Campbell, 1965 MEX (GE ME MI MO OA PU) Charisius mexicanus Campbell, 1965: 49. Charisius picturatus Champion, 1893 MEX (GE ME OA) Charisius picturatus Champion, 1893a: 565. Charisius punctatus Campbell, 2014 GUA Charisius punctatus Campbell, 2014a: 290. Charisius salvini Champion, 1888 GUA SAL HON NIC Charisius salvini Champion, 1888: 423. Charisius subalatus (Champion, 1888) GUA SAL Narses subalatus Champion, 1888: 424. Charisius zunilensis Champion, 1888 MEX (CI VE) GUA HON Charisius zunilensis Champion, 1888: 422. Charisius interstitialis Champion, 1888: 422. Synonymy: Campbell (2014a : 285). Charisius floridanus Linell, 1899: 184. Synonymy (with C. interstitialis Champion): Campbell (1965 : 51). Genus Diopoenus Champion, 1888 [M] Diopoenus Champion, 1888: 445. Type species: Diopoenus compressicornis Champion, 1888, monotypy. Diopoenus compressicornis Champion, 1888 MEX (PU) Diopoenus compressicornis Champion, 1888: 445. Genus Hymenorus Mulsant, 1852 [M] Hymenorus Mulsant, 1852: 68 [as Hymenophorus ]. Type species: Hymenorus doublieri Mulsant, 1852, monotypy. Note. See Bousquet et al. (2015 : 133) for precedence of the spelling Hymenorus over Hymenophorus . Hymenorus alienus Fall, 1931 USA (AZ) Hymenorus alienus Fall, 1931b: 217. Hymenorus americanus Champion, 1888 MEX (CL GE VE) GUA NIC Hymenorus americanus Champion, 1888: 438. Hymenorus anguillae Campbell, 1971 LAN (Anguilla) Hymenorus anguillae Campbell, 1971: 76. Hymenorus angustatus Champion, 1888 MEX (FD) GUA Hymenorus angustatus Champion, 1888: 436. Hymenorus antillensis Campbell, 1971 LAN Hymenorus antillensis Campbell, 1971: 77. Hymenorus apacheanus Casey, 1891 USA (AZ CA) Hymenorus apacheanus Casey, 1891: 99. Hymenorus arkansanus Fall, 1931 USA (FL AR) Hymenorus arkansanus Fall, 1931b: 183. Hymenorus atratus Fall, 1931 USA (AZ) Hymenorus atratus Fall, 1931b: 189. Hymenorus badius Champion, 1888 MEX (VE) Hymenorus badius Champion, 1888: 433. Hymenorus bahamensis Campbell, 1971 BAH CUB Hymenorus bahamensis Campbell, 1971: 88. Hymenorus balli Campbell, 2014 MEX (CI) GUA Hymenorus balli Campbell, 2014b: 299. Hymenorus bifurcatus Campbell, 2014 GUA Hymenorus bifurcatus Campbell, 2014b: 301. Hymenorus bitumescens Fall, 1931 USA (AZ) Hymenorus bitumescens Fall, 1931b: 194. Hymenorus brevicornis Champion, 1888 MEX (FD VE) Hymenorus brevicornis Champion, 1888: 426. Hymenorus brevipes Champion, 1888 MEX (GE) Hymenorus brevipes Champion, 1888: 435. Hymenorus brevis Fall, 1931 USA (AZ) Hymenorus brevis Fall, 1931b: 230. Hymenorus caducus Fall, 1931 USA (AL FL) Hymenorus caducus Fall, 1931b: 213. Hymenorus canaliculatus Champion, 1888 MEX (VE) Hymenorus canaliculatus Champion, 1888: 428. Hymenorus capensis Fall, 1931 MEX (BS) Hymenorus capensis Fall, 1931b: 205. Hymenorus castaneus Champion, 1888 MEX (DU) Hymenorus castaneus Champion, 1888: 434. Hymenorus cassus Fall, 1931 MEX (BC) Hymenorus cassus Fall, 1931b: 197. Hymenorus caurinus Fall, 1931 CAN (BC) USA (OR) Hymenorus caurinus Fall, 1931b: 185. Hymenorus chiriquensis Campbell, 1962 PAN Hymenorus chiriquensis Campbell, 1962: 95. Hymenorus colonoides Champion, 1888 MEX (GU JA PU VE) GUA Hymenorus colonoides Champion, 1888: 435. Hymenorus communis LeConte, 1866 USA (FL GA MD NC NY PA SC WI) Hymenorus communis LeConte, 1866b: 135. Hymenorus confertus LeConte, 1866 MEX (BS) Hymenorus confertus LeConte, 1866b: 136. Hymenorus conformis Fall, 1931 USA (TX) Hymenorus conformis Fall, 1931b: 199. Hymenorus conicicollis Fall, 1931 USA (GA SC) Hymenorus conicicollis Fall, 1931b: 239. Hymenorus convexus Casey, 1891 USA (FL TX) / BAH TUR CUB CAY Hymenorus convexus Casey, 1891: 106. Hymenorus corticarioides Champion, 1888 MEX (CL GE) Hymenorus corticarioides Champion, 1888: 441. Hymenorus crinitus Fall, 1931 USA (AZ) Hymenorus crinitus Fall, 1931b: 244. Hymenorus curticollis Casey, 1891 USA (AR IA IN MS PA) Hymenorus curticollis Casey, 1891: 95. Hymenorus cubensis Campbell, 1971 CUB Hymenorus cubensis Campbell, 1971: 81. Hymenorus darlingtoni Campbell, 1971 CUB Hymenorus darlingtoni Campbell, 1971: 83. Hymenorus densus LeConte, 1866 USA (AL FL GA IN NC SC TX) MEX (VE) / BAH Hymenorus densus LeConte, 1866b: 138. Hymenorus deplanatus Champion, 1888 USA (AZ) MEX (SO) Hymenorus deplanatus Champion, 1888: 440. Hymenorus gemellus Casey, 1891: 121. Synonymy: Fall (1931b : 231). Hymenorus depressus Champion, 1888 MEX (GE) Hymenorus depressus Champion, 1888: 435. Hymenorus dichrous Blatchley, 1919 USA (FL GA NC SC) Hymenorus dichrous Blatchley, 1919: 66. Hymenorus difficilis Casey, 1891 USA (NY) Hymenorus difficilis Casey, 1891: 94. Hymenorus digressus Fall, 1931 USA (AZ) Hymenorus digressus Fall, 1931b: 206. Hymenorus discrepans Casey, 1891 USA (CA) Hymenorus discrepans Casey, 1891: 98. Hymenorus discretus Casey, 1891 CAN (ON QC) USA (FL GA IN MA MD MN MO NC NE NJ NY PA RI SC VA WI) Hymenorus discretus Casey, 1891: 105. Hymenorus disparatus Fall, 1931 USA (AZ CO NM TX) Hymenorus disparatus Fall, 1931b: 215. Hymenorus dissensus Casey, 1891 USA (TX) Hymenorus dissensus Casey, 1891: 109. Hymenorus distinctus Fall, 1931 USA (AL FL GA MS SC) Hymenorus distinctus Fall, 1931b: 179. Hymenorus dorsalis Schwarz, 1878 USA (AL FL GA NC SC) Hymenorus dorsalis Schwarz, 1878: 370. Hymenorus sabalensis Blatchley, 1919: 67. Synonymy: Fall (1931b : 212). Hymenorus dubius Fall, 1931 USA (AL FL GA MS SC) Hymenorus dubius Fall, 1931b: 184. Hymenorus durangoensis Champion, 1888 MEX (DU) Hymenorus durangoensis Champion, 1888: 426. Hymenorus emmenastoides Champion, 1888 MEX (VE) GUA Hymenorus emmenastoides Champion, 1888: 436. Hymenorus excavatus Campbell, 2014 GUA Hymenorus excavatus Campbell, 2014b: 305. Hymenorus exiguus Casey, 1891 USA (AZ CA TX) Hymenorus exiguus Casey, 1891: 100. Hymenorus exilis Fall, 1931 USA (AZ) Hymenorus exilis Fall, 1931b: 233. Hymenorus facetus Fall, 1931 MEX (BS) Hymenorus facetus Fall, 1931b: 234. Hymenorus farri Campbell, 1971 USA (FL) MEX (VE) GUA BEL / BAH TUR CUB CAY JAM PRI LAN Hymenorus farri Campbell, 1971: 84. Hymenorus flohri Champion, 1888 MEX (FD MO) Hymenorus flohri Champion, 1888: 429. Hymenorus floridanus Casey, 1891 USA (FL) Hymenorus floridanus Casey, 1891: 116. Hymenorus forreri Champion, 1888 MEX (DU) Hymenorus forreri Champion, 1888: 431. Hymenorus foveiventris Champion, 1888 GUA Hymenorus foveiventris Champion, 1888: 432. Hymenorus fuscipennis Fall, 1931 USA (FL) Hymenorus fuscipennis Fall, 1931b: 211. Hymenorus fusculus Casey, 1891 USA (CA) Hymenorus fusculus Casey, 1891: 117. Hymenorus fusicornis Casey, 1891 USA (CA) Hymenorus fusicornis Casey, 1891: 112. Hymenorus grandicollis Champion, 1888 USA (AZ) MEX (SO) Hymenorus grandicollis Champion, 1888: 429. Hymenorus granulatus Blatchley, 1912 USA (FL) Hymenorus granulatus Blatchley, 1912: 331. Hymenorus guatemalensis Champion, 1888 GUA Hymenorus guatemalensis Champion, 1888: 439. Hymenorus haitellus Campbell, 1971 HAI Hymenorus haitellus Campbell, 1971: 94. Hymenorus haitius Campbell, 1971 HAI DOM Hymenorus haitius Campbell, 1971: 93. Hymenorus helvinus Casey, 1891 USA (TX) Hymenorus helvinus Casey, 1891: 101. Hymenorus heteropygus Fall, 1931 USA (FL GA MS) Hymenorus heteropygus Fall, 1931b: 241. Hymenorus hispaniolensis Campbell, 1971 HAI DOM Hymenorus hispaniolensis Campbell, 1971: 78. Hymenorus hispidulus Champion, 1888 MEX (VE) Hymenorus hispidulus Champion, 1888: 431. Hymenorus horrescens Fall, 1931 USA (NM TX) Hymenorus horrescens Fall, 1931b: 235. Hymenorus humeralis LeConte, 1866 USA (AL FL KY MD OH PA SC TN) Hymenorus humeralis LeConte, 1866b: 135. Hymenorus idoneus Fall, 1931 USA (AZ) Hymenorus idoneus Fall, 1931b: 218. Hymenorus igualensis Champion, 1888 MEX (GE JA) Hymenorus igualensis Champion, 1888: 434. Hymenorus illusus Fall, 1931 USA (AL FL GA MD SC) Hymenorus illusus Fall, 1931b: 192. Hymenorus inaequalis Casey, 1891 USA (AZ) Hymenorus inaequalis Casey, 1891: 114. Hymenorus incertus Fall, 1931 USA (AZ) Hymenorus incertus Fall, 1931b: 220. Hymenorus indutus Casey, 1891 USA (AZ NM TX) Hymenorus indutus Casey, 1891: 119. Hymenorus infuscatus Casey, 1891 USA (CA) Hymenorus infuscatus Casey, 1891: 90. Hymenorus inopiatus Fall, 1931 USA (FL GA MD SC) Hymenorus inopiatus Fall, 1931b: 242. Hymenorus inquilinus Casey, 1891 USA (CA) Hymenorus inquilinus Casey, 1891: 112. Hymenorus insularis Campbell, 1971 BAH Hymenorus insularis Campbell, 1971: 91. Hymenorus intermedius Casey, 1891 USA (AZ TX) Hymenorus intermedius Casey, 1891: 102. Hymenorus inutilis Fall, 1931 USA (AZ NM NV) Hymenorus inutilis Fall, 1931b: 208. Hymenorus irritus Fall, 1931 USA (AZ CA) Hymenorus irritus Fall, 1931b: 199. Hymenorus jacobinus Fall, 1931 USA (CA) Hymenorus jacobinus Fall, 1931b: 206. Hymenorus jamaicensis Campbell, 1971 CAY JAM Hymenorus jamaicensis Campbell, 1971: 79. Hymenorus laticollis Champion, 1888 MEX (FD GE JA) Hymenorus laticollis Champion, 1888: 429. Hymenorus longicollis Champion, 1888 MEX (VE) Hymenorus longicollis Champion, 1888: 434. Hymenorus macilentus Fall, 1931 USA (NM) Hymenorus macilentus Fall, 1931b: 188. Hymenorus maritimus Champion, 1888 GUA Hymenorus maritimus Champion, 1888: 437. Hymenorus melsheimeri Casey, 1891 USA (MI NY SC) Hymenorus melsheimeri Casey, 1891: 92. Hymenorus milleporus Fall, 1931 USA (AZ) Hymenorus milleporus Fall, 1931b: 236. Hymenorus minutus Campbell, 1971 BAH Hymenorus minutus Campbell, 1971: 98. Hymenorus molestus Fall, 1931 CAN (NB NS ON PE QC) USA (IN LA PA WI) Hymenorus molestus Fall, 1931b: 182. Hymenorus montivagus Fall, 1931 USA (CA) Hymenorus montivagus Fall, 1931b: 207. Hymenorus nevadensis Fall, 1931 USA (NV) Hymenorus nevadensis Fall, 1931b: 236. Hymenorus niger (Melsheimer, 1846) CAN (MB NB NS ON PE QC) USA (FL GA IN MA MD MI MN MS NC NY PA SC TX WI) Mycetocharus niger Melsheimer, 1846: 59. Hymenorus nitidipennis Casey, 1891 USA (AZ) Hymenorus nitidipennis Casey, 1891: 113. Hymenorus obesus Casey, 1891 CAN (MB NB NS ON QC) USA (AL FL GA IN LA MA MD MI MO NC NJ NY PA SC TX VA WI) Hymenorus obesus Casey, 1891: 93. Hymenorus oblivius Fall, 1931 USA (TX) Hymenorus oblivius Fall, 1931b: 216. Hymenorus obscurus (Say, 1826) USA (FL GA IN MA MD NJ NY PA SC TX VA WI) Cistela obscura Say, 1826: 242. Hymenorus occidentalis Champion, 1888 USA (TX) MEX (GU VE) Hymenorus occidentalis Champion, 1888: 425. Hymenorus oculatus Champion, 1888 MEX (VE) GUA Hymenorus oculatus Champion, 1888: 427. Hymenorus pallidus Champion, 1888 MEX (DU GE) Hymenorus pallidus Champion, 1888: 439. Hymenorus panamensis Campbell, 1962 PAN Hymenorus panamensis Campbell, 1962: 93. Hymenorus papagonis Fall, 1931 USA (AZ) Hymenorus papagonis Fall, 1931b: 201. Hymenorus parvicollis Champion, 1888 MEX (DU) Hymenorus parvicollis Champion, 1888: 440. Hymenorus parvus Fall, 1931 USA (CA) MEX (BS) Hymenorus parvus Fall, 1931b: 203. Hymenorus perforatus Casey, 1891 USA (GA IA IN MD NC PA SC) Hymenorus perforatus Casey, 1891: 95. Hymenorus picipennis Casey, 1891 CAN (NS ON QC) USA (AL MD MI NH NY PA SC WI) Hymenorus picipennis Casey, 1891: 90. Hymenorus pilosus (Melsheimer, 1846) CAN (NS ON QC) USA (AL AR FL GA IA IN KS LA MA MD MI MS NC NJ NY OH PA SC SD VA WI) Allecula pilosa Melsheimer, 1846: 58. Hymenorus pini Champion, 1888 GUA Hymenorus pini Champion, 1888: 428. Hymenorus planulus Horn, 1894 MEX (BS) Hymenorus planulus Horn, 1894b: 434. Hymenorus porosicornis Casey, 1891 USA (NM TX) Hymenorus porosicornis Casey, 1891: 101. Hymenorus prolixus Casey, 1891 USA (AZ NM NV TX UT) Hymenorus prolixus Casey, 1891: 103. Hymenorus protibialis Fall, 1931 USA (AZ CA) Hymenorus protibialis Fall, 1931b: 196. Hymenorus punctatissimus LeConte, 1866 USA (AZ CA NV TX UT) MEX (SO) Hymenorus punctatissimus LeConte, 1866b: 138. Hymenorus macer Casey, 1891: 118. Synonymy: Fall (1931b : 221). Hymenorus punctulatus (LeConte, 1859) USA (CA) Allecula punctulata LeConte, 1859b: 78. Hymenorus pygmaeus Campbell, 1971 BAH Hymenorus pygmaeus Campbell, 1971: 99. Hymenorus quietus Fall, 1931 USA (FL MO) Hymenorus quietus Fall, 1931b: 239. Hymenorus rotundicollis Casey, 1891 USA (AZ) Hymenorus rotundicollis Casey, 1891: 111. Hymenorus rufescens Champion, 1888 MEX (VE YU) Hymenorus rufescens Champion, 1888: 433. Hymenorus ruficollis Champion, 1888 USA (AZ) MEX (BC BS SO) Hymenorus ruficollis Champion, 1888: 438. Hymenorus rufohumeralis Campbell, 1982 USA (CA) Hymenorus rufohumeralis Campbell, 1982: 131. Hymenorus rufovalis Fall, 1931 USA (AZ) Hymenorus rufovalis Fall, 1931b: 230. Hymenorus segnis Champion, 1888 MEX (GE) Hymenorus segnis Champion, 1888: 430. Hymenorus semirufus Fall, 1931 USA (FL) Hymenorus semirufus Fall, 1931b: 203. Hymenorus seriatus Casey, 1891 USA (AZ) Hymenorus seriatus Casey, 1891: 109. Hymenorus setosus Hatch, 1965 USA (OR) Hymenophorus setosus Hatch, 1965: 185. Hymenorus significans Fall, 1931 USA (TX) Hymenorus significans Fall, 1931b: 237. Hymenorus similis Champion, 1888 MEX (DU MO) Hymenorus similis Champion, 1888: 432. Hymenorus simiolus Fall, 1931 USA (TX) Hymenorus simiolus Fall, 1931b: 232. Hymenorus sinuatus ebeninus Fall, 1931 USA (CA) Hymenorus sinuatus var. ebeninus Fall, 1931b: 188. Hymenorus sinuatus sinuatus Fall, 1931 CAN (BC) USA (CA ID OR WA) Hymenorus sinuatus Fall, 1931b: 187. Hymenophorus megops Hatch, 1965: 185. New synonymy [YB]. Telesicles magnus Hatch, 1965: 185. New synonymy [YB]. Hymenorus sobrinus Casey, 1891 USA (FL MD NJ SC WI) Hymenorus sobrinus Casey, 1891: 115. Hymenorus sordidus Champion, 1888 MEX (VE) GUA Hymenorus sordidus Champion, 1888: 427. Hymenorus sparsepunctatus Campbell, 1971 CUB Hymenorus sparsepunctatus Campbell, 1971: 97. Hymenorus spinifer Horn, 1894 USA (AZ) Hymenorus spinifer Horn, 1894b: 434. Hymenorus striatus (Pic, 1930) HAI DOM Cistelopsis striata Pic, 1930: 26. Hymenorus tarsalis Champion, 1888 GUA Hymenorus tarsalis Champion, 1888: 426. Hymenorus tenellus Casey, 1891 USA (FL GA MD NJ SC) Hymenorus tenellus Casey, 1891: 115. Hymenorus elbertae Blatchley, 1918: 57. Synonymy: Fall (1931b : 227). Hymenorus tenuistriatus Fall, 1931 USA (AL FL NC SC) Hymenorus tenuistriatus Fall, 1931b: 226. Hymenorus testaceus Casey, 1891 USA (AZ) Hymenorus testaceus Casey, 1891: 110. Hymenorus texensis Fall, 1931 USA (TX) Hymenorus texensis Fall, 1931b: 241. Hymenorus thoracicus Fall, 1931 USA (CA) Hymenorus thoracicus Fall, 1931b: 214. Hymenorus tibialis Champion, 1888 GUA Hymenorus tibialis Champion, 1888: 430. Hymenorus torridus Champion, 1888 MEX (GE) Hymenorus torridus Champion, 1888: 436. Hymenorus transversus Campbell, 1971 BAH Hymenorus transversus Campbell, 1971: 92. Hymenorus tritus Fall, 1931 USA (AZ) Hymenorus tritus Fall, 1931b: 219. Hymenorus trivialis Fall, 1931 MEX (BC) Hymenorus trivialis Fall, 1931b: 210. Hymenorus ulomoides Fall, 1931 USA (CA) Hymenorus ulomoides Fall, 1931b: 187. Hymenorus uniseriatus Casey, 1891 USA (CA) Hymenorus uniseriatus Casey, 1891: 115. Hymenorus vigilax Fall, 1931 USA (AZ) Hymenorus vigilax Fall, 1931b: 200. Hymenorus villosus Champion, 1888 MEX (JA MO) Hymenorus villosus Champion, 1888: 440. Hymenorus wolcotti Campbell, 1971 PRI VIS Hymenorus wolcotti Campbell, 1971: 74. Genus Knausia Fall, 1931 [F] Knausia Fall, 1931a: 15. Type species: Knausia crassicornis Fall, 1931, monotypy. Knausia crassicornis Fall, 1931 USA (NM TX) Knausia crassicornis Fall, 1931a: 16. Genus Latacula Campbell, 1971 [F] Latacula Campbell, 1971: 103. Type species: Latacula beckeri Campbell, 1971, original designation. Latacula beckeri Campbell, 1971 JAM Latacula beckeri Campbell, 1971: 105. Latacula insularis Campbell, 1971 JAM Latacula insularis Campbell, 1971: 106. Genus Lobopoda Solier, 1835 [F] Lobopoda Solier, 1835a: 233. Type species: Lobopoda striata Solier, 1835, subsequent designation ( Bousquet et al. 2015 : 134). Subgenus Flavipoda Campbell, 1966 Flavipoda Campbell, 1966: 21. Type species: Helops flavipes Fabricius, 1792 [as Allecula flavipes Jacquelin duVal, 1857], original designation. Lobopoda androsi Campbell, 1971 BAH Lobopoda androsi Campbell, 1971: 33. Lobopoda badia Campbell, 1971 CUB Lobopoda badius Campbell, 1971: 31. Lobopoda bahamensis Campbell, 1966 BAH CUB Lobopoda bahamensis Campbell, 1966: 27. Lobopoda bicolor Campbell, 1966 CUB Lobopoda bicolor Campbell, 1966: 28. Lobopoda cayamasensis Campbell, 1966 CUB Lobopoda cayamasensis Campbell, 1966: 33. Lobopoda deyrupi Steiner, 2006 BAH Lobopoda deyrupi Steiner, 2006: 32. Lobopoda emarginata Campbell, 1966 CUB Lobopoda emarginata Campbell, 1966: 32. Lobopoda flavifemoralis Campbell, 1966 CUB Lobopoda flavifemoralis Campbell, 1966: 29. Lobopoda flavipes (Fabricius, 1792) 64 CUB Helops flavipes Fabricius, 1792a: 122 [secondary homonym of Cistela flavipes Fabricius, 1792b: 45]. Cistela fuscula Schönherr, 1808: 336. Replacement name for Cistela flavipes (Fabricius, 1792a). Lobopoda nesiotica Campbell, 1971 BAH Lobopoda nesiotica Campbell, 1971: 36. Lobopoda quadratinota Campbell, 1971 CUB Lobopoda quadratinota Campbell, 1971: 28. Lobopoda schwarzi Campbell, 1971 CUB Lobopoda schwarzi Campbell, 1971: 29. Lobopoda tibiodentata Campbell, 1966 CUB Lobopoda tibiodentata Campbell, 1966: 30. Lobopoda villasensis Campbell, 1971 CUB Lobopoda villasensis Campbell, 1971: 35. Subgenus Glabrilobopoda Campbell, 1966 Glabrilobopoda Campbell, 1966: 46. Type species: Lobopoda glabrata Champion, 1888, original designation. Lobopoda aeneipennis Champion, 1888 PAN Lobopoda aeneipennis Champion, 1888: 408. Lobopoda cariniventris Champion, 1888 PAN Lobopoda cariniventris Champion, 1888: 408. Lobopoda coronadensis Campbell, 1966 CRI Lobopoda coronadensis Campbell, 1966: 54. Lobopoda darlingtoni Campbell, 1971 DOM Lobopoda darlingtoni Campbell, 1971: 40. Lobopoda glabrata Champion, 1888 PAN Lobopoda glabrata Champion, 1888: 409. Lobopoda impunctata Campbell, 1966 CRI Lobopoda impunctata Campbell, 1966: 52. Lobopoda irazuensis Champion, 1888 CRI Lobopoda irazuensis Champion, 1888: 406. Lobopoda nitens Champion, 1888 CRI Lobopoda nitens Champion, 1888: 406. Lobopoda nitida Champion, 1888 PAN Lobopoda nitida Champion, 1888: 407. Lobopoda obsoleta Champion, 1888 MEX (VE) GUA Lobopoda obsoleta Champion, 1888: 409. Lobopoda portobellensis Campbell, 1966 PAN Lobopoda portobellensis Campbell, 1966: 58. Lobopoda tilaranensis Campbell, 1966 CRI Lobopoda tilaranensis Campbell, 1966: 50. Lobopoda viridipennis Champion, 1888 PAN Lobopoda viridipennis Champion, 1888: 407. Subgenus Lobopoda Solier, 1835 Lobopoda Solier, 1835a: 233. Type species: Lobopoda striata Solier, 1835, subsequent designation ( Bousquet et al. 2015 : 134). Lobopoda acuticauda Campbell, 1966 NIC CRI PAN Lobopoda acuticauda Campbell, 1966: 77. Lobopoda aeneotincta Champion, 1888 CRI PAN Lobopoda aeneotincta Champion, 1888: 405. Lobopoda alutacea Campbell, 1971 CUB Lobopoda alutacea Campbell, 1971: 58. Lobopoda apicalis Champion, 1888 GUA Lobopoda apicalis Champion, 1888: 393. Lobopoda atrata Champion, 1888 NIC PAN Lobopoda atrata Champion, 1888: 394. Lobopoda attenuata Champion, 1888 GUA NIC CRI Lobopoda attenuata Champion, 1888: 397. Lobopoda calcarata Champion, 1893 MEX (OA) Lobopoda calcarata Champion, 1893a: 563. Lobopoda championi Campbell, 1966 CRI PAN Lobopoda championi Campbell, 1966: 105. Lobopoda chontalensis Champion, 1888 NIC CRI Lobopoda chontalensis Champion, 1888: 399. Lobopoda colona Campbell, 1971 HAI Lobopoda colona Campbell, 1971: 56. Lobopoda convexicollis Champion, 1888 MEX (VE YU) GUA Lobopoda convexicollis Champion, 1888: 395. Lobopoda cordata Campbell, 1971 BAH Lobopoda cordata Campbell, 1971: 47. Lobopoda costaricensis Campbell, 1966 CRI Lobopoda costaricensis Campbell, 1966: 103. Lobopoda cubensis Campbell, 1966 CUB Lobopoda cubensis Campbell, 1966: 157. Lobopoda distans Campbell, 1971 CUB Lobopoda distans Campbell, 1971: 48. Lobopoda diversicauda Campbell, 1966 CRI Lobopoda diversicauda Campbell, 1966: 106. Lobopoda erythrocnemis (Germar, 1823) USA (AL AR FL GA KY LA MD MS NC SC TN TX) Allecula erythrocnemis Germar, 1823: 164. Lobopoda fallaciosa Campbell, 1971 CUB Lobopoda fallaciosa Campbell, 1971: 49. Lobopoda femoralis Champion, 1888 MEX (CI SL TB VE) GUA CRI PAN Lobopoda femoralis Champion, 1888: 398. Lobopoda foveata Champion, 1888 CRI PAN Lobopoda foveata Champion, 1888: 405. Lobopoda galapagoensis Linell, 1898 SAL NIC PAN / SA Lobopoda galapagoensis Linell, 1898: 266. Lobopoda brunneipennis Campbell, 1966: 98. Synonymy: Peck and Kukalová-Peck (1990 : 1637). Lobopoda granulata Campbell, 1966 CRI PAN LAN (Barbados) / SA Lobopoda granulata Campbell, 1966: 85. Lobopoda guatemalensis Campbell, 1966 GUA Lobopoda guatemalensis Campbell, 1966: 150. Lobopoda guerrerensis Campbell, 1966 MEX (GE) Lobopoda guerrerensis Campbell, 1966: 152. Lobopoda haitensis Campbell, 1966 HAI DOM Lobopoda haitensis Campbell, 1966: 158. Lobopoda hirta Champion, 1888 NIC Lobopoda hirta Champion, 1888: 400. Lobopoda hispaniolensis Campbell, 1971 DOM Lobopoda hispaniolensis Campbell, 1971: 46. Lobopoda insularis Champion, 1896 LAN Lobopoda insularis Champion, 1896: 33. Lobopoda jamaicensis Campbell, 1966 JAM Lobopoda jamaicensis Campbell, 1966: 161. Lobopoda laevicollis Champion, 1888 MEX (CI VE YU) Lobopoda laevicollis Champion, 1888: 401. Lobopoda meridensis Campbell, 1966 MEX (YU) Lobopoda meridensis Campbell, 1966: 147. Lobopoda micans Campbell, 1971 DOM Lobopoda micans Campbell, 1971: 50. Lobopoda minuta Champion, 1888 PAN Lobopoda minuta Champion, 1888: 403. Lobopoda monticola Campbell, 1966 USA (TX) Lobopoda monticola Campbell, 1966: 124. Lobopoda mucronata Champion, 1888 PAN Lobopoda mucronata Champion, 1888: 393. Lobopoda nigrans (Melsheimer, 1846) USA (AL CT DC FL GA IL IN KS LA MA MD MI MS NC NJ NY OH PA RI SC TX VA) Cistela atra Say, 1826: 242 [junior primary homonym of Cistela ater Fabricius, 1775 and Cistela atra Olivier, 1795]. Cistela nigrans Melsheimer, 1846: 60. Replacement name for Cistela atra Say, 1826. Lobopoda nigrissima Campbell, 1966 MEX (TA) Lobopoda nigrissima Campbell, 1966: 133. Lobopoda notapuncta Campbell, 1971 HAI DOM Lobopoda notapuncta Campbell, 1971: 54. Lobopoda oblonga Champion, 1888 MEX (YU) Lobopoda oblonga Champion, 1888: 396. Lobopoda opaca Champion, 1888 MEX (CI) GUA CRI PAN Lobopoda opaca Champion, 1888: 400. Lobopoda biolleyi Pic, 1927: 22. Synonymy: Campbell (1966 : 87). Lobopoda opacicollis Champion, 1888 USA (FL LA TX) MEX (GU NL SI TA VE) GUA BEL HON NIC Lobopoda opacicollis Champion, 1888: 400. Lobopoda subcuneata Casey, 1891: 79. Synonymy: Campbell (1966 : 83). Lobopoda panamensis Champion, 1888 PAN / SA Lobopoda panamensis Champion, 1888: 392. Lobopoda paracollis Campbell, 1971 CUB Lobopoda paracollis Campbell, 1971: 55. Lobopoda paracornis Campbell, 1971 CUB Lobopoda paracornis Campbell, 1971: 53. Lobopoda parvula Champion, 1888 MEX (JA VE) Lobopoda parvula Champion, 1888: 403. Lobopoda picipennis Campbell, 1971 HAI DOM Lobopoda picipennis Campbell, 1971: 61. Lobopoda pilosa Champion, 1888 MEX (CI) GUA Lobopoda pilosa Champion, 1888: 405. Lobopoda polita Campbell, 1971 CUB Lobopoda polita Campbell, 1971: 52. Lobopoda proxima Champion, 1888 MEX (CI VE YU) GUA Lobopoda proxima Champion, 1888: 402. Lobopoda puncticollis Champion, 1888 GUA Lobopoda puncticollis Champion, 1888: 396. Lobopoda punctulata (Melsheimer, 1846) USA (AL AR FL GA IA IL IN KS KY LA MD MO MS NC NJ NY OH OK PA SC TN TX VA WI) MEX (NL TA VE) Cistela punctulata Melsheimer, 1846: 59. Lobopoda jalapensis Champion, 1888: 402. Synonymy: Campbell (1966 : 119). Lobopoda oculatifrons Casey, 1891: 81. Synonymy: Campbell (1966 : 119). Lobopoda remoinsularis Campbell, 1966 CRI PAN Lobopoda remoinsularis Campbell, 1966: 99. Lobopoda sandersoni Campbell, 1971 DOM Lobopoda sandersoni Campbell, 1971: 60. Lobopoda sculpturata Champion, 1888 PAN Lobopoda sculpturata Champion, 1888: 401. Lobopoda seriata Champion, 1888 MEX (YU) Lobopoda seriata Champion, 1888: 395. Lobopoda simplex Champion, 1888 BEL Lobopoda simplex Champion, 1888: 399. Lobopoda subparallela Champion, 1888 MEX (CI GE MO OA VE) Lobopoda subparallela Champion, 1888: 394. Lobopoda substriatus Campbell, 1966 DOM Lobopoda substriatus Campbell, 1966: 159. Lobopoda sulcaticollis Pic, 1933 CUB Lobopoda sulcaticollis Pic, 1933: 1. Lobopoda tabogensis Campbell, 1966 PAN Lobopoda tabogensis Campbell, 1966: 78. Lobopoda teapensis Champion, 1893 MEX (TB) Lobopoda teapensis Champion, 1893a: 564. Lobopoda tenuicornis Champion, 1888 MEX (CI) PAN Lobopoda tenuicornis Champion, 1888: 403. Lobopoda terminalis Campbell, 1966 GUA Lobopoda terminalis Campbell, 1966: 145. Lobopoda thomasensis Campbell, 1971 VIS (St. Thomas) Lobopoda thomasensis Campbell, 1971: 50. Lobopoda tropicalis Champion, 1888 PAN Lobopoda tropicalis Champion, 1888: 398. Lobopoda veracruzensis Campbell, 1966 MEX (TA VE) Lobopoda veracruzensis Campbell, 1966: 150. Lobopoda viridis Champion, 1888 MEX (CI VE) NIC Lobopoda viridis Champion, 1888: 404. Lobopoda longipes Borchmann, 1937: 218. Synonymy: Campbell (1966 : 135). Lobopoda yucatanica Champion, 1888 MEX (YU) Lobopoda yucatanica Champion, 1888: 397. Lobopoda wittmeri Campbell, 1978 DOM Lobopoda wittmeri Campbell, 1978c: 204. Subgenus Mesolobopoda Campbell, 1966 Mesolobopoda Campbell, 1966: 34. Type species: Allecula socia LeConte, 1854, original designation. Lobopoda acutangula Champion, 1888 MEX (CI HI MI VE) GUA BEL NIC CRI PAN Lobopoda acutangula Champion, 1888: 390. Lobopoda antiguaensis Campbell, 1971 LAN (Antigua) Lobopoda antiguaensis Campbell, 1971: 39. Lobopoda ebenina Champion, 1896 LAN Lobopoda ebenina Champion, 1896: 34. Lobopoda socia (LeConte, 1854) USA (FL LA TX) MEX (JA NL SI SL TA TB VE YU) GUA BEL HON NIC Allecula socia LeConte, 1854a: 84. Lobopoda mexicana Champion, 1888: 392. Synonymy: Campbell (1966 : 42). Lobopoda trinidadensis Campbell, 1966 MEX (YU) / SA Lobopoda trinidadensis Campbell, 1966: 39. Lobopoda tristis Champion, 1888 CRI PAN Lobopoda tristis Champion, 1888: 391. Subgenus Monoloba Solier, 1835 Monoloba Solier, 1835a: 235. Type species: Lobopoda dircaeoides Solier, 1835, monotypy. Lobopoda asperula Champion, 1888 MEX (YU) Lobopoda asperula Champion, 1888: 390. Lobopoda gigantea Champion, 1888 MEX (VE) Lobopoda gigantea Champion, 1888: 388. Lobopoda grandis Champion, 1888 NIC PAN / SA Lobopoda grandis Champion, 1888: 389. Lobopoda tarsalis Fleutiaux and Sallé, 1890 LAN (Guadeloupe) Lobopoda tarsalis Fleutiaux and Sallé, 1890: 431. [incertae sedis] Lobopoda sordida (Horn, 1894) MEX (BC) Allecula sordida Horn, 1894b: 432. Genus Madreallecula Kanda, 2013 [F] Madreallecula Kanda, 2013: 587. Type species: Madreallecula mcclevei Kanda, 2013, original designation. Madreallecula mcclevei Kanda, 2013 USA (AZ) Madreallecula mcclevei Kanda, 2013: 588. Genus Menes Champion, 1888 [M] Menes Champion, 1888: 442. Type species: Menes meridanus Champion, 1888, subsequent designation ( Bousquet et al. 2015 : 134). Menes meridanus Champion, 1888 MEX (YU) Menes meridanus Champion, 1888: 442. Menes rotundatus Champion, 1888 MEX (VE) Menes rotundatus Champion, 1888: 443. Genus Menoeceus Champion, 1888 [M] Menoeceus Champion, 1888: 443. Type species: Menoeceus crassicornis Champion, 1888, subsequent designation ( Casey 1891 : 122). Menoeceus aequalis Champion, 1888 MEX (VE) Menoeceus aequalis Champion, 1888: 444. Menoeceus crassicornis Champion, 1888 MEX (GE JA MO PU VE) GUA Menoeceus crassicornis Champion, 1888: 444. Menoeceus texanus Champion, 1888 USA (TX) Menoeceus texanus Champion, 1888: 444. Genus Notacula Campbell, 1971 [F] Notacula Campbell, 1971: 107. Type species: Notacula howdenae Campbell, 1971, original designation. Notacula howdenae Campbell, 1971 JAM Notacula howdenae Campbell, 1971: 107. Genus Obesacula Campbell, 1971 [F] Obesacula Campbell, 1971: 109. Type species: Obesacula aptera Campbell, 1971, original designation. Obesacula aptera Campbell, 1971 JAM Obesacula aptera Campbell, 1971: 111. Cyrtosoma jamaicensis Marcuzzi, 1977: 42. Synonymy: Ivie (2005 : 70). Genus Parahymenorus Campbell, 1971 [M] Parahymenorus Campbell, 1971: 100. Type species: Parahymenorus metallicus Campbell, 1971, original designation. Parahymenorus metallicus caymanensis Campbell, 1971 CAY Parahymenorus metallicus caymanensis Campbell, 1971: 103. Parahymenorus metallicus metallicus Campbell, 1971 JAM Parahymenorus metallicus metallicus Campbell, 1971: 102. Genus Phedius Champion, 1888 [M] Phedius Champion, 1888: 447. Type species: Phedius chevrolati Champion, 1888, subsequent designation ( Lucas 1920 : 500). Phedius carbonarius Champion, 1888 MEX (HI) Phedius carbonarius Champion, 1888: 448. Phedius chevrolati Champion, 1888 MEX (VE) Phedius chevrolati Champion, 1888: 447. Phedius cylindricollis Champion, 1888 MEX (JA) Phedius cylindricollis Champion, 1888: 449. Phedius funereus Schaeffer, 1905 USA (AZ) MEX (SO) Phedius funereus Schaeffer, 1905b: 176. Phedius funestus Champion, 1888 MEX (OA PU TA) Phedius funestus Champion, 1888: 450. Phedius hidalgoensis Champion, 1888 MEX (HI) Phedius hidalgoensis Champion, 1888: 448. Phedius hirtus Champion, 1893 MEX (GE) Phedius hirtus Champion, 1893a: 568. Phedius lapidicola Champion, 1893 MEX (MO) Phedius lapidicola Champion, 1893a: 568. Phedius mexicanus Champion, 1888 MEX (GU) Phedius mexicanus Champion, 1888: 450. Phedius obovatus Champion, 1888 MEX (AG GU) Phedius obovatus Champion, 1888: 449. Phedius opaculus Horn, 1894 MEX (BS) Phedius opaculus Horn, 1894b: 431. Genus Pitholaus Champion, 1888 [M] Pitholaus Champion, 1888: 446. Type species: Pitholaus helopioides Champion, 1888, monotypy. Pitholaus helopioides Champion, 1888 GUA Pitholaus helopioides Champion, 1888: 446. Genus Polyidus Champion, 1888 [M] Polyidus Champion, 1888: 441. Type species: Polydius meridionalis Champion, 1888, monotypy. Polyidus meridionalis Champion, 1888 MEX (CI) GUA CRI Polyidus meridionalis Champion, 1888: 442. Genus Punctacula Campbell, 1971 [F] Punctacula Campbell, 1971: 112. Type species: Punctacula howdeni Campbell, 1971, original designation. Punctacula howdeni Campbell, 1971 JAM Punctacula howdeni Campbell, 1971: 114. Genus Stenochidus LeConte, 1862 [M] Stenochidus LeConte, 1862a: 244. Type species: Stenochia gracilis LeConte, 1851, subsequent designation ( Lucas 1920 : 608). Stenochidus cyanescens (LeConte, 1859) USA (CA NV OR) Prionychus cyanescens LeConte, 1859b: 78. Stenochidus cyanescens var. carbonarius Schaeffer, 1911: 126. Synonymy: Marshall (1967a : 2). Stenochidus gracilis (LeConte, 1851) USA (CA) Stenochia gracilis LeConte, 1851: 150. Stenochidus robustus Schaeffer, 1911 USA (CA) Stenochidus robustus Schaeffer, 1911: 125. Genus Telesicles Champion, 1888 [M] Telesicles Champion, 1888: 450. Type species: Telesicles cordatus Champion, 1888, monotypy. Telesicles cordatus Champion, 1888 USA (AZ CA CO NM TX UT) MEX (DU) Telesicles cordatus Champion, 1888: 451. Genus Temnes Champion, 1888 [M] Temnes Champion, 1888: 410. Type species: Temnes caeruleus Champion, 1888, monotypy. Temnes caeruleus Champion, 1888 PAN Temnes caeruleus Champion, 1888: 410. Genus Theatetes Champion, 1888 [M] Theatetes Champion, 1888: 420. Type species: Theatetes basicornis Champion, 1888, monotypy. Theatetes basicornis Champion, 1888 MEX (VE) Theatetes basicornis Champion, 1888: 420. Subtribe Gonoderina Seidlitz, 1896 Gonoderina Seidlitz, 1896: 83. Type genus: Gonodera Mulsant, 1856. Pseudocistelini Portevin, 1934: 39. Type genus: Pseudocistela Crotch, 1874. Genus Andrimus Casey, 1891 [M] Andrimus Casey, 1891: 155. Type species: Cteniopus murrayi LeConte, 1866, subsequent designation ( Lucas 1920 : 96). Andrimus murrayi (LeConte, 1866) USA (AL FL GA NC SC VA) Cteniopus murrayi LeConte, 1866b: 141. Andrimus brunneus Casey, 1891: 157. Synonymy: Peck and Thomas (1998 : 109). Andrimus concolor Casey, 1891: 158. New synonymy [based on Marshall (1964 : 156) unpublished thesis]. Andrimus nigrescens Casey, 1891: 159. Synonymy: Peck and Thomas (1998 : 109). Andrimus convergens Casey, 1891: 159. New synonymy [based on Marshall (1964 : 156) unpublished thesis]. Andrimus confusus Blatchley, 1912: 331. Synonymy: Peck and Thomas (1998 : 109). Andrimus parvulus Blatchley, 1919: 67. Synonymy: Peck and Thomas (1998 : 109). Genus Androchirus LeConte, 1862 [M] Androchirus LeConte, 1862a: 244. Type species: Cistela fuscipes Melsheimer, 1846 (= Cistela erythropa Kirby, 1837), original designation. Androchirus erythropus (Kirby, 1837) [Fig. 32 ] CAN (MB NB NS ON QC) USA (AL CT DC DE IA IL IN KS KY MA MD MI MN NC NH NJ NY OH PA SC VA VT WI) Figure 32. Androchirus erythropus (Kirby, 1837). Scale bar = 1 mm. Cistela erythropa Kirby, 1837: 239. Cistela fuscipes Melsheimer, 1846: 60. Synonymy: Casey (1891 : 169). Androchirus luteipes LeConte, 1862a: 245. Synonymy: Horn (1876b : 192). Androchirus femoralis (Olivier, 1791) USA (FL GA LA MS OH RI SC TX) Cistela femoralis Olivier, 1791: 6. Genus Capnochroa LeConte, 1862 [F] Capnochroa LeConte, 1862a: 244. Type species: Cistela fuliginosa Melsheimer, 1846, monotypy. Capnochroa fuliginosa (Melsheimer, 1846) CAN (NB NS ON PE QC) USA (CT DC DE GA IA IL IN KY MA MD ME MI MO NC NH NJ NY OH PA RI SC TN VA WI WV) Cistela fuliginosa Melsheimer, 1846: 59. Genus Chromatia LeConte, 1862 [F] Chromatia LeConte, 1862a: 244. Type species: Cistela amoena Say, 1824, monotypy. Chromatia amoena (Say, 1824) CAN (ON QC) USA (CT GA IN KS MA MD MN NH NJ NY OH PA SC TN TX WI) Cistela amoena Say, 1824a: 268. Genus Isomira Mulsant, 1856 [F] Isomira Mulsant, 1856: 52. Type species: Chrysomela murina Linnaeus, 1758, subsequent designation (C.G. Thomson 1859 : 119). Tedinus Casey, 1891: 153. Type species: Tedinus angustus Casey, 1891, monotypy. Synonymy: Bousquet and Campbell (1991 : 259). Isomira acuta Campbell, 1968 MEX (CI) GUA Isomira acuta Campbell, 1968: 460. Isomira alticola Campbell, 1968 GUA Isomira alticola Campbell, 1968: 465. Isomira angusta (Casey, 1891) USA (GA SC) Tedinus angustus Casey, 1891: 154. Isomira brevicollis Champion, 1888 MEX (VE) Isomira brevicollis Champion, 1888: 459. Isomira championi Campbell, 1968 MEX (NL) Isomira championi Campbell, 1968: 453. Isomira comstocki Papp, 1956 CAN (AB BC) USA (AZ CA ID NV OR UT WA WY) Isomira comstocki Papp, 1956: 147. Isomira damnata Marshall, 1970 USA (CA) Isomira damnata Marshall, 1970c: 4. Isomira evanescens Champion, 1888 GUA Isomira evanescens Champion, 1888: 458. Isomira howdeni hidalgoensis Campbell, 1968 MEX (HI) Isomira howdeni hidalgoensis Campbell, 1968: 455. Isomira howdeni howdeni Campbell, 1968 MEX (DU) Isomira howdeni howdeni Campbell, 1968: 454. Isomira iowensis Casey, 1891 CAN (ON) USA (AR FL GA IA IL KS MD MO NC OH PA SC TN TX VA) Isomira iowensis Casey, 1891: 145. Isomira luscitiosa Casey, 1891 USA (CA) Isomira luscitiosa Casey, 1891: 148. Isomira mexicana Campbell, 1968 MEX (CI GE OA VE) Isomira mexicanus Campbell, 1968: 457. Isomira monticola Casey, 1891 USA (CA) Isomira monticola Casey, 1891: 150. Isomira oblongula Casey, 1891 CAN (ON QC) USA (FL IL IN MI NC NY OH PA SC TX WI) Isomira oblongula Casey, 1891: 151. Isomira obsoleta Champion, 1888 MEX (GE OA VE) GUA Isomira obsoleta Champion, 1888: 457. Isomira pulla (Melsheimer, 1846) CAN (ON QC) USA (AL AR DC DE FL GA IA IL IN KY MA MD ME MI MN MS NC NJ NY OH PA RI SC TN VA WI) Cistela pulla Melsheimer, 1846: 60. Isomira ignora Blatchley, 1914: 144. Synonymy: Marshall (1970a : 44). Isomira quadristriata (Couper, 1865) CAN (MB NB NS ON PE QC SK) USA (CT FL GA IL IN MA MD ME MI MN NC NH NJ NY OH PA RI SC TN VA WI WV) Cistela quadristriata Couper, 1865: 62. Isomira velutina LeConte, 1866b: 139. Synonymy: Casey (1891 : 149). Isomira rotundata Campbell, 1968 MEX (SL TA) Isomira rotundata Campbell, 1968: 456. Isomira ruficollis Hamilton, 1893 USA (IN KY OH PA) Isomira ruficollis Hamilton, 1893: 308. Isomira sericea (Say, 1824) CAN (NB NS ON QC) USA (AR CT DC DE FL GA IA IL IN KY MA MD ME MI MN MO MS NC NH NJ NY OH PA RI SC TN VA WI WV) / BAH Cistela sericea Say, 1824a: 270. Isomira tenebrosa Casey, 1891: 146. Synonymy: Marshall (1970a : 46). Isomira subaenea guatemalensis Campbell, 1968 GUA Isomira subaenea guatemalensis Campbell, 1968: 464. Isomira subaenea punctata Campbell, 1968 GUA Isomira subaenea punctata Campbell, 1968: 464. Isomira subaenea soror Campbell, 1968 GUA Isomira subaenea soror Campbell, 1968: 463. Isomira subaenea subaenea Champion, 1888 MEX (CI) GUA Isomira subaenea Champion, 1888: 458. Isomira texana Casey, 1891 USA (TX) Isomira texana Casey, 1891: 153. Isomira valida Schwarz, 1878 CAN (ON) USA (AL AR FL GA IL IN KS MD NJ OH SC WI WV) Isomira valida Schwarz, 1878: 370. Isomira similis Blatchley, 1910: 1278. Synonymy: Marshall (1970a : 41). Isomira variabilis (Horn, 1875) USA (AZ CA) Cistela variabilis Horn, 1875: 156. Isomira discolor Casey, 1891: 145. Synonymy: Marshall (1970c : 2). Genus Onychomira Campbell, 1984 [F] Onychomira Campbell, 1984: 289. Type species: Onychomira floridensis Campbell, 1984, original designation. Onychomira floridensis Campbell, 1984 USA (FL) Onychomira floridensis Campbell, 1984: 291. Genus Pseudocistela Crotch, 1874 [F] Pseudocistela Crotch, 1874: 108. Type species: Cistela brevis Say, 1824, subsequent designation ( Novák and Pettersson 2008 : 327). Pseudocistela alternans (Champion, 1888) MEX (OA) Cistela alternans Champion, 1888: 456. Pseudocistela brevis (Say, 1824) CAN (NB ON QC) USA (CT DC FL IL IN MD MI MN MO NC NH NJ NY OH PA SC VA VT WI) Cistela brevis Say, 1824a: 269 [junior primary homonym of Cistela brevis Illiger, 1794 65 ]. Cistela erythroptera Ziegler, 1844: 46. Synonymy: Gemminger [in Gemminger and Harold] (1870 : 2047). Pseudocistela calida (Champion, 1888) PAN Cistela calida Champion, 1888: 453. Pseudocistela chiriquensis (Champion, 1888) PAN Cistela chiriquensis Champion, 1888: 454. Pseudocistela cinerascens (Champion, 1888) MEX (PU) Cistela cinerascens Champion, 1888: 453. Pseudocistela decepta (Champion, 1888) PAN Cistela decepta Champion, 1888: 454. Pseudocistela delitescens (Champion, 1888) GUA Cistela delitescens Champion, 1888: 455. Pseudocistela fragilicornis (Champion, 1888) GUA Cistela fragilicornis Champion, 1888: 457. Pseudocistela juquilae (Champion, 1888) MEX (OA) Cistela juquilae Champion, 1888: 456. Pseudocistela marginata (Ziegler, 1844) USA (CT GA MA MD NC NJ NY PA) Cistela marginata Ziegler, 1844: 46. Pseudocistela nigricornis (Champion, 1888) MEX (CI DU GE GU VE) NIC CRI PAN Cistela nigricornis Champion, 1888: 452. Pseudocistela occulta (Champion, 1888) GUA Cistela occulta Champion, 1888: 455. Pseudocistela opaca (LeConte, 1859) USA (CA ID NV) Xystropus opacus LeConte, 1859b: 78. Cistela thevenetii Horn, 1875: 156. Synonymy: Poole and Gentili (1996 : 441) [probably based on Marshall (1964 : 87) unpublished thesis]. Pseudocistela ovipennis (Champion, 1893) MEX (GE JA) Cistela ovipennis Champion, 1893a: 569. Pseudocistela pectinata Hopping, 1933 CAN (BC) Pseudocistela pectinata Hopping, 1933: 285. Pseudocistela pinguis (LeConte, 1859) CAN (BC) USA (CA CO ID NM NV OR WA) Xystropus pinguis LeConte, 1859a: 16. Pseudocistela pacifica Hopping, 1933: 284. Synonymy: Bousquet and Campbell (1991 : 260). Pseudocistela sandersoni Campbell, 1971 JAM Pseudocistela sandersoni Campbell, 1971: 17. Pseudocistela zunilensis (Champion, 1888) GUA Cistela zunilensis Champion, 1888: 452. Subtribe Mycetocharina Gistel, 1848 Mycetocharisidae Gistel, 1848: [10]. Type genus: Mycetochara Guérin-Méneville, 1827. Genus Hymenochara Campbell, 1978 [F] Hymenochara Campbell, 1978a: 435. Type species: Mycetophila rufipes J.E. LeConte, 1824, original designation. Hymenochara arizonensis Campbell, 1978 USA (AZ) Hymenochara arizonensis Campbell, 1978a: 440. Hymenochara rufipes (J.E. LeConte, 1824) CAN (ON QC) USA (AL FL IN KY MA MD MI MO MS NC NY OH PA SC) Mycetophila rufipes J.E. LeConte, 1824: 170. Genus Mycetochara Guérin-Méneville, 1827 [F] Mycetophila Gyllenhal, 1810: 541 [junior homonym of Mycetophila Meigen, 1803]. Type species: Cistela scapularis Illiger, 1805 (= Cistela humeralis Fabricius, 1787), subsequent designation ( Westwood 1838 : 32). Mycetochara Guérin-Méneville, 1827: 346. Replacement name for Mycetophila Gyllenhal, 1810. Note. See Bousquet et al. (2015 : 138) for authorship of this name. Mycetocharis Gyllenhal, 1827: 510. Replacement name for Mycetophila Gyllenhal, 1810. Mycetochares Latreille, 1829a: 42. Replacement name for Mycetophila Gyllenhal, 1810. Stigmatoma LeConte, 1862a: 244. Type species: Cistela fraterna Say, 1824, monotypy. Synonymy: LeConte (1866b : 139). Mycetochara analis (LeConte, 1878) CAN (AB BC MB NB ON QC SK) USA (AR AZ CO GA IN KS MA MD MI NE NJ NY OH OR PA SC WA WI) Mycetochares analis LeConte, 1878b: 618. Mycetochares lugubris LeConte, 1878b: 618. Synonymy: Campbell (1978b : 931). Mycetochara horni Dury, 1902: 172. Synonymy: Campbell (1978b : 931). Mycetochara davisi Hatch, 1965: 187. Synonymy: Campbell (1978b : 931). Mycetochara basillaris (Say, 1824) USA (PA) Cistela basillaris Say, 1824a: 269. Mycetochara bicolor (Couper, 1865) [Fig. 33 ] CAN (NB NS ON QC) USA (IA IN MA ME MI MO NC NH NY PA SC VA VT WI) Figure 33. Mycetochara bicolor (Couper, 1865). Scale bar = 1 mm. Mycetochares bicolor Couper, 1865: 62. Mycetochara binotata (Say, 1824) CAN (MB NB NS ON QC) USA (CT IN KY MA MD MI MN NC NH NY OH PA SC VA WI) Cistela binotata Say, 1824b: 285. Mycetochares marginata LeConte, 1878b: 618. Synonymy: Campbell (1978b : 932). Mycetochares longula LeConte, 1878b: 618. Synonymy: Campbell (1978b : 932). Mycetochara foveata (LeConte, 1866) CAN (NB ON QC) USA (IA IL IN MA MD MI MO NC NH NJ NY OH RI SC VA WI) Mycetochares foveata LeConte, 1866b: 140. Mycetochares tenuis LeConte, 1866b: 140. Synonymy: Campbell (1978b : 929). Mycetochares gracilis LeConte, 1878b: 615 [junior primary homonym of Mycetochares gracilis Falderman, 1837]. Synonymy: Campbell (1978b : 929). Mycetochara gilvipes Casey, 1891: 131. Synonymy: Campbell (1978b : 929). Mycetochara lecontei Borchmann, 1909: 714. Replacement name for Mycetochara gracilis (LeConte, 1878). Mycetochara fraterna (Say, 1824) CAN (AB BC MB NB NS ON QC SK) USA (CT DC IA IL IN MA MD ME NC NH NJ NY OH PA SC SD VA WI) Cistela fraterna Say, 1824a: 270. Mycetochares laticollis LeConte, 1878b: 617. Synonymy: Casey (1891 : 128). Mycetochara megalops Casey, 1891: 129. Synonymy: Campbell (1978b : 925). Mycetochara nigerrima Casey, 1891: 132. Synonymy: Campbell (1978b : 925). Mycetochara haldemani (LeConte, 1866) USA (FL GA IL IN MD NC NY OH PA VA WI) Mycetochares haldemani LeConte, 1866b: 140. Mycetochara lata Hatch, 1965 USA (OR) Mycetochara lata Hatch, 1965: 189. Mycetochara perplexata Marshall, 1970 USA (CA) Mycetochara perplexata Marshall, 1970b: 3. Mycetochara marshalli Campbell, 1978b: 934. New synonymy [YB]. Mycetochara procera Casey, 1891 CAN (BC) USA (AZ CA ID NV OR WA) Mycetochara procera Casey, 1891: 140. Mycetochara pacifica Casey, 1891: 139. Synonymy: Campbell (1978b : 936). Mycetochara nevadensis Casey, 1891: 142. Synonymy: Campbell (1978b : 936). Mycetochara crassulipes Casey, 1891: 142. Synonymy: Campbell (1978b : 936). Mycetochara downei Hatch, 1965: 187. Synonymy: Campbell (1978b : 937). Mycetochara angusta Hatch, 1965: 188. Synonymy: Campbell (1978b : 937). Mycetochara malkini Hatch, 1965: 188. Synonymy: Campbell (1978b : 937). Mycetochara caseyi Hatch, 1965: 188. Synonymy: Campbell (1978b : 937). Mycetochara pubipennis (LeConte, 1878) USA (CA) Mycetochares pubipennis LeConte, 1878b: 617. Mycetochara longipennis Casey, 1891: 139. Synonymy: Campbell (1978b : 935). Mycetochara ruficornis Melsheimer, 1846 USA (PA) Mycetocharus ruficornis Melsheimer, 1846: 59. Subtribe Xystropodina Solier, 1835 Xystropides Solier, 1835a: 229. Type genus: Xystropus Solier, 1835. Lystronychides Lacordaire, 1859: 512. Type genus: Lystronychus Latreille, 1829. Genus Anamphidora Casey, 1924 [F] Anamphidora Casey, 1924: 330. Type species: Anamphidora parvula Casey, 1924, original designation. Anamphidora campbelli Marshall, 1967 USA (TX) Anamphidora campbelli Marshall, 1967b: 209. Anamphidora kimberleei Marshall, 1970 MEX (BC) Anamphidora kimberleei Marshall, 1970d: 294. Anamphidora parvula Casey, 1924 MEX (DU) Anamphidora parvula Casey, 1924: 330. Genus Cteisa Solier, 1835 [F] Cteisa Solier, 1835a: 242. Type species: Cteisa hirta Solier, 1835, monotypy. Cteisa pedinoides Mäklin, 1875 MEX (VE) GUA PAN / JAM / SA Cteisa pedinoides Mäklin, 1875b: 681. Genus Erxias Champion, 1888 [M] Erxias Champion, 1888: 460. Type species: Erxias bicolor Champion, 1888, subsequent designation ( Bousquet et al. 2015 : 138). Erxias bicolor Champion, 1888 PAN / SA Erxias bicolor Champion, 1888: 460. Erxias violaceipennis Champion, 1888 NIC Erxias violaceipennis Champion, 1888: 460. Genus Lystronychus Latreille, 1829 [M] Lystronychus Latreille, 1829a: 41 [as Lystronichus ]. Type species: Helops equestris Fabricius, 1775, subsequent designation ( Saunders 1836 : 154). Note. Lystronychus is in prevailing usage (see Bouchard et al. 2011 : 427) and so deemed to be the correct original spelling. Subgenus Lystronychus Latreille, 1829 Lystronychus Latreille, 1829a: 41 [as Lystronichus ]. Type species: Helops equestris Fabricius, 1775, subsequent designation ( Saunders 1836 : 154). Lystronychus championi Horn, 1894 USA (TX) Lystronychus championi Horn, 1894b: 433. Lystronychus delauneyi (Fleutiaux and Sallé, 1890) LAN (Guadeloupe) Anaedus delauneyi Fleutiaux and Sallé, 1890: 428. Lystronychus piliferus Champion, 1888 USA (TX) MEX (CI CL JA OA PU VE) GUA NIC / SA Lystronychus piliferus Champion, 1888: 462. Lystronychus purpureipennis Champion, 1888 GUA Lystronychus purpureipennis Champion, 1888: 463. Lystronychus rufonotatus Champion, 1896 LAN (St. Vincent) Lystronychus rufonotatus Champion, 1896: 35. Lystronychus rufulus Borchmann, 1930 MEX (PU) Lystronychus rufulus Borchmann, 1930: 98. Lystronychus scapularis Champion, 1888 USA (AZ) MEX (YU) GUA NIC PAN Lystronychus scapularis Champion, 1888: 463. Lystronychus tuberculifer Champion, 1896 LAN (Grenadines) Lystronychus tuberculifer Champion, 1896: 34. Genus Prostenus Klug, 1829 [M] Prostenus Klug, 1829: 5. Type species (suggested): Prostenus periscelis Perty, 1830 (see Bousquet et al. 2015 : 139). Mecocerus Solier, 1835a: 241 [junior homonym of Mecocerus Schönherr, 1833]. Type species: Xystropus dejeanii Solier, 1835, monotypy. Synonymy: Lacordaire (1859 : 513). Prostenus panamensis Champion, 1888 PAN Prostenus panamensis Champion, 1888: 461. Genus Xystropus Solier, 1835 [M] Xystropus Solier, 1835a: 241. Type species: Xystropus pilosus Solier, 1835, monotypy. Note. See Bousquet et al. (2015 : 139) for available species originally included in this genus. Xystropus californicus (Horn, 1868) MEX (OA) NIC CRI PAN / SA Prostenus californicus Horn, 1868: 138. Xystropus fulgidus Mäklin, 1875b: 680. Synonymy: Casey (1891 : 74). Xystropus fallax Mäklin, 1875 PAN / SA Xystropus fallax Mäklin, 1875b: 677. Xystropus lebasii Mäklin, 1875 PAN / SA Xystropus lebasii Mäklin, 1875b: 679. Subfamily DIAPERINAE Latreille, 1802 Diaperialae Latreille, 1802: 161. Type genus: Diaperis Geoffroy, 1762. Tribe Crypticini Brullé, 1832 Crypticites Brullé, 1832: 190. Type genus: Crypticus Latreille, 1816. Genus Ellipsodes Wollaston, 1854 [M] Ellipsodes Wollaston, 1854: 485. Type species: Sphaeridium glabratum Fabricius, 1781, monotypy. Subgenus Anthrenopsis Koch, 1950 Anthrenopsis Koch, 1950: 74. Type species: Platydema scriptipenne Fairmaire, 1875 (= Basides ziczac Motschulsky, 1873), original designation. Ellipsodes ziczac (Motschulsky, 1873) LAN (Guadeloupe, Grenada) – Adventive Basides ziczac Motschulsky, 1873: 475. Platydema scriptipenne Fairmaire, 1875: xxxiii. Synonymy: Kaszab (1975 : 102). Genus Gondwanocrypticus Español, 1955 [M] Gondwanocrypticus Español, 1955: 10. Type species: Crypticus platensis Fairmaire, 1884, original designation. Gondwanocrypticus aterrimus (Champion, 1886) MEX (CI) GUA BEL SAL HON NIC CRI PAN / SA Crypticus aterrimus Champion, 1886: 138. Gondwanocrypticus filicornis (Chevrolat, 1878) JAM DOM LAN Platydema filicorne Chevrolat, 1878b: 222. Gondwanocrypticus maculatus (Champion, 1886) MEX (MO) GUA NIC Crypticus maculatus Champion, 1886: 138. Gondwanocrypticus mexicanus (Champion, 1886) MEX (VE) Crypticus mexicanus Champion, 1886: 137. Gondwanocrypticus obsoletus (Say, 1824) USA (DE FL GA LA MD MS NC SC TX VA) / CUB Crypticus obsoletus Say, 1824a: 265. Gondwanocrypticus ovatus (Champion, 1886) MEX (JA OA) GUA Crypticus ovatus Champion, 1886: 137. Gondwanocrypticus pictus (Gebien, 1928) USA i (AL FL GA MS NC SC) / SA Crypticus pictus Gebien, 1928a: 118. Gondwanocrypticus platensis (Fairmaire, 1884) USA (AL CA DE FL GA LA MD MS NC SC TX VA WV) / BAH CAY / SA – Adventive Crypticus platensis Fairmaire, 1884: 510. Gondwanocrypticus undatus (Champion, 1896) LAN Crypticus undatus Champion, 1896: 5. Genus Poecilocrypticus Gebien, 1928 [M] Poecilocrypticus Gebien, 1928a: 121. Type species: Poecilocrypticus formicophilus Gebien, 1928, monotypy. Poecilocrypticus formicophilus Gebien, 1928 USA i (AL FL GA LA MS NC OK SC TX) / BAH / SA Poecilocrypticus formicophilus Gebien, 1928a: 122. Tribe Diaperini Latreille, 1802 Diaperialae Latreille, 1802: 161. Type genus: Diaperis Geoffroy, 1762. Subtribe Adelinina LeConte, 1862 Alphitophagida Gistel, 1856b: 185 66 . Type genus: Alphitophagus Stephens, 1832. Adelinini LeConte, 1862a: 237. Type genus: Adelina Dejean, 1835. Schedarosini Reitter, 1876: 42. Type genus: Schedarosus Reitter, 1876 (= Adelina Dejean, 1835). Doliemini Reitter, 1917: 58. Type genus: Doliema Pascoe, 1860 (= Adelina Dejean, 1835). Gnathocerini Skopin, 1978: 228. Type genus: Gnatocerus Thunberg, 1814. Genus Adelina Dejean, 1835 [F] Adelina Dejean, 1835: 315. Type species: Cucujus planus Fabricius, 1801, monotypy. Doliema Pascoe, 1860: 50. Type species: Doliema platisoides Pascoe, 1860, monotypy. Synonymy: Fleutiaux and Sallé (1890 : 428, as Adelina LeConte). Schedarosus Reitter, 1876: 42. Type species: Schedarosus cucujiformis Reitter, 1876 (= Pytho pallida Say, 1823), subsequent designation ( Löbl et al. 2008b : 42). Synonymy (with Doliema Pascoe): Champion (1886 : 157). Adelina angustata (Champion, 1886) GUA NIC Doliema angustata Champion, 1886: 159. Adelina bacardi Steiner, 2006 BAH Adelina bacardi Steiner, 2006: 15. Adelina bidens (Schaeffer, 1915) USA (FL TX) GUA / BAH CUB CAY DOM Doliema bidens Schaeffer, 1915: 238. Adelina bifurcata (Champion, 1893) USA (AZ) MEX (BS JA OA VE YU) CRI Doliema bifurcata Champion, 1893a: 535. Adelina dominicana (Ardoin, 1977) DOM Doliema dominicana Ardoin, 1977a: 18. Adelina frontalis (Champion, 1886) BEL / SA Doliema frontalis Champion, 1886: 159. Adelina klapperichi (Ardoin, 1977) DOM Doliema klapperichi Ardoin, 1977a: 12. Adelina latiramosa Doyen, 1984 MEX (PU) Adelina latiramosa Doyen, 1984a: 777. Adelina maryjoae Steiner, 2005 BAH CAY Adelina maryjoae Steiner, 2005: 449. Adelina mystax Triplehorn and Ivie, 1983 VIS Adelina mystax Triplehorn and Ivie, 1983: 272. Adelina pallida (Say, 1824) USA (CA FL GA IN LA MD NC OH SC TN VA) MEX (CH HI VE YU) GUA BEL NIC / CUB PRI / SA Pytho pallida Say, 1824a: 271. Schedarosus cucujiformis Reitter, 1876: 43. Synonymy: Champion (1893a : 535). Adelina pici (Ardoin, 1977) BAH CUB CAY LAN / SA Doliema pici Ardoin, 1977a: 7. Adelina plana (Fabricius, 1801) USA (AZ CA FL IN NC) MEX (JA TB VE) GUA BEL HON NIC PAN / BAH CUB CAY DOM LAN / SA Cucujus planus Fabricius, 1801b: 94. Adelina depressa Erichson, 1847: 119. Synonymy (in doubt): Champion (1893a : 535). Adelina plana LeConte, 1851: 149 [junior secondary homonym of Adelina plana (Fabricius, 1801)]. Synonymy (in doubt): Champion (1886 : 157). Sitophagus lecontei Horn, 1870: 346. Replacement name for Sitophagus planus (LeConte, 1851). Schedarosus scidarius Reitter, 1876: 44. Synonymy: Champion (1886 : 158). Doliema diabolica Pic, 1923: 24. Synonymy: Ardoin (1977a : 3). Adelina quadridentata (Champion, 1893) MEX (JA OA) CRI Doliema quadridentata Champion, 1893a: 535. Genus Alphitophagus Stephens, 1832 [M] Alphitophagus Stephens, 1832: 12. Type species: Alphitophagus quadripustulatus Stephens, 1832 (= Diaperis bifasciata Say, 1824), monotypy. Phyletes Redtenbacher, 1845: 128. Type species: Phylethus populi Redtenbacher, 1848 (= Diaperis bifasciata Say, 1824), subsequent monotypy in Redtenbacher (1848 : 589, as Phylethus ). Synonymy: Seidlitz (1894 : 533). Alphitophagus bifasciatus (Say, 1824) [Fig. 34 ] CAN (MB ON QC SK) USA (AL AR CA DC GA IA ID IL IN KS LA MD MN MO MS NC NE NJ NY OH OR PA SC SD TN TX VA WA WI) – Adventive Figure 34. Alphitophagus bifasciatus (Say, 1824). Scale bar = 1 mm. Diaperis bifasciata Say, 1824a: 268. Alphitophagus quadripustulatus Stephens, 1832: 12. Synonymy: Horn (1870 : 385). Platydema lilliputanum Carter, 1937: 130. Synonymy: Hinton (1947b : 90). Genus Cynaeus LeConte, 1862 [M] Cynaeus LeConte, 1862a: 233. Type species: Platydema angustum LeConte, 1851, original designation. Cynaeus angustus (LeConte, 1851) [Fig. 35 ] CAN (AB BC MB ON QC SK) USA (AZ CA CO GA IA ID IL IN KS MD MI MN NC ND NM OH OR SD TN TX UT VA WA WI WY) MEX (BC SO) Figure 35. Cynaeus angustus (LeConte, 1851). Scale bar = 1 mm. Platydema angustum LeConte, 1851: 149. Cynaeus opacus Champion, 1886: 156. Synonymy: Blaisdell (1943 : 267). Cynaeus depressus Horn, 1870 USA (AZ CA) MEX (BC) Cynaeus depressus Horn, 1870: 369. Genus Doliodesmus Spilman, 1967 [M] Doliodesmus Spilman, 1967: 149. Type species: Doliodesmus charlesi Spilman, 1967, monotypy. Doliodesmus charlesi Spilman, 1967 USA (AZ) MEX (BC BS) Doliodesmus charlesi Spilman, 1967: 153. Genus Doliopines Horn, 1894 [M] Doliopines Horn, 1894b: 427. Type species: Doliopines cucujinus Horn, 1894, monotypy. Doliopines cucujinus Horn, 1894 MEX (BC BS SO) Doliopines cucujinus Horn, 1894b: 428. Genus Gnatocerus Thunberg, 1814 [M] Gnatocerus Thunberg, 1814: 47. Type species: Gnatocerus ruber Thunberg, 1814 (= Trogosita cornuta Fabricius, 1798), monotypy. Gnathocerus Agassiz, 1846: 164. Unjustified emendation of Gnatocerus Thunberg, 1814, not in prevailing usage. Subgenus Echocerus Horn, 1870 Echocerus Horn, 1870: 366. Type species: Trogosita maxillosa Fabricius, 1801, monotypy. Gnatocerus analis (Champion, 1886) GUA Echocerus analis Champion, 1886: 146. Gnatocerus angelicus (Blaisdell, 1923) MEX (BC) Echocerus angelicus Blaisdell, 1923: 277. Gnatocerus breviceps (Blaisdell, 1943) MEX (BS) Echocerus breviceps Blaisdell, 1943: 266. Gnatocerus curvicornis (Champion, 1893) USA (FL) MEX (JA OA YU) / BAH CUB CAY / LAN Echocerus curvicornis Champion, 1893a: 533. Echocerus recurvatus Chittenden, 1895a: 2. Synonymy: Chittenden (1895b : 331). Gnatocerus maxillosus (Fabricius, 1801) USA (CA FL GA KS MD MI OH SC TX WI) MEX (GU) GUA NIC / CUB PRI LAN / SA – Adventive Trogosita maxillosa Fabricius, 1801a: 155. Subgenus Gnatocerus Thunberg, 1814 Gnatocerus Thunberg, 1814: 47. Type species: Gnatocerus ruber Thunberg, 1814 (= Trogosita cornuta Fabricius, 1798), monotypy. Cerandria Dejean, 1834: 200. Type species: Trogosita cornuta Fabricius, 1798, subsequent designation ( Duponchel 1841 : 285). Synonymy: Schaum (1849 : 283). Sicinus Champion, 1886: 146. Type species: Sicinus guatemalensis Champion, 1886, subsequent designation ( Löbl et al. 2008b : 43). Synonymy: Leng (1920 : 233). Gnatocerus brevipes (Champion, 1886) GUA Sicinus brevipes Champion, 1886: 147. Gnatocerus cornutus (Fabricius, 1798) [Fig. 36 ] CAN (BC MB NS ON QC) USA (CA CT FL MA MD OH OR WA) MEX (GU VE) GUA / CUB PRI – Adventive Figure 36. Gnatocerus ( Gnatocerus ) cornutus (Fabricius, 1798). Scale bar = 1 mm. Trogosita cornuta Fabricius, 1798: 51. Trogossita maxillaris Palisot de Beauvois, 1812: 125; pl. 32 (fig. 4) 67 . Synonymy: Fauvel (1904 : 174). Gnatocerus guatemalensis (Champion, 1886) USA (FL IL KY MD OH PA TX) MEX GUA / BAH CUB LAN Sicinus guatemalensis Champion, 1886: 147. Echocerus dentiger Chittenden, 1895a: 1. Synonymy: Leng (1918 : 208). Genus Iccius Champion, 1886 [M] Iccius Champion, 1886: 147. Type species: Iccius cephalotes Champion, 1886, subsequent designation ( Gebien 1940 : 760). Iccius cephalotes Champion, 1886 MEX (VE) GUA PAN / CUB Iccius cephalotes Champion, 1886: 148. Iccius cylindricus Champion, 1886 USA (AZ LA TX) MEX (MO) GUA Iccius cylindricus Champion, 1886: 148. Iccius elongatus Kulzer, 1949 CRI Iccius elongatus Kulzer, 1949: 304. Iccius grenadensis Champion, 1896 LAN Iccius grenadensis Champion, 1896: 19. Iccius monoceros Ferrer and Ødegaard, 2005 PAN Iccius monoceros Ferrer and Ødegaard, 2005: 637. Iccius rufotestaceus Champion, 1896 LAN Iccius rufotestaceus Champion, 1896: 18. Hypophlaeus dufaui Pic, 1945: 7. Synonymy: Bremer and Triplehorn (1999 : 59). Genus Loxostethus Triplehorn, 1962 [M] Loxostethus Triplehorn, 1962: 504. Type species: Loxostethus fasciatus Triplehorn, 1962, original designation. Loxostethus erythroscelis Triplehorn and Merkl, 1997 HAI DOM Loxostethus erythroscelis Triplehorn and Merkl, 1997: 739. Loxostethus fasciatus Triplehorn, 1962 CUB Loxostethus fasciatus Triplehorn, 1962: 504. Loxostethus quadrimaculata Zayas, 1988: 93. Synonymy: Ivie (1991 : 400). Loxostethus gibbosus Triplehorn and Merkl, 1997 CUB Loxostethus gibbosus Triplehorn and Merkl, 1997: 738. Loxostethus gowdeyi (Pic, 1930) CUB JAM HAI DOM Pentaphyllus gowdeyi Pic, 1930: 33. Loxostethus jamaicensis Triplehorn, 1962: 506. Synonymy: Triplehorn and Merkl (1997 : 739). Loxostethus opacifrons Triplehorn, 1962: 506. Synonymy: Triplehorn and Merkl (1997 : 739). Heterophylus meszarosi Kaszab, 1977a: 123. Synonymy: Triplehorn and Merkl (1997 : 739). Loxostethus baracoae Garrido and Gutiérrez, 1995a: 7. Synonymy: Triplehorn and Merkl (1997 : 739). Loxostethus guadeloupensis (Kaszab, 1977) LAN (Guadeloupe) Heterophylus guadeloupensis Kaszab, 1977a: 122. Loxostethus oblongus Triplehorn and Merkl, 1997 DOM Loxostethus oblongus Triplehorn and Merkl, 1997: 737. Loxostethus unicolor Triplehorn, 1962 HAI PRI Loxostethus unicolor Triplehorn, 1962: 506. Heterophylus ruficornis Kaszab, 1981: 80. Synonymy: Triplehorn and Merkl (1997 : 737). Genus Mophis Champion, 1886 [M] Mophis Champion, 1886: 168. Type species: Mophis marginicollis Champion, 1886, subsequent designation ( Gebien 1940 : 1061). Mophis affinis Champion, 1886 MEX (GE OA VE) Mophis affinis Champion, 1886: 169. Mophis aterrimus Champion, 1886: 169. Synonymy: Champion (1893a : 536). Mophis cynaeoides (Champion, 1886) MEX (DU FD VE) Sitophagus cynaeoides Champion, 1886: 162. Mophis marginicollis Champion, 1886 GUA Mophis marginicollis Champion, 1886: 169. Genus Phayllus Champion, 1886 [M] Phayllus Champion, 1886: 167. Type species: Phayllus minutus Champion, 1886, monotypy. Phayllus minutus Champion, 1886 MEX (VE) GUA BEL NIC PAN / SA Phayllus minutus Champion, 1886: 167. Genus Saptine Champion, 1886 [F] Saptine Champion, 1886: 180. Type species: Saptine ovata Champion, 1886, monotypy. Saptine ovata Champion, 1886 MEX (VE) Saptine ovata Champion, 1886: 181. Genus Sitophagus Mulsant, 1854 [M] Sitophagus Mulsant, 1854: 264. Type species: Sitophagus solieri Mulsant, 1854 (= Uloma hololeptoides Laporte, 1840), monotypy. Sitophagus alveolatus Doyen, 1984 USA (AZ) Sitophagus alveolatus Doyen, 1984a: 779. Sitophagus dilatifrons Champion, 1886 GUA PAN Sitophagus dilatifrons Champion, 1886: 162. Sitophagus fuliginosus Champion, 1886 GUA Sitophagus fuliginosus Champion, 1886: 161. Sitophagus hololeptoides (Laporte, 1840) USA (AZ CA FL TX) MEX (DU PU VE YU) GUA BEL NIC CRI PAN / BAH CUB PRI LAN / SA Uloma hololeptoides Laporte, 1840: 220. Sitophagus solieri Mulsant, 1854: 265. Synonymy: Champion (1886 : 161). Adelina farinaria Wollaston, 1858: 414. Synonymy (with S. solieri Mulsant): Bates (1872 : 99). Sitophagus castaneus Reitter, 1877: 9. Synonymy: Champion (1886 : 161). Sitophagus laticollis Kulzer, 1961 MEX (OA) Sitophagus laticollis Kulzer, 1961b: 540. Sitophagus uniformis Doyen, 1990 MEX (GE JA OA PU) Sitophagus uniformis Doyen, 1990: 250. Subtribe Diaperina Latreille, 1802 Diaperialae Latreille, 1802: 161. Type genus: Diaperis Geoffroy, 1762. Pentaphyllaires Mulsant, 1854: 196. Type genus: Pentaphyllus Dejean, 1821. Platydeminae Reitter, 1917: 61. Type genus: Platydema Laporte and Brullé, 1831. Genus Ceropria Laporte and Brullé, 1831 [F] Ceropria Laporte and Brullé, 1831: 332, 396. Type species: Helops indutus Wiedemann, 1819, subsequent designation ( Gebien 1940 : 422). Epilampus Dejean, 1834: 198. Unnecessary replacement name for Ceropria Laporte and Brullé, 1831 (see Bousquet and Bouchard 2013 : 52). Ceropria induta (Wiedemann, 1819) USA (FL) – Adventive Helops indutus Wiedemann, 1819: 164. Genus Cosmonota Blanchard, 1842 [F] Cosmonota Blanchard, 1842: pl. 14. Type species: Cosmonota angustata Blanchard, 1842, subsequent designation ( Gebien 1940 : 417). Cosmonota nigripes Chevrolat, 1877 MEX (VE) GUA BEL NIC Cosmonota nigripes Chevrolat, 1877b: 173. Cosmonota pubescens Champion, 1886 NIC CRI PAN Cosmonota pubescens Champion, 1886: 210. Cosmonota silphoides (Laporte and Brullé, 1831) MEX (JA VE) GUA BEL NIC PAN / SA Platydema silphoides Laporte and Brullé, 1831: 369. Platydema agile Chevrolat, 1877b: 178. Synonymy: Gebien (1940 : 413). Genus Diaperis Geoffroy, 1762 [F] Diaperis Geoffroy, 1762: 337. Type species: Chrysomela boleti Linnaeus, 1758, subsequent designation ( Latreille 1810 : 429). Allophasia Pascoe, 1871: 351. Type species: Allophasia fryi Pascoe, 1871, monotypy. Synonymy: Triplehorn and Brendell (1985 : 14). Diaperis californica Blaisdell, 1929 USA (CA OR) Diaperis californica Blaisdell, 1929c: 60. Diaperis maculata Olivier, 1791 [Fig. 37 ] CAN (MB NB NS ON PE QC SK) USA (AL AR CT DC DE FL GA IA IL IN KS KY LA MA MD ME MI MN MO MS NC ND NE NH NJ NY OH OK PA RI SC SD TN TX VA WI WV WY) MEX (VE) GUA CRI PAN / BAH CUB CAY JAM DOM PRI LAN Figure 37. Diaperis maculata Olivier, 1791. Scale bar = 1 mm. Diaperis maculata Olivier, 1791: 273. Diaperis hydactina Fabricius, 1798: 178 68 . Synonymy: Latreille (1804 : 307, as D. hydni ). Diaperis suturalis Chevrolat, 1877a: 170. Synonymy: Champion (1886 : 174). Diaperis maculata var. floridana Blatchley, 1912: 332. Synonymy: Peck and Thomas (1998 : 106). Diaperis nigronotata Pic, 1926 USA (AL AR FL GA IA IN KS LA MD MN MO MS OH OK PA SC TN TX WI WV) Diaperis rufipes var. nigronotata Pic, 1926: 22. Diaperis rufipes var. bicoloriceps Pic, 1926: 22. Synonymy: Triplehorn (1965 : 373). Diaperis rufipes Horn, 1870 USA (AZ CA NM) MEX (BS) Diaperis rufipes Horn, 1870: 379. Genus Lelegeis Champion, 1886 [M] Lelegeis Champion, 1886: 209. Type species: Lelegeis aeneipennis Champion, 1886, monotypy. Lelegeis aeneipennis Champion, 1886 MEX (VE) Lelegeis aeneipennis Champion, 1886: 210. Lelegeis apicalis (Laporte and Brullé, 1831) CUB PRI Platydema apicalis Laporte and Brullé, 1831: 359. Lelegeis hispaniolae Triplehorn, 1962 HAI DOM Lelegeis hispaniolae Triplehorn, 1962: 503. Lelegeis nigrifrons (Chevrolat, 1878) MEX (VE) PAN / SA Platydema nigrifrons Chevrolat, 1878g: cxlviii. Genus Liodema Horn, 1870 [F] Liodema Horn, 1870: 385. Type species: Platydema laevis Haldeman, 1848, monotypy. Liodema connexa Bates, 1873 MEX (VE) GUA PAN / SA Liodema connexum Bates, 1873c: 236. Platydema nigro-fasciatum Chevrolat, 1878b: 215. Synonymy: Champion (1886 : 207). Liodema explanata Triplehorn, 1998 CRI PAN Liodema explanatum Triplehorn, 1998: 325. Liodema laevis (Haldeman, 1848) USA (FL GA MS NC SC TX) MEX (QU TA VE) GUA CRI PAN Platydema laevis Haldeman, 1848: 101. Liodema maculata (Fabricius, 1801) MEX (NA PU SL TA VE YU) GUA SAL HON NIC CRI PAN / SA Mycetophagus maculatus Fabricius, 1801b: 566. Platydema 4-notata Laporte and Brullé, 1831: 380. Synonymy (in doubt with L. kirschi Bates): Champion (1886 : 205). Liodema obydense Bates, 1873c: 235. Synonymy: Triplehorn (1998 : 327). Liodema kirschi Bates, 1873c: 235. Synonymy: Gebien (1940 : 417). Liodema fulvum Bates, 1873c: 236. Synonymy (in doubt with L. kirschi Bates): Champion (1886 : 205). Liodema horni Bates, 1873c: 236. Synonymy: Triplehorn (1998 : 328). Scaphidema tergocinctum Chevrolat, 1877b: 178. Synonymy (with L. kirshi Bates): Chevrolat (1878d : 243). Scaphidema proximum Chevrolat, 1877b: 178. Synonymy (with L. obydense Bates): Chevrolat (1878d : 243). Liodema inscriptum Chevrolat, 1878b: 222. Synonymy (with L. kirshi Bates): Champion (1886 : 205). Liodema serricornis Bates, 1873 MEX (TA VE) GUA SAL HON NIC CRI PAN / DOM / SA Liodema serricorne Bates, 1873c: 236. Platydema cruciatum Chevrolat, 1877c: 182. Synonymy: Triplehorn (1998 : 328). Platydema hamatiferum Chevrolat, 1878f: c. Synonymy: Triplehorn (1998 : 328). Platydema ramulosum Chevrolat, 1878f: c. Synonymy: Triplehorn (1998 : 328). Liodema zimmermani Champion, 1886: 206. Synonymy: Triplehorn (1998 : 328). Liodema flavo-variegatum Champion, 1886: 208. Synonymy: Triplehorn (1998 : 328). Genus Neomida Latreille, 1829 [F] Neomida Latreille, 1829a: 29. Type species: Ips haemorrhoidalis Fabricius, 1787, monotypy. Oplocephala Laporte and Brullé, 1831: 338. Type species: Ips haemorrhoidalis Fabricius, 1787, subsequent designation ( Motschulsky 1845a : 80). Synonymy: Dejean (1834 : 197). Arrhenoplita Kirby, 1837: 235. Type species: Ips haemorrhoidalis Fabricius, 1787, original designation. Synonymy: Duponchel and Chevrolat (1841 : 157). Hoplocephala Agassiz, 1846: 185. Unjustified emendation of Oplocephala Laporte and Brullé, 1831, not in prevailing usage. Evoplus LeConte, 1866b: 128. Type species: Evoplus ferruginea LeConte, 1866, monotypy. Synonymy (with Arrhenoplita Kirby): Champion (1886 : 175). Neomida acera Triplehorn, 1994 CRI PAN Neomida acera Triplehorn, 1994a: 426. Neomida aeneipennis Triplehorn, 1965 MEX (CI HI JA OA PU QR QU SL TA VE YU) GUA BEL SAL HON NIC CRI Neomida aeneipennis Triplehorn, 1965: 382. Neomida armata (Laporte and Brullé, 1831) CUB / SA Oplocephala armata Laporte and Brullé, 1831: 345. Neomida bicornis (Fabricius, 1777) CAN (MB NB NS ON PE QC) USA (AL AR CT DC DE FL GA IA IL IN KS KY LA MA MD ME MI MN MO MS NC NE NH NJ NY OH OK PA RI SC SD TN TX VA WI WV) / BAH BER CUB CAY JAM Hispa bicornis Fabricius, 1777: 215. Hispa cornigera Fabricius, 1781: 82. Synonymy: Triplehorn (1965 : 377). Diaperis viridipennis Fabricius, 1801b: 586. Synonymy: Triplehorn (1965 : 377). Blaps metallica Palisot de Beauvois, 1817: 140 [junior primary homonym of Blaps metallica Fabricius, 1801]. Synonymy: Horn (1885a : 88). Oplocephala virescens Laporte and Brullé, 1831: 341. Synonymy: Melsheimer (1853 : 137). Oplocephala capra Laporte and Brullé, 1831: 345. Synonymy (in doubt): Triplehorn (2006 : 332). Oplocephala gracilis Motschulsky, 1873: 467. Synonymy: Horn (1874b : 98). Neomida castanea (Bates, 1873) MEX (VE) NIC CRI PAN / SA Hoplocephala castanea Bates, 1873b: 204. Hoplocephala oblonga Chevrolat, 1878f: xcvii. Synonymy: Triplehorn (2006 : 325). Neomida cioides (Champion, 1886) MEX (CI QU VE) CRI PAN / DOM LAN / SA Arrhenoplita cioides Champion, 1886: 180. Neomida clavicornis (Champion, 1886) MEX (VE) CI Arrhenoplita clavicornis Champion, 1886: 176. Neomida deltocera Triplehorn, 1994 CRI PAN LAN / SA Neomida deltocera Triplehorn, 1994a: 423. Neomida distans (Champion, 1886) MEX (VE) CRI PAN / SA Arrhenoplita distans Champion, 1886: 178. Neomida divergicornis Triplehorn, 1994 MEX (CI VE) Neomida divergicornis Triplehorn, 1994a: 420. Neomida dolichocera Triplehorn, 1994 CRI Neomida dolichocera Triplehorn, 1994a: 417. Neomida ferruginea (LeConte, 1866) USA (AL FL GA LA TX) MEX (CL HI NA PU SI SL TA TB VE) GUA BEL CRI PAN / CUB CAY JAM HAI DOM / SA Evoplus ferruginea LeConte, 1866b: 128. Oplocephala castanea Motschulsky, 1873: 467. Synonymy (in doubt): Horn (1874b : 98) Neomida heterocera Triplehorn, 1994 CRI PAN Neomida heterocera Triplehorn, 1994a: 421. Neomida hoffmanseggii (Laporte and Brullé, 1831) MEX (CL) CRI PAN / SA Oplocephala hoffmanseggii Laporte and Brullé, 1831: 346. Neomida inermis (Champion, 1886) GUA CRI PAN / LAN (Guadeloupe) Arrhenoplita inermis Champion, 1886: 179. Neomida lateralis (Bates, 1873) MEX (PU VE) CRI PAN / SA Hoplocephala lateralis Bates, 1873b: 204. Hoplocephala dytiscoïdes Chevrolat, 1877a: 170. Synonymy: Triplehorn (2006 : 319). Hoplocephala lutea Chevrolat, 1878f: xcvii. Synonymy: Triplehorn (2006 : 333). Neomida lawrencei Triplehorn, 1994 MEX (HI OA) BEL CRI PAN Neomida lawrencei Triplehorn, 1994a: 419. Neomida lecontei (Bates, 1873) MEX (CI VE) BEL HON PAN / JAM DOM PRI LAN / SA Evoplus lecontii Bates, 1873c: 233. Uloma guadeloupensis Marcuzzi, 1971: 110. Synonymy: Soldati and Touroult (2014 : 102), confirmed by Ivie and Hart (2017a : 116). Neomida nigricornis (Champion, 1886) GUA BEL CRI PAN Arrhenoplita nigricornis Champion, 1886: 179. Neomida obsoleta (Champion, 1886) MEX (OA VE) BEL CRI PAN Arrhenoplita obsoleta Champion, 1886: 178. Neomida occidentalis (Champion, 1893) MEX (BS GE JA PU SI) HON CRI Arrhenoplita occidentalis Champion, 1893a: 537. Neomida myllocnema Triplehorn, 1965: 386. Synonymy: Triplehorn (2006 : 315). Neomida paurocera Triplehorn, 1994 SAL HON Neomida paurocera Triplehorn, 1994a: 424. Neomida pentaphylloides (Champion, 1886) GUA Arrhenoplita pentaphylloides Champion, 1886: 180. Neomida picea (Laporte and Brullé, 1831) GUA BEL CRI PAN / SA Oplocephala picea Laporte and Brullé, 1831: 344. Hoplocephala testaceipes Pic, 1926: 28. Synonymy: Triplehorn (2006 : 313). Neomida pogonocera Triplehorn, 1994 PAN / SA Neomida pogonocera Triplehorn, 1994a: 422. Neomida punctatissima (Champion, 1893) MEX (GE JA) Arrhenoplita punctatissima Champion, 1893a: 537. Neomida suilla (Champion, 1896) LAN / SA Arrhenoplita suilla Champion, 1896: 11. Neomida telecera Triplehorn, 2006 CRI Neomida telecera Triplehorn, 2006: 328. Genus Paniasis Champion, 1886 [M] Paniasis Champion, 1886: 208. Type species: Paniasis dilatipes Champion, 1886, monotypy. Pseudapsida Kulzer, 1961a: 219. Type species: Pseudapsida brasiliensis Kulzer, 1961, original designation. Synonymy: Ferrer and Ødegaard (2005 : 637). Paniasis dilatipes Champion, 1886 MEX (VE) / SA Paniasis dilatipes Champion, 1886: 209. Paniasis kulzeri Ferrer and Ødegaard, 2005 PAN Paniasis kulzeri Ferrer and Ødegaard, 2005: 637. Genus Pentaphyllus Dejean, 1821 [M] Pentaphyllus Dejean, 1821: 68. Type species: Mycetophagus testaceus Hellwig, 1792, monotypy. Iphicorynus Jacquelin du Val, 1861: 299. Type species: Pentaphyllus melanophthalmus Mulsant, 1854 (= Nitidula chrysomeloides Rossi, 1792), monotypy. Synonymy: Gemminger [in Gemminger and Harold] (1870 : 1956). Pentaphyllus californicus Horn, 1870 USA (CA) Pentaphyllus californicus Horn, 1870: 387. Pentaphyllus pallidus LeConte, 1866 CAN (ON QC) USA (CT GA IL IN KY MD MI NJ NY OH PA SC WI) Pentaphyllus pallidus LeConte, 1866b: 126. Pentaphyllus americanus Motschulsky, 1873: 482. Synonymy: Horn (1874b : 98). Pentaphyllus testaceus (Hellwig, 1792) CAN (ON) – Adventive Mycetophagus testaceus Hellwig, 1792: 400. Genus Platydema Laporte and Brullé, 1831 [F] Platydema Laporte and Brullé, 1831: 350. Type species: Diaperis violacea Fabricius, 1790, subsequent designation ( Westwood 1838 : 32). Typhobia Pascoe, 1869: 279. Type species: Typhobia fuliginea Pascoe, 1869, monotypy. Synonymy: Champion (1886 : 181). Histeropsis Chevrolat, 1878b: 221. Type species: Platydema americanum Laporte and Brullé, 1831, subsequent designation ( Löbl et al. 2008b : 42). Synonymy: Champion (1886 : 181). Platydema americana Laporte and Brullé, 1831 CAN (AB BC MB NB NS ON QC SK) USA (AZ CA CO CT IA ID IL KS MD ME MI MN MO MT NC NE NH NJ NM NV NY OH OR PA SC SD TX VA WA WI WY) Platydema americana Laporte and Brullé, 1831: 358. Platydema polita Laporte and Brullé, 1831: 361. Synonymy: Horn (1870 : 384). Platydema angulata Chevrolat, 1877 MEX Platydema angulatum Chevrolat, 1877d: 186. Platydema antennata Laporte and Brullé, 1831 BAH CUB CAY HAI Platydema antennata Laporte and Brullé, 1831: 366. Platydema apicenotata Champion, 1896 LAN Platydema apicenotatum Champion, 1896: 13. Platydema basicornis Chevrolat, 1877 CUB Platydema basicorne Chevrolat, 1877b: 178. Platydema bimaculata Champion, 1886 MEX (VE) GUA BEL NIC CRI PAN / SA Platydema bimaculatum Champion, 1886: 193. Platydema biplagiata Champion, 1886 MEX (VE) GUA NIC PAN Platydema biplagiatum Champion, 1886: 201. Platydema bisignata Chevrolat, 1877 MEX GUA / SA Platydema bisignatum Chevrolat, 1877c: 181. Platydema brevis Champion, 1886 MEX (VE) GUA PAN Platydema breve Champion, 1886: 200. Platydema concolor Champion, 1893 NIC PAN Platydema unicolor Champion, 1886: 203 [junior primary homonym of Platydema unicolor Chevrolat, 1878]. Platydema concolor Champion, 1893a: 539. Replacement name for Platydema unicolor Champion, 1886. Platydema cordovensis Champion, 1886 MEX (VE) GUA Platydema cordovense Champion, 1886: 203. Platydema cyanea Laporte and Brullé, 1831 "Amérique septentrionale" 69 Platydema cyanea Laporte and Brullé, 1831: 392. Platydema cyanescens Laporte and Brullé, 1831 USA (AL FL GA IN LA MS NC OH SC TN TX) Platydema cyanescens Laporte and Brullé, 1831: 356. Platydema dichrocera Triplehorn, 1962 CUB Platydema dichrocerum Triplehorn, 1962: 502. Platydema dimidiata Chevrolat, 1878 MEX (VE) GUA BEL HON Platydema dimidiatum Chevrolat, 1878a: 194. Platydema diophthalma Laporte and Brullé, 1831 MEX (DU VE YU) GUA BEL HON NIC CRI PAN / CUB Platydema diophthalma Laporte and Brullé, 1831: 383. Platydema luna Chevrolat, 1877d: 186. Synonymy: Champion (1886 : 193). Platydema elegans Chevrolat, 1878 MEX (PU VE) Platydema elegans Chevrolat, 1878a: 195. Platydema elliptica (Fabricius, 1798) CAN (ON) USA (AL AR CO CT DC DE FL GA IA IL IN KS KY LA MD MI MO MS NC NJ NY OH OK PA SC TN TX UT VA WI) Tenebrio ellipticus Fabricius, 1798: 49. Platydema erotyloides Chevrolat, 1878 PAN / SA New North American record Platydema ornatum Chevrolat, 1878b: 209 [junior primary homonym of Platydema ornatum Chevrolat, 1877]. Platydema erotyloides Chevrolat, 1878d: 243. Replacement name for Platydema ornatum Chevrolat, 1878. Platydema erythrocera Laporte and Brullé, 1831 USA (AL AR DC FL GA IL IN KS LA MD MO MS NC NY OH OK SC TN TX VA WV) MEX (CI HI) BEL / BAH Platydema erythrocera Laporte and Brullé, 1831: 355. Neomida flavicornis Motschulsky, 1873: 479. Synonymy: Horn (1874b : 98). Platydema hondurense Champion, 1886: 186. Synonymy: Triplehorn (1994b : 247). Platydema excavata (Say, 1824) CAN (ON QC) USA (AL AR AZ CT DC DE FL GA IA IL IN KS KY LA MA MD MI MN MO MS NC NE NH NJ NM NY OH OK PA RI SC SD TN TX VA WI WV) MEX (JA PU VE YU) GUA BEL HON NIC CRI PAN / BAH CUB CAY HAI DOM JAM PRI LAN / SA Diaperis excavata Say, 1824a: 267. Platydema tuberculata Laporte and Brullé, 1831: 352. Synonymy: Champion (1886 : 184). Platydema nigritum Motschulsky, 1873: 470. Synonymy: Horn (1874b : 98). Platydema fraternum Chevrolat, 1878b: 210. Synonymy: Champion (1886 : 184). Platydema parvulum Casey, 1884: 50. Synonymy: Casey (1885 : 195). Platydema fasciatocollis Chevrolat, 1878 MEX Platydema fasciato-colle Chevrolat, 1878a: 194. Platydema fasciata (Fabricius, 1801) MEX (PU VE) GUA BEL / SA Mycetophagus fasciatus Fabricius, 1801b: 567. Platydema ferruginea Chevrolat, 1877 MEX (CI VE YU) GUA PAN Platydema ferrugineum Chevrolat, 1877d: 186. Platydema bi-impressum Chevrolat, 1878b: 214. Synonymy: Champion (1886 : 190). Platydema flavipes (Fabricius, 1801) USA (AL AR DC DE FL GA ON KS KY LA MA MD MS NC ND NH NJ NY OH PA SC TN TX VA) Mycetophagus flavipes Fabricius, 1801b: 567. Platydema basalis Haldeman, 1848: 101. Synonymy: Horn (1870 : 382). Platydema flexuosa Chevrolat, 1877 CUB Platydema flexuosum Chevrolat, 1877b: 178. Platydema fuliginosa Laporte and Brullé, 1831 MEX Platydema fuliginosa Laporte and Brullé, 1831: 374. Platydema guatemalensis Champion, 1886 MEX (CI VE) GUA SAL CRI PAN / LAN / SA Platydema guatemalense Champion, 1886: 197. Platydema hoegei Champion, 1886 MEX (CI GE PU VE) Platydema högei Champion, 1886: 195. Platydema immaculata Champion, 1886 PAN Platydema immaculatum Champion, 1886: 192. Platydema inquilina Linell, 1899 USA (AZ) Platydema inquilinum Linell, 1899: 183. Platydema laevipes Haldeman, 1848 USA (AL AR DC FL GA IA IN KS LA MA MD MO MS NC NJ NY OH PA SC TX VA WI) Platydema laevipes Haldeman, 1848: 101. Platydema crenatum LeConte, 1878a: 422. Synonymy: Triplehorn (1965 : 407). Platydema lucens Champion, 1886 MEX (VE) Platydema lucens Champion, 1886: 202. Platydema maculipennis Champion, 1886 MEX (VE) GUA Platydema maculipenne Champion, 1886: 201. Platydema melancholica Champion, 1886 GUA Platydema melancholicum Champion, 1886: 190. Platydema mexicana Champion, 1886 USA (AZ NM) MEX (CH DU VE) Platydema mexicanum Champion, 1886: 187. Platydema micans Zimmerman, 1870 USA (AL AR DC FL GA IN KS LA MD MS NC SC TN TX VA) MEX (JA) / BAH TUR CUB CAY JAM HAI / SA Platydema micans Zimmerman [in Horn], 1870: 383. Platydema monilicornis Chevrolat, 1877 MEX Platydema monilicorne Chevrolat, 1877d: 186. Platydema neglecta Triplehorn, 1965 CAN (BC) USA (CA ID MT NV OR UT WA) Platydema neglectum Triplehorn, 1965: 404. Platydema nicaraguensis Champion, 1886 NIC Platydema nicaraguense Champion, 1886: 192. Platydema nigrata (Motschulsky, 1873) USA (AL AZ CA FL GA IN KS LA MS NC NM SC TX) MEX (MO SI VE YU) GUA BEL HON NIC CRI / BAH CUB Neomida nigrata Motschulsky, 1873: 478. Neomida texana Motschulsky, 1873: 478. Synonymy (in doubt with P. janus sensu Horn, 1870 = P. nigrita ): Horn (1874b : 98). Platydema nigromaculata Champion, 1886 BEL NIC PAN Platydema nigromaculatum Champion, 1886: 199. Platydema nitida (Chevrolat, 1877) MEX (YU) Scaphidema nitidum Chevrolat, 1877a: 170. Platydema oculata Champion, 1886 MEX (VE) Platydema oculatum Champion, 1886: 191. Platydema oregonensis LeConte, 1857 CAN (BC) USA (CA ID OR WA) Platydema oregonense LeConte, 1857: 51. Platydema ornata Chevrolat, 1877 MEX (VE) Platydema ornatum Chevrolat, 1877d: 186. Platydema panamensis Champion, 1886 PAN Platydema panamense Champion, 1886: 198. Platydema picicornis (Fabricius, 1792) CUB HAI PRI / SA Mycetophagus picicornis Fabricius, 1792b: 498. Platydema picilabrum Melsheimer, 1846 USA (AL AR FL GA IL IN KS KY LA MA MD MI MO MS NC NJ NY OH PA SC TN TX VA WI WV) Platydema picilabrum Melsheimer, 1846: 62. Platydema pilifera Champion, 1896 LAN Platydema piliferum Champion, 1896: 12. Platydema pretiosa Champion, 1886 BEL Platydema pretiosum Champion, 1886: 197. Platydema punctatostriata Chevrolat, 1877 CUB Platydema punctatostriatum Chevrolat, 1877b: 178. Platydema quadrimaculata Laporte and Brullé, 1831 USA (PA) 70 Platydema 4-maculata Laporte and Brullé, 1831: 383. Platydema quindecimmaculata (Chevrolat, 1878) GUA NIC PAN / SA Platydema 15-maculatum Chevrolat, 1878g: cxlix. Platydema rotundata Chevrolat, 1877 MEX (GE JA MO PU VE YU) GUA CRI Platydema rotundatum Chevrolat, 1877d: 186. Platydema ruficollis Laporte and Brullé, 1831 USA (AR FL GA IA IL KY MD MS NC NJ OK SC TX VA WI) Platydema ruficollis Laporte and Brullé, 1831: 375. Neomida sanguinicollis Melsheimer, 1846: 61. Synonymy: Haldeman (1848 : 102). Platydema ruficornis (Sturm, 1826) CAN (ON QC) USA (AL AR CO CT DC DE FL GA IA IL IN KS KY LA MA MD MI MN MO MS NC NE NJ NY OH OK PA SC TN TX VA WI WV) / BAH Diaperis ruficornis Sturm, 1826: 69. Platydema rufiventris Laporte and Brullé, 1831: 378. Synonymy: Melsheimer (1853 : 138). Platydema pallens Laporte and Brullé, 1831: 377. Synonymy (in doubt): Horn (1870 : 382) 71 . Neomida rufa Melsheimer, 1846: 62. Synonymy: Horn (1870 : 382). Platydema analis Haldeman, 1848: 101. Synonymy: Horn (1870 : 382). Platydema opaculum Casey, 1884: 51. Synonymy (with P. ruficorne var. anale Haldeman): Horn (1885b : 111). Platydema rugiceps Champion, 1886 MEX (VE) GUA NIC PAN Platydema rugiceps Champion, 1886: 191. Platydema sexmaculata Chevrolat, 1878 MEX Platydema sexmaculatum Chevrolat, 1878a: 194. Platydema sexnotata Chevrolat, 1878 MEX (JA VE) NIC CRI Platydema sexnotatum Chevrolat, 1878a: 194. Platydema sobrina Chevrolat, 1877 MEX (VE) GUA BEL NIC CRI PAN / SA Neomida discolor Motschulsky, 1873: 477 [ nomen dubium ]. Platydema sobrinum Chevrolat, 1877d: 186. Synonymy (in doubt): Champion (1886 : 189). Platydema subcostata Laporte and Brullé, 1831 CAN (ON QC) USA (AL AR CT DC DE FL GA IA IL IN KS KY LA MA MD MI MN MO MS NC NH NJ NY OH PA RI SC TN TX VA WI WV) Platydema subcostata Laporte and Brullé, 1831: 362. Platydema clypeatus Haldeman, 1848: 102. Synonymy: Horn (1870 : 384). Platydema oblongulum Motschulsky, 1873: 470. Synonymy: Horn (1874b : 98). Platydema subquadrata (Motschulsky, 1873) 72 "Amérique centrale" Neomida subquadrata Motschulsky, 1873: 477. Platydema pernigrum Casey, 1884: 49. Synonymy: Champion (1886 : 188). Platydema submaculata (Chevrolat, 1878) MEX (PU VE YU) BEL Platydema submaculatum Chevrolat, 1878f: xcix. Platydema teleops Triplehorn, 1965 CAN (NB NS ON QC) USA (CT DC IA IL IN KS MA MD ME MI MN MO NC NE NH NJ NY OH OK SC TN TX VA WI WV) Platydema teleops Triplehorn, 1965: 399. Platydema tibialis (Chevrolat, 1878) NIC PAN / SA Platydema tibiale Chevrolat, 1878g: cxlviii. Platydema transversa Laporte and Brullé, 1831 MEX (OA VE) GUA BEL PAN / SA Platydema transversa Laporte and Brullé, 1831: 381. Platydema tricolor Champion, 1886 GUA Platydema tricolor Champion, 1886: 200. Platydema undata Chevrolat, 1878 MEX (CI PU SL VE) GUA BEL SAL NIC CRI PAN / SA Neomida picta Motschulsky, 1873: 480 [junior secondary homonym of Platydema pictum (Ménétriés, 1832)]. Platydema undatum Chevrolat, 1878a: 194. Synonymy: Champion (1886 : 185). Platydema rodriguezi Champion, 1886: 185. Synonymy: Triplehorn (1994b : 249). Platydema venusta Champion, 1886 NIC PAN Platydema venustum Champion, 1886: 204. Platydema ventralis Chevrolat, 1877 MEX Platydema ventrale Chevrolat, 1877d: 186. Platydema versicolor Chevrolat, 1878 MEX (PU VE) Platydema versicolor Chevrolat, 1878a: 195. Platydema viriditincta Champion, 1886 MEX (OA) Platydema viriditinctum Champion, 1886: 186. Platydema wandae Triplehorn, 1965 USA (AZ NM) Platydema wandae Triplehorn, 1965: 427. Platydema woldai Triplehorn and Philips, 1998 USA i (FL) MEX GUA SAL HON PAN Platydema woldai Triplehorn and Philips [in Philips et al.], 1998 : 291. Genus Stenoscapha Bates, 1873 [F] Stenoscapha Bates, 1873c: 237. Type species: Stenoscapha tibialis Bates, 1873, monotypy. Stenoscapha jalapensis Champion, 1886 MEX (VE) Stenoscapha jalapensis Champion, 1886: 208. Genus Ulomoides Blackburn, 1888 [M] Ulomoides Blackburn, 1888: 274. Type species: Ulomoides humeralis Blackburn, 1888, monotypy. Palembus Casey, 1891: 65. Type species: Palembus ocularis Casey, 1891, monotypy. Synonymy: Doyen et al. (1990 : 237). Martianus Fairmaire, 1893: 540. Type species: Martianus castaneus Fairmaire, 1893 (= Palembus ocularis Casey, 1891), original designation. Synonymy (with Palembus Casey): Halstead (1974 : 241). Tenebriomimus Kolbe, 1901: 342. Type species: Tenebriomimus adansoniarum Kolbe, 1901 (= Palembus ocularis Casey, 1891), monotypy. Synonymy (with Martianus Fairmaire): Gebien (1922 : 268). Ulomoides ocularis (Casey, 1891) USA (FL) / BAH CUB CAY JAM DOM PRI LAN – Adventive Palembus ocularis Casey, 1891: 65. Martianus castaneus Fairmaire, 1893: 541. Synonymy: Halstead (1974 : 242). Tenebriomimus adansoniarum Kolbe, 1901: 342. Synonymy (with M. castaneus Fairmaire): Gebien (1922 : 268). Tribe Gnathidiini Gebien, 1921 Gnathidiini Gebien, 1921: 41. Type genus: Gnathidium Gebien, 1921. Subtribe Anopidiina Jeannel and Paulian, 1945 Anopidiini Jeannel and Paulian, 1945: 62. Type genus: Anopidium Jeannel and Paulian, 1945. Genus Caecophloeus Dajoz, 1972 [M] Caecophloeus Dajoz, 1972: 278. Type species: Caecophloeus franzi Dajoz, 1972, original designation. Caecophloeus darlingtoni Dajoz, 1975 HAI Caecophloeus darlingtoni Dajoz, 1975: 115. Caecophloeus distinctus Dajoz, 1975 MEX (CI) Caecophloeus distinctus Dajoz, 1975: 117. Caecophloeus franzi Dajoz, 1972 JAM Caecophloeus franzi Dajoz, 1972: 280. Caecophloeus ineditus Dajoz, 1975 MEX (CI) Caecophloeus ineditus Dajoz, 1975: 119. Caecophloeus pubescens Dajoz, 1975 PAN Caecophloeus pubescens Dajoz, 1975: 117. Genus Cryptozoon Schaufuss, 1882 [N] Cryptozoon Schaufuss, 1882: 47. Type species: Cryptozoon civile Schaufuss, 1882, present designation . Cryptozoon civile Schaufuss, 1882 PRI Cryptozoon civile Schaufuss, 1882: 47. Cryptozoon nitidicolle Schaufuss, 1882 PRI Cryptozoon nitidicolle Schaufuss, 1882: 47. Genus Menimopsis Champion, 1896 [F] Menimopsis Champion, 1896: 16. Type species: Menimopsis excaecus Champion, 1896, monotypy. Caecomenimopsis Kaszab, 1970: 198. Type species: Caecomenimopsis leleupi Kaszab, 1970, original designation. Synonymy: Peck (1990 : 370). Menimopsis excaeca Champion, 1896 LAN Menimopsis excaecus Champion, 1896: 17. Menimopsis franzi Kaszab, 1977 JAM Menimopsis franzi Kaszab, 1977a: 122. Menimopsis jamaicensis (Dajoz, 1975) JAM Caecomenimopsis jamaicensis Dajoz, 1975: 121. 73 Menimopsis jamaicensis Kaszab, 1977 JAM Menimopsis jamaicensis Kaszab, 1977a: 121 [junior secondary homonym of Menimopsis jamaicensis (Dajoz, 1975)]. 74 Genus Neanopidium Dajoz, 1975 [N] Neanopidium Dajoz, 1975: 93. Type species: Neanopidium mexicanum Dajoz, 1975, original designation. Neanopidium affine Dajoz, 1975 MEX (HI) Neanopidium affinis Dajoz, 1975: 107. Neanopidium convexum Dajoz, 1975 MEX (VE) Neanopidium convexum Dajoz, 1975: 104. Neanopidium curticorne Dajoz, 1975 MEX (CI) Neanopidium curticornis Dajoz, 1975: 100. Neanopidium dubium Dajoz, 1975 MEX (HI) Neanopidium dubium Dajoz, 1975: 108. Neanopidium humerale Dajoz, 1975 MEX (OA) Neanopidium humeralis Dajoz, 1975: 105. Neanopidium lawrencei Dajoz, 1975 MEX (VE) Neanopidium lawrencei Dajoz, 1975: 103. Neanopidium mexicanum Dajoz, 1975 MEX (OA) Neanopidium mexicanum Dajoz, 1975: 96. Neanopidium minutum Dajoz, 1975 MEX (VE) Neanopidium minutum Dajoz, 1975: 105. Neanopidium newtoni Dajoz, 1975 MEX (VE) Neanopidium newtoni Dajoz, 1975: 102. Neanopidium pubescens Dajoz, 1975 MEX (VE) Neanopidium pubescens Dajoz, 1975: 102. Neanopidium punctatum Dajoz, 1975 MEX (SL) Neanopidium punctatum Dajoz, 1975: 106. Neanopidium simile Dajoz, 1975 MEX (HI) Neanopidium similis Dajoz, 1975: 98. Neanopidium testaceum Dajoz, 1975 MEX (TA) Neanopidium testaceum Dajoz, 1975: 103. Genus Sphaerognathium Dajoz, 1975 [N] Sphaerognathium Dajoz, 1975: 112. Type species: Sphaerognathium globosum Dajoz, 1975, original designation. Sphaerognathium globosum Dajoz, 1975 HAI Sphaerognathium globosum Dajoz, 1975: 113. Genus Tyrtaeus Champion, 1913 [M] Tyrtaeus Champion, 1913: 76. Type species: Tyrtaeus rufus Champion, 1913, original designation. Tyrtaeus cribripennis Champion, 1913 PAN Tyrtaeus cribripennis Champion, 1913: 77. Tyrtaeus dobsoni Hinton, 1947 USA (FL) – Adventive Tyrtaeus dobsoni Hinton, 1947a: 852. Tyrtaeus rufus Champion, 1913 75 USA (FL) MEX (VE) GUA CRI PAN / CUB CAY LAN / SA Tyrtaeus rufus Champion, 1913: 77. Tyrtaeus guadalupensis Dajoz, 1981: 227. Synonymy: Hopp and Ivie (2008 : 429). Tribe Hypophlaeini Billberg, 1820 Hypophlaeides Billberg, 1820a: 33. Type genus: Hypophlaeus Fabricius, 1790 (= Corticeus Piller and Mitterpacher, 1783). Corticeini Boddy, 1965: 144. Type genus: Corticeus Piller and Mitterpacher, 1783. Genus Corticeus Piller and Mitterpacher, 1783 [M] Corticeus Piller and Mitterpacher, 1783: 87. Type species: Corticeus unicolor Piller and Mitterpacher, 1783, monotypy. Hypophlaeus Fabricius, 1790: 222. Type species: Hypophlaeus castaneus Fabricius, 1790 (= Corticeus unicolor Piller and Mitterpacher, 1783), subsequent designation ( Curtis 1832 : pl. 430). Synonymy: Crotch (1870 : 47). Subgenus Corticeus Piller and Mitterpacher, 1783 Corticeus Piller and Mitterpacher, 1783: 87. Type species: Corticeus unicolor Piller and Mitterpacher, 1783, monotypy. Corticeus coynei Triplehorn, 1970 HON NIC Corticeus coynei Triplehorn [in Triplehorn and Moser], 1970: 47. Corticeus crassicornis Champion, 1886 GUA Corticeus crassicornis Champion, 1886: 173. Corticeus longicornis Champion, 1886 MEX Corticeus longicornis Champion, 1886: 172. Corticeus mexicanus mexicanus Reitter, 1878 MEX (VE) GUA NIC PAN / SA Corticeus mexicanus Reitter, 1878: 191. Corticeus cylindricus Reitter, 1878: 192 [junior primary homonym of Corticeus cylindricus Reitter, 1877]. Synonymy: Bremer and Triplehorn (1999 : 57). Corticeus erratus Reitter, 1894: 16. Replacement name for Corticeus cylindricus Reitter, 1878. Hypophloeus meridanus Pic, 1914: 15. Synonymy: Bremer and Triplehorn (1999 : 57). Corticeus opaculus (LeConte, 1878) USA (AZ CA) MEX GUA Hypophloeus opaculus LeConte, 1878a: 423. Corticeus pallidipennis Champion, 1886 MEX (VE) GUA Corticeus pallidipennis Champion, 1886: 173. Corticeus parallelus (Melsheimer, 1846) CAN (MB ON QC) USA (AL AR DC DE FL GA IL IN KY LA MA MD ME MI MN MO MS NC ND NE NH NJ NY OH PA SC TN TX VA WI WV) Hypophloeus parallelus Melsheimer, 1846: 63. Corticeus paulostriatus (Pic, 1945) USA (AR FL TN) / CUB HAI DOM PRI Hypophlaeus paulostriatus Pic, 1945: 8. Corticeus tensicollis Triplehorn, 1979: 46. Synonymy: Bremer and Triplehorn (1999 : 58). Corticeus praetermissus (Fall, 1926) CAN (AB BC MB NB NF NS NT ON QC SK YT) USA (AK AZ CA CO ID MA ME MI MN NE NH NM NV NY OR PA SD TX UT WA WI WV WY) MEX Hypophloeus praetermissus Fall, 1926: 199. Corticeus puncticollis Champion, 1886 GUA Corticeus puncticollis Champion, 1886: 172. Corticeus rosei Triplehorn, 1970 USA (AZ) MEX (CH DU JA ME NL PU) HON Corticeus rosei Triplehorn [in Triplehorn and Moser], 1970: 49. Corticeus rufipes (Fabricius, 1801) MEX (OA TB VE) GUA BEL NIC PAN / CUB PRI LAN / SA Hypophloeus rufipes Fabricius, 1801b: 558. Corticeus sordidus Champion, 1913 GUA Corticeus sordidus Champion, 1913: 162. Corticeus strublei Blaisdell, 1934 USA (AZ CA CO ID NM OR SD UT WA WY) [ MEX ] Corticeus strublei Blaisdell, 1934a: 188. Corticeus subopacus (Wallis, 1933) CAN (AB BC) USA (AK CO ID ME MI MT NC NH NY PA WA WI WV WY) Hypophloeus subopacus Wallis, 1933: 247. Corticeus substriatus (LeConte, 1878) CAN (BC) USA (AZ CA CO ID MT NM NV OR SD UT WA) MEX (BC) Hypophloeus substriatus LeConte, 1878a: 423. Corticeus tenuis (LeConte, 1878) CAN (AB BC NB NS ON QC) USA (AZ CA ID MA ME MI MN MT NH NY OR PA VA WA WI WV WY) Hypophlocus [sic!] tenuis LeConte, 1878a: 424. Hypophloeus minor Wallis, 1933: 248. Synonymy: Triplehorn (1990 : 294). Hypophloeus occidentalis Wallis, 1933: 249. Synonymy: Triplehorn (1990 : 294). Subgenus Pogonophloeus Bremer, 1998 Pogonophloeus Bremer, 1998: 9. Type species: Hypophloeus thoracicus Melsheimer, 1846, original designation. Corticeus cavus (LeConte, 1866) USA (AL DC IA KS KY MD MO MS NC OH OK PA TX VA WV WI) Hypophloeus cavus LeConte, 1866b: 129. Corticeus hatchi Boddy, 1957 USA (AZ CA CO NM OR) Corticeus hatchi Boddy, 1957: 197. Corticeus thoracicus (Melsheimer, 1846) CAN (ON) USA (AL AR DC DE FL GA IN KY LA MD MN MO MS NC NJ NY OH OK PA SC TN TX VA WI WV) / BAH Hypophloeus thoracicus Melsheimer, 1846: 63. Hypophloeus piliger LeConte, 1878a: 422. Synonymy: Triplehorn (1990 : 292). Subgenus Tylophloeus Bremer, 1998 Tylophloeus Bremer, 1998: 10. Type species: Hypophloeus flavipennis Motschulsky, 1860, original designation. Corticeus glaber (LeConte, 1878) USA (AL AR DC FL GA LA MD MS NC NJ OH SC TN TX VA WV) / BAH Hypophloeus glaber LeConte, 1878a: 422. Genus Myonophloeus Bremer and Lillig, 2017 [M] Myonophloeus Bremer and Lillig, 2017: 68. Type species: Corticeus tuberculatus Triplehorn, 1979, original designation. Myonophloeus tuberculatus (Triplehorn, 1979) CUB Corticeus tuberculatus Triplehorn, 1979: 48. Tribe Myrmechixenini Jacquelin du Val, 1858 Myrméchixénites Jacquelin du Val, 1858: 223. Type genus: Myrmechixenus Chevrolat, 1835. Genus Myrmechixenus Chevrolat, 1835 [M] Myrmechixenus Chevrolat, 1835: 267. Type species: Myrmechixenus subterraneus Chevrolat, 1835, monotypy. Myrmecoxenus Agassiz, 1846: 243. Unjustified emendation of Myrmechixenus Chevrolat, 1835, not in prevailing usage. Myrmechixenus latridioides Crotch, 1873 USA (CA DC SC TX WA) – Adventive Myrmecoxenus latridioides Crotch, 1873: 363. Tribe Phaleriini Blanchard, 1845 Phalériides Blanchard, 1845: 29. Type genus: Phaleria Latreille, 1802. Sepedonastidae Gistel, 1856a: 382. Type genus: Sepedonastes Gistel, 1856 (= Phaleria Latreille, 1802). Cataphronetini Reitter, 1917: 57. Type genus: Cataphronetis Lucas, 1846 (= Phtora Germar, 1836). Genus Phaleria Latreille, 1802 [F] Phaleria Latreille, 1802: 162. Type species: Tenebrio cadaverinus Fabricius, 1792, subsequent designation ( Westwood 1838 : 32) (see ICZN 1975). Sepedonastes Gistel, 1856a: 382. Type species: Tenebrio bimaculatus Herbst, 1799 (= Dytiscus bimaculatus Linnaeus, 1767), subsequent designation ( Bouchard et al. 2005 : 501). Synonymy: Bouchard et al. (2005 : 501). Halophalerus Crotch, 1874: 107. Type species: Phaleria rotundata LeConte, 1851, present designation . Synonymy: Austin (1880 : 38). Phaleria championi Triplehorn and Watrous, 1980 MEX (CL NA SI) Phaleria championi Triplehorn and Watrous, 1980: 56. Phaleria debilis LeConte, 1866 USA (CA) MEX (BC JA NA SO) GUA NIC Phaleria debilis LeConte, 1866b: 126. Phaleria neotropicalis Champion, 1886: 220. Synonymy: Triplehorn (1991 : 267). Phaleria insularis Champion, 1886: 221. Synonymy: Triplehorn and Watrous (1979 : 288). Phaleria fulva Fleutiaux and Sallé, 1890 DOM LAN / SA Phaleria fulva Fleutiaux and Sallé, 1890: 423. Phaleria gracilipes Casey, 1890 USA (AL LA TX) MEX (TB VE) Phaleria gracilipes Casey, 1890b: 484. Phaleria lodingi Blaisdell, 1932c: 116. Synonymy: Triplehorn and Watrous (1979 : 292). Phaleria guatemalensis Champion, 1886 MEX (GE JA OA SI) GUA / SA Phaleria guatemalensis Champion, 1886: 218. Phaleria lata Blaisdell, 1923 MEX (BC BS SO) Phaleria latus Blaisdell, 1923: 276. Phaleria pacifica Champion, 1886 MEX (NA) GUA NIC Phaleria pacifica Champion, 1886: 220. Phaleria panamensis Champion, 1886 MEX (BC CO GE JA MI NA SI SO TB) GUA BEL NIC PAN Phaleria panamensis Champion, 1886: 218. Phaleria dytiscoides Champion, 1886: 218. Synonymy: Triplehorn (1991 : 263). Phaleria marginipennis Champion, 1886: 219. Synonymy: Triplehorn (1991 : 263). Phaleria opacicollis Champion, 1886: 219. Synonymy: Triplehorn (1991 : 263). Phaleria picipes Say, 1824 USA (FL GA MD NC NJ SC VA) MEX (MO QR YU) BEL HON PAN / BAH CAY CUB HAI JAM PRI LAN / SA Phaleria picipes Say, 1824b: 280. Phaleria pilatei Chevrolat, 1879: ccxlix. Synonymy: Watrous and Triplehorn (1982 : 19). Phaleria variabilis Quedenfeldt, 1886: 128. Synonymy: Watrous and Triplehorn (1982 : 20). Phaleria caymanensis Marcuzzi, 1977: 34. Synonymy: Watrous and Triplehorn (1982 : 19). Phaleria pilifera LeConte, 1866 MEX (BC BS SO) Phaleria pilifera LeConte, 1866b: 125. Phaleria punctipes LeConte, 1878 USA (FL) MEX (QR) BEL / BAH BAR CUB CAY JAM LAN Phaleria punctipes LeConte, 1878a: 421. Phaleria guadeloupensis Fleutiaux and Sallé, 1890: 423. Synonymy: Watrous and Triplehorn (1982 : 13). Phaleria jamaicensis Marcuzzi, 1977: 36. Synonymy: Watrous and Triplehorn (1982 : 13). Phaleria rotundata LeConte, 1851 USA (CA) MEX (BC BS) Phaleria rotundata LeConte, 1851: 148. Phaleria limbata Horn, 1870: 375. Synonymy: Fall (1901 : 173, as limbalis ). Phaleria testacea Say, 1824 USA (CT DE FL GA LA MA MD ME MS NC NH NJ NY RI SC TX VA) MEX (QR) / BAH CUB JAM HIS PRI LAN / SA Phaleria testacea Say, 1824b: 280. Phaleria brasiliensis Laporte, 1840: 219. Synonymy: Watrous and Triplehorn (1982 : 19). Phaleria cayennensis Laporte, 1840: 219. Synonymy: Triplehorn (1991 : 267). Phaleria longula LeConte, 1866b: 125. Synonymy: Triplehorn and Watrous (1979 : 289). Phaleria angustata Chevrolat, 1879: ccxlviii. Synonymy: Watrous and Triplehorn (1982 : 19). Phaleria chevrolati Fleutiaux and Sallé, 1890: 422. Synonymy: Watrous and Triplehorn (1982 : 19). Phaleria chevrolati var. thoracica Fleutiaux and Sallé, 1890: 423. Synonymy: Watrous and Triplehorn (1982 : 19). Phaleria chevrolati var. quadrinotata Fleutiaux and Sallé, 1890: 423. Synonymy: Watrous and Triplehorn (1982 : 19). Phaleria maculipennis Marcuzzi, 1962: 37. Synonymy: Watrous and Triplehorn (1982 : 19). Phaleria thinophila Watrous and Triplehorn, 1982 CRI / JAM DOM PRI LAN / SA Phaleria thinophila Watrous and Triplehorn, 1982: 15. Genus Phaleromela Reitter, 1916 [F] Phaleromela Reitter, 1916: 4. Type species: Phaleria subhumeralis Marseul, 1876, monotypy. Phaleromela humeralis (Laporte, 1840) USA (CA) Phaleria humeralis Laporte, 1840: 219. Phaleromela picta (Mannerheim, 1843) CAN (BC) USA (AK CA OR WA) Phaleria picta Mannerheim, 1843: 277. Phaleria globosa LeConte, 1857: 51. New synonymy [YB]. Phaleromela prohumeralis Triplehorn, 1961 USA (CA) Phaleria humeralis Horn, 1870: 377 [junior primary homonym of Phaleria humeralis Laporte, 1840]. Phaleromela prohumeralis Triplehorn, 1961: 127. Replacement name for Phaleromela humeralis (Horn, 1870). Phaleromela variegata Triplehorn, 1961 [Fig. 38 ] CAN (AB BC NT SK YT) USA (CA ID OR WA) Figure 38. Phaleromela variegata Triplehorn, 1961. Scale bar = 1 mm. Scaphidema pictum Horn, 1874a: 36 [junior secondary homonym of Phaleromela picta (Mannerheim, 1843)]. Phaleromela variegata Triplehorn, 1961: 126. Replacement name for Phaleromela picta (Horn, 1874). Tribe Scaphidemini Reitter, 1922 Scaphidemini Reitter, 1922: 2. Type genus: Scaphidema Redtenbacher, 1848. Genus Scaphidema Redtenbacher, 1848 [F] Scaphidema Redtenbacher, 1848: 591. Type species: Scaphidium bicolor Fabricius, 1798, monotypy. Nelites LeConte, 1850: 232. Type species: Nelites aeneolus LeConte, 1850, monotypy. Synonymy: LeConte (1862a : 237). Microbasanus Pic, 1921: 1. Type species: Microbasanus jureceki Pic, 1921, monotypy. Synonymy: Löbl et al. (2008a : 318) (see also Schawaller 2008 : 384). Scaphidema aeneola (LeConte, 1850) CAN (AB BC LB MB NB NS ON PE QC SK YT) USA (AK CT IL MA MI MN NH NY OH PA WI WY) Nelites aeneolus LeConte, 1850: 232. Tribe Trachyscelini Blanchard, 1845 Trachyscélides Blanchard, 1845: 28. Type genus: Trachyscelis Latreille, 1809. Genus Trachyscelis Latreille, 1809 [F] Trachyscelis Latreille, 1809: 379. Type species: Trachyscelis aphodioides Latreille, 1809, monotypy. Trachyscelis aphodioides Latreille, 1809 USA 76 (FL LA MD NC VA) / BAH TUR PRI LAN – Adventive Trachyscelis aphodioides Latreille, 1809: 379. Trachyscelis flavipes Melsheimer, 1846: 61. Synonymy: Steiner (2004 : 335). Subfamily STENOCHIINAE Kirby, 1837 Stenochiadae Kirby, 1837: 238. Type genus: Stenochia Kirby, 1819 (= Strongylium Kirby, 1819). Tribe Cnodalonini Oken, 1843 Cnodaliden Oken, 1843: 484. Type genus: Cnodalon Latreille, 1797. Coelometopidae Schaum, 1859: 71. Type genus: Coelometopus Solier, 1848. Upidae C.G. Thomson, 1859: 116. Type genus: Upis Fabricius, 1792. Catapiestides Lacordaire, 1859: 381. Type genus: Catapiestus Perty, 1831. Polypleuri LeConte, 1862a: 229. Type genus: Polypleurus Eschscholtz, 1831. Hegemonini Reitter, 1922: 3. Type genus: Hegemona Laporte, 1840. Camariinae Anonymous, 1924: 164. Type genus: Camaria Lepeletier and Audinet-Serville, 1828. Genus Alobates Motschulsky, 1872 [M] Alobates Motschulsky, 1872: 25. Type species: Tenebrio pensylvanicus DeGeer, 1775, original designation. Alobates barbatus (Knoch, 1801) 77 USA (AL AR DC DE FL GA IL IN KS KY LA MA MD MI MO MS NC NJ NY OH RI SC TN TX VA WI WV) Upis glabra Herbst, 1799: 32 [ nomen dubium ]. Tenebrio barbatus Knoch, 1801: 166. Synonymy (in doubt): LeConte (1866a : 61). Nyctobates intermedia Haldeman, 1852: 376. Synonymy: Horn (1870 : 333). Alobates pensylvanicus (DeGeer, 1775) CAN (MB NB NS ON QC) USA (AL AZ CA CT DC DE FL GA IA IN KS KY LA MA MD ME MI MN MS NC NH NJ OH OR PA SC SD TN TX VA WI WV) MEX (NL) Tenebrio pensylvanicus DeGeer, 1775: 53. Upis chrysops Herbst, 1797: 236. Synonymy (in doubt): Knoch (1801 : 168). Tenebrio sublaevis Palisot de Beauvois, 1817: 163. Synonymy: LeConte (1866a : 61). Tenebrio striatellus Drapiez, 1820: 327. Synonymy: Horn (1889 : 212). Nyctobates inermis Mannerheim, 1843: 284. Synonymy: Horn (1870 : 333). Alobates pennsylvanicus Champion, 1892: 522. Unjustified emendation of Alobates pensylvanicus (DeGeer, 1775), not in prevailing usage. Genus Apsida Lacordaire, 1859 [F] Apsida Lacordaire, 1859: 309. Type species: Apsida chrysomelina Lacordaire, 1859, original designation. Hapsida Gemminger [in Gemminger and Harold], 1870: 1955. Unjustified emendation of Apsida Lacordaire, 1859, not in prevailing usage. Apsida belti Bates, 1873 USA (TX) MEX (TA TB VE YU) GUA NIC CRI Apsida belti Bates, 1873e: 16. Apsida boucardi Bates, 1873 MEX (VE) GUA BEL CRI PAN Apsida boucardi Bates, 1873e: 17. Cosmonota geminata Chevrolat, 1877b: 173. Synonymy: Champion (1886 : 215). Cosmonota grammica Chevrolat, 1877b: 173. Synonymy: Champion (1886 : 215). Apsida chrysomelina Lacordaire, 1859 MEX (CI VE) GUA BEL NIC CRI PAN / SA Apsida chrysomelina Lacordaire, 1859: 309. Apsida chrysomelina Bates, 1873e: 15 [junior primary homonym of Apsida chrysomelina Lacordaire, 1859]. Synonymy: Champion (1886 : 211). Apsida gibbosa Champion, 1886 MEX (VE) GUA BEL CRI Hapsida gibbosa Champion, 1886: 212. Apsida lustrans Triplehorn, 1970 CRI Apsida lustrans Triplehorn, 1970: 569. Apsida punctipennis Champion, 1886 GUA Hapsida punctipennis Champion, 1886: 213. Apsida purpureomicans Bates, 1873 MEX (VE) GUA BEL CRI PAN / SA Apsida purpureomicans Bates, 1873e: 16. Apsida aeneomicans Bates, 1873e: 16. Synonymy: Triplehorn (1970 : 571). Apsida seriatopunctata Champion, 1886 MEX (VE) Hapsida seriato-punctata Champion, 1886: 212. Apsida simulatrix Ferrer and Ødegaard, 2005 PAN Apsida simulatrix Ferrer and Ødegaard, 2005: 640. Apsida terebrans Champion, 1886 MEX (TB) GUA BEL NIC CRI PAN Hapsida terebrans Champion, 1886: 214. Genus Blapida Perty, 1830 [F] Blapida Perty, 1830: 58. Type species: Blapida okeni Perty, 1830, monotypy. Ryssochiton Gray [in Griffith and Pidgeon], 1832: pl. 50. Type species: Rhyssochiton politus Gray, 1832, monotypy. Synonymy: Lacordaire (1859 : 425). Blapida alternata Gebien, 1919 CRI PAN Blapida alternata Gebien, 1919: 137. Blapida castaneipennis Champion, 1896 LAN Blapida castaneipennis Champion, 1896: 28. Blapida neotropicalis Champion, 1886 GUA NIC Blapida neotropicalis Champion, 1886: 247. Genus Bothynocephalus Doyen, 1988 [M] Bothynocephalus Doyen, 1988: 315. Type species: Bothynocephalus cristatus Doyen, 1988, original designation. Bothynocephalus cristatus Doyen, 1988 HON Bothynocephalus cristatus Doyen, 1988: 316. Bothynocephalus foveolatus Doyen, 1995 MEX (OA) Bothynocephalus foveolatus Doyen, 1995: 12. Bothynocephalus ribardoi Doyen, 1995 CRI Bothynocephalus ribardoi Doyen, 1995: 13. Bothynocephalus thoracicus Doyen, 1995 CRI Bothynocephalus thoracicus Doyen, 1995: 12. Genus Brosimapsida Ferrer and Ødegaard, 2005 [F] Brosimapsida Ferrer and Ødegaard, 2005: 640. Type species: Brosimapsida gonospoides Ferrer and Ødegaard, 2005, original designation. Brosimapsida gonospoides Ferrer and Ødegaard, 2005 PAN Brosimapsida gonospoides Ferrer and Ødegaard, 2005: 640. Genus Calydonella Doyen, 1995 [F] Calydonella Doyen, 1995: 8. Type species: Calydonella lisa Doyen, 1995, original designation. Calydonella lisa Doyen, 1995 CRI PAN Calydonella lisa Doyen, 1995: 10. Genus Camaria Lepeletier and Audinet-Serville, 1828 [F] Camaria Lepeletier and Audinet-Serville, 1828: 454. Type species: Camaria nitida Lepeletier and Audinet-Serville, 1828, monotypy. Camaria laevis Gebien, 1919 "Zentralamerika" / SA Camaria laevis Gebien, 1919: 52. Camaria parallela Champion, 1886 PAN Camaria parallela Champion, 1886: 246. Genus Choastes Champion, 1893 [M] Choaspes Champion, 1885: 118 [junior homonym of Choaspes Moore, 1881]. Type species: Choaspes purpureus Champion, 1885, subsequent designation ( Gebien 1941 : 338). Choastes Champion, 1893a: 526. Replacement name for Choaspes Champion, 1885. Choastes angulicollis (Champion, 1885) NIC PAN Choaspes angulicollis Champion, 1885: 119. Choastes purpureus (Champion, 1885) GUA BEL NIC PAN Choaspes purpureus Champion, 1885: 119. Genus Cibdelis Mannerheim, 1843 [F] Cibdelis Mannerheim, 1843: 282. Type species: Cibdelis blaschkii Mannerheim, 1843, monotypy. Scotera Motschulsky, 1845b: 365. Type species: Scotera gibbosa Motschulsky, 1845, monotypy. 78 Synonymy: Motschulsky (1845b : 365). Cibdelis bachei LeConte, 1861 USA (CA) Cibdelis bachei LeConte, 1861b: 353. Cibdelis blaschkii Mannerheim, 1843 USA (CA) Cibdelis blaschkii Mannerheim, 1843: 284. Cibdelis cylindrica Casey, 1924 USA (CA) Cibdelis cylindrica Casey, 1924: 323. Cibdelis gibbosa (Motschulsky, 1845) USA (CA) Scotera gibbosa Motschulsky, 1845b: 365. Cibdelis laevigata Casey, 1891: 60. Synonymy: Leng (1920 : 235). Cibdelis ventricosa Casey, 1924 USA (CA) Cibdelis ventricosa Casey, 1924: 322. Genus Cnephalura Doyen, 1988 [F] Cnephalura Doyen, 1988: 313. Type species: Cnephalura umbrata Doyen, 1988, original designation. Cnephalura umbrata Doyen, 1988 MEX (CI) Cnephalura umbrata Doyen, 1988: 313. Genus Cnodalon Latreille, 1797 [N] Cnodalon Latreille, 1797: 23. Type species: Cnodalum viride Latreille, 1804, subsequent monotypy in Latreille (1804 : 321). Cnodalum Agassiz, 1846: 91. Unjustified emendation for Cnodalon Latreille, 1797, not in prevailing usage. Cnodalon viride Latreille, 1804 HAI Cnodalum viride Latreille, 1804: 321. Genus Coelocnemis Mannerheim, 1843 [F] Coelocnemis Mannerheim, 1843: 280. Type species: Coelocnemis dilaticollis Mannerheim, 1843, subsequent designation ( Lucas 1920 : 194). Coelocnemis dilaticollis Mannerheim, 1843 79 CAN (AB BC) USA (CA ID MT NV OR UT WA WY) Coelocnemis dilaticollis Mannerheim, 1843: 282. Coelocnemis californica Mannerheim, 1843: 282. Synonymy: Horn (1870 : 336). Coelocnemis rugosa Linell, 1899: 181. Synonymy: Doyen (1973 : 93). Coelocnemis columbiana Casey, 1924: 314. Synonymy: Boddy (1965 : 175). Coelocnemis rauca Casey, 1924: 315. Synonymy: Doyen (1973 : 93). Coelocnemis ovipennis Casey, 1924: 315. Synonymy: Doyen (1973 : 93). Coelocnemis utensis Casey, 1924: 316. Synonymy: Doyen (1973 : 93). Coelocnemis spaldingi Casey, 1924: 316. Synonymy: Doyen (1973 : 93). Coelocnemis basalis Casey, 1924: 316. Synonymy: Doyen (1973 : 93). Coelocnemis idahoensis Casey, 1924: 318. Synonymy: Boddy (1965 : 175). Coelocnemis barretti Blaisdell, 1928: 163. Synonymy: Doyen (1973 : 93). Coelocnemis lucia Doyen, 1973 USA (CA) Coelocnemis lucia Doyen, 1973: 96. Coelocnemis magna LeConte, 1851 USA (AZ CA NM) MEX (BC) Coelocnemis magna LeConte, 1851: 150. Coelocnemis obesa LeConte, 1851: 150. Synonymy: Doyen (1973 : 87). Coelocnemis caudicalis Casey, 1924: 316. Synonymy: Doyen (1973 : 87). Coelocnemis caudicalis deserta Casey, 1924: 316. Synonymy: Doyen (1973 : 87). Coelocnemis antennalis Casey, 1924: 317. Synonymy: Doyen (1973 : 87). Coelocnemis aequalis Casey, 1924: 318. Synonymy: Doyen (1973 : 87). Coelocnemis smithiana Casey, 1924: 318. Synonymy: Doyen (1973 : 87). Coelocnemis rotundicollis Casey, 1924: 319. Synonymy: Doyen (1973 : 87). Coelocnemis longicollis Casey, 1924: 319. Synonymy: Doyen (1973 : 87). Coelocnemis punctata LeConte, 1854 USA (AZ CA CO ID NM NV OR UT) Coelocnemis punctatus LeConte, 1854c: 225. Coelocnemis angusta Casey, 1924: 320. Synonymy: Doyen (1973 : 98). Coelocnemis tanneri Blaisdell, 1928: 164. Synonymy: Doyen (1973 : 98). Coelocnemis rugulosa Doyen, 1973 USA (CA OR) Coelocnemis rugulosa Doyen, 1973: 97. Coelocnemis slevini Blaisdell, 1925 MEX (BC) Coelocnemis slevini Blaisdell, 1925b: 337. Coelocnemis sulcata Casey, 1895 USA (AZ CA ID NV UT) Coelocnemis sulcata Casey, 1895: 615. Genus Cyrtosoma Perty, 1830 [N] Cyrtosoma Perty, 1830: 59. Type species: Cyrtosoma unicolor Perty, 1830, monotypy. Cyrtosoma arimense Marcuzzi, 1999 LAN Cyrtosoma arimensis Marcuzzi, 1999: 85. Cyrtosoma decemlineatum Champion, 1886 MEX (VE) BEL NIC PAN Cyrtosoma decem-lineatum Champion, 1886: 244. Cyrtosoma denticolle Chevrolat, 1878 MEX (VE) GUA BEL NIC CRI PAN / SA Cyrtosoma denticolle Chevrolat, 1878e: 273. Cyrtosoma grenadense Marcuzzi, 1999 LAN (Grenada) Cyrtosoma grenadensis Marcuzzi, 1999: 85. Cyrtosoma lherminierii (Guérin-Méneville, 1844) LAN Cnodalon atrum Guérin-Méneville, 1833: pl. 31 [junior primary homonym of Cnodalon atrum Lepeletier and Audinet-Serville, 1825]. Cnodalon l'herminierii Guérin-Méneville 80 , 1844: 123. Replacement name for Cnodalon atrum Guérin-Méneville, 1833. Cyrtosoma martiniquense Marcuzzi, 1999 LAN (Martinique) Cyrtosoma martiniquensis Marcuzzi, 1999: 83. Cyrtosoma piceum (Laporte and Brullé, 1831) LAN (Guadeloupe) Platydema picea Laporte and Brullé, 1831: 362. Cyrtosoma williamsi Marcuzzi, 1992 PAN Cyrtosoma williamsi Marcuzzi, 1992: 237. Genus Dinomus Brême, 1842 [M] Dinomus Brême, 1842: 113. Type species: Dinomus perforatus Brême, 1842, monotypy. Dinomus perforatus Brême, 1842 MEX Dinomus perforatus Brême, 1842: 114. Genus Elomosda Bates, 1870 [F] Elomosda Bates, 1870: 273. Type species: Elomosda beltii Bates, 1870, monotypy. Elomosda beltii Bates, 1870 GUA NIC CRI Elomosda beltii Bates, 1870: 275. Genus Epicalla Champion, 1886 [F] Epicalla Champion, 1886: 249. Type species: Epicalla varipes Champion, 1886, subsequent designation ( Lucas 1920 : 268). Epicalla aenipes Ferrer and Ødegaard, 2005 PAN Epicalla aenipes Ferrer and Ødegaard, 2005: 642. Epicalla agnata Gebien, 1928 CRI Epicalla agnata Gebien, 1928b: 217. Epicalla avia Gebien, 1928 BEL Epicalla avia Gebien, 1928b: 209. Epicalla cupreonitens Champion, 1886 PAN Epicalla cupreo-nitens Champion, 1886: 250. Epicalla elongata Ferrer and Ødegaard, 2005 PAN Epicalla elongata Ferrer and Ødegaard, 2005: 642. Epicalla famula Gebien, 1928 CRI Epicalla famula Gebien, 1928b: 216. Epicalla hera Gebien, 1928 CRI Epicalla hera Gebien, 1928b: 207. Epicalla instriata Pic, 1921 PAN Epicalla instriata Pic, 1921b: 28. Epicalla juvenca Gebien, 1928 NIC CRI Epicalla juvenca Gebien, 1928b: 218. Epicalla lata Champion, 1886 MEX (JA SI) Epicalla lata Champion, 1886: 250. Epicalla nevermanni Gebien, 1928 CRI Epicalla nevermanni Gebien, 1928b: 215. Epicalla pygmaea Ferrer and Ødegaard, 2005 PAN Epicalla pygmaea Ferrer and Ødegaard, 2005: 642. Epicalla varipes Champion, 1886 NIC Epicalla varipes Champion, 1886: 249. Genus Glyptotus LeConte, 1858 [M] Glyptotus LeConte, 1858b: 75. Type species: Glyptotus cribratus LeConte, 1858, monotypy. Glyptotus cribratus LeConte, 1858 USA (AL FL GA MS NC SC TX VA) MEX / BAH Glyptotus cribratus LeConte, 1858b: 75. Glyptotus nitidus Champion, 1885 MEX (VE) NIC Glyptotus nitidus Champion, 1885: 113. Glyptotus yucatanus Champion, 1892 MEX (YU) Glyptotus yucatanus Champion, 1892: 524. Genus Gonospa Champion, 1886 [F] Gonospa Champion, 1886: 216. Type species: Gonospa phaedonoides Champion, 1886, subsequent designation ( Gebien 1940 : 426). Gonospa phaedonoides Champion, 1886 PAN Gonospa phaedonoides Champion, 1886: 217. Gonospa similis Ferrer and Ødegaard, 2005 PAN Gonospa similis Ferrer and Ødegaard, 2005: 639. Genus Haplandrus LeConte, 1862 [M] Haplandrus LeConte, 1862a: 230. Type species: Helops femoratus Fabricius, 1798 (= Upis fulvipes Herbst, 1797), monotypy. Haplandrus deyruporum Steiner, 2016 USA (FL) Haplandrus deyruporum Steiner, 2016: 537. Haplandrus fulvipes (Herbst, 1797) CAN (NS ON QC) USA (CT FL GA IA IN MD MI NC NY OH PA RI SC TN VA WI) Upis fulvipes Herbst, 1797: 238. Helops femoratus Fabricius, 1798: 53. Synonymy: Schönherr (1806 : 157). Genus Hegemona Laporte, 1840 [F] Hegemona Laporte, 1840: 230. Type species: Hegemona resplendens Laporte, 1840, monotypy. Eucamptus Germar, 1842: 444 [junior homonym of Eucamptus Chevrolat, 1833]. Type species: Eucamptus iridis Germar, 1842 (= Hegemona resplendens Laporte, 1840), monotypy. Synonymy: Duponchel (1845 : 498). Eusarca Chevrolat, 1845: 526. Type species: Eusarca iridipennis Chevrolat, 1845 (= Hegemona resplendens Laporte, 1840), monotypy. Synonymy: Duponchel (1845 : 498). Hegemona alternata Pic, 1936 GUA Hegemona alternatus Pic, 1936: 15. Hegemona angustata Champion, 1887 GUA Hegemona angustatus Champion, 1887: 272. Hegemona bicaudata Champion, 1887 GUA Hegemona bicaudatus Champion, 1887: 270. Hegemona chiriquensis Champion, 1887 CRI PAN Hegemona chiriquensis Champion, 1887: 273. Hegemona compressa Allard, 1877 MEX GUA Hegemona compressus Allard, 1877b: 61, 254. Hegemona costaricensis Champion, 1887 CRI Hegemona costaricensis Champion, 1887: 275. Hegemona elongata Allard, 1877 MEX (YU) Hegemona elongatus Allard, 1877b: 61, 253. Hegemona flibuster (J. Thomson, 1856) MEX GUA BEL NIC CRI Eucamptus flibuster J. Thomson, 1856: 475. Hegemona filibuster Champion, 1887: 275. Unjustified emendation of Hegemona flibuster (J. Thomson, 1856), not in prevailing usage. Hegemona furcillata Allard, 1877 MEX Hegemona furcillatus Allard, 1877b: 61, 252. Hegemona guatemalensis Champion, 1887 GUA Hegemona guatemalensis Champion, 1887: 274. Hegemona hondurensis Champion, 1887 BEL Hegemona hondurensis Champion, 1887: 269. Hegemona interrupta Champion, 1887 CRI Hegemona interruptus Champion, 1887: 275. Hegemona lineata Champion, 1887 GUA BEL Hegemona lineatus Champion, 1887: 271. Hegemona mexicana Champion, 1887 MEX (PU) Hegemona mexicanus Champion, 1887: 274. Hegemona nigra Champion, 1887 GUA HON Hegemona niger Champion, 1887: 271. Hegemona refulgens Champion, 1893 GUA Hegemona refulgens Champion, 1893a: 549. Hegemona resplendens Laporte, 1840 MEX (VE YU) Hegemona resplendens Laporte, 1840: 230. Eucamptus iridis Germar, 1842: 444. Synonymy: Duponchel (1845 : 498). Eusarca iridipennis Chevrolat, 1845: 526. Synonymy: Duponchel (1845 : 498). Hegemona retrodentata Allard, 1877 MEX (CI OA) Hegemona retrodentatus Allard, 1877b: 61, 253. Hegemona zunilensis Champion, 1887 GUA Hegemona zunilensis Champion, 1887: 272. Genus Hesiodus Champion, 1885 [M] Hesiodus Champion, 1885: 115. Type species: Hesiodus longitarsus Champion, 1885, subsequent designation ( Lucas 1920 : 323). Hesiodus caraibus Fleutiaux and Sallé, 1890 LAN (Guadeloupe) Hesiodus caraibus Fleutiaux and Sallé, 1890: 424. Hesiodus conspurcatus Champion, 1885 PAN Hesiodus conspurcatus Champion, 1885: 116. Hesiodus debilis Champion, 1885 GUA Hesiodus debilis Champion, 1885: 117. Hesiodus ellipticus Champion, 1893 NIC Hesiodus ellipticus Champion, 1893a: 525. Hesiodus jansoni Champion, 1885 MEX (YU) NIC Hesiodus jansoni Champion, 1885: 116. Hesiodus longitarsis Champion, 1885 MEX (VE) BEL NIC Hesiodus longitarsis Champion, 1885: 115. Hesiodus sordidus Champion, 1885 MEX (VE) Hesiodus sordidus Champion, 1885: 116. Genus Hicetaon Champion, 1885 [M] Hicetaon Champion, 1885: 111. Type species: Hicetaon frontalis Champion, 1885, monotypy. Hicetaon frontalis Champion, 1885 MEX (VE YU) BEL Hicetaon frontalis Champion, 1885: 112. Genus Ilus Champion, 1885 [M] Ilus Champion, 1885: 117. Type species: Ilus apicicornis Champion, 1885, monotypy. Ilus apicicornis Champion, 1885 BEL CRI Ilus apicicornis Champion, 1885: 118. Genus Iphthiminus Spilman, 1973 [M] Iphthiminus Spilman, 1973: 42. Type species: Iphthimus italicus Truqui, 1857, original designation. Iphthiminus lewisii (Horn, 1870) USA (AZ CA CO NM NV TX UT WY) MEX (BC CH) Iphthimus lewisii Horn, 1870: 335. Iphthimus laevissimus Casey, 1890b: 408. Synonymy: Gardiner and Pollock (2015 : 364). Iphthiminus opacus (LeConte, 1866) CAN (AB MB NB NS ON QC SK) USA (AZ CA CT IN MA ME MI MN MT NH NY OH PA SD VA VT WI WY) Iphthimus opacus LeConte, 1866b: 121. Iphthiminus serratus (Mannerheim, 1843) [Fig. 39 ] CAN (AB BC) USA (CA ID MT NE NV OR WA WY) Figure 39. Iphthiminus serratus (Mannerheim, 1843). Scale bar = 1 mm. Nyctobates serrata Mannerheim, 1843: 284. Nyctobates sublaevis Bland, 1865: 382. Synonymy: Gardiner and Pollock (2015 : 356). Iphthinus servilis Walker, 1866: 326. Synonymy: Horn (1870 : 334). Iphthinus servator Walker, 1866: 327. Synonymy: Horn (1870 : 334). Iphthinus subligatus Walker, 1866: 327. Synonymy: Horn (1870 : 334). Iphthimus salebrosus Casey, 1924: 327. Synonymy: Gardiner and Pollock (2015 : 356). Genus Isaminas Champion, 1887 [M] Isaminas Champion, 1887: 266. Type species: Isaminas gibbipennis Champion, 1887, subsequent designation ( Gebien 1943 : 401). Pteroglymmius Gebien, 1928b: 223. Type species: Pteroglymmius erotyloides Gebien, 1928, monotypy. Synonymy: Doyen (1988 : 301). Isaminas breedlovei Doyen, 1988 MEX (CI) Isaminas breedlovei Doyen, 1988: 303. Isaminas brevicollis Champion, 1887 MEX (CI) GUA Isaminas brevicollis Champion, 1887: 267. Isaminas erotyloides (Gebien, 1928) HON Pteroglymmius erotyloides Gebien, 1928b: 224. Isaminas gibbipennis Champion, 1887 NIC CRI Isaminas gibbipennis Champion, 1887: 267. Isaminas reticuloides Doyen, 1988 MEX (CI) Isaminas reticuloides Doyen, 1988: 303. Isaminas sullivani Doyen, 1988 CRI Isaminas sullivani Doyen, 1988: 306. Genus Isicerdes Champion, 1885 [M] Isicerdes Champion, 1885: 113. Type species: Isicerdes occultus Champion, 1885, subsequent designation ( Lucas 1920 : 353). Isicerdes funebris Champion, 1885 GUA Isicerdes funebris Champion, 1885: 114. Isicerdes occultus Champion, 1885 MEX (VE) GUA BEL PAN Isicerdes occultus Champion, 1885: 114. Isicerdes vicinus Champion, 1892 MEX (JA YU) CRI Isicerdes vicinus Champion, 1892: 524. Genus Lenkous Kaszab, 1973 [M] Lenkous Kaszab, 1973: 315. Type species: Lenkous myrmecophilus Kaszab, 1973, original designation. Lenkous ibisca Ferrer and Ødegaard, 2005 PAN Lenkous ibisca Ferrer and Ødegaard, 2005: 639. Genus Merinus LeConte, 1862 [M] Merinus LeConte, 1862a: 230. Type species: Tenebrio laevis Olivier, 1795, original designation. Merinus laevis (Olivier, 1795) [Fig. 40 ] CAN (ON QC) USA (AL CT FL DC GA IA IL IN KS MD MI MO MS NC NJ NY OH OK PA RI SC TN VA WI WV) Figure 40. Merinus laevis (Olivier, 1795). Scale bar = 1 mm. Tenebrio laevis Olivier, 1795: [57] 10. Genus Mitys Champion, 1885 [M] Mitys Champion, 1885: 97. Type species: Mitys inflatus Champion, 1885, subsequent designation ( Gebien 1943 : 402). Mitys inflatus Champion, 1885 MEX (OA VE) Mitys inflatus Champion, 1885: 97. Mitys opacus Champion, 1885 MEX (TA) Mitys opacus Champion, 1885: 98. Mitys politus (Brême, 1842) MEX (PU VE) Sphoerotus politus Brême, 1842: 109. Mitys laevis Champion, 1885: 98. Synonymy: Champion (1892 : 520). Genus Moeon Champion, 1886 [N] 81 Moeon Champion, 1886: 251. Type species: Moeon isthmicus Champion, 1886, subsequent designation ( Gebien 1942 : 330). Moeon isthmicum Champion, 1886 PAN Moeon isthmicus Champion, 1886: 251. Moeon panamense Champion, 1886 PAN Moeon panamensis Champion, 1886: 251. Genus Mophon Champion, 1886 [M] Mophon Champion, 1886: 247. Type species: Mophon tinctipennis Champion, 1886, monotypy. Mophon tinctipennis Champion, 1886 NIC PAN Mophon tinctipennis Champion, 1886: 248. Genus Mylaris Pallas, 1781 [F] Mylaris Pallas, 1781: 37 82 . Type species: Tenebrio gigas Linnaeus, 1763, subsequent designation ( Guérin-Méneville 1844 : 120). Iphthinus Dejean, 1834 [30 June]: 203. Type species: Tenebrio gigas Linnaeus, 1763, subsequent designation ( Spilman 1973 : 42). Synonymy: Spilman (1973 : 42). Cecrops Gistel, 1834 [23 September]: 21. Type species: Tenebrio gigas Linnaeus, 1763, subsequent designation ( Bousquet and Bouchard 2017a : 131). Synonymy: Bousquet and Bouchard (2017a : 132). Nyctobates Guérin-Méneville, 1834 ["31 December"]: 33. Type species: Tenebrio gigas Linnaeus, 1763, original designation. Synonymy: Ferrer and Siliansky (2007: 186). Mylaris gigas (Linnaeus, 1763) MEX GUA NIC CRI PAN / SA Tenebrio gigas Linnaeus, 1763: 13. Mylaris gigantea Pallas, 1781: 37. Synonymy: Pallas (1781 : 37). Tenebrio laminatus Fabricius, 1787: 211. Synonymy: Fabricius (1801a : 144). Mylaris procera (Champion, 1885) MEX (SI VE) GUA BEL PAN / SA Nyctobates procerus Champion, 1885: 107. Genus Nesocyrtosoma Marcuzzi, 1976 [N] Nesocyrtosoma Marcuzzi, 1976: 137. Type species: Cyrtosoma inflatum Marcuzzi, 1976, designation of the International Commission on Zoological Nomenclature ( ICZN 2017 ). Pachycyrtosoma Marcuzzi, 1999: 81. Type species: Cyrtosoma merkli Marcuzzi, 1999, original designation. Synonymy: Hopp and Ivie (2009 : 13). Serrania Garrido, 2003: 50. Type species: Diaperis viridula Zayas, 1988 (= Platydema virens Laporte and Brullé, 1831), monotypy. Synonymy: Hopp and Ivie (2009 : 13). Nesocyrtosoma altagracia Hopp and Ivie, 2009 DOM Nesocyrtosoma altagracia Hopp and Ivie, 2009: 40. Nesocyrtosoma bankense Hopp and Ivie, 2009 PRI LAN Nesocyrtosoma bankense Hopp and Ivie, 2009: 57. Nesocyrtosoma basilense Hopp and Ivie, 2009 HAI Nesocyrtosoma basilense Hopp and Ivie, 2009: 41. Nesocyrtosoma bestiola Hopp and Ivie, 2009 DOM Nesocyrtosoma bestiola Hopp and Ivie, 2009: 20. Nesocyrtosoma crenulatum Hopp and Ivie, 2009 HAI DOM Nesocyrtosoma crenulatum Hopp and Ivie, 2009: 44. Nesocyrtosoma bromelicolus Garrido and Varela, 2010: 33. Synonymy: Hopp (2011 : 242). Nesocyrtosoma critalense (Zayas, 1988) CUB Cnodalon critalensis Zayas, 1988: 99. Nesocyrtosoma cubanense (Kulzer, 1961) CUB Apsida cubanensis Kulzer, 1961a: 217. Nesocyrtosoma cuproso (Zayas, 1988) 83 CUB Cnodalon cuproso Zayas, 1988: 98. Nesocyrtosoma curvum Hopp and Ivie, 2009 PRI Nesocyrtosoma curvum Hopp and Ivie, 2009: 68. Nesocyrtosoma darlingtoni Hopp and Ivie, 2009 HAI Nesocyrtosoma darlingtoni Hopp and Ivie, 2009: 47. Nesocyrtosoma dentatum Hopp and Ivie, 2009 CUB Nesocyrtosoma dentatum Hopp and Ivie, 2009: 76. Nesocyrtosoma dolosum Hopp and Ivie, 2009 HAI Nesocyrtosoma dolosum Hopp and Ivie, 2009: 42. Nesocyrtosoma elongatum (Zayas, 1988) CUB Cnodalon elongatus Zayas, 1988: 101. Nesocyrtosoma fernandoi Hopp and Ivie, 2009 CUB Nesocyrtosoma fernandoi Hopp and Ivie, 2009: 59. Nesocyrtosoma ferrugineum (Garrido and Gutiérrez, 1996) CUB Cyrtosoma ferruginea Garrido and Gutiérrez, 1996c: 282. Nesocyrtosoma garridoi Hopp and Ivie, 2009 CUB Nesocyrtosoma garridoi Hopp and Ivie, 2009: 60. Nesocyrtosoma gebieni (Marcuzzi, 1976) CUB Cyrtosoma gebieni Marcuzzi, 1976: 139. Cnodalon punctatum Zayas, 1988: 103. Synonymy: Garrido and Gutiérrez (1996c : 282). Nesocyrtosoma guerreroi Hopp and Ivie, 2009 DOM Nesocyrtosoma guerreroi Hopp and Ivie, 2009: 69. Nesocyrtosoma hispaniolae (Marcuzzi, 1999) DOM Cyrtosoma hispaniolae Marcuzzi, 1999: 83. Nesocyrtosoma inflatum (Marcuzzi, 1976) CUB Cyrtosoma inflatum Marcuzzi, 1976: 138. Cnodalon trinitatis Zayas, 1988: 102 [junior secondary homonym of Cyrtosoma trinitatis Marcuzzi, 1976]. Synonymy: Hopp and Ivie (2009 : 31). Cyrtosoma iviei Marcuzzi, 1998a: 160. Replacement name for Cyrtosoma trinitatis (Zayas, 1988). Nesocyrtosoma lacrima Hopp and Ivie, 2009 LAN Nesocyrtosoma lacrima Hopp and Ivie, 2009: 36. Nesocyrtosoma larseni Hopp and Ivie, 2009 CUB DOM Nesocyrtosoma larseni Hopp and Ivie, 2009: 50. Nesocyrtosoma merkli (Marcuzzi, 1999) DOM Cyrtosoma merkli Marcuzzi, 1999: 82. Nesocyrtosoma mutabile Hopp and Ivie, 2009 DOM Nesocyrtosoma mutabile Hopp and Ivie, 2009: 48. Nesocyrtosoma nearnsi Hopp and Ivie, 2009 HAI DOM Nesocyrtosoma nearnsi Hopp and Ivie, 2009: 65. Nesocyrtosoma neibaense Hopp and Ivie, 2009 HAI DOM Nesocyrtosoma neibaense Hopp and Ivie, 2009: 29. Nesocyrtosoma otus Hopp and Ivie, 2009 HAI DOM Nesocyrtosoma otus Hopp and Ivie, 2009: 45. Nesocyrtosoma parallelum (Zayas, 1988) CUB Cnodalon parallelus Zayas, 1988: 96. Nesocyrtosoma productum Hopp and Ivie, 2009 DOM Nesocyrtosoma productum Hopp and Ivie, 2009: 67. Nesocyrtosoma puertoricense Hopp and Ivie, 2009 PRI Nesocyrtosoma puertoricense Hopp and Ivie, 2009: 61. Nesocyrtosoma purpureum Hopp and Ivie, 2009 DOM Nesocyrtosoma purpureum Hopp and Ivie, 2009: 43. Nesocyrtosoma scabrosum Hopp and Ivie, 2009 HAI Nesocyrtosoma scabrosum Hopp and Ivie, 2009: 35. Nesocyrtosoma serratum Hopp and Ivie, 2009 DOM Nesocyrtosoma serratum Hopp and Ivie, 2009: 62. Nesocyrtosoma skelleyi Hopp and Ivie, 2009 DOM Nesocyrtosoma skelleyi Hopp and Ivie, 2009: 63. Nesocyrtosoma simplex Hopp and Ivie, 2009 HAI DOM Nesocyrtosoma simplex Hopp and Ivie, 2009: 37. Nesocyrtosoma teresitae Hopp and Ivie, 2009 CUB Nesocyrtosoma teresitae Hopp and Ivie, 2009: 67. Nesocyrtosoma tumefactum (Marcuzzi, 1976) CUB Cyrtosoma tumefactum Marcuzzi, 1976: 138. Cnodalon tumefactum Zayas, 1988: 95 [junior secondary homonym of Cyrtosoma tumefactum Marcuzzi, 1976]. Synonymy: Garrido and Gutiérrez (1996c : 282). Cnodalon inflatum Zayas, 1988: 101 [junior secondary homonym of Cyrtosoma inflatum Marcuzzi, 1976]. Synonymy: Hopp and Ivie (2009 : 24). Cyrtosoma zayasi Marcuzzi, 1998a: 160. Replacement name for Cyrtosoma tumefactum (Zayas, 1988). Cyrtosoma gundlachi Marcuzzi, 1998a: 160. Replacement name for Cyrtosoma inflatum (Zayas, 1988). Nesocyrtosoma turquinense (Zayas, 1988) CUB Cnodalon turquinensis Zayas, 1988: 96. Nesocyrtosoma virens (Laporte and Brullé, 1831) CUB PRI Platydema virens Laporte and Brullé, 1831: 391. Hoplocephala flavicornis Chevrolat, 1877a: 170. Synonymy: Chevrolat (1878d : 243). Diaperis viridula Zayas, 1988: 92. Synonymy: Hopp and Ivie (2009 : 71). Genus Nuptis Motschulsky, 1872 [M] Nuptis Motschulsky, 1872: 25. Type species: Nuptis tenuis Motschulsky, 1872, original designation. Nuptis caliginosus Champion, 1885 MEX (VE YU) Nuptis caliginosus Champion, 1885: 109. Nuptis cornutus Champion, 1885 GUA NIC CRI PAN / SA Nuptis cornutus Champion, 1885: 108. Nuptis corticalis Champion, 1885 NIC PAN Nuptis corticalis Champion, 1885: 110. Nuptis inquinatus Champion, 1885 MEX (CI JA) GUA NIC Nuptis inquinatus Champion, 1885: 109. Nuptis laticollis Champion, 1892 PAN Nuptis laticollis Champion, 1892: 523. Nuptis tenebrosus Champion, 1885 MEX (VE) GUA PAN Nuptis tenebrosus Champion, 1885: 110. Nuptis tenuis Motschulsky, 1872 NIC Nuptis tenuis Motschulsky, 1872: 32. Nuptis validus Champion, 1885 MEX (VE) GUA Nuptis validus Champion, 1885: 110. Genus Oeatus Champion, 1885 [M] Oeatus Champion, 1885: 111. Type species: Oeatus chevrolati Champion, 1885, subsequent designation ( Gebien 1941 : 342). Oeatus chevrolati Champion, 1885 MEX (VE) GUA BEL Oeatus chevrolati Champion, 1885: 111. Oeatus similis Champion, 1892 MEX (CI OA) GUA BEL CRI Oeatus similis Champion, 1892: 523. Genus Oenopion Champion, 1885 [M] Oenopion Champion, 1885: 98. Type species: Oenopion gibbosus Champion, 1885, monotypy. Oenopion adeptus Doyen, 1971 MEX (NL PU) Oenopion adeptus Doyen, 1971: 114. Oenopion gibbosus Champion, 1885 MEX (VE) Oenopion gibbosus Champion, 1885: 99. Oenopion zopheroides (Horn, 1874) USA (NM TX) MEX (SL) Iphthimus zopheroides Horn, 1874a: 34. Genus Othryoneus Champion, 1886 [M] Othryoneus Champion, 1886: 245. Type species: Othryoneus erotyloides Champion, 1886, subsequent designation ( Gebien 1942 : 315). Gaurobates Gebien, 1928b: 184. Type species: Gaurobates pictus Gebien, 1928, monotypy. Synonymy: Ferrer (2010 : 82). Othryoneus erotyloides Champion, 1886 NIC Othryoneus erotyloides Champion, 1886: 246. Othryoneus triplehorni Ferrer and Ødegaard, 2005 PAN Othryoneus triplehorni Ferrer and Ødegaard, 2005: 637. Genus Oxidates Champion, 1886 [M] Oxidates Champion, 1886: 263. Type species: Oxidates planicollis Champion, 1886, subsequent designation ( Gebien 1943 : 402). Oxidates aurichalceus Champion, 1887 MEX Oxidates aurichalceus Champion, 1887: 265. Oxidates elongatus Champion, 1893 MEX (GE) Oxidates elongatus Champion, 1893a: 548. Oxidates gibbus Champion, 1893 MEX (VE) Oxidates gibbus Champion, 1893a: 548. Oxidates gravidus (Brême, 1842) MEX (VE) Sphoerotus gravidus Brême, 1842: 109. Oxidates mexicanus (Brême, 1842) MEX (VE) Sphoerotus mexicanus Brême, 1842: 110. Oxidates planicollis Champion, 1886 MEX (VE) Oxidates planicollis Champion, 1886: 264. Oxidates princeps Champion, 1887 MEX (OA VE) Oxidates princeps Champion, 1887: 265. Oxidates puncticeps Champion, 1887 MEX (VE) Oxidates puncticeps Champion, 1887: 266. Oxidates thoracicus (Brême, 1842) MEX (VE) Sphoerotus thoracicus Brême, 1842: 110. Genus Polopinus Casey, 1924 [M] Polopinus Casey, 1924: 326. Type species: Polypleurus nitidus LeConte, 1866, original designation. Polopinus hubbelli Kritsky, 1989 USA (FL) Polopinus hubbelli Kritsky, 1989: 132. Polopinus ingens Casey, 1924 USA (FL GA) Polopinus ingens Casey, 1924: 327. Polopinus nitidus (LeConte, 1866) USA (FL) Polypleurus nitidus LeConte, 1866b: 118. Polopinus nitidus subdepressus Casey, 1924: 327. Synonymy: Kritsky (1989 : 128). Polopinus nitidus brevior Casey, 1924: 327. Synonymy: Kritsky (1989 : 128). Polopinus youngi Kritsky, 1989 USA (FL) Polopinus youngi Kritsky, 1989: 130. Genus Polypleurus Eschscholtz, 1831 [M] Polypleurus Eschscholtz, 1831: 10, 11. Type species: Polypleurus geminatus Eschscholtz, 1831, monotypy. Polypleurus geminatus Eschscholtz, 1831 USA (FL GA) Polypleurus geminatus Eschscholtz, 1831: 11. Polypleurus perforatus (Germar, 1823) USA (AL AR FL GA IL LA MD MO MS NC NJ OK PA SC TX VA WV) Upis perforata Germar, 1823: 148. Polypleurus punctatus Solier, 1838: 197. Synonymy: LeConte (1866a : 61). Genus Saziches Champion, 1886 [M] Saziches Champion, 1886: 261. Type species: Saziches subcaudatus Champion, 1886, monotypy. Saziches giesberti Doyen, 1988 CRI Saziches giesberti Doyen, 1988: 310. Saziches subcaudatus Champion, 1886 GUA Saziches subcaudatus Champion, 1886: 262. Genus Sthenoboea Champion, 1885 [F] Sthenoboea Champion, 1885: 112. Type species: Sthenoboea apicalis Champion, 1885, monotypy. Sthenoboea apicalis Champion, 1885 MEX Sthenoboea apicalis Champion, 1885: 113. Genus Upis Fabricius, 1792 [M] Upis Fabricius, 1792b: 515. Type species: Attelabus ceramboides Linnaeus, 1758, monotypy. Upis ceramboides (Linnaeus, 1758) [Fig. 41 ] CAN (AB BC MB NB NF NS NT ON QC PE SK YT) USA (AK ID ME MI NH NY OH OR PA SD VT WA WI WY) – Holarctic Figure 41. Upis ceramboides (Linnaeus, 1758). Scale bar = 1 mm. Attelabus ceramboides Linnaeus, 1758: 388. Tenebrio variolosus DeGeer, 1775: 32. Synonymy: DeGeer (1775 : 32). Tenebrio reticulatus Say, 1824b: 279. Synonymy: Dejean (1834 : 204). Genus Xenius Champion, 1886 [M] Xenius Champion, 1886: 224. Type species: Xenius scabripennis Champion, 1886, monotypy. Xenius scabripennis Champion, 1886 NIC PAN Xenius scabripennis Champion, 1886: 224. Genus Xylopinus LeConte, 1862 [M] Xylopinus LeConte, 1862a: 230. Type species: Tenebrio anthracinus Knoch, 1801 (= Tenebrio saperdoides Olivier, 1795), subsequent designation ( Gebien 1941 : 336). Taenobates Motschulsky, 1872: 25. Type species: Tenebrio saperdoides Olivier, 1795, original designation. Synonymy: C.O. Waterhouse (1876 : 288). Xylopinus aenescens LeConte, 1866 CAN (NB ON QC) USA (AL CT DC FL GA IA IN LA MA MD MI NC NJ NY OH PA RI SC TN VA WI) Xylopinus aenescens LeConte, 1866b: 120. Xylopinus saperdoides (Olivier, 1795) CAN (NB NS ON QC) USA (AL AR CT DC DE FL GA IA IL IN KS LA MD MI MN MO MS NC NH NY OH OK PA SC TN TX VA VT WI WV) Tenebrio saperdoides Olivier, 1795: [57] 11. Helops spinipes Fabricius, 1798: 53. Synonymy: Illiger (1802 : 344). Tenebrio anthracinus Knoch, 1801: 169. Synonymy (with H. spinipes Fabricius): Illiger (1802 : 344). Tenebrio rufipes Say, 1825: 203. Synonymy: Melsheimer (1853 : 139). Tribe Stenochiini Kirby, 1837 Stenochiadae Kirby, 1837: 238. Type genus: Stenochia Kirby, 1819 (= Strongylium Kirby, 1819). Strongyliides Lacordaire, 1859: 478. Type genus: Strongylium Kirby, 1819. Genus Cuphotes Champion, 1887 [M] Spheniscus Kirby, 1819: 421 [junior homonym of Spheniscus Moehring, 1758]. Type species: Spheniscus erotyloides Kirby, 1819, monotypy. Cuphotes Champion, 1887: 332. Replacement name for Spheniscus Kirby, 1819. Phygoscotus Schulz, 1902: 134. Replacement name for Spheniscus Kirby, 1819. Cuphotes cinctus (Olivier, 1795) NIC CRI PAN / SA Helops cinctus Olivier, 1795 [58]: 13. Erotylus unifasciatus Fabricius, 1798: 101. Synonymy: Chevrolat (1843 : 80). Spheniscus 4maculatus Erichson, 1847: 120. Synonymy: Champion (1887 : 334). Spheniscus 4-plagiatus Kirsch, 1866: 202. Synonymy: Champion (1887 : 334). Cuphotes corallifer (J. Thomson, 1859) PAN / SA Spheniscus corallifer J. Thomson, 1859: 108. Cuphotes elongatus (J. Thomson, 1859) NIC PAN / SA Spheniscus elongatus J. Thomson, 1859: 112. Cuphotes jansoni Champion, 1887 NIC Cuphotes jansoni Champion, 1887: 333. Cuphotes multimaculatus Pic, 1918 CRI Cuphotes multimaculatus Pic, 1918b: 7. Cuphotes nigromaculatus marginicollis (J. Thomson, 1859) MEX GUA Spheniscus marginicollis J. Thomson, 1859: 110. Cuphotes nigromaculatus nigromaculatus (J. Thomson, 1859) MEX (JA VE) GUA NIC CRI PAN Spheniscus nigro-maculatus J. Thomson, 1859: 110. Cuphotes unicolor Champion, 1887 NIC Cuphotes unicolor Champion, 1887: 334. Genus Mentes Champion, 1893 [M] Mentes Champion, 1893a: 559. Type species: Mentes ruficollis Champion, 1893, subsequent designation ( Lucas 1920 : 404). Mentes aeneopiceus Champion, 1896 LAN Mentes aeneopiceus Champion, 1896: 30. Mentes cisteloides Doyen, 1990 MEX (JA) Mentes cisteloides Doyen, 1990: 254. Mentes fusiformis Champion, 1893 GUA Mentes fusiformis Champion, 1893a: 560. Mentes ruficollis Champion, 1893 PAN Mentes ruficollis Champion, 1893a: 559. Mentes setipennis Champion, 1893 GUA Mentes setipennis Champion, 1893a: 560. Genus Oploptera Chevrolat, 1844 [F] Oploptera Chevrolat [in Guérin-Méneville], 1844: 126. Type species: Strongylium serraticorne Guérin-Méneville, 1834, monotypy. Otocerus Mäklin, 1867: 484. Unnecessary replacement name for Oploptera Chevrolat, 1844. Hoploptera Gemminger [in Gemminger and Harold], 1870: 2037. Unjustified emendation of Oploptera Chevrolat, 1844, not in prevailing usage. Subgenus Oploptera Chevrolat, 1844 [F] Oploptera Chevrolat [in Guérin-Méneville], 1844: 126. Type species: Strongylium serraticorne Guérin-Méneville, 1834, monotypy. Oploptera angelicae (Ferrer and Ødegaard, 2005) PAN Otocerus angelicae Ferrer and Ødegaard, 2005: 644. Oploptera chamelensis (Doyen, 1990) MEX (JA) Otocerus chamelensis Doyen, 1990: 256. Oploptera delicata (Ferrer and Ødegaard, 2005) PAN Otocerus delicatus Ferrer and Ødegaard, 2005: 649. Oploptera dilaticornis (Champion, 1888) PAN Otocerus dilaticornis Champion, 1888: 378. Oploptera hamata (Champion, 1888) NIC Otocerus hamatus Champion, 1888: 381. Oploptera interrupta (Champion, 1888) PAN Otocerus interruptus Champion, 1888: 380. Oploptera microps (Champion, 1888) NIC Otocerus microps Champion, 1888: 381. Oploptera nicaraguensis (Champion, 1888) NIC Otocerus nicaraguensis Champion, 1888: 379. Oploptera torolae (Champion, 1888) GUA Otocerus torolae Champion, 1888: 378. Subgenus Plicatocerus Pic, 1918 Plicatocerus Pic, 1918b: 11. Type species: Otocerus impressipennis Champion, 1888, monotypy. Oploptera impressipennis (Champion, 1888) PAN Otocerus impressipennis Champion, 1888: 382. Genus Poecilesthus Dejean, 1834 [M] Poecilesthus Dejean, 1834: 207. Type species: Erotylus fasciatus Fabricius, 1781, subsequent designation ( Hope 1841 : 133). Diestica Pascoe, 1868: xii. Type species: Diestica viridipennis Pascoe, 1868, monotypy. Synonymy: Gebien (1911b : 589). Poecilesthus cupripennis Champion, 1893 PAN Poecilesthus cupripennis Champion, 1893a: 562. Poecilesthus fragilicornis Champion, 1887 CRI PAN Poecilesthus fragilicornis Champion, 1887: 338. Poecilesthus guatemalensis Champion, 1887 GUA Poecilesthus guatemalensis Champion, 1887: 339. Poecilesthus immaculatus Champion, 1887 PAN Poecilesthus immaculatus Champion, 1887: 340. Poecilesthus laeviceps Champion, 1887 PAN Poecilesthus laeviceps Champion, 1887: 340. Poecilesthus laticollis Champion, 1887 MEX (CI VE) GUA Poecilesthus laticollis Champion, 1887: 339. Poecilesthus latus Champion, 1887 NIC PAN Poecilesthus latus Champion, 1887: 338. Poecilesthus maklini Champion, 1887 GUA Poecilesthus mäklini Champion, 1887: 341. Poecilesthus nigropunctatus Champion, 1887 MEX PAN / SA Poecilesthus nigro-punctatus Champion, 1887: 336. Poecilesthus variipes Champion, 1887 NIC PAN Poecilesthus variipes Champion, 1887: 337. Genus Pseudotocerus Champion, 1888 [M] Pseudotocerus Champion, 1888: 383. Type species: Stenochia longipes Lucas, 1859, subsequent designation ( Gebien 1948 : 542). Pseudotocerus attenuatus Champion, 1888 NIC Pseudotocerus attenuatus Champion, 1888: 383 84 . Genus Strongylium Kirby, 1819 [N] Strongylium Kirby, 1819: 417. Type species: Strongylium chalconatum Kirby, 1819, monotypy. Stenochia Kirby, 1819: 423. Type species: Stenochia rufipes Kirby, 1819, subsequent designation ( Hope 1841 : 133). Synonymy: Latreille (1829b : 683). Gentinadis Laporte, 1840: 240. Type species: Stenochia caerulea Laporte, 1840 (= Helops azureus Germar, 1823), monotypy. Synonymy: Lacordaire (1859 : 484). Saerangodes Sturm, 1843: 163. Type species: Helops interpunctatus Germar, 1823, monotypy. Synonymy (with Stenochia Kirby): Blanchard (1845 : 33). Reminius Casey, 1924: 321. Type species: Reminius ocularis Casey, 1924 (= Tenebrio terminatus Say, 1824), original designation. Synonymy: Spilman (1959 : 63). Strongylium acraeum Garrido and Armas, 2012 PRI Strongylium acraeum Garrido and Armas, 2012b: 76. Strongylium amethystinum (Guérin-Méneville, 1838) CUB Stenochia amethystina Guérin-Méneville [in Guérin-Méneville and Chevrolat], 1838: 281. Strongylium angustulum Mäklin, 1867 PAN / SA Strongylium angustulum Mäklin, 1867: 314. Strongylium antennale Mäklin, 1867 CUB Strongylium antennale Mäklin, 1867: 270. Strongylium anthrax Schwarz, 1878 USA (FL) Strongylium anthrax Schwarz, 1878: 369. Strongylium apache Triplehorn and Spilman, 1973 USA (AZ NM) MEX [SO] Strongylium apache Triplehorn and Spilman, 1973: 10. Strongylium apicicorne Mäklin, 1867 MEX (VE) Strongylium apicicorne Mäklin, 1867: 324. Strongylium armatum Mäklin, 1867 MEX (CI VE) GUA PAN Strongylium armatum Mäklin, 1867: 311. Strongylium atrum Champion, 1888 USA (AZ NM) MEX (CH DU SI) Strongylium atrum Champion, 1888: 360. Strongylium aulicum Mäklin, 1867 USA (FL TX) MEX (OA VE) GUA NIC PAN Strongylium aulicum Mäklin, 1867: 363. Strongylium auratum (Laporte, 1840) MEX (CI GE PU VE YU) GUA BEL NIC CRI PAN / SA Stenochia aurata Laporte, 1840: 240. Stenochia auratum var. hilaris Mäklin, 1867: 402. Synonymy: Champion (1888 : 360). Strongylium azureum (Germar, 1823) CUB / SA Helops azureus Germar, 1823: 153. Stenochia caerulea Laporte, 1840: 240. Synonymy: Mäklin (1867 : 404). Strongylium baetianum Garrido and Armas, 2012 HAI Strongylium baetianum Garrido and Armas, 2012a: 64. Strongylium basiclavis Zayas, 1988 CUB Strongylium basiclavis Zayas, 1988: 110. Strongylium belti Champion, 1888 NIC Strongylium belti Champion, 1888: 358. Strongylium bivittatum Champion, 1888 MEX (OA) Strongylium bivittatum Champion, 1888: 361. Strongylium blandum Mäklin, 1867 MEX (VE) Strongylium blandum Mäklin, 1867: 341. Strongylium brevipes Champion, 1888 NIC PAN Strongylium brevipes Champion, 1888: 372. Strongylium canaliculatum Champion, 1887 MEX GUA Strongylium canaliculatum Champion, 1887: 346. Strongylium cancellatum Mäklin, 1867 MEX (VE) BEL Strongylium cancellatum Mäklin, 1867: 320. Strongylium carinipenne Champion, 1888 PAN Strongylium carinipenne Champion, 1888: 374. Strongylium chalcopterum Mäklin, 1867 LAN (Martinique) Strongylium chalcopterum Mäklin, 1867: 431. Strongylium championi Gebien, 1948 USA (TX) MEX (JA VE) GUA BEL Strongylium varians Champion, 1888: 365 [junior secondary homonym of Strongylium varians (Pascoe, 1883)]. Strongylium championi Gebien, 1948: 532. Replacement name for Strongylium varians Champion, 1888. Strongylium chiriquense Champion, 1887 PAN Strongylium chiriquense Champion, 1887: 351. Strongylium chontalense Champion, 1887 NIC Strongylium chontalense Champion, 1887: 344. Strongylium cinctum Mäklin, 1867 MEX (VE) Strongylium cinctum Mäklin, 1867: 337. Strongylium clavicorne Champion, 1893 MEX (VE) Strongylium clavicorne Champion, 1893a: 562. Strongylium colombianum Champion, 1888 PAN / SA Strongylium colombianum Champion, 1888: 354. Strongylium conicicolle Mäklin, 1867 NIC CRI PAN Strongylium conicicolle Mäklin, 1867: 447. Strongylium conradti Champion, 1893 GUA Strongylium conradti Champion, 1893a: 563. Strongylium costaricense Champion, 1888 CRI Strongylium costaricense Champion, 1888: 353. Strongylium crassicorne Champion, 1887 NIC Strongylium crassicorne Champion, 1887: 347. Strongylium crenatum Mäklin, 1867 USA (AL AR FL GA IA KS LA MD MO MS NC OH OK SC TN TX VA) Strongylium crenatum Mäklin, 1867: 307. Strongylium cribripes Mäklin, 1867 MEX (VE) NIC PAN Strongylium cribripes Mäklin, 1867: 275. Strongylium cruentatum Mäklin, 1867 MEX (VE) Strongylium cruentatum Mäklin, 1867: 335. Strongylium cultellatum Mäklin, 1867 USA (FL) – Adventive Strongylium cultellatum Mäklin, 1867: 453. Strongylium cupeyal Zayas, 1988 CUB Strongylium cupeyal Zayas, 1988: 110. Strongylium cuproso Garrido, 2004 CUB Strongylium cuproso Garrido, 2004d: 52. Strongylium curticorne Champion, 1888 MEX (CI) Strongylium curticorne Champion, 1888: 369. Strongylium decoratum Mäklin, 1867 NIC CRI PAN / SA Strongylium decoratum Mäklin, 1867: 365. Strongylium delauneyi Fleutiaux and Sallé, 1890 LAN Strongylium delauneyi Fleutiaux and Sallé, 1890: 429. Strongylium dentatum Champion, 1887 NIC Strongylium dentatum Champion, 1887: 348. Strongylium discoidale Mäklin, 1867 MEX (VE) Strongylium discoidale Mäklin, 1867: 339. Strongylium elongatum Garrido and Armas, 2012 DOM Strongylium elongatum Garrido and Armas, 2012a: 65. Strongylium eminens Mäklin, 1867 MEX (JA VE) Strongylium eminens Mäklin, 1867: 374. Strongylium erraticum Champion, 1888 NIC Strongylium erraticum Champion, 1888: 373. Strongylium exaratum Champion, 1887 GUA PAN Strongylium exaratum Champion, 1887: 350. Strongylium excavatum Mäklin, 1867 MEX (OA VE) GUA NIC PAN Strongylium excavatum Mäklin, 1867: 274. Strongylium eximium Mäklin, 1867 CUB Strongylium eximium Mäklin, 1867: 269. Strongylium fossifrons Mäklin, 1867 PAN / SA Strongylium fossifrons Mäklin, 1867: 285. Strongylium fragile Champion, 1888 PAN Strongylium fragile Champion, 1888: 377. Strongylium frontale Champion, 1888 PAN Strongylium frontale Champion, 1888: 357. Strongylium funestum Mäklin, 1867 MEX Strongylium funestum Mäklin, 1867: 295. Strongylium gerstaeckeri Mäklin, 1867 MEX (VE) GUA NIC CRI PAN Strongylium gerstaeckeri Mäklin, 1867: 277. Strongylium gibbum Mäklin, 1867 MEX GUA Strongylium gibbum Mäklin, 1867: 252. Strongylium gregarium Champion, 1888 PAN Strongylium gregarium Champion, 1888: 373. Strongylium guadeloupense Gebien, 1911 LAN (Guadeloupe) Strongylium inaequale Fleutiaux and Sallé, 1890: 430 [junior primary homonym of Strongylium inaequale Mäklin, 1867]. Strongylium guadeloupense Gebien, 1911b: 596. Replacement name for Strongylium inaequale Fleutiaux and Sallé, 1890. Strongylium hemistriatum Triplehorn and Spilman, 1973 USA (TX) Strongylium hemistriatum Triplehorn and Spilman, 1973: 20. Strongylium hoepfneri chevrolatii Mäklin, 1867 MEX Strongylium chevrolatii Mäklin, 1867: 235. Strongylium hoepfneri hoepfneri Mäklin, 1867 MEX (VE) GUA Strongylium hoepfneri Mäklin, 1867: 232. Strongylium hoepfneri immundum Mäklin, 1867 MEX Strongylium immundum Mäklin, 1867: 234. Strongylium hoepfneri pectorale Mäklin, 1867 MEX NIC Strongylium pectorale Mäklin, 1867: 233. Strongylium hoepfneri scutellare Mäklin, 1867 MEX Strongylium scutellare Mäklin, 1867: 233. Strongylium ignitum Champion, 1887 NIC PAN Strongylium ignitum Champion, 1887: 348. Strongylium impressicolle Mäklin, 1867 MEX (DU JA VE) GUA NIC CRI Strongylium impressicolle Mäklin, 1867: 301. Strongylium languidum Mäklin, 1867 MEX (VE) GUA Strongylium languidum Mäklin, 1867: 312. Strongylium langurioides Champion, 1888 NIC Strongylium langurioides Champion, 1888: 355. Strongylium laterale Mäklin, 1867 MEX (VE) Strongylium laterale Mäklin, 1867: 334. Strongylium limitatum Mäklin, 1867 MEX (VE) Strongylium limitatum Mäklin, 1867: 342. Strongylium lucidum Mäklin, 1867 CRI Strongylium lucidum Mäklin, 1867: 283. Strongylium maculicolle Champion, 1887 NIC CRI PAN Strongylium maculicolle Champion, 1887: 342. Strongylium maisi Garrido, 2004 CUB Strongylium maisi Garrido, 2004d: 52. Strongylium marginale Mäklin, 1867 MEX (VE) Strongylium marginale Mäklin, 1867: 338. Strongylium misantlae Champion, 1888 MEX (OA VE) GUA Strongylium misantlae Champion, 1888: 367. Strongylium montebarreto Garrido, 2004 CUB Strongylium montebarreto Garrido, 2004d: 50. Strongylium nigrum Zayas, 1988 CUB Strongylium nigra Zayas, 1988: 110. Strongylium nitidiceps Champion, 1888 MEX (VE) Strongylium nitidiceps Champion, 1888: 364. Strongylium nubeculosum Mäklin, 1867 MEX (YU) NIC Strongylium nubeculosum Mäklin, 1867: 336. Strongylium oculatum Champion, 1888 GUA NIC Strongylium oculatum Champion, 1888: 371. Strongylium opacipenne Champion, 1888 MEX (VE) Strongylium opacipenne Champion, 1888: 361. Strongylium paddai Ivie and Triplehorn, 1986 VIS Strongylium paddai Ivie and Triplehorn, 1986: 423. Strongylium panamense Champion, 1888 PAN Strongylium panamense Champion, 1888: 363. Strongylium permodicum Mäklin, 1867 GUA NIC PAN / LAN / SA Strongylium permodicum Mäklin, 1867: 320. Strongylium preciosus Zayas, 1988 85 CUB Strongylium preciosus Zayas, 1988: 108. Strongylium pulvinatum Mäklin, 1867 PRI Strongylium pulvinatum Mäklin, 1867: 265. Strongylium pumilum Garrido and Armas, 2012 PRI Strongylium pumilum Garrido and Armas, 2012b: 73. Strongylium punctifrons Mäklin, 1867 MEX (CI VE) BEL Strongylium punctifrons Mäklin, 1867: 296. Strongylium punctipes Champion, 1888 MEX (JA) GUA Strongylium punctipes Champion, 1888: 375. Strongylium quisqueyanum Garrido and Armas, 2012 DOM Strongylium quisqueyanum Garrido and Armas, 2012a: 66. Strongylium ramosum Mäklin, 1867 MEX (VE) Strongylium ramosum Mäklin, 1867: 340. Strongylium sallei Mäklin, 1867 MEX (VE) GUA NIC Strongylium sallei Mäklin, 1867: 257. Strongylium sallaei Champion, 1887: 345. Unjustified emendation of Strongylium sallei Mäklin, 1867, not in prevailing usage. Strongylium semistriatum Mäklin, 1867 MEX (VE) Strongylium semistriatum Mäklin, 1867: 251. Strongylium simplicipes Pic, 1918 PAN Strongylium simplicipes Pic, 1918b: 15. Strongylium simplicicolle LeConte, 1878 USA (AL FL GA MS NC SC TN VA) Strongylium simplicicolle LeConte, 1878a: 424. Strongylium subcostatum Mäklin, 1867 MEX GUA Strongylium subcostatum Mäklin, 1867: 316. Strongylium suturale Mäklin, 1867 MEX (VE) GUA Strongylium suturale Mäklin, 1867: 337. Strongylium tenuicolle (Say, 1826) [Fig. 42 ] CAN (MB ON QC) USA (AL AR CO CT DC DE FL GA IA IL IN KS KY LA MA MD MI MN MO MS NC NH NJ NY OH OK PA RI SC SD TN TX VA WI WV) Figure 42. Strongylium tenuicolle (Say, 1826). Scale bar = 1 mm. Helops tenuicollis Say, 1826: 241. Strongylium terminatum (Say, 1824) USA (AL DC FL IA IL IN KS KY LA MD MI MO MS NC NE NJ NY OH OK PA SC SD TX VA WI) Tenebrio terminatus Say, 1824a: 267. Reminius ocularis Casey, 1924: 322. Synonymy: Spilman (1959 : 63). Strongylium tinctipes Champion, 1887 NIC PAN Strongylium tinctipes Champion, 1887: 349. Strongylium turquinense Zayas, 1988 CUB Strongylium turquinensis Zayas, 1988: 107. Strongylium variicorne Champion, 1887 PAN Strongylium variicorne Champion, 1887: 352. Strongylium ventrale Champion, 1888 PAN Strongylium ventrale Champion, 1888: 356. Strongylium venustum Zayas, 1988 CUB Strongylium venusta Zayas, 1988: 109. Strongylium verde Garrido and Armas, 2012 PRI Strongylium verde Garrido and Armas, 2012b: 74. Strongylium vikenae Ferrer and Ødegaard, 2005 PAN Strongylium vikenae Ferrer and Ødegaard, 2005: 644. Strongylium virescens Zayas, 1988 CUB Strongylium virescens Zayas, 1988: 110. Strongylium viridipes Mäklin, 1867 MEX (PU VE) Strongylium viridipes Mäklin, 1867: 274. Strongylium viriditinctum Champion, 1888 GUA Strongylium viriditinctum Champion, 1888: 359. Strongylium woodruffi Garrido and Armas, 2012 DOM Strongylium woodruffi Garrido and Armas, 2012a: 67. Tribe Talanini Champion, 1887 Dignamptini LeConte and Horn, 1883: 385. Type genus: Dignamptus LeConte, 1878 (= Talanus Jacquelin du Val, 1857). Talanides Champion, 1887: 321. Type genus: Talanus Jacquelin du Val, 1857. Note. Family-group name conserved over Dignamptini (see Bouchard et al. 2005 ). Genus Talanus Jacquelin du Val, 1857 [M] Talanus Jacquelin du Val, 1857: 156. Type species: Talanus cribrarius Jacquelin du Val, 1857, monotypy. Dignamptus LeConte, 1878a: 421. Type species: Dignamptus stenochinus LeConte, 1878, present designation . Synonymy: Champion (1887 : 321). Talanus aeneipennis Champion, 1887 MEX (TB VE) BEL Talanus aeneipennis Champion, 1887: 327. Talanus apterus Champion, 1887 GUA PAN Talanus apterus Champion, 1887: 328. Talanus ater Champion, 1887 PAN Talanus ater Champion, 1887: 327. Talanus columbianus Mäklin, 1878 PAN / SA Talanus columbianus Mäklin, 1878a: 99. Talanus cribrarius Jacquelin du Val, 1857 CUB Talanus cribrarius Jacquelin du Val, 1857: 156. Talanus ferrugineus Champion, 1896 LAN Talanus ferrugineus Champion, 1896: 31. Talanus guadeloupensis Fleutiaux and Sallé, 1890 LAN Talanus guadeloupensis Fleutiaux and Sallé, 1890: 430. Talanus guatemalensis Champion, 1887 GUA Talanus guatemalensis Champion, 1887: 326. Talanus insularis Mäklin, 1878 PRI LAN Talanus insularis Mäklin, 1878a: 98. Talanus interstitialis Champion, 1887 ME (CI JA) GUA NIC Talanus interstitialis Champion, 1887: 324. Talanus laevicollis Champion, 1896 LAN Talanus laevicollis Champion, 1896: 32. Talanus laevipennis Champion, 1887 GUA Talanus laevipennis Champion, 1887: 322. Talanus langurinus (LeConte, 1878) USA (FL TX) Dignamptus langurinus LeConte, 1878a: 421. Talanus laticeps Champion, 1887 PAN Talanus laticeps Champion, 1887: 325. Talanus lecontei Champion, 1887 MEX (TB VE YU) GUA BEL Talanus lecontei Champion, 1887: 323. Talanus longicornis Champion, 1887 GUA BEL NIC PAN Talanus longicornis Champion, 1887: 328. Talanus mecoscelis Triplehorn, 1968 USA (TX) Talanus mecoscelis Triplehorn, 1968a: 33. Talanus neotropicalis Champion, 1887 MEX (JA VE YU) GUA CRI PAN / SA Talanus neotropicalis Champion, 1887: 322. Talanus spilmani Triplehorn, 1968 USA (FL) Talanus spilmani Triplehorn, 1968a: 35. Talanus stenochinus (LeConte, 1878) USA (FL LA) Dignamptus stenochinus LeConte, 1878a: 421. Talanus okeechobensis Blatchley, 1914: 143. Synonymy: Fall (1932 : 148). Talanus subexaratus Mäklin, 1878 MEX (CI GE SI VE) GUA BEL NIC CRI PAN / SA Talanus subexaratus Mäklin, 1878a: 102. Talanus subopacus Champion, 1887 MEX (VE) BEL Talanus subopacus Champion, 1887: 323. Talanus victori Garrido and Gutiérrez, 2004 PRI Talanus victori Garrido and Gutiérrez, 2004: 63. Incertae sedis: Tenebrionidae Tenebrio calculensis Scudder, 1895 CAN (ON) Tenebrio calculensis Scudder, 1895: 31 86 .

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350210/

Technological Approaches for Improving Vaccination Compliance and Coverage

Vaccination has been well recognised as a critically important tool in preventing infectious disease, yet incomplete immunisation coverage remains a major obstacle to achieving disease control and eradication. As medical products for global access, vaccines need to be safe, effective and inexpensive. In line with these goals, continuous improvements of vaccine delivery strategies are necessary to achieve the full potential of immunisation. Novel technologies related to vaccine delivery and route of administration, use of advanced adjuvants and controlled antigen release (single-dose immunisation) approaches are expected to contribute to improved coverage and patient compliance. This review discusses the application of micro- and nano-technologies in the alternative routes of vaccine administration (mucosal and cutaneous vaccination), oral vaccine delivery as well as vaccine encapsulation with the aim of controlled antigen release for single-dose vaccination. 1. Introduction Estimated global immunisation coverage varies per country and per vaccine, with some vaccines such as pneumococcal and rotavirus ranging between 35–47%, falling significantly short of the WHO's recommended coverage of >90%. Although many different factors affect these figures, vaccination compliance is one of the major obstacles to broader immunisation coverage globally. Pain that is felt during parenteral vaccine administration is one of the most frequently reported causes of vaccine hesitancy. Success of the oral polio vaccine in bringing the disease close to eradication (notwithstanding recent issues related to the vaccine itself [ 1 ]), and the increasing uptake of the nasal flu vaccine for children, indicate that non-injectable routes of vaccination have a significant impact on improving coverage and compliance. Vaccines also have a critical role in slowing down and preventing the spread of antibiotic resistance worldwide [ 2 ]. Approaches to broaden vaccination coverage include the development of new technologies for the mode and route of vaccine administration, and formulations for cutaneous, oral and mucosal delivery ( Figure 1 ). We discuss here the targeted strategies and key innovations of different vaccination technologies, highlighting their potential to overcome common vaccination-related limitations and improve compliance. 2. Vaccine Formulations for Pulmonary and Nasal Delivery Mucosal immunity is the first and foremost line of defence against pathogens that enter the body through the oral mucosa and the nasal cavity, such as influenza and viruses causing meningitis, measles or whooping cough. Estimates of the surface area of the mucus membranes lining the lungs vary between 50–75 square meters, representing the largest epithelial surface exposed to the outside environment. However, most licensed vaccines are administered by intramuscular injection, which preferentially induces systemic immune responses [ 3 ]. Protective mucosal responses can be effectively elicited by mucosal immunisation [ 4 ]; furthermore, mucosal vaccines are attractive by being non-invasive and needle-free. The airway mucosa is the site of substantial immunological activity where constant immune monitoring recruits highly professional innate and adaptive immune cells, to protect the host from microbial and environmental insults. Nasopharynx-associated lymphoid tissue (NALT) is an organised mucosal immune system that consists of lymphoid tissue, B cells, T cells and antigen presenting cells (APCs), covered by an epithelial layer containing microfold (M) cells [ 5 ]. M cells in the epithelial cell layers have a specialised role in transporting antigens across the epithelium [ 5 , 6 ]. Following mucosal administration, immunity is induced at mucosal as well as serosal surfaces and in both local and distal mucosa. Compared to conventional injectable vaccines, mucosal vaccines have many other advantages: ease of administration, better patient compliance, lower costs, avoidance of needle stick injuries and needle waste, and the scope for mass immunisation [ 7 ]. The interconnection of various mucosal sites through a common mucosal immune system allows the possibility to immunise via the nose against infectious diseases that originate from distal mucosal sites [ 8 ]. Intranasal administration of a vaccine allows the induction of a strong systemic and local immune response. In a recent phase I clinical trial of a novel intranasal respiratory syncytial virus (RSV) F protein vaccine, which was linked to an immunostimulatory bacterium-like particle, persistent nasal IgA and serum IgG responses were observed for up to six months [ 9 ]. In addition, the enzymatic activity in the nasal cavity is relatively weak, compared to the oral route, which can be a reason why some vaccines are more efficient when administered intranasally. In a study that compared the mucosal delivery of the intranasal and oral Bordetella bronchiseptica vaccine, the intranasal vaccine conferred stronger clinical immunity [ 10 ]. However, antigens can also be rapidly removed from the nasal cavity or poorly absorbed by epithelial cells, potentially leading to reduced immunogenicity [ 11 ]. Nasal vaccines can be delivered in the form of powder, aerosol, gel or drops. Each of these forms have their advantages and disadvantages, as reviewed previously [ 12 ]. Mucosal vaccination can protect against several mucosally transmitted bacterial and viral diseases, and a few oral and nasal mucosal vaccines have been authorised for use in humans e.g., sabin polio, rotavirus, and nasal influenza vaccine [ 7 ]. All of these mucosal vaccines are based on the entire pathogen, either killed or live attenuated. Thus, they are associated with many disadvantages including laborious and expensive production and distribution, and the risk of reversion to the virulent form. Conversely, the new generation of subunit vaccines that are based on single or multiple highly purified pathogenic antigens, such as peptides, proteins, polysaccharides, and nucleic acids, represent safer alternatives. Such subunit vaccines are inherently poorly immunogenic and require the inclusion of an adjuvant. This is especially relevant for mucosal delivery routes where the targeted mucosal epithelium, which naturally is in contact with many possible antigens, requires a strong immune-potentiating signal in order not to induce tolerance [ 13 ]. Subunit vaccines for pulmonary and nasal immunisation, when combined with adjuvants, can overcome many of the shortcomings of conventional vaccines. Table 1 summarises the vaccines delivered through pulmonary and nasal routes in different phases of clinical testing. Adjuvants comprise structurally diverse compounds, which can be categorised as delivery systems or immunopotentiators, or a combination of both [ 14 ]. Delivery systems enhance the immune response against the co-delivered antigen by protecting it from degradation and allowing sustained antigen release. Different classes of delivery systems that can be used as a platform technology for delivering vaccines include liposomes, polymer particles, inorganic particles, bacterial or viral vectors, outer membrane vesicles, immunostimulating complexes, emulsions, and virus-like particles ( Table 2 ). These vaccine carriers can extend the residence time of the antigen on the mucosa, which increases its chances of getting into the deeper layers and reaching the immune cells [ 15 ]. Different strategies can be used to deliver vaccine antigen to the mucosa: mucoadhesion, M cell targeting or targeting APCs. Mucoadhesion can be achieved by employing positively charged carriers such as chitosan or liposomes. Endocine™ is a mucosal adjuvant based on endogenous lipids found in the human body that possess a negative charge. It was shown to be both safe and effective in inducing a humoral and cell-mediated immune (CMI) response after intranasal administration in animal models, including in aged mice [ 16 ]. A positively charged oil-in-water nanoemulsion, when combined with the H5 hemagglutinin antigen, was recently shown to be effective in protecting against influenza challenge in ferrets [ 17 ]. A carrier composed of liposomes covered with chitosan induced both systemic and mucosal immunity in mice when administered intranasally together with an epitope from group A Streptococcus protein [ 18 ]. There are many specific targets on the M cell surface that can be used for designing an efficient vaccine carrier [ 19 ]. Khan et al. used a conjugate of GB-1 M cell ligand and F and G protein fragments from RSV to vaccinate mice intranasally and reported an efficient mucosal and systemic response, as well as protection against nasal challenge with RSV [ 20 ]. The conjugation of chitosan with mannose increases its engulfment by dendritic cells (DCs) and macrophages, as these cells express the mannose receptor on their surface. Mannosylated chitosan used in a DNA vaccine against M. tuberculosis administered intranasally to mice-induced secretory IgA (sIgA) in the broncho-alveolar lavage (BAL) fluid and provided improved protection in challenge experiments [ 21 ]. Another example is the use of β-glucan which is recognised by the Dectin-1 receptor localised on DCs [ 22 ]. The addition of β-glucan to chitosan-HbsAg vaccine significantly increased the anti-HbsAg antibody titre in immunised mice [ 23 ]. It has to be underlined that in some cases the exact mechanism of APC targeting is unknown. DOTAP/DC-chol liposomes administered intranasally with pneumococcal surface protein A provided protection against pneumococcal infection, and were shown to be specifically engulfed by DCs, even though these particles do not possess any known DC ligands [ 24 ]. Immunopotentiators activate the immune system through pattern-recognition receptors (PRRs) expressed by APCs. Immunopotentiators can be bacterial or viral toll-like receptor (TLR) agonists, stimulator of interferon genes (STING) agonists, and cytokines ( Table 3 ). Delivery systems and immunopotentiators together determine the magnitude and quality of the innate immune response and the subsequent adaptive immune response specific to the co-delivered vaccine antigen. Vaccine antigens are delivered to dendritic cells, the most specialised APCs, initiating the differentiation of T-helper cell subsets, which in turn interact with B cells, eventually resulting in the production of antibodies (sIgA) at mucosal sites [ 8 ]. Dry Powders for Pulmonary Immunisation Most vaccine antigens are macromolecules, such as polysaccharides, proteins, peptides, and nucleic acids, and usually are at a great risk of chemical and physical degradation in liquid formulations [ 25 ]. All vaccines lose potency over time, and the rate of potency loss is dependent on the handling and storage temperature. The delivery of vaccine antigens in the form of dry powder particles to the lungs is recognised as a potential immunisation strategy that improves vaccine stability in comparison to liquid vaccine formulations [ 26 ]. The thermostability of vaccine antigens can be further improved by formulating them as dry powder microparticles in the presence of sugars as stabilising excipients [ 26 ]. A number of drying methods such as spray-drying, freeze-drying, and spray freeze-drying are used to prepare dry powder vaccine particles [ 26 ]. These approaches to develop thermostable vaccine formulations that are resistant to damage caused by freezing or overheating also eliminate the dependence on a cold chain. Thus, dry powder-based inhalable vaccine formulations for pulmonary immunisation not only induce systemic and mucosal immune responses [ 8 ], but also have logistical advantages over injectable vaccines [ 26 ]. Dry powder-based vaccine formulations have been designed and pre-clinically tested against several infectious diseases, progressing into clinical trials ( Table 1 ). An inhalable vaccine formulation of alginate-coated live Mycobacterium microparticles was more immunogenic than liquid aerosols, and provided better protection in mice against experimental Mycobacterium tuberculosis infection [ 27 ]. In another study, an intrapulmonary-delivered Advax-adjuvanted influenza vaccine induced higher memory B and T cell responses than intranasal or intramuscular immunisation and conferred superior disease protection [ 28 ]. The development of inhalable dry powder vaccines is thus a promising new strategy for pulmonary immunisation. However, a number of parameters defines the optimal performance of dry powder vaccines such as aerodynamic particle size, aerosolisation performance, antigen stability, controlled release, drug delivery device, safety, and the scale-up of manufacturing. Advancements in pharmaceutical and nano-technologies enabling the development and testing of dry powder vaccines for pulmonary immunisation should help to lay the groundwork for the successful commercialisation of the first aerosolised mucosal vaccine. Dry Powders for Pulmonary Immunisation Most vaccine antigens are macromolecules, such as polysaccharides, proteins, peptides, and nucleic acids, and usually are at a great risk of chemical and physical degradation in liquid formulations [ 25 ]. All vaccines lose potency over time, and the rate of potency loss is dependent on the handling and storage temperature. The delivery of vaccine antigens in the form of dry powder particles to the lungs is recognised as a potential immunisation strategy that improves vaccine stability in comparison to liquid vaccine formulations [ 26 ]. The thermostability of vaccine antigens can be further improved by formulating them as dry powder microparticles in the presence of sugars as stabilising excipients [ 26 ]. A number of drying methods such as spray-drying, freeze-drying, and spray freeze-drying are used to prepare dry powder vaccine particles [ 26 ]. These approaches to develop thermostable vaccine formulations that are resistant to damage caused by freezing or overheating also eliminate the dependence on a cold chain. Thus, dry powder-based inhalable vaccine formulations for pulmonary immunisation not only induce systemic and mucosal immune responses [ 8 ], but also have logistical advantages over injectable vaccines [ 26 ]. Dry powder-based vaccine formulations have been designed and pre-clinically tested against several infectious diseases, progressing into clinical trials ( Table 1 ). An inhalable vaccine formulation of alginate-coated live Mycobacterium microparticles was more immunogenic than liquid aerosols, and provided better protection in mice against experimental Mycobacterium tuberculosis infection [ 27 ]. In another study, an intrapulmonary-delivered Advax-adjuvanted influenza vaccine induced higher memory B and T cell responses than intranasal or intramuscular immunisation and conferred superior disease protection [ 28 ]. The development of inhalable dry powder vaccines is thus a promising new strategy for pulmonary immunisation. However, a number of parameters defines the optimal performance of dry powder vaccines such as aerodynamic particle size, aerosolisation performance, antigen stability, controlled release, drug delivery device, safety, and the scale-up of manufacturing. Advancements in pharmaceutical and nano-technologies enabling the development and testing of dry powder vaccines for pulmonary immunisation should help to lay the groundwork for the successful commercialisation of the first aerosolised mucosal vaccine. 3. Oral (Gastrointestinal) Vaccines Oral delivery is the most patient-friendly route of administration, and consequently, oral vaccines have the potential to improve vaccine efficacy by enhancing their accessibility and distribution, which may lead to better vaccine coverage [ 3 ]. Oral vaccination is also regarded as the optimal means to fight infections caused by enteric pathogens as it induces intestinal immunity through the gut-associated mucosal tissues [ 62 ]. The first successfully implemented oral vaccine was the oral polio vaccine developed in the 1950s by Albert Sabin. It had the ability to induce protective sIgA responses in the intestinal mucosa, the main site of poliovirus entry and multiplication. This significantly reduced viral transmission, leading towards the global eradication of polio [ 8 ]. Other licensed oral vaccines target diseases induced by enteric pathogens such as Vibrio cholerae , Salmonella typhi and rotavirus, causing cholera, typhoid fever and gastroenteritis, respectively [ 63 ]. Despite the clear benefits of oral vaccines, only a few have been successfully developed. Oral vaccines have to overcome difficult challenges linked to the gastrointestinal biology: the acidic environment in the stomach, the proteolytic enzymes necessary for protein degradation, the presence of mucus, low intestinal permeability and the generally poor immunogenicity of orally delivered antigens [ 64 ]. Consequently, an efficient oral vaccine should ideally be (1) stable in a highly enzymatic environment and resistant to site-specific pH; (2) delivered to specific immune-induction sites (e.g., Peyer's patches in the small intestine); (3) adapted to interactions with mucus; (4) able to be transported through the intestinal epithelial barrier; (5) captured by appropriate APCs and able to evade mucosal tolerance [ 19 ]. A number of oral drug delivery technologies are currently under pre-clinical and clinical development to overcome these challenges [ 65 ]. Various particle-, liposomal- or adenoviral-based systems have been evaluated as vehicles to deliver the vaccine antigens [ 62 ]. Other promising formulations are site-specific delivery systems, which are often capsule- or tablet-based. These systems can facilitate antigen protection and the delivery of vaccines to specific areas of the gastrointestinal tract and particularly to the key sampling sites such as the Peyer's patches [ 19 ]. Site-specific release can be achieved through the application of pH-dependent coating such as shellac, cellulose acetate phthalate, cellulose acetate trimellitate, poly(vinyl acetate phthalate), or hydroxypropyl methylcellulose phthalate [ 66 ]. These delivery systems play an important role in the stability and the delivery efficacy of vaccine components to the intestine. However, additional targeted strategies to specifically induce intestinal immune responses may be beneficial. Ligand-mediated vaccine delivery systems have been shown to direct antigens to specific receptors expressed on intestinal M cells, epithelial cells or intestinal APCs [ 19 ]. Given the pivotal role of M cells in antigen sampling, several lectin-, antibody- and peptide-based targeted strategies have been developed to specifically engage these cells. Plant lectin-based ligands, facilitating the bioadhesion to glycans expressed on M cells, have been tested. Ulex europaeus agglutinin-1 (UEA-1) was shown to specifically bind to α-L-fucose residues expressed on mouse Peyer's patch M cells [ 67 , 68 ] and able to target polystyrene microparticles [ 69 ] or liposomes [ 70 ] to them. Since many intestinal pathogens gain entry to the host through M cells, some M cell receptors used by bacteria have also been evaluated. For example, glycoprotein 2 (GP2) is expressed on murine and human intestinal M cells, and Escherichia coli ( E. coli ) and Salmonella enterica typhimurium are able to bind GP2 through FimH, a pili component on the bacterial outer membrane [ 71 ]. The conjugation of an anti-GP2 monoclonal antibody to ovalbumin (OVA) resulted in effective M cell targeting and oral immunisation with this system triggering enhanced faecal OVA-specific sIgA responses compared to the antigen alone in mice [ 72 ]. Other M cell-specific antibodies have been analysed. The conjugation of the anti-M cell antibody 5B11 to polystyrene particles enhanced their uptake by rabbit intestinal M cells in an ileal loop model [ 73 ], while oral vaccination with a conjugate of the NKM 16-2-4 antibody to botulinum toxoid (BT) enhanced BT-specific serum IgG and mucosal IgA responses as well as protective immunity against lethal challenge with BT in mice [ 74 ]. Finally, some peptides targeting M cells have been tested by oral route. The tripeptide Arginine-Glycine-Aspartic Acid (RGD) motif, which could bind to β-integrins on M cells, was shown to enhance antigen-specific serum IgG responses in mice [ 75 ] and the tetragalloyl-D-lysine dendrimer (TGDK) targeting murine, human, and nonhuman primate M cells was demonstrated to enhance faecal antigen-specific IgA responses in macaques [ 76 ]. In addition to targeted strategies to engage specific cells, including M cells, the choice of antigen and adjuvant is pivotal in developing efficient oral vaccines. The current licensed oral vaccines are composed of either live-attenuated or killed organisms, sometimes in combination with protein subunit components [ 77 ]. To enhance the antigen immunogenicity, novel recombinant enterotoxigenic E. coli (ETEC) [ 78 ] and Vibrio cholerae [ 79 ] strains, overexpressing antigens or expressing multiple antigens, have been developed and were successfully tested in animal models and in clinical trials. Considering that much vaccine development is currently focused on subunit vaccines, the addition of adjuvants to the formulation may be crucial to overcome intestinal tolerance. However, there are currently no licensed adjuvanted oral vaccines for human use. Cholera toxin (CT) and the heat-labile enterotoxin of E. coli (LT) have been shown to be potent mucosal adjuvants in pre-clinical studies. However, the native forms of these toxins are too toxic for use in humans, leading some research groups to develop toxin or toxin subunit mutants [ 77 ]. For instance, double-mutant labile toxin (dmLT) has been tested in pre-clinical studies [ 80 ] and evaluated in a Phase I clinical trial as part of a prototype oral ETEC vaccine [ 81 ]. Interestingly, dmLT was shown to promote Th17 responses but also protective sIgA responses in pre-clinical studies [ 82 ]. A non-toxic CT derivative was also developed as CTA1-DD which is a fusion between the A subunit of CT and the D-fragment of the Staphylococcus aureus protein A [ 83 ]. CTA1-DD was shown to be safe and enhanced the immunogenicity of various antigens in animal models [ 84 ]. Unconventional T cells such as invariant natural killer T (iNKT) cells or mucosal-associated invariant T (MAIT) cells have been considered as potential adjuvant targets: Intestinal sites are enriched in these cells which are at the interface between innate and adaptive immunity and can modulate APCs [ 85 ]. Some iNKT cell agonists have been investigated as adjuvants to enhance immune responses in immunotherapy and vaccination strategies [ 86 ]. Lavelle and colleagues demonstrated the potential of the iNKT cell activator α-Galactosylceramide (α-GalCer) as an oral adjuvant to enhance intestinal immune responses induced by experimental whole-cell killed ETEC [ 87 ], Vibrio cholerae [ 88 ] and Helicobacter pylori [ 89 ] antigens in mouse models. In addition, a novel oral delivery-integrated system named Single Multiple Pill ® (SmPill ® ), containing oil-in-water emulsions formulated as 1 mm minispheres, was reported to effectively protect and enhance the release of various drugs in targeted intestinal regions [ 90 ]. The SmPill ® integrated system, incorporating a recombinant formalin-killed whole-cell E. coli overexpressing the colonisation factor antigen I (CFA/I) and the orally active adjuvant α-GalCer, was shown to facilitate a controlled and sustained antigen release at intestinal pH [ 87 ]. Furthermore, this vaccine delivery system was able to enhance the intestinal CFA/I-specific sIgA responses in mice. It was also shown that a novel whole-cell killed Vibrio cholerae strain and recombinant cholera toxin subunit B (CTB) could be successfully loaded as antigens in SmPill ® minispheres. Consistent with the previous findings, Davitt and colleagues demonstrated that combining these antigens and α-GalCer in SmPill ® minispheres enhanced intestinal lipopolysaccharide and CTB-specific IgA responses and induced intestinal antigen-specific Th1 responses ( Figure 2 ) [ 88 ]. Finally, a key concern in vaccine formulation is stability. Indeed, the development of thermostable vaccine formulations may improve vaccine coverage, especially in low-income countries. The stability of this integrated oral delivery system containing formalin-killed whole-cell E. coli overexpressing CFA/I, and the orally active adjuvant α-GalCer, was evaluated under various temperature and humidity conditions. Longet and colleagues determined that SmPill ® minispheres maintained both the antigenicity of CFA/I and the immunostimulatory activity of the α-GalCer adjuvant after the storage of SmPill ® minispheres under room temperature and extreme storage conditions for several months [ 91 ]. Collectively, these results support the potential of the SmPill ® minisphere approach to enhance the immunogenicity of orally delivered antigens [ 87 ] and maintain the stability of oral vaccine formulations [ 91 ]. This exemplifies the potential to use integrated strategies to overcome challenges in developing oral vaccines. 4. Cutaneous Immunisation Skin or (trans)cutaneous vaccination is a route of immunisation mediated by topical, intradermal (ID) or intraepidermal delivery [ 92 ]. In line with global aspirations to expand vaccination coverage, cutaneous vaccines are regarded as a promising option for overcoming diverse issues, from vaccine safety and reactogenicity to patient preferences [ 93 ]. As an immunocompetent and multi-functional organ, the skin appears to be highly susceptible to certain vaccine adjuvants, resulting in enhanced immunogenicity and allowing the reduction in antigen dose and immunisation frequency [ 94 ]. Therefore, it is important that the immunogenicity profiles of cutaneous vaccines are not significantly inferior to other routes of vaccination [ 93 ]. Among the many technological approaches for cutaneous vaccination, several deserve a more detailed overview. Electroporation is an electro-permeabilisation method, being intensively explored in different fields: DNA manipulation in vitro, drug delivery and gene therapy. It is based on the transitory structural perturbation of lipid membranes (such as the cell membrane) through the application of high-voltage electrical impulses. Typically, electroporation involves the short-term exposure (few µs to ms) to high-voltage pulses (50–1500 V) with up to 1 s intervals. It is hypothesised that a structural rearrangement in the lipid bilayer occurs, consequently forming transient pores and facilitating molecular transport for both small molecules and biologics [ 95 ]. In the context of skin vaccine delivery, the CELLECTRA ® electroporation device (developed by Inovio Pharmaceuticals) has shown good safety and efficacy [ 96 ] and is currently being assessed in a Phase III clinical study for DNA-based immunotherapy of cervical cancer (NCT03721978). It has more recently also been applied to a DNA-based coronavirus vaccine (NCT04336410). Interestingly, there are findings suggesting that electroporation alone may act as a physical adjuvant, by stimulating the 'trickling' of Langerhans cells (LCs) away from the treatment site (presumably to lymph nodes) and inducing a certain level of pro-inflammatory cytokines [ 97 ]. Among the different thermal microporation technologies, fractional infrared laser ablation stands out as particularly suitable for bypassing the skin barrier properties. It enables the disruption of the stratum corneum in a highly controlled and adjustable fashion, simultaneously providing an intrinsic adjuvanting effect. Consequently, this technology has been intensively investigated for the pain-free prophylactic and therapeutic treatment of type I allergies and tumours [ 92 , 98 , 99 ]. Despite a two-step administration process (microporation followed by antigen application), laser ablation could potentially be valuable in mass vaccination campaigns, particularly in combination with dry vaccine patches, offering advantages such as heat stability, the avoidance of needle-related injuries, and improved uptake, at a cost similar to conventional vaccination [ 99 ]. Currently, the major challenge is how to standardise the pore depth and ensure sufficient reproducibility, due to the variability of skin thickness depending on body site, age and ethnicity [ 92 ]. The available results encourage further investigations in healthy adults to evaluate the safety and efficacy of laser microporation prior to, for example, vaccine patch application [ 98 ]. Microneedle arrays represent another interesting approach for intradermal vaccine delivery. Although these devices comprise needles, their length (10–2000 μm) offers relatively pain-free application [ 100 ]. Among the many microneedle types, future widespread vaccine delivery is expected for solid and dissolvable needles [ 93 , 101 ]. However, attaining reproducible coating and mechanical properties remain among the frequently noted critical attributes. Preclinical studies conducted to date imply that microneedles may generate immunogenicity comparable to intradermal or intramuscular vaccination, with some studies reporting higher and more durable antibody and cellular responses [ 101 ]. Satisfactory immune responses may be attained even without adjuvant addition or with considerably lower adjuvant doses than otherwise required, which is an important safety asset [ 92 ]. Therefore, microneedle patches are relatively cost effective, easy to produce and accepted well by the patients [ 102 ]. The fact that they allow self-administration may be the most significant factor in the further prospects for this mode of vaccine delivery [ 101 ]. In the search for an improved needle-free vaccination strategy, the application of dry vaccine powders using ballistic (powder) injectors seems to be a promising approach for delivering antigens to the skin, owing to improved vaccine stability and cold-chain independent logistics [ 103 , 104 ]. As a result, this technology has been recognised as potentially suitable for mass vaccination campaigns in developing countries [ 104 ]. Numerous studies show that DNA and RNA vaccines, as well as conventional vaccines in a dry state could be administered using the powder injectors [ 92 ]. However, in order to achieve the successful delivery of a particulate vaccine into the skin, apart from the design of ballistic devices, the powder properties (composition, particle size, shape, density) have to be carefully adjusted [ 104 ]. It should be emphasised that, although adequate immune responses have been observed in numerous preclinical and clinical studies, there are currently no ballistic injection products authorised for human use. The main unsolved issues are the high cost, cutaneous adverse reactions (e.g., erythema, petechiae, skin discoloration, oedema and skin flaking) and pain, that could lead to reduced patient adherence [ 105 , 106 ]. Jet injection is another needle-free approach that delivers liquid vaccine formulations in a 2–500 μL range using a highly pressured propulsion system connected to a needle-free syringe or nozzle [ 92 , 107 ]. The delivery of the vaccine can occur intradermally but also subcutaneously or even intramuscularly, forming a depot, depending on the applied velocity and overall design. Apart from a variation in the depth of delivery, other safety-related issues are successfully circumvented by the fact that this method now utilises prefilled disposable delivery units, thus avoiding contamination. If future research focuses on achieving more cost-effective manufacturing, jet injectors may be a part of promising multi-platform systems (e.g., coupled with nanoparticulate formulations) for driving further the cutaneous vaccination approach [ 107 , 108 ]. The aforementioned physical devices, although efficient in antigen delivery into the skin, may lead to some skin barrier damage, making them less suitable for mass vaccination under critical hygienic conditions [ 108 , 109 ]. As a result, there has been an increasing interest in passive delivery strategies, particularly nanocarriers, enabling antigen application to intact skin as well as improved antigen stability, sustained antigen release and increased antigenicity by mimicking the size of microorganisms [ 108 , 110 , 111 ]. Until now, different nanoparticles have been studied for this purpose, including vesicular nanocarriers (transfersomes, ethosomes, liposomes, niosomes, nanoemulsions) and solid nanoparticles (polymeric nanoparticles, silica-based nanoparticles) [ 92 , 110 ]. However, although nanoparticles may lead to a superior immune response compared to conventional intramuscular immunisation (particularly with an appropriate adjuvant), progress towards clinical settings has been negligible, due to difficulties in ensuring the accurate, reproducible and efficient delivery of antigen-loaded nanoparticles into the epidermal and dermal tissue [ 109 , 111 ]. Interestingly, in recent years, vaccination via the follicular route using nanoparticles, particularly non-flexible ones, has been recognised as a promising approach. It offers a CD8 + T cell-biased immune response (due to a large number of perifollicular APCs) that could be beneficial for the development of vaccines against intracellular pathogens, viruses and cancers [ 112 ]. However, it is important to emphasise that research in this area is still at an experimental stage, due to numerous factors affecting trans-follicular immunisation with nanoparticles, including particle size, surface properties (charge and composition of surface layer) and hair cycling [ 109 ]. 5. Controlled Antigen Release Delivery Systems for Single-Dose Immunisation Since the early 1990s, there has been extensive research on controlled release delivery systems for vaccine applications [ 113 , 114 ]. Prior to this, controlled delivery technologies were developed for a sustained delivery of drug molecules and provided the basic understanding of controlled release applications. This paved the way for delayed drug delivery technologies based on "smart" polymers, such as biodegradable microparticles, solid implants or in-site gel-forming implants [ 115 ] and the notion of developing biodegradable microparticles to accommodate prime-boost vaccine regimens within a single-dose formulation [ 116 ]. Encapsulating the booster vaccine into polymer particles should enable the pulsatile or continued release of the booster dose, that when combined with a free priming vaccine, can mimic a prime-boost regimen within a single immunisation. This approach would alleviate logistical challenges, the costs and the pressure on resources to deliver the booster doses, leading to increased vaccination compliance and coverage globally. Vaccine encapsulation also addresses the need for antigen sparing by improving immune responses through antigen shielding, controlled antigen release and adjuvanting effect due to the particulate nature of the encapsulated vaccine delivery system. The biodegradable polymer of choice is the FDA-approved poly lactic-co-glycolic acid (PLGA), which can be formulated into nano- and microparticles using a range of methods, the most common being water-in-oil-in-water (W/O/W) double emulsion solvent evaporation. An advantage of using polymer particles for vaccine delivery is that they can be adapted in size and/or structure to resemble a pathogen or to achieve the targeted delivery to promote humoral and CMI responses [ 117 , 118 ]. Particle size influences the immune response. For example, an efficient phagocytosis by macrophages may induce a more rapid immune response, while larger particles avoid the direct uptake by APCs and prolong antigen release [ 119 , 120 , 121 , 122 ]. Encapsulated antigens are protected from enzymatic degradation or rapid elimination in vivo, further contributing to an enhanced immune response [ 117 , 118 ]. The tuneable characteristics of the particulate vaccine delivery systems and the possibility for co-encapsulation or surface attachment of immunostimulatory agents play an important role in achieving the desired immune responses or adjuvant effects [ 123 , 124 ]. Various administration routes showing pre-clinical success have been reported for the oral, intranasal, parenteral and dermal delivery of antigens encapsulated in PLGA particles. These vaccine candidates, formulated using the W/O/W method, show great potential as a versatile vaccine delivery system and can achieve various immunologic requirements such as boosting antibody titres and inducing a CMI response. Although PLGA-based microparticles may no longer be a novel approach for vaccine delivery, there is a need for technological innovation to achieve more efficient, cost-effective, preferably solvent-free manufacturing methods of polymer-based controlled antigen-release delivery systems. Progress of PLGA Polymer Vaccine Delivery Systems The main method to achieve controlled antigen release has been the encapsulation into a polymer matrix by the W/O/W method. To this day, it remains the classic method that has been used to produce a wide array of particles with varying characteristics [ 125 ]. In general, for W/O/W, the primary emulsion consists of the antigen in an aqueous buffer emulsified into an 'oil' phase containing the polymer of choice in a selected solvent. This is followed by a secondary emulsification of the primary emulsion into an aqueous solution containing stabilising surfactants. Lastly, during a solidification phase the particles precipitate and harden as the solvent is evaporated. The manufacturing parameter of this method can be modified at various steps to obtain particles with the desired physicochemical characteristics. PLGA has been the most popular choice of biodegradable polymer and extensive research has demonstrated the influence of the co-polymer ratio, polymer viscosity, molecular weight, volume ratios and end caps on antigen release [ 116 , 126 , 127 ]. It is important to consider that it is not necessarily the PLGA polymer itself, but the particulate nature of the delivery system that allows for its immune-enhancing activity. The particulates are recognised as foreign material, triggering an immune response involving phagocytosis, the production of cytokines and the further activation of T cells. The potential of encapsulated vaccines was initially demonstrated using ovalbumin (OVA). Superior IgG responses were detected in mice receiving subcutaneous immunisation of OVA encapsulated in PLGA particles (10 µm) compared to OVA combined with Freund's adjuvant [ 113 ]. Liu et al. demonstrated that more efficient cross-presentation was achieved with OVA adsorbed or encapsulated into lipid-PLGA nanoparticles, exemplifying the potential to use integrated delivery strategies [ 128 ]. Numerous studies have confirmed the influence of particle size on both release kinetics and the resulting immune response [ 119 , 121 ]. Studies with smaller particles (0.3–7 µm) [ 129 ] and larger particles (100–150 µm) [ 130 ] both reported superior IgG titres after intraperitoneal administration. This highlights the difficulty of obtaining direct size and effect correlations for encapsulated vaccine delivery systems. Depending on the antigen and the desired immune response, the optimal particle size may differ greatly. Additionally, differences in the route of administration, animal models, and encapsulation efficiencies should be considered. Alternative polymer materials have also been explored. Microparticle formulation strategies based on poly (D,L-lactic acid) (PLA) have been investigated in encapsulating various antigens including rotavirus, tetanus toxoid and Vibrio cholerae [ 131 ]. PLA particles (2–8 µm) with encapsulated tetanus toxoid, administered intramuscularly to Wistar rats, induced high antibody titres. A further increase was observed when co-administered with Alhydrogel adjuvant [ 120 , 132 ]. The incorporation of poly (ethylene glycol) (PEG) was investigated with Vibrio cholerae -loaded PLA/PEG microparticles (4–5 µm), inducing high antibody titres as well as protection against a lethal challenge after oral administration in CD-1 outbred mice [ 133 ]. The W/O/W method development includes combining PLGA with polysaccharides, or using polysaccharides alone, to formulate antigen delivery systems. A combination of PLGA and sodium alginate investigated for the intradermal delivery of encapsulated malaria peptides demonstrated that particles of 1 µm induced a balanced Th1/Th2 response in BALB/c mice. In combination with immunostimulatory peptides (Arg–Gly–Asp), a strong CMI response was measured compared to the antigen alone or encapsulated in PLGA only [ 134 ]. Cationic polymers, such as chitosan, have been explored to formulate particles, however, these require emulsification with internal or external gelation techniques. When combined into PLGA particles, chitosan takes on an adjuvant role due to its inherent positive charge [ 135 ]. Novel functionalised dextrans have also been investigated in vaccine delivery systems [ 136 ]. The co-delivery of the encapsulated antigen and an adjuvant to enhance antigen presentation has been one of the focal points in the last decade, as characterisation methods have become increasingly advanced. Microparticle formulations containing aluminium salts and TLR9 agonist CpG oligonucleotides have shown that combining the antigen and adjuvant into a particulate delivery system can result in robust immune responses for single-dose vaccines compared to multi-immunisation schedules [ 124 , 132 , 137 ]. As mentioned above, other approaches have formulated the particles with ligands such as mannose to achieve DC targeting [ 21 , 135 ]. Multi-platform systems such as the single-dose pandemic influenza vaccine of recombinant outer membrane vesicles (rOMVs) encapsulated in PLGA demonstrated equivalent IgG titres to a prime and boost regimen, and protected mice against a challenge with a lethal dose of H1N1, six months post the initial vaccination. These microparticles (10–20 µm), manufactured using W/O/W, had a prolonged antigen release over 30 days [ 138 ]. This work demonstrates that significant results can be achieved when combining the classic W/O/W method with novel vaccine antigen technologies. Progress of PLGA Polymer Vaccine Delivery Systems The main method to achieve controlled antigen release has been the encapsulation into a polymer matrix by the W/O/W method. To this day, it remains the classic method that has been used to produce a wide array of particles with varying characteristics [ 125 ]. In general, for W/O/W, the primary emulsion consists of the antigen in an aqueous buffer emulsified into an 'oil' phase containing the polymer of choice in a selected solvent. This is followed by a secondary emulsification of the primary emulsion into an aqueous solution containing stabilising surfactants. Lastly, during a solidification phase the particles precipitate and harden as the solvent is evaporated. The manufacturing parameter of this method can be modified at various steps to obtain particles with the desired physicochemical characteristics. PLGA has been the most popular choice of biodegradable polymer and extensive research has demonstrated the influence of the co-polymer ratio, polymer viscosity, molecular weight, volume ratios and end caps on antigen release [ 116 , 126 , 127 ]. It is important to consider that it is not necessarily the PLGA polymer itself, but the particulate nature of the delivery system that allows for its immune-enhancing activity. The particulates are recognised as foreign material, triggering an immune response involving phagocytosis, the production of cytokines and the further activation of T cells. The potential of encapsulated vaccines was initially demonstrated using ovalbumin (OVA). Superior IgG responses were detected in mice receiving subcutaneous immunisation of OVA encapsulated in PLGA particles (10 µm) compared to OVA combined with Freund's adjuvant [ 113 ]. Liu et al. demonstrated that more efficient cross-presentation was achieved with OVA adsorbed or encapsulated into lipid-PLGA nanoparticles, exemplifying the potential to use integrated delivery strategies [ 128 ]. Numerous studies have confirmed the influence of particle size on both release kinetics and the resulting immune response [ 119 , 121 ]. Studies with smaller particles (0.3–7 µm) [ 129 ] and larger particles (100–150 µm) [ 130 ] both reported superior IgG titres after intraperitoneal administration. This highlights the difficulty of obtaining direct size and effect correlations for encapsulated vaccine delivery systems. Depending on the antigen and the desired immune response, the optimal particle size may differ greatly. Additionally, differences in the route of administration, animal models, and encapsulation efficiencies should be considered. Alternative polymer materials have also been explored. Microparticle formulation strategies based on poly (D,L-lactic acid) (PLA) have been investigated in encapsulating various antigens including rotavirus, tetanus toxoid and Vibrio cholerae [ 131 ]. PLA particles (2–8 µm) with encapsulated tetanus toxoid, administered intramuscularly to Wistar rats, induced high antibody titres. A further increase was observed when co-administered with Alhydrogel adjuvant [ 120 , 132 ]. The incorporation of poly (ethylene glycol) (PEG) was investigated with Vibrio cholerae -loaded PLA/PEG microparticles (4–5 µm), inducing high antibody titres as well as protection against a lethal challenge after oral administration in CD-1 outbred mice [ 133 ]. The W/O/W method development includes combining PLGA with polysaccharides, or using polysaccharides alone, to formulate antigen delivery systems. A combination of PLGA and sodium alginate investigated for the intradermal delivery of encapsulated malaria peptides demonstrated that particles of 1 µm induced a balanced Th1/Th2 response in BALB/c mice. In combination with immunostimulatory peptides (Arg–Gly–Asp), a strong CMI response was measured compared to the antigen alone or encapsulated in PLGA only [ 134 ]. Cationic polymers, such as chitosan, have been explored to formulate particles, however, these require emulsification with internal or external gelation techniques. When combined into PLGA particles, chitosan takes on an adjuvant role due to its inherent positive charge [ 135 ]. Novel functionalised dextrans have also been investigated in vaccine delivery systems [ 136 ]. The co-delivery of the encapsulated antigen and an adjuvant to enhance antigen presentation has been one of the focal points in the last decade, as characterisation methods have become increasingly advanced. Microparticle formulations containing aluminium salts and TLR9 agonist CpG oligonucleotides have shown that combining the antigen and adjuvant into a particulate delivery system can result in robust immune responses for single-dose vaccines compared to multi-immunisation schedules [ 124 , 132 , 137 ]. As mentioned above, other approaches have formulated the particles with ligands such as mannose to achieve DC targeting [ 21 , 135 ]. Multi-platform systems such as the single-dose pandemic influenza vaccine of recombinant outer membrane vesicles (rOMVs) encapsulated in PLGA demonstrated equivalent IgG titres to a prime and boost regimen, and protected mice against a challenge with a lethal dose of H1N1, six months post the initial vaccination. These microparticles (10–20 µm), manufactured using W/O/W, had a prolonged antigen release over 30 days [ 138 ]. This work demonstrates that significant results can be achieved when combining the classic W/O/W method with novel vaccine antigen technologies. 6. Advanced Vaccine Encapsulation Methods The classic W/O/W emulsification method has a number of shortcomings that have impeded the successful commercial development of an encapsulated antigen in polymer microparticles for single-dose vaccines. Formulation issues and the lack of particle uniformity lead to uneven antigen release profiles, including an initial burst (release of antigen upon injection) [ 139 ]. Considerable progress has been made to develop encapsulation technologies that address the issues of antigen stability, encapsulation efficiency, particle size and distribution and the suitable release profiles [ 140 , 141 ]. To encapsulate antigens, stability is critical and the encapsulation method needs to be optimised for each vaccine. Antigen stability may be impaired due to mechanical and chemical stress induced during emulsification steps. Novel encapsulation technologies that use milder processing methods are likely to mitigate the mechanical stress and reduce solvent-surface interactions; one such approach is coaxial electrospraying [ 142 ]. Furthermore, antigen stability can be achieved with the co-encapsulation of stabilising additives such as hydrophilic PEG, surfactants, sugars, or protein serum albumin [ 131 , 133 , 142 ]. The co-encapsulation of cationic excipients such as Eudragit E, poly(L-lysine) and branched polyethylenimine has shown promising results for inactivated polio vaccine encapsulation by W/O/W into PLGA microparticles [ 143 ]. The very adaptable process parameters of the W/O/W method have allowed the optimisation of encapsulation processes to reach suitable encapsulation efficiencies. Nonetheless, the (double) emulsification step means that the encapsulation process occurs at random. The spontaneous emulsification (SE) solvent diffusion technique has been suggested as an improved method for controlled protein encapsulation. With SE, a more homogenous distribution of the encapsulated antigen is achieved, thus reducing the initial burst effect [ 139 ]. The SE method was validated by encapsulating bovine serum albumin (BSA): the in vitro release kinetics demonstrated a pulsatile-release of BSA and comparable antibody titres after a single-dose subcutaneous administration to BALB/c mice [ 144 ]. Technologies such as microfluidics and spray-drying have demonstrated the feasibility of more controlled encapsulation processes and may be critical in achieving the necessary encapsulation efficiencies for large scale vaccine manufacturing. It has been demonstrated that the W/O/W method can be adapted to produce particles of desired size, however, it is quite difficult to achieve an extremely narrow size distribution (monodispersity). The size variation is largely due to the emulsification step, during which the particles have different droplet sizes, followed by variable precipitation rates during the solidification phase. The uniformity of particle size and distribution is also an important factor regarding the reproducibility and feasibility to scale up. Sieving the particles after emulsification can result in more uniform particle preparations. This is referred to as extrusion and achieved with Shirasu porous glass (SPG) beads [ 119 , 124 , 145 ]. Alternatively, encapsulation using novel microfluidic technologies may be a promising approach to achieve both adaptable particle sizes and a narrow size distribution. There remains a concern of translating laboratory-scale success to commercial-scale manufacture. Conventional emulsification methods may be inflated to a large scale of more than hundreds of litres per hour, however, there are numerous process parameters that need to be optimised individually [ 146 ]. This is an important aspect to take into consideration when validating novel antigen encapsulation technologies. There is an increasing interest in developing core-shell particles with hydrophilic cores to maintain the microenvironment and therefore the conformation and biological integrity of the antigen [ 147 ]. They can be formulated using W/O/W method, microfluidics, or by StampEd assembly of polymer layers (SEAL), a recently reported microfabrication approach based on 3D printing [ 148 ]. Core:shell microparticles (25 µm) with an acceptable size distribution (span) of 1.4 were manufactured using the W/O/W method combined with ionotropic gelation to form a sodium alginate-based hydro-core ( Figure 3 a,b) (Lemoine et al., unpublished). Using fluorophore labelling and confocal imaging, the core:shell structure of these particles was demonstrated and a variable distribution of the alginate cores was observed. Comparatively, core:shell particles with different shell thicknesses were manufactured by a microfluidics method. Using two microfluidic flow-focusing designs consecutively, the core and shell size of the particles were modified independently to produce two different populations, namely "thin-" and "thick-" shell particles. Both populations were highly monodispersed, with the coefficient of variation less than 5% (Guyon et al., unpublished). Precise and identical size parameters across a population of particles could contribute to a sharper burst release of the payload, as particles are likely to exhibit the same behaviour if they are monodispersed. The identification of other significant factors affecting the release is also more tractable when the size of a particle batch is controlled. 7. Approaches to Encapsulation Using Microfluidics Microfluidics involves the manipulation of fluid flows at a microscale, giving the fluids a laminar behaviour, and is used in specific applications such as droplet microfluidics. Thus, microfluidics can produce highly monodispersed droplets in a controlled and repeatable manner, a feature utilised in drug delivery, and in particular for vaccine encapsulation. In microfluidics, droplets are generated by intersection designs, where two immiscible or partially miscible phases are put into contact, and subsequently produce droplets by the combined actions of shear stress, viscous forces, and interfacial tension. This process can be conducted in capillaries assembled coaxially in "co-flow" type intersections or in microfluidic chips, often made with polydimethylsiloxane (PDMS)—a silicon polymer poured onto a mould and hardened to form microfluidic chips—or with glass, where the "T-junction" or "flow-focusing" designs are embedded. Multiple emulsions are formed with the different fluids flowing through successive intersections, generating successive layers of the multiple emulsion (see Figure 4 ). These template multiple emulsions, either produced by capillary microfluidics [ 149 , 150 ], or chip microfluidics [ 151 , 152 ], are then converted into microcapsules. 7.1. Benefits of Using Microfluidics As mentioned previously, the droplets generated by microfluidics exhibit an unmatched uniformity. The usual coefficient of variation (CV) of the droplet diameter is below 5% [ 153 , 154 ]. This property is essential for vaccine encapsulation as the uniform size not only ensures uniform antigen loading and dosing but also enables to assess the relationship between the particle size and the antigen release kinetics without the noise brought by polydispersity. Microfluidics also demonstrate high encapsulation efficiencies (EE) as the emulsification process is not random but happens in a user-controlled way. Thus, Pessi et al. reported a 84% EE for BSA in polycaprolactone microcapsules produced with capillary microfluidic double emulsion [ 155 ]. Moreover, as the droplet generation occurs repeatably at a defined intersection, it minimizes the contact between the solvent phase and the antigen phase, while removing any agitation stress that takes place in classic batch emulsification methods, thus lowering the risks of loss of antigenicity. Droplet microfluidic methods exhibit a user-friendly modularity, where the size and frequency generation can be optically monitored and changed by modifying the flow rates of the injected fluids in the microfluidic setup, as experimental law has been reported between the flow rates ratio and the size of the droplets produced [ 156 ]. This reduces the amount of post-production controls and potential waste by out-of-range production. 7.2. Towards Implementing Microfluidics for Vaccine Delivery Systems Despite all the benefits of microfluidics outlined here, no direct use of this technology in the production of encapsulated vaccines for delayed delivery has been reported yet. Difficulties in setting up adequate microfluidics platform and processes could be the main reason. Indeed, microfluidics applications require both an accurate fluid flow controller, such as pressure pumps and connected reservoirs, or syringe pumps, and an imaging system, composed of a microscope, or a similar set of lenses and light, and a high-speed camera. These components are not frequently found in biology laboratories, and their setup and use also involves an engineering skill set. Moreover, the microfluidic element itself necessitates manufacturing that has not been upscaled for mass production yet by the industry. Thus, capillary microfluidics systems are homemade, with many challenges to face in the precise assembly, while microfluidic chips are in most cases produced by soft lithography, involving clean room facilities and expertise. The inability of microfluidics systems to achieve the yields of production needed by the drug manufacturing industry is often mentioned as one of the major drawbacks of microfluidics approaches. Indeed, although microfluidic droplet generation rate can reach several kilohertz, the total volume encapsulated is limited due to the inherent microscale aspect of the technology. However, parallelisation approaches, where multiple droplet microfluidic processes are performed simultaneously with the adequate designs, have been published and demonstrated more than acceptable throughputs [ 146 , 157 , 158 ]. Thus, Yadavali et al. reported a production rate of 277 g per hour of polycaprolactone microparticles using a silicon and glass parallelised microfluidic device [ 158 ]. Finally, the cost of vaccination must be kept in consideration, in particular when developing sophisticated technologies for vaccine formulation and delivery. The mobilisation of global funds for vaccine development, led by the WHO and dedicated organisations such as the Gavi Alliance, along with the scalability of methods and increasing the vaccine production capacity in the developing countries which significantly reduces the cost per dose [ 159 ], can help accelerate access to vaccines where they are most needed. 7.1. Benefits of Using Microfluidics As mentioned previously, the droplets generated by microfluidics exhibit an unmatched uniformity. The usual coefficient of variation (CV) of the droplet diameter is below 5% [ 153 , 154 ]. This property is essential for vaccine encapsulation as the uniform size not only ensures uniform antigen loading and dosing but also enables to assess the relationship between the particle size and the antigen release kinetics without the noise brought by polydispersity. Microfluidics also demonstrate high encapsulation efficiencies (EE) as the emulsification process is not random but happens in a user-controlled way. Thus, Pessi et al. reported a 84% EE for BSA in polycaprolactone microcapsules produced with capillary microfluidic double emulsion [ 155 ]. Moreover, as the droplet generation occurs repeatably at a defined intersection, it minimizes the contact between the solvent phase and the antigen phase, while removing any agitation stress that takes place in classic batch emulsification methods, thus lowering the risks of loss of antigenicity. Droplet microfluidic methods exhibit a user-friendly modularity, where the size and frequency generation can be optically monitored and changed by modifying the flow rates of the injected fluids in the microfluidic setup, as experimental law has been reported between the flow rates ratio and the size of the droplets produced [ 156 ]. This reduces the amount of post-production controls and potential waste by out-of-range production. 7.2. Towards Implementing Microfluidics for Vaccine Delivery Systems Despite all the benefits of microfluidics outlined here, no direct use of this technology in the production of encapsulated vaccines for delayed delivery has been reported yet. Difficulties in setting up adequate microfluidics platform and processes could be the main reason. Indeed, microfluidics applications require both an accurate fluid flow controller, such as pressure pumps and connected reservoirs, or syringe pumps, and an imaging system, composed of a microscope, or a similar set of lenses and light, and a high-speed camera. These components are not frequently found in biology laboratories, and their setup and use also involves an engineering skill set. Moreover, the microfluidic element itself necessitates manufacturing that has not been upscaled for mass production yet by the industry. Thus, capillary microfluidics systems are homemade, with many challenges to face in the precise assembly, while microfluidic chips are in most cases produced by soft lithography, involving clean room facilities and expertise. The inability of microfluidics systems to achieve the yields of production needed by the drug manufacturing industry is often mentioned as one of the major drawbacks of microfluidics approaches. Indeed, although microfluidic droplet generation rate can reach several kilohertz, the total volume encapsulated is limited due to the inherent microscale aspect of the technology. However, parallelisation approaches, where multiple droplet microfluidic processes are performed simultaneously with the adequate designs, have been published and demonstrated more than acceptable throughputs [ 146 , 157 , 158 ]. Thus, Yadavali et al. reported a production rate of 277 g per hour of polycaprolactone microparticles using a silicon and glass parallelised microfluidic device [ 158 ]. Finally, the cost of vaccination must be kept in consideration, in particular when developing sophisticated technologies for vaccine formulation and delivery. The mobilisation of global funds for vaccine development, led by the WHO and dedicated organisations such as the Gavi Alliance, along with the scalability of methods and increasing the vaccine production capacity in the developing countries which significantly reduces the cost per dose [ 159 ], can help accelerate access to vaccines where they are most needed. 8. Conclusions From the above considerations, it is evident that non-invasive vaccination systems are in development as well as are advanced delivery systems that may prove to be an essential part of promising multi-platform vaccine strategies. However, it has yet to be shown whether the presented technologies may provide reliable and cost-effective approaches to vaccination, yielding more efficient vaccines and improved patient compliance to immunisation programmes in both the developed and the developing countries.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10526800/

Diverse Roles of Protein Palmitoylation in Cancer Progression, Immunity, Stemness, and Beyond

Protein S-palmitoylation, a type of post-translational modification, refers to the reversible process of attachment of a fatty acyl chain—a 16-carbon palmitate acid—to the specific cysteine residues on target proteins. By adding the lipid chain to proteins, it increases the hydrophobicity of proteins and modulates protein stability, interaction with effector proteins, subcellular localization, and membrane trafficking. Palmitoylation is catalyzed by a group of zinc finger DHHC-containing proteins (ZDHHCs), whereas depalmitoylation is catalyzed by a family of acyl-protein thioesterases. Increasing numbers of oncoproteins and tumor suppressors have been identified to be palmitoylated, and palmitoylation is essential for their functions. Understanding how palmitoylation influences the function of individual proteins, the physiological roles of palmitoylation, and how dysregulated palmitoylation leads to pathological consequences are important drivers of current research in this research field. Further, due to the critical roles in modifying functions of oncoproteins and tumor suppressors, targeting palmitoylation has been used as a candidate therapeutic strategy for cancer treatment. Here, based on recent literatures, we discuss the progress of investigating roles of palmitoylation in regulating cancer progression, immune responses against cancer, and cancer stem cell properties. 1. Introduction Discovered in the early 1970s [ 1 , 2 , 3 , 4 ], protein S-palmitoylation (hereinafter referred to as protein palmitoylation) is a type of protein post-translational modification. It refers to the reversible covalent attachment of a fatty acyl chain—specifically, a 16-carbon palmitate acid—to the specific cysteine residues on target proteins via a thioester bond ( Figure 1 ). By attaching the saturated fatty acid to proteins, palmitoylation increases the hydrophobicity of proteins and plays critical roles in regulating protein stability, interaction with effector proteins, sub-cellular localization, enzymatic activity, membrane trafficking, and many other aspects of cellular processes. The reversible nature of palmitoylation enables palmitoylation to impact protein function in a spatiotemporal and dynamic manner. For example, it has been reported that palmitoylation enables ionotropic glutamate receptors, responsible for the glutamate-mediated postsynaptic excitation of neurons, to interact with their downstream partners and, thus, transduce signaling by attaching their tails to the plasma membrane [ 5 ]. Palmitoylation has also been shown to protect the protective antigen receptor, which is involved in anthrax toxin, against premature degradation by blocking ubiquitination [ 6 ]. The dynamic cycle of palmitoylation takes seconds to hours to modulate protein biological functions [ 7 , 8 , 9 , 10 ]. Identification of the palmitoyl transferases, the enzymes that govern protein palmitoylation, was accomplished in 2002 [ 11 , 12 ]. Since these enzymes feature a conserved Asp-His-His-Cys (DHHC) motif responsible for their catalytic activity, they are also known as zinc finger DHHC-type containing (ZDHHC) proteins. To date, 23 different ZDHHCs [ 8 , 13 ], designated as ZDHHC1 to ZDHHC24 and skipping ZDHHC10, have been identified in mammalian cells. They are polytopic membrane proteins, and most of them are located in the ER or the Golgi membrane, with ZDHHC5, 20, and 21 positioned in the plasma membrane [ 14 , 15 ]. ZDHHC isoforms consist of four to six transmembrane (TM) domains. The catalytic ZDHHC domain is located in the cytosolic part in between the second and third TMs [ 16 ]. The ZDHHCs catalyze protein palmitoylation in a two-step process, which involves auto palmitoylation of themselves to form an acyl–enzyme intermediate and the subsequent transfer of Acyl-CoA to the targeted cysteine residues in substrate proteins [ 17 ]. Despite the similar zinc-finger-like motif, ZDHHCs display distinct preferences in substrate proteins and uneven levels of catalytic efficiency [ 18 , 19 , 20 ]. Palmitoylated proteins may respond to more than one ZDHHC enzyme; however, one particular ZDHHC enzyme often has a stronger effect than others on substrate palmitoylation in the cell. The regulatory mechanisms underlying how ZHHDCs select candidates for modification and their functional redundancy are not entirely clear. On the contrary, the enzymatic thioester hydrolysis that removes palmitate from palmitoylated proteins is catalyzed by a family of serine hydrolases, including acyl-protein thioesterases (APT1, APT2) [ 21 , 22 , 23 ], palmitoyl protein thioesterases (PPT1, PPT2) [ 24 , 25 ], α/β hydrolase domain-containing proteins 17A/B/C (ABHD17A/B/C) [ 26 , 27 , 28 ], and ABHD10 [ 29 ]. While APT1 and APT2 are responsible for depalmitoylation of the cytosolic proteins [ 21 , 23 ], PPT1 and PPT2 control protein degradation in lysosomes [ 30 ]. The binding affinity of APT1 and APT2 on palmitoylated proteins and their catalytic rates rely on the amino acid sequences surrounding the palmitoylation sites [ 31 , 32 , 33 ]. Further, although significant overlapping of substrates was observed in APT1 and APT2, selectivity in depalmitoylating substrates was also identified. In addition, this selectivity observed in APTs is mainly achieved through hydrolysis rate but not binding affinity. For example, APT1 has a better hydrolysis rate to short peptide than APT2. Several methodologies have been developed for detecting palmitoylated proteins. Among these, acyl-biotin exchange (ABE) [ 34 ] and acyl-resin-assisted capture (acyl-Rac) [ 35 ] are widely used ( Figure 2 ). In these methods, free cysteines of palmitoylated proteins are capped and thioester linkages are subsequently cleaved to generate new thiols. These thiols are then selectively labeled by biotin for ABE or thiol-reactive resin for acyl-Rac, allowing further enrichment and detection of palmitoylated proteins. A variant of this method called acyl-PEG exchange (APE) exploits PEG labeling of newly generated thiols as a mass tag for mobility-shift-based assays to identify levels of protein palmitoylation [ 36 ]. Other methods developed for analysis of specific palmitoylated proteins include in-cell imaging based on bio-orthogonal fatty acid labeling and in situ proximity ligation or quantification of palmitoylation levels by gas/liquid chromatography and mass spectrometry [ 37 , 38 ]. A broad spectrum of proteins including enzymes, cancer promoters, cancer suppressors, and transcription factors have been identified to be palmitoylated. Palmitoylome-proteomic studies have identified thousands of palmitoylated proteins [ 10 , 39 ]. The 'SwissPalm' database indicates that more than 10% of the entire human proteome is susceptible to palmitoylation [ 40 , 41 ]; among those, over 1000 substrates have already been characterized experimentally. More recently, it has also been shown that among 299 cancer drivers identified in humans, 79 are palmitoylated [ 8 , 42 ]. Functional investigations have shown that palmitoylation is essential for the pathophysiological functions of the many oncoproteins and tumor suppressors, such as RAS, EGFR, and Hedgehog [ 43 , 44 , 45 ]. Consequently, dysregulation of protein palmitoylation has been implicated in all aspects of cancer hallmark functions, cancer metabolism, and regulation of tumor micro-environment [ 46 , 47 , 48 ]. Due to the functions of ZDHHCs in palmitoylating cancer-related proteins, roles of palmitoyl transferases in cancer have also been revealed. Aberrant ZDHHC activity has also been shown to be strongly correlated with various types of cancer [ 49 ]. For example, copy number variation of a chromosome region including the gene of ZDHHC11 is observed in lung and bladder cancers [ 50 ]; upregulation of ZDHHC9 has been found in colorectal tumors [ 51 ]; and upregulation of ZDHHC5, as an oncogenic factor, was reported in p53-mutant gliomas [ 52 ]. In breast cancer, patients associated with an elevated expression of ZDHHC3 were correlated with lower patient survival [ 53 ]. Further, functional investigations have also validated the essential roles of ZDHHCs in cancer. For example, an RNAi-based screen targeting, all 23 members of the ZDHHC family in non-small lung cancer (NSCLC) identified ZDHHC5 to be essential for the growth of NSCLC but not normal human bronchial epithelial cells [ 54 ]. In gliomas, inactivation of ZDHHC15 blocked glioma cells proliferation by decreasing activation of Signal Transducer and Activator of Transcription 3 (STAT3) [ 55 ] and knockdown of ZDHHC12 reduced the growth, migration, and invasion capabilities of glioma cells [ 56 , 57 ]. In pancreatic cancer, knockdown of ZDHHC3 impaired tumor progression in a xenograft mice model [ 58 ], whereas inactivation of ZDHHC9 modified the tumor microenvironment from an immunosuppressive to a proinflammatory environment and, thus, suppressed tumor growth [ 59 ]. DHHC9 is also essential for leukemogenesis by palmitoylating oncogenic NRAS [ 60 ]. Study of palmitoylation is often conducted from two aspects: palmitoylated proteins and palmitoyl transferases, the enzymes modulating palmitoylation ( Figure 1 b,c). When the studies are focused on the palmitoylated proteins, several questions can be addressed: 1—if a specific protein is palmitoylated; 2—where the palmitoylation sites are or which cysteine residues are palmitoylated; 3—which ZDHHC is the main palmitoyl transferase responsible for the palmitoylation of the specific protein; 4—how palmitoylation affects functions of the specific protein; 5—if palmitoylation leads to pathological conditions or contributes to physiological conditions by modulating protein function; 6—how the palmitoylation is regulated. When the studies are focused on ZDHHCs, questions to be addressed are as follows: 1—what the biological function of the ZDHHCs is in physiological or specific pathological contexts; 2—for a particular ZDHHC, what the substrates are; 3—if inhibitors can be developed to target the ZDHHCs; 4—how expression of ZDHHCs and ZDHHCs-mediated palmitoylation are regulated. Despite some reviews summarizing functions of palmitoylated proteins in cancer [ 8 , 61 , 62 ], a review discussing the recent progress on understanding roles of palmitoylation in cancer immunology and cancer stem cell maintenance is lacking. Here, focusing on literatures that were published in the last five years, we discuss the critical roles of palmitoylation in cancer, speculate the future direction of the palmitoylation research field, and discuss the obstacles the field is facing. 2. Palmitoylation in Cancer 2.1. Palmitoylation in Regulating Growth Signaling 2.1.1. Regulation of AKT Signaling by Palmitoylation Activated by phosphatidylinositol-3-kinase (PI3K), AKT plays a central role in a variety of cellular events including growth, proliferation, glucose uptake, metabolism, and cell survival [ 63 ]. Not surprisingly, a variety of human cancers exhibit upregulated AKT activity and several mouse models with activated AKT develop cancer. Palmitoylation of AKT at cysteine 344 (Cys344) was identified in both HEK293T and preadipocyte HeLa Kyoto cells, which indicated a new layer of regulation for AKT [ 64 ]. Mutation of Cys344 resulted in reduction of AKT308 phosphorylation and recruitment of AKT to lysosomes, a process stimulated by inducers of oxidative stress and autophagy ( Figure 3 a). These observations indicate that palmitoylation is essential for AKT activation and prevents degradation of AKT. Studies to understand roles of AKT palmitoylation in cancer initiation and progression, and to identify the palmitoyl transferase that catalyzes AKT palmitoylation, would be of interest. In addition to palmitoylating AKT directly to increase AKT signaling, palmitoylation is able to regulate AKT signaling in an indirect manner. For example, by attenuating the stability of mTOR, a kinase complex that phosphorylates AKT, palmitoylation of mTOR mediated by ZDHHC22 reduced AKT signaling in breast cancer cells [ 65 ]. Site-directed mutagenesis identified Cys361 and Cys362 as the main palmitoylation sites of mTOR. On the contrary, in liver cancer, palmitoylation of proprotein convertase subtilisin/kexin type 9 (PCSK9), a key enzyme regulating cholesterol metabolism, increased affinity of PCSK9 in binding with phosphatase and tensin homolog (PTEN) [ 66 ]. Consequently, binding of PCSK9 led to degradation of PTEN and, thus, released its inhibitory function on AKT signaling. Together, regulation of AKT is context-dependent and it is mediated in several layers. 2.1.2. Regulation of Wnt Signaling by Palmitoylation Wnt signaling is essential for organ formation during development and for organ homeostasis in postnatal life [ 67 , 68 ]. In osteosarcoma, the most frequent malignant primary bone tumor in children and young adults, ZDHHC19 was found to be highly expressed, and knockdown of ZDHHC19 led to downregulated expression of Wnt/β-catenin [ 69 ] ( Figure 3 b). These observations established the connection between ZDHHC19 and Wnt/β-catenin signaling. Downstream of Wnt, low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) act as co-receptors to activate β-catenin pathway. Palmitoylation of LRP6 was reported in 2008 [ 70 ]. Mutation of Cys1394 and Cys1399, two LRP6 palmitoylation sites, diminished the distribution of LRP6 to the plasma membrane by retaining LRP6 in the endoplasmic reticulum (ER). As a result, due to the lack of LRP6 on the plasma membrane, Wnt/β-catenin signaling cannot be transduced efficiently. These data revealed the second layer of regulation on Wnt/β-catenin signaling provided by palmitoylation. Wnt signaling can be antagonized by dickkopf1 (DKK1), which binds and, thus, degrades LRP6, one of the Wnt receptors [ 71 , 72 ]. Furthermore, DKK1 and LRP6 can form a ternary complex with cytoskeleton-associated protein 4 (CKAP4), a cell surface receptor that activates PI3K/AKT signaling, to enhance DKK1-dependent cancer cell proliferation [ 73 , 74 , 75 ]. In human cervical carcinoma cells (HeLa), CKAP4 was identified as a substrate of ZDHHC2 from the palmitoylome scale [ 76 ]. Palmitoylation of CKAP4 at Cys100 was confirmed by ABE assay, and the direct interaction between ZDHHC2 and CKAP4 was confirmed by Co-immunoprecipitation (Co-IP) ( Figure 3 b). Mechanistically, palmitoylation by ZDHHC2 is required for CKAP4 trafficking from the ER to the plasma membrane and, thus, mediates PI3K/AKT signaling [ 77 , 78 ]. Indeed, expression of CKAP4-WT but not CKAP4-C100S rescued the reduction in tumor growth induced by knockout of CKAP4. Interestingly, DKK1 induces depalmitoylation of CKAP4 through APT1/2 [ 79 ]. Together, palmitoylation of CKAP4 translocated CKAP4 to the plasma membrane, enabling DKK1 to antagonize Wnt signaling. 2.1.3. Regulation of IGF-1/IGF-1R Signaling by Palmitoylation Flotillin-1 (FLOT-1) is a lipid raft-associated protein that has been implicated in the progression of cancers [ 80 , 81 , 82 ] and insulin signaling to trigger glucose transporter redistribution in adipocytes [ 83 ]. In the process of understanding how FLOT-1, a non-transmembrane protein, sustains insulin signaling on plasma membrane (PM), Morrow et al. found that plasma membrane association of FLOT-1 requires palmitoylation [ 84 ]. Palmitoylation of Flot-1 occurs at Cys-34 in the prohibitin-like domain (PHB) at the N terminus of flotillin ( Figure 3 c). Further study found that the palmitoylation turnover of FLOT-1 in the plasma membrane was induced by Insulin-like growth factor-1 (IGF-1) [ 85 ]. As a result, although the mechanism is not fully characterized, palmitoylation of FLOT-1 mediated by ZDHHC19 prevents desensitization of IGF-1R via endocytosis and lysosomal degradation, leading to excessive IGF-1R-mediated signaling and, thus, increased migration and invasion of cervical cancer cells [ 86 ]. Although palmitoylation of IGF-1R was not reported, these studies provided solid evidence showing the importance of palmitoylation in mediating IGF-1/IGF-1R signaling. 2.2. Roles of Palmitoylation in Cancer Immunology 2.2.1. Regulation of IFNγ/IFNGR1-Mediated PD-L1/PD-1 Signaling by Palmitoylation Stimulated by interferon-γ (IFNγ) ligand, IFNγ receptor 1 (IFNGR1) activates JAK/STAT signaling and, thus, induces transcription of programmed death protein ligand 1 (PD-L1) [ 87 ] ( Figure 4 ). Downstream of PD-L1, programmed death protein 1 (PD-1), the receptor of PD-L1, delivers inhibitory signals to regulate the balance between T cell activation, tolerance, and immunopathology. PD-1 is expressed on the surface of activated T cells as an inhibitory receptor, while its ligand PD-L1 is mainly expressed in antigen-presenting cells and tumor cells. By binding to its receptor PD-1 on T cells, expression of PD-L1 on tumor cells inhibits T cell activation and, thus, drives the escape of tumor cells from immune surveillance. Therefore, components of the IFNγ/IFNGR1-mediated PD-L1/PD-1 signaling pathway play a critical role in the success or failure of immune checkpoint blockades. Several studies have reported that palmitoylation provides regulatory roles for IFNγ/IFNGR1 signaling by modifying functions of the components. For example, in colorectal cancer cells, stability of IFNGR1 was found to be negatively regulated by palmitoylation; although, the specific ZDHHC that palmitoylates IFNGR1 was not identified [ 88 ]. Mutation of Cys122, the palmitoylation site of IFNGR1, or treatment of a palmitoylation inhibitor 2-bromopalmitate (2-BP) blocked the degradation of IFNGR1. Furthermore, IFNGR1 palmitoylation is also essential for the interaction between IFNGR1 and its binding partner AP3D1. As a consequence, IFNGR1 is able to trigger its downstream immune response signaling to inhibit cancer growth. Further, palmitoylation modulates the immune response to cancer by directly altering PD-L1 functions. Palmitoylation of endogenous PD-L1 was firstly identified in breast cancer cell lines MDA-MB231 and BT549 [ 89 ]. Inhibition of PD-L1 palmitoylation by 2-BP decreased PD-L1's protein level. Mutation of Cys272 or knockdown of ZDHHC9, the palmitoyl transferase responsible for PD-L1, abolished PD-L1 palmitoylation and decreased PD-L1 cell surface distribution. More importantly, blocking PD-L1 palmitoylation sensitized tumor cells to T-cell killing and, thereby, impaired tumor growth in vivo. These observations indicate that, in breast cancer cells, palmitoylation facilitates immune suppression to cancer by stabilizing and maintaining PD-L1 on the cell surface. Further, palmitoylation of PD-L1 in colorectal cancer and lung adenocarcinoma was also reported [ 90 , 91 ]. Of note, unlike ZDHHC9-mediated PD-L1 palmitoylation in breast cancer cells, palmitoylation of PD-L1 in colorectal cancer cells is mediated by ZDHHC3 but not ZDHHC9, indicating that PD-L1 utilizes multiple ZDHHCs to ensure its palmitoylation in different contexts. Mechanistically, palmitoylation stabilizes PD-L1 by suppressing ubiquitination and degradation in lysosomes, thereby repressing anti-tumor immunity. As a treatment strategy, to increase the immune response against cancer, a designed peptide that contains the palmitoylation region of PD-L1 was developed as a competitive inhibitor to inhibit PD-L1 palmitoylation. More recently, roles of ZDHHC9 in anti-tumor immunity were also revealed in pancreatic cancer [ 59 ]. Different from the studies conducted on breast cancer, colorectal cancer, and lung adenocarcinoma, this study found that, in the pancreatic tumor xenograft model, tumors with ZDHHC9 knockdown led to a change in PD-L1 level on the surrounding immune cells. Although it is unclear how ZDHHC9 in the tumor cells affects PD-L1 expression level on the surrounding immune cells, this study reported the non-autonomous function of ZDHHC9 in regulating the cancer immune response for the first time. In addition to PD-L1, palmitoylation also affects functions of its receptor PD-1. In addition to being expressed on the T cells to function as an inhibitory receptor, expression of PD-1 in cancer cells has also been revealed. Different from the function of PD-1 on T cells, in cancer cells, PD-1 promotes tumor growth independently of the adaptive immune system by modulating mTOR signaling. In 2021, Yao et al. revealed the palmitoylation of PD-1 at Cys192 in a variety of cancer cells [ 92 ]. Inhibition of PD-1 palmitoylation by 2-BP blocked the interaction between PD-1 and Rab11, a key molecule in transporting the cargo proteins to the recycling endosomes. As a consequence, decreased storage of PD-1 in recycling endosomes led to increased PD-1 degradation in the lysosomes. These results suggest that palmitoylation attenuates degradation of PD-1 by facilitating transportation of PD-1 to the recycling endosomes. More importantly, palmitoylation of PD-1 is essential for cancer cell growth as it is required for PD-1 to interact with its downstream signaling mediators to transduce signaling. Therefore, palmitoylation of PD-1 in cancer cells promotes cancer cell growth by preventing degradation of PD-1 and promoting its interaction with its downstream signaling mediators. Similar to the competitive inhibitor of PD-L1, a peptide containing the palmitoylation region of PD-1 was developed as a competitive inhibitor for PD-1 palmitoylation. Moving forward, it would be interesting to investigate the possibility of PD-1 palmitoylation in T cells and if palmitoylation affects the function of PD-1 in adaptive immune system. 2.2.2. Palmitoylation Triggers Anti-Immune Response by Sorting Proteins into Extracellular Vesicles The roles of extracellular vesicles (EV), lipid-enclosed particles that circulate bioactive content, in cancer development and progression have been revealed for decades [ 93 ]. Recently, Mariscal et al. demonstrated that, by anchoring proteins to cellular membranes, palmitoylation plays an important role in sorting proteins into the EV [ 94 ]. By doing so, the anti-immune response is triggered. For example, by secreting palmitoylated proteins through EV, acute myeloid leukemia (AML) cells activate toll-like receptor 2 (TLR2), a receptor that mediates activation of the immune system [ 95 ]. Subsequently, TLR2 induced the differentiation of monocytes into T-cell inhibitory myeloid-derived suppressor cells (MDSC), a type of immunosuppressive cell [ 96 ], resulting in a blockage of immune-response-mediated AML cell removal. Indeed, although the identity of the palmitoylated proteins in EV was not identified and it is still unknown how palmitoylation triggers TLR2 activation, AML cells treated with 2-BP lost the capacity to generate MDSC and an immune-suppressive environment for their survival. 2.2.3. Regulation of the Innate Immune Response in Preventing Cancer by Palmitoylation STING (Stimulator of interferon genes) is an innate immune sensor of immune surveillance of viral/bacterial infection and is involved in the maintenance of an immune-friendly microenvironment to prevent tumorigenesis [ 97 , 98 , 99 , 100 ]. Palmitoylation of STING was initially reported in 2016 [ 101 ]. Dual mutations of Cys88/91 abolished the palmitoylation of STING and, thus, its role in activating expression of its downstream targets including IRF3, IFNβ, and NF-κB, suggesting that palmitoylation is required for activation of STING. Over-expression of ZDHHC3, ZDHHC7, and ZDHHC15 increased palmitoylation of STING, suggesting that they are responsible for STING palmitoylation. Further, another study found that palmitoylation of STING is essential for STING to interact with the mitochondrial voltage-dependent anion channel VDAC2 and, thus, prevent VDAC2-induced mitochondria dysfunction [ 102 ]. 2.3. Regulation of Cancer Stem Cell Potency by Palmitoylation Cancer stem cells are responsible for cancer treatment resistance, leading to relapse, disease progression, and eventually systemic disease [ 103 ]. Lately, functions of palmitoylation in cancer stem cells have received increasing attention. Mechanistic analysis found that palmitoylation not only affects the self-renewal capacity of cancer stem cells but also affects their tumorigenicity ( Figure 5 ). In the past five years, as the majority of studies were conducted on glioblastoma stem cells (GSCs), we focused on discussing roles of palmitoylation in GSCs here. Acting downstream of ERK and AKT, glycogen synthase kinase 3β (GSK3β), a serine/threonine protein kinase, is also considered as a key hub for promoting malignancy of GSCs [ 104 , 105 ]. Zhao et al. found that roles of GSK3β are affected by palmitoylation when ZDHHC4 palmitoylates GSK3β at the Cys14 residue [ 106 ]. Mechanistically, palmitoylation of GSK3β negatively affected binding of GSK3β to AKT1 and S6K kinases, thereby releasing the inhibitory roles of AKT1 and S6K on GSK3β. Downstream of GSK3β, activated GSK3β phosphorylates histone methyltransferase EZH2, which, in turn, regulates STAT3 methylation and phosphorylation, leading to increased expression of stem-cell-related genes [ 107 ]. More importantly, due to the increased GSK3β activity induced by palmitoylation, tumorigenicity of GSCs was increased and resistance to chemotherapy was generated. In addition to modifying EZH2 activity by palmitoylating GSK3β, EZH2 activity can also be affected by palmitoylation directly. For example, in p53-mutant GSCs, ZDHHC5 palmitoylated EZH2 at Cys571 and Cys576 [ 52 ]. After being palmitoylated, palmitoylation blocked phosphorylation of EZH2 and, thus, its activity in mediating methylation of H3K27me3. As a consequence, loss of H3K27me3 released the suppressed expression of stemness markers cluster of differentiation (CD)133 and SOX2, thereby increasing the neurosphere formation capacity of GSCs. Further studies found that ZDHHC5-mediated EZH2 palmitoylation is regulated by mutant p53 as it transcriptionally induced expression of ZDHHC5 by interacting with nuclear transcription factor NF-Y. More recently, it was observed that P53-induced ZDHHC5 expression can also be enhanced by treatment of propofol, an agent that induces local anesthetics [ 108 ]. Consequently, upregulated ZDHHC5 led to increased EZH2 palmitoylation, resulting in reduced H3K27me3 at promoter regions of genes that regulate stem cell potency and increase expression of these genes. Hyperactive transforming growth factor-beta (TGF-β) signaling has also been viewed as a signature event in mesenchymal glioblastoma [ 109 , 110 ]. Downstream of TGF-β, the TGF-β receptor phosphorylates transcription factor SMAD3, inducing expression of genes including leukemia inhibitory factor (LIF) and platelet-derived growth factor-beta (PDGF-β) [ 111 , 112 , 113 ]. By doing so, TGF-β induces self-renewal and promotes proliferation of glioma-initiating cells [ 112 , 113 , 114 ]. Recently, Fan et al. reported that palmitoylation of SMAD3 mediated by ZDHHC19 is essential in transducing TGF-β signaling in GSCs [ 115 ]. Specifically, palmitoylation of SMAD3 at Cys421 mediated the translocation from cytosol to the nucleus. OCT4A, also known as POU5F1, is a key transcription factor in the self-renewal, proliferation, and differentiation of stem cells [ 116 , 117 ]. Palmitoylation of OCT4A mediated by ZDHHC17 is essential for protecting OCT4A from degradation and, thus, maintaining its protein stability [ 118 ]. Consequently, palmitoylation of OCT4A retained the stemness of GSCs by binding to the enhancer of SOX2, a key gene for maintaining the tumorigenicity of GSCs; thus, it promoted SOX2 expression. Targeting OCT4A palmitoylation using a competitive inhibitor that contains the OCT4A palmitoylation sequence effectively inhibited palmitoylation of OCT4A and, thereby, reduced the tumorigenesis in vivo. Palmitoylation of OCT4A mediated by ZDHHC17 was identified as a promising therapeutic approach toward effectively eliminating cancer-initiating cells. The involvement of ZDHHC15 and ZDHHC18 in GSCs was revealed recently [ 119 , 120 ]. Knockdown of ZDHHC15 reduced the capacity of GSCs to form neurospheres, indicating that ZDHHC15 is essential for GSCs' self-renewal [ 119 ]. Immunoprecipitation-coupled with mass spectrometry analysis identified GP130, an IL-6 receptor subunit, as a substrate of ZDHHC15 in GSCs. Further mechanism analysis found that palmitoylation mediates the cell membrane localization of GP130 and, thus, activates IL-6/STAT3 signaling to maintain GSCs' renewal ability. Whereas, for ZDHHC18, although no palmitoylation targets were identified, high expression of ZDHHC18 was observed in mesenchymal GSCs [ 120 ]. Mechanistic investigation found that, by interacting with the E3 ligase of BMI1, ZDHHC18 blocked degradation of BMI1, a gene contributing to the maintenance and renewal of cancer-initiating stem cells [ 121 , 122 ], and, thus, maintained the self-renewal capacity of GSCs [ 120 ]. 2.4. Palmitoylation Regulates Cancer Cell Migration by Modifying Cytoskeleton-Related Proteins and Cell Adhesion Molecules In addition to regulating cancer cell growth, palmitoylation also contributes to cancer cell migration and invasion by modifying functions of actin cytoskeletal remodeling related proteins and cell adhesion molecules. For example, palmitoylation of RhoU, an atypical Rho GTPase, at Cys256 is essential for migration of prostate cancer cells [ 123 , 124 ]. Mechanistic analysis found that palmitoylated RhoU increased cell migration ability by interacting and, thus, stabilizing Cdc42, a gene that regulates cell spread area [ 124 , 125 ]. Palmitoylation of α-tubulin has also been reported [ 126 , 127 , 128 , 129 ]. For example, it was reported that palmitoylation at Cys377 mediates astral microtubule function during nuclear migration in the M phase of the cell cycle. More interestingly, it was revealed that palmitoylation level of α-tubulin can also been regulated. For example, androgen treatment increased α-tubulin palmitoylation to mediate proliferation of prostate cancer cells, although the molecular mechanism was not fully investigated [ 130 ]. Cancer cell migration can also be regulated by modifying palmitoylation of cell adhesion molecules. For example, palmitoylation of cell adhesion molecules CD44 and MCAM prevented melanoma cell invasion [ 131 ]. On the contrary, depalmitoylation of CD44 and MCAM mediated by APT1 resulted in increased invasion. 2.5. Cancer Cells Use Palmitoylation to Overcome Nutrient Deficiency GLUT1 is a widely expressed glucose transporter responsible for the constant uptake of glucose [ 132 ]. Numerous studies have also shown that GLUT1 is essential for cancer growth [ 133 , 134 , 135 ]. In glioblastoma cells, site-directed mutagenesis showed that GLUT1 is palmitoylated at Cys207 and disruption of palmitoylation abolished localization of GLUT1 on the plasma membrane [ 136 ]. Expression of GLUT1-WT but not the palmitoylation defective form of GLUT1, GLUT1-C207S, restored the reduction in glucose uptake induced by knockout of GLUT1. Furthermore, blocking GLUT1 palmitoylation mediated by knockout of ZDHHC9, the enzyme that palmitoylates GLUT1, impaired glycolysis and reduced GBM tumor growth. These observations indicate that palmitoylation is required for GLUT1 to localize on the plasma membrane and, thus, conduct its function in mediating glucose uptake and tumor growth. Malate dehydrogenase 2 (MDH2) catalyzes the reversible reaction of malate to oxaloacetate in the TCA cycle [ 137 ]. Aberrant MDH2 function has been found to be associated with malignancy of cancer [ 138 , 139 ]. Palmitoylation of MDH2 was firstly identified in 2008 from a palmitoylome-proteomic study [ 140 ]. Recently, Pei et al. found that palmitoylation of MDH2 at Cys138 increased its binding affinity with its coenzyme NAD+ in order to maintain the function of glycolysis and mitochondrial respiration in ovarian cancer cells [ 141 ]. MDH2 interacted with ZDHHC18 and exogenous expression of ZDHHC18 increased palmitoylation levels of MDH2, indicating that ZDHHC18 is the palmitoyl transferase responsible for MDH2 palmitoylation. Of note, palmitoylation of MDH2 mediated by ZDHHC18 was stimulated by glutamine deprivation. These data revealed how cancer cells use palmitoylation and mitochondrial respiration for its growth to adapt to the nutrition-deficient tumor microenvironment. 2.6. Palmitoylation Negatively Contributes to Cancer In addition to positively contributing to cancer progression, the negative function of palmitoylation in cancer growth has also been revealed. Palmitoylation conducts its cancer suppression function by suppressing the function of oncoproteins, maintaining functions of tumor suppressors, or modifying growth-signaling transduction. 2.6.1. Palmitoylation Negatively Regulates Oncoprotein Functions Astrocyte elevated gene-1 (AEG-1) is an oncogene that is over-expressed in a wide variety of cancers [ 142 , 143 , 144 , 145 ]. Palmitoylation of AEG-1 in several physiological contexts was initially reported by Zhou et al. [ 146 ] ( Figure 6 a). From this research, they found that AEG-1 is palmitoylated at Cys75 and a palmitoylation-defective form of AEG-1 enhanced hepatocellular carcinoma progression in vivo. Mechanistically, palmitoylation negatively regulates AEG-1 function by reducing the stability of AEG-1 and by reducing its affinity with its interacting proteins. Further, a different research group identified ZDHHC6 as the main palmitoyl transferase for AEG-1 and found that CRISPR/Cas9 knock-in mice with a palmitoylation-defective form of AEG-1 had increased signaling pathways and regulators that contribute to cell proliferation, motility, angiogenesis, and lipid accumulation [ 147 ]. Together, palmitoylation plays a negative regulatory role on AEG-1 function. By transducing PI3K/AKT and RAS/MAPK signaling, receptor tyrosine kinase FLT3 plays an important role in the development of hematopoietic progenitors [ 148 ]. Consequently, mutation of FLT3 is one of the causes of cancer. For example, internal tandem duplication within FLT3 (FLT3-ITD) confers constitutive activation of FLT3, represents one of the most frequent mutations in acute myeloid leukemia (AML), and correlates with a poor prognosis [ 149 ]. Palmitoylation of FLT3-ITD mediated by ZDHHC6 was identified in AML, and palmitoylation redirected the localization of FLT3-ITD from the plasma membrane to ER ( Figure 6 b) [ 150 ]. Mutation of Cys563 maintained FLT3-ITD on the plasma membrane to conduct its function in activating downstream signaling. Further, inhibition of depalmitoylation with palmostatin B (palm B), a pan-depalmitoylase inhibitor, not only reduced proliferation of FLT3-ITD+ AML cells but also synergized FLT3-ITD+ AML cells to gilteritinib, a FLT3 kinase inhibitor, treatment. Together, these results suggest that palmitoylation plays a repressive role for FLT3. 2.6.2. Palmitoylation Maintains Functions of Tumor Suppressors G protein-coupled receptors (GPCRs) are a group of membrane proteins that convert extracellular signals into intracellular responses, including responses to growth signaling; neurotransmitters; as well as responses to vision, olfaction, and taste signals [ 151 ]. Due to its critical roles in enhancing DNA repair, mutations of the melanocortin-1 receptor (MC1R), a GPCR, have been correlated to a higher risk of melanoma. Palmitoylation of MC1R at Cys78 and Cys315 was identified in melanocytes, and the C315S mutant, a palmitoylation-defective form of MC1R, promoted melanomagenesis [ 152 ] ( Figure 6 c). Palmitoylation of MC1R is mainly mediated by ZDHHC13, and hyper-palmitoylation of MC1R mediated by ZDHHC13 also prevented melanomagenesis. Further, patients with high ZDHHC13 are correlated with better survival, and inhibition of APT2, the MC1R depalmitoylation enzyme, effectively suppressed melanomagenesis by blocking MC1R palmitoylation [ 153 ]. Together, these results highlighted a central role of MC1R palmitoylation in protection against melanoma. TP53 is one of the most extensively studied tumor suppressor genes whose multifaceted mechanisms involve apoptosis, DNA repair, genomic stabilization, and angiogenesis [ 154 ]. A recent study found that TP53 is the key mediator for ZDHHC1-induced breast cancer suppression ( Figure 6 d). Five palmitoylation sites—Cys135, Cys176, Cys182, Cys275, and Cys277—were identified in TP53. Mutation of Cys135, Cys176, and Cys275 significantly blocked nuclear translocation of TP53 and the expression of TP53 downstream targets, e.g., P21 and BAX, resulting in the promotion of tumor growth. Together, by modifying TP53 nuclear translocation, palmitoylation conducts its tumor suppression function [ 155 ]. SET domain-containing 2 (SETD2) is a histone lysine methyltransferase. By mediating the methylation of H3K36me3, SETD2 contributes to DNA damage response (DDR) by recruiting RAD51 to DNA double strand break sites. As a functional DDR is critical for maintaining genome integrity and preventing tumor development [ 156 ], SETD2 is considered as a putative tumor suppressor gene in cancers [ 157 , 158 ]. In EGFR-amplified glioblastoma, palmitoylation of SETD2 mediated by ZDHHC16 protects SETD2 from degradation and, thereby, facilitates its role in mediating DNA damage response and repressing cancer initiation ( Figure 6 e) [ 159 ]. GNA13 encodes one of the alpha subunits of the heterotrimeric G proteins that transduce signals of GPCR. By negatively regulating the expression of BCL2, GNA13 has also been identified as a tumor suppressor in B-cell lymphoma [ 160 ]. Palmitoylation of GNA13 was initially reported in early 2000 ( Figure 6 f). The study revealed that GNA13 is palmitoylated at Cys14 and Cys18. The wild type but not the palmitoylation-defective form of GNA13 localizes at the plasma membrane and, thus, transduces Rho-dependent signaling [ 161 ]. Moreover, expression of GNA13WT but not the palmitoylation-defective form of GNA13 inhibited proliferation of B-cell lymphoma, suggesting that palmitoylation of GNA13 is required for its tumor suppression function [ 160 ]. 2.6.3. Palmitoylation Suppresses Cancer by Reducing Growth Signaling In addition to repressing tumors by directly affecting functions of cancer-related protein, palmitoylation can also block tumor growth by modifying growth signaling. For example, palmitoylation at Cys44, Cys45, and Cys47 of the small CTD phosphatase 1 (SCP1) leads to its translocation from the nuclear to plasma membranes [ 162 ]. As a result, membrane-located SCP1 dephosphorylates AKT at serine 473, leading to suppressed angiogenesis and decreased tumor growth of lung carcinoma in xenograft mice. 2.1. Palmitoylation in Regulating Growth Signaling 2.1.1. Regulation of AKT Signaling by Palmitoylation Activated by phosphatidylinositol-3-kinase (PI3K), AKT plays a central role in a variety of cellular events including growth, proliferation, glucose uptake, metabolism, and cell survival [ 63 ]. Not surprisingly, a variety of human cancers exhibit upregulated AKT activity and several mouse models with activated AKT develop cancer. Palmitoylation of AKT at cysteine 344 (Cys344) was identified in both HEK293T and preadipocyte HeLa Kyoto cells, which indicated a new layer of regulation for AKT [ 64 ]. Mutation of Cys344 resulted in reduction of AKT308 phosphorylation and recruitment of AKT to lysosomes, a process stimulated by inducers of oxidative stress and autophagy ( Figure 3 a). These observations indicate that palmitoylation is essential for AKT activation and prevents degradation of AKT. Studies to understand roles of AKT palmitoylation in cancer initiation and progression, and to identify the palmitoyl transferase that catalyzes AKT palmitoylation, would be of interest. In addition to palmitoylating AKT directly to increase AKT signaling, palmitoylation is able to regulate AKT signaling in an indirect manner. For example, by attenuating the stability of mTOR, a kinase complex that phosphorylates AKT, palmitoylation of mTOR mediated by ZDHHC22 reduced AKT signaling in breast cancer cells [ 65 ]. Site-directed mutagenesis identified Cys361 and Cys362 as the main palmitoylation sites of mTOR. On the contrary, in liver cancer, palmitoylation of proprotein convertase subtilisin/kexin type 9 (PCSK9), a key enzyme regulating cholesterol metabolism, increased affinity of PCSK9 in binding with phosphatase and tensin homolog (PTEN) [ 66 ]. Consequently, binding of PCSK9 led to degradation of PTEN and, thus, released its inhibitory function on AKT signaling. Together, regulation of AKT is context-dependent and it is mediated in several layers. 2.1.2. Regulation of Wnt Signaling by Palmitoylation Wnt signaling is essential for organ formation during development and for organ homeostasis in postnatal life [ 67 , 68 ]. In osteosarcoma, the most frequent malignant primary bone tumor in children and young adults, ZDHHC19 was found to be highly expressed, and knockdown of ZDHHC19 led to downregulated expression of Wnt/β-catenin [ 69 ] ( Figure 3 b). These observations established the connection between ZDHHC19 and Wnt/β-catenin signaling. Downstream of Wnt, low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) act as co-receptors to activate β-catenin pathway. Palmitoylation of LRP6 was reported in 2008 [ 70 ]. Mutation of Cys1394 and Cys1399, two LRP6 palmitoylation sites, diminished the distribution of LRP6 to the plasma membrane by retaining LRP6 in the endoplasmic reticulum (ER). As a result, due to the lack of LRP6 on the plasma membrane, Wnt/β-catenin signaling cannot be transduced efficiently. These data revealed the second layer of regulation on Wnt/β-catenin signaling provided by palmitoylation. Wnt signaling can be antagonized by dickkopf1 (DKK1), which binds and, thus, degrades LRP6, one of the Wnt receptors [ 71 , 72 ]. Furthermore, DKK1 and LRP6 can form a ternary complex with cytoskeleton-associated protein 4 (CKAP4), a cell surface receptor that activates PI3K/AKT signaling, to enhance DKK1-dependent cancer cell proliferation [ 73 , 74 , 75 ]. In human cervical carcinoma cells (HeLa), CKAP4 was identified as a substrate of ZDHHC2 from the palmitoylome scale [ 76 ]. Palmitoylation of CKAP4 at Cys100 was confirmed by ABE assay, and the direct interaction between ZDHHC2 and CKAP4 was confirmed by Co-immunoprecipitation (Co-IP) ( Figure 3 b). Mechanistically, palmitoylation by ZDHHC2 is required for CKAP4 trafficking from the ER to the plasma membrane and, thus, mediates PI3K/AKT signaling [ 77 , 78 ]. Indeed, expression of CKAP4-WT but not CKAP4-C100S rescued the reduction in tumor growth induced by knockout of CKAP4. Interestingly, DKK1 induces depalmitoylation of CKAP4 through APT1/2 [ 79 ]. Together, palmitoylation of CKAP4 translocated CKAP4 to the plasma membrane, enabling DKK1 to antagonize Wnt signaling. 2.1.3. Regulation of IGF-1/IGF-1R Signaling by Palmitoylation Flotillin-1 (FLOT-1) is a lipid raft-associated protein that has been implicated in the progression of cancers [ 80 , 81 , 82 ] and insulin signaling to trigger glucose transporter redistribution in adipocytes [ 83 ]. In the process of understanding how FLOT-1, a non-transmembrane protein, sustains insulin signaling on plasma membrane (PM), Morrow et al. found that plasma membrane association of FLOT-1 requires palmitoylation [ 84 ]. Palmitoylation of Flot-1 occurs at Cys-34 in the prohibitin-like domain (PHB) at the N terminus of flotillin ( Figure 3 c). Further study found that the palmitoylation turnover of FLOT-1 in the plasma membrane was induced by Insulin-like growth factor-1 (IGF-1) [ 85 ]. As a result, although the mechanism is not fully characterized, palmitoylation of FLOT-1 mediated by ZDHHC19 prevents desensitization of IGF-1R via endocytosis and lysosomal degradation, leading to excessive IGF-1R-mediated signaling and, thus, increased migration and invasion of cervical cancer cells [ 86 ]. Although palmitoylation of IGF-1R was not reported, these studies provided solid evidence showing the importance of palmitoylation in mediating IGF-1/IGF-1R signaling. 2.1.1. Regulation of AKT Signaling by Palmitoylation Activated by phosphatidylinositol-3-kinase (PI3K), AKT plays a central role in a variety of cellular events including growth, proliferation, glucose uptake, metabolism, and cell survival [ 63 ]. Not surprisingly, a variety of human cancers exhibit upregulated AKT activity and several mouse models with activated AKT develop cancer. Palmitoylation of AKT at cysteine 344 (Cys344) was identified in both HEK293T and preadipocyte HeLa Kyoto cells, which indicated a new layer of regulation for AKT [ 64 ]. Mutation of Cys344 resulted in reduction of AKT308 phosphorylation and recruitment of AKT to lysosomes, a process stimulated by inducers of oxidative stress and autophagy ( Figure 3 a). These observations indicate that palmitoylation is essential for AKT activation and prevents degradation of AKT. Studies to understand roles of AKT palmitoylation in cancer initiation and progression, and to identify the palmitoyl transferase that catalyzes AKT palmitoylation, would be of interest. In addition to palmitoylating AKT directly to increase AKT signaling, palmitoylation is able to regulate AKT signaling in an indirect manner. For example, by attenuating the stability of mTOR, a kinase complex that phosphorylates AKT, palmitoylation of mTOR mediated by ZDHHC22 reduced AKT signaling in breast cancer cells [ 65 ]. Site-directed mutagenesis identified Cys361 and Cys362 as the main palmitoylation sites of mTOR. On the contrary, in liver cancer, palmitoylation of proprotein convertase subtilisin/kexin type 9 (PCSK9), a key enzyme regulating cholesterol metabolism, increased affinity of PCSK9 in binding with phosphatase and tensin homolog (PTEN) [ 66 ]. Consequently, binding of PCSK9 led to degradation of PTEN and, thus, released its inhibitory function on AKT signaling. Together, regulation of AKT is context-dependent and it is mediated in several layers. 2.1.2. Regulation of Wnt Signaling by Palmitoylation Wnt signaling is essential for organ formation during development and for organ homeostasis in postnatal life [ 67 , 68 ]. In osteosarcoma, the most frequent malignant primary bone tumor in children and young adults, ZDHHC19 was found to be highly expressed, and knockdown of ZDHHC19 led to downregulated expression of Wnt/β-catenin [ 69 ] ( Figure 3 b). These observations established the connection between ZDHHC19 and Wnt/β-catenin signaling. Downstream of Wnt, low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) act as co-receptors to activate β-catenin pathway. Palmitoylation of LRP6 was reported in 2008 [ 70 ]. Mutation of Cys1394 and Cys1399, two LRP6 palmitoylation sites, diminished the distribution of LRP6 to the plasma membrane by retaining LRP6 in the endoplasmic reticulum (ER). As a result, due to the lack of LRP6 on the plasma membrane, Wnt/β-catenin signaling cannot be transduced efficiently. These data revealed the second layer of regulation on Wnt/β-catenin signaling provided by palmitoylation. Wnt signaling can be antagonized by dickkopf1 (DKK1), which binds and, thus, degrades LRP6, one of the Wnt receptors [ 71 , 72 ]. Furthermore, DKK1 and LRP6 can form a ternary complex with cytoskeleton-associated protein 4 (CKAP4), a cell surface receptor that activates PI3K/AKT signaling, to enhance DKK1-dependent cancer cell proliferation [ 73 , 74 , 75 ]. In human cervical carcinoma cells (HeLa), CKAP4 was identified as a substrate of ZDHHC2 from the palmitoylome scale [ 76 ]. Palmitoylation of CKAP4 at Cys100 was confirmed by ABE assay, and the direct interaction between ZDHHC2 and CKAP4 was confirmed by Co-immunoprecipitation (Co-IP) ( Figure 3 b). Mechanistically, palmitoylation by ZDHHC2 is required for CKAP4 trafficking from the ER to the plasma membrane and, thus, mediates PI3K/AKT signaling [ 77 , 78 ]. Indeed, expression of CKAP4-WT but not CKAP4-C100S rescued the reduction in tumor growth induced by knockout of CKAP4. Interestingly, DKK1 induces depalmitoylation of CKAP4 through APT1/2 [ 79 ]. Together, palmitoylation of CKAP4 translocated CKAP4 to the plasma membrane, enabling DKK1 to antagonize Wnt signaling. 2.1.3. Regulation of IGF-1/IGF-1R Signaling by Palmitoylation Flotillin-1 (FLOT-1) is a lipid raft-associated protein that has been implicated in the progression of cancers [ 80 , 81 , 82 ] and insulin signaling to trigger glucose transporter redistribution in adipocytes [ 83 ]. In the process of understanding how FLOT-1, a non-transmembrane protein, sustains insulin signaling on plasma membrane (PM), Morrow et al. found that plasma membrane association of FLOT-1 requires palmitoylation [ 84 ]. Palmitoylation of Flot-1 occurs at Cys-34 in the prohibitin-like domain (PHB) at the N terminus of flotillin ( Figure 3 c). Further study found that the palmitoylation turnover of FLOT-1 in the plasma membrane was induced by Insulin-like growth factor-1 (IGF-1) [ 85 ]. As a result, although the mechanism is not fully characterized, palmitoylation of FLOT-1 mediated by ZDHHC19 prevents desensitization of IGF-1R via endocytosis and lysosomal degradation, leading to excessive IGF-1R-mediated signaling and, thus, increased migration and invasion of cervical cancer cells [ 86 ]. Although palmitoylation of IGF-1R was not reported, these studies provided solid evidence showing the importance of palmitoylation in mediating IGF-1/IGF-1R signaling. 2.2. Roles of Palmitoylation in Cancer Immunology 2.2.1. Regulation of IFNγ/IFNGR1-Mediated PD-L1/PD-1 Signaling by Palmitoylation Stimulated by interferon-γ (IFNγ) ligand, IFNγ receptor 1 (IFNGR1) activates JAK/STAT signaling and, thus, induces transcription of programmed death protein ligand 1 (PD-L1) [ 87 ] ( Figure 4 ). Downstream of PD-L1, programmed death protein 1 (PD-1), the receptor of PD-L1, delivers inhibitory signals to regulate the balance between T cell activation, tolerance, and immunopathology. PD-1 is expressed on the surface of activated T cells as an inhibitory receptor, while its ligand PD-L1 is mainly expressed in antigen-presenting cells and tumor cells. By binding to its receptor PD-1 on T cells, expression of PD-L1 on tumor cells inhibits T cell activation and, thus, drives the escape of tumor cells from immune surveillance. Therefore, components of the IFNγ/IFNGR1-mediated PD-L1/PD-1 signaling pathway play a critical role in the success or failure of immune checkpoint blockades. Several studies have reported that palmitoylation provides regulatory roles for IFNγ/IFNGR1 signaling by modifying functions of the components. For example, in colorectal cancer cells, stability of IFNGR1 was found to be negatively regulated by palmitoylation; although, the specific ZDHHC that palmitoylates IFNGR1 was not identified [ 88 ]. Mutation of Cys122, the palmitoylation site of IFNGR1, or treatment of a palmitoylation inhibitor 2-bromopalmitate (2-BP) blocked the degradation of IFNGR1. Furthermore, IFNGR1 palmitoylation is also essential for the interaction between IFNGR1 and its binding partner AP3D1. As a consequence, IFNGR1 is able to trigger its downstream immune response signaling to inhibit cancer growth. Further, palmitoylation modulates the immune response to cancer by directly altering PD-L1 functions. Palmitoylation of endogenous PD-L1 was firstly identified in breast cancer cell lines MDA-MB231 and BT549 [ 89 ]. Inhibition of PD-L1 palmitoylation by 2-BP decreased PD-L1's protein level. Mutation of Cys272 or knockdown of ZDHHC9, the palmitoyl transferase responsible for PD-L1, abolished PD-L1 palmitoylation and decreased PD-L1 cell surface distribution. More importantly, blocking PD-L1 palmitoylation sensitized tumor cells to T-cell killing and, thereby, impaired tumor growth in vivo. These observations indicate that, in breast cancer cells, palmitoylation facilitates immune suppression to cancer by stabilizing and maintaining PD-L1 on the cell surface. Further, palmitoylation of PD-L1 in colorectal cancer and lung adenocarcinoma was also reported [ 90 , 91 ]. Of note, unlike ZDHHC9-mediated PD-L1 palmitoylation in breast cancer cells, palmitoylation of PD-L1 in colorectal cancer cells is mediated by ZDHHC3 but not ZDHHC9, indicating that PD-L1 utilizes multiple ZDHHCs to ensure its palmitoylation in different contexts. Mechanistically, palmitoylation stabilizes PD-L1 by suppressing ubiquitination and degradation in lysosomes, thereby repressing anti-tumor immunity. As a treatment strategy, to increase the immune response against cancer, a designed peptide that contains the palmitoylation region of PD-L1 was developed as a competitive inhibitor to inhibit PD-L1 palmitoylation. More recently, roles of ZDHHC9 in anti-tumor immunity were also revealed in pancreatic cancer [ 59 ]. Different from the studies conducted on breast cancer, colorectal cancer, and lung adenocarcinoma, this study found that, in the pancreatic tumor xenograft model, tumors with ZDHHC9 knockdown led to a change in PD-L1 level on the surrounding immune cells. Although it is unclear how ZDHHC9 in the tumor cells affects PD-L1 expression level on the surrounding immune cells, this study reported the non-autonomous function of ZDHHC9 in regulating the cancer immune response for the first time. In addition to PD-L1, palmitoylation also affects functions of its receptor PD-1. In addition to being expressed on the T cells to function as an inhibitory receptor, expression of PD-1 in cancer cells has also been revealed. Different from the function of PD-1 on T cells, in cancer cells, PD-1 promotes tumor growth independently of the adaptive immune system by modulating mTOR signaling. In 2021, Yao et al. revealed the palmitoylation of PD-1 at Cys192 in a variety of cancer cells [ 92 ]. Inhibition of PD-1 palmitoylation by 2-BP blocked the interaction between PD-1 and Rab11, a key molecule in transporting the cargo proteins to the recycling endosomes. As a consequence, decreased storage of PD-1 in recycling endosomes led to increased PD-1 degradation in the lysosomes. These results suggest that palmitoylation attenuates degradation of PD-1 by facilitating transportation of PD-1 to the recycling endosomes. More importantly, palmitoylation of PD-1 is essential for cancer cell growth as it is required for PD-1 to interact with its downstream signaling mediators to transduce signaling. Therefore, palmitoylation of PD-1 in cancer cells promotes cancer cell growth by preventing degradation of PD-1 and promoting its interaction with its downstream signaling mediators. Similar to the competitive inhibitor of PD-L1, a peptide containing the palmitoylation region of PD-1 was developed as a competitive inhibitor for PD-1 palmitoylation. Moving forward, it would be interesting to investigate the possibility of PD-1 palmitoylation in T cells and if palmitoylation affects the function of PD-1 in adaptive immune system. 2.2.2. Palmitoylation Triggers Anti-Immune Response by Sorting Proteins into Extracellular Vesicles The roles of extracellular vesicles (EV), lipid-enclosed particles that circulate bioactive content, in cancer development and progression have been revealed for decades [ 93 ]. Recently, Mariscal et al. demonstrated that, by anchoring proteins to cellular membranes, palmitoylation plays an important role in sorting proteins into the EV [ 94 ]. By doing so, the anti-immune response is triggered. For example, by secreting palmitoylated proteins through EV, acute myeloid leukemia (AML) cells activate toll-like receptor 2 (TLR2), a receptor that mediates activation of the immune system [ 95 ]. Subsequently, TLR2 induced the differentiation of monocytes into T-cell inhibitory myeloid-derived suppressor cells (MDSC), a type of immunosuppressive cell [ 96 ], resulting in a blockage of immune-response-mediated AML cell removal. Indeed, although the identity of the palmitoylated proteins in EV was not identified and it is still unknown how palmitoylation triggers TLR2 activation, AML cells treated with 2-BP lost the capacity to generate MDSC and an immune-suppressive environment for their survival. 2.2.3. Regulation of the Innate Immune Response in Preventing Cancer by Palmitoylation STING (Stimulator of interferon genes) is an innate immune sensor of immune surveillance of viral/bacterial infection and is involved in the maintenance of an immune-friendly microenvironment to prevent tumorigenesis [ 97 , 98 , 99 , 100 ]. Palmitoylation of STING was initially reported in 2016 [ 101 ]. Dual mutations of Cys88/91 abolished the palmitoylation of STING and, thus, its role in activating expression of its downstream targets including IRF3, IFNβ, and NF-κB, suggesting that palmitoylation is required for activation of STING. Over-expression of ZDHHC3, ZDHHC7, and ZDHHC15 increased palmitoylation of STING, suggesting that they are responsible for STING palmitoylation. Further, another study found that palmitoylation of STING is essential for STING to interact with the mitochondrial voltage-dependent anion channel VDAC2 and, thus, prevent VDAC2-induced mitochondria dysfunction [ 102 ]. 2.2.1. Regulation of IFNγ/IFNGR1-Mediated PD-L1/PD-1 Signaling by Palmitoylation Stimulated by interferon-γ (IFNγ) ligand, IFNγ receptor 1 (IFNGR1) activates JAK/STAT signaling and, thus, induces transcription of programmed death protein ligand 1 (PD-L1) [ 87 ] ( Figure 4 ). Downstream of PD-L1, programmed death protein 1 (PD-1), the receptor of PD-L1, delivers inhibitory signals to regulate the balance between T cell activation, tolerance, and immunopathology. PD-1 is expressed on the surface of activated T cells as an inhibitory receptor, while its ligand PD-L1 is mainly expressed in antigen-presenting cells and tumor cells. By binding to its receptor PD-1 on T cells, expression of PD-L1 on tumor cells inhibits T cell activation and, thus, drives the escape of tumor cells from immune surveillance. Therefore, components of the IFNγ/IFNGR1-mediated PD-L1/PD-1 signaling pathway play a critical role in the success or failure of immune checkpoint blockades. Several studies have reported that palmitoylation provides regulatory roles for IFNγ/IFNGR1 signaling by modifying functions of the components. For example, in colorectal cancer cells, stability of IFNGR1 was found to be negatively regulated by palmitoylation; although, the specific ZDHHC that palmitoylates IFNGR1 was not identified [ 88 ]. Mutation of Cys122, the palmitoylation site of IFNGR1, or treatment of a palmitoylation inhibitor 2-bromopalmitate (2-BP) blocked the degradation of IFNGR1. Furthermore, IFNGR1 palmitoylation is also essential for the interaction between IFNGR1 and its binding partner AP3D1. As a consequence, IFNGR1 is able to trigger its downstream immune response signaling to inhibit cancer growth. Further, palmitoylation modulates the immune response to cancer by directly altering PD-L1 functions. Palmitoylation of endogenous PD-L1 was firstly identified in breast cancer cell lines MDA-MB231 and BT549 [ 89 ]. Inhibition of PD-L1 palmitoylation by 2-BP decreased PD-L1's protein level. Mutation of Cys272 or knockdown of ZDHHC9, the palmitoyl transferase responsible for PD-L1, abolished PD-L1 palmitoylation and decreased PD-L1 cell surface distribution. More importantly, blocking PD-L1 palmitoylation sensitized tumor cells to T-cell killing and, thereby, impaired tumor growth in vivo. These observations indicate that, in breast cancer cells, palmitoylation facilitates immune suppression to cancer by stabilizing and maintaining PD-L1 on the cell surface. Further, palmitoylation of PD-L1 in colorectal cancer and lung adenocarcinoma was also reported [ 90 , 91 ]. Of note, unlike ZDHHC9-mediated PD-L1 palmitoylation in breast cancer cells, palmitoylation of PD-L1 in colorectal cancer cells is mediated by ZDHHC3 but not ZDHHC9, indicating that PD-L1 utilizes multiple ZDHHCs to ensure its palmitoylation in different contexts. Mechanistically, palmitoylation stabilizes PD-L1 by suppressing ubiquitination and degradation in lysosomes, thereby repressing anti-tumor immunity. As a treatment strategy, to increase the immune response against cancer, a designed peptide that contains the palmitoylation region of PD-L1 was developed as a competitive inhibitor to inhibit PD-L1 palmitoylation. More recently, roles of ZDHHC9 in anti-tumor immunity were also revealed in pancreatic cancer [ 59 ]. Different from the studies conducted on breast cancer, colorectal cancer, and lung adenocarcinoma, this study found that, in the pancreatic tumor xenograft model, tumors with ZDHHC9 knockdown led to a change in PD-L1 level on the surrounding immune cells. Although it is unclear how ZDHHC9 in the tumor cells affects PD-L1 expression level on the surrounding immune cells, this study reported the non-autonomous function of ZDHHC9 in regulating the cancer immune response for the first time. In addition to PD-L1, palmitoylation also affects functions of its receptor PD-1. In addition to being expressed on the T cells to function as an inhibitory receptor, expression of PD-1 in cancer cells has also been revealed. Different from the function of PD-1 on T cells, in cancer cells, PD-1 promotes tumor growth independently of the adaptive immune system by modulating mTOR signaling. In 2021, Yao et al. revealed the palmitoylation of PD-1 at Cys192 in a variety of cancer cells [ 92 ]. Inhibition of PD-1 palmitoylation by 2-BP blocked the interaction between PD-1 and Rab11, a key molecule in transporting the cargo proteins to the recycling endosomes. As a consequence, decreased storage of PD-1 in recycling endosomes led to increased PD-1 degradation in the lysosomes. These results suggest that palmitoylation attenuates degradation of PD-1 by facilitating transportation of PD-1 to the recycling endosomes. More importantly, palmitoylation of PD-1 is essential for cancer cell growth as it is required for PD-1 to interact with its downstream signaling mediators to transduce signaling. Therefore, palmitoylation of PD-1 in cancer cells promotes cancer cell growth by preventing degradation of PD-1 and promoting its interaction with its downstream signaling mediators. Similar to the competitive inhibitor of PD-L1, a peptide containing the palmitoylation region of PD-1 was developed as a competitive inhibitor for PD-1 palmitoylation. Moving forward, it would be interesting to investigate the possibility of PD-1 palmitoylation in T cells and if palmitoylation affects the function of PD-1 in adaptive immune system. 2.2.2. Palmitoylation Triggers Anti-Immune Response by Sorting Proteins into Extracellular Vesicles The roles of extracellular vesicles (EV), lipid-enclosed particles that circulate bioactive content, in cancer development and progression have been revealed for decades [ 93 ]. Recently, Mariscal et al. demonstrated that, by anchoring proteins to cellular membranes, palmitoylation plays an important role in sorting proteins into the EV [ 94 ]. By doing so, the anti-immune response is triggered. For example, by secreting palmitoylated proteins through EV, acute myeloid leukemia (AML) cells activate toll-like receptor 2 (TLR2), a receptor that mediates activation of the immune system [ 95 ]. Subsequently, TLR2 induced the differentiation of monocytes into T-cell inhibitory myeloid-derived suppressor cells (MDSC), a type of immunosuppressive cell [ 96 ], resulting in a blockage of immune-response-mediated AML cell removal. Indeed, although the identity of the palmitoylated proteins in EV was not identified and it is still unknown how palmitoylation triggers TLR2 activation, AML cells treated with 2-BP lost the capacity to generate MDSC and an immune-suppressive environment for their survival. 2.2.3. Regulation of the Innate Immune Response in Preventing Cancer by Palmitoylation STING (Stimulator of interferon genes) is an innate immune sensor of immune surveillance of viral/bacterial infection and is involved in the maintenance of an immune-friendly microenvironment to prevent tumorigenesis [ 97 , 98 , 99 , 100 ]. Palmitoylation of STING was initially reported in 2016 [ 101 ]. Dual mutations of Cys88/91 abolished the palmitoylation of STING and, thus, its role in activating expression of its downstream targets including IRF3, IFNβ, and NF-κB, suggesting that palmitoylation is required for activation of STING. Over-expression of ZDHHC3, ZDHHC7, and ZDHHC15 increased palmitoylation of STING, suggesting that they are responsible for STING palmitoylation. Further, another study found that palmitoylation of STING is essential for STING to interact with the mitochondrial voltage-dependent anion channel VDAC2 and, thus, prevent VDAC2-induced mitochondria dysfunction [ 102 ]. 2.3. Regulation of Cancer Stem Cell Potency by Palmitoylation Cancer stem cells are responsible for cancer treatment resistance, leading to relapse, disease progression, and eventually systemic disease [ 103 ]. Lately, functions of palmitoylation in cancer stem cells have received increasing attention. Mechanistic analysis found that palmitoylation not only affects the self-renewal capacity of cancer stem cells but also affects their tumorigenicity ( Figure 5 ). In the past five years, as the majority of studies were conducted on glioblastoma stem cells (GSCs), we focused on discussing roles of palmitoylation in GSCs here. Acting downstream of ERK and AKT, glycogen synthase kinase 3β (GSK3β), a serine/threonine protein kinase, is also considered as a key hub for promoting malignancy of GSCs [ 104 , 105 ]. Zhao et al. found that roles of GSK3β are affected by palmitoylation when ZDHHC4 palmitoylates GSK3β at the Cys14 residue [ 106 ]. Mechanistically, palmitoylation of GSK3β negatively affected binding of GSK3β to AKT1 and S6K kinases, thereby releasing the inhibitory roles of AKT1 and S6K on GSK3β. Downstream of GSK3β, activated GSK3β phosphorylates histone methyltransferase EZH2, which, in turn, regulates STAT3 methylation and phosphorylation, leading to increased expression of stem-cell-related genes [ 107 ]. More importantly, due to the increased GSK3β activity induced by palmitoylation, tumorigenicity of GSCs was increased and resistance to chemotherapy was generated. In addition to modifying EZH2 activity by palmitoylating GSK3β, EZH2 activity can also be affected by palmitoylation directly. For example, in p53-mutant GSCs, ZDHHC5 palmitoylated EZH2 at Cys571 and Cys576 [ 52 ]. After being palmitoylated, palmitoylation blocked phosphorylation of EZH2 and, thus, its activity in mediating methylation of H3K27me3. As a consequence, loss of H3K27me3 released the suppressed expression of stemness markers cluster of differentiation (CD)133 and SOX2, thereby increasing the neurosphere formation capacity of GSCs. Further studies found that ZDHHC5-mediated EZH2 palmitoylation is regulated by mutant p53 as it transcriptionally induced expression of ZDHHC5 by interacting with nuclear transcription factor NF-Y. More recently, it was observed that P53-induced ZDHHC5 expression can also be enhanced by treatment of propofol, an agent that induces local anesthetics [ 108 ]. Consequently, upregulated ZDHHC5 led to increased EZH2 palmitoylation, resulting in reduced H3K27me3 at promoter regions of genes that regulate stem cell potency and increase expression of these genes. Hyperactive transforming growth factor-beta (TGF-β) signaling has also been viewed as a signature event in mesenchymal glioblastoma [ 109 , 110 ]. Downstream of TGF-β, the TGF-β receptor phosphorylates transcription factor SMAD3, inducing expression of genes including leukemia inhibitory factor (LIF) and platelet-derived growth factor-beta (PDGF-β) [ 111 , 112 , 113 ]. By doing so, TGF-β induces self-renewal and promotes proliferation of glioma-initiating cells [ 112 , 113 , 114 ]. Recently, Fan et al. reported that palmitoylation of SMAD3 mediated by ZDHHC19 is essential in transducing TGF-β signaling in GSCs [ 115 ]. Specifically, palmitoylation of SMAD3 at Cys421 mediated the translocation from cytosol to the nucleus. OCT4A, also known as POU5F1, is a key transcription factor in the self-renewal, proliferation, and differentiation of stem cells [ 116 , 117 ]. Palmitoylation of OCT4A mediated by ZDHHC17 is essential for protecting OCT4A from degradation and, thus, maintaining its protein stability [ 118 ]. Consequently, palmitoylation of OCT4A retained the stemness of GSCs by binding to the enhancer of SOX2, a key gene for maintaining the tumorigenicity of GSCs; thus, it promoted SOX2 expression. Targeting OCT4A palmitoylation using a competitive inhibitor that contains the OCT4A palmitoylation sequence effectively inhibited palmitoylation of OCT4A and, thereby, reduced the tumorigenesis in vivo. Palmitoylation of OCT4A mediated by ZDHHC17 was identified as a promising therapeutic approach toward effectively eliminating cancer-initiating cells. The involvement of ZDHHC15 and ZDHHC18 in GSCs was revealed recently [ 119 , 120 ]. Knockdown of ZDHHC15 reduced the capacity of GSCs to form neurospheres, indicating that ZDHHC15 is essential for GSCs' self-renewal [ 119 ]. Immunoprecipitation-coupled with mass spectrometry analysis identified GP130, an IL-6 receptor subunit, as a substrate of ZDHHC15 in GSCs. Further mechanism analysis found that palmitoylation mediates the cell membrane localization of GP130 and, thus, activates IL-6/STAT3 signaling to maintain GSCs' renewal ability. Whereas, for ZDHHC18, although no palmitoylation targets were identified, high expression of ZDHHC18 was observed in mesenchymal GSCs [ 120 ]. Mechanistic investigation found that, by interacting with the E3 ligase of BMI1, ZDHHC18 blocked degradation of BMI1, a gene contributing to the maintenance and renewal of cancer-initiating stem cells [ 121 , 122 ], and, thus, maintained the self-renewal capacity of GSCs [ 120 ]. 2.4. Palmitoylation Regulates Cancer Cell Migration by Modifying Cytoskeleton-Related Proteins and Cell Adhesion Molecules In addition to regulating cancer cell growth, palmitoylation also contributes to cancer cell migration and invasion by modifying functions of actin cytoskeletal remodeling related proteins and cell adhesion molecules. For example, palmitoylation of RhoU, an atypical Rho GTPase, at Cys256 is essential for migration of prostate cancer cells [ 123 , 124 ]. Mechanistic analysis found that palmitoylated RhoU increased cell migration ability by interacting and, thus, stabilizing Cdc42, a gene that regulates cell spread area [ 124 , 125 ]. Palmitoylation of α-tubulin has also been reported [ 126 , 127 , 128 , 129 ]. For example, it was reported that palmitoylation at Cys377 mediates astral microtubule function during nuclear migration in the M phase of the cell cycle. More interestingly, it was revealed that palmitoylation level of α-tubulin can also been regulated. For example, androgen treatment increased α-tubulin palmitoylation to mediate proliferation of prostate cancer cells, although the molecular mechanism was not fully investigated [ 130 ]. Cancer cell migration can also be regulated by modifying palmitoylation of cell adhesion molecules. For example, palmitoylation of cell adhesion molecules CD44 and MCAM prevented melanoma cell invasion [ 131 ]. On the contrary, depalmitoylation of CD44 and MCAM mediated by APT1 resulted in increased invasion. 2.5. Cancer Cells Use Palmitoylation to Overcome Nutrient Deficiency GLUT1 is a widely expressed glucose transporter responsible for the constant uptake of glucose [ 132 ]. Numerous studies have also shown that GLUT1 is essential for cancer growth [ 133 , 134 , 135 ]. In glioblastoma cells, site-directed mutagenesis showed that GLUT1 is palmitoylated at Cys207 and disruption of palmitoylation abolished localization of GLUT1 on the plasma membrane [ 136 ]. Expression of GLUT1-WT but not the palmitoylation defective form of GLUT1, GLUT1-C207S, restored the reduction in glucose uptake induced by knockout of GLUT1. Furthermore, blocking GLUT1 palmitoylation mediated by knockout of ZDHHC9, the enzyme that palmitoylates GLUT1, impaired glycolysis and reduced GBM tumor growth. These observations indicate that palmitoylation is required for GLUT1 to localize on the plasma membrane and, thus, conduct its function in mediating glucose uptake and tumor growth. Malate dehydrogenase 2 (MDH2) catalyzes the reversible reaction of malate to oxaloacetate in the TCA cycle [ 137 ]. Aberrant MDH2 function has been found to be associated with malignancy of cancer [ 138 , 139 ]. Palmitoylation of MDH2 was firstly identified in 2008 from a palmitoylome-proteomic study [ 140 ]. Recently, Pei et al. found that palmitoylation of MDH2 at Cys138 increased its binding affinity with its coenzyme NAD+ in order to maintain the function of glycolysis and mitochondrial respiration in ovarian cancer cells [ 141 ]. MDH2 interacted with ZDHHC18 and exogenous expression of ZDHHC18 increased palmitoylation levels of MDH2, indicating that ZDHHC18 is the palmitoyl transferase responsible for MDH2 palmitoylation. Of note, palmitoylation of MDH2 mediated by ZDHHC18 was stimulated by glutamine deprivation. These data revealed how cancer cells use palmitoylation and mitochondrial respiration for its growth to adapt to the nutrition-deficient tumor microenvironment. 2.6. Palmitoylation Negatively Contributes to Cancer In addition to positively contributing to cancer progression, the negative function of palmitoylation in cancer growth has also been revealed. Palmitoylation conducts its cancer suppression function by suppressing the function of oncoproteins, maintaining functions of tumor suppressors, or modifying growth-signaling transduction. 2.6.1. Palmitoylation Negatively Regulates Oncoprotein Functions Astrocyte elevated gene-1 (AEG-1) is an oncogene that is over-expressed in a wide variety of cancers [ 142 , 143 , 144 , 145 ]. Palmitoylation of AEG-1 in several physiological contexts was initially reported by Zhou et al. [ 146 ] ( Figure 6 a). From this research, they found that AEG-1 is palmitoylated at Cys75 and a palmitoylation-defective form of AEG-1 enhanced hepatocellular carcinoma progression in vivo. Mechanistically, palmitoylation negatively regulates AEG-1 function by reducing the stability of AEG-1 and by reducing its affinity with its interacting proteins. Further, a different research group identified ZDHHC6 as the main palmitoyl transferase for AEG-1 and found that CRISPR/Cas9 knock-in mice with a palmitoylation-defective form of AEG-1 had increased signaling pathways and regulators that contribute to cell proliferation, motility, angiogenesis, and lipid accumulation [ 147 ]. Together, palmitoylation plays a negative regulatory role on AEG-1 function. By transducing PI3K/AKT and RAS/MAPK signaling, receptor tyrosine kinase FLT3 plays an important role in the development of hematopoietic progenitors [ 148 ]. Consequently, mutation of FLT3 is one of the causes of cancer. For example, internal tandem duplication within FLT3 (FLT3-ITD) confers constitutive activation of FLT3, represents one of the most frequent mutations in acute myeloid leukemia (AML), and correlates with a poor prognosis [ 149 ]. Palmitoylation of FLT3-ITD mediated by ZDHHC6 was identified in AML, and palmitoylation redirected the localization of FLT3-ITD from the plasma membrane to ER ( Figure 6 b) [ 150 ]. Mutation of Cys563 maintained FLT3-ITD on the plasma membrane to conduct its function in activating downstream signaling. Further, inhibition of depalmitoylation with palmostatin B (palm B), a pan-depalmitoylase inhibitor, not only reduced proliferation of FLT3-ITD+ AML cells but also synergized FLT3-ITD+ AML cells to gilteritinib, a FLT3 kinase inhibitor, treatment. Together, these results suggest that palmitoylation plays a repressive role for FLT3. 2.6.2. Palmitoylation Maintains Functions of Tumor Suppressors G protein-coupled receptors (GPCRs) are a group of membrane proteins that convert extracellular signals into intracellular responses, including responses to growth signaling; neurotransmitters; as well as responses to vision, olfaction, and taste signals [ 151 ]. Due to its critical roles in enhancing DNA repair, mutations of the melanocortin-1 receptor (MC1R), a GPCR, have been correlated to a higher risk of melanoma. Palmitoylation of MC1R at Cys78 and Cys315 was identified in melanocytes, and the C315S mutant, a palmitoylation-defective form of MC1R, promoted melanomagenesis [ 152 ] ( Figure 6 c). Palmitoylation of MC1R is mainly mediated by ZDHHC13, and hyper-palmitoylation of MC1R mediated by ZDHHC13 also prevented melanomagenesis. Further, patients with high ZDHHC13 are correlated with better survival, and inhibition of APT2, the MC1R depalmitoylation enzyme, effectively suppressed melanomagenesis by blocking MC1R palmitoylation [ 153 ]. Together, these results highlighted a central role of MC1R palmitoylation in protection against melanoma. TP53 is one of the most extensively studied tumor suppressor genes whose multifaceted mechanisms involve apoptosis, DNA repair, genomic stabilization, and angiogenesis [ 154 ]. A recent study found that TP53 is the key mediator for ZDHHC1-induced breast cancer suppression ( Figure 6 d). Five palmitoylation sites—Cys135, Cys176, Cys182, Cys275, and Cys277—were identified in TP53. Mutation of Cys135, Cys176, and Cys275 significantly blocked nuclear translocation of TP53 and the expression of TP53 downstream targets, e.g., P21 and BAX, resulting in the promotion of tumor growth. Together, by modifying TP53 nuclear translocation, palmitoylation conducts its tumor suppression function [ 155 ]. SET domain-containing 2 (SETD2) is a histone lysine methyltransferase. By mediating the methylation of H3K36me3, SETD2 contributes to DNA damage response (DDR) by recruiting RAD51 to DNA double strand break sites. As a functional DDR is critical for maintaining genome integrity and preventing tumor development [ 156 ], SETD2 is considered as a putative tumor suppressor gene in cancers [ 157 , 158 ]. In EGFR-amplified glioblastoma, palmitoylation of SETD2 mediated by ZDHHC16 protects SETD2 from degradation and, thereby, facilitates its role in mediating DNA damage response and repressing cancer initiation ( Figure 6 e) [ 159 ]. GNA13 encodes one of the alpha subunits of the heterotrimeric G proteins that transduce signals of GPCR. By negatively regulating the expression of BCL2, GNA13 has also been identified as a tumor suppressor in B-cell lymphoma [ 160 ]. Palmitoylation of GNA13 was initially reported in early 2000 ( Figure 6 f). The study revealed that GNA13 is palmitoylated at Cys14 and Cys18. The wild type but not the palmitoylation-defective form of GNA13 localizes at the plasma membrane and, thus, transduces Rho-dependent signaling [ 161 ]. Moreover, expression of GNA13WT but not the palmitoylation-defective form of GNA13 inhibited proliferation of B-cell lymphoma, suggesting that palmitoylation of GNA13 is required for its tumor suppression function [ 160 ]. 2.6.3. Palmitoylation Suppresses Cancer by Reducing Growth Signaling In addition to repressing tumors by directly affecting functions of cancer-related protein, palmitoylation can also block tumor growth by modifying growth signaling. For example, palmitoylation at Cys44, Cys45, and Cys47 of the small CTD phosphatase 1 (SCP1) leads to its translocation from the nuclear to plasma membranes [ 162 ]. As a result, membrane-located SCP1 dephosphorylates AKT at serine 473, leading to suppressed angiogenesis and decreased tumor growth of lung carcinoma in xenograft mice. 2.6.1. Palmitoylation Negatively Regulates Oncoprotein Functions Astrocyte elevated gene-1 (AEG-1) is an oncogene that is over-expressed in a wide variety of cancers [ 142 , 143 , 144 , 145 ]. Palmitoylation of AEG-1 in several physiological contexts was initially reported by Zhou et al. [ 146 ] ( Figure 6 a). From this research, they found that AEG-1 is palmitoylated at Cys75 and a palmitoylation-defective form of AEG-1 enhanced hepatocellular carcinoma progression in vivo. Mechanistically, palmitoylation negatively regulates AEG-1 function by reducing the stability of AEG-1 and by reducing its affinity with its interacting proteins. Further, a different research group identified ZDHHC6 as the main palmitoyl transferase for AEG-1 and found that CRISPR/Cas9 knock-in mice with a palmitoylation-defective form of AEG-1 had increased signaling pathways and regulators that contribute to cell proliferation, motility, angiogenesis, and lipid accumulation [ 147 ]. Together, palmitoylation plays a negative regulatory role on AEG-1 function. By transducing PI3K/AKT and RAS/MAPK signaling, receptor tyrosine kinase FLT3 plays an important role in the development of hematopoietic progenitors [ 148 ]. Consequently, mutation of FLT3 is one of the causes of cancer. For example, internal tandem duplication within FLT3 (FLT3-ITD) confers constitutive activation of FLT3, represents one of the most frequent mutations in acute myeloid leukemia (AML), and correlates with a poor prognosis [ 149 ]. Palmitoylation of FLT3-ITD mediated by ZDHHC6 was identified in AML, and palmitoylation redirected the localization of FLT3-ITD from the plasma membrane to ER ( Figure 6 b) [ 150 ]. Mutation of Cys563 maintained FLT3-ITD on the plasma membrane to conduct its function in activating downstream signaling. Further, inhibition of depalmitoylation with palmostatin B (palm B), a pan-depalmitoylase inhibitor, not only reduced proliferation of FLT3-ITD+ AML cells but also synergized FLT3-ITD+ AML cells to gilteritinib, a FLT3 kinase inhibitor, treatment. Together, these results suggest that palmitoylation plays a repressive role for FLT3. 2.6.2. Palmitoylation Maintains Functions of Tumor Suppressors G protein-coupled receptors (GPCRs) are a group of membrane proteins that convert extracellular signals into intracellular responses, including responses to growth signaling; neurotransmitters; as well as responses to vision, olfaction, and taste signals [ 151 ]. Due to its critical roles in enhancing DNA repair, mutations of the melanocortin-1 receptor (MC1R), a GPCR, have been correlated to a higher risk of melanoma. Palmitoylation of MC1R at Cys78 and Cys315 was identified in melanocytes, and the C315S mutant, a palmitoylation-defective form of MC1R, promoted melanomagenesis [ 152 ] ( Figure 6 c). Palmitoylation of MC1R is mainly mediated by ZDHHC13, and hyper-palmitoylation of MC1R mediated by ZDHHC13 also prevented melanomagenesis. Further, patients with high ZDHHC13 are correlated with better survival, and inhibition of APT2, the MC1R depalmitoylation enzyme, effectively suppressed melanomagenesis by blocking MC1R palmitoylation [ 153 ]. Together, these results highlighted a central role of MC1R palmitoylation in protection against melanoma. TP53 is one of the most extensively studied tumor suppressor genes whose multifaceted mechanisms involve apoptosis, DNA repair, genomic stabilization, and angiogenesis [ 154 ]. A recent study found that TP53 is the key mediator for ZDHHC1-induced breast cancer suppression ( Figure 6 d). Five palmitoylation sites—Cys135, Cys176, Cys182, Cys275, and Cys277—were identified in TP53. Mutation of Cys135, Cys176, and Cys275 significantly blocked nuclear translocation of TP53 and the expression of TP53 downstream targets, e.g., P21 and BAX, resulting in the promotion of tumor growth. Together, by modifying TP53 nuclear translocation, palmitoylation conducts its tumor suppression function [ 155 ]. SET domain-containing 2 (SETD2) is a histone lysine methyltransferase. By mediating the methylation of H3K36me3, SETD2 contributes to DNA damage response (DDR) by recruiting RAD51 to DNA double strand break sites. As a functional DDR is critical for maintaining genome integrity and preventing tumor development [ 156 ], SETD2 is considered as a putative tumor suppressor gene in cancers [ 157 , 158 ]. In EGFR-amplified glioblastoma, palmitoylation of SETD2 mediated by ZDHHC16 protects SETD2 from degradation and, thereby, facilitates its role in mediating DNA damage response and repressing cancer initiation ( Figure 6 e) [ 159 ]. GNA13 encodes one of the alpha subunits of the heterotrimeric G proteins that transduce signals of GPCR. By negatively regulating the expression of BCL2, GNA13 has also been identified as a tumor suppressor in B-cell lymphoma [ 160 ]. Palmitoylation of GNA13 was initially reported in early 2000 ( Figure 6 f). The study revealed that GNA13 is palmitoylated at Cys14 and Cys18. The wild type but not the palmitoylation-defective form of GNA13 localizes at the plasma membrane and, thus, transduces Rho-dependent signaling [ 161 ]. Moreover, expression of GNA13WT but not the palmitoylation-defective form of GNA13 inhibited proliferation of B-cell lymphoma, suggesting that palmitoylation of GNA13 is required for its tumor suppression function [ 160 ]. 2.6.3. Palmitoylation Suppresses Cancer by Reducing Growth Signaling In addition to repressing tumors by directly affecting functions of cancer-related protein, palmitoylation can also block tumor growth by modifying growth signaling. For example, palmitoylation at Cys44, Cys45, and Cys47 of the small CTD phosphatase 1 (SCP1) leads to its translocation from the nuclear to plasma membranes [ 162 ]. As a result, membrane-located SCP1 dephosphorylates AKT at serine 473, leading to suppressed angiogenesis and decreased tumor growth of lung carcinoma in xenograft mice. 3. Targeting Protein Palmitoylation for Cancer Treatment Palmitoylation has been proposed to be targeted from three directions: targeting ZDHHC enzymes, blocking substrate palmitoylation, and preventing depalmitoylation, although challenges exist. Targeting ZDHHCs for disease treatment has attracted increased attention. However, as introduced above, many proteins can be palmitoylated by more than one ZDHHC enzyme. Therefore, inhibiting a single ZDHHC cannot fully block the palmitoylation of the substrates. One potential solution to this problem is to search for pan-ZDHHC inhibitors that can block the function of several enzymes required for the palmitoylation of a single target. Also, a complementary ZDHHC-agnostic approach to eliminate the problem of functional redundancy between individual ZDHHC enzymes is to identify compounds that directly prevent substrate palmitoylation via irreversible covalent modification of individual cysteine residues. Further, although ZDHHCs contain a conserved zinc finger (DHHC) domain, few ZDHHC inhibitors are available and no therapeutic drugs that target specific ZDHHCs have been approved to date. The few reported broad-spectrum ZDHHC inhibitors include compound V [ 163 ], tunicamycin [ 164 ], cerulenin [ 165 ], and 2-bromopalmitate (2-BP) [ 166 ] ( Table 1 ). Among these inhibitors, 2-BP has been used in pre-clinical studies to validate the concept that ZDHHC protein inhibition can promote cancer death [ 43 , 167 , 168 ]. However, 2-BP is not selective for individual ZDHHC enzymes, and off-target acylation of other intracellular proteins increases the risk of unknown side effects and limits its potential as a therapeutic candidate for disease treatment [ 169 , 170 , 171 , 172 ]. In 2021, another broad-spectrum ZDHHC inhibitor, cyano-myracrylamide (CMA), was identified [ 173 ]. Although developing treatments to target ZDHHCs has limitations, as described above, researchers continually make new discoveries to overcome the challenges. For example, last year, Qiu et al. identified that artemisinin (ART), a clinically approved antimalarial endoperoxide nature product, can be used as a ZDHHC6 inhibitor; although, it is unclear if ART also inhibits the function of other ZDHHCs [ 174 ]. Rather than inhibiting protein palmitoylation, in some cases, it may be more effective to prevent depalmitoylation, although each acyl-protein thioesterase can depalmitoylate more than one protein ( Table 1 ). For example, inhibition of APT enzymes suppressed tumor formation by promoting the proper localization of SCRIB and enhancing activity of MC1R [ 152 , 186 , 187 ]. APTs, in general, are highly druggable targets, and a handful of inhibitors have been developed to target APTs. Among those, palmostatin B (Palm B) [ 176 ] and hexadecylfluorophosphonate (HDFP) [ 10 ] are being widely used in research as they are broad-spectrum serine hydrolase inhibitors that target all depalmitoylases. Although significant overlapping in substrates is observed in different APTs, selectivity in substrates among different APTs has also been revealed [ 31 , 32 , 33 ]. Furthermore, inhibitors targeting different APTs in the selective way are also well known. For example, in 2013, the first selective inhibitors for APT1 and APT2, ML348 and ML349, were identified [ 9 , 188 ]. More recently, several inhibitors have been revealed to inhibit specific depalmitoylase selectively. For instance, in oral squamous cell carcinomas (OSCC), the expression level of PPT1 can be reduced by erianin, a natural bibenzyl compound, although the molecular mechanism was not fully characterized [ 189 ]. GNS561, a specific PPT1 inhibitor, by itself, is able to effectively inhibit the progression of liver cancer [ 177 , 190 ]. Furthermore, another selective and potent PPT1 inhibitor, DC661, not only impaired tumor growth [ 180 ] but also enhanced the response of liver cancer cells to treatment of sorafenib, a multi-kinase inhibitor [ 191 ], and enhanced the antitumor activity of the anti-PD-1 antibody in melanoma [ 192 ]. Dimeric quinacrines 661 (DQ661) was also identified as an inhibitor for PPT1 and, thus, can be used to inhibit cancer growth [ 181 ]. More recently, from a serine-hydrolase-directed compound library screening, ABD957 was identified to have selective inhibition on ABHD17 [ 179 ]. To target palmitoylated proteins specifically, recently, a few studies have developed customized peptides to compete with the palmitoylation of specific proteins and, thus, block the palmitoylation of specific proteins. For example, Chen et al. showed a cell-penetrating peptide that contains the palmitoylated sequence of OCT4A—a key transcription factor in the self-renewal, proliferation, and differentiation of stem cells—could function as a competitive inhibitor to effectively inhibit the palmitoylation of OCT4A [ 118 ]. Similarly, Yao et al. developed a competitive inhibitor of PD-L1 palmitoylation by fusing green fluorescent protein (GFP) to the palmitoylation motif of PD-L1 [ 90 ]. Using the same concept, a peptide that fuses GFP with the palmitoylation motif of PCSK9, a gene that plays a critical role in anti-tumor immune responses, also showed inhibition on palmitoylation of PCSK9 both in vitro and in vivo [ 66 ]. Different from the competitive inhibitors, inhibitors targeting the palmitoylation pocket have also been reported. For example, several inhibitors have been developed to target the transcriptional enhanced associate domain (TEAD) palmitoylation pocket [ 185 , 193 , 194 , 195 ] ( Table 1 ). Serving as the receptor for the downstream effectors of the Hippo pathway, YAP and TAZ, TEAD upregulates the expression of multiple genes involved in organ size control and tumorigenesis. Palmitoylation of TEAD at conserved cysteine residues was shown to be required for binding of TEAD to YAP and TAZ in a variety of cancer cells [ 196 , 197 ]. Structural analysis found that TEAD palmitoylation is critical for protein folding and stability as the lipid tail extends into a conserved hydrophobic core of the protein [ 198 ]. Targeting the palmitoylation pocket of TEAD, several inhibitors have been identified. For example, compounds TM2 and MGH-CP1 were identified as inhibitors that bind to the palmitate-binding pocket (PBP) to suppress TEAD palmitoylation and, thus, function [ 183 , 184 , 185 ]. Further, from a structure-based virtual ligand screening, JM17 was identified as an inhibitor for TEAD palmitoylation. Treatment of JM17 reduced the stability of TEAD, leading to impaired proliferation; colony formation; and migration of mesothelioma (NCI-H226), breast, and ovarian cancer cells [ 182 ]. 4. Future Perspectives Although we have learned significantly about palmitoylation, many critical issues remain. For example, although we have showed that the functions of numerous proteins are regulated by palmitoylation, we have little understanding on how palmitoylation and depalmitoylation are regulated. Recently, several studies have suggested that palmitoylation can be regulated by altering the expression level, protein stability, or distribution of enzymes involved in palmitoylation [ 131 , 159 , 199 , 200 , 201 , 202 ]. Other studies have suggested that palmitoylation can be regulated by extracellular stimulators [ 130 , 203 , 204 ]. Studying the regulation of palmitoylation can help us understand the roles of palmitoylation in leading to pathological diseases. Although ZDHHCs display different preferences for different substrate proteins [ 17 , 18 , 205 , 206 ], no consensus on palmitoylation sequence motifs has been reached and the mechanism of enzyme–substrate pairs has not been established [ 207 ]. Palmitoylated proteins are often substrates for more than one ZDHHC enzyme, while one particular ZDHHC enzyme often has a stronger effect than others on substrate palmitoylation in the cell [ 19 , 20 , 207 ]. ZDHHCs vary substantially in their palmitoylation activities and their affinities in binding to their cognate substrate proteins [ 205 ]. Although ZDHHCs have a similar structure, a few ZDHHCs do have some unique signatures. For example, ZDHHC5 and ZDHHC8 have a long and highly disordered C-terminal tail [ 208 , 209 ], whereas ZDHHC13 and ZDHHC17 possess an ankyrin-repeat domain [ 205 , 210 ]. In the past few years, a new technology leveraging high-density CRISPR screens has been used to identify novel functional domains on a protein [ 211 , 212 , 213 ]. We expect that this technology can possibly be used to study protein structures of ZDHHCs for palmitoylation research fields. In terms of relevant diseases, it would be interesting to test if cells under certain pathological conditions have a different list of palmitoylated proteins compared with cells under physiological conditions and, if so, if the palmitoylation state of individual cysteine residues within a given protein vary over time when cells undergo malignant changes. In cancers, 79 out of the 299 cancer drivers have been identified to be palmitoylated [ 8 , 42 ]. For example, palmitoylation of GNAQ/11, a G protein α subunit q/11 polypeptide, was reported in uveal melanoma (UM) that contains a GNAQ/11 mutation in over 85% of patients [ 214 ]. Specifically, by controlling the location of GNAQ/11, palmitoylation is essential for GNAQ/11 in mediating growth signaling and, thus, malignant progression of UM. As the research moves forward, we speculate that there will be more studies focused on understanding if palmitoylation affects functions of the critical cancer drivers. For example, similar to UM, over 85% of patients with Ewing sarcoma, a type of bone and soft tissue cancer, contain fusion protein EWSR1-FLI1. It would be of interest to test if palmitoylation also affects the function of EWSR1-FLI1 and if palmitoylation is one of the contributing factors in EWSR1-FLI1-mediated Ewing sarcoma. Although the lack of antibodies recognizing palmitoylated proteins is hindering our understanding of the implication of a dynamic lipid modification of proteins in cell signaling and regulation, the development of chemical approaches to study protein palmitoylation has revolutionized the understanding of the field [ 34 , 36 , 215 ]. We foresee that identification of palmitoylated proteins at the proteome level [ 216 ] will help the field in understanding the roles of palmitoylation in leading to pathological consequences at the systematic level. Further, genetic screens, e.g., CRISPR screens, have been used to identify vulnerable targets for cancer treatment [ 217 ]; applying genetic screens in palmitoylation research would help us test if enzymes involved in palmitoylation can be vulnerable cancer treatment targets in various contexts.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2907578/

Proprotein Convertase Inhibition Results in Decreased Skin Cell Proliferation, Tumorigenesis, and Metastasis 1 2

PACE4 is a proprotein convertase (PC) responsible for cleaving and activating proteins that contribute to enhance tumor progression. PACE4 overexpression significantly increased the susceptibility to carcinogenesis, leading to enhanced tumor cell proliferation and premature degradation of the basement membrane. In the present study, we sought to evaluate a novel approach to retard skin tumor progression based on the inhibition of PACE4. We used decanoyl-RVKR-chloromethylketone (CMK), a small-molecule PC inhibitor, for in vitro and in vivo experiments. We found that CMK-dependent blockage of PACE4 activity in skin squamous cell carcinoma cell lines resulted in impaired insulin-like growth factor 1 receptor maturation, diminished its intrinsic tyrosine kinase activity, and decreased tumor cell proliferation. Two-stage skin chemical carcinogenesis experiments, together with topical applications of CMK, demonstrated that this PC inhibitor markedly reduced tumor incidence, tumor multiplicity, and metastasis, pointing to a significant delay in tumor progression in wild-type and PACE4 transgenic mice. These results identify PACE4, together with other PCs, as suitable targets to slow down or block tumor progression, suggesting that PC inhibition is a potential approach for therapy for solid tumors.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172777/

The cytosolic entry of diphtheria toxin catalytic domain requires a host cell cytosolic translocation factor complex

In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650–800-fold purified from human T cell and yeast extracts, respectively. The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast. Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu. These results confirm and extend earlier observations of diphtheria toxin C-domain unfolding and refolding that must occur before and after vesicle membrane translocation. In addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain. Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes. Introduction Diphtheria toxin (DT; * 58 kD) is a typical single-chain AB toxin composed of three functional domains: the amino-terminal catalytic (C) domain corresponds to fragment A (21 kD), and the transmembrane (T) and carboxy-terminal receptor-binding domains comprise fragment B (37 kD) of the toxin ( Choe et al., 1992 ). A disulfide bond between Cys186 and Cys201 subtends a protease-sensitive loop and connects fragment A with fragment B. Furin-mediated cleavage within this loop and retention of the disulfide bond have been shown to be prerequisites for intoxication of eukaryotic cells ( Ariansen et al., 1993 ; Tsuneoka et al., 1993 ). Substitution of the native receptor domain with human interleukin-2 (IL-2) has resulted in the formation of a fusion protein toxin, DAB 389 IL-2, whose cytotoxic action is specifically targeted only to cells expressing the high affinity IL-2 receptors ( Bacha et al., 1988 ; Waters et al., 1990 ; vanderSpek 2002>Ratts and vanderSpek, 2002 ). Because DAB 389 IL-2 binds with greater affinity to its receptor compared with native DT, this fusion protein toxin has proven to be an effective and novel probe for studying internalization of the C-domain by target cells ( Williams et al., 1990 ). Although much is known about the mechanisms of receptor-binding and receptor-mediated endocytosis of native DT and the DT-related fusion proteins, little is known about the precise molecular mechanisms of C-domain translocation across the endosomal membrane and its release into the cytosol. Unfolding of the DT C-domain was first postulated as a prerequisite for translocation by Kagan et al. (1981) and Donovan et al. (1981) . The necessity for complete denaturation of the DT C-domain before translocation was then indirectly demonstrated by Wiedlocha et al. (1992) and by Falnes et al. (1994) . At present, there are two conflicting hypotheses for translocation of denatured DT C-domain across the early endosomal membrane. Studies using artificial lipid bilayers suggest that the DT T-domain itself exhibits chaperonin-like properties and is solely sufficient to promote C-domain delivery across the bilayer ( Oh et al., 1999 ; Ren et al., 1999 ). In contrast, studies using partially purified early endosomes that were preloaded with toxin suggest that C-domain translocation across the vesicle membrane is dependent on ATP and the presence of cytosolic components which include β-COP ( Lemichez et al., 1997 ). Because protease digestion patterns of DT inserted into planar lipid bilayers differ from those of DT inserted into the plasma membrane ( Moskaug et al., 1991 ; Cabiaux et al., 1994 ), it seems likely that interaction(s) between the toxin and proteins associated with the endosomal membrane (e.g., receptor) influence the orientation and/or stoichiometry of insertion of the T-domain and translocation of the C-domain. In addition, Ren et al. (1999) and Hammond et al. (2002) have shown that although the DT T-domain has chaperonin-like properties, it has a significantly greater affinity for other molten globule-like polypeptides compared with its own C-domain. To further define the requirements of C-domain translocation across the endosomal membrane, we have used an in vitro C-domain translocation assay essentially as described by Lemichez et al. (1997) . This assay uses purified early endosomes that have been preloaded with DAB 389 IL-2 and monitors the translocation of ADP-ribosyltransferase activity from the endosomal lumen to the external milieu. We have used translocated ADP-ribosyltransferase activity to monitor the purification of cytosolic components that are required for this process. In the present paper, we demonstrate by mass spectrometry (MS) sequence identification and the effect of specific inhibitors that the chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase (TrR-1) are components of a cytosolic translocation factor (CTF) complex that is essential for the translocation and release of C-domain from early endosomes. Furthermore, the identification of CTF complex homologues in partially purified yeast extracts suggests that DT C-domain translocation may proceed by a fundamental mechanism of entry. Results Partial purification of the DT C-domain CTF complex The requirements for DT C-domain translocation across the early endosomal membrane and release into the external milieu were monitored using an in vitro translocation assay modified from Umata et al. (1990) and Lemichez et al. (1997) . The early endosomal compartment of HUT102 6TG cells was preloaded with DAB 389 IL-2 in the presence of bafilomycin A1. Early endosomes were purified by sucrose density gradient centrifugation, and then incubated in the presence of ATP and cytosolic extracts from either HUT102 6TG cells or yeast. After incubation at 37°C, translocation of the C-domain across the endosomal membrane and release into the external medium was monitored by ADP-ribosyltransferase activity of both the pellet and supernatant fluid fractions after ultracentrifugation. The [ 32 P]ADP ribosylation of elongation factor 2 (EF-2) was measured by autoradiography of SDS-PAGE of reaction mixtures ( Chung and Collier, 1977 ). The limit of sensitivity of this assay is in the range of 10 −14 –10 −16 M C-domain, a level well below that of detection by immunoblot (unpublished data). As shown in Fig. 1 A, on dilution of bafilomycin A1 and the addition of both ATP and cytosolic extracts to the reaction mixture, the C-domain is translocated across the endosomal membrane and released into the external medium. Moreover, preboiling the cytosolic extracts before their addition to the reaction mixture abolishes C-domain translocation. These results suggest that the C-domain translocation across the membrane of early endosomes requires cytosolic protein(s). The time course of C-domain translocation was examined using the epitope-labeled fusion protein toxin DAB 189(VSV-G) B 389 IL-2. The cytotoxic potency of the epitope-tagged fusion toxin is almost identical to that of DAB 389 IL-2 (IC 50 = 3 × 10 −11 M vs. 4 × 10 −12 M). As shown in Fig. 1 B, the ADP-ribosyltransferase activity as measured by densitometry of the combined 32 P-labeled EF-2 from each paired pellet and supernatant fluid fraction is plotted as percentage of ADP-ribosyltransferase activity in the supernatant fluid. As can be seen, translocation of the C-domain is linear for up to 45 min, at which time ∼80% of the total activity is found in the supernatant fluid fraction. As reported by Lemichez et al. (1997) , whereas ADP-ribosyltransferase activity was translocated to the external medium, cointernalized HRP activity was found to remain in the pellet fraction throughout the incubation period (unpublished data). These results strongly suggest that C-domain translocation is specific, and not the result of spontaneous endosomal lysis during the incubation period. Finally, in the presence of added ATP and cytosolic extracts, the translocation of the C-domain is dependent on membrane fluidity and does not occur at temperatures below 15°C. Figure 1. Partial purification of cytosolic proteins required to mediate DT C-domain translocation from the lumen of early endosomes in vitro. (A) Early endosomes from human T cells (HUT102/6TG), preloaded with DAB 389 IL-2, were incubated for 30 min at 37°C with 2 mM ATP (A) and/or 4 μg HUT102/6TG crude cytosol (C). C* denotes heat inactivation of cytosol before incubation with endosomes. Endosomes were pelleted, and the supernatant fraction (S) was assayed for DT C-domain ADP-ribosyltransferase activity by measuring the incorporation of [ 32 P]NAD + into AD[ 32 P]ribosyl-EF-2 after 7% SDS-PAGE and autoradiography. (B) Time course for translocation of ADP-ribosyltransferase activity from DA 189(VSV-G) B 389 IL-2 across the early endosomal membrane. Translocations were performed as described above, except the reactions were incubated at different temperatures (0, 15, and 37°C) for 15, 30, and 45 min. Both the supernatant and the pellet fractions were assayed for ADP-ribosyltransferase activity, and the autoradiographic signals were measured by densitometry. The sum of densitometry units from each pair of supernatant fluid and pellet fractions is plotted as the percentage of activity in the supernatant fractions at that time point. ( n = 3; error bar denotes SD). To rule out the possibility that the crude T cell and yeast extracts contained an allosteric regulator(s) of vesicular ATPase activity rather than protein(s) that are required for C-domain translocation, early endosomes were charged with a 70-kD dextran conjugated with the pH-sensitive fluorescent dye, SNARF-1. As shown in Fig. 2 A, compared with pH 7.5, the fluorescence emission of 1 ng/ml SNARF-1 is decreased approximately fourfold at pH 4.5. As measured by the quenching of fluorescence emission of SNARF-1, in vitro acidification of the early endosomal lumen occurs on dilution of bafilomycin A1 and requires the addition of 2 mM ATP to the reaction mixture ( Fig. 2 B). Moreover, the time course for the acidification of early endosomes in vitro is virtually identical after the addition of either 2 mM ATP or 2 mM ATP plus partially purified T cell CTF complex. Figure 2. The in vitro acidification of early endosomes requires ATP and does not require any cytosolic protein factors. (A) Fluorescence emission of 1 ng/ml SNARF-1 70 kD dextran conjugate standards at pH 7.5 and 4.5 was measured at an excitation wavelength of 534 nM and an emission wavelength of 645 nM. (B) Purified early endosomes preloaded with the pH-sensitive SNARF-1 70 kD dextran conjugates were incubated in translocation assay buffer for 20 min at 37°C with 2 mM ATP and/or 0.1 μg/μL of Mono Q-purified cytosol. In each instance, assays were performed in triplicate and fluorescence was monitored using a fluorescence detector (model 650S; PerkinElmer). Error bars denote SD. Partial purification of human T cell and yeast cytosolic factors required for the in vitro translocation of ADP-ribosyltransferase activity across the membrane of early endosomes Because C-domain translocation across the endosomal vesicle membrane requires the addition of cytosolic components to the reaction mixture, we used translocation of ADP-ribosyltransferase activity to monitor the partial purification of the active component(s) from both human T cell (HUT102/6TG) and yeast (NLY22 − ) extracts. After DEAE anion exchange chromatography, translocation-active fractions (150 mM–190 mM NaCl) were pooled and applied to a Sephacryl ® 200 sizing column. The translocation-active fractions (250–100 kD) were pooled and further fractionated by Mono Q HPLC under conditions free of reducing agents. The translocation-active fraction was found to elute from the Mono Q column at 27.3 mS. As shown in Fig. 3 A, after fractionation on Mono Q, CTF complex activity from human T cell and yeast cell extracts was increased by 650-fold and 800-fold, respectively. Further analysis of the Mono Q-pooled fractions by SDS-PAGE and colloidal Coomassie staining revealed multiple protein bands ranging in apparent molecular mass from ∼12–100 kD ( Fig. 3 B). Figure 3. The partial purification of CTFs results in the increase of translocation in vitro specific activity. (A) Translocation in vitro specific activity of CTFs increases after each stage of purification. Reactions were performed as described in Fig. 1 , and only the ADP-ribosyltransferase activity of the supernatant fluid fractions is shown. CE, crude extract; DEAE, DEAE-Sepharose anion exchange chromatography (150–190 mM NaCl fractions); S200, Sephacryl ® 200 sizing chromatography (250–100-kD fractions); MQ, Mono Q anion exchange chromatography (27.3-mS fractions). (B) Colloidal Coomassie stained 10% SDS-PAGE protein band profiles after Mono Q anion exchange chromatography. Partially purified CTF complex fractions from both T cells and yeast cells were eluted at a conductance of 27.3 mS. Identification of individual components of the CTF complex Tryptic peptides from "in-gel" digestion of individual protein bands resolved by SDS-PAGE were subjected to analysis by mass spectroscopy using matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) and nano-electrospray ionization (ESI) quadrupole/orthogonal TOF spectrometers ( Jensen et al., 1999 ). Peptide maps and tandem MS sequence data allowed for the unequivocal identification of Hsp 90 (α and β) and TrR-1 in the partially purified CTF complex mixture from human T cells ( Fig. 4 ; Table I ). Importantly, the corresponding yeast homologues, Hsp 82 and TrR-1, as well as thioredoxin peroxidase, were identified in the partially purified CTF complex from yeast cells ( Table I ). The cumulative peptide coverage for each protein identified through LC-MS/MS sequencing was between 65 and 85% of the total protein (Table S1, available at www.jcb.org/cgi/content/full/jcb.200210028/DC1 ). Ions unassigned in the LC-MS/MS spectra were indicative of truncation, sequence variation, and/or post-translational modification. Figure 4. Identification of putative CTF(s) using MS. (A) Representative total ion chromatogram from an online capillary liquid chromatography mass spectrometric analysis of the in-gel tryptic digest after immunoprecipitation of the 84-kD band ( Fig. 2 B and Fig. 5 B) from human partially purified CTFs using rabbit polyclonal anti-Hsp 90 antibodies. (B) Mass spectrum from LC-MS elution at time 19.5 to 20.5 min, as indicated by the shaded region in (A). Peaks are labeled with the m/z value, the charge state, the corresponding amino acid segment, and specification of the Hsp 90 isoform. (C) Tandem mass spectrum for m/z 575.98 4+ (redundant sequence from Hsp 90 α and β; see inset on B). Complementary a and b (NH 2 -terminal derived) as well as y (COOH-terminal derived) ions are labeled in the spectrum with m/z value and charge state. All observed a, b, and y ions are indicated in the peptide diagram. Data were analyzed using BioAnalyst™ (Applied Biosystems) reconstruction algorithms. For initial screening and searches, acquired mass values were compared with theoretical protein digests using the Mascot search engine (Matrix Science Ltd.). Table I. Summary of the data obtained for each putative CTF component identified in this work: MALDI, ESI-MS/MS, LC-MS/MS, Western blot (WB), in vitro translocation assay (TA), and mammalian cell cytotoxicity assay (CA) Putative CTFs Data obtained Human T cell (Hut 102/6TG) Hsp 90 α MALDI, ESI-MS/MS, LC-MS/MS, WB, TA, CA Hsp 90 β MALDI, ESI-MS/MS, LC-MS/MS, WB, TA, CA TrR-1 MALDI, ESI-MS/MS, WB, TA, CA Yeast (NLY22 − ) Hsp 82 MALDI, ESI-MS/MS, WB, TA, CA TrR-1 MALDI, LC-MS/MS, TA, CA Thioredoxin peroxidase (AHP1) MALDI, ESI-MS/MS Hsp 90 is essential (but not sufficient) for C-domain translocation in vitro To determine whether Hsp 90 was a component of the CTF complex and to establish a functional role for this chaperonin in C-domain translocation, we conducted a series of experiments to examine the effects of using both polyclonal anti-Hsp 90 antibodies and specific inhibitors. As shown in Fig. 5 A, either immunoprecipitation of Hsp 90 from the CTF complex or addition of anti-Hsp 90 to the CTF complex before initiation of the translocation reaction resulted in marked loss of ADP-ribosyltransferase activity in the supernatant fluid fraction. There was no significant loss of ADP-ribosyltransferase activity in the pellet fractions, which argues against the possibility of any translocated but nonrefolded pool of DT C-domain. Because attempts to reconstitute the immunodepleted CTF complex with human recombinant (hr) Hsp 90 failed to restore translocation activity, we conclude that additional, as yet unknown, protein(s) required for translocation was also removed by immunoprecipitation ( Fig. 5 B). Figure 5. Hsp 90 is a component of the CTF complex. (A) Hut 102/6TG (H) and yeast NLY22 − (Y) partially purified CTF complexes were coincubated with 1 μg anti-Hsp 90 and 1 μg anti-Hsp 82 antibodies, respectively, for 10 min at RT before standard analysis in the in vitro translocation assay. Analogously, CTF complexes were depleted of Hsp 90 or Hsp 82 by immunoprecipitation and assayed for translocation activity in vitro. Either partially purified CTF complex or hrHsp 90 was added back to Hsp 90- depleted CTF complexes as indicated, and translocation activity in vitro was assayed. (B) Colloidal Coomassie-stained 10% SDS-PAGE protein band profiles of immunoprecipitated human Hsp 90 (IP), marker (M). Arrow indicates Hsp 90 as identified by ESI LC-MS/MS analysis. Because geldanamycin and radicicol are well-known inhibitors of Hsp 90, we next examined the effect of these two agents on C-domain translocation in vitro. These agents are known to bind to the ATPase site of the chaperone and block ATP hydrolysis, thereby inhibiting refolding and release of substrate ( Grenert et al., 1997 ; Schulte et al., 1998 ). Fig. 6 shows that neither the addition of geldanamycin nor radicicol alone was capable of inhibiting C-domain translocation. However, when both inhibitors were used in combination, C-domain translocation was inhibited. There are several reports demonstrating the synergistic inhibitory effects of geldanamycin and radicicol on Hsp 90, and inhibition is thought to result from either the disruption of substrate binding or the interaction with cochaperonins ( Rosenhagen et al., 2001 ). This phenomenon appears to be Hsp 90-specific because the addition of hrHsp 90 to geldanamycin/radicicol-treated human T cell CTF complexes restored C-domain translocation ( Fig. 6 ). Interestingly, the addition of rhHsp 90 to the geldanamycin/radicicol treated CTF complex from yeast only partially restored C-domain translocation in vitro, suggesting that these agents disrupt a species-specific Hsp 82 cochaperone interaction necessary for reconstitution of translocation activity. Figure 6. Hsp 90 is essential for mediating DAB 389 IL-2 C-domain translocation from the lumen of early endosomes to the external milieu. The Hsp 90-specific inhibitors, geldanamycin and radicicol, were preincubated with partially purified CTF complex as indicated for 15 min at RT before assaying for translocation activity in vitro. Excess partially purified HUT 102/6TG CTF complex and hrHsp 90 were added to geldanamycin/radicicol treated CTF complexes as indicated and assayed for translocation activity in vitro. TrR-1 is essential (but not sufficient) for DT C-domain translocation in vitro Because TrR-1 was also identified by MS sequence analysis of CTF complexes from human T cell and yeast extracts, we have used both immunoprecipitation and specific inhibitors to demonstrate a functional role of TrR-1 in the translocation and/or release of the C-domain from early endosomes. As shown in Fig. 7 A, immunoprecipitation of TrR-1 from human CTF complexes and 2′,5′-ADP-Sepharose affinity chromatographic depletion of yeast TrR-1 from CTF complex mixtures abolished C-domain translocation in vitro. Because there was no significant loss of ADP-ribosyltransferase activity in the pellet fractions (unpublished data), we conclude that there is no pool of translocated but nonrefolded DT C-domain. Reconstitution experiments in which bovine recombinant (br) TrR-1 was added back to TrR-1-depleted CTF complexes, from both T cell and yeast, failed to restore C-domain translocation (unpublished data). Because these experiments were performed under conditions known to reduce the interchain disulfide bond between the C- and T-domains, these results suggest that TrR-1 is a component of a complex and that another factor(s) essential for translocation was codepleted with TrR-1 ( Fig. 7 B). Figure 7. TrR-1 is a component of the CTF complex. (A) Hut 102/6TG (H) and yeast NLY22 − (Y) partially purified CTF complexes were depleted of TrR-1 by immunoprecipitation with anti-human TrR-1 antibodies or affinity purification using 2′,5′ ADP-Sepharose. TrR-1–depleted CTF complexes were then assayed for translocation activity in vitro under reducing conditions. Either partially purified CTF complex or brTrR-1 was added back to TrR-1–depleted CTF complexes as indicated, and translocation activity in vitro was assayed. (B) Colloidal Coomassie-stained 10% SDS-PAGE protein band profiles of 2′,5′ ADP-Sepharose affinity-purified yeast TrR-1 (AP), marker (M). Arrow indicates yeast TrR-1 as identified by ESI LC-MS/MS analysis. Next, we examined the effect of the TrR-1 stereo-specific inhibitor cis-13-retinoic acid on C-domain translocation ( Schallreuter and Wood, 1989 ). The addition of cis-13-retinoic acid, but not trans-13-retinoic acid, to either human or yeast CTF complex mixtures resulted in the complete inhibition of C-domain translocation in vitro under nonreducing conditions ( Fig. 8 ). Importantly, the addition of excess brTrR-1 to cis-13-retinoic–treated complex restored C-domain translocation activity in vitro, suggesting that cis-13-retinoic acid inhibition is TrR-1 specific. Finally, when assayed under reducing conditions (20 mM DTT), cis-13-retinoic acid had no effect on C-domain translocation (unpublished data). Together, these results indicate that TrR-1 activity plays an essential role in the translocation and/or release of the C-domain from early endosomes. Figure 8. TrR-1 function is essential for mediating DAB 389 IL-2 C-domain translocation from the lumen of early endosomes to the external milieu under nonreducing conditions. Partially purified CTF complex, both human and yeast, were assayed for translocation activity in vitro under nonreducing conditions using translocation buffer containing 10 μM NADPH without DTT. The TrR-1 stereospecific inhibitor cis-13-retinoic acid and the inactive trans-13-retinoic acid isomer were preincubated with partially purified CTF complex as indicated for 15 min at RT before assaying for translocation activity in vitro under nonreducing conditions. Excess brHsp 90 was added to c13RA-treated CTF complexes as indicated, and translocation activity in vitro was assayed under nonreducing conditions. Geldanamycin/radicicol and cis-13-retinoic acid protect HUT102 6TG cells from the cytotoxic action of DAB 389 IL-2 Because geldanamycin and radicicol were found to have a synergistic effect in blocking the in vitro translocation of ADP-ribosyltransferase from purified early endosomes, we examined the ability of these agents to protect intact cells from DAB 389 IL-2. A series of dose-response experiments showed that neither the addition of 10 nM geldanamycin nor 10 nM radicicol alone confer protection against the fusion protein toxin. However, as seen in in vitro translocation assays, these agents in combination (10 nM each) were able to affect a two-log shift in the DAB 389 IL-2 dose-response curve (IC 50 ≈ 10 −8 M) compared with the untreated control (IC 50 ≈ 5 × 10 −10 M). In a similar fashion, cis-13-retinoic acid was found to affect a similar dose-dependent shift in the DAB 389 IL-2 dose-response curve for HUT102 6TG cells (unpublished data), confirming and extending early observations made by Sandvig and Olsnes (1981) . Partial purification of the DT C-domain CTF complex The requirements for DT C-domain translocation across the early endosomal membrane and release into the external milieu were monitored using an in vitro translocation assay modified from Umata et al. (1990) and Lemichez et al. (1997) . The early endosomal compartment of HUT102 6TG cells was preloaded with DAB 389 IL-2 in the presence of bafilomycin A1. Early endosomes were purified by sucrose density gradient centrifugation, and then incubated in the presence of ATP and cytosolic extracts from either HUT102 6TG cells or yeast. After incubation at 37°C, translocation of the C-domain across the endosomal membrane and release into the external medium was monitored by ADP-ribosyltransferase activity of both the pellet and supernatant fluid fractions after ultracentrifugation. The [ 32 P]ADP ribosylation of elongation factor 2 (EF-2) was measured by autoradiography of SDS-PAGE of reaction mixtures ( Chung and Collier, 1977 ). The limit of sensitivity of this assay is in the range of 10 −14 –10 −16 M C-domain, a level well below that of detection by immunoblot (unpublished data). As shown in Fig. 1 A, on dilution of bafilomycin A1 and the addition of both ATP and cytosolic extracts to the reaction mixture, the C-domain is translocated across the endosomal membrane and released into the external medium. Moreover, preboiling the cytosolic extracts before their addition to the reaction mixture abolishes C-domain translocation. These results suggest that the C-domain translocation across the membrane of early endosomes requires cytosolic protein(s). The time course of C-domain translocation was examined using the epitope-labeled fusion protein toxin DAB 189(VSV-G) B 389 IL-2. The cytotoxic potency of the epitope-tagged fusion toxin is almost identical to that of DAB 389 IL-2 (IC 50 = 3 × 10 −11 M vs. 4 × 10 −12 M). As shown in Fig. 1 B, the ADP-ribosyltransferase activity as measured by densitometry of the combined 32 P-labeled EF-2 from each paired pellet and supernatant fluid fraction is plotted as percentage of ADP-ribosyltransferase activity in the supernatant fluid. As can be seen, translocation of the C-domain is linear for up to 45 min, at which time ∼80% of the total activity is found in the supernatant fluid fraction. As reported by Lemichez et al. (1997) , whereas ADP-ribosyltransferase activity was translocated to the external medium, cointernalized HRP activity was found to remain in the pellet fraction throughout the incubation period (unpublished data). These results strongly suggest that C-domain translocation is specific, and not the result of spontaneous endosomal lysis during the incubation period. Finally, in the presence of added ATP and cytosolic extracts, the translocation of the C-domain is dependent on membrane fluidity and does not occur at temperatures below 15°C. Figure 1. Partial purification of cytosolic proteins required to mediate DT C-domain translocation from the lumen of early endosomes in vitro. (A) Early endosomes from human T cells (HUT102/6TG), preloaded with DAB 389 IL-2, were incubated for 30 min at 37°C with 2 mM ATP (A) and/or 4 μg HUT102/6TG crude cytosol (C). C* denotes heat inactivation of cytosol before incubation with endosomes. Endosomes were pelleted, and the supernatant fraction (S) was assayed for DT C-domain ADP-ribosyltransferase activity by measuring the incorporation of [ 32 P]NAD + into AD[ 32 P]ribosyl-EF-2 after 7% SDS-PAGE and autoradiography. (B) Time course for translocation of ADP-ribosyltransferase activity from DA 189(VSV-G) B 389 IL-2 across the early endosomal membrane. Translocations were performed as described above, except the reactions were incubated at different temperatures (0, 15, and 37°C) for 15, 30, and 45 min. Both the supernatant and the pellet fractions were assayed for ADP-ribosyltransferase activity, and the autoradiographic signals were measured by densitometry. The sum of densitometry units from each pair of supernatant fluid and pellet fractions is plotted as the percentage of activity in the supernatant fractions at that time point. ( n = 3; error bar denotes SD). To rule out the possibility that the crude T cell and yeast extracts contained an allosteric regulator(s) of vesicular ATPase activity rather than protein(s) that are required for C-domain translocation, early endosomes were charged with a 70-kD dextran conjugated with the pH-sensitive fluorescent dye, SNARF-1. As shown in Fig. 2 A, compared with pH 7.5, the fluorescence emission of 1 ng/ml SNARF-1 is decreased approximately fourfold at pH 4.5. As measured by the quenching of fluorescence emission of SNARF-1, in vitro acidification of the early endosomal lumen occurs on dilution of bafilomycin A1 and requires the addition of 2 mM ATP to the reaction mixture ( Fig. 2 B). Moreover, the time course for the acidification of early endosomes in vitro is virtually identical after the addition of either 2 mM ATP or 2 mM ATP plus partially purified T cell CTF complex. Figure 2. The in vitro acidification of early endosomes requires ATP and does not require any cytosolic protein factors. (A) Fluorescence emission of 1 ng/ml SNARF-1 70 kD dextran conjugate standards at pH 7.5 and 4.5 was measured at an excitation wavelength of 534 nM and an emission wavelength of 645 nM. (B) Purified early endosomes preloaded with the pH-sensitive SNARF-1 70 kD dextran conjugates were incubated in translocation assay buffer for 20 min at 37°C with 2 mM ATP and/or 0.1 μg/μL of Mono Q-purified cytosol. In each instance, assays were performed in triplicate and fluorescence was monitored using a fluorescence detector (model 650S; PerkinElmer). Error bars denote SD. Partial purification of human T cell and yeast cytosolic factors required for the in vitro translocation of ADP-ribosyltransferase activity across the membrane of early endosomes Because C-domain translocation across the endosomal vesicle membrane requires the addition of cytosolic components to the reaction mixture, we used translocation of ADP-ribosyltransferase activity to monitor the partial purification of the active component(s) from both human T cell (HUT102/6TG) and yeast (NLY22 − ) extracts. After DEAE anion exchange chromatography, translocation-active fractions (150 mM–190 mM NaCl) were pooled and applied to a Sephacryl ® 200 sizing column. The translocation-active fractions (250–100 kD) were pooled and further fractionated by Mono Q HPLC under conditions free of reducing agents. The translocation-active fraction was found to elute from the Mono Q column at 27.3 mS. As shown in Fig. 3 A, after fractionation on Mono Q, CTF complex activity from human T cell and yeast cell extracts was increased by 650-fold and 800-fold, respectively. Further analysis of the Mono Q-pooled fractions by SDS-PAGE and colloidal Coomassie staining revealed multiple protein bands ranging in apparent molecular mass from ∼12–100 kD ( Fig. 3 B). Figure 3. The partial purification of CTFs results in the increase of translocation in vitro specific activity. (A) Translocation in vitro specific activity of CTFs increases after each stage of purification. Reactions were performed as described in Fig. 1 , and only the ADP-ribosyltransferase activity of the supernatant fluid fractions is shown. CE, crude extract; DEAE, DEAE-Sepharose anion exchange chromatography (150–190 mM NaCl fractions); S200, Sephacryl ® 200 sizing chromatography (250–100-kD fractions); MQ, Mono Q anion exchange chromatography (27.3-mS fractions). (B) Colloidal Coomassie stained 10% SDS-PAGE protein band profiles after Mono Q anion exchange chromatography. Partially purified CTF complex fractions from both T cells and yeast cells were eluted at a conductance of 27.3 mS. Identification of individual components of the CTF complex Tryptic peptides from "in-gel" digestion of individual protein bands resolved by SDS-PAGE were subjected to analysis by mass spectroscopy using matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) and nano-electrospray ionization (ESI) quadrupole/orthogonal TOF spectrometers ( Jensen et al., 1999 ). Peptide maps and tandem MS sequence data allowed for the unequivocal identification of Hsp 90 (α and β) and TrR-1 in the partially purified CTF complex mixture from human T cells ( Fig. 4 ; Table I ). Importantly, the corresponding yeast homologues, Hsp 82 and TrR-1, as well as thioredoxin peroxidase, were identified in the partially purified CTF complex from yeast cells ( Table I ). The cumulative peptide coverage for each protein identified through LC-MS/MS sequencing was between 65 and 85% of the total protein (Table S1, available at www.jcb.org/cgi/content/full/jcb.200210028/DC1 ). Ions unassigned in the LC-MS/MS spectra were indicative of truncation, sequence variation, and/or post-translational modification. Figure 4. Identification of putative CTF(s) using MS. (A) Representative total ion chromatogram from an online capillary liquid chromatography mass spectrometric analysis of the in-gel tryptic digest after immunoprecipitation of the 84-kD band ( Fig. 2 B and Fig. 5 B) from human partially purified CTFs using rabbit polyclonal anti-Hsp 90 antibodies. (B) Mass spectrum from LC-MS elution at time 19.5 to 20.5 min, as indicated by the shaded region in (A). Peaks are labeled with the m/z value, the charge state, the corresponding amino acid segment, and specification of the Hsp 90 isoform. (C) Tandem mass spectrum for m/z 575.98 4+ (redundant sequence from Hsp 90 α and β; see inset on B). Complementary a and b (NH 2 -terminal derived) as well as y (COOH-terminal derived) ions are labeled in the spectrum with m/z value and charge state. All observed a, b, and y ions are indicated in the peptide diagram. Data were analyzed using BioAnalyst™ (Applied Biosystems) reconstruction algorithms. For initial screening and searches, acquired mass values were compared with theoretical protein digests using the Mascot search engine (Matrix Science Ltd.). Table I. Summary of the data obtained for each putative CTF component identified in this work: MALDI, ESI-MS/MS, LC-MS/MS, Western blot (WB), in vitro translocation assay (TA), and mammalian cell cytotoxicity assay (CA) Putative CTFs Data obtained Human T cell (Hut 102/6TG) Hsp 90 α MALDI, ESI-MS/MS, LC-MS/MS, WB, TA, CA Hsp 90 β MALDI, ESI-MS/MS, LC-MS/MS, WB, TA, CA TrR-1 MALDI, ESI-MS/MS, WB, TA, CA Yeast (NLY22 − ) Hsp 82 MALDI, ESI-MS/MS, WB, TA, CA TrR-1 MALDI, LC-MS/MS, TA, CA Thioredoxin peroxidase (AHP1) MALDI, ESI-MS/MS Hsp 90 is essential (but not sufficient) for C-domain translocation in vitro To determine whether Hsp 90 was a component of the CTF complex and to establish a functional role for this chaperonin in C-domain translocation, we conducted a series of experiments to examine the effects of using both polyclonal anti-Hsp 90 antibodies and specific inhibitors. As shown in Fig. 5 A, either immunoprecipitation of Hsp 90 from the CTF complex or addition of anti-Hsp 90 to the CTF complex before initiation of the translocation reaction resulted in marked loss of ADP-ribosyltransferase activity in the supernatant fluid fraction. There was no significant loss of ADP-ribosyltransferase activity in the pellet fractions, which argues against the possibility of any translocated but nonrefolded pool of DT C-domain. Because attempts to reconstitute the immunodepleted CTF complex with human recombinant (hr) Hsp 90 failed to restore translocation activity, we conclude that additional, as yet unknown, protein(s) required for translocation was also removed by immunoprecipitation ( Fig. 5 B). Figure 5. Hsp 90 is a component of the CTF complex. (A) Hut 102/6TG (H) and yeast NLY22 − (Y) partially purified CTF complexes were coincubated with 1 μg anti-Hsp 90 and 1 μg anti-Hsp 82 antibodies, respectively, for 10 min at RT before standard analysis in the in vitro translocation assay. Analogously, CTF complexes were depleted of Hsp 90 or Hsp 82 by immunoprecipitation and assayed for translocation activity in vitro. Either partially purified CTF complex or hrHsp 90 was added back to Hsp 90- depleted CTF complexes as indicated, and translocation activity in vitro was assayed. (B) Colloidal Coomassie-stained 10% SDS-PAGE protein band profiles of immunoprecipitated human Hsp 90 (IP), marker (M). Arrow indicates Hsp 90 as identified by ESI LC-MS/MS analysis. Because geldanamycin and radicicol are well-known inhibitors of Hsp 90, we next examined the effect of these two agents on C-domain translocation in vitro. These agents are known to bind to the ATPase site of the chaperone and block ATP hydrolysis, thereby inhibiting refolding and release of substrate ( Grenert et al., 1997 ; Schulte et al., 1998 ). Fig. 6 shows that neither the addition of geldanamycin nor radicicol alone was capable of inhibiting C-domain translocation. However, when both inhibitors were used in combination, C-domain translocation was inhibited. There are several reports demonstrating the synergistic inhibitory effects of geldanamycin and radicicol on Hsp 90, and inhibition is thought to result from either the disruption of substrate binding or the interaction with cochaperonins ( Rosenhagen et al., 2001 ). This phenomenon appears to be Hsp 90-specific because the addition of hrHsp 90 to geldanamycin/radicicol-treated human T cell CTF complexes restored C-domain translocation ( Fig. 6 ). Interestingly, the addition of rhHsp 90 to the geldanamycin/radicicol treated CTF complex from yeast only partially restored C-domain translocation in vitro, suggesting that these agents disrupt a species-specific Hsp 82 cochaperone interaction necessary for reconstitution of translocation activity. Figure 6. Hsp 90 is essential for mediating DAB 389 IL-2 C-domain translocation from the lumen of early endosomes to the external milieu. The Hsp 90-specific inhibitors, geldanamycin and radicicol, were preincubated with partially purified CTF complex as indicated for 15 min at RT before assaying for translocation activity in vitro. Excess partially purified HUT 102/6TG CTF complex and hrHsp 90 were added to geldanamycin/radicicol treated CTF complexes as indicated and assayed for translocation activity in vitro. TrR-1 is essential (but not sufficient) for DT C-domain translocation in vitro Because TrR-1 was also identified by MS sequence analysis of CTF complexes from human T cell and yeast extracts, we have used both immunoprecipitation and specific inhibitors to demonstrate a functional role of TrR-1 in the translocation and/or release of the C-domain from early endosomes. As shown in Fig. 7 A, immunoprecipitation of TrR-1 from human CTF complexes and 2′,5′-ADP-Sepharose affinity chromatographic depletion of yeast TrR-1 from CTF complex mixtures abolished C-domain translocation in vitro. Because there was no significant loss of ADP-ribosyltransferase activity in the pellet fractions (unpublished data), we conclude that there is no pool of translocated but nonrefolded DT C-domain. Reconstitution experiments in which bovine recombinant (br) TrR-1 was added back to TrR-1-depleted CTF complexes, from both T cell and yeast, failed to restore C-domain translocation (unpublished data). Because these experiments were performed under conditions known to reduce the interchain disulfide bond between the C- and T-domains, these results suggest that TrR-1 is a component of a complex and that another factor(s) essential for translocation was codepleted with TrR-1 ( Fig. 7 B). Figure 7. TrR-1 is a component of the CTF complex. (A) Hut 102/6TG (H) and yeast NLY22 − (Y) partially purified CTF complexes were depleted of TrR-1 by immunoprecipitation with anti-human TrR-1 antibodies or affinity purification using 2′,5′ ADP-Sepharose. TrR-1–depleted CTF complexes were then assayed for translocation activity in vitro under reducing conditions. Either partially purified CTF complex or brTrR-1 was added back to TrR-1–depleted CTF complexes as indicated, and translocation activity in vitro was assayed. (B) Colloidal Coomassie-stained 10% SDS-PAGE protein band profiles of 2′,5′ ADP-Sepharose affinity-purified yeast TrR-1 (AP), marker (M). Arrow indicates yeast TrR-1 as identified by ESI LC-MS/MS analysis. Next, we examined the effect of the TrR-1 stereo-specific inhibitor cis-13-retinoic acid on C-domain translocation ( Schallreuter and Wood, 1989 ). The addition of cis-13-retinoic acid, but not trans-13-retinoic acid, to either human or yeast CTF complex mixtures resulted in the complete inhibition of C-domain translocation in vitro under nonreducing conditions ( Fig. 8 ). Importantly, the addition of excess brTrR-1 to cis-13-retinoic–treated complex restored C-domain translocation activity in vitro, suggesting that cis-13-retinoic acid inhibition is TrR-1 specific. Finally, when assayed under reducing conditions (20 mM DTT), cis-13-retinoic acid had no effect on C-domain translocation (unpublished data). Together, these results indicate that TrR-1 activity plays an essential role in the translocation and/or release of the C-domain from early endosomes. Figure 8. TrR-1 function is essential for mediating DAB 389 IL-2 C-domain translocation from the lumen of early endosomes to the external milieu under nonreducing conditions. Partially purified CTF complex, both human and yeast, were assayed for translocation activity in vitro under nonreducing conditions using translocation buffer containing 10 μM NADPH without DTT. The TrR-1 stereospecific inhibitor cis-13-retinoic acid and the inactive trans-13-retinoic acid isomer were preincubated with partially purified CTF complex as indicated for 15 min at RT before assaying for translocation activity in vitro under nonreducing conditions. Excess brHsp 90 was added to c13RA-treated CTF complexes as indicated, and translocation activity in vitro was assayed under nonreducing conditions. Geldanamycin/radicicol and cis-13-retinoic acid protect HUT102 6TG cells from the cytotoxic action of DAB 389 IL-2 Because geldanamycin and radicicol were found to have a synergistic effect in blocking the in vitro translocation of ADP-ribosyltransferase from purified early endosomes, we examined the ability of these agents to protect intact cells from DAB 389 IL-2. A series of dose-response experiments showed that neither the addition of 10 nM geldanamycin nor 10 nM radicicol alone confer protection against the fusion protein toxin. However, as seen in in vitro translocation assays, these agents in combination (10 nM each) were able to affect a two-log shift in the DAB 389 IL-2 dose-response curve (IC 50 ≈ 10 −8 M) compared with the untreated control (IC 50 ≈ 5 × 10 −10 M). In a similar fashion, cis-13-retinoic acid was found to affect a similar dose-dependent shift in the DAB 389 IL-2 dose-response curve for HUT102 6TG cells (unpublished data), confirming and extending early observations made by Sandvig and Olsnes (1981) . Discussion In the present report, we demonstrate that the in vitro translocation of the DAB 389 IL-2 ADP-ribosyltransferase activity across the membrane of early endosomes and its release into the external milieu requires a CTF complex. Using translocated ADP-ribosyltransferase activity as an assay, we have partially purified the CTF complex from both human T cell and yeast extracts. MS sequencing of individual protein bands revealed by colloidal Coomassie staining of SDS-polyacrylamide gels has allowed the identification of Hsp 90 and TrR-1 from human T cells, and the homologous Hsp 82 and TrR-1 from yeast extracts. A functional role for these proteins in the translocation and/or cytosolic release of ADP-ribosyltransferase activity was established through immunoprecipitation and the use of specific inhibitors. After depletion of either Hsp 90 or TrR-1 from partially purified human T cell and yeast CTF complexes, we were not successful in reconstituting in vitro translocation of the C-domain by the addition of hrHsp 90 or brTrR-1, either alone or in combination. These results suggest that Hsp 90 chaperonin and TrR-1 are components of a complex(es) that is (are) necessary for facilitating C-domain translocation across the early endosomal membrane. In marked contrast, we were able to reconstitute in vitro C-domain translocation activity in either geldanamycin/radicicol or cis-13-retinoic acid treated CTF complexes by the addition of recombinant proteins to the mixture. Together, these observations lead us to conclude that both the chaperonin Hsp 90 and TrR-1 are required for C-domain translocation, but are not in themselves sufficient. Hsp 90 is ubiquitously expressed and is known to be a component of several multi-molecular chaperonin complexes that are highly conserved in eukaryotes ( Chang and Lindquist, 1994 ). The interaction of Hsp 90 with other cochaperonins and the formation of discrete complexes is known to mediate Hsp 90 substrate recognition ( Caplan, 1999 ). Although Hsp 90 does not usually directly bind nor refold nascent polypeptides, it is known to refold a growing list of newly synthesized proteins including membrane-associated protein kinases ( Bijlmakers and Marsh, 2000 ). In addition to its refolding activity, Hsp 90 complexes are also known to regulate the trafficking of membrane-associated proteins through interactions with cytoskeleton motors ( Pratt et al., 1999 ). The CTF complex is capable of refolding thermally denatured DT fragment A in vitro, and refolding requires the ATPase activity of Hsp 90 (unpublished data). However, the inhibition of Hsp 90 ATPase activity by either geldanamycin or radicicol alone does not inhibit translocation of ADP-ribosyltransferase activity across the early endosomal membrane. As such, it would appear that refolding of denatured C-domain into an active conformation and translocation are mutually exclusive events. The synergistic effects of geldanamycin and radicicol on the inhibition of ADP-ribosyltransferase translocation are of interest, and is consistent with previous reports ( Rosenhagen et al., 2001 ). It is possible that when used in combination, these inhibitors result in either a disruption of Hsp90 substrate recognition and/or the disruption of Hsp 90–cochaperone interactions, thereby leading to an inhibition of C-domain translocation. Although a firm conclusion cannot yet be reached, the inability to reconstitute yeast CTF complexes with mammalian factors supports the later hypothesis. We are currently investigating whether or not Hsp 90 interacts directly with the fusion protein toxin during translocation or is simply an architectural component of the complex. After furin-mediated nicking of the α-carbon backbone of either DT or DAB 389 IL-2, retention of the interchain disulfide bond between the C- and T-domains of the toxin presumably is essential for insertion and threading of the denatured C-domain into and through the nascent channel formed by the T-domain ( vanderSpek et al., 1994 ). Moreover, post-translocation reduction of this disulfide bond is also required for the release of the C-domain into the cytosol because unreduced C-domain and membrane-inserted T-domain are both targeted for proteolytic degradation ( Moskaug et al., 1993 ; Madshus et al., 1994 ). Indeed, the pivotal role of this event is underscored by the observation that reduction of this interchain disulfide bond is the rate-limiting step in the diphtherial intoxication of eukaryotic cells ( Papini et al., 1993 ). Observations reported here confirm and extend these earlier findings, and strongly suggest that TrR-1 is a component of the CTF complex required for the release of the C-domain from the early endosome. These observations also confirm and extend the earlier observations of Sandvig and Olsnes (1981) , who reported that retinoic acids inhibit the action of several AB toxins, including DT, on eukaryotic cells. Although the data reported here clearly demonstrate that TrR-1 activity is required for at least the cytosolic release of the DAB 389 IL-2 C-domain from purified early endosomes, we cannot conclude whether or not TrR-1 is directly involved in the reduction of the interchain disulfide bond. Because we have identified thioredoxin peroxidase in CTF complexes purified from yeast, it is possible that TrR-1 functions indirectly through a cascade of reductases (e.g., thioredoxin; Moskaug et al., 1987 ). It is widely accepted that anthrax lethal toxin and edema factor, as well as the botulinum neurotoxins, must pass through an acidic early endosomal compartment in order to deliver their respective C-domain into the cytosol of targeted cells. The unfolding of the C-domains of anthrax lethal factor ( Wesche et al., 1998 ) and botulinum toxin serotype D ( Bade et al., 2002 ), as well as the TrR-1–mediated reduction of the botulinum neurotoxins ( Kistner and Habermann, 1992 ; Bigalke and Shoer, 2000 ), have been postulated to be prerequisites for their delivery to the cytosol. Accordingly, the findings reported here may have wider implications. Importantly, several protein complexes of similar composition have been described in protein-trapping proteomic analysis of yeast. For example, Ho et al. (2002) has shown that cyclophilin-trapped complexes from yeast contain Hsp 82, TrR-1, and Sec 27. Moreover, cyclophilin is required for the cytosolic entry of HIV ( Braaten et al., 1996 ), the vacuolar import of fructose-1,6-bisphophatase ( Brown et al., 2001 ), and the activation of peroxiredoxins ( Lee et al., 2001 ). It should also be noted that trafficking mechanisms mediated by cyclophilin–Hsp 90 complexes are synergistically affected by geldanamycin and radicicol ( Meyer et al., 2000 ). In aggregate, observations reported here confirm and extend the hypothesis that multiple pathogens from diverse phylogenetic backgrounds, as well as many of their virulence determinants have convergently evolved to recruit host cell proteins (e.g., CTF complexes) in order to facilitate their membrane translocation and release into the cytosol of eukaryotic cells. Materials and methods Cell culture HUT1026TG cells (TIB 1620; American Type Culture Collection), were maintained in RPMI 1640 (BioWhittaker) supplemented with 10% FBS (HyClone), 2 mM glutamine (BioWhittaker), 50 IU/ml penicillin, and 50 μg/ml streptomycin (BioWhittaker) at 37°C in 5% CO 2 . Yeast strain NLY22 − (a gift from Dr. Kevin Jarrell, Modular Genetics Inc., Lincoln, MA) was maintained in YPD media (Difco ® ) and on YPD agar plates at 30°C. Purification of EF-2 EF-2 was partially purified using a procedure by Chung and Collier (1977) . After purification, fractions containing EF-2 were identified by ADP-ribosyltransferase using DAB 389 IL-2 (see In vitro ribosylation assay). EF-2 was further purified by DEAE-Sepharose (Reactifs IBF) anion exchange chromatography. EF-2 was eluted with a linear gradient, 0–200 mM NaCl, in 50 mM Tris-HCl, pH 8.0, 50 mM Mg(OAc) 2 , 0.1 M KCl, 4 mM CaCl 2 , 5 mM 2-ME and 1 μg PMSF (Sigma-Aldrich) per ml. Fractions containing EF-2 were identified as above. Aliquots were adjusted to a final concentration of 2 mM DTT, 5% glycerol, and stored at −70°C. Purified EF-2 was ∼80% homogeneous as resolved by 7% SDS-PAGE and stained with colloidal Coomassie (Invitrogen). Protein concentration was determined by Bradford Assay according to standard protocols using Coomassie Protein Assay Reagent (Pierce Chemical Co.). Purification of early endosomes Early endosomes were isolated from HUT102/6TG cells according to a protocol by Duprez and Dautry-Varsat (1986) . The early endosomal compartment was loaded with 1 μM DAB 389 IL-2, 1 μM DA 189(VSV-G) B 389 IL-2, 8 mg/ml 70-kD SNARF1-dextran conjugate (Molecular Probes, Inc.), and/or 5 mg/ml HRP (Sigma-Aldrich) using 1 μM bafilomycin A1–primed cells (Sigma-Aldrich). Purification of HUT102/6TG CTF complex Crude cytosolic extract was isolated from HUT 102/6TG cells according to the protocol modified from Bomsel et al. (1990) . In brief, cells were washed three times with cold PBS containing 5 mg/ml BSA, once with cold PBS alone, and twice with cold cytosol buffer (CB; 3% sucrose in 100 mM Hepes-KOH, pH 7.9, 1.4 M KCl, 30 mM MgCl 2 , 2 mM EDTA, and 5 mM DTT). Cells were lysed by 20 passages through a 25 G needle in CB containing protease inhibitors as follows: 10 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml antipain, and 1 μm PMSF (all obtained from Sigma-Aldrich) The lysate was centrifuged at 1,000 g for 15 min at 4°C. The post-nuclear supernatant was then centrifuged at 170,000 g for 1 h at 4°C. The supernatant fraction was dialyzed overnight at 4°C against cytosol dialysis buffer (CDB; 1% sucrose in 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, and 2 mM 2-ME) containing protease inhibitors as described in CB. Crude cytosol was fractionated according to standard chromatographic protocols. In brief, crude extract was loaded onto an in-house packed DEAE-Sepharose (Reactifs IBF) XK 26 column (Amersham Biosciences) for anion exchange chromatography. A peristaltic FPLC pump (P-1; Amersham Biosciences) and Single Path Monitor (UV-1; Amersham Biosciences) were used during chromatography. The column was preequilibrated with buffer B3 (containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 5 mM 2-mercaptoethanol, and 1 μg PMSF per ml), and "loaded" sample was washed using the same buffer. CTFs were eluted with a linear gradient, 0–400 mM NaCl, in buffer B3 at a flow rate of 5 ml/min. Fractions containing CTFs were identified using an in vitro translocation assay and in vitro ribosylation assay in series (see Materials and methods). Fractions containing in vitro translocation activity eluted between 150 to 190 mM NaCl, and were pooled and concentrated using Centriplus Centrifugal Filters (YM-10; Amicon) according to manufacturer's directions. Protein concentration was determined as described by Bradford assay. Next, CTFs were fractionated by size exclusion chromatography using Sephacryl ® S200 (Amersham Biosciences) XK 26 column (Amersham Biosciences) equilibrated with buffer B3. A Single Path Monitor (UV-1; Amersham Biosciences) was used to monitor chromatography. Sample loads of 5 ml were isocratically eluted in buffer B3. Flow rate was gravitationally determined at ∼2 ml per min. Resolution of the mobile phase was monitored by 7–12% SDS-PAGE and staining with colloidal Coomassie. CTFs were identified using an in vitro translocation assay and an in vitro ribosylation assay in series, and correlated with elution of 100 to 250 kD sized proteins, but contained proteins as small as 20–25 kD when visualized by 7%-12% SDS-PAGE and stained with colloidal Coomassie. Partially purified CTFs were further purified by anion exchange chromatography using a column (Mono Q HR 5/5; Amersham Biosciences) on an HPLC (Biosys2000; Beckman Coulter). The column was preequilibrated with buffer B4 (containing 50 mM Tris-HCl, pH 8.0, and 1 mM EDTA). Sample loads of 2 ml were washed using buffer B4 and CTFs were eluted using serial hyperbolic step gradients, 0 to 1.0 M NaCl, in buffer B4 at a flow rate 2 ml/min. CTFs were identified using an in vitro translocation assay and an in vitro ribosylation assay in series and eluted at a conductance of 27.3 mS. Translocation in vitro-competent fractions were pooled, dialyzed against 50 mM Tris-HCl, pH 7.4, and 1% sucrose overnight at 4°C, and then concentrated using Microcon Centrifugal Filters (YM-10; Amicon) according to manufacturer's directions. Protein concentration was determined as by Bradford assay. Controls indicated that the purified CTF complex had no intrinsic ADP-ribosyltransferase activity. Purification of NLY22 − CTF complex Yeast crude cytosolic extract was isolated using the same procedure described above for HUT 102/6TG cells, except NLY22 − cells were lysed by vortexing cells with 212–300 micrometer glass beads (Sigma-Aldrich). Cell lysis was monitored by decrease in exclusion of Trypan Blue dye (GIBCO BRL). Controls indicated that the purified CTF complex had no intrinsic ADP-ribosyltransferase activity. In vitro translocation assay Translocation of the C-domain was performed using protocol modified by Lemichez et al. (1997) as follows: 25-μl reaction mixtures containing 4 μl early endosomes in translocation buffer (TB; 50 mM Tris-HCl, pH 7.4, and 25 mM EDTA). For reducing conditions, TB contained 20 mM DTT. For nonreducing conditions, TB contained 10 μM NADPH (Qbiogene). ATP and cytosol were added to 2 mM and 5.0 to 0.09 μg/μl as indicated, respectively. Translocation mixtures were incubated at 37°C for 30 min, and the supernatant fluid and pellet were separated by ultracentrifugation at 180,000 g at 4°C for 20 min. The pellet fraction was resuspended in 25 μl TB containing 0.2% Triton X-100 (Sigma-Aldrich), and both the lysed pellet and supernatant fluid were boiled for 5 min. The inhibitors geldanamycin (Alomone Labs), radicicol (Sigma-Aldrich), cis-13-retinoic acid (Sigma-Aldrich), and trans-13-retinoic acid (Sigma-Aldrich) were added as indicated. hrHsp 90 (StressGen Biotechnologies), brTrR-1 (American Diagnostica, Inc.), and hrTrx (American Diagnostica, Inc.) were added as indicated. The membrane integrity of purified early endosomes in the assay system was verified using HRP as described by Lemichez et al. (1997) . In vitro ADP-ribosylation assay The in vitro NAD + -dependent ADP-ribosylation of EF-2 was performed according to a protocol by Chung and Collier (1977) . Reaction mixtures contained 3 pM [ 32 P]-NAD + (800 μCi/mmol; Dupont-NEN Life Science Products), and when indicated 1 mM ATP and/or 0.5 mg/ml crude HUT102/6TG cytosol. Where indicated, autoradiographic signals on X-OMAT AR film (Kodak) were analyzed by ImageQuant™ software (Molecular Dynamics) and Kodak ID software (Kodak) according to manufacturer's directions. Immunoprecipitation and affinity chromatography Immunoprecipitation of human Hsp 90 (both α and β), yeast Hsp 82, and human TrR-1 were performed according to standard protocols using rabbit IgG polyclonal anti–human Hsp 90 antibodies (Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-Hsp82 antiserum (a gift from S. Lindquist, Massachusetts Institute of Technology, Cambridge, MA), and rabbit polyclonal anti–human TrR-1 antibodies (Upstate Biotechnology). Antibody was first cross-linked to Protein A Agarose (Santa Cruz Biotechnology, Inc.) before immunoprecipitation. In each instance, 2–4 μg rabbit polyclonal was incubated with 100 μl or 200 μl of resuspended volume of Protein A Agarose in 50 mM Tris-HCl and 1 mM EDTA containing 1% NP-40 and 100 mM NaCl on a rocker overnight at 4°C. Bound antibody was collected by centrifugation at 1,000 g for 5 min at 4°C, and washed 2× with 10× current volume with 0.2 M sodium borate (Sigma-Aldrich), pH 9.0, for 5 min at 25°C. Dimethyl Pimelimidate.2HCl (Sigma-Aldrich) was added to a final concentration of 20 mM, and the reaction mixture was incubated for 30 min at 25°C. Cross-linked antibody was pelleted by centrifugation at 1,000 g for 5 min at 4°C, and the pellet was washed 2× with 10× current volume 0.2 M ethanolamine (Sigma-Aldrich) for 30 min at 25°C, and 2× with PBS for 30 min at 25°C. Immunoprecipitations using the cross-linked antibody agarose conjugates were performed according to standard protocols. In brief, 200 μl of Mono Q partially purified CTFs (∼0.1 μg/μl) in 50 mM Tris-HCl and 1% sucrose, containing 1% NP-40 and 25 mM NaCl, was incubated with 20 μl of antibody-agarose conjugate on a rocker overnight at 4°C. Immunoprecipitates were collected by centrifugation at 1,000 g for 5 min at 4°C, and supernatant fluid was evaluated in the in vitro translocation assay. Pellet was washed 3× with 100 μl cold 50 mM Tris-HCl and 1 mM EDTA containing 1% NP-40 and 50 mM NaCl, and resuspended in 50 μl 1× SDS-PAGE loading buffer and boiled for 5 min. Antibody-agarose beads were pelleted by centrifugation at 1,000 g for 5 min at 25°C and the supernatant was analyzed by 10% SDS-PAGE, stained with colloidal Coomassie, and selected bands were evaluated by LC-MS/MS. Yeast TrR-1 was affinity purified using 2′,5′ ADP-Sepharose agarose (Amersham Biosciences) using a protocol modified from Hunt et al. (1983) . In brief, 20 μg of 2′,5′ ADP-Sepharose agarose was washed 2× with 200 μl 50 mM Tris-HCl and 1 mM EDTA for 20 min. Mono Q partially purified CTFs (200 μl of ∼0.1 μg/μl) in 50 mM Tris-HCl, 1 mM EDTA, 1% sucrose, and 25 mM NaCl was incubated with 2′,5′ ADP-Sepharose on a rocker overnight at 4°C. Affinity-purified TrR-1 was collected by centrifugation at 1,000 g for 5 min at 4°C. The supernatant fluid was assayed for translocation activity in vitro. The pellet was washed 2× in 100 μl 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 1% sucrose, and was then resuspended in 50 μl 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 1% sucrose containing 20 μM NADPH and incubated for 2 h at 25°C. The supernatant fluid was collected after centrifugation at 1,000 g for 5 min at 4°C, and the supernatant fluid was analyzed by 10% SDS-PAGE, stained with colloidal Coomassie, and selected bands were evaluated by LC-MS/MS. Western blots Confirmation of CTF identification by MS was performed by Western blot analysis according to standard protocols. In addition to using antibodies (see Immunoprecipitation and affinity chromatography), horse polyclonal anti-DT antibody (Massachusetts Antitoxin and Vaccine Laboratories) was used. In brief, samples were analyzed by 7–12% SDS-PAGE, transferred to Immobilon-P (Millipore), probed with the appropriate primary and secondary antibodies, and detected using either 3,3′-DAB (Sigma-Aldrich) or ECL (Amersham Biosciences) according to the manufacturer's directions. In-gel reduction, alkylation, and digestion of partially purified CTFs The preparation of partially purified CTFs for identification by MS was performed using a modified procedure from Shevchenko et al. (1996) . In brief, partially purified CTFs were separated by 10% SDS-PAGE, stained with colloidal Coomassie, and selected bands were excised and chopped into small pieces. Gel pieces were washed 3× in 50 mM ammonium bicarbonate (Sigma-Aldrich) in 50% acetonitrile (ACN; Acros) for 20 min at 25°C. Gel pieces were washed with 100% ACN for 10 min at 25°C. Supernatant was discarded, and the gel pieces were dried in a SpeedVac ® for 15 min. Gel pieces were reduced in 20 mM DTT, 50 mM ammonium bicarbonate, and 5% ACN for 1 h at 55°C. Supernatant was discarded and the pieces were washed with 100 μl 50 mM ammonium bicarbonate for 10 min at 25°C and subsequently with 100 μl 100% ACN for 10 min at 25°C. Gel pieces were alkylated in 100 μl 100 mM iodoacetamide (ICN Biomedicals) and 50 mM ammonium bicarbonate for 30 min in the dark at 25°C. Supernatant was discarded and the pieces were washed with 100 μl 50 mM ammonium bicarbonate for 10 min at 25°C and subsequently dried with 100 μl 100% ACN for 10 min at 25°C. The washing and drying steps were repeated before drying the pieces in a SpeedVac ® for 15 min. Gel pieces were rehydrated in digestion buffer (50 mM ammonium bicarbonate) and MS Sequencing Grade Trypsin (Roche) at an estimated 1:100 enzyme to substrate ratio on ice for 45 min. 50 mM Ammonium bicarbonate was added when necessary to keep the gel pieces wet. Digestions were incubated for 6–8 h at 37°C. Peptides were extracted from the gel pieces using 100 μl 20 mM ammonium bicarbonate for 20 min, followed by 2× 200 μl 1% TFA in 50% ACN for 20 min, and finally 1× 100 μl 100% ACN for 10 min. Supernatant fluids were pooled and dried in a SpeedVac ® . The pellets were resuspended in 0.1% TFA and desalted using ZipTip ® C18 pipette tips (Millipore) according to manufacturer's directions. Capillary HPLC of tryptic peptides HPLC was performed using a capillary LC system (LC Packings; Dionex Corp.) composed of a Famous autosampler, a Switchos microcolumn switching unit and an Ultimate pump. Sample loads of 5 μl were preconcentrated and desalted online with a "small molecule" C 18 CapTrap™ (Michrom BioResources) using a solution of 5% FA and 0.1% TFA at a flow rate of 50 μl per min for 4 min. Capillary HPLC columns were prepared in house as follows: 300 μm ID × 15-cm fused silica capillaries were pressure bomb–packed (Mass Evolution, Inc.) at 2,000 PSI with Magic C 18 3-μm 200 à pore-reversed phase-packing material (Michrom BioResources) using 2-propanol as a carrier solvent. Columns were washed with 10% acetic acid, followed by methanol, then the HPLC mobile phase before use at a flow rate of 2 μl per min. Elution was by linear gradient; 95% A (5% ACN, 0.1% formic acid) to 55% B (85% ACN, 10% 2-propanol, 0.1% formic acid) over 50 min followed by 60 min of column regeneration. MALDI and ESI MS, tandem MS, and LC-MS/MS MALDI MS was acquired in positive polarity on a mass spectrometer (Reflex IV; Bruker) with delayed extraction in the reflectron mode using a UV nitrogen laser. A laser power of 28–45% was used, and 50–100 laser shots were summed for each spectrum. The matrix used was 2,5-dihydroxybenzoic acid (Sigma-Aldrich). Data were analyzed using BioAnalyst™ (Applied Biosystems) reconstruction algorithms. For initial screening and searches, acquired mass values were compared with theoretical protein digests using the Mascot search engine (Matrix Science, Ltd.). Reported scores, based on a probability of match, were statistically significant for each protein identified in Table I . ESI MS and MS/MS were performed using an ESI quadrupole/orthogonal acceleration time-of-flight mass spectrometer (QSTARi ® Pulsar; Applied Biosystems). MS and MS/MS were acquired in the positive polarity mode over the range of m/z 320–1800 (MS) and m/z 100–1800 (MS/MS) with resolution >1:9,000 (full width half maximum) and better than 50 ppm mass accuracy (external calibration). For nanospray, a Protana source was used using uncoated glass nanospray tips pulled in house to 1 μm ID using a capillary puller (Sutter Instrument Co.) ESI was initiated at ∼1,200 V via a Pt wire inserted into the glass tip. Tandem mass spectra were acquired using Ar as the collision gas and sufficient collision energy to obtain complete sequence information of the precursor. Pulsed ion enhancement of product ions was used for MS/MS of low S/N precursors. For LC-MS, the LC was coupled to the mass spectrometer using 50 μm ID distal coated nanospray tips pulled to 15 μm ID, 75 μm OD at the tip (New Objectives Inc.). ESI was performed at 4,500 V. Information-dependent acquisition was used to obtain MS/MS spectra of peaks during elution from the LC system. MS peaks that exceeded a threshold of 10 counts/s were subjected to MS/MS using preset collision energies proportional to the m/z value of the precursor (∼18–60 V, lab frame). Pulsed ion enhancement was used for all LC-MS/MS spectra. Cytotoxicity assays Cytotoxicity assays for the fusion protein toxins were performed essentially as described by vander Spek et al. (1994) . Cytotoxicity assays to evaluate the affects of geldanamycin, radicicol, and retinoic acid on DAB 389 IL-2 intoxication were modified from vander Spek et al. (1994) as such: cells were seeded at 5 × 10 4 cells per well and preincubated with inhibitors geldanamycin, radicicol, cis-13-retinoic acid, for 30 min at 37°C, 5% CO 2 and subsequently incubated with varying concentrations of DAB 389 IL-2 and inhibitor for 15 min at 37°C, 5% CO 2 . Cells were pelleted and washed free of toxin with media containing inhibitor and incubated for 8–12 h at 37°C, 5% CO 2 . Cells were then washed and pulsed with minimal media (leucine depleted; BioWhittaker) containing [ 14 C]leucine (NEN Life Science Products) for 2 h at 37°C, 5% CO 2 , and protein synthesis was analyzed according to vander Spek et al. (1994) . Media alone and media plus inhibitor alone served as controls. Assays were performed in quadruplicate. Online supplemental material Table of peptide coverage from LC-MS/MS is available as online supplemental material at http://www.jcb.org/cgi/content/full/jcb.200210028/DC1 . Cell culture HUT1026TG cells (TIB 1620; American Type Culture Collection), were maintained in RPMI 1640 (BioWhittaker) supplemented with 10% FBS (HyClone), 2 mM glutamine (BioWhittaker), 50 IU/ml penicillin, and 50 μg/ml streptomycin (BioWhittaker) at 37°C in 5% CO 2 . Yeast strain NLY22 − (a gift from Dr. Kevin Jarrell, Modular Genetics Inc., Lincoln, MA) was maintained in YPD media (Difco ® ) and on YPD agar plates at 30°C. Purification of EF-2 EF-2 was partially purified using a procedure by Chung and Collier (1977) . After purification, fractions containing EF-2 were identified by ADP-ribosyltransferase using DAB 389 IL-2 (see In vitro ribosylation assay). EF-2 was further purified by DEAE-Sepharose (Reactifs IBF) anion exchange chromatography. EF-2 was eluted with a linear gradient, 0–200 mM NaCl, in 50 mM Tris-HCl, pH 8.0, 50 mM Mg(OAc) 2 , 0.1 M KCl, 4 mM CaCl 2 , 5 mM 2-ME and 1 μg PMSF (Sigma-Aldrich) per ml. Fractions containing EF-2 were identified as above. Aliquots were adjusted to a final concentration of 2 mM DTT, 5% glycerol, and stored at −70°C. Purified EF-2 was ∼80% homogeneous as resolved by 7% SDS-PAGE and stained with colloidal Coomassie (Invitrogen). Protein concentration was determined by Bradford Assay according to standard protocols using Coomassie Protein Assay Reagent (Pierce Chemical Co.). Purification of early endosomes Early endosomes were isolated from HUT102/6TG cells according to a protocol by Duprez and Dautry-Varsat (1986) . The early endosomal compartment was loaded with 1 μM DAB 389 IL-2, 1 μM DA 189(VSV-G) B 389 IL-2, 8 mg/ml 70-kD SNARF1-dextran conjugate (Molecular Probes, Inc.), and/or 5 mg/ml HRP (Sigma-Aldrich) using 1 μM bafilomycin A1–primed cells (Sigma-Aldrich). Purification of HUT102/6TG CTF complex Crude cytosolic extract was isolated from HUT 102/6TG cells according to the protocol modified from Bomsel et al. (1990) . In brief, cells were washed three times with cold PBS containing 5 mg/ml BSA, once with cold PBS alone, and twice with cold cytosol buffer (CB; 3% sucrose in 100 mM Hepes-KOH, pH 7.9, 1.4 M KCl, 30 mM MgCl 2 , 2 mM EDTA, and 5 mM DTT). Cells were lysed by 20 passages through a 25 G needle in CB containing protease inhibitors as follows: 10 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml antipain, and 1 μm PMSF (all obtained from Sigma-Aldrich) The lysate was centrifuged at 1,000 g for 15 min at 4°C. The post-nuclear supernatant was then centrifuged at 170,000 g for 1 h at 4°C. The supernatant fraction was dialyzed overnight at 4°C against cytosol dialysis buffer (CDB; 1% sucrose in 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, and 2 mM 2-ME) containing protease inhibitors as described in CB. Crude cytosol was fractionated according to standard chromatographic protocols. In brief, crude extract was loaded onto an in-house packed DEAE-Sepharose (Reactifs IBF) XK 26 column (Amersham Biosciences) for anion exchange chromatography. A peristaltic FPLC pump (P-1; Amersham Biosciences) and Single Path Monitor (UV-1; Amersham Biosciences) were used during chromatography. The column was preequilibrated with buffer B3 (containing 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 5 mM 2-mercaptoethanol, and 1 μg PMSF per ml), and "loaded" sample was washed using the same buffer. CTFs were eluted with a linear gradient, 0–400 mM NaCl, in buffer B3 at a flow rate of 5 ml/min. Fractions containing CTFs were identified using an in vitro translocation assay and in vitro ribosylation assay in series (see Materials and methods). Fractions containing in vitro translocation activity eluted between 150 to 190 mM NaCl, and were pooled and concentrated using Centriplus Centrifugal Filters (YM-10; Amicon) according to manufacturer's directions. Protein concentration was determined as described by Bradford assay. Next, CTFs were fractionated by size exclusion chromatography using Sephacryl ® S200 (Amersham Biosciences) XK 26 column (Amersham Biosciences) equilibrated with buffer B3. A Single Path Monitor (UV-1; Amersham Biosciences) was used to monitor chromatography. Sample loads of 5 ml were isocratically eluted in buffer B3. Flow rate was gravitationally determined at ∼2 ml per min. Resolution of the mobile phase was monitored by 7–12% SDS-PAGE and staining with colloidal Coomassie. CTFs were identified using an in vitro translocation assay and an in vitro ribosylation assay in series, and correlated with elution of 100 to 250 kD sized proteins, but contained proteins as small as 20–25 kD when visualized by 7%-12% SDS-PAGE and stained with colloidal Coomassie. Partially purified CTFs were further purified by anion exchange chromatography using a column (Mono Q HR 5/5; Amersham Biosciences) on an HPLC (Biosys2000; Beckman Coulter). The column was preequilibrated with buffer B4 (containing 50 mM Tris-HCl, pH 8.0, and 1 mM EDTA). Sample loads of 2 ml were washed using buffer B4 and CTFs were eluted using serial hyperbolic step gradients, 0 to 1.0 M NaCl, in buffer B4 at a flow rate 2 ml/min. CTFs were identified using an in vitro translocation assay and an in vitro ribosylation assay in series and eluted at a conductance of 27.3 mS. Translocation in vitro-competent fractions were pooled, dialyzed against 50 mM Tris-HCl, pH 7.4, and 1% sucrose overnight at 4°C, and then concentrated using Microcon Centrifugal Filters (YM-10; Amicon) according to manufacturer's directions. Protein concentration was determined as by Bradford assay. Controls indicated that the purified CTF complex had no intrinsic ADP-ribosyltransferase activity. Purification of NLY22 − CTF complex Yeast crude cytosolic extract was isolated using the same procedure described above for HUT 102/6TG cells, except NLY22 − cells were lysed by vortexing cells with 212–300 micrometer glass beads (Sigma-Aldrich). Cell lysis was monitored by decrease in exclusion of Trypan Blue dye (GIBCO BRL). Controls indicated that the purified CTF complex had no intrinsic ADP-ribosyltransferase activity. In vitro translocation assay Translocation of the C-domain was performed using protocol modified by Lemichez et al. (1997) as follows: 25-μl reaction mixtures containing 4 μl early endosomes in translocation buffer (TB; 50 mM Tris-HCl, pH 7.4, and 25 mM EDTA). For reducing conditions, TB contained 20 mM DTT. For nonreducing conditions, TB contained 10 μM NADPH (Qbiogene). ATP and cytosol were added to 2 mM and 5.0 to 0.09 μg/μl as indicated, respectively. Translocation mixtures were incubated at 37°C for 30 min, and the supernatant fluid and pellet were separated by ultracentrifugation at 180,000 g at 4°C for 20 min. The pellet fraction was resuspended in 25 μl TB containing 0.2% Triton X-100 (Sigma-Aldrich), and both the lysed pellet and supernatant fluid were boiled for 5 min. The inhibitors geldanamycin (Alomone Labs), radicicol (Sigma-Aldrich), cis-13-retinoic acid (Sigma-Aldrich), and trans-13-retinoic acid (Sigma-Aldrich) were added as indicated. hrHsp 90 (StressGen Biotechnologies), brTrR-1 (American Diagnostica, Inc.), and hrTrx (American Diagnostica, Inc.) were added as indicated. The membrane integrity of purified early endosomes in the assay system was verified using HRP as described by Lemichez et al. (1997) . In vitro ADP-ribosylation assay The in vitro NAD + -dependent ADP-ribosylation of EF-2 was performed according to a protocol by Chung and Collier (1977) . Reaction mixtures contained 3 pM [ 32 P]-NAD + (800 μCi/mmol; Dupont-NEN Life Science Products), and when indicated 1 mM ATP and/or 0.5 mg/ml crude HUT102/6TG cytosol. Where indicated, autoradiographic signals on X-OMAT AR film (Kodak) were analyzed by ImageQuant™ software (Molecular Dynamics) and Kodak ID software (Kodak) according to manufacturer's directions. Immunoprecipitation and affinity chromatography Immunoprecipitation of human Hsp 90 (both α and β), yeast Hsp 82, and human TrR-1 were performed according to standard protocols using rabbit IgG polyclonal anti–human Hsp 90 antibodies (Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-Hsp82 antiserum (a gift from S. Lindquist, Massachusetts Institute of Technology, Cambridge, MA), and rabbit polyclonal anti–human TrR-1 antibodies (Upstate Biotechnology). Antibody was first cross-linked to Protein A Agarose (Santa Cruz Biotechnology, Inc.) before immunoprecipitation. In each instance, 2–4 μg rabbit polyclonal was incubated with 100 μl or 200 μl of resuspended volume of Protein A Agarose in 50 mM Tris-HCl and 1 mM EDTA containing 1% NP-40 and 100 mM NaCl on a rocker overnight at 4°C. Bound antibody was collected by centrifugation at 1,000 g for 5 min at 4°C, and washed 2× with 10× current volume with 0.2 M sodium borate (Sigma-Aldrich), pH 9.0, for 5 min at 25°C. Dimethyl Pimelimidate.2HCl (Sigma-Aldrich) was added to a final concentration of 20 mM, and the reaction mixture was incubated for 30 min at 25°C. Cross-linked antibody was pelleted by centrifugation at 1,000 g for 5 min at 4°C, and the pellet was washed 2× with 10× current volume 0.2 M ethanolamine (Sigma-Aldrich) for 30 min at 25°C, and 2× with PBS for 30 min at 25°C. Immunoprecipitations using the cross-linked antibody agarose conjugates were performed according to standard protocols. In brief, 200 μl of Mono Q partially purified CTFs (∼0.1 μg/μl) in 50 mM Tris-HCl and 1% sucrose, containing 1% NP-40 and 25 mM NaCl, was incubated with 20 μl of antibody-agarose conjugate on a rocker overnight at 4°C. Immunoprecipitates were collected by centrifugation at 1,000 g for 5 min at 4°C, and supernatant fluid was evaluated in the in vitro translocation assay. Pellet was washed 3× with 100 μl cold 50 mM Tris-HCl and 1 mM EDTA containing 1% NP-40 and 50 mM NaCl, and resuspended in 50 μl 1× SDS-PAGE loading buffer and boiled for 5 min. Antibody-agarose beads were pelleted by centrifugation at 1,000 g for 5 min at 25°C and the supernatant was analyzed by 10% SDS-PAGE, stained with colloidal Coomassie, and selected bands were evaluated by LC-MS/MS. Yeast TrR-1 was affinity purified using 2′,5′ ADP-Sepharose agarose (Amersham Biosciences) using a protocol modified from Hunt et al. (1983) . In brief, 20 μg of 2′,5′ ADP-Sepharose agarose was washed 2× with 200 μl 50 mM Tris-HCl and 1 mM EDTA for 20 min. Mono Q partially purified CTFs (200 μl of ∼0.1 μg/μl) in 50 mM Tris-HCl, 1 mM EDTA, 1% sucrose, and 25 mM NaCl was incubated with 2′,5′ ADP-Sepharose on a rocker overnight at 4°C. Affinity-purified TrR-1 was collected by centrifugation at 1,000 g for 5 min at 4°C. The supernatant fluid was assayed for translocation activity in vitro. The pellet was washed 2× in 100 μl 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 1% sucrose, and was then resuspended in 50 μl 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 1% sucrose containing 20 μM NADPH and incubated for 2 h at 25°C. The supernatant fluid was collected after centrifugation at 1,000 g for 5 min at 4°C, and the supernatant fluid was analyzed by 10% SDS-PAGE, stained with colloidal Coomassie, and selected bands were evaluated by LC-MS/MS. Western blots Confirmation of CTF identification by MS was performed by Western blot analysis according to standard protocols. In addition to using antibodies (see Immunoprecipitation and affinity chromatography), horse polyclonal anti-DT antibody (Massachusetts Antitoxin and Vaccine Laboratories) was used. In brief, samples were analyzed by 7–12% SDS-PAGE, transferred to Immobilon-P (Millipore), probed with the appropriate primary and secondary antibodies, and detected using either 3,3′-DAB (Sigma-Aldrich) or ECL (Amersham Biosciences) according to the manufacturer's directions. In-gel reduction, alkylation, and digestion of partially purified CTFs The preparation of partially purified CTFs for identification by MS was performed using a modified procedure from Shevchenko et al. (1996) . In brief, partially purified CTFs were separated by 10% SDS-PAGE, stained with colloidal Coomassie, and selected bands were excised and chopped into small pieces. Gel pieces were washed 3× in 50 mM ammonium bicarbonate (Sigma-Aldrich) in 50% acetonitrile (ACN; Acros) for 20 min at 25°C. Gel pieces were washed with 100% ACN for 10 min at 25°C. Supernatant was discarded, and the gel pieces were dried in a SpeedVac ® for 15 min. Gel pieces were reduced in 20 mM DTT, 50 mM ammonium bicarbonate, and 5% ACN for 1 h at 55°C. Supernatant was discarded and the pieces were washed with 100 μl 50 mM ammonium bicarbonate for 10 min at 25°C and subsequently with 100 μl 100% ACN for 10 min at 25°C. Gel pieces were alkylated in 100 μl 100 mM iodoacetamide (ICN Biomedicals) and 50 mM ammonium bicarbonate for 30 min in the dark at 25°C. Supernatant was discarded and the pieces were washed with 100 μl 50 mM ammonium bicarbonate for 10 min at 25°C and subsequently dried with 100 μl 100% ACN for 10 min at 25°C. The washing and drying steps were repeated before drying the pieces in a SpeedVac ® for 15 min. Gel pieces were rehydrated in digestion buffer (50 mM ammonium bicarbonate) and MS Sequencing Grade Trypsin (Roche) at an estimated 1:100 enzyme to substrate ratio on ice for 45 min. 50 mM Ammonium bicarbonate was added when necessary to keep the gel pieces wet. Digestions were incubated for 6–8 h at 37°C. Peptides were extracted from the gel pieces using 100 μl 20 mM ammonium bicarbonate for 20 min, followed by 2× 200 μl 1% TFA in 50% ACN for 20 min, and finally 1× 100 μl 100% ACN for 10 min. Supernatant fluids were pooled and dried in a SpeedVac ® . The pellets were resuspended in 0.1% TFA and desalted using ZipTip ® C18 pipette tips (Millipore) according to manufacturer's directions. Capillary HPLC of tryptic peptides HPLC was performed using a capillary LC system (LC Packings; Dionex Corp.) composed of a Famous autosampler, a Switchos microcolumn switching unit and an Ultimate pump. Sample loads of 5 μl were preconcentrated and desalted online with a "small molecule" C 18 CapTrap™ (Michrom BioResources) using a solution of 5% FA and 0.1% TFA at a flow rate of 50 μl per min for 4 min. Capillary HPLC columns were prepared in house as follows: 300 μm ID × 15-cm fused silica capillaries were pressure bomb–packed (Mass Evolution, Inc.) at 2,000 PSI with Magic C 18 3-μm 200 à pore-reversed phase-packing material (Michrom BioResources) using 2-propanol as a carrier solvent. Columns were washed with 10% acetic acid, followed by methanol, then the HPLC mobile phase before use at a flow rate of 2 μl per min. Elution was by linear gradient; 95% A (5% ACN, 0.1% formic acid) to 55% B (85% ACN, 10% 2-propanol, 0.1% formic acid) over 50 min followed by 60 min of column regeneration. MALDI and ESI MS, tandem MS, and LC-MS/MS MALDI MS was acquired in positive polarity on a mass spectrometer (Reflex IV; Bruker) with delayed extraction in the reflectron mode using a UV nitrogen laser. A laser power of 28–45% was used, and 50–100 laser shots were summed for each spectrum. The matrix used was 2,5-dihydroxybenzoic acid (Sigma-Aldrich). Data were analyzed using BioAnalyst™ (Applied Biosystems) reconstruction algorithms. For initial screening and searches, acquired mass values were compared with theoretical protein digests using the Mascot search engine (Matrix Science, Ltd.). Reported scores, based on a probability of match, were statistically significant for each protein identified in Table I . ESI MS and MS/MS were performed using an ESI quadrupole/orthogonal acceleration time-of-flight mass spectrometer (QSTARi ® Pulsar; Applied Biosystems). MS and MS/MS were acquired in the positive polarity mode over the range of m/z 320–1800 (MS) and m/z 100–1800 (MS/MS) with resolution >1:9,000 (full width half maximum) and better than 50 ppm mass accuracy (external calibration). For nanospray, a Protana source was used using uncoated glass nanospray tips pulled in house to 1 μm ID using a capillary puller (Sutter Instrument Co.) ESI was initiated at ∼1,200 V via a Pt wire inserted into the glass tip. Tandem mass spectra were acquired using Ar as the collision gas and sufficient collision energy to obtain complete sequence information of the precursor. Pulsed ion enhancement of product ions was used for MS/MS of low S/N precursors. For LC-MS, the LC was coupled to the mass spectrometer using 50 μm ID distal coated nanospray tips pulled to 15 μm ID, 75 μm OD at the tip (New Objectives Inc.). ESI was performed at 4,500 V. Information-dependent acquisition was used to obtain MS/MS spectra of peaks during elution from the LC system. MS peaks that exceeded a threshold of 10 counts/s were subjected to MS/MS using preset collision energies proportional to the m/z value of the precursor (∼18–60 V, lab frame). Pulsed ion enhancement was used for all LC-MS/MS spectra. Cytotoxicity assays Cytotoxicity assays for the fusion protein toxins were performed essentially as described by vander Spek et al. (1994) . Cytotoxicity assays to evaluate the affects of geldanamycin, radicicol, and retinoic acid on DAB 389 IL-2 intoxication were modified from vander Spek et al. (1994) as such: cells were seeded at 5 × 10 4 cells per well and preincubated with inhibitors geldanamycin, radicicol, cis-13-retinoic acid, for 30 min at 37°C, 5% CO 2 and subsequently incubated with varying concentrations of DAB 389 IL-2 and inhibitor for 15 min at 37°C, 5% CO 2 . Cells were pelleted and washed free of toxin with media containing inhibitor and incubated for 8–12 h at 37°C, 5% CO 2 . Cells were then washed and pulsed with minimal media (leucine depleted; BioWhittaker) containing [ 14 C]leucine (NEN Life Science Products) for 2 h at 37°C, 5% CO 2 , and protein synthesis was analyzed according to vander Spek et al. (1994) . Media alone and media plus inhibitor alone served as controls. Assays were performed in quadruplicate. Online supplemental material Table of peptide coverage from LC-MS/MS is available as online supplemental material at http://www.jcb.org/cgi/content/full/jcb.200210028/DC1 . Supplemental Material [Supplemental Material Index]

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6711143/

The prevalence and impact of lysogeny among oral isolates of Enterococcus faecalis

ABSTRACT Bacterial phenotypic properties are frequently influenced by the uptake of extrachromosomal genetic elements, such as plasmids and bacteriophage genomes. Such modifications can result in enhanced pathogenicity due to toxin production, increased toxin release, altered antigenicity, and resistance to antibiotics. In the case of bacteriophages, the phage genome can stably integrate into the bacterial chromosome as a prophage, to produce a lysogenic cell. Oral enterococcal strains have been isolated from subgingival plaque and the root canals of endodontically-treated teeth that have failed to heal. Previously, we isolated a bacteriophage, phage ɸEf11, induced from a lysogenic Enterococcus faecalis strain recovered from the root canal of a failed endodontic case. PCR analysis using phage ɸEf11-specific oligonucleotide primers, disclosed that lysogens containing ɸEf11 prophages were commonly found among oral E. faecalis strains, being detected in 19 of 61 (31%) strains examined. Furthermore, in comparison to an isogenic cured strain, cultures of a lysogen harboring an ɸEf11 prophage exhibited altered phenotypic characteristics, such as increased persistence at high density, enhanced biofilm formation, and resistance to a bacteriophage lytic enzyme. From these results we conclude that lysogeny is common among oral E. faecalis strains, and that it alters properties of the lysogenic cell. Methods and materials Microorganisms The source and relevant characteristics of the 61 oral E. faecalis strains used in this study are listed in Table 1 . All strains were grown in Brain Heart Infusion Broth @ at 37°C in stationary cultures. In addition, we included a panel of unrelated bacterial species to test the specificity of the primers that we used to detect the ɸEf11 sequence. These included: Streptococcus mutans, S. sanguis, Staphylococcus aureus, Finegoldia (Peptostreptococcus) magna (magnus), Clostridium perfringens, Actinomyces israelii , and Eggerthella (Eubacterium) lenta (lentum) . 10.1080/20002297.2019.1643207-T0001 Table 1. E. faecalis strains used and presence/absence of ɸEf11 ORF43. Strain Source Antibiotic status ORF 43 Present Reference or Origin E. faecalis TUSoD1 Root canal Em (r) , TC r a E. faecalis TUSoD2 Root canal Em (r) + a E. faecalis TUSoD3 Root canal Em (r) + a E. faecalis TUSoD9 Root canal Em (r) ,TC r + a E. faecalis TUSoD10 Root canal Em (r) a E. faecalis TUSoD11 Root canal Em (r) ,TC r + a E. faecalis TUSoD12 Root canal TC r + a E. faecalis TUSoD15 Root canal TC r + a E. faecalis TUSoD17 Root canal a E. faecalis TUSoD18 Root canal a E. faecalis GS1 Root canal b E. faecalis GS2 Root canal + b E. faecalis GS3 Root canal b E. faecalis GS4 Root canal b E. faecalis GS6 Root canal b E. faecalis GS7 Root canal b E. faecalis GS8 Root canal TC r + b E. faecalis GS9 Root canal TC r + b E. faecalis GS10 Root canal b E. faecalis GS12 Root canal b E. faecalis GS13 Root canal b E. faecalis GS14 Root canal b E. faecalis GS15 Root canal b E. faecalis GS16 Root canal b E. faecalis GS17 Root canal b E. faecalis GS18 Root canal b E. faecalis GS19 Root canal b E. faecalis GS21 Root canal b E. faecalis GS22 Root canal + b E. faecalis GS23 Root canal + b E. faecalis GS24 Root canal TC r b E. faecalis GS25 Root canal + b E. faecalis GS26 Root canal b E. faecalis GS27 Root canal b E. faecalis GS28 Root canal b E. faecalis GS29 Root canal + b E. faecalis GS30 Root canal TC r b E. faecalis GS31 Root canal TC r b E. faecalis GS32 Root canal b E. faecalis GS33 Root canal + b E. faecalis GS34 Tongue c E. faecalis AA-OR3 Oral Cl (r) , Cm r d E. faecalis AA-OR4 Oral Cm (r) , TC r d E. faecalis AA-OR26 Oral Cl (r) , Cm r , Em r , Gm r , TC r d E. faecalis AA-OR34 Oral Cl (r) , Cm (r) + d E. faecalis AA-T4 tongue Cm (r) , TC r d E. faecalis AA-T26 Tongue Cm r , Em r , Gm r , TC r d E. faecalis OS16 Oral e E. faecalis OS25 Tongue e E. faecalis E1 Oral isolate Cl r ,Cm r , Em r , TC (r) f E. faecalis E2 Oral isolate Amp (r) , Cl r , Cm r , Em (r), TC r + f E. faecalis E3 Oral isolate Cl r , Em (r) + f E. faecalis E4 Oral isolate Cl r , Em (r), Tc r + f E. faecalis E5 Oral isolate Cl r + f E. faecalis E6 Oral isolate Cl r f E. faecalis E7 Oral isolate f E. faecalis E8 Oral isolate Cl r , Tc r f E. faecalis E10 Oral isolate Amp (r) , Cl r , Tc r f E. faecalis E11 Oral isolate Cl r f E. faecalis ER3/25 Root canal c E. faecalis ER5/1 Root canal c r = resistant, (r) = intermediate resistance Amp = ampicillin, Cm = chloramphenicol, Cl = clindamycin, Em = erythromycin, Gm = gentamicin, Tc = tetracycline a-Stevens et al. 2009 [ 12 ], b-Sedgley et al. 2005a [ 11 ], c-Johnson et al. 2006 [ 79 ], d-Sedgley et al. 2006 [ 80 ], e-Sedgley et al. 2005b [ 81 ], f-Sedgley et al. 2004 [ 4 ]. Primers and PCR conditions Our previous sequencing of the genome of ɸEf11, a bacteriophage induced and isolated from an E. faecalis strain (TUSoD11) recovered from an infected root canal, demonstrated a genome of 42,822 bp distributed among 65 open reading frames (ORFs) [ 13 ]. Within that genome, ORF43 was designated as coding for a 'hypothetical protein'. Our search of several databases failed to disclose any genes homologous to ɸEf11 ORF43. The sequence uniqueness of this target gene provided specificity of PCR amplicons produced by ORF43-specific primers, for the presence of the ɸEf11 DNA. ɸEf11 ORF43-specific oligonucleotide primers [forward (ɸEf11 F) and reverse (ɸEf11 R)] were designed which were predicted to produce a 165 bp amplicon in PCRs with phage ɸEf11 DNA templates ( Table 2 , Figure 1 ). Template DNA was prepared by suspending cells of each strain in lysis buffer [1% (v/v) Triton X-100, 20 mM Tris-HCl (pH 8.5), 2 mM EDTA], heating to 100°C for 10 min., and then recovering the released DNA in the supernatant following centrifugation. PCR mixtures contained: 5 μl (= 5 nmol) each of forward (ɸEf11 F) and reverse (ɸEf11 R) primer, 5 μl of DNA template solution (≈ 85ng DNA), 20 μl 2 x GoTaq PCR master mix (Promega), and 5 μl dH 2 O. In addition to the ORF43-specific forward and reverse oligonucleotide primers, an E. faecalis species-specific primer set (forward: 1F, reverse: 1R), and a universal primer set (forward: RRN4, reverse: RRN5) were used in control PCRs ( Table 2 ). The E. faecalis -specific primers [ 14 ] were used as positive controls in PCRs for all the E. feacalis strains tested. Similarly, the universal primer set, which recognizes two highly conserved regions of eubacterial 16S rRNA genes [ 15 ], was used as an internal positive control in PCRs involving DNA templates from non-enterococcal bacterial species. Additional (control) PCRs were prepared using E. faecalis -specific (1F and 1R) or universal primers (RRN4 and RRN5) instead of the phage ɸEf11 ORF43-specific primers. PCR conditions for reactions containing the ɸEf11 ORF43-specific primers (ɸEf11F and ɸEf11R) and E. faecalis -specific primers (1F and 1R) were: 97°C for 1min, followed by 26 cycles of (i) 94°C for 1 min, (ii) 50°C for 45 sec, and (iii) 72°C for 1 min. This was followed by an additional 4 min at 72°C. For PCRs utilizing the universal primers (RRN4 and RRN5), the reaction conditions were: 97°C for 1 min, followed by 25 cycles of (i) 95°C for 30 sec, (ii) 55°C for 30 sec and (iii) 72°C for 30 sec, followed by an additional 4 min at 72°C. Following PCR, amplification products were detected by agarose [2%(w/v)] gel electrophoresis and ethidium bromide staining. 10.1080/20002297.2019.1643207-T0002 Table 2. Primer sets used for PCR amplification. Primer Set Sequence Purpose Predicted Amplicon Size Reference ɸEf11 F ɸEf11 R 5ʹ-GAGAGTGGAAGTGGATTCAATG-3ʹ 5ʹ-GCACTTTCATCTAAACTCTCG-3' Amplification of ɸEf11 ORF 43 165 bp This study 1F 1R 5ʹ-GTTTATGCCGCATGGCATAAGAG-3ʹ 5ʹ-CCGTCAGGGGACGTTCAG-3' E. faecalis -specific primers 310 bp a RRN4 RRN5 5ʹ-CAGGATTAGATACCCTGGTAGTCCACGC-3ʹ 5ʹ-GACGGGCGGTGTGTACAAGGCCCGGGAACG-3' Universal (16S rDNA) primers 625 bp b a-Siqueira et al. 2004 [ 14 ], b-Goncharoff et al. 1993 [ 15 ] 10.1080/20002297.2019.1643207-F0001 Figure 1. Phage ɸEf11 ORF43 sequence (237 bp). Primer binding sites are underlined. The 165 bp PCR amplicon product is shown in bold type. Generation of a cured derivative strain of lysogenic E. faecalis strain TUSoD11 In a previous communication we reported our generation and isolation of a cured E. faecalis strain [ 16 ]. Briefly, allelic exchange mutagenesis was employed to delete a module of six lysogeny-related genes and insert a selectable antibiotic resistance gene (erythromycin) into the ɸEf11 prophage of lysogenic E. faecalis TUSoD11. PCR screening of the recombinant transformant clones selected on erythromycin plates confirmed the absence of the targeted prophage genes in the cells of the recovered colonies. Surprisingly, in addition to the deletion of the six genes of the targeted lysogeny gene module, the cells of a few of the recovered colonies also lacked any other of the ɸEf11 prophage genes, for which they were screened, as well. Because the phage ɸEf11 genome is circularly permuted, deletion of the entire prophage from the TUSoD11 chromosome could have occurred through homologous recombination between the gene exchange vector that was used and homologous regions that could be positioned at either end of the ɸEf11 prophage. PCR screening was conducted using ɸEf11 prophage-specific primers and template DNA from presumptive recombinant clones selected on the antibiotic (erythromycin) plates. Those clones, no longer possessing any detectable ɸEf11 prophage genes, were considered cured of the prophage, and designated E. faecalis TUSoD11(ΔɸEf11). By this process we have obtained the isogenic pair of lysogenic and non-lysogenic E. faecalis strains [TUSoD11 and TUSoD11(ΔɸEf11)], differing only in the presence or absence of the ɸEf11 prophage. Growth rate assay Cultures of lysogenic E. faecalis TUSoD11 and its cured isogenic derivative TUSoD11(ΔɸEf11) were grown overnight at 37°C in BHI broth. Portions of each culture were inoculated into fresh BHI broth to produce suspensions having an OD 600 of 0.1. Samples of each suspension were placed into wells of a flat-bottomed 96 well microtiter plate (200 μl/well). Control wells contained uninoculated BHI broth. The plate was loaded into a microplate reader (Synergy HT), and incubated at 37°C for 24 h, during which the OD 600 of each well was measured. The result of triplicate assays was recorded. Biofilm assay Biofilms were established and assayed colorimetrically as described by Knezevic and Petrovic [ 17 ]. Cultures of E. faecalis TUSoD11 and TUSoD11(ΔɸEf11) were grown overnight at 37°C in modified LB broth (0.5% yeast extract, 1.0% Tryptone, 1.0% NaCl, 0.5% glucose). Each culture was diluted to OD 600 of 0.1 using modified LB broth. Samples of 200 μl of each culture were inoculated into the wells of a flat bottomed 96 well microtiter plate. After incubation at 37°C for 48 h, the medium and planktonic cells in each well were removed and the wells were washed twice with PBS (0.072% Na 2 PO 4 , 0.021% KH 2 PO 4 , 0.765% NaCl, pH 7.2). The attached cells in each well were left to air dry, and then fixed by incubation in absolute methanol (200 μl per well) for 15 min. The fixative was then removed, the wells were allowed to air dry, and then crystal violet (0.4%) was added (200 μl per well). The stain was removed after 15 min and the plate was washed under a stream of tap water. After allowing the wells to air dry, 200 μl of 33% acetic acid was added to each well and, after 20 min, the OD 595 of each well was read in a microplate reader (Synergy HT). Control wells were prepared using uninoculated modified LB broth. The results are the mean of five replicate cultures (± SE). Additional biofilm assays were conducted following procedures modified from Merritt et al. [ 18 ]. Here, the colony forming units (CFUs) recovered from biofilms were directly enumerated by plating onto an agar medium. Briefly, biofilms of E. faecalis TUSoD11 or TUSoD11(ΔɸEf11) were established on sterilized circular (12 mm diameter) glass cover slips placed in wells of a 24 well plate. The culture medium, containing the planktonic cells, was removed and the wells containing the biofilm-coated cover slips were washed six times with 2 ml of sterile PBS. Each cover slip was then aseptically transferred to a sterile glass tube containing 4 ml of PBS whereupon it was sonicated (MSE, Soniprep 150 plus) for 8 sec at ~ 50% amplitude (≈ 7microns) and a power output of ~ 5 watts. Each sonicated cover slip (in 4 ml PBS) was vigorously vortexed for 5 sec, and the titer of the resulting E. faecalis suspension was determined by plating dilutions onto plates of Thallous Acetate Agar Medium, which is selective for enterococci [ 19 ]. Plate lysis and turbidity reduction assays for detection of sensitivity to bacteriophage ɸEf11 endolysin In the course of a productive infection, many bacteriophages (phages) synthesize muralytic enzymes (endolysins) to lyse the infected host cell and enable the release of the progeny virions. The external application of endolysins to strains of most (Gram positive) bacteria will also cause cell wall degradation and result in cell lysis from without. Previously, we isolated and characterized an endolysin produced by E. faecalis bacteriophage ɸEf11 [ 20 ]. Preparations of this endolysin (ORF28 endolysin) were used to test the sensitivity of the isogenic pair of lysogenic (TUSoD11) and cured [TUSoD11(ΔɸEf11)] E. faecalis strains. For plate lysis assays, 0.1 ml of an overnight BHI broth culture of each of the two paired E. faecalis strains was inoculated into 3 ml of molten (45°C) soft agar (BHI broth containing 0.7% agar). This mixture was rapidly poured into plates containing a solid layer of BHI agar (BHI broth containing 1.5% agar), and allowed to solidify and air dry. The endolysin preparation (3 μl) was then spotted onto the center of the solidified soft agar layer, and this was allowed to air dry. The plates were then incubated at 37°C overnight, whereupon they were inspected for clear zones in the bacterial lawn where the spots were originally placed, indicating lytic activity. For turbidity reduction assays, overnight 10 ml BHI broth cultures of the lysogenic (TUSoD11) and isogenic cured strain [TUSoD11(ΔɸEf11)] of E. faecalis were grown, and the cells were collected by centrifugation (7,500 x g x 10 min). The cells were resuspended in 5 ml of PBS, and 1.5 ml of this suspension was transferred into sterile, clear tubes. Each tube then received 30 μl of a filter sterilized preparation of the phage ɸEf11 ORF28 endolysin [ 20 ]. Control tubes received 30 μl of PBS instead of the endolysin. The tubes were incubated at 37°C and observed for changes in turbidity. Microorganisms The source and relevant characteristics of the 61 oral E. faecalis strains used in this study are listed in Table 1 . All strains were grown in Brain Heart Infusion Broth @ at 37°C in stationary cultures. In addition, we included a panel of unrelated bacterial species to test the specificity of the primers that we used to detect the ɸEf11 sequence. These included: Streptococcus mutans, S. sanguis, Staphylococcus aureus, Finegoldia (Peptostreptococcus) magna (magnus), Clostridium perfringens, Actinomyces israelii , and Eggerthella (Eubacterium) lenta (lentum) . 10.1080/20002297.2019.1643207-T0001 Table 1. E. faecalis strains used and presence/absence of ɸEf11 ORF43. Strain Source Antibiotic status ORF 43 Present Reference or Origin E. faecalis TUSoD1 Root canal Em (r) , TC r a E. faecalis TUSoD2 Root canal Em (r) + a E. faecalis TUSoD3 Root canal Em (r) + a E. faecalis TUSoD9 Root canal Em (r) ,TC r + a E. faecalis TUSoD10 Root canal Em (r) a E. faecalis TUSoD11 Root canal Em (r) ,TC r + a E. faecalis TUSoD12 Root canal TC r + a E. faecalis TUSoD15 Root canal TC r + a E. faecalis TUSoD17 Root canal a E. faecalis TUSoD18 Root canal a E. faecalis GS1 Root canal b E. faecalis GS2 Root canal + b E. faecalis GS3 Root canal b E. faecalis GS4 Root canal b E. faecalis GS6 Root canal b E. faecalis GS7 Root canal b E. faecalis GS8 Root canal TC r + b E. faecalis GS9 Root canal TC r + b E. faecalis GS10 Root canal b E. faecalis GS12 Root canal b E. faecalis GS13 Root canal b E. faecalis GS14 Root canal b E. faecalis GS15 Root canal b E. faecalis GS16 Root canal b E. faecalis GS17 Root canal b E. faecalis GS18 Root canal b E. faecalis GS19 Root canal b E. faecalis GS21 Root canal b E. faecalis GS22 Root canal + b E. faecalis GS23 Root canal + b E. faecalis GS24 Root canal TC r b E. faecalis GS25 Root canal + b E. faecalis GS26 Root canal b E. faecalis GS27 Root canal b E. faecalis GS28 Root canal b E. faecalis GS29 Root canal + b E. faecalis GS30 Root canal TC r b E. faecalis GS31 Root canal TC r b E. faecalis GS32 Root canal b E. faecalis GS33 Root canal + b E. faecalis GS34 Tongue c E. faecalis AA-OR3 Oral Cl (r) , Cm r d E. faecalis AA-OR4 Oral Cm (r) , TC r d E. faecalis AA-OR26 Oral Cl (r) , Cm r , Em r , Gm r , TC r d E. faecalis AA-OR34 Oral Cl (r) , Cm (r) + d E. faecalis AA-T4 tongue Cm (r) , TC r d E. faecalis AA-T26 Tongue Cm r , Em r , Gm r , TC r d E. faecalis OS16 Oral e E. faecalis OS25 Tongue e E. faecalis E1 Oral isolate Cl r ,Cm r , Em r , TC (r) f E. faecalis E2 Oral isolate Amp (r) , Cl r , Cm r , Em (r), TC r + f E. faecalis E3 Oral isolate Cl r , Em (r) + f E. faecalis E4 Oral isolate Cl r , Em (r), Tc r + f E. faecalis E5 Oral isolate Cl r + f E. faecalis E6 Oral isolate Cl r f E. faecalis E7 Oral isolate f E. faecalis E8 Oral isolate Cl r , Tc r f E. faecalis E10 Oral isolate Amp (r) , Cl r , Tc r f E. faecalis E11 Oral isolate Cl r f E. faecalis ER3/25 Root canal c E. faecalis ER5/1 Root canal c r = resistant, (r) = intermediate resistance Amp = ampicillin, Cm = chloramphenicol, Cl = clindamycin, Em = erythromycin, Gm = gentamicin, Tc = tetracycline a-Stevens et al. 2009 [ 12 ], b-Sedgley et al. 2005a [ 11 ], c-Johnson et al. 2006 [ 79 ], d-Sedgley et al. 2006 [ 80 ], e-Sedgley et al. 2005b [ 81 ], f-Sedgley et al. 2004 [ 4 ]. Primers and PCR conditions Our previous sequencing of the genome of ɸEf11, a bacteriophage induced and isolated from an E. faecalis strain (TUSoD11) recovered from an infected root canal, demonstrated a genome of 42,822 bp distributed among 65 open reading frames (ORFs) [ 13 ]. Within that genome, ORF43 was designated as coding for a 'hypothetical protein'. Our search of several databases failed to disclose any genes homologous to ɸEf11 ORF43. The sequence uniqueness of this target gene provided specificity of PCR amplicons produced by ORF43-specific primers, for the presence of the ɸEf11 DNA. ɸEf11 ORF43-specific oligonucleotide primers [forward (ɸEf11 F) and reverse (ɸEf11 R)] were designed which were predicted to produce a 165 bp amplicon in PCRs with phage ɸEf11 DNA templates ( Table 2 , Figure 1 ). Template DNA was prepared by suspending cells of each strain in lysis buffer [1% (v/v) Triton X-100, 20 mM Tris-HCl (pH 8.5), 2 mM EDTA], heating to 100°C for 10 min., and then recovering the released DNA in the supernatant following centrifugation. PCR mixtures contained: 5 μl (= 5 nmol) each of forward (ɸEf11 F) and reverse (ɸEf11 R) primer, 5 μl of DNA template solution (≈ 85ng DNA), 20 μl 2 x GoTaq PCR master mix (Promega), and 5 μl dH 2 O. In addition to the ORF43-specific forward and reverse oligonucleotide primers, an E. faecalis species-specific primer set (forward: 1F, reverse: 1R), and a universal primer set (forward: RRN4, reverse: RRN5) were used in control PCRs ( Table 2 ). The E. faecalis -specific primers [ 14 ] were used as positive controls in PCRs for all the E. feacalis strains tested. Similarly, the universal primer set, which recognizes two highly conserved regions of eubacterial 16S rRNA genes [ 15 ], was used as an internal positive control in PCRs involving DNA templates from non-enterococcal bacterial species. Additional (control) PCRs were prepared using E. faecalis -specific (1F and 1R) or universal primers (RRN4 and RRN5) instead of the phage ɸEf11 ORF43-specific primers. PCR conditions for reactions containing the ɸEf11 ORF43-specific primers (ɸEf11F and ɸEf11R) and E. faecalis -specific primers (1F and 1R) were: 97°C for 1min, followed by 26 cycles of (i) 94°C for 1 min, (ii) 50°C for 45 sec, and (iii) 72°C for 1 min. This was followed by an additional 4 min at 72°C. For PCRs utilizing the universal primers (RRN4 and RRN5), the reaction conditions were: 97°C for 1 min, followed by 25 cycles of (i) 95°C for 30 sec, (ii) 55°C for 30 sec and (iii) 72°C for 30 sec, followed by an additional 4 min at 72°C. Following PCR, amplification products were detected by agarose [2%(w/v)] gel electrophoresis and ethidium bromide staining. 10.1080/20002297.2019.1643207-T0002 Table 2. Primer sets used for PCR amplification. Primer Set Sequence Purpose Predicted Amplicon Size Reference ɸEf11 F ɸEf11 R 5ʹ-GAGAGTGGAAGTGGATTCAATG-3ʹ 5ʹ-GCACTTTCATCTAAACTCTCG-3' Amplification of ɸEf11 ORF 43 165 bp This study 1F 1R 5ʹ-GTTTATGCCGCATGGCATAAGAG-3ʹ 5ʹ-CCGTCAGGGGACGTTCAG-3' E. faecalis -specific primers 310 bp a RRN4 RRN5 5ʹ-CAGGATTAGATACCCTGGTAGTCCACGC-3ʹ 5ʹ-GACGGGCGGTGTGTACAAGGCCCGGGAACG-3' Universal (16S rDNA) primers 625 bp b a-Siqueira et al. 2004 [ 14 ], b-Goncharoff et al. 1993 [ 15 ] 10.1080/20002297.2019.1643207-F0001 Figure 1. Phage ɸEf11 ORF43 sequence (237 bp). Primer binding sites are underlined. The 165 bp PCR amplicon product is shown in bold type. Generation of a cured derivative strain of lysogenic E. faecalis strain TUSoD11 In a previous communication we reported our generation and isolation of a cured E. faecalis strain [ 16 ]. Briefly, allelic exchange mutagenesis was employed to delete a module of six lysogeny-related genes and insert a selectable antibiotic resistance gene (erythromycin) into the ɸEf11 prophage of lysogenic E. faecalis TUSoD11. PCR screening of the recombinant transformant clones selected on erythromycin plates confirmed the absence of the targeted prophage genes in the cells of the recovered colonies. Surprisingly, in addition to the deletion of the six genes of the targeted lysogeny gene module, the cells of a few of the recovered colonies also lacked any other of the ɸEf11 prophage genes, for which they were screened, as well. Because the phage ɸEf11 genome is circularly permuted, deletion of the entire prophage from the TUSoD11 chromosome could have occurred through homologous recombination between the gene exchange vector that was used and homologous regions that could be positioned at either end of the ɸEf11 prophage. PCR screening was conducted using ɸEf11 prophage-specific primers and template DNA from presumptive recombinant clones selected on the antibiotic (erythromycin) plates. Those clones, no longer possessing any detectable ɸEf11 prophage genes, were considered cured of the prophage, and designated E. faecalis TUSoD11(ΔɸEf11). By this process we have obtained the isogenic pair of lysogenic and non-lysogenic E. faecalis strains [TUSoD11 and TUSoD11(ΔɸEf11)], differing only in the presence or absence of the ɸEf11 prophage. Growth rate assay Cultures of lysogenic E. faecalis TUSoD11 and its cured isogenic derivative TUSoD11(ΔɸEf11) were grown overnight at 37°C in BHI broth. Portions of each culture were inoculated into fresh BHI broth to produce suspensions having an OD 600 of 0.1. Samples of each suspension were placed into wells of a flat-bottomed 96 well microtiter plate (200 μl/well). Control wells contained uninoculated BHI broth. The plate was loaded into a microplate reader (Synergy HT), and incubated at 37°C for 24 h, during which the OD 600 of each well was measured. The result of triplicate assays was recorded. Biofilm assay Biofilms were established and assayed colorimetrically as described by Knezevic and Petrovic [ 17 ]. Cultures of E. faecalis TUSoD11 and TUSoD11(ΔɸEf11) were grown overnight at 37°C in modified LB broth (0.5% yeast extract, 1.0% Tryptone, 1.0% NaCl, 0.5% glucose). Each culture was diluted to OD 600 of 0.1 using modified LB broth. Samples of 200 μl of each culture were inoculated into the wells of a flat bottomed 96 well microtiter plate. After incubation at 37°C for 48 h, the medium and planktonic cells in each well were removed and the wells were washed twice with PBS (0.072% Na 2 PO 4 , 0.021% KH 2 PO 4 , 0.765% NaCl, pH 7.2). The attached cells in each well were left to air dry, and then fixed by incubation in absolute methanol (200 μl per well) for 15 min. The fixative was then removed, the wells were allowed to air dry, and then crystal violet (0.4%) was added (200 μl per well). The stain was removed after 15 min and the plate was washed under a stream of tap water. After allowing the wells to air dry, 200 μl of 33% acetic acid was added to each well and, after 20 min, the OD 595 of each well was read in a microplate reader (Synergy HT). Control wells were prepared using uninoculated modified LB broth. The results are the mean of five replicate cultures (± SE). Additional biofilm assays were conducted following procedures modified from Merritt et al. [ 18 ]. Here, the colony forming units (CFUs) recovered from biofilms were directly enumerated by plating onto an agar medium. Briefly, biofilms of E. faecalis TUSoD11 or TUSoD11(ΔɸEf11) were established on sterilized circular (12 mm diameter) glass cover slips placed in wells of a 24 well plate. The culture medium, containing the planktonic cells, was removed and the wells containing the biofilm-coated cover slips were washed six times with 2 ml of sterile PBS. Each cover slip was then aseptically transferred to a sterile glass tube containing 4 ml of PBS whereupon it was sonicated (MSE, Soniprep 150 plus) for 8 sec at ~ 50% amplitude (≈ 7microns) and a power output of ~ 5 watts. Each sonicated cover slip (in 4 ml PBS) was vigorously vortexed for 5 sec, and the titer of the resulting E. faecalis suspension was determined by plating dilutions onto plates of Thallous Acetate Agar Medium, which is selective for enterococci [ 19 ]. Plate lysis and turbidity reduction assays for detection of sensitivity to bacteriophage ɸEf11 endolysin In the course of a productive infection, many bacteriophages (phages) synthesize muralytic enzymes (endolysins) to lyse the infected host cell and enable the release of the progeny virions. The external application of endolysins to strains of most (Gram positive) bacteria will also cause cell wall degradation and result in cell lysis from without. Previously, we isolated and characterized an endolysin produced by E. faecalis bacteriophage ɸEf11 [ 20 ]. Preparations of this endolysin (ORF28 endolysin) were used to test the sensitivity of the isogenic pair of lysogenic (TUSoD11) and cured [TUSoD11(ΔɸEf11)] E. faecalis strains. For plate lysis assays, 0.1 ml of an overnight BHI broth culture of each of the two paired E. faecalis strains was inoculated into 3 ml of molten (45°C) soft agar (BHI broth containing 0.7% agar). This mixture was rapidly poured into plates containing a solid layer of BHI agar (BHI broth containing 1.5% agar), and allowed to solidify and air dry. The endolysin preparation (3 μl) was then spotted onto the center of the solidified soft agar layer, and this was allowed to air dry. The plates were then incubated at 37°C overnight, whereupon they were inspected for clear zones in the bacterial lawn where the spots were originally placed, indicating lytic activity. For turbidity reduction assays, overnight 10 ml BHI broth cultures of the lysogenic (TUSoD11) and isogenic cured strain [TUSoD11(ΔɸEf11)] of E. faecalis were grown, and the cells were collected by centrifugation (7,500 x g x 10 min). The cells were resuspended in 5 ml of PBS, and 1.5 ml of this suspension was transferred into sterile, clear tubes. Each tube then received 30 μl of a filter sterilized preparation of the phage ɸEf11 ORF28 endolysin [ 20 ]. Control tubes received 30 μl of PBS instead of the endolysin. The tubes were incubated at 37°C and observed for changes in turbidity. Results Specificity of ɸEf11 F/ɸEf11 R primer set TUSoD11 is the lysogenic E. faecalis strain from which phage ɸEf11 was originally induced and isolated [ 12 ]. Consequently we considered the TUSoD11 DNA to be a positive control for the presence of the phage ɸEf11 ORF43 sequence. E. faecalis strain JH2-2 supports lytic infection by phage ɸEf11 [ 12 ], and therefore, by reason of superinfection exclusion, this strain would not be expected to harbor the phage ɸEf11 genome. ORF43-specific primers produced an amplicon of the predicted size (165 bp) when used in PCRs with template DNA from E. faecalis TUSoD11 ( Figure 2(a ), lane 2). Furthermore, no amplicon of the predicted size was produced using the ORF43-specific primers in PCRs with templates from E. faecalis JH2-2 ( Figure 2(a ), lane 3), or any of the unrelated bacterial species tested ( Figure 2(a ), lanes 4–10). In contrast, PCRs using template from JH2-2 or any of the unrelated bacterial species with either E. faecalis -specific primers 1F and 1R (for JH2-2) or universal primers RRN4 and RRN5 (for the unrelated bacterial species) did yield amplicons of the expected size, ≈ 310 bp and ≈ 625 bp, respectively ( Figures 2(b ), 3 (a)). These results demonstrate the specificity of the ɸEf11F/ɸEf11R primer set for the ɸEf11 ORF43 DNA sequence. 10.1080/20002297.2019.1643207-F0002 Figure 2. (a). Specificity of ORF43 primers for ɸEf11prophage. PCR reactions performed with ϕEf11F and ϕEf11R primers and template DNA from positive (lane 2 ) and negative (lanes 3 – 10 ) controls. Lanes: 1 , Bench Top Marker (Promega); 2 , E. faecalis TUSoD 11 (positive control); 3 , E. faecalis JH2-2 (negative control); 4 , S. mutans ; 5 , S. aureus ; 6 , S. sanguinis ; 7 , F. magna ; 8 , C. perfringens ; 9 , A. israelii ; 10 , E. lentum . Note that the 165 bp ϕEf11 bacteriophage-specific amplicon was only produced in the reaction containing template DNA from E. faecalis TUSoD 11 (lane 2 ) that is known to harbor the ϕEf11 bacteriophage genome. (b). Presence of template DNA from each bacterial species in PCRs. PCR reactions performed with universal primers (RRN4 and RRN5) and template DNA from E. faecalis TUSoD11 (lanes 2, 3), S. mutans (lanes 4,5), F. magna (lanes 6,7). Lane 8 blank. Lane 1, Bench Top Marker (Promega), Lane 9, 100 bp ladder. Amplicons of the expected size (625 bp) confirmed presence of bacterial template DNA in each of the PCRs. 10.1080/20002297.2019.1643207-F0003 Figure 3. Presence of ɸEf11 prophage in oral E. faecalis strains. PCR reactions performed with (a) E. faecalis -specific primers (IF and 1R), or (b) ɸEf11 ORF 43-specific primers (ɸEf11F and ɸEf11R). Lanes: 1, E. faecalis GS25; 2, E. faecalis GS29; 3, E. faecalis GS33; 4, E. faecalis AA-OR34; 5, E. faecalis TUSoD11 (positive control); 6, E. faecalis JH2-2 (negative control); 7, Bench Top 1kb DNA ladder (Promega). 310 bp amplicons in A indicate the presence of E. faecalis DNA from each of the strains tested, and the 165 bp amplicons in B indicate the presence of phage ɸEf11 ORF43 in the DNA from each the same strains (with the exception of JH2-2, negative control). Prevalence of ORF43 among 61 oral E. faecalis strains We next used the ORF43-specific primers in PCRs with template DNA from 61 E. faecalis strains isolated from the human oral cavity. An example of the results from these reactions can be seen in Figure 3(b ) where five of the E. faecalis strains tested produced amplicons of the predicted size. All in all, 19 (31%) of the 61 oral E. faecalis strains were found to be positive for the ɸEf11 ORF43 DNA sequence ( Table 1 ). Cured, recombinant clones of TUSoD11 were generated lacking ɸEf11 prophage genes Primer sets specific for numerous ɸEf11 prophage genes were used in PCR to screen for the presence of the prophage in TUSoD11 clones that had been transformed with a gene exchange vector. As can be seen in the example shown in Figure 4 , clones were identified in which none of the 14 ɸEf11 prophage genes examined could be detected by PCR. These clones were considered to be cured, and were designated TUSoD11(ΔɸEf11). 10.1080/20002297.2019.1643207-F0004 Figure 4. Deletion of ɸEf11 genes from cured derivative of E. faecalis strain TUSoD11(ΔɸEf11). (a). Representation of phage ɸEf11 genome (ORFs1-65). Packaging, Head morphogenesis, Tail morphogenesis, Lysis, Integration, Lysogeny establishment and maintenance, Lytic cycle regulation, DNA replication and modification. ORFs labeled X could not be detected in the cured strain. (b). PCR Screening of recombinant TUSoD11 clone for the presence of prophage ɸEf11 genes. AGE analysis of PCR amplicons produced with ɸEf11 ORF-specific primers (ORFs 1, 16, 17, 20, 23, 31, 32, 35, 36, 44, 47, 54, 61 and 65) and templates from either E. faecalis TUSoD11 (lysogenic strain) or TUSoD11(ΔɸEf11)(cured strain). Lane numbers refer to ORF specificity of primer set. M = molecular mass standard (GeneRuler1kb DNA ladder), Ef = E. faecalis -specific primer set. Amplicons in upper half of the figure were generated using lysogenic E. faecalis TUSoD11 template, PCRs in lower half of the figure were conducted with template from (cured) TUSoD11(ΔɸEf11). Lysogeny promoted the maintenance of a higher cell density relative to the cured strain Growth curves of the lysogenic strain (TUSoD11) and the cured strain [TUSoD11(ΔɸEf11)], were compared. As seen in Figure 5 , the growth rates of the two strains were quite similar for the first 12 h of incubation, however after 12 h, the cell density of the lysogenic strain suspension was maintained, and even increased over the next 12 h. In contrast, the cell density of the suspension of the cured strain steadily decreased between 12 and 24 h of incubation. 10.1080/20002297.2019.1643207-F0005 Figure 5. Comparative growth curves of Lysogenic (TUSoD11) and Cured [TUSoD11(ΔɸEf11)] strains of E. faecalis . Each strain was grown in triplicate. Biofilm formation is enhanced in the lysogen compared to the cured E. faecalis strain In comparison with the cured E. faecalis strain [TUSoD11(ΔɸEf11)], the isogenic, lysogenic strain (TUSoD11) formed approximately 50% more biofilm ( Figure 6 ). Furthermore, enumeration of the bacteria recovered from biofilms disclosed that the lysogenic E. faecalis strain (TUSoD11) yielded approximately six times the colony forming unit count of its isogenic cured counterpart [TUSoD11(ΔɸEf11)] ( Figure 7 ). This suggests that the enhanced biofilm formation produced by the lysogenic strain observed by crystal violet staining, is due, at least in part, to a higher bacterial population. 10.1080/20002297.2019.1643207-F0006 Figure 6. Biofilm assay by crystal violet staining. Colorimetric measurement of biofilm formation by lysogenic (TUSoD11) and cured [TUSoD11(ΔɸEf11)] E. faecalis strains. Biofilms formed by lysogenic and cured strains were stained with crystal violet. Values are the mean of five replicate cultures ±SE. 10.1080/20002297.2019.1643207-F0007 Figure 7. Biofilm assay by direct bacterial enumeration. Colony Forming Units (CFUs) recovered from biofilms produced by lysogenic (TUSoD11) and cured [TUSoD11(ΔɸEf11)] E. faecalis strains. Biofilms formed by lysogenic and cured strains were sonicated, and the suspended cells were recovered and tittered. The values are mean of three replicate cultures ±SE. The presence of a prophage renders the lysogenic cell resistant to the lytic action of an externally applied bacteriophage endolysin Spotting 3 μl of the phage ɸEf11 endolysin preparation onto a lawn of the cured E. faecalis strain [TUSoD11(ΔɸEf11)] produced a clear lytic zone, whereas no such effect was seen in a lawn of the lysogenic strain, TUSoD11 ( Figure 8 ) Furthermore, addition of the ɸEf11 ORF28 endolysin to a suspension of the cured E. faecalis strain [TUSoD11(ΔɸEf11)] resulted in a rapid and pronounced clearing ( Figure 9 ). In contrast, a suspension of the lysogenic strain (TUSoD11) exhibited no overt change following the addition of the endolysin ( Figure 9 ). 10.1080/20002297.2019.1643207-F0008 Figure 8. Plate lysis assay for sensitivity of E. faecalis strains to phage ɸEf11 endolysin. Layers of either (a) E. faecalis TUSoD11 (lysogen) or (b) TUSoD11(ΔɸEf11) (cured) were prepared. A drop of endolysin suspension was placed on the center of each layer, and the plates were incubated at 37°C overnight. Note the lytic zone in the layer of the cured strain. 10.1080/20002297.2019.1643207-F0009 Figure 9. Effect of phage endolysin on suspensions of lysogenic (TUSoD11) and cured [TUSoD11(ΔɸEf11)] E. faecalis strains. (a). Suspensions at 0 time. (b). Suspensions after incubation with the endolysin for 30 min. Specificity of ɸEf11 F/ɸEf11 R primer set TUSoD11 is the lysogenic E. faecalis strain from which phage ɸEf11 was originally induced and isolated [ 12 ]. Consequently we considered the TUSoD11 DNA to be a positive control for the presence of the phage ɸEf11 ORF43 sequence. E. faecalis strain JH2-2 supports lytic infection by phage ɸEf11 [ 12 ], and therefore, by reason of superinfection exclusion, this strain would not be expected to harbor the phage ɸEf11 genome. ORF43-specific primers produced an amplicon of the predicted size (165 bp) when used in PCRs with template DNA from E. faecalis TUSoD11 ( Figure 2(a ), lane 2). Furthermore, no amplicon of the predicted size was produced using the ORF43-specific primers in PCRs with templates from E. faecalis JH2-2 ( Figure 2(a ), lane 3), or any of the unrelated bacterial species tested ( Figure 2(a ), lanes 4–10). In contrast, PCRs using template from JH2-2 or any of the unrelated bacterial species with either E. faecalis -specific primers 1F and 1R (for JH2-2) or universal primers RRN4 and RRN5 (for the unrelated bacterial species) did yield amplicons of the expected size, ≈ 310 bp and ≈ 625 bp, respectively ( Figures 2(b ), 3 (a)). These results demonstrate the specificity of the ɸEf11F/ɸEf11R primer set for the ɸEf11 ORF43 DNA sequence. 10.1080/20002297.2019.1643207-F0002 Figure 2. (a). Specificity of ORF43 primers for ɸEf11prophage. PCR reactions performed with ϕEf11F and ϕEf11R primers and template DNA from positive (lane 2 ) and negative (lanes 3 – 10 ) controls. Lanes: 1 , Bench Top Marker (Promega); 2 , E. faecalis TUSoD 11 (positive control); 3 , E. faecalis JH2-2 (negative control); 4 , S. mutans ; 5 , S. aureus ; 6 , S. sanguinis ; 7 , F. magna ; 8 , C. perfringens ; 9 , A. israelii ; 10 , E. lentum . Note that the 165 bp ϕEf11 bacteriophage-specific amplicon was only produced in the reaction containing template DNA from E. faecalis TUSoD 11 (lane 2 ) that is known to harbor the ϕEf11 bacteriophage genome. (b). Presence of template DNA from each bacterial species in PCRs. PCR reactions performed with universal primers (RRN4 and RRN5) and template DNA from E. faecalis TUSoD11 (lanes 2, 3), S. mutans (lanes 4,5), F. magna (lanes 6,7). Lane 8 blank. Lane 1, Bench Top Marker (Promega), Lane 9, 100 bp ladder. Amplicons of the expected size (625 bp) confirmed presence of bacterial template DNA in each of the PCRs. 10.1080/20002297.2019.1643207-F0003 Figure 3. Presence of ɸEf11 prophage in oral E. faecalis strains. PCR reactions performed with (a) E. faecalis -specific primers (IF and 1R), or (b) ɸEf11 ORF 43-specific primers (ɸEf11F and ɸEf11R). Lanes: 1, E. faecalis GS25; 2, E. faecalis GS29; 3, E. faecalis GS33; 4, E. faecalis AA-OR34; 5, E. faecalis TUSoD11 (positive control); 6, E. faecalis JH2-2 (negative control); 7, Bench Top 1kb DNA ladder (Promega). 310 bp amplicons in A indicate the presence of E. faecalis DNA from each of the strains tested, and the 165 bp amplicons in B indicate the presence of phage ɸEf11 ORF43 in the DNA from each the same strains (with the exception of JH2-2, negative control). Prevalence of ORF43 among 61 oral E. faecalis strains We next used the ORF43-specific primers in PCRs with template DNA from 61 E. faecalis strains isolated from the human oral cavity. An example of the results from these reactions can be seen in Figure 3(b ) where five of the E. faecalis strains tested produced amplicons of the predicted size. All in all, 19 (31%) of the 61 oral E. faecalis strains were found to be positive for the ɸEf11 ORF43 DNA sequence ( Table 1 ). Cured, recombinant clones of TUSoD11 were generated lacking ɸEf11 prophage genes Primer sets specific for numerous ɸEf11 prophage genes were used in PCR to screen for the presence of the prophage in TUSoD11 clones that had been transformed with a gene exchange vector. As can be seen in the example shown in Figure 4 , clones were identified in which none of the 14 ɸEf11 prophage genes examined could be detected by PCR. These clones were considered to be cured, and were designated TUSoD11(ΔɸEf11). 10.1080/20002297.2019.1643207-F0004 Figure 4. Deletion of ɸEf11 genes from cured derivative of E. faecalis strain TUSoD11(ΔɸEf11). (a). Representation of phage ɸEf11 genome (ORFs1-65). Packaging, Head morphogenesis, Tail morphogenesis, Lysis, Integration, Lysogeny establishment and maintenance, Lytic cycle regulation, DNA replication and modification. ORFs labeled X could not be detected in the cured strain. (b). PCR Screening of recombinant TUSoD11 clone for the presence of prophage ɸEf11 genes. AGE analysis of PCR amplicons produced with ɸEf11 ORF-specific primers (ORFs 1, 16, 17, 20, 23, 31, 32, 35, 36, 44, 47, 54, 61 and 65) and templates from either E. faecalis TUSoD11 (lysogenic strain) or TUSoD11(ΔɸEf11)(cured strain). Lane numbers refer to ORF specificity of primer set. M = molecular mass standard (GeneRuler1kb DNA ladder), Ef = E. faecalis -specific primer set. Amplicons in upper half of the figure were generated using lysogenic E. faecalis TUSoD11 template, PCRs in lower half of the figure were conducted with template from (cured) TUSoD11(ΔɸEf11). Lysogeny promoted the maintenance of a higher cell density relative to the cured strain Growth curves of the lysogenic strain (TUSoD11) and the cured strain [TUSoD11(ΔɸEf11)], were compared. As seen in Figure 5 , the growth rates of the two strains were quite similar for the first 12 h of incubation, however after 12 h, the cell density of the lysogenic strain suspension was maintained, and even increased over the next 12 h. In contrast, the cell density of the suspension of the cured strain steadily decreased between 12 and 24 h of incubation. 10.1080/20002297.2019.1643207-F0005 Figure 5. Comparative growth curves of Lysogenic (TUSoD11) and Cured [TUSoD11(ΔɸEf11)] strains of E. faecalis . Each strain was grown in triplicate. Biofilm formation is enhanced in the lysogen compared to the cured E. faecalis strain In comparison with the cured E. faecalis strain [TUSoD11(ΔɸEf11)], the isogenic, lysogenic strain (TUSoD11) formed approximately 50% more biofilm ( Figure 6 ). Furthermore, enumeration of the bacteria recovered from biofilms disclosed that the lysogenic E. faecalis strain (TUSoD11) yielded approximately six times the colony forming unit count of its isogenic cured counterpart [TUSoD11(ΔɸEf11)] ( Figure 7 ). This suggests that the enhanced biofilm formation produced by the lysogenic strain observed by crystal violet staining, is due, at least in part, to a higher bacterial population. 10.1080/20002297.2019.1643207-F0006 Figure 6. Biofilm assay by crystal violet staining. Colorimetric measurement of biofilm formation by lysogenic (TUSoD11) and cured [TUSoD11(ΔɸEf11)] E. faecalis strains. Biofilms formed by lysogenic and cured strains were stained with crystal violet. Values are the mean of five replicate cultures ±SE. 10.1080/20002297.2019.1643207-F0007 Figure 7. Biofilm assay by direct bacterial enumeration. Colony Forming Units (CFUs) recovered from biofilms produced by lysogenic (TUSoD11) and cured [TUSoD11(ΔɸEf11)] E. faecalis strains. Biofilms formed by lysogenic and cured strains were sonicated, and the suspended cells were recovered and tittered. The values are mean of three replicate cultures ±SE. The presence of a prophage renders the lysogenic cell resistant to the lytic action of an externally applied bacteriophage endolysin Spotting 3 μl of the phage ɸEf11 endolysin preparation onto a lawn of the cured E. faecalis strain [TUSoD11(ΔɸEf11)] produced a clear lytic zone, whereas no such effect was seen in a lawn of the lysogenic strain, TUSoD11 ( Figure 8 ) Furthermore, addition of the ɸEf11 ORF28 endolysin to a suspension of the cured E. faecalis strain [TUSoD11(ΔɸEf11)] resulted in a rapid and pronounced clearing ( Figure 9 ). In contrast, a suspension of the lysogenic strain (TUSoD11) exhibited no overt change following the addition of the endolysin ( Figure 9 ). 10.1080/20002297.2019.1643207-F0008 Figure 8. Plate lysis assay for sensitivity of E. faecalis strains to phage ɸEf11 endolysin. Layers of either (a) E. faecalis TUSoD11 (lysogen) or (b) TUSoD11(ΔɸEf11) (cured) were prepared. A drop of endolysin suspension was placed on the center of each layer, and the plates were incubated at 37°C overnight. Note the lytic zone in the layer of the cured strain. 10.1080/20002297.2019.1643207-F0009 Figure 9. Effect of phage endolysin on suspensions of lysogenic (TUSoD11) and cured [TUSoD11(ΔɸEf11)] E. faecalis strains. (a). Suspensions at 0 time. (b). Suspensions after incubation with the endolysin for 30 min. Discussion The development and introduction of culture-independent, molecular methods of microbial detection and identification has resulted in a more thorough appreciation of the microbial diversity throughout the human body. Compared to the cultural methods previously used, 16S RNA gene amplification and sequencing can provide a more complete assessment of microbial population composition. The microbiomes of the oral cavity [ 21 ] and several individual ecological niches within the oral cavity, such as the saliva [ 22 – 24 ], the periodontal pocket [ 25 – 27 ], the dorsum of the tongue [ 28 ] and the infected root canal [ 29 – 31 ] have been explored using this technique. However, it is also true that 16S RNA gene sequencing technology is not well suited to provide information on intraspecies strain variation. This is of some concern since it is well established that virulence properties of many pathogenic bacteria are due to the uptake of exogenous genetic information, such as plasmids and phage genomes, thereby modifying the genome of individual strains of a given species. Such modifications could go undetected by a simple enumeration of species present in a given microbiome. Other studies, using pyrosequencing methods have described the virome of the human oral (salivary) environment [ 32 ]. This study determined that > 99% of the assembled contigs were homologous to bacteriophage sequences, suggesting that the vast majority of the human salivary virome was composed of bacteriophages. The metabolic gene profile of the viral DNA population was dominated by genes that were identified as coding for functions related to nucleic acid metabolism and virulence factors. In addition, approximately 10% of the viral contigs had integrase homologs, suggesting a temperate bacteriophage origin of a substantial portion of the oral virome genome. However, these data do not provide information on the proportion of the oral (bacterial) microbiome that has actually been influenced by the horizontal gene transmission of these viral genes through lysogeny. In an analysis of the salivary bacteriophage transcriptome in health versus periodontal disease, it was found that many bacteriophage genes are expressed in the oral cavity in both health and disease [ 33 ], however, here again, it cannot be ascertained to what extent the resident bacterial population is chronically infected by a virus, or how the host bacteria are altered (in the case of lysogenic infection). Here, we identified a strain variability within one species of oral bacteria (E. faecalis ) in terms of the presence or absence of a prophage in the bacterial genome, and illustrate some of the ways in which this variation (lysogeny) has impacted the properties of the host cell. We used PCR to detect an ɸEf11 phage-specific sequence (ORF43) in a panel of oral E. faecalis strains. The ORF43 sequence was selected as a phage ɸEf11 indicator due to its uniqueness among all the sequences searched in databases. The specificity of the ORF43 PCR primer set was validated using negative controls including template DNA from unrelated bacterial species, and E. faecalis strains, such as JH2-2, that supported lytic infection by phage ɸEf11. Lysogenic E. faecalis strains harboring an ɸEf11 prophage do not support lytic infection by phage ɸEf11 due to superinfection immunity. In contrast, template from a positive control strain, E. faecalis TUSoD11, the strain from which phage ɸEf11 was originally induced, did produce an amplicon of the predicted size (165 bp) when used in PCR with the ORF43 primers. Using these primers, we found that lysogeny among the oral E. faecalis strains tested was fairly common, with 19 out of the 61 (31%) strains displaying evidence of harboring an ɸEf11 prophage. Furthermore, it is likely that this incidence of lysogeny (31%) is an underestimation of the prevalence of lysogeny in oral E. faecalis strains since our procedures would only allow the detection of ɸEf11 prophages in the strains examined, and there have been several different E. faecalis phages detected in human oral samples [ 34 , 35 ]. Lysogens containing these prophages would not have been detected in our study. The prevalence of lysogeny among our panel of oral E. faecalis strains is not unexpected. Lysogeny is widespread in nature and can be anticipated to be a common feature of oral bacterial strains, as suggested by the findings of the previously mentioned study of human salivary virome [ 32 ]. It has been reported that approximately half of all sequenced bacterial genomes contain one or more prophages [ 36 – 39 ]. The high incidence of putative integrase-related sequences in the salivary virome reported by Pride et al. [ 32 ] suggests the likelihood that the oral cavity harbors a high proportion of temperate bacteriophages potentially capable of producing lysogenic infections. In this regard, we previously demonstrated that lysogeny was widespread in strains of another oral bacterium, Aggregatibacter (Actinobacillus) actinomycetemcomitans , a species associated with aggressive localized periodontitis [ 40 ]. Among the 42 A. actinomycetemcomitans strains tested 14 (34%) were found to be lysogenic. Lysogeny often results in altered phenotypic properties of the host bacteria [ 41 – 43 ]. These alterations include: resistance to superinfection by subsequent phages, enhanced virulence, and increased fitness (improved ability to outcompete nonlysogenic strains). Alterations can be due to expression of prophage genes in the lysogen [ 44 ] or to modifications of host gene expression as a consequence of the insertion or excision of the prophage into or out of the bacterial chromosome [ 45 ]. In either case, the new phenotype may provide a selective advantage for the lysogen. Our results suggest that lysogenic infection of E. faecalis by phage ɸEf11 results in an elevated persistent yield, a denser biofilm formation, and resistance to a phage-coded lytic enzyme. The increase in growth (higher cell density maintained during stationary phage) seen in the lysogenic E. faecalis strain is in agreement with previous studies reporting that the presence of a prophage confers the ability for the lysogen to grow at higher rates and produce higher yields in stationary growth phase [ 39 ]. Lysogens of Escherichia coli [ 46 – 48 ] and Streptococcus suis [ 49 ] have been shown to grow more rapidly and maintain higher stationary phase titers than their nonlysogenic counterparts. Similarly, our finding that lysogeny increases biofilm production in E. faecalis is in accord with several previous studies demonstrating the biofilm-promoting effect of the presence of a prophage [ 48 , 50 – 52 ]. Both of these properties may play a role in the fitness of the E. faecalis strain to compete in the oral environment. The differential sensitivity to the phage endolysin between the lysogenic and cured E. faecalis strains is somewhat surprising. The endolysin is a murein hydrolase that is produced by a phage in the course of a lytic infection. It is required by the virus in order to lyse the infected cell and permit the release of the progeny virions. Therefore, it might be expected that the lysogenic strain (TUSoD11), harboring the ɸEf11 prophage, should be sensitive to the endolysin. The apparent lack of sensitivity of the lysogenic strain to the endolysin might be due to the fact that in our assay, the endolysin was applied externally to the cell layer, whereas during lytic infection, the source of the endolysin is internal, from within the infected cell. It is conceivable that lysogeny results in surface modification of the infected cell, rendering it resistant or inaccessible to externally applied endolysin. Prophage-mediated cell surface modification has been well documented for many other phage-host systems [ 53 – 66 ]. Cell wall polysaccharides and capsules are known to be produced by many E. faecalis strains [ 67 – 69 ], especially those of oral origin [ 70 – 72 ]. If these cell-surface components are modified due to lysogenic conversion, and they no longer serve a vital function needed for lysin activity, then as we observed with lysogenic strain (TUSoD11), no lytic activity would be detected. The cured strain [TUSoD11(ΔɸEf11)], lacking any phage-mediated cell surface modification, would (and did) remain sensitive to the lytic effects of the endolysin. It is not immediately clear whether this explanation is accurate for the ɸEf11 lysogens. Although many (33/65) of the bacteriophage ɸEf11 genes have been annotated [ 13 ], none of these appear to be related to cell surface polysaccharide or capsule modification. It is possible that one of the remaining uncharacterized phage genes plays a role in modification of the host cell, however this remains to be determined. Further studies are needed to determine the capsular status of both the lysogenic (TUSoD11), and cured TUSoD11(ΔɸEf11)], strains. While the present investigation compares properties of pure/individual cultures of an isogenic pair of lysogenic and cured E. faecalis strains in vitro, in vivo lysogeny may impact mixed cultures as well. Phage produced as a result of induction of lysogenic strains may infect and kill susceptible nonlysogenic strains, resulting in the competitive advantage of the remaining uninduced lysogens, which are immune to superinfection. If a similar relationship exists for the many other bacterial species of the oral microbiome, then the oral microbial composition may be markedly shaped by lysogeny, and the bacterial viruses produced by the induction of lysogens. This investigation demonstrated the impact of lysogeny on just three phenotypic characteristics of E. faecalis . Clearly, there are numerous other features that are known to be modifiable due to lysogenic conversion in other bacterial species [ 3 , 43 , 45 , 73 – 76 ] and many of these deserve to be examined in relation to E. faecalis infection. To date, relatively few studies have been conducted to identify phage-mediated modifications of E. faecalis . Two such phenotypic modifications of E. faecalis due to lysogenic conversion that have previously been demonstrated are the production of homologs of platelet binding proteins PblA/PblB of Streptococcus mitis phage SM1 [ 77 ] and enhanced intestinal colonization [ 78 ]. E. faecalis lysogens harboring prophages encoding PblA/PblB homologs exhibit enhanced platelet adherence compared to strains lacking these prophages [ 77 ]. Mixed infection by lysogenic and phage-sensitive E. faecalis strains in mice resulted in a 1.5 fold enrichment of the lysogen in the colon [ 78 ]. In light of the widespread incidence of lysogeny in oral E. faecalis strains, further studies are needed to evaluate the potential modulation of other properties due to the presence of a prophage. Furthermore, to more thoroughly understand the factors driving oral microbiome establishment and pathogenic potential, more investigation is needed into the prevalence and significance of lysogeny among the many other members of this microbial community. Acknowledgements Publication of this article was funded in part by the Temple University Libraries Open Access Publishing Fund. Disclosure statement No potential conflict of interest was reported by the authors.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3002119/

Power to Detect Spatial Disturbances under Different Levels of Geographic Aggregation

Objective Spatio and/or temporal surveillance systems are designed to monitor the ongoing appearance of disease cases in space and time, and to detect potential disturbances in either dimension. Patient addresses are sometimes reported at some level of geographic aggregation, for example by ZIP code or census tract. While this aggregation has the advantage of protecting patient privacy, it also risks compromising statistical efficiency. This paper investigated the variation in power to detect a change in the spatial distribution in the presence of spatial aggregation. Methods The authors generated 400,000 spatial datasets with varying location and spread of simulated spatial disturbances, both on a purely synthetic uniform population, and on a heterogeneous population, representing hospital admissions to three community hospitals in Cape Cod, Massachusetts. The authors evaluated the power of the M- statistic to detect spatial disturbances, comparing the use of exact spatial locations versus twelve different levels of aggregation, where the M- statistic is a comparison of two distributions of interpoint distances between locations. Results When the spread of simulated spatial disturbances was contained to a small portion of the study region or affects a large proportion of the population at risk, power was highest when exact locations were reported. If the spatial disturbance was a more modest signal, the best power was attained at an aggregated level. Conclusions The precision at which patients' locations are reported has the potential to affect the power of detection significantly. Objective Spatio and/or temporal surveillance systems are designed to monitor the ongoing appearance of disease cases in space and time, and to detect potential disturbances in either dimension. Patient addresses are sometimes reported at some level of geographic aggregation, for example by ZIP code or census tract. While this aggregation has the advantage of protecting patient privacy, it also risks compromising statistical efficiency. This paper investigated the variation in power to detect a change in the spatial distribution in the presence of spatial aggregation. Methods The authors generated 400,000 spatial datasets with varying location and spread of simulated spatial disturbances, both on a purely synthetic uniform population, and on a heterogeneous population, representing hospital admissions to three community hospitals in Cape Cod, Massachusetts. The authors evaluated the power of the M- statistic to detect spatial disturbances, comparing the use of exact spatial locations versus twelve different levels of aggregation, where the M- statistic is a comparison of two distributions of interpoint distances between locations. Results When the spread of simulated spatial disturbances was contained to a small portion of the study region or affects a large proportion of the population at risk, power was highest when exact locations were reported. If the spatial disturbance was a more modest signal, the best power was attained at an aggregated level. Conclusions The precision at which patients' locations are reported has the potential to affect the power of detection significantly.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9782046/

Dynamics and Patterning of 5-Hydroxytryptamine 2 Subtype Receptors in JC Polyomavirus Entry

The organization and dynamics of plasma membrane receptors are a critical link in virus-receptor interactions, which finetune signaling efficiency and determine cellular responses during infection. Characterizing the mechanisms responsible for the active rearrangement and clustering of receptors may aid in developing novel strategies for the therapeutic treatment of viruses. Virus-receptor interactions are poorly understood at the nanoscale, yet they present an attractive target for the design of drugs and for the illumination of viral infection and pathogenesis. This study utilizes super-resolution microscopy and related techniques, which surpass traditional microscopy resolution limitations, to provide both a spatial and temporal assessment of the interactions of human JC polyomavirus (JCPyV) with 5-hydroxytrypamine 2 receptors (5-HT 2 Rs) subtypes during viral entry. JCPyV causes asymptomatic kidney infection in the majority of the population and can cause fatal brain disease, and progressive multifocal leukoencephalopathy (PML), in immunocompromised individuals. Using Fluorescence Photoactivation Localization Microscopy (FPALM), the colocalization of JCPyV with 5-HT 2 receptor subtypes (5-HT 2A , 5-HT 2B , and 5-HT 2C ) during viral attachment and viral entry was analyzed. JCPyV was found to significantly enhance the clustering of 5-HT 2 receptors during entry. Cluster analysis of infected cells reveals changes in 5-HT 2 receptor cluster attributes, and radial distribution function (RDF) analyses suggest a significant increase in the aggregation of JCPyV particles colocalized with 5-HT 2 receptor clusters in JCPyV-infected samples. These findings provide novel insights into receptor patterning during viral entry and highlight improved technologies for the future development of therapies for JCPyV infection as well as therapies for diseases involving 5-HT 2 receptors. 1. Introduction The plasma membrane serves as the initial site of response generation between living cells and various external signals. External ligands engage membrane-localized receptors, which bind to specific target stimuli and generate signaling cascades across the membrane into the cell that will decide the fate of cells in response to the stimulus [ 1 ]. Receptors cluster in nanoscopic domains on the plasma membrane where they control ligand sensitivity to improve efficiency and protein interaction [ 2 ]. Examples include G-protein coupled receptors (GPCRs) [ 3 ], immune-cell receptors [ 1 ], as well as receptors hijacked by microbial toxins [ 4 ] or viruses [ 5 ]. During the past couple of decades, research has emphasized the importance of the spatial localization of receptors such as GPCRs in their response to specific signaling pathways crucial for physiological pathways [ 6 , 7 ]. Furthermore, as human diseases are correlated to aberrations in the distribution of membrane-bound receptors and/or their activation, it is important to characterize and understand the mechanisms underlying the dynamic rearrangement and clustering of receptors, as it may aid in developing novel strategies for therapeutic treatment of diseases [ 8 ]. Recent work has established super-resolution microscopy as a useful technique for the nanoscale study of cellular receptors, including the formation of homo- and hetero-oligomers and more extended clusters [ 9 , 10 ]. Advancements in microscopy have been pivotal for biological studies, especially in addressing membrane organization and ligand-receptor interactions. These advancements include groundbreaking localization-based super-resolution microscopy techniques; fluorescence photoactivation localization microscopy (FPALM) [ 11 ], stochastic optical reconstruction microscopy (STORM) [ 12 ], and photoactivated localization microscopy (PALM) [ 13 ], which utilize the photochemical properties of specific fluorescent probes by stochastically activating sparse subsets of them, imaging those visible molecules, and determining their locations from their diffraction-limited images [ 11 ]. Ultra-resolution structures are then generated by rendering the images of multiple fluorophores localized over time [ 11 , 14 ]. Thus, FPALM and similar techniques can be used to map biological components and their dynamics at the nanoscale level, helping to provide a more resolved and accurate representation of critical biological events. In the past two decades, super-resolution microscopy techniques have been employed in a variety of virological studies, lending new perspectives with improved levels of confirmation. For example, super-resolution microscopy has led to insights into the redistribution of envelope proteins in human immunodeficiency virus (HIV) virion maturation [ 15 ] and specific recruitment of HIV envelope proteins to assembly sites for virion formation [ 16 ]; the assembly of the replication complex by yellow fever viruses [ 17 ]; the identification of the dual function of CD81 in influenza virus uncoating and budding processes [ 18 ]; the organization of hepatitis C virus (HCV) structural proteins in lipid droplets [ 19 ]; the tracking of adenovirus genomes in infected host cells [ 20 ]; the mapping of viral architecture in vaccinia virus [ 21 ] and the visualization of morphological changes in the endoplasmic reticulum during Zika virus infection [ 22 ]. Further, FPALM imaging has been utilized in conformational studies of biological structures such as actin at the nanoscale [ 23 ]; the organization of caveolin-1 below the diffraction limit in a living vertebrate animal model [ 24 ], and the modulation of clustering of the cellular lipid phosphatidylinositol 4,5-bisphosphate (PIP2) in the presence of influenza hemagglutinin [ 25 ]. These advances using super-resolution microscopy have provided a deeper understanding of biological events and provide the groundwork for further discovery in the field of virus-host cell interactions. Considering the size of viruses and the scale of viral–host cell interactions [ 14 ], the diffraction limit of conventional microscopy techniques hampers the identification of these events [ 26 ]. Yet, super-resolution studies of virus–receptor interactions have so far been limited. Currently, there is little knowledge about the rearrangements of host-cell receptors in the context of JCPyV infection. Therefore, in the present study, we employed super-resolution microscopy to bridge this gap to understand how JC polyomavirus (JCPyV) alters the dynamics of host-cell serotonin 5-hydoxytryptamine (5-HT 2 ) receptors in the context of JCPyV infection. JCPyV is the causative agent of progressive multifocal leukoencephalopathy (PML), a viral disease that is rare but often becomes debilitating and fatal due to the lysis of glial cells in the central nervous system (CNS) [ 27 , 28 , 29 ]. Although PML is a rare disease, nearly 80% of adults worldwide are seropositive for JCPyV [ 30 , 31 , 32 , 33 , 34 ]. In the majority of cases, JCPyV causes an asymptomatic infection in the kidney [ 32 , 35 , 36 , 37 ]. In immunocompromised individuals such as patients with HIV/AIDS or those undergoing immunomodulatory treatments, the infection can spread within the CNS, infecting and lysing oligodendrocytes and astrocytes, eventually causing PML [ 38 , 39 , 40 ]. Due to the successful use of combination antiretroviral therapy (cART) treatments, the occurrence of PML in individuals with HIV/AIDS has dropped (~0.1%) compared to the prevalence of PML in ~5–8% of HIV patients prior to cART [ 41 ]. Although rare, PML can also occur in people with hematological malignancies, such as lymphoma and leukemia. Additionally, PML incidence has risen in the past two decades in those receiving prolonged immunomodulatory therapies such as natalizumab, rituximab, and efalizumab [ 42 , 43 , 44 , 45 , 46 ]. The incidence of PML in these patient populations led to the withdrawal of natalizumab and efalizumab from the market, yet natalizumab was later reintroduced with significant safety warnings and risk stratification [ 41 , 47 ]. Despite the significant reduction in incidence rate in the past two decades, the survival rate of up to 1 year for PML remains at ~75% in natalizumab-related PML patients [ 48 ] and ~70% in HIV-related patients under treatment [ 49 , 50 , 51 , 52 , 53 ]. In addition, the limited treatments available typically involve restoring the immune system which can cause other fatal CNS complications such as immune reconstitution inflammatory syndrome [ 49 , 54 , 55 ]. Therefore, it is vital to understand how JCPyV establishes infection in human cells. Human JCPyV is a member of the viral Polyomaviridae family, characterized by non-enveloped virions containing a double-stranded DNA genome [ 56 , 57 ]. The JCPyV capsid is approximately 40 nm in diameter and is comprised of three viral capsid proteins: viral protein 1 (VP1), VP2, and VP3 [ 58 , 59 ]. JCPyV initiates infection either through extracellular vesicles [ 60 ] or by directly binding to α2,6-linked sialic acid receptors [ 61 , 62 , 63 ] and/or non-sialylated glycosaminoglycans (GAGs) on host cells via the VP1 protein [ 64 ]. Subsequently, the virus enters the cell through the utilization of the 5-hydroxytryptamine 2 (5-HT 2 ) serotonin receptors [ 65 , 66 ]. Among the different isoforms of 5-HT receptors, only the subtype 2 receptors, 5-HT 2A R, 5-HT 2B R, and 5-HT 2C R, support JCPyV entry by clathrin-mediated endocytosis [ 67 ]. A transient interaction has been observed between JCPyV and each of the 5-HT 2 R subtypes at 5 min post-infection [ 68 ]. However, the underlying mechanism and spatial distribution of 5-HT 2 receptors during JCPyV entry and infection remains unclear and hindered by microscopy resolution limits [ 66 ]. Expressed in the kidneys [ 69 , 70 ] and CNS, 5-HT 2 receptors are classified as GPCRs, which mediate a large array of physiological and behavioral functions in humans, including mood regulation and social cognition [ 71 ], neurogenesis [ 72 ], memory [ 73 ], and depression [ 74 ]. GPCRs comprise 14 different isoforms [ 75 ], and specifically, 5-HT 2 receptors respond to ligand stimuli by desensitization using receptor internalization via clathrin-mediated endocytosis [ 76 , 77 ]. Upon agonist activation, GPCRs can go through substantial reorganization from their base levels, including cluster formation (as in μ opioid receptors [ 78 ]) or modulation of receptor diffusion kinetics (as in metabotropic glutamate receptor mGluR3 [ 79 ]), which changes the redistribution of receptors in clathrin-coated pits [ 80 ]. Variability in patient response to antidepressant drugs was shown to depend on changes in the clustering of serotonin transporters as well as 5-HT 2A receptors [ 81 ]. While spatial reorganization and clustering of GPCRs can mediate their function [ 3 ], not enough is known about the clustering of 5-HT 2 receptors and their nanoscale distribution and dynamics in living cells, which are important for treatments. 5-HT 2 receptors are necessary for a crucial step in the infectious viral lifecycle of JCPyV, facilitating entry into host cells. Still, the clustering and dynamics of the receptors that facilitate viral entry are unknown. This study exploits FPALM to provide crucial insight into spatial and temporal interactions between JCPyV and the 5-HT 2 receptors to define how these interactions promote viral infection at the nanoscale. Our results indicate that viral localization varies between the 5-HT 2 receptor subtypes necessary for viral internalization at different time points postinfection. These data also reveal the cluster properties of 5-HT 2 receptors in response to the virus, furthering our understanding of the pattern and dynamics of host cell receptors in the presence of JCPyV and providing a novel view of virus–host interactions. Additionally, these findings demonstrate the cluster dynamics of 5-HT 2 receptors in response to ligands, including neurotransmitters and target therapeutics, and provide new information about GPCRs [ 82 ]. Altogether, this research enhances our knowledge of viral entry and understanding of JCPyV invasion of host cells, which could inform the development of potential antiviral therapies for PML. 2. Materials and Methods 2.1. Cells, Viruses, Antibodies, and Reagents HEK293A cells were grown in Dulbecco's minimal essential medium (DMEM, Corning, Corning, NY, USA) with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (P/S, Mediatech, Inc., Manassas, VA, USA) and 0.2% plasmocin prophylactic (InvivoGen, San Diego, CA, USA). HEK293A stable cells expressing 5-HT 2 R subtypes (5-HT 2A , 5-HT 2B, and 5-HT 2C ) in fusion with YFP [ 67 ] were maintained in DMEM with, 10% FBS, 1% P/S, 0.2% plasmocin prophylactic (InvivoGen), and 500 µg of G418 (Corning). Cell lines were graciously provided by the Atwood laboratory (Brown University, Providence, RI, USA). All cell types were cultured at 37 °C with 5% CO 2 in a humidified incubator. JCPyV strain Mad-1/SVEΔ was kindly provided by Atwood laboratory (Brown University). The generation of JCPyV was described previously [ 83 , 84 ] and the proliferation of JCPyV was performed using published protocols [ 85 ]. Alexa Fluor 647-labeled JCPyV (JCPyV-647) was prepared as described [ 63 , 67 ]. Viral titers were determined through fluorescent focus unit (FFU) infectivity assays in SVG-A cells [ 67 ]. Viral infectivity FFU assays were performed using antibodies derived from two hybridoma-derived supernatants, one containing a monoclonal antibody specific for JCPyV VP1 (PAB596) that can cross-react with SV40 VP1 (generously provided by Ed Harlow) and a monoclonal antibody that is exclusively specific against JCPyV large T antigen (PAB962) (generously provided by Tevethia laboratory, Penn State University, State College, PA, USA). Additionally, an anti-pan cadherin antibody (1:200, Abcam) and DAPI nuclear counter strain (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) was used for confocal immunofluorescence imaging. 2.2. Generation of 5-HT 2 R-Dendra2 Fusion Constructs The constructs for 5-HT 2A R-YFP, 5-HT 2B R-YFP, 5-HT 2C R-YFP, and pEYFP-N1 (Clontech, Mountain View, CA, USA) were graciously provided by Atwood laboratory (Brown University). To generate the 5-HT 2A R-Dendra2 constructs, both the 5-HT 2A R-YFP and HA-Dendra2 [ 86 ] constructs were double digested with BamHI (NEB) and NotI-HF (NEB). The digested products were gel eluted on a 1% agarose gel (Invitrogen) using a silica bead-based DNA gel extraction kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's protocol. The gel-purified Dendra2 gene and 5-HT 2A R containing pEYFP-plasmid (without YFP gene) were ligated to generate a C-terminal fusion of the 5-HT 2A receptor to the Dendra2 gene. The empty Dendra2 construct was generated in the same manner using BamHI and NotI enzymes to replace the YFP gene with the Dendra2 gene in the pEYFP plasmid. To generate the 5-HT 2B R-Dendra2 fusion construct, a cDNA fragment of the 5-HT 2B R gene was PCR amplified with a 40 nucleotide-5′ primer containing a HindIII site followed by a Kozak sequence and the first 25 nucleotides specific to 5-HT 2B R open reading frame (ORF) (Table 1) and a 3′ primer complementary sequence to the last 23 nucleotides of the ORF with a BamHI site in place of the stop codon (Table 1). Similarly, for generating the 5-HT 2C R-Dendra2 and Dopamine Receptor D2 (DRD2)-Dendra2 constructs, cDNA for both genes were amplified using individual 5′ primers with an XhoI site followed by a Kozak sequence and 20 nucleotides specific to the start sequence for each ORF, respectively, and 3′ primers complementary sequences of 26 nucleotides and 24 nucleotides, respectively, followed by a BamHI site replacing the stop codon. In the 5′ primers of DRD2 cDNA, a degenerate mutation was introduced at the 2nd codon (T→C) to remove the BamHI site. The PCR cDNA products of all three 5-HT 2B R, 5-HT 2C R, and DRD2 ORFs were purified using a silica bead DNA extraction kit (Thermo Scientific), double digested along with the empty Dendra2 plasmid for each ORF using a respective pair of restriction enzymes and gel extracted using a 1% agarose gel (Invitrogen) and a silica bead DNA extraction kit (Thermo Scientific). The gel-eluted products of each gene were ligated with the respective Dendra2 plasmids that were digested with the same pair of restriction enzymes, such that the Dendra2 gene is fused to the C-terminus of the appropriate receptor. All plasmids were sequence verified for orientation and confirmed using a CMV-F universal forward primer and a Dendra2-N reverse primer (Table 1) at The University of Maine DNA sequencing facility and analyzed using FinchTV (Version 1.4.0) software. 2.3. Generation of HEK293A Stable Cell Lines for 5-HT 2 R-Dendra2 HEK293A cells plated in a T-25 flask (25 cm 2 -Cell Star) up to 80% confluence were transfected with 13 µg of 5-HT 2A R-, 5-HT 2B R-, 5-HT 2C R–Dendra2, and DRD2-Dendra2 fusion or empty Dendra2 constructs using Fugene HD transfection reagent (Promega) according to the manufacturers' instructions. The cells were incubated at 37 °C for 48 h in DMEM containing 10% FBS, 1% P/S (Mediatech, Inc., Manassas, VA, USA), and 0.2% plasmocin prophylactic (InvivoGen). The medium was then replaced with selective media, consisting of DMEM containing 10% FBS, 1% P/S (Mediatech, Inc.), 0.2% plasmocin prophylactic (InvivoGen) and 500 µg of Geneticin G418 (Corning) to select for cells transfected with the Dendra2 constructs containing the Geneticin resistance cassette. The medium was then replaced every 3 days for 1 week. Subsequently, the cells were expanded in a T-75 flask (75-cm 2 Cell Star) under selective pressure for an additional week and confirmed for percent transfection and viability. Cells were maintained under a selective medium and the expression of constructs was verified by epifluorescence microscopy at the start and through the duration of each experiment. 2.4. Indirect Immunofluorescence Detection and Quantification of JCPyV Infection HEK293A stable 5-HT 2A R-Dendra2, 5-HT 2B R-Dendra2, 5-HT 2C R-Dendra2, and DRD2-Dendra2 cells were plated to 70% confluency in 96-well plates and infected with JCPyV at a multiplicity of infection (MOI) of 2 FFU/cell at 37 °C for 1 h and then replenished with complete DMEM and incubated at 37 °C. At 48 h postinfection, cells were washed with 1× phosphate buffer saline (PBS) and fixed using 4% paraformaldehyde (PFA) at room temperature (RT) for 10 min. After fixation, stable cells were permeabilized with 1× TBS-1% Triton X-100 for 15 min and blocked in 20% goat serum (Vector Labs, Newark, CA, USA) in 1× PBS. Following permeabilization, cells were washed and stained with PAB962 (T-antigen Ab, 1:2) at 37 °C for 1 h. Cells were then washed with 1× PBS and stained with an anti-mouse Alexa Fluor 633 secondary antibody (Thermo Fisher) at 37 °C for 1 h and stained for nuclei using DAPI stain (Thermo Fisher) at RT for 5 min. Virus infectivity was quantified by counting the number of T-antigen positive cells per five visual fields (per well, per sample), using a Nikon Eclipse Ti epifluorescence microscope (Micro Video Instruments, Inc., Avon, MA, USA). Infectivity was determined by dividing the number of infected cells per visual field by the total number of DAPI-positive nuclei per visual field as previously described [ 87 , 88 ]. The average percent infection was normalized to the control sample 5-HT 2A R-YFP (100%) [ 66 , 89 ]. Data were plotted as a bar graph faceted by the three 5-HT subtypes and the negative control using R (version 4.0.5) using the package ggplot2 [ 90 ]. 2.5. Cell-Surface Expression of 5-HT 2 R-Dendra2 Fusion Proteins in HEK293A Stable Cells 5-HT 2 R-Dendra2 fusion protein-expressing HEK293A stable cells were plated to 60% confluency in a 6 mm 96 wells (#1.5H) glass bottom plate (CellVis) along with control samples in complete DMEM with 500 µg of G418 (Corning). At 24 h post-plating, cells were washed with 1× PBS and fixed using 4% PFA (Invitrogen) for 10 min and washed again three times with 1× PBS. After washing, cells were incubated with blocking buffer (2% goat serum, 0.2% Triton X-100 and 0.1% BSA in 1× PBS) at RT for 1 h and then stained with an anti-pan-cadherin antibody (1:200, Abcam) in blocking buffer t at 4 °C O/N. Cells were washed three times with 1× PBS and stained with a secondary anti-mouse Alexa Fluor-647 antibody (1:1000) in blocking reagent at RT for 1 h. Furthermore, cells were washed and stained for nuclear marker using DAPI counterstain (1:1000) at RT for 5 min. After the final washes, cells were observed by confocal microscopy using an Olympus laser scanning confocal microscope (model IX81; Olympus America, Inc., Westborough, MA, USA) at 60× magnification (oil immersion) using FluoView software (version 04.01.01.05). Fields of view were visualized through z-sectioning using the DAPI channel, and fluorescence images were obtained for cell nuclei (blue), receptor/control (green) and anti-pan cadherin (magenta) channels using 405/635-, 543/488-nm multiline argon laser, and a 633 HeNe laser, respectively. Cell-surface expression of receptors was determined using ImageJ by defining Manders' overlap coefficient utilizing colocalization threshold analysis (FIJI) [ 91 ]. Each field of view in comparable z-planes was analyzed and represents the percentage of overlap between the pan-cadherin cell-surface marker and Dendra2/YFP expression for expressed receptors. Data were plotted in a box graph and statistics were calculated using GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA). 2.6. Analysis of JCPyV Attachment in Stable Cells 5-HT 2 R-Dendra2 fusion protein-expressing HEK293A stable cells were plated in 12-well plates and were removed upon 100% confluency using CellStripper (Corning, Corning, NY, USA), followed by centrifugation at 414× g at 4 °C for 5 min to make pellets. Cells were washed with 1× PBS, pelleted again, and resuspended in 10% complete phenol-free MEM (Corning). Cells were incubated on the ice at 4 °C for 45 min. Cells were pelleted and resuspended in 200 μL of 10% complete phenol-free MEM containing Alexa Fluor 647-labeled JCPyV and were further incubated on ice at 4 °C for 1 h to permit viral attachment. Cells were pelleted, washed with 1× PBS, fixed in 4% PFA for 10 min, and then resuspended in 300 μL of 1× PBS. For the analysis of viral attachment in fixed cells, flow cytometry was carried out by using a BD FACSCanto instrument (BD Biosciences) equipped with a 633-nm AP-C laser line (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA) for 10,000 events before analysis with BD FACSDiva (Becton, Dickinson, and Company) and FlowJo software (TreeStar, Inc., Ashland, OR, USA). To exclude complex and dead cells from the samples, FlowJo Software was used to gate the samples. 2.7. Quantification of JCPyV Entry in Stable Cells Cells seeded in a 96-well, #1.5 glass-bottomed plate (CellVis) to ~70% confluence were chilled on ice at 4 °C for 45 min prior to infection. Cells were then incubated with Alexa Fluor 647-labeled JCPyV (HEK293A cells) in MEM containing 2% FBS and 1% P/S on ice at 4 °C for 1 h for viral attachment. To allow for viral entry, the cells were further incubated at 37 °C for 1.5 h. Subsequently, the cells were washed in 1× PBS and fixed in 4% PFA for 10 min. Using 60× objective (oil immersion), cells with Dendra2 expression were viewed to define the field of view, and images were taken for differential interference contrast (DIC), Dendra2, and JCPyV-647 expression in respective channels. Viral entry was quantified by making masks in ImageJ software and generating regions of interest (ROIs) (adjusted to remove plasma membrane of cells) for at least 30 Dendra2-expressing cells in each sample type, which were further used to measure the mean fluorescent units of JCPyV-647 per cell in a background-corrected sample within an applied threshold intensity. Cross-sections were measured for at least three independent experiments. 2.8. Preparing Samples for Two-Color FPALM Experiment HEK293A stable cells were plated up to 60% confluency in three separate 6 mm, 96-well (#1.5H) glass bottom plates (CellVis) along with control samples in complete DMEM with 500 µg of G418 (Corning). After 24 h, plates were incubated at 4 °C for 1 h to chill the cells, then cells were infected with JCPyV-647 at an MOI of 3 FFU/cell. Plates were incubated at 37 °C and fixed at 0-, 5-, and 15-min post-infection (mpi) using 4% PFA at RT for 10 min. Fixed plates were washed three times with 1× PBS before processing for imaging. 2.9. Super-Resolution Fluorescence Photoactivation Localization Microscopy (FPALM) Samples were processed and imaged as described previously [ 92 , 93 ]. In brief, the experimental setup was performed using an Olympus (Tokyo, Japan) IX71 inverted microscope with 60X 1.45 NA total internal reflection fluorescence (TIRF) microscopy objective lens, 2X telescope in the detection path, and an Andor iXon + electron-multiplying charge-coupled device (DU897-DCS-#BV; Andor, Belfast, UK). Excitation was achieved by employing a 558 nm laser (CrystaLaser, Reno, NV, USA) and activation using 405 nm (FBB-405- 050-FSFS-100; RGBlase LLC, Fremont, CA, USA) in either widefield or TIRF illumination. The corresponding lasers were aligned together with mirrors and a dichroic (Z405RDC; Chroma, Rockingham, VT, USA) and directed into an f = 350 mm convex lens (Thorlabs, Newton, NJ, USA) at one focal length from the objective back aperture plane. FPALM image acquisition was carried out with a frame rate of 30–50 Hz and an electron multiplication gain of 200. For Dendra2 single-channel data acquisition, the detection filters consisted of both a T565LP (Chroma) or a 561RU dichroic (Semrock, Rochester, NY, USA), a 405 nm and two 561 nm notch filters (NF03-405E-25 and NF561E-25; Semrock), and a 605/70 emission filter (Chroma). Raw data were then processed and analyzed with custom MATLAB software (The MathWorks, Natick, MA, USA). For the determination of parameters, including fluorescent molecule locations, a nonlinear least-squares algorithm was used for fitting PSFs to two-dimensional Gaussians (an approximation of the PSF). Acquired camera frames were processed according to standard algorithms for the identification and localization of individual emitters [ 86 ]. 2.10. Glucose Oxidase Imaging Buffer For imaging, the organic dye Alexa 647, a glucose oxidase buffer (GLOX) was needed to allow the organic dye to blink for super-resolution imaging. GLOX was prepared on the day of imaging based on the protocol for dSTORM [ 12 , 94 ] and according to the following reagent concentrations: 10 µg/mL Catalase from bovine liver (Sigma-Aldrich, C9322, St. Louis, MO, USA), 50 µg/mL glucose oxidase from Aspergillus niger (Sigma-Aldrich, G2133), 10 mM Tris-HCL, 10 mM MEA, and 10% glucose diluted into PBS. All reagents were prepared individually and combined only just prior to imaging. New GLOX buffer was made every hour of imaging, as the effects of the GLOX buffer were noticeably reduced after this time. 2.11. Multicolor Widefield FPALM Imaging of JCPyV and Mock Infected Samples The experimental protocol for image acquisition was performed as described previously [ 86 , 93 ]. Laser illumination alignment setup included 405 and 558 nm lasers along with far-red 638 lasers (CrystaLaser, Reno, NV, USA) for JCPyV-647 treated samples and the illumination trail was always co-aligned with all the beams. The sets of filters and dichroic employed in the current setup comprised the following: dichroic in the microscope turret, DiO1R 405/488/561/635 (Semrock); notch filters in detection path (2× 405 nm, 2× 561 nm, and a 635 nm; all Semrock); dichroic in the two-color detection module, FF580-FDi01 (Semrock); emission filter in the red channel, a Brightline 664 LP (Semrock), and in the orange channel, and FF-01 585/40 (Semrock). Fluorescent beads (Tetraspeck microspheres, 0.1 mm; Invitrogen (Carlsbad, CA, USA)/ThermoFisher Scientific (Waltham, MA, USA)) were used for spatial orientation and calibration during image acquisition in both target channels concurrently. The final images were also double-checked systematically by using a previously described algorithm [ 86 ] to determine the transformation that best aligned the two channels. After background subtraction using a temporal median filter [ 95 ], the localization of fluorophores was performed with a pixel intensity threshold (typically 12–20 photons) for the merged pair of channels or using separate thresholds for each channel when one label was significantly brighter than the other (as for Alexa-647 and Dendra2). Localizations were scrutinized for a good fit, the number of photons per localization (within the range of 10–3000 photons), point spread function width (1/ e 2 radius 140–600 nm), localization uncertainty ( 0.80. Bleedthrough rates for this combination of probes were also estimated from the α histograms to be 0.3% (average for Dendra2 and Alexa Fluro-647) and 0.80. Bleedthrough rates for this combination of probes were also estimated from the α histograms to be 0.3% (average for Dendra2 and Alexa Fluro-647) and <1% for Dendra2 and Alexa Fluro-647. When calculating RDFs and Manders' Coefficients, bleedthrough correction was applied using the method from Dahan Kim et al., 2013. Individual molecules are binned into a small grid (20 nm) and then the number count is corrected in each pixel according to equation: n A C o r r = n A M e a s − k B A n A M e a s − k B A n B M e a s 1 − k A B − k B A n B C o r r = n B M e a s − k A B n A M e a s − k A B n B M e a s 1 − k A B − k B A where n A M e a s and n B M e a s are the binned "intensities" (number counts of molecules of different types) before bleed-through correction, n A C o r r and n B C o r r are the bleedthrough corrected values, and k A B and k B A are the rates of bleed-through from channel A into channel B and channel B to channel A, respectively. For each experiment, the magnification of the microscope was determined by using the image of a standard calibrated scale and measured using ImageJ. The calculation of the pixel-value-to-photon conversion factor was conducted using previously published methods [ 23 ]. 3. Results 3.1. 5-HT 2 R-Dendra2 Forms Clusters on the Cell Surface of Stably Transfected Cells To better define receptor clustering and the role of 5-HT 2 Rs in JCPyV infection, localization-based super-resolution microscopy was employed to visualize 5-HT 2 Rs at the plasma membrane. Plasmid constructs that express 5-HT 2 R subtypes tagged with the photoactivatable fluorescent protein Dendra2 [ 96 ] (5-HT 2A R-Dendra2, 5-HT 2B R-Dendra2, and 5-HT 2C R-Dendra2) and, for comparison, the dopamine receptor tagged with Dendra2 (DRD2) were created for use in FPALM ( Figure S1 ). DRD2 was selected because it is also a GPCR but is not required by JCPyV for entry or infection [ 65 ]. Furthermore, the expression of 5-HT 2A R, 5-HT 2B R, and 5-HT 2C R in poorly-permissive HEK293A cells restores infection [ 66 ]. Thus, to generate stable cell lines, 5-HT 2 R-Dendra2 constructs were transfected into HEK293A cells and sustained in antibiotic-selective media, as shown in the schematic representation ( Figure S1 ) and 5-HT 2 R-Dendra2 construct-expressing cells were confirmed for restoration of JCPyV infection ( Figure S2 ). To confirm cell-surface expression, the stable cell lines were plated, fixed after 24 h and stained for the cell membrane marker pan-cadherin [ 67 ]. Cell-surface expression of receptors was confirmed and measured in stable HEK293A cells expressing 5-HT 2 Rs (5-HT 2A , 5-HT 2B, and 5-HT 2C ) and DRD2 using confocal microscopy and image analysis ( Figure 1 A). Expression levels of 5-HT 2 Rs and DRD2 stable cells were compared to previously published 5-HT 2A R-YFP, 5-HT 2B R-YFP, and 5-HT 2C R-YFP stable cells that support an increase in JCPyV entry and infection compared to control HEK293A cells [ 66 , 67 , 89 ]. The stable cells demonstrated a distinct cell-surface expression in all 5-HT 2 R sample types and the DRD2 sample ( Figure 1 A). Images were analyzed to quantify the cell-surface expression of each receptor by measuring the percent overlap between the fluorescence intensities of the receptor and the stain for the cell-surface cadherins using ImageJ [ 67 ] ( Figure 1 B). The expression levels of each serotonin receptor are similar to the comparable 5-HT 2 R-YFP stable cell lines ( Figure 1 B). Altogether, the Dendra-5-HT 2 R-expressing cells represent a model system in which each 5-HT 2 R receptor type is fluorescently labeled and present at the cell surface. 3.2. Cell-Surface Characterization of 5-HT 2 Rs Using FPALM-TIRF Microscopy Proper expression of membrane-bound receptors is vital to the utilization of the receptors by ligands and downstream events [ 97 ] including viral infection [ 98 ]. To define the spatial distribution of 5-HT 2 Rs in HEK293A cells, FPALM-TIRF microscopy was employed to visualize and characterize the Dendra2-fused receptors on the plasma membrane ( Figure 2 ). Cell lines stably expressing receptors were plated on 96-well glass-bottom dishes and fixed 24 h after plating. FPALM-TIRF imaging of the Dendra2-expressing 5-HT 2 Rs revealed a clustered distribution of receptors on the plasma membrane for each 5-HT 2 R subtype (5-HT 2A , 5-HT 2B , and 5-HT 2C ) ( Figure 2 ). The observed distribution in the plasma membrane suggests that the receptors are (a) present on the cell surface and (b) clustered in a manner similar to that of other cell membrane-bound receptors viewed via TIRF microscopy in previous studies [ 99 ]. 3.3. Manders' Colocalization Analysis: 5-HT 2 Rs Colocalize with JCPyV during Virus Entry Previous studies and work from the Maginnis laboratory demonstrate that 5-HT 2 receptors are not required for JCPyV attachment, yet they are essential for virus internalization [ 66 , 67 ]. Assetta et al. utilized a proximity ligation assay (PLA) to show transient interactions between JCPyV and 5-HT 2A , 5-HT 2B , and 5-HT 2C receptor subtypes at 5 min post-infection (mpi) [ 68 ]; however, no interactions were observed at 0 and 15 mpi, and the nanoscale distribution of these 5-HT receptors during entry and infection has not been studied. To further define the interactions between JCPyV and 5-HT 2 Rs, we used two-color super-resolution FPALM to image and analyze colocalization between Alexa647-labeled purified JCPyV (JCPyV-647) with 5-HT 2A R-, 5-HT 2B R-, and 5-HT 2C R-Dendra2 fusion proteins, DRD2, and the Dendra2-only expressing stable cell lines at 0, 5, and 15 mpi ( Figure 3 ). DRD2 and Dendra2 only were used for comparison as they did not support JCPyV infection ( Figure S2 ). Colocalization of JCPyV with all the 5-HT 2 Rs was observed across all three time points ( Figure 3 B, Table 1 ). Quantification of the colocalization events was evaluated using Manders' Coefficient of Colocalization (MCC) between the Dendra2-tagged receptor and JCPyV-647, for all three timepoints, with at least three independent experiments of at least ten cells each (i.e., a total of ≥ 30 cells for each timepoint) ( Figure 3 B, Table 1 ). These results demonstrate JCPyV colocalization with 5-HT 2 R-Dendra2 for all three subtypes, 5-HT 2A R, 5-HT 2B R, and 5-HT 2C R, as well as with the DRD2 receptor through all three time points ( Figure 3 B, Table 1 )), even though DRD2 receptors are not required for viral infection ( Figure S2 ) [ 65 ]. JCPyV colocalization with receptors varies with different time points postinfection; however, significant differences were only observed between the virus and Dendra2-tagged 5-HT 2 R subtypes and the DRD2 receptor at 5 mpi ( Figure 3 B). At 0 mpi, JCPyV-647 colocalizes with 5-HT 2 Rs (5-HT 2A , 5-HT 2B , and 5-HT 2C ) as well as DRD2, and no significant difference was observed between them ( Figure 3 B, Table 1 and Table 2 ). At 5 mpi, a significant increase (58% ± 6%; p < 0.001) was observed in JCPyV-647 colocalization with 5-HT 2A R-Dendra2 when compared to JCPyV-647 colocalization with DRD2, with significance determined using a Wilcoxon Rank Sum Test ( Table 1 ). Similarly, a significant increase of 45% ± 6% ( p < 0.001) in JCPyV-647 colocalization with 5-HT 2B R-Dendra2 and a 69% ± 6% ( p < 0.001) increase in JCPyV-647 colocalization with 5-HT 2C R were observed in comparison to JCPyV-647 colocalization with DRD2 ( Figure 3 B, Table 1 and Table 2 ). At the 15 mpi, similar levels of colocalization were observed in JCPyV-647 with 5-HT 2 R subtypes compared to DRD2; however, among 5-HT 2 Rs, there was a significant increase (9%± 3%; p < 0.05) in JCPyV-647 colocalization with 5-HT 2C R compared to colocalization with 5-HT 2A R, and a 17%± 3% increase in JCPyV-647 colocalization with 5-HT 2C R compared to JCPyV-647 colocalization with 5-HT 2B R ( p < 0.001) ( Figure 3 B, Table 1 and Table 2 ). Cumulatively, the Manders' colocalization analysis demonstrates that JCPyV colocalizes with 5-HT 2 R subtypes as well as the DRD2 receptor at all three timepoints, but there is a significant increase in JCPyV colocalization with 5-HT 2 R subtypes at 5 mpi when compared to the DRD2 receptor ( Table 1 and Table 2 ). 3.4. JCPyV Attachment Is Not Enhanced in Cells Expressing 5-HT 2 Receptors JCPyV binds to host cells through α2,6-linked sialic acid receptor motifs and/or non-sialylated glycosaminoglycans (GAGs) via the attachment protein VP1 [ 62 , 63 , 64 , 100 ], and internalization is mediated by 5-HT 2 receptors [ 65 , 66 ]. Previous research has demonstrated that viral attachment to host cells remains unaffected upon the expression of 5-HT 2 receptors when compared to control cells [ 66 ], suggesting that attachment and entry constitute a two-step process. A JCPyV infectivity assay using cell lines stably expressing 5-HT 2 Rs, DRD2, or Dendra2 showed a significant increase in JCPyV infectivity of 5-HT 2 Rs-expressing cells compared to DRD2- and Dendra2-expressing cells ( Figure S2 ), confirming previous findings [ 66 ]. To test whether stable cell lines expressing Dendra2-tagged 5-HT 2 receptors show enhanced JCPyV attachment, flow cytometry was used to quantify viral attachment as a function of receptor subtype. Stable cells expressing 5-HT 2 Rs, DRD2, or Dendra2 were plated, incubated for 24 h, removed from plates, and incubated with JCPyV-647 on ice for 1 h. JCPyV-647 attachment was measured in virus-treated and mock samples using flow cytometry ( Figure 4 ). Equivalent levels of JCPyV-647 mean fluorescence intensities were observed in cells expressing 5-HT 2 Rs, DRD2, and Dendra2, suggesting that JCPyV attachment is not significantly affected by overexpression of 5-HT 2 Rs or DRD2. This confirms that the stable cells do not enhance or reduce viral attachment to host cells, and these data are consistent with previously-reported results with 5-HT 2 R-YFP [ 66 ]. 3.5. JCPyV Entry Is Enhanced in 5-HT 2 R-Expressing Cells Previous studies have established that JCPyV infection is increased when 5-HT 2 Rs are overexpressed in semi-permissible cells, such as HEK293A cells [ 66 ] and Figure S2 . Further, the 5-HT 2 R-mediated increase in JCPyV infection is attributed to a significant enhancement of viral internalization [ 65 , 66 , 67 ]. To determine whether the expression of Dendra-tagged 5-HT 2 Rs or DRD2 increases the internalization of JCPyV in stable cells, viral entry was measured using confocal microscopy. Stable cell lines expressing Dendra2-tagged 5-HT 2 Rs, DRD2, or Dendra2 were plated and incubated with JCPyV-647 on ice to synchronize viral attachment, then further incubated at 37 °C for 1.5 h for viral entry. Cells were fixed and viral internalization was measured by confocal microscopy. The quantification of the relative fluorescence intensity of JCPyV-647 within individual cells was determined using ImageJ [ 67 , 101 ]. Representative confocal images of individual samples for viral internalization ( Figure 5 A) demonstrate an increase in JCPyV-647 fluorescence in 5-HT 2 R-expressing samples compared to DRD2 and Dendra2 samples. Quantification was performed to measure internalized JCPyV-647 by generating cell masks in ImageJ, and then by measuring fluorescence intensities inside individual cells, excluding the cell membrane ( Figure 5 B) (similar to [ 101 ] but with the mask generation performed manually). Stable cells expressing 5-HT 2 Rs demonstrated a significant increase in viral internalization when compared to DRD2- and Dendra2-expressing stable cells. Taken together, these data demonstrate that the stable cell lines expressing Dendra2-tagged 5-HT 2 Rs support and enhance JCPyV internalization with equivalent efficiency. These data further suggest that while JCPyV localizes in membrane areas where DRD2 is expressed ( Figure 3 , Table 1 ), DRD2 does not enhance viral entry [ 66 ]. 3.6. JCPyV Changes Cluster Properties for 5-HT 2 Receptor Subtypes in Infected Cells The clustering of receptors at the plasma membrane and interactions between receptors and ligands results in the translation of exogenous signals into a cellular response [ 102 ]. Previous studies suggested that viral attachment to cell-surface receptors involved in endocytosis would cause spatial confinement of virus particles immediately following attachment [ 2 ]. Recent advancements in microscopic techniques have improved the analysis of spatial arrangements and patterning of cell membrane receptors and suggest that aggregation of receptor clusters can induce functional consequences that are not predictable from individual components [ 1 ]. To characterize the spatial dynamics and aggregation of 5-HT 2 receptors in the presence of JCPyV, widefield FPALM images obtained were analyzed and quantified for cluster properties of receptors in 5-HT 2 Rs, DRD2, and Dendra2 in JCPyV-infected cells at three timepoints (0, 5, and 15 mpi). The Dendra2 sample was used as a negative control for receptor cluster densities. Individual clusters were identified using both the radial distribution function and single linkage cluster analysis, and the physical properties of clusters were analyzed for cluster density, circularity, and area [ 103 ]. Figure 6 illustrates the cluster properties of 5-HT 2 R subtypes and DRD2 in cells infected and imaged at three timepoints post-infection (data shown are the average of three independent replicates with at least 10 images per sample per replicate). Comparing individual cluster properties for each sample across the three timepoints post-infection, a significant (68% ± 12%; p < 0.05) increase was observed in the median cluster density of 5-HT 2A R from 0 to 5 mpi, and a subsequent decrease in density of (−36% ± 12%; p < 0.05) was observed from the 5 min to the 15 min timepoint ( Figure 6 ). Similarly, an increase in median cluster density by 52% ± 10% ( p < 0.05) was observed for 5-HT 2B R from 0 to 5 mpi, and a decrease in density of −30% ± 9% ( p < 0.05) was observed from 5 mpi to 15 mpi. The trend was also observed in the 5-HT 2C R sample, with an increase of 49% ± 10% ( p < 0.05) in median cluster density at 5 min compared to 0 mpi, while a decrease of −28% ± 10% ( p < 0.05) was observed in mean cluster density from 5 to 15 mpi. Significance was determined using a Kruskal–Wallis test. No such significant change in median cluster density was observed between the three time points for the control DRD2 and Dendra2 samples. Considering cluster area, the only significant change was observed in the 5-HT 2B R sample, in which the median cluster area decreased −9% ± 5% ( p < 0.05) from 0 mpi to 5 mpi. Furthermore, a significant change in cluster circularity was only observed in the 5-HT 2B R sample, with median cluster circularity decreasing −12% ± 3% ( p < 0.05) from the 5 min to the 15 min timepoint. Collectively, these data represent a significant increase in median cluster densities of 5-HT 2 receptor subtypes from 0 mpi to 5 mpi, and a subsequent decrease in those same densities from 5 mpi to 15 mpi. This significant increase in median receptor-cluster density was only observed in cells stably expressing Dendra2-tagged 5-HT 2 Rs and was not observed in the DRD2 or control (Dendra2) samples. Our data demonstrate that JCPyV changes the cluster properties of 5-HT 2 receptors in the plasma membrane of host cells during viral attachment and entry, suggesting that alterations in 5-HT 2 receptor patterning are associated with JCPyV entry. 3.7. Radial Distribution Function Analysis: JCPyV Aggregation within and Adjacent to 5-HT 2 Receptor Clusters Recent data highlight the importance of the characterization of spatial patterning of cell surface receptor signaling clusters, as their patterns can reveal aspects of their function which are invisible to non-imaging methods [ 1 ]. To characterize the spatial distribution of JCPyV localized in or adjacent to 5-HT 2 R receptor clusters of infected cells, the radial distribution functions (RDFs), denoted by amplitude (g) as a function of radius (r), were calculated for the identified clusters of 5-HT 2 R subtypes and DRD2 in JCPyV-infected cells, as well as for JCPyV-647 particles in the vicinity. JCPyV-647 RDF calculations were performed for infected cells expressing 5-HT 2 Rs and DRD2 at 0, 5, and 15 mpi ( Figure 7 ). Each row represents JCPyV-RDFs for all samples at the given timepoint (i.e., Figure 7 A represents JCPyV-RDFs at 0 min, Figure 7 B represents JCPyV-RDFs at 5 min, and Figure 7 C shows JCPyV-RDFs for all samples at 15 mpi). In individual graphs, the g(r) value (y-axis) indicates the probability of an average JCPyV being found at a given distance r (x-axis) from the center of the cluster, with g(r) = 1 expected for a random distribution of the virus. Results demonstrate the concentration of JCPyV molecules toward the center of a 5-HT 2 R cluster at all three timepoints ( Figure 7 ). Significant changes in the RDFs of JCPyV particles within 5-HT 2 R or DRD2 receptor clusters were not observed at 0 mpi. However, at 5 mpi, a significant increase ( p < 0.01 with the Kruskal–Wallis test) in the density of JCPyV particles adjacent to or within the receptor clusters was observed in JCPyV-RDFs when comparing infected samples expressing 5-HT 2 R-Dendra2 with infected samples expressing DRD2-Dendra2. No significant difference was observed for JCPyV-RDFs between infected cells expressing various 5-HT 2 R subtypes. Furthermore, no significant difference was observed between the JCPyV-RDFs comparing infected cells expressing 5-HT 2 Rs or DRD2 samples at 15 mpi ( Figure 7 ). Altogether, these data show a significant increase in the relative density of JCPyV particles adjacent to or within 5-HT 2 Rs clusters in infected cells at 5 mpi when compared to infected cells expressing DRD2. These results demonstrate the enhanced aggregation of virus particles within 5HT 2 receptor clusters at 5 mpi, which is a time consistent with the colocalization of JCPyV with clathrin and viral entry [ 67 ]. 3.1. 5-HT 2 R-Dendra2 Forms Clusters on the Cell Surface of Stably Transfected Cells To better define receptor clustering and the role of 5-HT 2 Rs in JCPyV infection, localization-based super-resolution microscopy was employed to visualize 5-HT 2 Rs at the plasma membrane. Plasmid constructs that express 5-HT 2 R subtypes tagged with the photoactivatable fluorescent protein Dendra2 [ 96 ] (5-HT 2A R-Dendra2, 5-HT 2B R-Dendra2, and 5-HT 2C R-Dendra2) and, for comparison, the dopamine receptor tagged with Dendra2 (DRD2) were created for use in FPALM ( Figure S1 ). DRD2 was selected because it is also a GPCR but is not required by JCPyV for entry or infection [ 65 ]. Furthermore, the expression of 5-HT 2A R, 5-HT 2B R, and 5-HT 2C R in poorly-permissive HEK293A cells restores infection [ 66 ]. Thus, to generate stable cell lines, 5-HT 2 R-Dendra2 constructs were transfected into HEK293A cells and sustained in antibiotic-selective media, as shown in the schematic representation ( Figure S1 ) and 5-HT 2 R-Dendra2 construct-expressing cells were confirmed for restoration of JCPyV infection ( Figure S2 ). To confirm cell-surface expression, the stable cell lines were plated, fixed after 24 h and stained for the cell membrane marker pan-cadherin [ 67 ]. Cell-surface expression of receptors was confirmed and measured in stable HEK293A cells expressing 5-HT 2 Rs (5-HT 2A , 5-HT 2B, and 5-HT 2C ) and DRD2 using confocal microscopy and image analysis ( Figure 1 A). Expression levels of 5-HT 2 Rs and DRD2 stable cells were compared to previously published 5-HT 2A R-YFP, 5-HT 2B R-YFP, and 5-HT 2C R-YFP stable cells that support an increase in JCPyV entry and infection compared to control HEK293A cells [ 66 , 67 , 89 ]. The stable cells demonstrated a distinct cell-surface expression in all 5-HT 2 R sample types and the DRD2 sample ( Figure 1 A). Images were analyzed to quantify the cell-surface expression of each receptor by measuring the percent overlap between the fluorescence intensities of the receptor and the stain for the cell-surface cadherins using ImageJ [ 67 ] ( Figure 1 B). The expression levels of each serotonin receptor are similar to the comparable 5-HT 2 R-YFP stable cell lines ( Figure 1 B). Altogether, the Dendra-5-HT 2 R-expressing cells represent a model system in which each 5-HT 2 R receptor type is fluorescently labeled and present at the cell surface. 3.2. Cell-Surface Characterization of 5-HT 2 Rs Using FPALM-TIRF Microscopy Proper expression of membrane-bound receptors is vital to the utilization of the receptors by ligands and downstream events [ 97 ] including viral infection [ 98 ]. To define the spatial distribution of 5-HT 2 Rs in HEK293A cells, FPALM-TIRF microscopy was employed to visualize and characterize the Dendra2-fused receptors on the plasma membrane ( Figure 2 ). Cell lines stably expressing receptors were plated on 96-well glass-bottom dishes and fixed 24 h after plating. FPALM-TIRF imaging of the Dendra2-expressing 5-HT 2 Rs revealed a clustered distribution of receptors on the plasma membrane for each 5-HT 2 R subtype (5-HT 2A , 5-HT 2B , and 5-HT 2C ) ( Figure 2 ). The observed distribution in the plasma membrane suggests that the receptors are (a) present on the cell surface and (b) clustered in a manner similar to that of other cell membrane-bound receptors viewed via TIRF microscopy in previous studies [ 99 ]. 3.3. Manders' Colocalization Analysis: 5-HT 2 Rs Colocalize with JCPyV during Virus Entry Previous studies and work from the Maginnis laboratory demonstrate that 5-HT 2 receptors are not required for JCPyV attachment, yet they are essential for virus internalization [ 66 , 67 ]. Assetta et al. utilized a proximity ligation assay (PLA) to show transient interactions between JCPyV and 5-HT 2A , 5-HT 2B , and 5-HT 2C receptor subtypes at 5 min post-infection (mpi) [ 68 ]; however, no interactions were observed at 0 and 15 mpi, and the nanoscale distribution of these 5-HT receptors during entry and infection has not been studied. To further define the interactions between JCPyV and 5-HT 2 Rs, we used two-color super-resolution FPALM to image and analyze colocalization between Alexa647-labeled purified JCPyV (JCPyV-647) with 5-HT 2A R-, 5-HT 2B R-, and 5-HT 2C R-Dendra2 fusion proteins, DRD2, and the Dendra2-only expressing stable cell lines at 0, 5, and 15 mpi ( Figure 3 ). DRD2 and Dendra2 only were used for comparison as they did not support JCPyV infection ( Figure S2 ). Colocalization of JCPyV with all the 5-HT 2 Rs was observed across all three time points ( Figure 3 B, Table 1 ). Quantification of the colocalization events was evaluated using Manders' Coefficient of Colocalization (MCC) between the Dendra2-tagged receptor and JCPyV-647, for all three timepoints, with at least three independent experiments of at least ten cells each (i.e., a total of ≥ 30 cells for each timepoint) ( Figure 3 B, Table 1 ). These results demonstrate JCPyV colocalization with 5-HT 2 R-Dendra2 for all three subtypes, 5-HT 2A R, 5-HT 2B R, and 5-HT 2C R, as well as with the DRD2 receptor through all three time points ( Figure 3 B, Table 1 )), even though DRD2 receptors are not required for viral infection ( Figure S2 ) [ 65 ]. JCPyV colocalization with receptors varies with different time points postinfection; however, significant differences were only observed between the virus and Dendra2-tagged 5-HT 2 R subtypes and the DRD2 receptor at 5 mpi ( Figure 3 B). At 0 mpi, JCPyV-647 colocalizes with 5-HT 2 Rs (5-HT 2A , 5-HT 2B , and 5-HT 2C ) as well as DRD2, and no significant difference was observed between them ( Figure 3 B, Table 1 and Table 2 ). At 5 mpi, a significant increase (58% ± 6%; p < 0.001) was observed in JCPyV-647 colocalization with 5-HT 2A R-Dendra2 when compared to JCPyV-647 colocalization with DRD2, with significance determined using a Wilcoxon Rank Sum Test ( Table 1 ). Similarly, a significant increase of 45% ± 6% ( p < 0.001) in JCPyV-647 colocalization with 5-HT 2B R-Dendra2 and a 69% ± 6% ( p < 0.001) increase in JCPyV-647 colocalization with 5-HT 2C R were observed in comparison to JCPyV-647 colocalization with DRD2 ( Figure 3 B, Table 1 and Table 2 ). At the 15 mpi, similar levels of colocalization were observed in JCPyV-647 with 5-HT 2 R subtypes compared to DRD2; however, among 5-HT 2 Rs, there was a significant increase (9%± 3%; p < 0.05) in JCPyV-647 colocalization with 5-HT 2C R compared to colocalization with 5-HT 2A R, and a 17%± 3% increase in JCPyV-647 colocalization with 5-HT 2C R compared to JCPyV-647 colocalization with 5-HT 2B R ( p < 0.001) ( Figure 3 B, Table 1 and Table 2 ). Cumulatively, the Manders' colocalization analysis demonstrates that JCPyV colocalizes with 5-HT 2 R subtypes as well as the DRD2 receptor at all three timepoints, but there is a significant increase in JCPyV colocalization with 5-HT 2 R subtypes at 5 mpi when compared to the DRD2 receptor ( Table 1 and Table 2 ). 3.4. JCPyV Attachment Is Not Enhanced in Cells Expressing 5-HT 2 Receptors JCPyV binds to host cells through α2,6-linked sialic acid receptor motifs and/or non-sialylated glycosaminoglycans (GAGs) via the attachment protein VP1 [ 62 , 63 , 64 , 100 ], and internalization is mediated by 5-HT 2 receptors [ 65 , 66 ]. Previous research has demonstrated that viral attachment to host cells remains unaffected upon the expression of 5-HT 2 receptors when compared to control cells [ 66 ], suggesting that attachment and entry constitute a two-step process. A JCPyV infectivity assay using cell lines stably expressing 5-HT 2 Rs, DRD2, or Dendra2 showed a significant increase in JCPyV infectivity of 5-HT 2 Rs-expressing cells compared to DRD2- and Dendra2-expressing cells ( Figure S2 ), confirming previous findings [ 66 ]. To test whether stable cell lines expressing Dendra2-tagged 5-HT 2 receptors show enhanced JCPyV attachment, flow cytometry was used to quantify viral attachment as a function of receptor subtype. Stable cells expressing 5-HT 2 Rs, DRD2, or Dendra2 were plated, incubated for 24 h, removed from plates, and incubated with JCPyV-647 on ice for 1 h. JCPyV-647 attachment was measured in virus-treated and mock samples using flow cytometry ( Figure 4 ). Equivalent levels of JCPyV-647 mean fluorescence intensities were observed in cells expressing 5-HT 2 Rs, DRD2, and Dendra2, suggesting that JCPyV attachment is not significantly affected by overexpression of 5-HT 2 Rs or DRD2. This confirms that the stable cells do not enhance or reduce viral attachment to host cells, and these data are consistent with previously-reported results with 5-HT 2 R-YFP [ 66 ]. 3.5. JCPyV Entry Is Enhanced in 5-HT 2 R-Expressing Cells Previous studies have established that JCPyV infection is increased when 5-HT 2 Rs are overexpressed in semi-permissible cells, such as HEK293A cells [ 66 ] and Figure S2 . Further, the 5-HT 2 R-mediated increase in JCPyV infection is attributed to a significant enhancement of viral internalization [ 65 , 66 , 67 ]. To determine whether the expression of Dendra-tagged 5-HT 2 Rs or DRD2 increases the internalization of JCPyV in stable cells, viral entry was measured using confocal microscopy. Stable cell lines expressing Dendra2-tagged 5-HT 2 Rs, DRD2, or Dendra2 were plated and incubated with JCPyV-647 on ice to synchronize viral attachment, then further incubated at 37 °C for 1.5 h for viral entry. Cells were fixed and viral internalization was measured by confocal microscopy. The quantification of the relative fluorescence intensity of JCPyV-647 within individual cells was determined using ImageJ [ 67 , 101 ]. Representative confocal images of individual samples for viral internalization ( Figure 5 A) demonstrate an increase in JCPyV-647 fluorescence in 5-HT 2 R-expressing samples compared to DRD2 and Dendra2 samples. Quantification was performed to measure internalized JCPyV-647 by generating cell masks in ImageJ, and then by measuring fluorescence intensities inside individual cells, excluding the cell membrane ( Figure 5 B) (similar to [ 101 ] but with the mask generation performed manually). Stable cells expressing 5-HT 2 Rs demonstrated a significant increase in viral internalization when compared to DRD2- and Dendra2-expressing stable cells. Taken together, these data demonstrate that the stable cell lines expressing Dendra2-tagged 5-HT 2 Rs support and enhance JCPyV internalization with equivalent efficiency. These data further suggest that while JCPyV localizes in membrane areas where DRD2 is expressed ( Figure 3 , Table 1 ), DRD2 does not enhance viral entry [ 66 ]. 3.6. JCPyV Changes Cluster Properties for 5-HT 2 Receptor Subtypes in Infected Cells The clustering of receptors at the plasma membrane and interactions between receptors and ligands results in the translation of exogenous signals into a cellular response [ 102 ]. Previous studies suggested that viral attachment to cell-surface receptors involved in endocytosis would cause spatial confinement of virus particles immediately following attachment [ 2 ]. Recent advancements in microscopic techniques have improved the analysis of spatial arrangements and patterning of cell membrane receptors and suggest that aggregation of receptor clusters can induce functional consequences that are not predictable from individual components [ 1 ]. To characterize the spatial dynamics and aggregation of 5-HT 2 receptors in the presence of JCPyV, widefield FPALM images obtained were analyzed and quantified for cluster properties of receptors in 5-HT 2 Rs, DRD2, and Dendra2 in JCPyV-infected cells at three timepoints (0, 5, and 15 mpi). The Dendra2 sample was used as a negative control for receptor cluster densities. Individual clusters were identified using both the radial distribution function and single linkage cluster analysis, and the physical properties of clusters were analyzed for cluster density, circularity, and area [ 103 ]. Figure 6 illustrates the cluster properties of 5-HT 2 R subtypes and DRD2 in cells infected and imaged at three timepoints post-infection (data shown are the average of three independent replicates with at least 10 images per sample per replicate). Comparing individual cluster properties for each sample across the three timepoints post-infection, a significant (68% ± 12%; p < 0.05) increase was observed in the median cluster density of 5-HT 2A R from 0 to 5 mpi, and a subsequent decrease in density of (−36% ± 12%; p < 0.05) was observed from the 5 min to the 15 min timepoint ( Figure 6 ). Similarly, an increase in median cluster density by 52% ± 10% ( p < 0.05) was observed for 5-HT 2B R from 0 to 5 mpi, and a decrease in density of −30% ± 9% ( p < 0.05) was observed from 5 mpi to 15 mpi. The trend was also observed in the 5-HT 2C R sample, with an increase of 49% ± 10% ( p < 0.05) in median cluster density at 5 min compared to 0 mpi, while a decrease of −28% ± 10% ( p < 0.05) was observed in mean cluster density from 5 to 15 mpi. Significance was determined using a Kruskal–Wallis test. No such significant change in median cluster density was observed between the three time points for the control DRD2 and Dendra2 samples. Considering cluster area, the only significant change was observed in the 5-HT 2B R sample, in which the median cluster area decreased −9% ± 5% ( p < 0.05) from 0 mpi to 5 mpi. Furthermore, a significant change in cluster circularity was only observed in the 5-HT 2B R sample, with median cluster circularity decreasing −12% ± 3% ( p < 0.05) from the 5 min to the 15 min timepoint. Collectively, these data represent a significant increase in median cluster densities of 5-HT 2 receptor subtypes from 0 mpi to 5 mpi, and a subsequent decrease in those same densities from 5 mpi to 15 mpi. This significant increase in median receptor-cluster density was only observed in cells stably expressing Dendra2-tagged 5-HT 2 Rs and was not observed in the DRD2 or control (Dendra2) samples. Our data demonstrate that JCPyV changes the cluster properties of 5-HT 2 receptors in the plasma membrane of host cells during viral attachment and entry, suggesting that alterations in 5-HT 2 receptor patterning are associated with JCPyV entry. 3.7. Radial Distribution Function Analysis: JCPyV Aggregation within and Adjacent to 5-HT 2 Receptor Clusters Recent data highlight the importance of the characterization of spatial patterning of cell surface receptor signaling clusters, as their patterns can reveal aspects of their function which are invisible to non-imaging methods [ 1 ]. To characterize the spatial distribution of JCPyV localized in or adjacent to 5-HT 2 R receptor clusters of infected cells, the radial distribution functions (RDFs), denoted by amplitude (g) as a function of radius (r), were calculated for the identified clusters of 5-HT 2 R subtypes and DRD2 in JCPyV-infected cells, as well as for JCPyV-647 particles in the vicinity. JCPyV-647 RDF calculations were performed for infected cells expressing 5-HT 2 Rs and DRD2 at 0, 5, and 15 mpi ( Figure 7 ). Each row represents JCPyV-RDFs for all samples at the given timepoint (i.e., Figure 7 A represents JCPyV-RDFs at 0 min, Figure 7 B represents JCPyV-RDFs at 5 min, and Figure 7 C shows JCPyV-RDFs for all samples at 15 mpi). In individual graphs, the g(r) value (y-axis) indicates the probability of an average JCPyV being found at a given distance r (x-axis) from the center of the cluster, with g(r) = 1 expected for a random distribution of the virus. Results demonstrate the concentration of JCPyV molecules toward the center of a 5-HT 2 R cluster at all three timepoints ( Figure 7 ). Significant changes in the RDFs of JCPyV particles within 5-HT 2 R or DRD2 receptor clusters were not observed at 0 mpi. However, at 5 mpi, a significant increase ( p < 0.01 with the Kruskal–Wallis test) in the density of JCPyV particles adjacent to or within the receptor clusters was observed in JCPyV-RDFs when comparing infected samples expressing 5-HT 2 R-Dendra2 with infected samples expressing DRD2-Dendra2. No significant difference was observed for JCPyV-RDFs between infected cells expressing various 5-HT 2 R subtypes. Furthermore, no significant difference was observed between the JCPyV-RDFs comparing infected cells expressing 5-HT 2 Rs or DRD2 samples at 15 mpi ( Figure 7 ). Altogether, these data show a significant increase in the relative density of JCPyV particles adjacent to or within 5-HT 2 Rs clusters in infected cells at 5 mpi when compared to infected cells expressing DRD2. These results demonstrate the enhanced aggregation of virus particles within 5HT 2 receptor clusters at 5 mpi, which is a time consistent with the colocalization of JCPyV with clathrin and viral entry [ 67 ]. 4. Discussion While previous work has detailed structures of viral proteins in complex with their receptors [ 104 , 105 , 106 ], gaps remain in our understanding of the dynamics of these interactions. Super-resolution microscopy techniques have enabled the nanoscale visualization of viruses and interactions with specific host cell receptors in living cells [ 14 ]. Plasma membrane expression and arrangement/organization of cell surface receptors in nanoscale domains are also crucial for proper induction and spatiotemporal control of signaling pathways associated with these receptors. GPCRs tend to generate clusters in nanoscopic domains, which are essential for adjusting ligand sensitivity, which controls protein interactions and signaling [ 3 ]. Previous studies have established that JCPyV can enter cells in a receptor-independent manner using extracellular vesicles [ 60 ] or in a receptor-dependent mechanism [ 66 , 67 , 68 ]. In a receptor-supported infection, JCPyV binds to α2,6-linked sialic acid receptors including lactoseries tetrasaccharide c (LSTc) [ 61 , 62 , 63 ] and/or non-sialylated glycosaminoglycans (GAGs) on host cells via the capsid protein VP1 [ 64 ] to initiate infection, and JCPyV then utilizes 5-HT 2 receptors to enter the host cell [ 65 , 66 , 67 ]. However, it is yet to be demonstrated how JCPyV reorganizes 5-HT 2 receptors to drive the internalization of virus particles. The present study was designed to observe the spatiotemporal patterning of 5-HT 2 receptors in the presence of JCPyV, which can serve as a model for other virus-receptor or ligand-5-HT 2 receptor interactions. Using super-resolution microscopy (FPALM) [ 11 ], our findings illuminate the dynamics of 5-HT 2 receptors during virus internalization ( Figure 8 ). In this study, cells stably expressing 5-HT 2 R (5-HT 2A R-Dendra2, 5-HT 2B R-Dendra2 , and 5-HT 2C R-Dendra2) subtypes resulted in increased JCPyV infectivity compared to dopamine-receptor-Dendra2 (DRD2) and control Dendra2-expressing cells ( Figure S2 ), consistent with previously published results [ 66 , 67 , 89 ]. Furthermore, 5-HT 2 Rs labeled with Dendra2 (5-HT 2A , 5-HT 2B, and 5-HT 2C ) were observed on the cell surface, and expression of 5-HT 2 receptors did not impact JCPyV attachment but rather increased viral internalization compared to controls ( Figure 4 , Figure 5 and Figure 8 ), as expected [ 66 , 67 ]. Using super-resolution microscopy, we demonstrate the nanoscale colocalization of JCPyV with the three 5-HT 2 receptor subtypes (5-HT 2A , 5-HT 2B, and 5-HT 2C ) during the initial steps of viral attachment and viral entry ( Figure 3 ), suggesting that the viral utilization of host receptors might be dependent on localization and clustering in the proximity of 5-HT 2 receptors. Transient colocalization of JCPyV with 5-HT 2 receptor subtypes has been previously reported using the proximity ligation assay [ 65 , 68 ]; however, our data further highlight the differences between JCPyV colocalization with individual 5-HT 2 receptors and their nanoscale cluster formation at all three timepoints using super-resolution microscopy. Furthermore, the data presented herein demonstrate significant increases in the mean cluster density of 5-HT 2 receptors in JCPyV-infected cells at 5 mpi ( Figure 6 ) during which time JCPyV has been shown to induce endocytosis. Additionally, this study identified an increase in aggregation of JCPyV particles colocalized with the 5-HT 2 receptor clusters at 5 mpi ( Figure 7 and Figure 8 ). The findings presented enhance our understanding of cell-surface receptor cluster properties in response to viral infection and illuminate how receptor reorganization can influence critical steps in viral infection. Data from FPALM images of infected 5-HT 2 -expressing cells show that JCPyV colocalizes with 5-HT 2 receptors during the viral attachment and entry process. Even though JCPyV colocalization was observed at all three time points (0, 5, and 15 min) postinfection, the colocalization of JCPyV was observed to be significantly higher with 5-HT 2 receptors compared to DRD2-receptors at 5 mpi ( Figure 3 ). Published data strengthen our results showing higher colocalization of JCPyV with 5-HT 2 receptors at 5 mpi [ 65 , 68 ]. These data suggest the persistent colocalization of JCPyV with 5-HT 2 receptors through viral attachment and entry might cause changes in the clustering of 5-HT 2 receptors. In recent years, advancements in microscopy have proven to be vital for studying such nanoscopic domains of receptor clusters, providing deeper insight into signal initiation and transduction mechanisms [ 107 , 108 , 109 ]. Our data emphasize high-resolution details for virus–receptor studies which are currently limited. Previous findings demonstrated that some agonists could cause a redistribution of GPCRs in the plasma membrane, such as μ opioid receptor clusters upon activation with the ligand DAMGO, leading to receptor clustering followed by endocytosis [ 78 , 80 ]. Our data highlight that JCPyV induces clustering of 5-HT 2 receptors at length scales inaccessible to diffraction-limited microscopy. Upon cluster analysis of infected cells, changes in cluster attributes, such as mean cluster density, mean cluster circularity and mean cluster area was investigated for 5-HT 2 receptor clusters. A significant increase in the mean cluster density of 5-HT 2 receptor clusters at 5 mpi compared to the cluster densities at 0 mpi was observed ( Figure 6 ). Such trends were not observed for DRD2 and Dendra2 samples. Furthermore, to confirm that the increase in the cluster density was due to JCPyV, RDF analyses were performed to measure the probability distribution of molecules in receptor clusters. Results show a significant increase in the aggregation of JCPyV particles within and adjacent to the 5-HT 2 receptor clusters in infected cells at 5 mpi, with no significant increase observed in the control sample ( Figure 7 ). These findings suggest that JCPyV aggregation in the cluster of 5-HT 2 receptors might be responsible for the increase in receptor cluster density which eventually leads to endocytosis of the virus-receptor complex. Previous studies established that JCPyV utilizes clathrin-mediated endocytosis for internalization and JCPyV co-localizes with clathrin at 5 mpi [ 67 , 110 ]. Furthermore, the 5-HT 2A receptor takes 2–10 min to internalize during the receptor recycling process [ 111 ]. These findings suggest that JCPyV-5-HT 2 receptor complexes may be endocytosed between 5 and 15 mpi; subsequently, as the higher density clusters are presumably internalized, the mean cluster density of 5-HT 2 receptor clusters and the density of JCPyV particles within the 5-HT 2 receptor clusters decreases ( Figure 8 ). Plasma membrane receptors form clusters in dynamic nano-domains on the cell surface that regulate ligand sensitivity to finetune signaling efficiency and control protein interactions [ 2 ], examples including GPCRs [ 3 ], immune-cell receptors [ 1 ], as well as receptors hijacked by microbial toxins [ 4 ] or viruses [ 5 ]. As human diseases are correlated to the aberrations in the distribution of membrane-bound receptors and/or their activation, it is important to characterize and understand the mechanisms underlying the dynamic rearrangement and clustering of receptors, as it may aid in developing novel strategies for therapeutic treatment of diseases [ 8 ]. In the past two decades, several studies have emphasized the importance of the spatial localization of GPCRs to the response of specific signaling pathways [ 6 , 7 ]. Recent studies demonstrate how the organization of GPCRs at the plasma membrane in intracellular membranes provides platforms for distinctive signaling, which are critical for key physiological functions [ 6 ]. 5-HT receptors, like other GPCRs, are known to cluster in membrane domains where they interact with other proteins, such as scaffolding protein postsynaptic density 95 (PSD95) and caveolin-1, which are regulated for the internalization process [ 112 , 113 ]. Increased use of super-resolution microscopy techniques has been helpful for the nanoscale study of GPCRs, including the formation of homo- and hetero-oligomers and other kinds of clusters [ 9 , 10 ]. While 5-HTRs are capable of homo- and hetero-oligomerization [ 114 , 115 , 116 ], the current study focused only on the expression of a single 5-HT 2 R subtype. Further, HEK293A cells utilized in this study, express very low levels of 5-HT 2 Rs [ 66 , 89 ], and thus it is not expected that expression of endogenous 5-HT 2 Rs would have driven significant homo- or hetero-oligomerization to impact the findings reported herein. DRD2 was included in this study because it is a GPCR but is not supportive of JCPyV infection ( Figure S2 ) [ 65 ]. However, we observed colocalization of JCPyV-647 with DRD2-Dendra2 at surface levels comparable to the 5-HT 2 Rs at all three timepoints ( Figure 3 ), yet DRD2 did not enhance viral entry into the cell ( Figure 5 ). Furthermore, while the cluster properties and colocalization of JCPyV with 5-HT 2 Rs changed dynamically with time, these trends were not observed for DRD2. These data suggest that JCPyV localization with DRD2 could be either due to a specific interaction between DRD2 and JCPyV or due to heterodimerization between the GPCRs, DRD2 and 5-HT 2 R [ 117 ], that serve as a target for JCPyV during infection [ 117 , 118 ]. An alternative explanation is that DRD2-5-HT 2 R heterodimers may act as a decoy receptor to limit host-cell infection. Furthermore, our results indicate that there are alterations in cluster properties of 5-HT 2 Rs upon infection. Using RDF and cluster analysis, changes in cluster properties and significant increases in the density of JCPyV particles adjacent to or within receptor clusters were observed at 5 mpi for 5-HT 2 Rs, a time consistent with endocytosis [ 111 ], while JCPyV particle density was not significantly increased in DRD2 clusters ( Figure 7 ). These data are supported by previous work [ 68 ] and suggest that aggregation of the virus at 5 mpi within receptor clusters may be due to viral endocytosis via 5-HT 2 Rs, while JCPyV interactions with 5-HT 2 Rs or DRD2 at 0 and 15 mpi could activate signaling events necessary for entry and infection. Additional studies would be required to further define whether DRD2 plays a specific role in JCPyV infection and to define whether GPCR-induced signals orchestrate JCPyV infection. Understanding the spatial arrangement and dynamics of viral and host cell components in cellular membranes is essential to elucidating virus-host interactions and their functional consequences [ 1 ]. Insight into the JCPyV attachment and entry processes can provide valuable knowledge for the development of treatments for the fatal disease PML and other viral-mediated diseases. With advancements in optics, spectroscopy, and nanoscale surface patterning, novel methodologies have brought a new perspective in understanding how cells respond to the environment and can help clarify mechanisms of molecular interaction and signaling that are not feasible by other methods [ 2 ].

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9816396/

Spondyloarthritides: Theories and beyond

Spondyloarthritides (SpA) are a family of interrelated rheumatic disorders with a typical disease onset ranging from childhood to middle age. If left untreated, they lead to a severe decrease in patients' quality of life. A succesfull treatment strategy starts with an accurate diagnosis which is achieved through careful analysis of medical symptoms. Classification criterias are used to this process and are updated on a regular basis. Although there is a lack of definite knowledge on the disease etiology of SpA, several studies have paved the way for understanding plausible risk factors and developing treatment strategies. The significant increase of HLA-B27 positivity in SpA patients makes it a strong candidate as a predisposing factor and several theories have been proposed to explain HLA-B27 driven disease progression. However, the presence of HLA-B27 negative patients underlines the presence of additional risk factors. The current treatment options for SpAs are Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), TNF inhibitors (TNFis), Disease-Modifying Anti-Rheumatic Drugs (DMARDs) and physiotherapy yet there are ongoing clinical trials. Anti IL17 drugs and targeted synthetic DMARDs such as JAK inhibitors are also emerging as treatment alternatives. This review discusses the current diagnosis criteria, treatment options and gives an overview of the previous findings and theories to clarify the possible contributors to SpA pathogenesis with a focus on Ankylosing Spondylitis (AS) and enthesitis-related arthritis (ERA). Introduction SpA is a group of immune system related disorders predominantly causing sterile inflammation at sacroiliac joints. In adults, patients often meet the definition of axial spondyloarthritis. In addition to AS, reactive arthritis, psoriatic arthritis, enteropathic arthritis, Reiter's syndrome, Inflammatory Bowel Disease (IBD)-associated arthritis and undifferentiated SpA can be included within this disease subset ( 1 ). The classification and terminology of juvenile SpA (JSpA) patients differ from the adults. Childhood onset patients are classified as ERA and juvenile onset psoriatic arthritis (JPsA) and juvenile idiopathic arthritis (JIA) is used as an umbrella term for these arthritides ( 2 ). In Europe and North America 10% of JIA patients are diagnosed as ERA ( 3 , 4 ) whereas this ratio increases further to 35%–40% in Asia ( 5 – 8 ). SpAs affects up to 2% of the population ( 1 ). The prevalence of the disease is highest in Europe followed by an Asian population whereas it is uncommon in Africans ( 9 ). SpA patients suffer from a significant decrease in their quality of life and may even need surgical operations as a remedy. The therapeutic agents used for the disease may cause side-effects (i.e., infection) and a certain portion of patients fail to respond to therapy ( 10 , 11 ). Overall, it is clear that the development of alternative treatment strategies are necessary however, the obscure disease etiology plays a negative role in this process. Although there are studies underlining the possible contribution of HLA-B27 allele in disease pathogenesis, the presence of HLA-B27 negative SpA patients indicates the presence of extra risk factors ( 12 ). Clinical features Similar to other diseases the early diagnosis of SpA is crucial. Delays may result in increased disease activity, irreversible structural damage, low therapy response and limited mobility ( 13 ). Physicians and patients should work hand in hand for early diagnosis to eliminate undesirable long-term effects. Diagnosis might be stalled if patients delay visiting a doctor due to limited access or in the belief that their symptoms will disappear spontaneously. Moreover, seeing other specialists rather than a rheumatologist might not only cause a delay but also may result in misdiagnosis ( 14 ). Thorough and distinctive analysis of the symptoms plays a fundamental role in the validity of diagnosis. SpA patients display several common clinical and laboratory findings such as arthritis ( Figure 1A ), psoriasis ( Figure 1A ), enthesitis, anterior uveitis ( Figure 1B ), inflammatory low back pain and family history of HLA-B27-related disease. Although the common features remain the same, the clinical phenotype differs across the ages in certain aspects with peripheral arthritis being predominant in JSpA and axial manifestations being more common in adult-onset disease ( 15 ). Inflammatory back pain is the most common complaint of SpA suggesting axial involvement. Shoulder and hip joint involvement is also more common in ERA ( 16 ). In fact the differences between childhood and adult- onset disease have been highlighted in a number of studies. Both are more common in males. Childhood cases typically present in adolescent years with arthritis in the big joints and often enthesitis ( 17 ). The most frequently involved joints are the knee (40%–50%), hip (30%–40%) and ankle (25%–40%) ( 16 , 18 ). Axial disease and back pain are less than expected in adult-onset disease. The frequency of axial involvement differs between studies. In a systematic review of the literature, comparing juvenile-onset AS (JoAS) and adult-onset AS (AoAS) cohorts showed that axial disease is significantly more frequent in AoAS than JoAS cases (4.3%–74% vs. 56%–95%) ( 19 ). Again family history seems to be more common in childhood-onset disease and may suggest a higher genetic load associated with the disease. Despite these differences, the pathogenesis of these two different onsets are similar; thanks to the recent emerging data we know the main pathways involved and it may not be appropriate to classify the childhood-onset disease separately from the adult one, under the "idiopathic" term anymore. Figure 1 Clinical findings in SpA patients. ( A ) Black arrow shows the arthritis and white arrow shows the psoriasis. ( B ) A patient with anterior uveitis. In recent years MRI has improved the assessment and diagnosis of axial disease. Axial disease was probably underestimated before the widespread use of MRI both in children and adults. Approximately half of the patients are known to be first been diagnosed with sacroiliitis on MRI ( 20 ). Classification criteria Most juvenile SpA are classified as enthesitis-related arthritis or undifferentiated arthritis, depending on whether psoriasis is present in the patient or their family. International League of Associations for Rheumatology (ILAR) criteria are used for the classification of JIA subtypes including ERA ( Supplementary Table S1 ) ( 2 , 21 ). According to the ILAR, ERA classification criteria is arthritis plus enthesitis or arthritis or enthesitis plus two of the following: (1) Sacroiliac joint tenderness and/or inflammatory back pain, (2) HLA-B27 positivity, (3) >6 years old boy and (4) Acute anterior uveitis and (5) Family history in at least one first degree relative of HLA-B27 associated disease like ankylosing spondylitis, ERA, sacroiliitis with IBD, reactive arthritis or acute anterior uveitis. In adults the diagnosis is based on the Assessment of SpA International Society (ASAS) classification ( Supplementary Table S1 ). ASAS criteria include both imaging and clinical findings: if sacroiliitis is present on imaging [by radiographs or magnetic resonance imaging (MRI)] ( Figure 2 ) only one other SpA feature is sufficent for classification. However, if imaging evidence of sacroiliitis is absent, positive HLA-B27 along with at least two other SpA features is required for the patient to be classified as having axial SpA. ASAS criteria for peripheral spondyloarthritis include peripheral arthritis and/or enthesitis and/or dactylitis plus 1 SpA feature (uveitis, psoriasis, Crohn's/colitis, preceding infection, HLA-B27, sacroiliitis on imaging) or ≥2 other SpA features (arthritis, enthesitis, dactylitis, inflammatory back pain, family history of SpA). Figure 2 MRI findings in SpA patients. ( A ) On T2-weighted fat-suppressed coronal sections, areas of bone marrow edema consistent with acute sacroiliitis are observed on the facing sides of the bilateral sacroiliac joint (white arrows). ( B ) On T1-weighted fat-free coronal sections, erosion and irregularities at the level of the left sacroiliac joint (white arrow) and pseudo-widening (star) of the joint space are observed. In the right sacroiliac joint, there is an appearance of fat replacement (black arrow) in the sacral region. The ASAS and ILAR criteria set indeed overlap in the defined features and they share several clinical and laboratory findings for classification. However, there are also important differences between the two. Firstly, the ILAR system does not specifically address children who have SpA by fulfilling the criteria for ankylosing spondylitis, or who have coexisting conditions such as inflammatory bowel disease ( 22 ). Reactive arthritis, IBD-related arthritis, and psoriatic arthritis are not among the diagnostic criteria in ERA. While psoriatic arthritis in children is a different subgroup of JIA, both psoriatic arthritis, reactive arthritis, and IBD are included in the SpA group in adults ( 23 ). Secondly, the ILAR classification criteria focus on the importance of extra-axial manifestations such as peripheral arthritis and enthesitis, while SpA classification pays attention to the presence of axial and spinal involvement. Finally, elevated inflammatory markers is one of the criteria in SpA, whereas that is not the case in ERA. Indeed the ILAR criteria has important limitations regarding the classification of patients in the spondyloarthropathy group. Enthesitis-related JIA was considered an undifferentiated SpA in ILAR, whereas all the different forms of adult SpA can be found in children, with the major difference being the higher proportion of undifferentiated forms in children. Thus a new classification criteria have been proposed by researchers from PRINTO, suggesting fundamental changes, in the classification of enthesitis-associated arthritis ( 24 ). This new criteria was called "Enthesitis/spondylitis-related JIA and included the following criteria: peripheral arthritis and enthesitis, or arthritis or enthesitis, plus ≥3 months of inflammatory back pain and sacroiliitis on imaging, or arthritis or enthesitis plus 2 of the following: (1) sacroiliac joint tenderness; (2) inflammatory back pain; (3) presence of HLA-B27 antigen; (4) acute (symptomatic) anterior uveitis; and (5) history of a SpA in a first-degree relative. Of note, if peripheral arthritis is present, it should persist for at least 6 weeks. The current PRINTO definition has been partly harmonized with the adult one, and an imaging criterion for radiographs ( 25 ) or magnetic resonance imaging ( 26 ) has been introduced. The adult definition of inflammatory back pain has been adopted. Because the term ERA could wrongly suggest the existence of a form of SpA that is specific to childhood, it was initially proposed to name this condition juvenile SpA and was later changed to enthesitis/spondylitis-related JIA. Furthermore one of the main differences of PRINTO classification criteria is that sacroiliitis on imaging was added among the list. Albeit definition of sacroiliitis on MRI for adult patients well-defined, the use of this definition of MRI findings for pediatric cases may cause false-positive results due to the physiologic bone marrow changes of growing bone. Recently, MRI definitions for active and structural sacroiliac joint lesions in juvenile cases are published ( 27 ). Although there is a lack of substantial molecular omics studies, most pediatricians would regard ERA more like a juvenile AS, especially once sacroiliitis is detected. There is a need for more follow-up data on patients with peripheral disease -fulfilling the ERA criteria, to understand whether they constitute a separate pediatric group. Moreover, sophisticated studies are crucial to understand whether axial-ERA is truly an early onset AS or SpA. Etiology The lack of knowledge on SpA etiology has been a major concern in diagnosis and disease treatment. The diagnosis is dependent on clinical manifestations which shows heterogeneity between patients whereas the therapeutic interventions were developed based on observational studies. In this section we will discuss the possible risk factors for SpAs and theories related with them. HLA-B27 The immune system acts as a safeguard to protect our body from the invasion of harmful intruders. These foreign entities' proteins should be presented as peptides to our immune cells to activate a potent immune response. Peptides loaded on Major histocompatibility complex (MHC) molecules located on cell surface can be recognized by T cells which in turn cause their activation. All nucleated cells have MHC class I molecules that take part in the presentation of intracellular antigens (i.e., viral, tumor) to CD8+ T lymphocytes and their heavy chains are encoded by genes at HLA-A, HLA-B and HLA-C loci ( 28 ). Antigen presenting cells (APCs) play a significant role in activating the adaptive immune system and are specialized cells. MHC class II molecules are expressed on these cells and are involved in the presentation of exogenous peptides (i.e., bacteria, parasites) to CD4+ T cells. These molecules are encoded by HLA-DR, HLA-DP and HLA-DQ ( 29 ). The first report showing the association of the MHC class I molecule HLA-B27 with SpAs was published in 1973 ( 30 ). Since then many studies were conducted to obtain more information on disease etiology and underlying mechanisms. HLA-B27 positive population constitutes 6%–8% of the general population whereas this ratio increases to more than 80% in AS patients ( 31 , 32 ) implementing its strong plausible contribution to disease etiology. HLA-B27 has different variants with aminoacid substitutions mostly in their peptide binding cleft ( 33 ). Among these variants, HLA-B*27:05, HLA-B*27:02 and HLA-B*27:04 show association with SpA whereas this is not the case for HLA-B*27:06 and HLA-B*27:09 ( 34 , 35 ). HLA-B*27:05 is more common in Caucasian, HLA-B*27:04 in Chinese and HLA-B*27:02 in Mediterranean population ( 36 ). The importance of HLA-B27 in SpA etiology was also recapitulated using animal models. Rats having high levels of HLA-B27*05 and human beta 2 microglobulin (B27-Tg) partially phenocopy the human disease with inflammatory bowel condition, inflammatory peripheral arthritis and skin lesions ( 37 ). Interestingly, genetic factors seems to play a role in the process based on the fact that SpA related symptoms are only manifested in rats having Lewis or Fischer background but not in Dark Agouti background. Mice with same genetic modifications also display spontaneous arthritis ( 38 ) and the lack of β2-microglobulin (β2m) or TAP1 gene does not impair the manifestation of disease related phenotype ( 39 , 40 ). The background of the mice has been found to be relative for the development of the disease as well ( 41 ). The HLA-B27 levels seems to be a pivotial factor regulating disease susceptibility. Higher levels of HLA-B27 are typically seen in the peripheral blood mononuclear cells (PBMCs) of patients compared to healthy controls positive for this allele ( 42 ). Moreover, individuals homozygote for HLA-B27 are associated with an increased risk of AS development compared to heterozygotes ( 43 ). The same phenomenon is also observed in animal models. Disease susceptibility shows a positive correlation with HLA-B27 copy number and its relative expression in lymphoid cells ( 44 ) that can be upregulated via pro-inflammatory stimuli. Of note, this dose dependent effect might also explain why only 2% of HLA-B27 positive patients develop the disease. The positivity of HLA-B27 also has an influence on disease manifestation. In more than 80% of AS patients, symptoms emerge at ≤30 years of age. Interestingly, HLA-B27 positive AS patients show an earlier disease onset compared to negative ones ( 45 ) and have a worse prognosis with elevated disease activity and duration ( 46 ). The frequency of specific symptoms also depends on HLA-B27 status. Psoriasis and IBD are more common in HLA-B27 negative patients whereas peripheral arthiritis and uveitis are observed more frequently in HLA-B27 positive ones ( 47 ). Overall, it is clear that HLA-B27 somehow plays a role in disease pathogenesis. Its possible contribution to disease progression and related theories are discussed below. Arthritogenic peptide/molecular mimicry hypothesis The mature MHC I molecule is composed of a heavy chain (HC), a β2m light chain and a peptide, 8–10 amino acids in length. Its formation involves a series of protein assembly and disassembly within the complex. First, newly synthesized heavys chains are translocated to the endoplasmic reticulum (ER) and glycosylated. This post-translational modification acts as a signal for incomplete folding which in turn triggers HCs interactions with chaperones calnexin and calreticulin. As HCs gain the correct tertiary structure, they associate with β2m resulting in the dissociation of calnexin ( 48 , 49 ). Next, the complex further interacts with a transporter associated with antigen processing (TAP) via tapasin which is bound to ERp57 to form the peptide loading complex ( 48 ). Eventually, Erp57 and calreticulin dissociate to allow the binding of peptides to the MHC I. Although, MHC class I molecules are responsible for the presentation of the peptides to CD8+ T cells, these peptides should be trimmed before they are loaded on the complex. For that, purpose proteasome performs the initial trimming process causing the formation of peptides ∼15 aa in length. These peptides enter ER through TAP transporter and furher cleaved by ERAP1 and ERAP2 to have the optimal length for the loading ( 50 ). Finally, as MHC I is loaded with the peptide, the complex is sent to the surface of nucleated cells in particular APCs to perform a successful round of peptide presentation. The plausible contribution of APCs in disease pathogenesis has been the center of many studies. The increased abundance of macrophages in sacroiliac ( 51 ) and enthesis ( 52 ) biopsies of AS patients attain a possible role for these cells. The level of circulating CD141+ dendritic cells (DCs) show a positive correlation with BASDAI in AS patients ( 53 ). Moreover, lower levels of MHC class II expression in DCs of AS patients ( 54 ) and animal model ( 55 ) implies that distortions in antigen presentation might very well be a key factor in disease pathogenesis. Previous studies suggested that HLA-B27 binds to a distinctive set of peptides that show similarity to self-peptides ( Figure 3 ). Their presentation to CD8+ T lymphocytes triggers the breakdown of self tolerance which in turn activates a destructive immune response in affected sites ( 56 ). In support of this notion, HLA-B27-restricted CD8+ T cells were detected in the synovial fluid of AS patients ( 57 , 58 ). They are also found to be directed against self-peptides derived from vasoactive intestinal peptide type 1 receptor (VIP1R, aminoacids 400–408) and glucagon receptor (GR, aminoacids 412–420) ( 59 , 60 ). A controversial finding pointed out that HLA-B*27:09 subtype that is not associated with the disease also presents the VIP1R-derived peptide ( 61 ). However, further investigations revealed that peptide's conformation differs from the one presented by the disease relevant variant HLA-B*27:05 ( 59 ). Figure 3 HLA-B27 related theories in SpA pathogenesis. The molecular mimicry between HLA-B27 and gram-negative bacteria was also suggested to be a key element in autoreactive T cell activation and autoimmune reaction. Indeed, the sequence homology between HLA-B27 and arthritogenic bacterias Klebsiella pneumoniae ( 62 ), Yersinia enterocolitica , Salmonella typhimurium , Shigella flexneri and Shigella sonnei was described ( 63 ). This theory is further supported by a study by Ramos et al. showing that a peptide derived from the intracytoplasmic tail of HLA-B27 shows similarity to Chlamydia trachomatis and acts as a ligand only for disease associated HLA-B27 variants ( 64 ). Although presentation of unusual peptides by HLA-B27 was suggested in disease pathogenesis, the ongoing presentation of disease related symptoms in CD8+ T cell depleted B27-Tg rats or TAP1 −/− mice argues strongly against the importance of antigen presentation in disease etiology ( 65 , 66 ) thus other theories were also developed ( 40 , 65 , 66 ). Homodimeric HLA-B27 molecule As mentioned above, the major function of a MHC class I molecule which is composed of a heavy chain, β2m and a peptide, is to present these peptides to CD8+ T cells. Rather, HLA-B27 was reported to be recognized by Natural Killer Cells (NKs) and CD4+ T lymphocytes in the form of β2m-free homodimers ( 67 ) which is established through an unpaired cysteine at position 67 ( 68 ) ( Figure 3 ). Strikingly, the B27-Tg rats with functional NK cells do not present disease symptoms indicating that these cells act in concert with lymphoctes in disease manifestation ( 69 ). As a matter of fact the critical involvement of CD4+ T cells in disease progression was recapitulated in many studies. Transfer of different T lymphocyte populations to athymic nude B27-Tg mice revealed that CD4+ T cells are the major cell population inducing colitis ( 70 ). Moreover, their levels shows an increment in the lymph nodes draining the sites of inflammation in animal model ( 71 ) and in peripheral blood of SpA patients ( 72 ). Higher levels of activated CD4+ T cells is also observed in B27-Tg rats compared to healthy ones ( 65 ). Studies aiming to understand the involvement of dimer formation in disease pathogenesis unearthed that HLA-B27 variants associated with SpA have an increased tendency for dimer formation ( 73 ). The receptors for HLA-B27 homodimers were found to be KIR3DL1, LILRB2 ( 74 ), KIR3DL2 ( 75 ) and LIR6 ( 68 ) and expressed on lymphocytes, monocytes and Natural Killer Cells (NKs) ( 75 ). The level of KIR3DL2 positive NK and CD4+ T cells increases in the peripheral blood and synovial fluid of SpA and ERA patients. The receptor engagement also shows a survival and activation profile in NKs and T cells respectively ( 73 , 76 ) whereas the dissociation between KIR3DL2 and HLA-B27 decreases the survival/proliferation of NKs and the release of disease related cytokine IL17 from the PBMCs of AS patients ( 77 ). In line with this finding, KIR3DL2+ CD4+ T cells collected from the synovial fluid of SpA patients displays enhanced levels of IL17 secretion ( 78 ). Unfolded protein response (UPR) activation UPR activation was suggested to be a major explanation in SpA pathogenesis. Proteins are biomolecules that orchestrate many cellular processes. To fulfill their task, they need to be folded properly in the organelle called Endoplasmic Reticulum (ER). Proteins with folding deficits can be removed via autophagy or Endoplasmic Reticulum Associated Degradation (ERAD) ( 79 ). However, the accumulation of misfolded proteins may also take place which in turn activates a stress response machinery namely UPR. This stress is regulated through 3 transmembrane proteins located on ER: Inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK) and Activating Transcription Factor 6 (ATF6). Upon its activation, IRE1 cleaves Xbp mRNA leading to the formation of an active transcription factor sXbp. This factor is responsible for the synthesis of chaperones and ERAD components to achieve cellular homeostasis whereas an increase in magnitude and duration of stress results in the activation of the apoptotic IRE1-JNK pathway. Activation of PERK leads to the phosphorylation of eIF2α which in turn put a halt to translation whereas activating selective translation of the ATF4 transcription factor. Similar to IRE1 pathway, ATF4 is also responsible for the activation of homeostatic genes yet unresolved stress promotes the expression of pro-apoptotic CHOP. ATF6 is translocated to Golgi upon UPR and cleaved with S1P and S2P proteases. The newly formed cytosolic domain of ATF6 is a transcription factor and is involved in the transcription of chaperones ( 80 ). HLA-B27 is unique in a way that it misfolds even in the presence of β2m and peptides which in turn activate UPR ( Figure 3 ). There are several bodies of evidence proving this aberrant feature of HLA-B27: - Folding rate: HLA-B27 has a slow folding nature which in turn induces homodimer formation and its retention in the ER. These molecules can then activate the UPR. The B pocket which is located at the peptide binding groove of HLA-B27 seems to be crucial for this machinery. Altering residues in this region not only enhances HLA-B27's folding but also alleviates homodimer formation ( 81 ). - ERAD: Another clue showing the misfolded nature of HLA-B27 is its enhanced predisposition to undergo ERAD ( 82 ) in which EDEM1, and HRD1 were found to be pivotal regulators ( 83 , 84 ). In support of this notion, the use of ERAD blocking reagents results in an increment in the levels of HLA-B27 dimers/oligomers. - Interaction with chaperones: The chaperones help misfolded proteins to gain a proper tertiary structure. The prolonged interaction between HLA-B27 multimers and chaperone Bip indicates the improper folding of HLA-B27 which in turn activates the stress response ( 85 ). In addition, the enhanced interaction between HLA-B27 and oxidoreductase Erp57 is also involved in dimer formation which again may turn on UPR ( 86 ). Macrophages residing in the peripheral joints of AS patients have increased levels of Bip compared to osteoarthritis patients ( 87 ) and mononuclear cells collected from the synovial fluid of SpA patients shows an activation state for UPR ( 88 ). In B27-Tg rats, bone marrow derived macrophages shows prominent UPR activation status which shows a positive correlation with HLA-B27 levels ( 89 , 90 ). The UPR induction was also observed in B27-Tg rats' dendritic cells ( 55 ). Strikingly, ERAP1 deficient B27-Tg rats remained healthy due to the blockade of UPR activation ( 91 ). The pathogenesis of SpA clearly involves the activation of the immune system thus UPR-driven immune modulation has also been the subject of extensive investigation. NF κ B was shown to be activated during UPR ( 92 ) that mediates Th17 differentiation via IL23, a cytokine that is elevated in the serum and synovial fluid of SpA patients ( 93 , 94 ). The activated Th17 cells in turn produce cytokines such as IL17, TNF and IL6 ( 95 ). A strong activation for IL23/IL17 axis was detected in the colon of B27-Tg rats ( 96 ) and overexpression of IL23 causes a disease phenotype similar to AS in mice ( 97 ). In addition, Th17 cells were found to be enriched in the peripheral blood of AS patients ( 98 ). Furthermore, DCs, a major source for IL23, contribute to Th17 cells' expansion in the B27-Tg animal model ( 71 ). Although, macrophages with prominent UPR activation are destined to produce higher levels of IL23 ( 96 ), there are also studies showing that IL23 production is independent of UPR activation thus further studies are warranted ( 99 ). Another NF κ B dependent cytokine TNF-α is also a critical component of the disease and also used as a target for therapy. Similar to IL23, overexpression of TNF results in spondyloarthritis formation in mice and this process was found to be regulated through mesenchymal cells ( 100 ). The level of Bip in the macrophages collected from the synovial fluid shows a positive correlation with TNF levels indicating that immune modulation by UPR might be the basis for elevated TNF levels in disease ( 87 ). TNF is detected in the inflamed tissues of SpA patients and also is elevated in PBMCs and serum ( 101 ). Other susceptibility genes Studies on families revealed that SpAs may have a heritable component. For JIA the recurrence risk in first cousins was determined to be 5.8 fold whereas the sibling relative risk is estimated to be 11.6 fold ( 102 ). AS's heritability is ≥90% ( 103 , 104 ) with a sibling recurrence risk of 8.2% ( 105 ). The prevalence increases dramatically with the presence of a first degree relative suffering from the disease ( 106 , 107 ) and the concordance rate was determined to be 25%–75% and 4%–15% in monozygotic and dizygotic twins respectively ( 103 , 104 , 108 ). Overall, these findings strongly indicate that genetic factors are key determinants in disease pathogenesis. As mentioned above the presence of HLA-B27 showed the highest association with disease susceptibility. However, the fact that only 2% of the HLA-B27 positive population develops SpA is a strong indicator that there are additional genetic risk factors for the development of the disease ( 109 ). To understand this phenomenon better genome wide association studies (GWAS) were performed in SpA patients. The second well established susceptibility locus for AS and ERA was found to be ERAP1 ( 110 , 111 ) which is involved in the presentation of peptides with optimal length. Several studies made it apparent that defects in ERAP function might be involved in disease pathogenesis. ERAP1 variants with a loss of function shows a protective effect for disease which provides strong evidence for the involvement of atypical processing of antigenic peptides ( 112 ). Indeed, HLA-B27 was shown to bind extended peptides with protruding C-terminus ( 113 ) which in turn may activate a potent T cell response thus leading to SpA development. The deficits in peptide trimming might also decrease the level of peptide- loaded MHC molecules which in turn increase the levels of misfolded HLA-B27 molecules, UPR activation and disease progression. Elevated UPR levels might also be regulated through other mechanisms including damaged ubiquitin-ERAD machinery. Previous studies clearly show that ubiquitin conjugating enzyme UBE2J1 is involved in targeting of MHC class I molecules for ERAD ( 83 ) thus further studies aiming to unveil the link between another susceptibility gene UBE2E3 ( 114 ) and ERAD in disease etiology would be valuable. Shaping immune response is indispensable in AS pathogenesis. As mentioned above, the IL23/IL17 axis is a crucial component of this machinery. Its importance was also verified in GWAS studies. Molecules related with this pathway (IL23R, IL12B, IL6R, IL1R1, IL1R2, TYK2, IL27A, STAT3, JAK2) are among the gene loci that shows association with AS ( 83 , 114 ). This also holds true for the TNF-α pathway. Research revealed the presence of disease associated SNPs near to/in TNFRSF1A ( 83 , 114 ), TNFSF15 ( 115 ) and TRADD ( 116 ). Genes related to T cell regulation, RUNX3, IL7R, EOMES, ZMIZ1, ICOSLG, SH2B3 and BACH2, are also among the AS risk loci ( 9 ). Other genes showing association with AS are GPR25, GPR65, GPR35, TBKBP1, PTGER4, BACH2, NOS2, FCGR2A, NKX2-3 ( 9 ), CARD9 ( 117 , 118 ), KIF21B ( 119 ), ANTXR2 ( 120 ), ANO6 ( 121 ). For JIA, ERAP1and IL23R are among the disease susceptibility genes for ERA and juvenile psoriatic arthritis respectively ( 111 ). The lower prevalence of JIA subtypes hampers the construction of well-powered cohorts for GWAS analysis. Thus combining all JIA subtypes rather than investigating them separately was used to detect genetic associations. PTPN2, ANGPT1, COG6 ( 122 ), CD80, JMJD1C ( 123 ), TRAF1-C5 ( 124 ), VTCN1 ( 125 ), IL2RA, IL2RB, STAT4 ( 126 ), TNFAIP3 and TRAF1/C5 ( 127 ) were found to be JIA-predisposing loci. Gender Many rheumatic diseases display gender predominance. Both the incidence of AS ( 109 ) and ERA ( 128 ) are higher in males. However, the male to female ratio has showed decrement over time ( 129 ). This gender predominance indicates that sex-specific factors might play a role in SpA pathogenesis. Among these factors the impact of hormones in SpA progression was extensively analyzed. Of note, the age interval for ERA patients is 6–16 and it is well known that the level of sex hormones increases with puberty thus the following observations may partially explain the male dominance of ERA. The effect of TNF inhibitors on SpA progression underlines TNF's importance in disease progression. Interestingly, estrogen was shown to decrease inflammation in SpA patients via downregulating TNF alpha levels ( 130 ). Estrogen supplementation was also shown to decrease disease severity both in human ( 131 ) and animal female subjects ( 132 ) whereas there are other studies showing no evident association ( 133 ). Testosterone levels on the other hand did not show any difference between SpA patients and healthy controls and were not likely to regulate disease progression ( 131 , 134 ). Sex hormones were also shown to regulate the microbiome ( 135 ) and immune system ( 136 ). The fact that both of these factors play a role in disease progression (see below) underlines the presence of a possible hormone-driven microbiome and immune system related axis however, further studies are needed. The gut and microbiome The gut is one of the affected sites in SpA. Inflammatory bowel disease (Crohn's disease and ulcerative colitis) occur concomitantly in up to 10% of SpA positive population ( 137 ). Patients display inflammatory lesions at intestinal mucosa ( 138 ) and the gut is also inflamed in the animal model ( 139 ) indicating that an active immune response in the gut and SpA might be interlinked. In support of this notion, macrophages expressing the CD163 scavenger receptor increases in the colonic mucosa of SpA patients ( 140 ) and IL23/IL17 axis is exacerbated in the colon of B27-Tg rats ( 96 ). The relation between SpA development and microbiota has been the center of attention for decades. In animal models, housing of the animals in pathogen-free conditions alleviated the formation of several disease related symptoms including colitis and arthritis. However, their transfer to conventional conditions caused their manifestation. Moreover, treating B27-Tg rats with antibiotics hampered colitis formation ( 141 ) indicating that the microbiome is a key player in disease pathogenesis ( 39 , 142 ). Indeed, there are studies showing the differences in microbiome of SpA patients and healthy controls ( 143 – 145 ) and ileal biopsies from AS patients revealed the presence of adherent and invasive bacteria which is accompanied by the decreased barrier function of the gut ( 146 ). Mucins play a major role in barrier function. Mucin-degrading Akkermansia muciniphila species was found to be elevated in B27-Tg rats indicating that SpA related dysbiosis may be involved in impaired gut barrier ( 147 ). T cells are another key player for maintaining the tolerance against commensal bacteria ( 148 ). Interestingly, CD4+ T cells isolated from B27-Tg rats produces higher levels of IFN-γ in response to antigens derived from these organisms implying that there might be a loss of tolerance for the microbiome ( 149 , 150 ). Moreover, the defective stimulation of T cells by APCs might also contribute to the loss of tolerance for microbial flora ( 151 , 152 ). The link between treatment response and microbiome was also investigated. Patients receiving 3 months of anti-TNF therapy did not show a significant difference in their microbiata composition. However, having higher levels of Burkholderiales prior to therapy and an increment in genus Dialister after therapy was observed in responders ( 153 ). Diet Diet plays a crucial role in the development and progression of many diseases. Diet has also been investigated in SpA however, most studies were not replicated. A study by Haugen et al. indicated that many AS patients reported that diet plays a role in the manifestation and severity of their symptoms ( 154 ) and they follow certain diets to decrease their intensity ( 155 ). Starch consumption was suggested to be an exacerbating factor in SpAs and a low starch diet was found to lower disease activity whereas there are also studies showing no impact ( 155 , 156 ). Salt and dietary fat consumption did not show any correlation with the severity of the symptoms ( 155 , 157 ). Although quitting dairy products seem to have an ameliorating role in disease ( 158 ) there are also studies showing no effect ( 157 ). In human subjects, the impact of prebiotic uptake in SpA progression was analyzed. SpA patients with concomitant quiescent ulcerative colitis receiving Lactobacillus acidophilus and Lactobacillus salivarius displayed lower disease activity ( 159 ). In contrast, a meta-analysis by Sanchez et al. opposed this finding ( 160 ). In animal model the severity of colitis was diminished with the supplementation of diet with prebiotics ( 161 ). The constituent, fructo-oligosaccharides was found to have the greatest anti-inflammatory effect in this regard ( 162 , 163 ). Fibre-rich diets also showed a remedial effect on disease by upregulating short chain fatty acids. Indeed, administration of propionate to B27-Tg animals attenuates intestinal inflammation ( 164 ). HLA-B27 The immune system acts as a safeguard to protect our body from the invasion of harmful intruders. These foreign entities' proteins should be presented as peptides to our immune cells to activate a potent immune response. Peptides loaded on Major histocompatibility complex (MHC) molecules located on cell surface can be recognized by T cells which in turn cause their activation. All nucleated cells have MHC class I molecules that take part in the presentation of intracellular antigens (i.e., viral, tumor) to CD8+ T lymphocytes and their heavy chains are encoded by genes at HLA-A, HLA-B and HLA-C loci ( 28 ). Antigen presenting cells (APCs) play a significant role in activating the adaptive immune system and are specialized cells. MHC class II molecules are expressed on these cells and are involved in the presentation of exogenous peptides (i.e., bacteria, parasites) to CD4+ T cells. These molecules are encoded by HLA-DR, HLA-DP and HLA-DQ ( 29 ). The first report showing the association of the MHC class I molecule HLA-B27 with SpAs was published in 1973 ( 30 ). Since then many studies were conducted to obtain more information on disease etiology and underlying mechanisms. HLA-B27 positive population constitutes 6%–8% of the general population whereas this ratio increases to more than 80% in AS patients ( 31 , 32 ) implementing its strong plausible contribution to disease etiology. HLA-B27 has different variants with aminoacid substitutions mostly in their peptide binding cleft ( 33 ). Among these variants, HLA-B*27:05, HLA-B*27:02 and HLA-B*27:04 show association with SpA whereas this is not the case for HLA-B*27:06 and HLA-B*27:09 ( 34 , 35 ). HLA-B*27:05 is more common in Caucasian, HLA-B*27:04 in Chinese and HLA-B*27:02 in Mediterranean population ( 36 ). The importance of HLA-B27 in SpA etiology was also recapitulated using animal models. Rats having high levels of HLA-B27*05 and human beta 2 microglobulin (B27-Tg) partially phenocopy the human disease with inflammatory bowel condition, inflammatory peripheral arthritis and skin lesions ( 37 ). Interestingly, genetic factors seems to play a role in the process based on the fact that SpA related symptoms are only manifested in rats having Lewis or Fischer background but not in Dark Agouti background. Mice with same genetic modifications also display spontaneous arthritis ( 38 ) and the lack of β2-microglobulin (β2m) or TAP1 gene does not impair the manifestation of disease related phenotype ( 39 , 40 ). The background of the mice has been found to be relative for the development of the disease as well ( 41 ). The HLA-B27 levels seems to be a pivotial factor regulating disease susceptibility. Higher levels of HLA-B27 are typically seen in the peripheral blood mononuclear cells (PBMCs) of patients compared to healthy controls positive for this allele ( 42 ). Moreover, individuals homozygote for HLA-B27 are associated with an increased risk of AS development compared to heterozygotes ( 43 ). The same phenomenon is also observed in animal models. Disease susceptibility shows a positive correlation with HLA-B27 copy number and its relative expression in lymphoid cells ( 44 ) that can be upregulated via pro-inflammatory stimuli. Of note, this dose dependent effect might also explain why only 2% of HLA-B27 positive patients develop the disease. The positivity of HLA-B27 also has an influence on disease manifestation. In more than 80% of AS patients, symptoms emerge at ≤30 years of age. Interestingly, HLA-B27 positive AS patients show an earlier disease onset compared to negative ones ( 45 ) and have a worse prognosis with elevated disease activity and duration ( 46 ). The frequency of specific symptoms also depends on HLA-B27 status. Psoriasis and IBD are more common in HLA-B27 negative patients whereas peripheral arthiritis and uveitis are observed more frequently in HLA-B27 positive ones ( 47 ). Overall, it is clear that HLA-B27 somehow plays a role in disease pathogenesis. Its possible contribution to disease progression and related theories are discussed below. Arthritogenic peptide/molecular mimicry hypothesis The mature MHC I molecule is composed of a heavy chain (HC), a β2m light chain and a peptide, 8–10 amino acids in length. Its formation involves a series of protein assembly and disassembly within the complex. First, newly synthesized heavys chains are translocated to the endoplasmic reticulum (ER) and glycosylated. This post-translational modification acts as a signal for incomplete folding which in turn triggers HCs interactions with chaperones calnexin and calreticulin. As HCs gain the correct tertiary structure, they associate with β2m resulting in the dissociation of calnexin ( 48 , 49 ). Next, the complex further interacts with a transporter associated with antigen processing (TAP) via tapasin which is bound to ERp57 to form the peptide loading complex ( 48 ). Eventually, Erp57 and calreticulin dissociate to allow the binding of peptides to the MHC I. Although, MHC class I molecules are responsible for the presentation of the peptides to CD8+ T cells, these peptides should be trimmed before they are loaded on the complex. For that, purpose proteasome performs the initial trimming process causing the formation of peptides ∼15 aa in length. These peptides enter ER through TAP transporter and furher cleaved by ERAP1 and ERAP2 to have the optimal length for the loading ( 50 ). Finally, as MHC I is loaded with the peptide, the complex is sent to the surface of nucleated cells in particular APCs to perform a successful round of peptide presentation. The plausible contribution of APCs in disease pathogenesis has been the center of many studies. The increased abundance of macrophages in sacroiliac ( 51 ) and enthesis ( 52 ) biopsies of AS patients attain a possible role for these cells. The level of circulating CD141+ dendritic cells (DCs) show a positive correlation with BASDAI in AS patients ( 53 ). Moreover, lower levels of MHC class II expression in DCs of AS patients ( 54 ) and animal model ( 55 ) implies that distortions in antigen presentation might very well be a key factor in disease pathogenesis. Previous studies suggested that HLA-B27 binds to a distinctive set of peptides that show similarity to self-peptides ( Figure 3 ). Their presentation to CD8+ T lymphocytes triggers the breakdown of self tolerance which in turn activates a destructive immune response in affected sites ( 56 ). In support of this notion, HLA-B27-restricted CD8+ T cells were detected in the synovial fluid of AS patients ( 57 , 58 ). They are also found to be directed against self-peptides derived from vasoactive intestinal peptide type 1 receptor (VIP1R, aminoacids 400–408) and glucagon receptor (GR, aminoacids 412–420) ( 59 , 60 ). A controversial finding pointed out that HLA-B*27:09 subtype that is not associated with the disease also presents the VIP1R-derived peptide ( 61 ). However, further investigations revealed that peptide's conformation differs from the one presented by the disease relevant variant HLA-B*27:05 ( 59 ). Figure 3 HLA-B27 related theories in SpA pathogenesis. The molecular mimicry between HLA-B27 and gram-negative bacteria was also suggested to be a key element in autoreactive T cell activation and autoimmune reaction. Indeed, the sequence homology between HLA-B27 and arthritogenic bacterias Klebsiella pneumoniae ( 62 ), Yersinia enterocolitica , Salmonella typhimurium , Shigella flexneri and Shigella sonnei was described ( 63 ). This theory is further supported by a study by Ramos et al. showing that a peptide derived from the intracytoplasmic tail of HLA-B27 shows similarity to Chlamydia trachomatis and acts as a ligand only for disease associated HLA-B27 variants ( 64 ). Although presentation of unusual peptides by HLA-B27 was suggested in disease pathogenesis, the ongoing presentation of disease related symptoms in CD8+ T cell depleted B27-Tg rats or TAP1 −/− mice argues strongly against the importance of antigen presentation in disease etiology ( 65 , 66 ) thus other theories were also developed ( 40 , 65 , 66 ). Homodimeric HLA-B27 molecule As mentioned above, the major function of a MHC class I molecule which is composed of a heavy chain, β2m and a peptide, is to present these peptides to CD8+ T cells. Rather, HLA-B27 was reported to be recognized by Natural Killer Cells (NKs) and CD4+ T lymphocytes in the form of β2m-free homodimers ( 67 ) which is established through an unpaired cysteine at position 67 ( 68 ) ( Figure 3 ). Strikingly, the B27-Tg rats with functional NK cells do not present disease symptoms indicating that these cells act in concert with lymphoctes in disease manifestation ( 69 ). As a matter of fact the critical involvement of CD4+ T cells in disease progression was recapitulated in many studies. Transfer of different T lymphocyte populations to athymic nude B27-Tg mice revealed that CD4+ T cells are the major cell population inducing colitis ( 70 ). Moreover, their levels shows an increment in the lymph nodes draining the sites of inflammation in animal model ( 71 ) and in peripheral blood of SpA patients ( 72 ). Higher levels of activated CD4+ T cells is also observed in B27-Tg rats compared to healthy ones ( 65 ). Studies aiming to understand the involvement of dimer formation in disease pathogenesis unearthed that HLA-B27 variants associated with SpA have an increased tendency for dimer formation ( 73 ). The receptors for HLA-B27 homodimers were found to be KIR3DL1, LILRB2 ( 74 ), KIR3DL2 ( 75 ) and LIR6 ( 68 ) and expressed on lymphocytes, monocytes and Natural Killer Cells (NKs) ( 75 ). The level of KIR3DL2 positive NK and CD4+ T cells increases in the peripheral blood and synovial fluid of SpA and ERA patients. The receptor engagement also shows a survival and activation profile in NKs and T cells respectively ( 73 , 76 ) whereas the dissociation between KIR3DL2 and HLA-B27 decreases the survival/proliferation of NKs and the release of disease related cytokine IL17 from the PBMCs of AS patients ( 77 ). In line with this finding, KIR3DL2+ CD4+ T cells collected from the synovial fluid of SpA patients displays enhanced levels of IL17 secretion ( 78 ). Unfolded protein response (UPR) activation UPR activation was suggested to be a major explanation in SpA pathogenesis. Proteins are biomolecules that orchestrate many cellular processes. To fulfill their task, they need to be folded properly in the organelle called Endoplasmic Reticulum (ER). Proteins with folding deficits can be removed via autophagy or Endoplasmic Reticulum Associated Degradation (ERAD) ( 79 ). However, the accumulation of misfolded proteins may also take place which in turn activates a stress response machinery namely UPR. This stress is regulated through 3 transmembrane proteins located on ER: Inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK) and Activating Transcription Factor 6 (ATF6). Upon its activation, IRE1 cleaves Xbp mRNA leading to the formation of an active transcription factor sXbp. This factor is responsible for the synthesis of chaperones and ERAD components to achieve cellular homeostasis whereas an increase in magnitude and duration of stress results in the activation of the apoptotic IRE1-JNK pathway. Activation of PERK leads to the phosphorylation of eIF2α which in turn put a halt to translation whereas activating selective translation of the ATF4 transcription factor. Similar to IRE1 pathway, ATF4 is also responsible for the activation of homeostatic genes yet unresolved stress promotes the expression of pro-apoptotic CHOP. ATF6 is translocated to Golgi upon UPR and cleaved with S1P and S2P proteases. The newly formed cytosolic domain of ATF6 is a transcription factor and is involved in the transcription of chaperones ( 80 ). HLA-B27 is unique in a way that it misfolds even in the presence of β2m and peptides which in turn activate UPR ( Figure 3 ). There are several bodies of evidence proving this aberrant feature of HLA-B27: - Folding rate: HLA-B27 has a slow folding nature which in turn induces homodimer formation and its retention in the ER. These molecules can then activate the UPR. The B pocket which is located at the peptide binding groove of HLA-B27 seems to be crucial for this machinery. Altering residues in this region not only enhances HLA-B27's folding but also alleviates homodimer formation ( 81 ). - ERAD: Another clue showing the misfolded nature of HLA-B27 is its enhanced predisposition to undergo ERAD ( 82 ) in which EDEM1, and HRD1 were found to be pivotal regulators ( 83 , 84 ). In support of this notion, the use of ERAD blocking reagents results in an increment in the levels of HLA-B27 dimers/oligomers. - Interaction with chaperones: The chaperones help misfolded proteins to gain a proper tertiary structure. The prolonged interaction between HLA-B27 multimers and chaperone Bip indicates the improper folding of HLA-B27 which in turn activates the stress response ( 85 ). In addition, the enhanced interaction between HLA-B27 and oxidoreductase Erp57 is also involved in dimer formation which again may turn on UPR ( 86 ). Macrophages residing in the peripheral joints of AS patients have increased levels of Bip compared to osteoarthritis patients ( 87 ) and mononuclear cells collected from the synovial fluid of SpA patients shows an activation state for UPR ( 88 ). In B27-Tg rats, bone marrow derived macrophages shows prominent UPR activation status which shows a positive correlation with HLA-B27 levels ( 89 , 90 ). The UPR induction was also observed in B27-Tg rats' dendritic cells ( 55 ). Strikingly, ERAP1 deficient B27-Tg rats remained healthy due to the blockade of UPR activation ( 91 ). The pathogenesis of SpA clearly involves the activation of the immune system thus UPR-driven immune modulation has also been the subject of extensive investigation. NF κ B was shown to be activated during UPR ( 92 ) that mediates Th17 differentiation via IL23, a cytokine that is elevated in the serum and synovial fluid of SpA patients ( 93 , 94 ). The activated Th17 cells in turn produce cytokines such as IL17, TNF and IL6 ( 95 ). A strong activation for IL23/IL17 axis was detected in the colon of B27-Tg rats ( 96 ) and overexpression of IL23 causes a disease phenotype similar to AS in mice ( 97 ). In addition, Th17 cells were found to be enriched in the peripheral blood of AS patients ( 98 ). Furthermore, DCs, a major source for IL23, contribute to Th17 cells' expansion in the B27-Tg animal model ( 71 ). Although, macrophages with prominent UPR activation are destined to produce higher levels of IL23 ( 96 ), there are also studies showing that IL23 production is independent of UPR activation thus further studies are warranted ( 99 ). Another NF κ B dependent cytokine TNF-α is also a critical component of the disease and also used as a target for therapy. Similar to IL23, overexpression of TNF results in spondyloarthritis formation in mice and this process was found to be regulated through mesenchymal cells ( 100 ). The level of Bip in the macrophages collected from the synovial fluid shows a positive correlation with TNF levels indicating that immune modulation by UPR might be the basis for elevated TNF levels in disease ( 87 ). TNF is detected in the inflamed tissues of SpA patients and also is elevated in PBMCs and serum ( 101 ). Arthritogenic peptide/molecular mimicry hypothesis The mature MHC I molecule is composed of a heavy chain (HC), a β2m light chain and a peptide, 8–10 amino acids in length. Its formation involves a series of protein assembly and disassembly within the complex. First, newly synthesized heavys chains are translocated to the endoplasmic reticulum (ER) and glycosylated. This post-translational modification acts as a signal for incomplete folding which in turn triggers HCs interactions with chaperones calnexin and calreticulin. As HCs gain the correct tertiary structure, they associate with β2m resulting in the dissociation of calnexin ( 48 , 49 ). Next, the complex further interacts with a transporter associated with antigen processing (TAP) via tapasin which is bound to ERp57 to form the peptide loading complex ( 48 ). Eventually, Erp57 and calreticulin dissociate to allow the binding of peptides to the MHC I. Although, MHC class I molecules are responsible for the presentation of the peptides to CD8+ T cells, these peptides should be trimmed before they are loaded on the complex. For that, purpose proteasome performs the initial trimming process causing the formation of peptides ∼15 aa in length. These peptides enter ER through TAP transporter and furher cleaved by ERAP1 and ERAP2 to have the optimal length for the loading ( 50 ). Finally, as MHC I is loaded with the peptide, the complex is sent to the surface of nucleated cells in particular APCs to perform a successful round of peptide presentation. The plausible contribution of APCs in disease pathogenesis has been the center of many studies. The increased abundance of macrophages in sacroiliac ( 51 ) and enthesis ( 52 ) biopsies of AS patients attain a possible role for these cells. The level of circulating CD141+ dendritic cells (DCs) show a positive correlation with BASDAI in AS patients ( 53 ). Moreover, lower levels of MHC class II expression in DCs of AS patients ( 54 ) and animal model ( 55 ) implies that distortions in antigen presentation might very well be a key factor in disease pathogenesis. Previous studies suggested that HLA-B27 binds to a distinctive set of peptides that show similarity to self-peptides ( Figure 3 ). Their presentation to CD8+ T lymphocytes triggers the breakdown of self tolerance which in turn activates a destructive immune response in affected sites ( 56 ). In support of this notion, HLA-B27-restricted CD8+ T cells were detected in the synovial fluid of AS patients ( 57 , 58 ). They are also found to be directed against self-peptides derived from vasoactive intestinal peptide type 1 receptor (VIP1R, aminoacids 400–408) and glucagon receptor (GR, aminoacids 412–420) ( 59 , 60 ). A controversial finding pointed out that HLA-B*27:09 subtype that is not associated with the disease also presents the VIP1R-derived peptide ( 61 ). However, further investigations revealed that peptide's conformation differs from the one presented by the disease relevant variant HLA-B*27:05 ( 59 ). Figure 3 HLA-B27 related theories in SpA pathogenesis. The molecular mimicry between HLA-B27 and gram-negative bacteria was also suggested to be a key element in autoreactive T cell activation and autoimmune reaction. Indeed, the sequence homology between HLA-B27 and arthritogenic bacterias Klebsiella pneumoniae ( 62 ), Yersinia enterocolitica , Salmonella typhimurium , Shigella flexneri and Shigella sonnei was described ( 63 ). This theory is further supported by a study by Ramos et al. showing that a peptide derived from the intracytoplasmic tail of HLA-B27 shows similarity to Chlamydia trachomatis and acts as a ligand only for disease associated HLA-B27 variants ( 64 ). Although presentation of unusual peptides by HLA-B27 was suggested in disease pathogenesis, the ongoing presentation of disease related symptoms in CD8+ T cell depleted B27-Tg rats or TAP1 −/− mice argues strongly against the importance of antigen presentation in disease etiology ( 65 , 66 ) thus other theories were also developed ( 40 , 65 , 66 ). Homodimeric HLA-B27 molecule As mentioned above, the major function of a MHC class I molecule which is composed of a heavy chain, β2m and a peptide, is to present these peptides to CD8+ T cells. Rather, HLA-B27 was reported to be recognized by Natural Killer Cells (NKs) and CD4+ T lymphocytes in the form of β2m-free homodimers ( 67 ) which is established through an unpaired cysteine at position 67 ( 68 ) ( Figure 3 ). Strikingly, the B27-Tg rats with functional NK cells do not present disease symptoms indicating that these cells act in concert with lymphoctes in disease manifestation ( 69 ). As a matter of fact the critical involvement of CD4+ T cells in disease progression was recapitulated in many studies. Transfer of different T lymphocyte populations to athymic nude B27-Tg mice revealed that CD4+ T cells are the major cell population inducing colitis ( 70 ). Moreover, their levels shows an increment in the lymph nodes draining the sites of inflammation in animal model ( 71 ) and in peripheral blood of SpA patients ( 72 ). Higher levels of activated CD4+ T cells is also observed in B27-Tg rats compared to healthy ones ( 65 ). Studies aiming to understand the involvement of dimer formation in disease pathogenesis unearthed that HLA-B27 variants associated with SpA have an increased tendency for dimer formation ( 73 ). The receptors for HLA-B27 homodimers were found to be KIR3DL1, LILRB2 ( 74 ), KIR3DL2 ( 75 ) and LIR6 ( 68 ) and expressed on lymphocytes, monocytes and Natural Killer Cells (NKs) ( 75 ). The level of KIR3DL2 positive NK and CD4+ T cells increases in the peripheral blood and synovial fluid of SpA and ERA patients. The receptor engagement also shows a survival and activation profile in NKs and T cells respectively ( 73 , 76 ) whereas the dissociation between KIR3DL2 and HLA-B27 decreases the survival/proliferation of NKs and the release of disease related cytokine IL17 from the PBMCs of AS patients ( 77 ). In line with this finding, KIR3DL2+ CD4+ T cells collected from the synovial fluid of SpA patients displays enhanced levels of IL17 secretion ( 78 ). Unfolded protein response (UPR) activation UPR activation was suggested to be a major explanation in SpA pathogenesis. Proteins are biomolecules that orchestrate many cellular processes. To fulfill their task, they need to be folded properly in the organelle called Endoplasmic Reticulum (ER). Proteins with folding deficits can be removed via autophagy or Endoplasmic Reticulum Associated Degradation (ERAD) ( 79 ). However, the accumulation of misfolded proteins may also take place which in turn activates a stress response machinery namely UPR. This stress is regulated through 3 transmembrane proteins located on ER: Inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK) and Activating Transcription Factor 6 (ATF6). Upon its activation, IRE1 cleaves Xbp mRNA leading to the formation of an active transcription factor sXbp. This factor is responsible for the synthesis of chaperones and ERAD components to achieve cellular homeostasis whereas an increase in magnitude and duration of stress results in the activation of the apoptotic IRE1-JNK pathway. Activation of PERK leads to the phosphorylation of eIF2α which in turn put a halt to translation whereas activating selective translation of the ATF4 transcription factor. Similar to IRE1 pathway, ATF4 is also responsible for the activation of homeostatic genes yet unresolved stress promotes the expression of pro-apoptotic CHOP. ATF6 is translocated to Golgi upon UPR and cleaved with S1P and S2P proteases. The newly formed cytosolic domain of ATF6 is a transcription factor and is involved in the transcription of chaperones ( 80 ). HLA-B27 is unique in a way that it misfolds even in the presence of β2m and peptides which in turn activate UPR ( Figure 3 ). There are several bodies of evidence proving this aberrant feature of HLA-B27: - Folding rate: HLA-B27 has a slow folding nature which in turn induces homodimer formation and its retention in the ER. These molecules can then activate the UPR. The B pocket which is located at the peptide binding groove of HLA-B27 seems to be crucial for this machinery. Altering residues in this region not only enhances HLA-B27's folding but also alleviates homodimer formation ( 81 ). - ERAD: Another clue showing the misfolded nature of HLA-B27 is its enhanced predisposition to undergo ERAD ( 82 ) in which EDEM1, and HRD1 were found to be pivotal regulators ( 83 , 84 ). In support of this notion, the use of ERAD blocking reagents results in an increment in the levels of HLA-B27 dimers/oligomers. - Interaction with chaperones: The chaperones help misfolded proteins to gain a proper tertiary structure. The prolonged interaction between HLA-B27 multimers and chaperone Bip indicates the improper folding of HLA-B27 which in turn activates the stress response ( 85 ). In addition, the enhanced interaction between HLA-B27 and oxidoreductase Erp57 is also involved in dimer formation which again may turn on UPR ( 86 ). Macrophages residing in the peripheral joints of AS patients have increased levels of Bip compared to osteoarthritis patients ( 87 ) and mononuclear cells collected from the synovial fluid of SpA patients shows an activation state for UPR ( 88 ). In B27-Tg rats, bone marrow derived macrophages shows prominent UPR activation status which shows a positive correlation with HLA-B27 levels ( 89 , 90 ). The UPR induction was also observed in B27-Tg rats' dendritic cells ( 55 ). Strikingly, ERAP1 deficient B27-Tg rats remained healthy due to the blockade of UPR activation ( 91 ). The pathogenesis of SpA clearly involves the activation of the immune system thus UPR-driven immune modulation has also been the subject of extensive investigation. NF κ B was shown to be activated during UPR ( 92 ) that mediates Th17 differentiation via IL23, a cytokine that is elevated in the serum and synovial fluid of SpA patients ( 93 , 94 ). The activated Th17 cells in turn produce cytokines such as IL17, TNF and IL6 ( 95 ). A strong activation for IL23/IL17 axis was detected in the colon of B27-Tg rats ( 96 ) and overexpression of IL23 causes a disease phenotype similar to AS in mice ( 97 ). In addition, Th17 cells were found to be enriched in the peripheral blood of AS patients ( 98 ). Furthermore, DCs, a major source for IL23, contribute to Th17 cells' expansion in the B27-Tg animal model ( 71 ). Although, macrophages with prominent UPR activation are destined to produce higher levels of IL23 ( 96 ), there are also studies showing that IL23 production is independent of UPR activation thus further studies are warranted ( 99 ). Another NF κ B dependent cytokine TNF-α is also a critical component of the disease and also used as a target for therapy. Similar to IL23, overexpression of TNF results in spondyloarthritis formation in mice and this process was found to be regulated through mesenchymal cells ( 100 ). The level of Bip in the macrophages collected from the synovial fluid shows a positive correlation with TNF levels indicating that immune modulation by UPR might be the basis for elevated TNF levels in disease ( 87 ). TNF is detected in the inflamed tissues of SpA patients and also is elevated in PBMCs and serum ( 101 ). Other susceptibility genes Studies on families revealed that SpAs may have a heritable component. For JIA the recurrence risk in first cousins was determined to be 5.8 fold whereas the sibling relative risk is estimated to be 11.6 fold ( 102 ). AS's heritability is ≥90% ( 103 , 104 ) with a sibling recurrence risk of 8.2% ( 105 ). The prevalence increases dramatically with the presence of a first degree relative suffering from the disease ( 106 , 107 ) and the concordance rate was determined to be 25%–75% and 4%–15% in monozygotic and dizygotic twins respectively ( 103 , 104 , 108 ). Overall, these findings strongly indicate that genetic factors are key determinants in disease pathogenesis. As mentioned above the presence of HLA-B27 showed the highest association with disease susceptibility. However, the fact that only 2% of the HLA-B27 positive population develops SpA is a strong indicator that there are additional genetic risk factors for the development of the disease ( 109 ). To understand this phenomenon better genome wide association studies (GWAS) were performed in SpA patients. The second well established susceptibility locus for AS and ERA was found to be ERAP1 ( 110 , 111 ) which is involved in the presentation of peptides with optimal length. Several studies made it apparent that defects in ERAP function might be involved in disease pathogenesis. ERAP1 variants with a loss of function shows a protective effect for disease which provides strong evidence for the involvement of atypical processing of antigenic peptides ( 112 ). Indeed, HLA-B27 was shown to bind extended peptides with protruding C-terminus ( 113 ) which in turn may activate a potent T cell response thus leading to SpA development. The deficits in peptide trimming might also decrease the level of peptide- loaded MHC molecules which in turn increase the levels of misfolded HLA-B27 molecules, UPR activation and disease progression. Elevated UPR levels might also be regulated through other mechanisms including damaged ubiquitin-ERAD machinery. Previous studies clearly show that ubiquitin conjugating enzyme UBE2J1 is involved in targeting of MHC class I molecules for ERAD ( 83 ) thus further studies aiming to unveil the link between another susceptibility gene UBE2E3 ( 114 ) and ERAD in disease etiology would be valuable. Shaping immune response is indispensable in AS pathogenesis. As mentioned above, the IL23/IL17 axis is a crucial component of this machinery. Its importance was also verified in GWAS studies. Molecules related with this pathway (IL23R, IL12B, IL6R, IL1R1, IL1R2, TYK2, IL27A, STAT3, JAK2) are among the gene loci that shows association with AS ( 83 , 114 ). This also holds true for the TNF-α pathway. Research revealed the presence of disease associated SNPs near to/in TNFRSF1A ( 83 , 114 ), TNFSF15 ( 115 ) and TRADD ( 116 ). Genes related to T cell regulation, RUNX3, IL7R, EOMES, ZMIZ1, ICOSLG, SH2B3 and BACH2, are also among the AS risk loci ( 9 ). Other genes showing association with AS are GPR25, GPR65, GPR35, TBKBP1, PTGER4, BACH2, NOS2, FCGR2A, NKX2-3 ( 9 ), CARD9 ( 117 , 118 ), KIF21B ( 119 ), ANTXR2 ( 120 ), ANO6 ( 121 ). For JIA, ERAP1and IL23R are among the disease susceptibility genes for ERA and juvenile psoriatic arthritis respectively ( 111 ). The lower prevalence of JIA subtypes hampers the construction of well-powered cohorts for GWAS analysis. Thus combining all JIA subtypes rather than investigating them separately was used to detect genetic associations. PTPN2, ANGPT1, COG6 ( 122 ), CD80, JMJD1C ( 123 ), TRAF1-C5 ( 124 ), VTCN1 ( 125 ), IL2RA, IL2RB, STAT4 ( 126 ), TNFAIP3 and TRAF1/C5 ( 127 ) were found to be JIA-predisposing loci. Gender Many rheumatic diseases display gender predominance. Both the incidence of AS ( 109 ) and ERA ( 128 ) are higher in males. However, the male to female ratio has showed decrement over time ( 129 ). This gender predominance indicates that sex-specific factors might play a role in SpA pathogenesis. Among these factors the impact of hormones in SpA progression was extensively analyzed. Of note, the age interval for ERA patients is 6–16 and it is well known that the level of sex hormones increases with puberty thus the following observations may partially explain the male dominance of ERA. The effect of TNF inhibitors on SpA progression underlines TNF's importance in disease progression. Interestingly, estrogen was shown to decrease inflammation in SpA patients via downregulating TNF alpha levels ( 130 ). Estrogen supplementation was also shown to decrease disease severity both in human ( 131 ) and animal female subjects ( 132 ) whereas there are other studies showing no evident association ( 133 ). Testosterone levels on the other hand did not show any difference between SpA patients and healthy controls and were not likely to regulate disease progression ( 131 , 134 ). Sex hormones were also shown to regulate the microbiome ( 135 ) and immune system ( 136 ). The fact that both of these factors play a role in disease progression (see below) underlines the presence of a possible hormone-driven microbiome and immune system related axis however, further studies are needed. The gut and microbiome The gut is one of the affected sites in SpA. Inflammatory bowel disease (Crohn's disease and ulcerative colitis) occur concomitantly in up to 10% of SpA positive population ( 137 ). Patients display inflammatory lesions at intestinal mucosa ( 138 ) and the gut is also inflamed in the animal model ( 139 ) indicating that an active immune response in the gut and SpA might be interlinked. In support of this notion, macrophages expressing the CD163 scavenger receptor increases in the colonic mucosa of SpA patients ( 140 ) and IL23/IL17 axis is exacerbated in the colon of B27-Tg rats ( 96 ). The relation between SpA development and microbiota has been the center of attention for decades. In animal models, housing of the animals in pathogen-free conditions alleviated the formation of several disease related symptoms including colitis and arthritis. However, their transfer to conventional conditions caused their manifestation. Moreover, treating B27-Tg rats with antibiotics hampered colitis formation ( 141 ) indicating that the microbiome is a key player in disease pathogenesis ( 39 , 142 ). Indeed, there are studies showing the differences in microbiome of SpA patients and healthy controls ( 143 – 145 ) and ileal biopsies from AS patients revealed the presence of adherent and invasive bacteria which is accompanied by the decreased barrier function of the gut ( 146 ). Mucins play a major role in barrier function. Mucin-degrading Akkermansia muciniphila species was found to be elevated in B27-Tg rats indicating that SpA related dysbiosis may be involved in impaired gut barrier ( 147 ). T cells are another key player for maintaining the tolerance against commensal bacteria ( 148 ). Interestingly, CD4+ T cells isolated from B27-Tg rats produces higher levels of IFN-γ in response to antigens derived from these organisms implying that there might be a loss of tolerance for the microbiome ( 149 , 150 ). Moreover, the defective stimulation of T cells by APCs might also contribute to the loss of tolerance for microbial flora ( 151 , 152 ). The link between treatment response and microbiome was also investigated. Patients receiving 3 months of anti-TNF therapy did not show a significant difference in their microbiata composition. However, having higher levels of Burkholderiales prior to therapy and an increment in genus Dialister after therapy was observed in responders ( 153 ). Diet Diet plays a crucial role in the development and progression of many diseases. Diet has also been investigated in SpA however, most studies were not replicated. A study by Haugen et al. indicated that many AS patients reported that diet plays a role in the manifestation and severity of their symptoms ( 154 ) and they follow certain diets to decrease their intensity ( 155 ). Starch consumption was suggested to be an exacerbating factor in SpAs and a low starch diet was found to lower disease activity whereas there are also studies showing no impact ( 155 , 156 ). Salt and dietary fat consumption did not show any correlation with the severity of the symptoms ( 155 , 157 ). Although quitting dairy products seem to have an ameliorating role in disease ( 158 ) there are also studies showing no effect ( 157 ). In human subjects, the impact of prebiotic uptake in SpA progression was analyzed. SpA patients with concomitant quiescent ulcerative colitis receiving Lactobacillus acidophilus and Lactobacillus salivarius displayed lower disease activity ( 159 ). In contrast, a meta-analysis by Sanchez et al. opposed this finding ( 160 ). In animal model the severity of colitis was diminished with the supplementation of diet with prebiotics ( 161 ). The constituent, fructo-oligosaccharides was found to have the greatest anti-inflammatory effect in this regard ( 162 , 163 ). Fibre-rich diets also showed a remedial effect on disease by upregulating short chain fatty acids. Indeed, administration of propionate to B27-Tg animals attenuates intestinal inflammation ( 164 ). Treatment and outcome There are several treatment options used in clinics for SpAs. However, the current therapy options do not always result in full remission. Treatment of ERA varies according to whether the disease is axial or peripheral, the number of active joints, the presence of risk factors, and accompanying extra-articular features ( Table 1 ). NSAIDs are used as the first-line treatment in enthesitis and sacroiliitis because of their analgesic and anti-inflammatory effects. For peripheral disease, DMARDs, especially methotrexate or salazopyrin are recommended. Sulfasalazine or methotrexate is used for enthesitis or active peripheral arthritis ( 165 ). The response to these non-biologic DMARDs varies in a wide range ( 166 ). Non-biologic DMARDs can also be used to prevent the development of anti-drug monoclonal antibodies against TNF inhibitors (TNFis) ( 167 ). Methotrexate and Salazopyrin monotherapy is not recommended in active sacroiliitis whereas they can be used as an adjunct therapy. If arthritis does not respond to non-biologic DMARDs or for patients who develop the axial disease then biologic DMARDs would be indicated, often along with the NSAID treatment. Among these, anti-TNF drugs are the first choice. Since etanercept and adalimumab are licensed for pediatric use, the present data is mainly focused on the effectiveness and safety of these two monoclonal anti-TNF drugs ( 168 , 169 ). Table 1 ERA treatment algorithm. Recently anti-IL17 has become an alternative treatment in ERA as well. A total of 86 patients (52 ERA, 34 JSpA 34 patients; median age, 14 years) were enrolled for an open-label secukinumab trial in the first treatment period. In the second period, responders received secukinumab or placebo. Secukinumab demonstrated a significantly longer time to disease flare and a consistent safety profile similar to adults ( 170 ). Moreover, there are ongoing clinical trials for ixekizumab (NCT04527380). Bridging therapy with systemic glucocorticoids might be used during the initiation or escalation of therapy. Intraarticular glucocorticoid injections of the sacroiliac joints as an adjunct therapy are conditionally recommended ( 165 ). Physiotherapy is also a crucial element in the treatment process thus should be offered to all SpA and JIA patients. Another important aspect of the treatment is to monitor the side-effects of the drugs. For NSAIDs, gastrointestinal problems may arise thus proton pump inhibitors might also be prescribed. On the other hand, adequate fluid intake is essential to circumvent renal injury ( 171 ). Anti-TNF drugs make patients prone to infections thus in countries where tuberculosis is still encountered, routine screening should be performed. Disease activity has to be followed to evaluate the response to treatment. For JIA patients, Juvenile Arthritis Disease Activity Score (JADAS) and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) are screened whereas BASDAI and Ankylosing Spondylitis Disease Activity (ASDAS) are used for the assessment of therapy success in adult SpAs ( 172 , 173 ). These are applied for childhood diseases with axial involvement as well. Finally, Weiss et al. have developed and validated the first disease activity assessment for JSpA through international input and consensus formation techniques: this new criterion was called the Juvenile Spondyloarthritis Disease Activity (JSpADA) Index. This outcome tool had a good performance in discriminating between subjects with active vs. inactive disease and responded well to changes in the disease activity ( 174 ). For adults, many therapeutics have been used and published. The reader is referred to excellent reviews on the subject. The primary treatment for SpA is NSAIDs and TNFis (certolizumab, etanercept, infliximab, adalimumab, and golimumab). Of note, SpA patients have higher levels of TNF-α ( 101 ) and HLA-B27 positive patients have a better response rate to TNF therapy ( 175 ) that might be explained by higher TNF levels in these patients ( 176 ). Therefore having TNF levels above a certain threshold value may help to estimate a better response and analysis of TNF levels before treatment might be beneficial. wIL17 inhibitors (secukinumab and ixekizumab) can also be used for patients. Anti-IL17 is not recommended in patients with IBD or recurrent uveitis. If the patient has tuberculosis or recurrent infections, sulfasalazine is preferred over secukinumab and ixekizumab. Tofacitinib (a JAK inhibitor) is a second-line option for patients with contraindications to TNFi or anti-IL17. Co-treatment with low-dose methotrexate is not generally recommended except with infliximab ( 177 ). Brodalumab (IL17RA), bimekizumab (dual inhibition of IL17A and IL17F), and upadacitinib (selective JAK1 inhibitor) demonstrated improvement in active axial SpA ( 178 – 180 ). Although IL23 inhibitors (tildrakizumab, risankizumab and guselkumab) are effective in the treatment of psoriatic arthritis ( 181 ), in phase 2 and phase 3 studies, the use of ustekinumab and risankizumab did not show any improvement on SpA disease activity ( 182 , 183 ). Of note, discontinuation of these biologic disease-modifying drugs (DMARDs) is not recommended due to the risk of flare ( 177 ) and all these biological DMARDs may be studied in adolescent patients as well. Conclusion As summarized above, studies ongoing for more than 4 decades have led to the discovery of many risk factors for SpA development. Among these factors HLA-B27 seems to be the spearhead helping us to better understand the etiology of the disease. HLA-B27 driven mechanisms are thought to involve UPR activation and switching on the IL23/IL17 axis. The fact that only a part of HLA-B27 positive people develop SpA indicates that there are additional factors contributing to disease pathogenesis. Although the threshold effect for HLA-B27 might be a possible explanation for this observation, it is most likely that further investigation of factors other than HLA-B27 is required that will also pave the way for the development of alternative therapies. The current treatment regimen involves NSAIDs, TNF inhibitors and possibly DMARDs. However, only some patients respond to the treatment which in turn causes a significant decrease in the non-responders' quality of life. Therapies targeting UPR and IL23/IL17 axis have recently gained attention but clinical trials are needed for further validation. Author contributions All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. Conflict of interest UK has received consultancy fees and/or speaker's bureau from Abbvie, Novartis, UCB, Lilly and Pfizer. SÖ has received consultancy fees and/or speaker's bureau from Novartis and SOBI. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Supplementary material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fped.2022.1074239/full#supplementary-material .

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4737579/

Generation and characterization of non-competitive furin-inhibiting nanobodies

The PC (proprotein convertase) furin cleaves a large variety of proproteins and hence plays a major role in many pathologies. Therefore furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy-chain-derived nanobodies against catalytically active furin were developed as specific furin inhibitors. The nanobodies bound only to furin but not to other PCs. Upon overexpression in cell lines, they inhibited the cleavage of two different furin substrates, TGFβ (transforming growth factor β) and GPC3 (glypican 3). Purified nanobodies could inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but did not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode-of-action based on steric hindrance. The dissociation constant of purified nanobody 14 is in the nanomolar range. The nanobodies were non-competitive inhibitors with an inhibitory constant in the micromolar range as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies could protect HEK (human embryonic kidney)-293T cells from diphtheria-toxin-induced cytotoxicity as efficiently as the PC inhibitor nona- d -arginine. In conclusion, these antibody-based single-domain nanobodies represent the first generation of highly specific non-competitive furin inhibitors.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661007/

Host cell cytotoxicity and cytoskeleton disruption by CerADPr, an ADP-ribosyltransferase of Bacillus cereus G9241

Bacillus cereus G9241 was isolated from a welder suffering from an anthrax-like inhalation illness. B. cereus G9241 encodes two megaplasmids, pBCXO1 and pBC210, which are analogous to the toxin- and capsule-encoding virulence plasmids of B. anthracis . Protein modeling predicted that the pBC210 LF homolog contained an ADP-ribosyltransferase (ADPr) domain. This putative bacterial ADP-ribosyltransferase domain was denoted CerADPr. Iterative modeling showed that CerADPr possessed several conserved ADP-ribosyltransferase features, including an α-3 helix, an ADP-ribosyltransferase turn-turn loop, and a "Gln-XXX-Glu" motif. CerADPr ADP-ribosylated a ~120kDa protein in HeLa cell lysates and intact cells. EGFP-CerADPr rounded HeLa cells, elicited cytoskeletal changes, and yielded a cytotoxic phenotype, indicating that CerADPr disrupts cytoskeletal signaling. CerADPr(E431D) did not possess ADP-ribosyltransferase or NAD glycohydrolase activities and did not elicit a phenotype in HeLa cells, implicating Glu431 as a catalytic residue. These experiments identify CerADPr as a cytotoxic ADP-ribosyltransferase that disrupts the host cytoskeleton.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10038131/

Dual Function Antibody Conjugates for Multimodal Imaging and Photoimmunotherapy of Cancer Cells

Precision imaging, utilizing molecular targeted agents, is an important tool in cancer diagnostics and guiding therapies. While there are limitations associated with single mode imaging probes, multimodal molecular imaging probes enabling target visualization through complementary imaging technologies provides an attractive alternative. However, there are several challenges associated with designing molecular probes carrying contrast agents for complementary multi-modal imaging. Here, we propose a dual function antibody conjugate (DFAC) comprising an FDA approved photosensitizer Benzoporphyrin derivative (BPD) and a naphthalocyanine-based photoacoustic dye (SiNc(OH)) for multimodal infrared (IR) imaging. While fluorescence imaging, through BPD, provides sensitivity, complementing it with photoacoustic imaging, through SiNc(OH), provides a depth-resolved spatial resolution much beyond the optical diffusion limits of fluorescence measurements. Through a series of in vitro experiments, we demonstrate the development and utilization of DFACs for multimodal imaging and photodynamic treatment of squamous cell carcinoma (A431) cell line. The proposed DFACs have potential use in precision imaging applications such as guiding tumor resection surgeries and photodynamic treatment of residual microscopic disease thereby minimizing local recurrence. The data demonstrated in this study merits further investigation for its preclinical and clinical translation.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7427085/

Reengineering anthrax toxin protective antigen for improved receptor-specific protein delivery

Background To increase the size of the druggable proteome, it would be highly desirable to devise efficient methods to translocate designed binding proteins to the cytosol, as they could specifically target flat and hydrophobic protein-protein interfaces. If this could be done in a manner dependent on a cell surface receptor, two layers of specificity would be obtained: one for the cell type and the other for the cytosolic target. Bacterial protein toxins have naturally evolved such systems. Anthrax toxin consists of a pore-forming translocation unit (protective antigen (PA)) and a separate protein payload. When engineering PA to ablate binding to its own receptor and instead binding to a receptor of choice, by fusing a designed ankyrin repeat protein (DARPin), uptake in new cell types can be achieved. Results Prepore-to-pore conversion of redirected PA already occurs at the cell surface, limiting the amount of PA that can be administered and thus limiting the amount of delivered payload. We hypothesized that the reason is a lack of a stabilizing interaction with wild-type PA receptor. We have now reengineered PA to incorporate the binding domain of the anthrax receptor CMG2, followed by a DARPin, binding to the receptor of choice. This construct is indeed stabilized, undergoes prepore-to-pore conversion only in late endosomes, can be administered to much higher concentrations without showing toxicity, and consequently delivers much higher amounts of payload to the cytosol. Conclusion We believe that this reengineered system is an important step forward to addressing efficient cell-specific delivery of proteins to the cytosol. Background To increase the size of the druggable proteome, it would be highly desirable to devise efficient methods to translocate designed binding proteins to the cytosol, as they could specifically target flat and hydrophobic protein-protein interfaces. If this could be done in a manner dependent on a cell surface receptor, two layers of specificity would be obtained: one for the cell type and the other for the cytosolic target. Bacterial protein toxins have naturally evolved such systems. Anthrax toxin consists of a pore-forming translocation unit (protective antigen (PA)) and a separate protein payload. When engineering PA to ablate binding to its own receptor and instead binding to a receptor of choice, by fusing a designed ankyrin repeat protein (DARPin), uptake in new cell types can be achieved. Results Prepore-to-pore conversion of redirected PA already occurs at the cell surface, limiting the amount of PA that can be administered and thus limiting the amount of delivered payload. We hypothesized that the reason is a lack of a stabilizing interaction with wild-type PA receptor. We have now reengineered PA to incorporate the binding domain of the anthrax receptor CMG2, followed by a DARPin, binding to the receptor of choice. This construct is indeed stabilized, undergoes prepore-to-pore conversion only in late endosomes, can be administered to much higher concentrations without showing toxicity, and consequently delivers much higher amounts of payload to the cytosol. Conclusion We believe that this reengineered system is an important step forward to addressing efficient cell-specific delivery of proteins to the cytosol. Background Targeted therapy is nowadays employed to treat several kinds of diseases in which aberrant signaling plays an important role. The molecular targets are typically of two types. The first group are cell surface molecules that are targeted with antibodies, which display a variety of mechanisms of action, including the inhibition of signaling, recruitment of immune functions, or of other molecules, or they can be coupled to toxins and form antibody-drug conjugates (ADCs). The second group of drug targets is intracellular, exemplified by kinases, which are targeted by small molecules that are inherently cell-permeable, and bind to small pockets on their target protein. While all of these approaches have shown great promise, lack of a sufficient therapeutic window and rapid development of resistance are common problems [ 1 – 4 ]. In contrast to extracellular targets that are well accessible to antibodies or other binding proteins, intracellular protein-protein interactions represent a largely untapped resource of targets for cell-specific targeted therapy [ 1 , 2 ]. Small molecules can be developed with high specificity and affinity for many intracellular proteins that provide pockets, a success of decades of development of medicinal chemistry. However, small molecules can usually not inhibit protein-protein interactions, since they cannot bind with high enough specificity to hydrophobic and flat protein-protein interfaces that lack deep binding pockets [ 2 ]. Furthermore, small molecules can be target-specific, but not cell-specific. Binding proteins can be generated today against basically any target molecule, but as therapeutics are mostly limited to targets accessible on the cell surface due to the impermeability of the plasma membrane to biological macromolecules, including proteins. Various delivery methods based on naturally occurring systems as well as on non-natural systems are being developed to deliver proteins across the plasma membrane, yet with widely varying effectiveness, thus aiming to increase the druggable proteome [ 3 ]. Bacterial protein toxins, e.g., anthrax toxin (from Bacillus anthracis ), have evolved naturally to overcome this barrier, the plasma membrane, and are able to transport protein toxins to the cytosol of cells in a receptor-specific manner. Upon receptor binding and proteolytic activation in anthrax toxin-mediated delivery, the toxin complex gets internalized via receptor-mediated endocytosis. In the endosomes, cargo molecules get translocated directly to the cytosol or to intraluminal vesicles and they eventually reach the cytosol by back fusion of these vesicles [ 4 – 7 ]. Due to the modular structure of these toxins, domains can be engineered for altered cell specificity and translocated cargo, making it an adaptable system for protein delivery [ 8 – 12 ]. Recently, our group has developed a generic delivery system based on anthrax toxin, able to deliver a set of model binding proteins to the cytosol of cells [ 10 ]. For retargeting the cell-binding and translocation domain of anthrax toxin, a designed ankyrin repeat protein (DARPin) which binds to the epithelial cell adhesion molecule (EpCAM) was fused C-terminally to protective antigen (PA) with a receptor binding-ablated domain 4 (carrying mutations N682A, D683A), termed PA m , for mutated PA. The PA-binding domain of one of the two native anthrax toxin cargoes, lethal factor 1-254 (LF N ), was fused N-terminally to different cargo DARPins. With this retargeting strategy, we successfully delivered these cargo DARPins to the cytosol of EpCAM-expressing cells [ 10 ]. For retargeted PA, however, only low concentrations could be used, due to a cytotoxic effect of PA alone that occurred with higher concentrations (> 20 nM). Our aim was therefore to generate an in-depth understanding of the underlying mechanism of this toxicity and use this knowledge to design novel reengineered protective antigen variants that overcome the cytotoxic limitations of retargeting and thus to be able to deliver higher quantities of payload. Inspired by the notion that the interface between domains 2 and 4 in the wild-type PA prepore is stabilized by binding to its natural receptor [ 13 ], we rationalized that the cytotoxicity is most likely due to a premature prepore-to-pore conversion of PA, already at physiological pH [ 13 , 14 ]. To counter this effect, we now generated a stabilized version of PA, which contains PA in its wild-type form (PA wt ) with the wild-type soluble extracellular receptor-binding domain of PA, fused to a retargeting DARPin. Here, we provide a detailed protein characterization, confirm the elimination of the cytotoxicity, and show a higher uptake of cytosolically delivered proteins with the new fusion construct. We show that the amount of cytosolically delivered cargo was so far limited by the cytotoxicity of the translocation domain and that this rate-limiting step has now been overcome. Results Design of PA wt -sANTXR-Ac2 Retargeting of PA to various cell surface receptors has previously been achieved by fusing a binding protein to the C-terminus of PA, and we have developed such strategy using DARPins [ 10 ]. Having fused an EpCAM-targeting DARPin (Ac2) with an affinity of 1.3 × 10 −7 M [ 15 ] to the C-terminus of a mutated version of PA, ablating binding to its own receptors, capillary morphogenesis gene-2 (CMG2) and tumor endothelial marker-8 (TEM8) (Fig. 1 a), we generated a highly efficient, cell-specific, retargeted delivery system. Even with low concentrations (20 nM) of the retargeting fusion construct PA m -Ac2, we could detect the cytosolic presence of cargo DARPins [ 10 ]. When increasing the concentration of PA m -Ac2, however, we observed that our delivery system was highly toxic for Flp-In 293-EpCAM-BirA cells stably overexpressing the targeted receptor, without any toxic cargo being delivered. Therefore, we performed an in-depth analysis of PA m -Ac2 to search for the possible cytotoxic mechanism and measures to overcome this. Fig. 1 Ribbon representation of the structures of PA constructs shown in their activated/furin-cleaved PA 63 version. a Previously published, retargeted PA m -Ac2 [ 10 ]. b – d Schematic representation of the prepore-to-pore conversion at the respective pH of furin-cleaved PA wt ( b ), PA m fused to a retargeting DARPin, PA m -Ac2 ( c ), and PA wt fused to the wild-type receptor domain and the retargeting DARPin, PA wt -sANTXR-Ac2 ( d ). e Newly designed stabilized PA wt -sANTXR-Ac2 with PA wt , the wild-type receptor CMG2 VWA domain, and the retargeting DARPin; PA shown in red, EpCAM-retargeting DARPin Ac2 shown in blue, CMG2 receptor VWA domain (sANTXR) shown in green, and prepore-stabilizing interaction region highlighted in black oval. Protein structures were adapted from PDB ID: 1TZN (PA prepore binding sANTXR), 1ACC (PA), and 4YDW (DARPin) When domain 4 of PA wt binds to the wild-type receptor, it forms a metal-ion-dependent structural bridge between domain 4 and the von Willebrand factor A (VWA) region of the anthrax toxin receptor (CMG2 or TEM8) (Fig. 1 b). Especially two binding residues (N682, D683) within domain 4 are very important for PA binding [ 16 ]. Although receptor binding is mainly mediated by domain 4 of PA, parts of the VWA region also interact with domain 2. Binding to the 340-348 loop of PA prevents the rearrangement of the PA insertion loop and the contiguous 2β2 and 2β3 β-strands. It has been shown that the prepore-to pore conversion of PA wt occurs at different pH, depending on it being incubated with or without its wild-type receptor [ 13 , 17 ]. Using mutated PA (PA m ), which is unable to bind its wild-type receptor, the stabilizing interactions between domain 2 and the VWA region are lost, which otherwise prevent the conformational change at neutral pH. Thus, merely fusing a retargeting molecule to PA m does not fully replicate the mechanism of PA wt , which limits the conformational changes to occur in late endosomes. Hence, we propose that the prepore-to-pore conversion of PA m -Ac2 can occur immediately upon oligomerization on the cellular surface, already at physiological pH, thus assembling an open pore allowing ions and other substances to freely pass in and out of the cell (Fig. 1 c). To prevent this premature prepore-to-pore conversion, we designed a domain-2/domain-4 interface-stabilized version of PA (Fig. 1 d, e). To achieve this, we genetically fused the 19.5-kDa VWA domain of CMG2 (residues 40-217, C175A), which we termed sANTXR, to the C-terminus of PA wt . A long (G 4 S) 5 linker between PA wt and sANTXR with an approximate length of 88 à allows the correct orientation and functional interaction of the fusion partners. The covalent linker massively increases the local effective concentration of sANTXR, which in combination with the high affinity for the PA-binding domain is expected to effectively reduce off-target effects of PA wt binding to CMG2 or TEM8 on the cell surface [ 18 ]. This was deduced from the structure of the wild-type conformation of the PA prepore [ 13 ], PDB ID: 1TZN. C-terminally to the sANTXR receptor domain, we fused the EpCAM-targeting DARPin Ac2. We propose that the sANTXR domain impedes premature prepore-to-pore conversion by creating a very similar domain arrangement as in PA wt bound to its receptor CMG2. We thus expect that the pH where the prepore-to-pore conversion can occur shifts back to wild-type conditions (Fig. 1 b–d), conditions that are present only in the (late) endosomes. The cytotoxicity of a premature prepore-to-pore conversion on the cell surface thus should get diminished. To confirm that the stabilizing interaction is really due to the functional interaction of PA with the wild-type receptor domain, we designed a PA mutant construct, PA m -sANTXR-Ac2, with the mutations N682A and D683A (Additional file 1 : Figure S1), which should prevent binding of PA m and sANTXR, thus having no stabilizing interaction. As another control, we also designed a variant with a very short linker between PA wt and the sANTXR domain, restraining the sANTXR domain to an orientation in which binding of PA wt to sANTXR is sterically prevented. Comparing these constructs, a functional dependency of the stabilizing interaction and prepore-to-pore conversion was tested. PA wt -sANTXR-Ac2 reduces cytotoxicity and is dependent on functional interaction of PA wt with its wild-type receptor domain We tested the cytotoxicity of our previously developed construct, PA m -Ac2, in comparison to the new construct PA wt -sANTXR-Ac2. Upon incubation of Flp-In 293-EpCAM-BirA cells, which have been made to stably overexpress EpCAM, with increasing concentrations of PA wt -sANTXR-Ac2, no change in cellular viability was observed up to 500 nM, the maximal concentration tested (Fig. 2 a). Flp-In 293-EpCAM-BirA cells, when incubated with PA m -Ac2 (not containing the receptor domain fusion), however, showed a decrease in viability of ~ 50% already at 19 nM, and even down to only 10% viability at a concentration of 167 nM PA m -Ac2. PA m -sANTXR-Ac2 (which comprises the mutated, non-interacting domains) showed a similar reduction of viability for concentrations ≥ 56 nM, confirming the necessity of a functional interaction between the VWA domain and PA wt . PA m -sANTXR-Ac2 appears to be less toxic than PA m -Ac2, presumably due to steric hindrance of the slightly larger fusion construct, impeding pore formation. We observed in time-lapse imaging video microscopy that the cytotoxicity with this construct occurs at a later timepoint, as described below (Additional file 1 : Figure S2, showing the analysis of the videos of Additional files 2 and 3 ). To ensure that the toxicity was not due to the mutations associated with PA m , we used PA wt -Ac2 as a further control, which, in addition to binding CMG2, will bind EpCAM via Ac2. We expected a comparable toxicity of PA wt -Ac2 and PA m -Ac2 on Flp-In 293-EpCAM-BirA, since binding will be mostly via the highly overexpressed EpCAM without prepore stabilization, and only to a limited extent via CMG2 and TEM8. Indeed, PA wt -Ac2 shows a similar toxicity as PA m -Ac2. A non-targeted control, without the EpCAM binding DARPin Ac2, had no effect on the cells. Fig. 2 Cytotoxicity of different PA variants in comparison with PA wt -sANTXR-Ac2. a Viability assay of Flp-In 293-EpCAM-BirA with respective concentrations of PA wt -sANTXR-Ac2, PA m -sANTXR-Ac2, PA m -Ac2, PA wt -Ac2, and PA wt ( n = 3). Error bars indicate SEM. b Competition assay with Ac2 DARPin and 100 nM PA m -Ac2 with increasing amounts of competitor Ac2-Flag ( n = 3). Error bars indicate SEM. c Quantification of propidium iodide (PI)-positive Flp-In 293-EpCAM-BirA cells during time-lapse imaging, cells treated with either PA wt -sANTXR-Ac2 (black squares) or PA m -Ac2 (green circles) ( n = 6). Error bars indicate SEM. d PI (red) staining for permeable cells and LF N -eGFP staining (green) for binding to surface PA, comparing PA wt -sANTXR-Ac2 and PA m -Ac2 on Flp-In 293-EpCAM-BirA cells To confirm the receptor-specific cytotoxicity of the PA prepore, we incubated Flp-In 293-EpCAM-BirA cells with 100 nM PA m -Ac2, which showed clear toxic effects (Fig. 2 a), and titrated the DARPin Ac2 (Ac2-FLAG) as a binding competitor. With increasing concentrations of competitor, the cytotoxicity was reduced, and with a ~ 3-fold excess of Ac2 DARPin over PA m -Ac2, 100% viability was restored, indicating the cytotoxicity is due to the interaction of PA m -Ac2 with EpCAM and not due to a non-specific cytotoxic effect (Fig. 2 b). In addition to the cell proliferation assay, we performed time-lapse imaging over 18 h. Flp-In 293-EpCAM-BirA cells were treated with 100 nM PA wt -sANTXR-Ac2 or PA m -Ac2, propidium iodide (PI), a marker of cell death, and eGFP fused to the C-terminus of LF N , LF N -eGFP. Cells were imaged over time with an automated LionHeart FX microscope. We measured the increase in PI staining for PA wt -sANTXR-Ac2 and PA m -Ac2 (Fig. 2 c and Additional files 4 and 5 ). Up to 250 cells are PI positive in wells incubated with PA m -Ac2 in a time-dependent manner, while PA wt -sANTXR-Ac2 remained constant at the initial number of ~ 50 PI-positive cells. The lag in response time immediately after addition of PA variants can be attributed to the binding and pore formation on the cell surface, as well as the tolerance of cells to a certain number of pores formed on the plasma membrane (Additional file 1 : Figure S3). We also confirmed cell death by PI staining with confocal microscopy. Cells were treated with 100 nM of the respective constructs and incubated for 3 h before confocal imaging. PA wt -sANTXR-Ac2 shows no cytotoxicity and is thus indistinguishable from untreated control cells, while cells treated with PA m -Ac2 detach and stain highly positive for PI (Fig. 2 d). With the control PA m -sANTXR-Ac2 (without functional interface between these components), we observed a slight delay in cytotoxicity in initial time-lapse imaging compared to PA m -Ac2 (Additional file 1 : Figure S2). We propose that the slightly larger receptor fusion construct PA m -sANTXR-Ac2 sterically hinders rapid prepore-to-pore conversion on the cell surface. To further investigate the structure-function relationship, we designed a construct with a very short linker (SL) between PA wt and the wild-type receptor domain, preventing the correct orientation and binding of PA wt to the VWA domain. With this construct, PA wt-SL -sANTXR-Ac2 (Additional file 1 : Figure S1), we performed a viability assay and could observe a reduced cell viability to 63% at 580 nM (Additional file 1 : Figure S4a). The higher concentrations where a cytotoxic effect is observed compared to PA m -Ac2 could have a similar cause as PA m -sANTXR-Ac2: steric hindrance with respect to form functional intramolecular complexes. To test this hypothesis, we performed a delivery assay to see if it would be still capable of prepore assembly, prepore-to-pore conversion, and delivery (see next section) as discussed below. Even though PA wt-SL -sANTXR-Ac2 was provided as a fusion with N-terminal His 6 -MBP, we want to point out that His 6 -MBP will be cleaved off by furin and the fusion construct, His 6 -MBP-PA wt-SL -sANTXR-Ac2, has previously been shown to demonstrate equivalent delivery to PA m -Ac2 [ 19 ]. PA wt -sANTXR-Ac2 reduces cytotoxicity in a receptor expression level-dependent manner In order to understand to what extent the cytotoxic effects of premature prepore-to-pore conversion is a function of the receptor expression level, we tested our constructs on a panel of EpCAM-positive cells, having different levels of receptor expression: HT29, MCF7, SKBR3, with EpCAM-negative RD cells as control. First, we assessed the EpCAM expression levels via flow cytometry using an Alexa Fluor 488-labeled anti-EpCAM mouse mAb (Fig. 3 a, Additional file 1 : Figure S5). EpCAM has the highest expression levels in the constructed Flp-In 293-EpCAM-BirA cells stably expressing EpCAM, followed by HT29, MCF7, SKBR3, and the EpCAM-negative RD cell line with no detectable surface EpCAM. Since Chernyavska et al. [ 20 ] recently estimated EpCAM levels of MCF7 cells at about 5.3 × 10 5 receptors/cell, we can assume that levels of the high-expressing Flp-In 293-EpCAM-BirA cells are around 2 million receptors/cell, even though these numbers have considerable uncertainty. Fig. 3 Effects of PA on different cell lines expressing EpCAM. a EpCAM surface expression data assessed via flow cytometry using an Alexa Fluor 488-labeled anti-EpCAM mouse mAb ( n = 3). Error bars reflect SEM. b Confocal imaging of stained Flp-In 293-EpCAM-BirA cells with PA wt -sANTXR-Ac2 and LF N -eGFP to assess PA oligomerization. c Viability assays for a set of cell lines with PA wt -sANTXR-Ac2, PA m -sANTXR-Ac2, PA m -Ac2, PA wt -Ac2, and PA wt ( n = 3). Error bars reflect SEM We then assessed whether the receptor expression level correlates with the oligomerization and prepore formation of PA wt -sANTXR-Ac2. It is possible to visualize PA oligomers by saturating available binding sites with LF N -eGFP, which is not transported (Additional file 1 : Figure S6). Using confocal microscopy, we found that a higher receptor density resulted in more prepore formation, reflecting successful PA oligomerization (Fig. 3 b). The signal was highest for Flp-In 293-EpCAM-BirA cells, followed by HT29 cells. For MCF7 and especially SKBR3, however, very little signal can be detected, although the receptor expression levels are in similar ranges as for the HT29 cell line. No signal for RD cells was observed, the EpCAM-negative control cell line. For cells expressing EpCAM, we detected a membrane-like staining pattern when incubated with LF N -eGFP and PA wt -sANTXR-Ac2. For Flp-In 293-EpCAM-BirA cells and HT29 cells, we further detected a dotted staining within cellular compartments, showing endo-/lysosomal localization. Endosomal entrapment of LF N -eGFP has been confirmed with the BirA assay (Additional file 1 : Figure S6). The detection of an endosomal-like staining for LF N -eGFP in MCF7 and SKBR3 cells is not evident due to the detection threshold of the microscope in combination with the limited numbers of receptors. We propose that the non-linear dependency of PA prepore formation on receptor density is due to a receptor-level threshold below which pore formation becomes less efficient. Additionally, varying mobilities of the receptors or different internalization and degradation rates of EpCAM in the different cell lines as well as different efficiency of furin activation may also contribute to these differences [ 21 ]. We then performed a viability assay with the panel of cell lines with PA wt -sANTXR-Ac2, PA m -sANTXR-Ac2, PA m -Ac2, PA wt -Ac2, and the non-targeted control, PA wt (Fig. 3 c). A reduced cell viability can be observed for HT29 cells (Fig. 3 c) with concentrations of 167 nM of PA wt -Ac2 and 500 nM of PA m -Ac2, leading to a viability of 46% and 33%, respectively. For MCF7, SKBR3, and RD cells, no cytotoxicity could be observed, which is in agreement with the lower expression levels of the receptor and it correlates to the expected lower levels of prepore formation on these cells. Lower toxicity of PA wt -sANTXR-Ac2 enables greater cytosolic protein delivery Previously, we have shown that PA m -Ac2 can efficiently deliver various cargoes to the cytosol of Flp-In 293-EpCAM-BirA cells stably overexpressing EpCAM [ 10 ]. Our goal in this study was to increase the amount of cytosolically delivered cargo molecules, which previously was not possible, since concentrations higher than 20 nM of the pore-forming PA m -Ac2 drastically reduced cellular viability even within the short 4-h incubation time (Additional file 2 ). Our newly designed, prepore-stabilizing PA wt -sANTXR-Ac2 was therefore next tested for efficient protein delivery with the biotin ligase assay [ 22 ]. We incubated Flp-In 293-EpCAM-BirA cells with PA wt -sANTXR-Ac2, PA m -sANTXR-Ac2, and PA m -Ac2 for 4 h in the presence of the proteasome inhibitor MG-132. MG-132 was included to assess the delivery systems independently of proteasomal degradation. As cargo proteins, we tested three different DARPins, varying in size and thermostability, which have previously shown to be effectively translocated [ 10 ]. These cargo molecules contain the biotin-acceptor avi-tag and an HA-tag at their C-terminus and are fused with their N-terminus to LF N . Cytosolically localized cargo proteins are biotinylated by a cytoplasmically encoded BirA of Flp-In 293-EpCAM-BirA cells [ 22 ]. Cargo molecules which are trapped within the endosome, not reaching the cytosol, are not biotinylated. The HA-tag is used to determine total cellular uptake, located in the cytosol and in any other cellular compartment, allowing the determination of the intracellular localization of a cargo molecule. After cell harvest and western blotting, biotinylated cargoes were detected with streptavidin IRDye 680LT and total cellular uptake was measured via an HA-tag antibody [ 22 ]. For quantification of cytosolically present cargo molecules, the protein(s) detected at around 70 kDa, which we hypothesized earlier to be endogenous heat shock protein 70 (HSP70), were chosen as a loading control [ 10 ]. With increasing concentrations of PA wt -sANTXR-Ac2, total cellular uptake (Fig. 4 b, d) and cytosolic delivery (Fig. 4 a, c) of the smallest DARPin NI 1 C increase. An increase in cytosolically present cargo can be seen up to an external concentration of 200 nM. Further increases in the concentration of PA wt -sANTXR-Ac2 did not yield higher amounts of delivered DARPin (Fig. 4 c, d), presumably due to a saturation of the receptors exploited for delivery. Fig. 4 Western blots of the BirA assay showing increased delivery of LF N -cargo constructs with PA wt -sANTXR-Ac2 on Flp-In 293-EpCAM-BirA cells. Cytosolically delivered cargo proteins are biotinylated by a cytoplasmically encoded BirA and stained with Streptavidin IRDye 680LT. Total cellular uptake measured via HA-tag on the LF N -cargo. a , b Increasing concentrations of respective PA constructs incubated with a 5-fold excess of LF N -NI 1 C. Boxes indicate the bands of interest. c , d Quantification of western blot bands from a and b . Black bars, PA wt -sANTXR-Ac2; red bars, PA m -sANTXR-Ac2; green bars, PA m -Ac2. The dotted line represents background signal (i.e., cells only), and the dashed line shows the signal of cargo at 20 nM PA wt -sANTXR-Ac2. e , f Cytosolic localization ( e ) and total cellular uptake ( f ) of three different cargo DARPins delivered with 20 nM (lanes 1–3) and 100 nM (lanes 4–6) of PA wt -sANTXR-Ac2 or 100 nM (lanes 7–9) for PA m -Ac2; lanes 10 and 11 represent cells incubated with 100 nM LF N -cargo without PA. "dest." refers to rationally destabilized versions of NI 2 C and NI 3 C DARPins [ 10 ]. Boxes indicate the bands of interest At 20 nM, similar delivery efficiencies can be observed for PA wt -sANTXR-Ac2, PA m -sANTXR-Ac2, and PA m -Ac2, but an increase to 100 nM does not lead to an increase in cytosolically present cargo for PA m -sANTXR-Ac2 and PA m -Ac2, as it does for PA wt -sANTXR-Ac2, likely due to the premature prepore-to-pore conversion of PA m -Ac2 and PA m -sANTXR-Ac2 on the cell surface. This lack of functional pores renders the cells unable to unfold and translocate LF N -cargo proteins (Fig. 4 c, d). Slightly higher total cellular uptake of LF N -cargo can be observed with PA m -sANTXR-Ac2 than for PA m -Ac2, probably due to the delayed cytotoxicity compared to PA m -Ac2. Similar results have been observed for LF N -NI 2 C dest. as well as LF N -NI 3 C dest. , constructs that have been slightly destabilized to facilitate their unfolding and refolding during transport through the pore [ 10 , 19 ] (Fig. 4 e, f). With increasing concentrations (20 nM and 100 nM), an increase in cytosolic cargo delivery can be observed. The BirA assay for His 6 -MBP-PA wt-SL -sANTXR-Ac2 (Additional file 1 : Figure S4b) showed a reduced amount of total cellular uptake, suggesting a steric inhibition effect already at the start of the internalization process. The results for this construct are in line with the results for PA m -sANTXR-Ac2 and confirm the functional dependency of PA on interactions with the sANTXR domain. Design of PA wt -sANTXR-Ac2 Retargeting of PA to various cell surface receptors has previously been achieved by fusing a binding protein to the C-terminus of PA, and we have developed such strategy using DARPins [ 10 ]. Having fused an EpCAM-targeting DARPin (Ac2) with an affinity of 1.3 × 10 −7 M [ 15 ] to the C-terminus of a mutated version of PA, ablating binding to its own receptors, capillary morphogenesis gene-2 (CMG2) and tumor endothelial marker-8 (TEM8) (Fig. 1 a), we generated a highly efficient, cell-specific, retargeted delivery system. Even with low concentrations (20 nM) of the retargeting fusion construct PA m -Ac2, we could detect the cytosolic presence of cargo DARPins [ 10 ]. When increasing the concentration of PA m -Ac2, however, we observed that our delivery system was highly toxic for Flp-In 293-EpCAM-BirA cells stably overexpressing the targeted receptor, without any toxic cargo being delivered. Therefore, we performed an in-depth analysis of PA m -Ac2 to search for the possible cytotoxic mechanism and measures to overcome this. Fig. 1 Ribbon representation of the structures of PA constructs shown in their activated/furin-cleaved PA 63 version. a Previously published, retargeted PA m -Ac2 [ 10 ]. b – d Schematic representation of the prepore-to-pore conversion at the respective pH of furin-cleaved PA wt ( b ), PA m fused to a retargeting DARPin, PA m -Ac2 ( c ), and PA wt fused to the wild-type receptor domain and the retargeting DARPin, PA wt -sANTXR-Ac2 ( d ). e Newly designed stabilized PA wt -sANTXR-Ac2 with PA wt , the wild-type receptor CMG2 VWA domain, and the retargeting DARPin; PA shown in red, EpCAM-retargeting DARPin Ac2 shown in blue, CMG2 receptor VWA domain (sANTXR) shown in green, and prepore-stabilizing interaction region highlighted in black oval. Protein structures were adapted from PDB ID: 1TZN (PA prepore binding sANTXR), 1ACC (PA), and 4YDW (DARPin) When domain 4 of PA wt binds to the wild-type receptor, it forms a metal-ion-dependent structural bridge between domain 4 and the von Willebrand factor A (VWA) region of the anthrax toxin receptor (CMG2 or TEM8) (Fig. 1 b). Especially two binding residues (N682, D683) within domain 4 are very important for PA binding [ 16 ]. Although receptor binding is mainly mediated by domain 4 of PA, parts of the VWA region also interact with domain 2. Binding to the 340-348 loop of PA prevents the rearrangement of the PA insertion loop and the contiguous 2β2 and 2β3 β-strands. It has been shown that the prepore-to pore conversion of PA wt occurs at different pH, depending on it being incubated with or without its wild-type receptor [ 13 , 17 ]. Using mutated PA (PA m ), which is unable to bind its wild-type receptor, the stabilizing interactions between domain 2 and the VWA region are lost, which otherwise prevent the conformational change at neutral pH. Thus, merely fusing a retargeting molecule to PA m does not fully replicate the mechanism of PA wt , which limits the conformational changes to occur in late endosomes. Hence, we propose that the prepore-to-pore conversion of PA m -Ac2 can occur immediately upon oligomerization on the cellular surface, already at physiological pH, thus assembling an open pore allowing ions and other substances to freely pass in and out of the cell (Fig. 1 c). To prevent this premature prepore-to-pore conversion, we designed a domain-2/domain-4 interface-stabilized version of PA (Fig. 1 d, e). To achieve this, we genetically fused the 19.5-kDa VWA domain of CMG2 (residues 40-217, C175A), which we termed sANTXR, to the C-terminus of PA wt . A long (G 4 S) 5 linker between PA wt and sANTXR with an approximate length of 88 à allows the correct orientation and functional interaction of the fusion partners. The covalent linker massively increases the local effective concentration of sANTXR, which in combination with the high affinity for the PA-binding domain is expected to effectively reduce off-target effects of PA wt binding to CMG2 or TEM8 on the cell surface [ 18 ]. This was deduced from the structure of the wild-type conformation of the PA prepore [ 13 ], PDB ID: 1TZN. C-terminally to the sANTXR receptor domain, we fused the EpCAM-targeting DARPin Ac2. We propose that the sANTXR domain impedes premature prepore-to-pore conversion by creating a very similar domain arrangement as in PA wt bound to its receptor CMG2. We thus expect that the pH where the prepore-to-pore conversion can occur shifts back to wild-type conditions (Fig. 1 b–d), conditions that are present only in the (late) endosomes. The cytotoxicity of a premature prepore-to-pore conversion on the cell surface thus should get diminished. To confirm that the stabilizing interaction is really due to the functional interaction of PA with the wild-type receptor domain, we designed a PA mutant construct, PA m -sANTXR-Ac2, with the mutations N682A and D683A (Additional file 1 : Figure S1), which should prevent binding of PA m and sANTXR, thus having no stabilizing interaction. As another control, we also designed a variant with a very short linker between PA wt and the sANTXR domain, restraining the sANTXR domain to an orientation in which binding of PA wt to sANTXR is sterically prevented. Comparing these constructs, a functional dependency of the stabilizing interaction and prepore-to-pore conversion was tested. PA wt -sANTXR-Ac2 reduces cytotoxicity and is dependent on functional interaction of PA wt with its wild-type receptor domain We tested the cytotoxicity of our previously developed construct, PA m -Ac2, in comparison to the new construct PA wt -sANTXR-Ac2. Upon incubation of Flp-In 293-EpCAM-BirA cells, which have been made to stably overexpress EpCAM, with increasing concentrations of PA wt -sANTXR-Ac2, no change in cellular viability was observed up to 500 nM, the maximal concentration tested (Fig. 2 a). Flp-In 293-EpCAM-BirA cells, when incubated with PA m -Ac2 (not containing the receptor domain fusion), however, showed a decrease in viability of ~ 50% already at 19 nM, and even down to only 10% viability at a concentration of 167 nM PA m -Ac2. PA m -sANTXR-Ac2 (which comprises the mutated, non-interacting domains) showed a similar reduction of viability for concentrations ≥ 56 nM, confirming the necessity of a functional interaction between the VWA domain and PA wt . PA m -sANTXR-Ac2 appears to be less toxic than PA m -Ac2, presumably due to steric hindrance of the slightly larger fusion construct, impeding pore formation. We observed in time-lapse imaging video microscopy that the cytotoxicity with this construct occurs at a later timepoint, as described below (Additional file 1 : Figure S2, showing the analysis of the videos of Additional files 2 and 3 ). To ensure that the toxicity was not due to the mutations associated with PA m , we used PA wt -Ac2 as a further control, which, in addition to binding CMG2, will bind EpCAM via Ac2. We expected a comparable toxicity of PA wt -Ac2 and PA m -Ac2 on Flp-In 293-EpCAM-BirA, since binding will be mostly via the highly overexpressed EpCAM without prepore stabilization, and only to a limited extent via CMG2 and TEM8. Indeed, PA wt -Ac2 shows a similar toxicity as PA m -Ac2. A non-targeted control, without the EpCAM binding DARPin Ac2, had no effect on the cells. Fig. 2 Cytotoxicity of different PA variants in comparison with PA wt -sANTXR-Ac2. a Viability assay of Flp-In 293-EpCAM-BirA with respective concentrations of PA wt -sANTXR-Ac2, PA m -sANTXR-Ac2, PA m -Ac2, PA wt -Ac2, and PA wt ( n = 3). Error bars indicate SEM. b Competition assay with Ac2 DARPin and 100 nM PA m -Ac2 with increasing amounts of competitor Ac2-Flag ( n = 3). Error bars indicate SEM. c Quantification of propidium iodide (PI)-positive Flp-In 293-EpCAM-BirA cells during time-lapse imaging, cells treated with either PA wt -sANTXR-Ac2 (black squares) or PA m -Ac2 (green circles) ( n = 6). Error bars indicate SEM. d PI (red) staining for permeable cells and LF N -eGFP staining (green) for binding to surface PA, comparing PA wt -sANTXR-Ac2 and PA m -Ac2 on Flp-In 293-EpCAM-BirA cells To confirm the receptor-specific cytotoxicity of the PA prepore, we incubated Flp-In 293-EpCAM-BirA cells with 100 nM PA m -Ac2, which showed clear toxic effects (Fig. 2 a), and titrated the DARPin Ac2 (Ac2-FLAG) as a binding competitor. With increasing concentrations of competitor, the cytotoxicity was reduced, and with a ~ 3-fold excess of Ac2 DARPin over PA m -Ac2, 100% viability was restored, indicating the cytotoxicity is due to the interaction of PA m -Ac2 with EpCAM and not due to a non-specific cytotoxic effect (Fig. 2 b). In addition to the cell proliferation assay, we performed time-lapse imaging over 18 h. Flp-In 293-EpCAM-BirA cells were treated with 100 nM PA wt -sANTXR-Ac2 or PA m -Ac2, propidium iodide (PI), a marker of cell death, and eGFP fused to the C-terminus of LF N , LF N -eGFP. Cells were imaged over time with an automated LionHeart FX microscope. We measured the increase in PI staining for PA wt -sANTXR-Ac2 and PA m -Ac2 (Fig. 2 c and Additional files 4 and 5 ). Up to 250 cells are PI positive in wells incubated with PA m -Ac2 in a time-dependent manner, while PA wt -sANTXR-Ac2 remained constant at the initial number of ~ 50 PI-positive cells. The lag in response time immediately after addition of PA variants can be attributed to the binding and pore formation on the cell surface, as well as the tolerance of cells to a certain number of pores formed on the plasma membrane (Additional file 1 : Figure S3). We also confirmed cell death by PI staining with confocal microscopy. Cells were treated with 100 nM of the respective constructs and incubated for 3 h before confocal imaging. PA wt -sANTXR-Ac2 shows no cytotoxicity and is thus indistinguishable from untreated control cells, while cells treated with PA m -Ac2 detach and stain highly positive for PI (Fig. 2 d). With the control PA m -sANTXR-Ac2 (without functional interface between these components), we observed a slight delay in cytotoxicity in initial time-lapse imaging compared to PA m -Ac2 (Additional file 1 : Figure S2). We propose that the slightly larger receptor fusion construct PA m -sANTXR-Ac2 sterically hinders rapid prepore-to-pore conversion on the cell surface. To further investigate the structure-function relationship, we designed a construct with a very short linker (SL) between PA wt and the wild-type receptor domain, preventing the correct orientation and binding of PA wt to the VWA domain. With this construct, PA wt-SL -sANTXR-Ac2 (Additional file 1 : Figure S1), we performed a viability assay and could observe a reduced cell viability to 63% at 580 nM (Additional file 1 : Figure S4a). The higher concentrations where a cytotoxic effect is observed compared to PA m -Ac2 could have a similar cause as PA m -sANTXR-Ac2: steric hindrance with respect to form functional intramolecular complexes. To test this hypothesis, we performed a delivery assay to see if it would be still capable of prepore assembly, prepore-to-pore conversion, and delivery (see next section) as discussed below. Even though PA wt-SL -sANTXR-Ac2 was provided as a fusion with N-terminal His 6 -MBP, we want to point out that His 6 -MBP will be cleaved off by furin and the fusion construct, His 6 -MBP-PA wt-SL -sANTXR-Ac2, has previously been shown to demonstrate equivalent delivery to PA m -Ac2 [ 19 ]. PA wt -sANTXR-Ac2 reduces cytotoxicity in a receptor expression level-dependent manner In order to understand to what extent the cytotoxic effects of premature prepore-to-pore conversion is a function of the receptor expression level, we tested our constructs on a panel of EpCAM-positive cells, having different levels of receptor expression: HT29, MCF7, SKBR3, with EpCAM-negative RD cells as control. First, we assessed the EpCAM expression levels via flow cytometry using an Alexa Fluor 488-labeled anti-EpCAM mouse mAb (Fig. 3 a, Additional file 1 : Figure S5). EpCAM has the highest expression levels in the constructed Flp-In 293-EpCAM-BirA cells stably expressing EpCAM, followed by HT29, MCF7, SKBR3, and the EpCAM-negative RD cell line with no detectable surface EpCAM. Since Chernyavska et al. [ 20 ] recently estimated EpCAM levels of MCF7 cells at about 5.3 × 10 5 receptors/cell, we can assume that levels of the high-expressing Flp-In 293-EpCAM-BirA cells are around 2 million receptors/cell, even though these numbers have considerable uncertainty. Fig. 3 Effects of PA on different cell lines expressing EpCAM. a EpCAM surface expression data assessed via flow cytometry using an Alexa Fluor 488-labeled anti-EpCAM mouse mAb ( n = 3). Error bars reflect SEM. b Confocal imaging of stained Flp-In 293-EpCAM-BirA cells with PA wt -sANTXR-Ac2 and LF N -eGFP to assess PA oligomerization. c Viability assays for a set of cell lines with PA wt -sANTXR-Ac2, PA m -sANTXR-Ac2, PA m -Ac2, PA wt -Ac2, and PA wt ( n = 3). Error bars reflect SEM We then assessed whether the receptor expression level correlates with the oligomerization and prepore formation of PA wt -sANTXR-Ac2. It is possible to visualize PA oligomers by saturating available binding sites with LF N -eGFP, which is not transported (Additional file 1 : Figure S6). Using confocal microscopy, we found that a higher receptor density resulted in more prepore formation, reflecting successful PA oligomerization (Fig. 3 b). The signal was highest for Flp-In 293-EpCAM-BirA cells, followed by HT29 cells. For MCF7 and especially SKBR3, however, very little signal can be detected, although the receptor expression levels are in similar ranges as for the HT29 cell line. No signal for RD cells was observed, the EpCAM-negative control cell line. For cells expressing EpCAM, we detected a membrane-like staining pattern when incubated with LF N -eGFP and PA wt -sANTXR-Ac2. For Flp-In 293-EpCAM-BirA cells and HT29 cells, we further detected a dotted staining within cellular compartments, showing endo-/lysosomal localization. Endosomal entrapment of LF N -eGFP has been confirmed with the BirA assay (Additional file 1 : Figure S6). The detection of an endosomal-like staining for LF N -eGFP in MCF7 and SKBR3 cells is not evident due to the detection threshold of the microscope in combination with the limited numbers of receptors. We propose that the non-linear dependency of PA prepore formation on receptor density is due to a receptor-level threshold below which pore formation becomes less efficient. Additionally, varying mobilities of the receptors or different internalization and degradation rates of EpCAM in the different cell lines as well as different efficiency of furin activation may also contribute to these differences [ 21 ]. We then performed a viability assay with the panel of cell lines with PA wt -sANTXR-Ac2, PA m -sANTXR-Ac2, PA m -Ac2, PA wt -Ac2, and the non-targeted control, PA wt (Fig. 3 c). A reduced cell viability can be observed for HT29 cells (Fig. 3 c) with concentrations of 167 nM of PA wt -Ac2 and 500 nM of PA m -Ac2, leading to a viability of 46% and 33%, respectively. For MCF7, SKBR3, and RD cells, no cytotoxicity could be observed, which is in agreement with the lower expression levels of the receptor and it correlates to the expected lower levels of prepore formation on these cells. Lower toxicity of PA wt -sANTXR-Ac2 enables greater cytosolic protein delivery Previously, we have shown that PA m -Ac2 can efficiently deliver various cargoes to the cytosol of Flp-In 293-EpCAM-BirA cells stably overexpressing EpCAM [ 10 ]. Our goal in this study was to increase the amount of cytosolically delivered cargo molecules, which previously was not possible, since concentrations higher than 20 nM of the pore-forming PA m -Ac2 drastically reduced cellular viability even within the short 4-h incubation time (Additional file 2 ). Our newly designed, prepore-stabilizing PA wt -sANTXR-Ac2 was therefore next tested for efficient protein delivery with the biotin ligase assay [ 22 ]. We incubated Flp-In 293-EpCAM-BirA cells with PA wt -sANTXR-Ac2, PA m -sANTXR-Ac2, and PA m -Ac2 for 4 h in the presence of the proteasome inhibitor MG-132. MG-132 was included to assess the delivery systems independently of proteasomal degradation. As cargo proteins, we tested three different DARPins, varying in size and thermostability, which have previously shown to be effectively translocated [ 10 ]. These cargo molecules contain the biotin-acceptor avi-tag and an HA-tag at their C-terminus and are fused with their N-terminus to LF N . Cytosolically localized cargo proteins are biotinylated by a cytoplasmically encoded BirA of Flp-In 293-EpCAM-BirA cells [ 22 ]. Cargo molecules which are trapped within the endosome, not reaching the cytosol, are not biotinylated. The HA-tag is used to determine total cellular uptake, located in the cytosol and in any other cellular compartment, allowing the determination of the intracellular localization of a cargo molecule. After cell harvest and western blotting, biotinylated cargoes were detected with streptavidin IRDye 680LT and total cellular uptake was measured via an HA-tag antibody [ 22 ]. For quantification of cytosolically present cargo molecules, the protein(s) detected at around 70 kDa, which we hypothesized earlier to be endogenous heat shock protein 70 (HSP70), were chosen as a loading control [ 10 ]. With increasing concentrations of PA wt -sANTXR-Ac2, total cellular uptake (Fig. 4 b, d) and cytosolic delivery (Fig. 4 a, c) of the smallest DARPin NI 1 C increase. An increase in cytosolically present cargo can be seen up to an external concentration of 200 nM. Further increases in the concentration of PA wt -sANTXR-Ac2 did not yield higher amounts of delivered DARPin (Fig. 4 c, d), presumably due to a saturation of the receptors exploited for delivery. Fig. 4 Western blots of the BirA assay showing increased delivery of LF N -cargo constructs with PA wt -sANTXR-Ac2 on Flp-In 293-EpCAM-BirA cells. Cytosolically delivered cargo proteins are biotinylated by a cytoplasmically encoded BirA and stained with Streptavidin IRDye 680LT. Total cellular uptake measured via HA-tag on the LF N -cargo. a , b Increasing concentrations of respective PA constructs incubated with a 5-fold excess of LF N -NI 1 C. Boxes indicate the bands of interest. c , d Quantification of western blot bands from a and b . Black bars, PA wt -sANTXR-Ac2; red bars, PA m -sANTXR-Ac2; green bars, PA m -Ac2. The dotted line represents background signal (i.e., cells only), and the dashed line shows the signal of cargo at 20 nM PA wt -sANTXR-Ac2. e , f Cytosolic localization ( e ) and total cellular uptake ( f ) of three different cargo DARPins delivered with 20 nM (lanes 1–3) and 100 nM (lanes 4–6) of PA wt -sANTXR-Ac2 or 100 nM (lanes 7–9) for PA m -Ac2; lanes 10 and 11 represent cells incubated with 100 nM LF N -cargo without PA. "dest." refers to rationally destabilized versions of NI 2 C and NI 3 C DARPins [ 10 ]. Boxes indicate the bands of interest At 20 nM, similar delivery efficiencies can be observed for PA wt -sANTXR-Ac2, PA m -sANTXR-Ac2, and PA m -Ac2, but an increase to 100 nM does not lead to an increase in cytosolically present cargo for PA m -sANTXR-Ac2 and PA m -Ac2, as it does for PA wt -sANTXR-Ac2, likely due to the premature prepore-to-pore conversion of PA m -Ac2 and PA m -sANTXR-Ac2 on the cell surface. This lack of functional pores renders the cells unable to unfold and translocate LF N -cargo proteins (Fig. 4 c, d). Slightly higher total cellular uptake of LF N -cargo can be observed with PA m -sANTXR-Ac2 than for PA m -Ac2, probably due to the delayed cytotoxicity compared to PA m -Ac2. Similar results have been observed for LF N -NI 2 C dest. as well as LF N -NI 3 C dest. , constructs that have been slightly destabilized to facilitate their unfolding and refolding during transport through the pore [ 10 , 19 ] (Fig. 4 e, f). With increasing concentrations (20 nM and 100 nM), an increase in cytosolic cargo delivery can be observed. The BirA assay for His 6 -MBP-PA wt-SL -sANTXR-Ac2 (Additional file 1 : Figure S4b) showed a reduced amount of total cellular uptake, suggesting a steric inhibition effect already at the start of the internalization process. The results for this construct are in line with the results for PA m -sANTXR-Ac2 and confirm the functional dependency of PA on interactions with the sANTXR domain. Discussion The druggable proteome is so far limited by requiring binding sites for small molecules. Macromolecular binding molecules, which do not have this restriction, are currently excluded by the lack of efficient, cell-specific cytosolic delivery systems. A solution to this problem would open up the intracellular target space for larger biological macromolecules, which can be created to almost any surface on the target. Since recombinant binding proteins are easily accessible today, a solution would drastically increase opportunities for targeted therapy approaches. Many molecules of great medical interest that are currently believed to be "undruggable targets," since they do not have a binding pocket for small molecules, could then be targeted. Large, flat, and hydrophobic protein-protein interaction surfaces would thus remain no longer undruggable. Previous studies have shown the utility of bacterial protein toxins as easily adaptable delivery systems. Such systems ultimately have two layers of specificity, the surface marker and the target in the cytosol, and may thus pave the way also to more specific treatments. In order to adapt bacterial protein toxins for cytosolic delivery, a thorough understanding of the wild-type delivery mechanism is absolutely necessary. The wild-type delivery mechanism of anthrax toxin has been studied in considerable detail [ 5 ]. Retargeting of anthrax toxin has been achieved by fusing different binding proteins to the C-terminus of a mutant version of PA (N682A, D683A), the anthrax toxin binding and internalization subunit, rendering it unable to bind its wild-type receptor [ 16 ]. However, the impact of this change in receptor specificity on the succeeding steps of the delivery process had not been studied. In this study, we have conducted an in-depth analysis of a DARPin-retargeted PA, which showed a clear cytotoxicity on targeted cells when high concentrations are matched with high receptor expression levels. We therefore rationally designed an improved PA variant. This new protein design allowed us to diminish the cytotoxicity, and it highlights the importance of the interaction between PA and its wild-type receptor in controlling the conformational changes during the internalization process, tightly linking it to the pH of the internal compartments. We deduced that the interaction of PA with sANTXR, now encoded in our improved PA variant itself and no more part of the actual interaction of PA wt with the surface VWA domain of CMG2 or TEM8, shifts the pH of the prepore-to-pore conversion to the wild-type conditions. The importance of this pH-sensing mechanism has been described before [ 13 ], but this knowledge had not been used in improved constructs. Furthermore, we showed that the mechanistic concept of cytotoxicity is valid and can be rescued across multiple EpCAM-expressing cell lines, and we confirmed that cytotoxicity is also dependent on the expression level of the targeted receptor. It remains still unclear, however, why there appears to be a threshold, above which PA m -Ac2 shows its toxic effect. However, there are multiple factors that may explain the differences across cell lines, including different furin activity on the cell surface or differences in receptor mobility, both involved in initiating oligomerization, and there may be others, some of which have already been discussed previously [ 19 ]. It has also been shown previously that retargeting of PA to HER2 could be achieved; however, the readout in this study was based on the cytotoxicity of the cargo component [ 11 ]. Our study clearly shows that cytotoxicity might arise from the delivery system itself even if no toxic cargo is present. Therefore, to advance the field, it is necessary to use an objective assay readout in order to properly evaluate and understand the capability of a delivery mechanism [ 3 , 22 ]. When using toxic cargoes, it is essential to exclude that prepore-to-pore conversion and its toxic effects would lead to an overestimation of the delivery of cargo. Conclusions The toxic effect, which originally hampered a further improvement of the retargeted delivery system, was greatly diminished by a rationally designed new PA variant, PA wt -sANTXR-Ac2. Higher total uptake and cytosolic delivery of cargo proteins confirmed the improvement of the system. Exemplarily, we have shown the increase with DARPins as cargo molecules; however, the system can also deliver other proteins which are able to pass through the PA pore. With this improved PA variant, we now aim for a broader range of applications with suitable intracellular drug targets. Methods Cell lines Flp-In 293 cells stably overexpressing EpCAM and BirA (Flp-In 293-EpCAM-BirA), RD cells stably overexpressing BirA (RD-BirA), and HT29 cells stably overexpressing BirA (HT29-BirA) were cultured using DMEM. MCF7-BirA and SKBR3-BirA cells were maintained in HAM/DMEM mix (50:50) and RPMI, respectively. All media were supplemented with 10% fetal calf serum and 100 IU/mL penicillin and 100 μg/mL streptomycin. G418 was added to the medium for 3 days after cells were taken in culture, to exclude cells that have lost BirA expression. The following G418 concentrations were used: HT29-BirA, 1000 μg/mL; MCF7-BirA, 400 μg/mL; SKBR3-BirA, 200 μg/mL; and RD-BirA, 200 μg/mL. Generation of stable BirA cell lines The generation of stable cell lines has been described before [ 10 , 19 ]. RD-BirA cells were generated as a stable pool as described in Verdurmen et al. [ 19 ] using 600 μg/mL G418. Cloning Cloning of most constructs used in this study has been described before [ 10 , 19 ]. LF N -eGFP-avi-HA was cloned by amplifying eGFP using primers containing a 5′ SpeI and a 3′ AgeI site for cloning into the SpeI/AgeI-restricted pQIq-LF N -avi-HA backbone. PA m -sANTXR-Ac2 was generated in the same way as PA wt -sANTXR-Ac2 [ 19 ]. The construct with a shorter linker between PA WT and sANTXR, termed His 6 -MBP-PA wt-SL -sANTXR-Ac2, has been cloned using sequence and ligation-independent cloning (SLIC) [ 23 ]. The following primers were used to amplify PCR products of PA wt -sANTXR-Ac2 in the linker region: 5′ GCAGG CGAAC GTACC TGGGC AGAAA CCATG GGTCT GAATA CCGCA GATAC 3′ and 5′AGGCT GGGTT TTATG ACCAG 3′ for the first PCR product and 5′ ATTGG TAGCC CTGGT CATAA AACCC AGCCT CGCCG TGCCT TTGAT CTG 3′ and 5′ CTTCC AGCAG TTTCT TACCC AGGTC GGATC CGCTC TGCGC CAGAA TGG 3′ for the second PCR product. The plasmid containing the sequence of PA wt -sANTXR-Ac2 was digested using NcoI and BamHI. The linker was shortened from SPGHK TQPGS (G 4 S) 5 GG to SPGHK TQP. Protein expression The E. coli strain BL21 was transformed with the described plasmids for the expression of the constructs. A single clone was picked the next day and used for inoculation of autoinduction medium [ 24 ]. The cultures were grown at 25 °C until a stable OD 600 was reached. Cultures were centrifuged for 10 min at 5000 g at 4 °C; the pellet washed with PBS, pH 7.4, shock-frozen, and stored at − 20 °C until purification. Protein purification All proteins, expressed as His 6 -MBP-PA variants and His 6 -MBP-LF N cargo constructs, were purified in a similar manner. All steps were performed at 4 °C. Tris-HCl buffers were adjusted to pH 8.0. Bacterial cell pellets were thawed and resuspended in lysis buffer (50 mM Tris-HCl, 0.5 mM EDTA, 0.4 mM 4-(2-aminoethyl)benzolsulfonyl fluoride (AEBSF), 500 mM NaCl, 10 mM MgCl 2 , 1 g/L lysozyme, 10% glycerol, 10 U/mL Pierce™ Universal Nuclease for Cell Lysis) (Thermo Scientific™ 88702). Cells were lysed by sonication and centrifuged for 45 min at 20,000 g , and the cleared lysate was filtered (pore size 0.22 μm). Proteins were purified by their His-tag via immobilized metal ion affinity chromatography (IMAC). Ni-NTA agarose (Qiagen) was packed in 7 mL benchtop columns (PD10), and columns were equilibrated in lysis buffer, not containing AEBSF and Pierce nuclease. Lysate was applied twice to the column, washed with 10 column volumes (CV) high-salt buffer (25 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole) and 10 CV low-salt buffer (25 mM Tris-HCl, 125 mM NaCl, 20 mM imidazole), and eluted with 2 CV elution buffer (25 mM Tris-HCl, 125 mM NaCl, 300 mM imidazole). Proteins were dialyzed overnight against anion exchange chromatography (AEX) equilibration buffer (25 mM Tris-HCl, 125 mM NaCl) with a 1:10 M ratio of his-tagged Tobacco etch virus (TEV) protease to cleave off His 6 -MBP. TEV protease, MBP, and residual uncleaved proteins were removed via reverse IMAC. For His 6 -MBP-PA wt-SL -sANTXR-Ac2, the His 6 -MBP tag was not cleaved off and no reverse IMAC was performed since equivalent delivery to His 6 -MBP cleaved variants of PA m -Ac2 has been shown before [ 19 ]. The unbound fraction of reverse IMAC was purified via AEX using a MonoQ 5/50 GL (GE Healthcare) on an ÄKTA Pure system (GE Healthcare). Proteins were eluted in a 40 CV gradient up to 50% AEX elution buffer (25 mM Tris-HCl, 1 M NaCl); protein-containing fractions were evaluated by SDS-PAGE, pooled and concentrated via Amicon Ultra-0.5 (Millipore; MWCO 30,000). Subsequently, proteins were polished and buffer exchanged to PBS (pH 7.4) via size exclusion chromatography (SEC) using a Superdex 200 10/300 GL (GE Healthcare). Monomeric fractions were pooled and concentrated as described before. LF N -cargo constructs, containing an avi-tag, were additionally incubated with streptavidin beads (Genscript) for 30 min at 4 °C while shaking in order to remove already biotinylated proteins. All proteins were snap-frozen in liquid N 2 and stored short term at − 20 °C. Purities and monomeric behavior were confirmed to be > 90% by Coomassie-stained SDS-PAGE and on an analytical SEC (Additional file 1 : Figure S7). Biotin ligase uptake assay To measure the total cellular uptake and cytosolic localization of cargo proteins, the biotin ligase assay was performed as described previously [ 22 ]. Viability assay XTT assays were used to evaluate cell viability. Cells were seeded in a flat 96-well plate 48 h before incubation with proteins. Twenty-five thousand cells were seeded for all cell lines. Cells were incubated with respective protein concentrations for 24 h under their normal culture conditions. Cell proliferation was measured with a Cell Proliferation XTT Kit (BIOFROXX) according to the manufacturer's instructions. Data were plotted as mean ± SEM ( n = 3). Flow cytometry analysis To determine receptor surface expression levels, cells were incubated on ice in PBS supplemented with 50 mM sodium azide and a 1:50 dilution of the respective antibody for 30 min. Cells were stained with Alexa Fluor 488-labeled anti-EpCAM mouse mAb (VU1D9, Cell Signaling) for EpCAM levels. A reference mouse mAb IgG1 isotype control labeled with Alexa Fluor 488 (MOPC-21, Cell Signaling) was used. EpCAM expression levels were determined on an LSR II Fortessa (BD Biosciences) on gated cells. Data ( n = 3) were analyzed with GraphPad Prism 8 and plotted as mean ± SEM. Confocal microscopy Forty-eight hours before confocal imaging, 60,000 cells were seeded in 8-well 15 μ-Slide glass bottom slides (Ibidi). PA (100 nM), LF N -eGFP (200 nM), and PI (1 μg/mL) were added to the cells with fresh medium. The panel of cell lines was incubated using 50 nM PA. Cells were imaged 3 h after addition of components at 37°C and 5% CO 2 on a Nikon Eclipse Ti-E inverted microscope with a Yokogawa spinning disc system W1, a 100x oil objective, and an incubation system for live cell imaging including a stage-top incubator for chambered cover glass. Time-lapse microscopy For time-lapse imaging, 25,000 cells were seeded in Nunc MicroWell 96-Well Optical Bottom Plate with Polymer Base (Thermo Scientific) 24 h before imaging. Fifty nanomolar PA, 100 nM LF N -eGFP, and PI (1 μg/mL) were added right before imaging to each well ( n = 6). Cells were imaged at 37 °C and 5% CO 2 using a LionHeart FX microscope with a 10x objective and the Gen5 software (3.05). Cellular analysis was performed with the Gen5 software in the Texas Red Channel. The threshold for cell detection was set to the image background levels, and object selection was set to 5 μm and 50 μm for min. and max. cell size, respectively. Data were plotted as mean ± SEM ( n = 6). Cell lines Flp-In 293 cells stably overexpressing EpCAM and BirA (Flp-In 293-EpCAM-BirA), RD cells stably overexpressing BirA (RD-BirA), and HT29 cells stably overexpressing BirA (HT29-BirA) were cultured using DMEM. MCF7-BirA and SKBR3-BirA cells were maintained in HAM/DMEM mix (50:50) and RPMI, respectively. All media were supplemented with 10% fetal calf serum and 100 IU/mL penicillin and 100 μg/mL streptomycin. G418 was added to the medium for 3 days after cells were taken in culture, to exclude cells that have lost BirA expression. The following G418 concentrations were used: HT29-BirA, 1000 μg/mL; MCF7-BirA, 400 μg/mL; SKBR3-BirA, 200 μg/mL; and RD-BirA, 200 μg/mL. Generation of stable BirA cell lines The generation of stable cell lines has been described before [ 10 , 19 ]. RD-BirA cells were generated as a stable pool as described in Verdurmen et al. [ 19 ] using 600 μg/mL G418. Cloning Cloning of most constructs used in this study has been described before [ 10 , 19 ]. LF N -eGFP-avi-HA was cloned by amplifying eGFP using primers containing a 5′ SpeI and a 3′ AgeI site for cloning into the SpeI/AgeI-restricted pQIq-LF N -avi-HA backbone. PA m -sANTXR-Ac2 was generated in the same way as PA wt -sANTXR-Ac2 [ 19 ]. The construct with a shorter linker between PA WT and sANTXR, termed His 6 -MBP-PA wt-SL -sANTXR-Ac2, has been cloned using sequence and ligation-independent cloning (SLIC) [ 23 ]. The following primers were used to amplify PCR products of PA wt -sANTXR-Ac2 in the linker region: 5′ GCAGG CGAAC GTACC TGGGC AGAAA CCATG GGTCT GAATA CCGCA GATAC 3′ and 5′AGGCT GGGTT TTATG ACCAG 3′ for the first PCR product and 5′ ATTGG TAGCC CTGGT CATAA AACCC AGCCT CGCCG TGCCT TTGAT CTG 3′ and 5′ CTTCC AGCAG TTTCT TACCC AGGTC GGATC CGCTC TGCGC CAGAA TGG 3′ for the second PCR product. The plasmid containing the sequence of PA wt -sANTXR-Ac2 was digested using NcoI and BamHI. The linker was shortened from SPGHK TQPGS (G 4 S) 5 GG to SPGHK TQP. Protein expression The E. coli strain BL21 was transformed with the described plasmids for the expression of the constructs. A single clone was picked the next day and used for inoculation of autoinduction medium [ 24 ]. The cultures were grown at 25 °C until a stable OD 600 was reached. Cultures were centrifuged for 10 min at 5000 g at 4 °C; the pellet washed with PBS, pH 7.4, shock-frozen, and stored at − 20 °C until purification. Protein purification All proteins, expressed as His 6 -MBP-PA variants and His 6 -MBP-LF N cargo constructs, were purified in a similar manner. All steps were performed at 4 °C. Tris-HCl buffers were adjusted to pH 8.0. Bacterial cell pellets were thawed and resuspended in lysis buffer (50 mM Tris-HCl, 0.5 mM EDTA, 0.4 mM 4-(2-aminoethyl)benzolsulfonyl fluoride (AEBSF), 500 mM NaCl, 10 mM MgCl 2 , 1 g/L lysozyme, 10% glycerol, 10 U/mL Pierce™ Universal Nuclease for Cell Lysis) (Thermo Scientific™ 88702). Cells were lysed by sonication and centrifuged for 45 min at 20,000 g , and the cleared lysate was filtered (pore size 0.22 μm). Proteins were purified by their His-tag via immobilized metal ion affinity chromatography (IMAC). Ni-NTA agarose (Qiagen) was packed in 7 mL benchtop columns (PD10), and columns were equilibrated in lysis buffer, not containing AEBSF and Pierce nuclease. Lysate was applied twice to the column, washed with 10 column volumes (CV) high-salt buffer (25 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole) and 10 CV low-salt buffer (25 mM Tris-HCl, 125 mM NaCl, 20 mM imidazole), and eluted with 2 CV elution buffer (25 mM Tris-HCl, 125 mM NaCl, 300 mM imidazole). Proteins were dialyzed overnight against anion exchange chromatography (AEX) equilibration buffer (25 mM Tris-HCl, 125 mM NaCl) with a 1:10 M ratio of his-tagged Tobacco etch virus (TEV) protease to cleave off His 6 -MBP. TEV protease, MBP, and residual uncleaved proteins were removed via reverse IMAC. For His 6 -MBP-PA wt-SL -sANTXR-Ac2, the His 6 -MBP tag was not cleaved off and no reverse IMAC was performed since equivalent delivery to His 6 -MBP cleaved variants of PA m -Ac2 has been shown before [ 19 ]. The unbound fraction of reverse IMAC was purified via AEX using a MonoQ 5/50 GL (GE Healthcare) on an ÄKTA Pure system (GE Healthcare). Proteins were eluted in a 40 CV gradient up to 50% AEX elution buffer (25 mM Tris-HCl, 1 M NaCl); protein-containing fractions were evaluated by SDS-PAGE, pooled and concentrated via Amicon Ultra-0.5 (Millipore; MWCO 30,000). Subsequently, proteins were polished and buffer exchanged to PBS (pH 7.4) via size exclusion chromatography (SEC) using a Superdex 200 10/300 GL (GE Healthcare). Monomeric fractions were pooled and concentrated as described before. LF N -cargo constructs, containing an avi-tag, were additionally incubated with streptavidin beads (Genscript) for 30 min at 4 °C while shaking in order to remove already biotinylated proteins. All proteins were snap-frozen in liquid N 2 and stored short term at − 20 °C. Purities and monomeric behavior were confirmed to be > 90% by Coomassie-stained SDS-PAGE and on an analytical SEC (Additional file 1 : Figure S7). Biotin ligase uptake assay To measure the total cellular uptake and cytosolic localization of cargo proteins, the biotin ligase assay was performed as described previously [ 22 ]. Viability assay XTT assays were used to evaluate cell viability. Cells were seeded in a flat 96-well plate 48 h before incubation with proteins. Twenty-five thousand cells were seeded for all cell lines. Cells were incubated with respective protein concentrations for 24 h under their normal culture conditions. Cell proliferation was measured with a Cell Proliferation XTT Kit (BIOFROXX) according to the manufacturer's instructions. Data were plotted as mean ± SEM ( n = 3). Flow cytometry analysis To determine receptor surface expression levels, cells were incubated on ice in PBS supplemented with 50 mM sodium azide and a 1:50 dilution of the respective antibody for 30 min. Cells were stained with Alexa Fluor 488-labeled anti-EpCAM mouse mAb (VU1D9, Cell Signaling) for EpCAM levels. A reference mouse mAb IgG1 isotype control labeled with Alexa Fluor 488 (MOPC-21, Cell Signaling) was used. EpCAM expression levels were determined on an LSR II Fortessa (BD Biosciences) on gated cells. Data ( n = 3) were analyzed with GraphPad Prism 8 and plotted as mean ± SEM. Confocal microscopy Forty-eight hours before confocal imaging, 60,000 cells were seeded in 8-well 15 μ-Slide glass bottom slides (Ibidi). PA (100 nM), LF N -eGFP (200 nM), and PI (1 μg/mL) were added to the cells with fresh medium. The panel of cell lines was incubated using 50 nM PA. Cells were imaged 3 h after addition of components at 37°C and 5% CO 2 on a Nikon Eclipse Ti-E inverted microscope with a Yokogawa spinning disc system W1, a 100x oil objective, and an incubation system for live cell imaging including a stage-top incubator for chambered cover glass. Time-lapse microscopy For time-lapse imaging, 25,000 cells were seeded in Nunc MicroWell 96-Well Optical Bottom Plate with Polymer Base (Thermo Scientific) 24 h before imaging. Fifty nanomolar PA, 100 nM LF N -eGFP, and PI (1 μg/mL) were added right before imaging to each well ( n = 6). Cells were imaged at 37 °C and 5% CO 2 using a LionHeart FX microscope with a 10x objective and the Gen5 software (3.05). Cellular analysis was performed with the Gen5 software in the Texas Red Channel. The threshold for cell detection was set to the image background levels, and object selection was set to 5 μm and 50 μm for min. and max. cell size, respectively. Data were plotted as mean ± SEM ( n = 6). Supplementary information Additional file 1: Figure S1. [Ribbon representation of constructs PA m -sANTXR-Ac2 and PA wt-SL -sANTXR-Ac2]. Figure S2. [Different imaging timepoints of time-lapse imaging experiments]. Figure S3. [Figure 2 c replotted with additional quantification of LF N -eGFP delivery]. Figure S4. [Toxicity and delivery of PA wt-SL -sANTXR-Ac2]. Figure S5. [Gating strategy for receptor expression analysis]. Figure S6. [Biotin ligase uptake assay of LF N -eGFP]. Figure S7. [HPLC analysis of protein purity and monomeric behavior]. Additional file 2. Additional file 3. Time-lapse microscopy of Flp-In 293-EpCAM-BirA cells incubated with PAm-sANTXR-Ac2. Additional file 4. Time-lapse microscopy of Flp-In 293-EpCAM-BirA cells incubated with PAm-Ac2. Additional file 5. Time-lapse microscopy of Flp-In 293-EpCAM-BirA cells incubated with PAwt-sANTXR-Ac2. Additional file 1: Figure S1. [Ribbon representation of constructs PA m -sANTXR-Ac2 and PA wt-SL -sANTXR-Ac2]. Figure S2. [Different imaging timepoints of time-lapse imaging experiments]. Figure S3. [Figure 2 c replotted with additional quantification of LF N -eGFP delivery]. Figure S4. [Toxicity and delivery of PA wt-SL -sANTXR-Ac2]. Figure S5. [Gating strategy for receptor expression analysis]. Figure S6. [Biotin ligase uptake assay of LF N -eGFP]. Figure S7. [HPLC analysis of protein purity and monomeric behavior]. Additional file 2. Additional file 3. Time-lapse microscopy of Flp-In 293-EpCAM-BirA cells incubated with PAm-sANTXR-Ac2. Additional file 4. Time-lapse microscopy of Flp-In 293-EpCAM-BirA cells incubated with PAm-Ac2. Additional file 5. Time-lapse microscopy of Flp-In 293-EpCAM-BirA cells incubated with PAwt-sANTXR-Ac2. Supplementary information Supplementary information accompanies this paper at 10.1186/s12915-020-00827-y.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6938160/

Hidden Aspects of Valency in Immune System Regulation

Valency can be defined as the number of discrete interactions a biomolecule can engage in. Valency can be critical for function, such as determining whether a molecule acts as a scaffold for assembling large supramolecular complexes or it forms a functional dimer. Here, we highlight the importance of the role of valency in regulating immune responses with a focus on innate immunity. We discuss some of the ways in which valency itself is regulated through transcriptional, post-transcriptional, and post-translational modifications. Finally, we propose that the valency model can be applied at the whole cell level to study differences in individual cell responses with relevance to putative therapeutic applications.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821843/

Growth and carcass characteristics of three Ethiopian indigenous goats fed concentrate at different supplementation levels

The study was carried out to evaluate the effect of genotypes and concentrate levels on growth performance and carcass characteristics of Bati, Hararghe highland (HH) and Short eared Somali (SS) goat types found in Ethiopia. A 3 × 2 factorial arrangement (3 genotype × 2 concentrate levels) was used to randomly allocate 36 goats (15.2 ± 0.30 kg initial weight); 12 goats from each genotype with age about 1 year were divided randomly into two groups for a feeding trial of 90 days. The two concentrate levels were L1 and L2, where L1 and L2 are levels fed to animals at the rate of 1 and 1.5 % BW, respectively. Hay was fed ad libitum with 20 % refusal rate. The mean daily dry matter intake of the goats was 520.5 g/day. The intake was about 67 g/day higher for L2 than L1 goats. Consequently, L2 goats had significantly (p  0.05) though HH and SS goats tended to have better efficiency than Bati goat. Table 2 Least square means for feed intakes, digestibility, and conversion efficiency of three Ethiopian goat types Variables Genotype (G) Concentrate level (C) p value Bati HH SS L1 L2 SEM G C G × C DM intake Concentrate (g/day) 200.4 a 196.0 ab 179.3 b 150.3 b 233.5 a 8.28 0.02  0.05) though HH and SS goats tended to have better efficiency than Bati goat. Table 2 Least square means for feed intakes, digestibility, and conversion efficiency of three Ethiopian goat types Variables Genotype (G) Concentrate level (C) p value Bati HH SS L1 L2 SEM G C G × C DM intake Concentrate (g/day) 200.4 a 196.0 ab 179.3 b 150.3 b 233.5 a 8.28 0.02 <0.01 0.88 Hay (g/day) 323.5 344.9 317.5 336.7 320.5 6.20 0.12 0.15 0.78 Total (g/day) 523.8 540.9 496.8 487.1 b 553.9 a 11.2 0.09 <0.01 0.92 Total (%BW) 2.86 b 2.96 ab 3.01 a 2.82 b 3.06 a 0.03 0.03 <0.01 0.18 CP intake Concentrate (g/day) 42.1 a 41.1 ab 37.6 b 31.5 b 48.9 a 1.74 0.02 <0.01 0.88 Hay (g/day) 26.1 27.7 25.6 27.2 25.8 0.49 0.12 0.15 0.78 Total (g/day) 68.1 68.9 63.2 58.5 b 74.8 a 1.89 0.05 <0.01 0.96 Total (%BW) 0.37 0.37 0.38 0.34 b 0.41 a 0.01 0.16 <0.01 0.05 Digestibility (%) Dry matter 71.7 68.3 69.1 67.3 b 72.1 a 1.03 0.29 0.01 0.29 Organic matter 72.3 68.9 69.7 67.9 b 72.7 a 1.02 0.28 0.01 0.29 Crude protein 77.4 74.3 74.6 73.2 b 77.6 a 0.87 0.18 <0.01 0.23 NDF 67.5 63.8 64.2 63.5 66.8 1.16 0.33 0.14 0.45 ADF 54.9 50.5 51.8 51.4 53.4 1.44 0.43 0.47 0.39 FCR 14.2 10.8 12.8 13.5 11.7 0.62 0.09 0.17 0.79 Least square means in a row with different subscript letter differ significantly (p < 0.05) HH Hararghe highland goat, SS Short-eared Somali goat, L1 hay + 1 % of body weight concentrate, L2 hay + 1.5 % of body weight concentrate, FCR feed conversion ratio, SEM pooled standard error of mean Body growth and dimensions Average daily gain (ADG) was significantly (p < 0.05) affected by level of concentrate but not by genotype (Table 3 ). Goats fed L2 gained 1 kg extra body weight compared to those fed L1 by growing at an average of 12 g greater rate per day. With increasing DM intake, there was a positive linear increase (r = 0.63; p < 0.01) in body weight change of animals. Bati and HH goats were heavier (p < 0.05) by more than 2 kg in their final weight (FW) and slaughter body weight (SBW) than SS goats. They also had higher (p < 0.05) height at withers and neck girths compared to SS goats. For other body linear measurements such as body length, heart girth and pelvic width, no significant differences were evidenced among genotype. Goats fed L2 had wider (p < 0.05) pelvic width than those consumed L1. They also tended (p = 0.10) to have better height at wither than L1 goats. Table 3 Least square means for body weight and dimensions of three Ethiopian goat types Variables Genotypes (G) Concentrate level (C) SEM p value Bati HH SS L1 L2 G C G × C Growth parameters IW (kg) 15.8 15.5 14.1 14.9 15.3 0.30 0.11 0.29 0.12 FW (kg) 19.6 a 20.1 a 17.8 b 18.4 b 19.9 a 0.38 <0.01 0.02 0.48 ADG (g/day) 42.1 51.4 41.3 38.7 b 50.9 a 2.42 0.17 0.02 0.98 SBW (kg) 18.8 a 19.3 a 17.1 b 17.8 b 19.0 a 0.37 <0.01 0.02 0.53 Dimensions (cm) Body length 51.7 51.2 50.5 50.7 51.6 0.38 0.47 0.32 0.96 Heart girth 65.8 65.9 64.3 64.9 65.8 0.41 0.19 0.28 0.84 Height at wither 60.8 a 61.4 a 58.4 b 59.6 60.8 0.46 <0.01 0.10 0.71 Pelvic width 10.7 11.0 10.5 10.4 b 11.1 a 0.16 0.34 0.04 0.61 Neck girth 34.0 a 34.0 a 32.7 b 33.4 33.7 0.24 0.03 0.51 0.53 Thigh circumference 20.5 20.8 20.3 20.3 20.8 0.15 0.34 0.12 0.53 Least square means in a row with different subscript letter differ significantly (p < 0.05) HH Hararghe highland goat, SS Short-eared Somali goat, L1 hay + 1 % of body weight concentrate, L2 hay + 1.5 % of body weight concentrate, IW initial weight, FW final weight, ADG average daily gain, SBW slaughter body weight, SEM pooled standard error of mean Carcass weight and dimensions Least square means for carcass weight and linear measurements of goats kept under two levels of feeding are set in Table 4 . HH goats had higher (p < 0.05) EBW and HCW than SS goats but had comparable carcass weight with that of Bati goats. The dressing percentage (DP) and rid-eye area of HH goat was higher DP of Bati goats but not different from that of SS goats. The DP of L2 goats was about 2.5 % higher than that of L1 goats. The average proportion of the leg relative to the weight of the carcass was about 32.6 % and varied between supplemented groups. A comparison between Bati and SS goats indicated that SS goat had significantly lighter fore-quarter part or shoulder. Goats receiving L2 had higher (p < 0.01) proportion of legs and ribs/racks compared to those receiving L1. Table 4 Least square means for slaughter and carcass characteristics of three Ethiopian goat types Variables Genotype (G) Concentrate level (C) SEM p value Bati HH SS L1 L2 G C G × C Carcass wt EBW (kg) 15.2 a 15.6 a 13.6 b 14.2 b 15.5 a 0.36 <0.01 <0.01 0.69 HCW (kg) 7.8 ab 8.3 a 7.2 b 7.3 b 8.4 a 0.22 <0.01 <0.01 0.21 DP (EBW basis) 51.2 b 54.4 a 53.0 ab 51.8 b 53.9 a 0.61 0.04 0.03 0.07 DP (SBW basis) 41.5 b 43.9 a 41.9 ab 41.1 b 43.8 a 0.54 0.03 <0.01 0.16 Dimensions (cm) Carcass length 50.9 47.8 45.4 47.7 48.4 1.01 0.10 0.74 0.74 Chest depth 28.4 28.2 27.1 27.4 28.3 0.30 0.12 0.11 0.44 Shoulder width 16.7 16.8 15.5 15.8 16.9 0.80 0.77 0.49 0.18 Hind leg width 11.1 ab 11.9 a 10.7 b 11.2 11.3 0.19 0.02 0.75 0.84 Rib-eye area (cm 2 ) 6.9 b 9.8 a 8.8 ab 7.9 9.1 0.31 <0.01 0.06 0.43 Fat thickness 1.14 1.17 1.22 1.1 1.2 0.03 0.45 0.09 0.77 Primal cuts (%) Legs 32.5 32.1 33.2 32.1 b 33.2 a 0.35 0.41 0.91 0.35 Loin 10.3 10.5 10.4 10.2 10.5 0.32 0.99 0.94 0.61 Ribs/racks 11.6 b 13.9 a 13.8 a 12.6 b 13.6 a 0.37 0.01 0.04 0.36 Shoulder 45.5 a 43.5 ab 42.5 b 43.3 44.7 0.44 0.02 0.24 0.43 Least square means in a row with different subscript letter differ significantly (p < 0.05) HH Hararghe highland goat, SS Short-eared Somali goat, L1 hay + 1 % of body weight concentrate, L2 hay + 1.5 % of body weight concentrate, SBW slaughter body weight, EBW empty body weight, HCW hot carcass weight, DP dressing percentage, SEM pooled standard error of mean Non-carcass components Table 5 shows least square means for non-carcass components of three Ethiopian goat types. The effects of genotype and feeding level were significant (p < 0.05) for some of the traits such as liver, heart plus lung, head, skin plus feet and total non-carcass/offals. SS goats had lower (p < 0.05) liver, skin plus feet, total offals and edible offals than Bati and HH goats. Goats fed L2 had heavier (p < 0.01) liver, heart plus lung, skin plus feet and empty GIT than those fed L1. In terms of fat deposition, SS goat which represented the lowland pastoral/agro-pastoral goat types had about 40 g heavier total internal fat than Bati and HH goats. Total non-carcass fat depositions obtained from goats receiving L2 were nearly twofold higher compared to those from goats fed L1. Bati and HH goats had heavier skins and feet than SS goats. The difference was also reflected between feeding levels where animals given L2 had significantly heavier skins and feet, total offals and edible offals than those receiving L1. Table 5 Least square means for non-carcass components (kg) of three Ethiopian goat types Non-carcass traits Genotype (G) Concentrate level (C) SEM p value Bati HH SS L1 L2 G C G × C Kidney 0.1 0.06 0.05 0.05 0.1 0.001 0.06 0.16 0.73 Liver 0.3 a 0.3 a 0.25 b 0.26 b 0.34 a 0.01 <0.01 <0.01 0.67 Heart + lung 1.0 a 0.9 ab 0.9 b 0.89 b 0.99 a 0.03 0.02 0.04 0.14 Head 1.2 1.3 1.09 1.2 1.21 0.03 0.05 0.51 0.62 Spleen 0.03 0.02 0.03 0.03 0.03 0.002 0.28 0.49 0.30 Skin + feet 2.5 a 2.5 a 2.2 b 2.3 b 2.5 a 0.05 <0.01 <0.01 0.20 GIT empty 1.2 1.3 1.2 1.1 b 1.28 a 0.03 0.25 <0.01 0.89 Digestive content 3.5 3.7 3.4 3.5 3.53 0.11 0.61 0.82 0.28 Genitalia 0.24 0.22 0.23 0.22 0.24 0.01 0.78 0.06 0.13 Non-carcass fat 0.29 0.31 0.35 0.23 b 0.40 a 0.02 0.25 <0.01 0.69 Total offals 10.4 a 10.8 a 9.7 b 9.9 b 10.6 a 0.19 0.02 0.03 0.40 Total edible offals 3.01 a 3.02 a 2.72 b 2.7 b 3.1 a 0.06 0.02 <0.01 0.83 Least square means in a row with different subscript letter differ significantly (p < 0.05) HH Hararghe highland goat, SS Short-eared Somali goat; L1 hay + 1 % of body weight concentrate, L2 hay + 1.5 % of body weight concentrate, GIT gastro-intestinal tract, SEM pooled standard error of mean Discussion Feed and nutrient intakes The daily DM intake (% live weight) of goat in this experiment is within the range of 2.6–3.2 % reported for Small East African goat and their crosses with Norwegian goat fed hay and supplemented with concentrate (William et al. 2013 ). For indigenous Ethiopian goats, DM intakes in the ranges of 2.9–3.9 and 2.9–3.2 % body weight were reported for Sidama (Solomon et al. 2008 ; Wondwosen et al. 2010 ; Tadesse et al. 2013 ) and for Hararghe highland goats (Wallie et al. 2012 ), respectively when they were supplemented with different levels of concentrates and other materials such as sweet potato vines and khat leftovers. Different from previous works, however, hay DM intake of goat in the present study was not significantly dropped as level of concentrate increased. This could be attributed to the relatively better quality of the hay which contained CP higher than the minimum of 70 g/kg DM CP required to support optimal microbial activity in the rumen (McDonald et al. 2002 ). Van Soest ( 1994 ) noted a rapidly declining trend of feed intake when the CP content of the consumed roughage is falling below 70 g/kg DM of the feed. The reason that L2 animals consumed more total DM and had better feed conversion efficiency is because they ingested about 27.4 % more CP and had higher rate of DM, OM and CP digestibility than L1 animals. As reported by Goodchild and McMeniman ( 1994 ), supplementation of poor quality forage with good protein feed has increased the availability of nitrogen in the rumen, thereby improving the rate of degradation and utilization of the feed. The average total CP intake of goats in the present study is in line with the recommended requirements of 65–70 g/day CP for goats weighing 20–25 kg (Kearl 1982 ). This indicates that the ration used in the present study can support better performance in goats. The estimated daily energy intakes were in the range of 4.8–5.9 MJ ME, which are within the recommended range of 3.25–6.47 MJ (Devendra and Burns 1983 ). Growth performance The difference in growth performance of goats fed different concentrate levels in the present study reflects the variations in feed conversion efficiency. The extra 1 kg weight gain of L2 over L1 goats can be due to their increased DM and protein intakes. The average daily weight gain recorded in the present study is within the range of 29–51 g reported for Small East African goats fed low and high levels of concentrate (Hango et al. 2007 ). Daily weight gains of 33–49 g/day were reported for HH goats fed different levels of leftover khat ( Catha edulis ) (Wallie et al. 2012 ), which are very close to the present values. Higher growth rates were also reported for Somali (Mengistu et al. 2007 ; Solomon and Simret 2008 ) and Sidama goats (Solomon et al. 2008 ) supplemented with concentrate and fed hay as basal diet. Body linear dimensions such as heart girth, height at wither, and body lengths are reported to be closely related to the size or weight of an animal (Vargas et al. 2007 ). In present study, only height at wither was found to be significantly higher for Bati and HH goats than SS goats, probably due to the fact that these goats are naturally taller than SS goats (FARM-Africa 1996 ). However, supplementation of goats with low to moderate quality basal diets did not affect body length and heart girth. The fact that SS goats had shorter height (p < 0.05) but comparable body length and heart girth with that of Bati goats indicates that SS goats have compact body than Bati goats. This is also confirmed by the result that SS goats tended to have relatively higher DP than Bati goats (Table 4 ). Mahgoub and Lu ( 1998 ) reported that Dhofari goat with smaller body size had higher carcass muscularity than Batina goat of larger body size. Carcass characteristics The carcass weight recorded in the present study is within the carcass weight of 6–9 kg reported for other Ethiopian goats (Hailu et al. 2005 ; Ameha et al. 2007 ). The effect of diet on carcass weight was clearly seen. The difference in carcass weight between supplement groups may be attributed to higher feed intake and, consequently, greater nutrient availability to promote weight gain and tissue development in goats fed L2 diet. Mushi et al. ( 2009a ) also reported higher weights and carcass yields in goats fed higher energy levels. The dressing percentage, expressed as percentage of full and empty body weight, were 42.4 and 52.8 %, respectively. The values are within the range of 39.5–41.8 and 53.3–56.6 % reported for Ethiopian (Ameha et al. 2007 ) and Omani goat breeds (Kadim et al. 2003 ). The reason for variation in DP among genotypes in the present study is because of greater difference in proportion of non-carcass components such as GIT. Dhanda et al. ( 1999 ) and Addisu et al. ( 2002 ) also reported a significant effect of genotype on DP and attributed this difference to variations in weight of digestive tract contents. On the other hand, the higher DP of L2 goat compared to that of L1 goat is likely associated to better development of muscle and fat tissue due to their increased DM and CP intakes (Safari et al. 2011 ). According to De Boer et al. ( 1974 ) linear carcass measurements are indices indirectly help to determine carcass conformation and affected by genotype, sex and feeding regimes. HH and SS goats had relatively wider (p < 0.05) rib-eye area than Bati goats implying that they have more desirable carcass. According to the report of Wolf et al. ( 1980 ), larger rib-eye area is associated with higher production of lean in the carcass and higher lean to bone ratio. The proportion of the legs (32–33 %) calculated in the present study is within 28–33 % reported for well-conformed goat breeds (Sen et al. 2004 ; Webb et al. 2005 ). The reason for SS goats to have equal proportions of primal cuts compared to that of Bati goat is because they deposited more muscle tissue relative to their body size. Mahgoub and Lu ( 1998 ) noted that small body sized goats had a higher carcass muscularity than large body sized ones. Non-carcass characteristics Information on yield of non-carcass components of animals is important especially in developing countries where they are more valuable for the households (Mushi et al. 2009b ). The presence of variations in internal fat deposition among different breeds of goat has been reported earlier by Mahgoub and Lu ( 1998 ) and Kadim et al. ( 2003 ). On average, about 0.32 kg total non-carcass fat was recorded in the present study, which is lower than 0.57 kg reported for other Ethiopian indigenous goats (Ameha et al. 2007 ). Among the three goat types, SS goats can be regarded as physiologically early maturing animals as they have higher deposition of internal fats, and also better composition of chemical fat in the muscle as indicated by Tadesse et al. ( 2015 ). According to Ameha et al. ( 2007 ), different animals can have different physiological maturities as a result of difference in chemical fat composition and total non-carcass fat contents. Higher proportions of total and edible offals were obtained from L2 than L1 goats, which is in agreement with the results of Solomon et al. ( 2008 ) and Solomon and Simret ( 2008 ). The total edible offals (2.8–3.2 kg) reported for HH (Asnakew and Berhan 2007 ) and Sidama goats (Tadesse et al. 2013 ) are also similar with finding of the present study. The fact that SS goats had lighter total offals in the present study indicates that this breed can produce more proportion of usable carcass components relative to their body size than Bati and HH goats. Goats fed L2 had heavier liver than those fed L1, may be due to their higher nutrient intake and, consequently, greater deposition of reserve substances such as glycogen as noted by Mushi et al. ( 2009b ). Because of their bigger body size, thicker skins and dense hair cover, HH and Bati goats produced heavier (p < 0.05) skin than SS goats. Similar observation has been noted by Kadim et al. ( 2003 ) on skins obtained from different Omani goat breeds. Feed and nutrient intakes The daily DM intake (% live weight) of goat in this experiment is within the range of 2.6–3.2 % reported for Small East African goat and their crosses with Norwegian goat fed hay and supplemented with concentrate (William et al. 2013 ). For indigenous Ethiopian goats, DM intakes in the ranges of 2.9–3.9 and 2.9–3.2 % body weight were reported for Sidama (Solomon et al. 2008 ; Wondwosen et al. 2010 ; Tadesse et al. 2013 ) and for Hararghe highland goats (Wallie et al. 2012 ), respectively when they were supplemented with different levels of concentrates and other materials such as sweet potato vines and khat leftovers. Different from previous works, however, hay DM intake of goat in the present study was not significantly dropped as level of concentrate increased. This could be attributed to the relatively better quality of the hay which contained CP higher than the minimum of 70 g/kg DM CP required to support optimal microbial activity in the rumen (McDonald et al. 2002 ). Van Soest ( 1994 ) noted a rapidly declining trend of feed intake when the CP content of the consumed roughage is falling below 70 g/kg DM of the feed. The reason that L2 animals consumed more total DM and had better feed conversion efficiency is because they ingested about 27.4 % more CP and had higher rate of DM, OM and CP digestibility than L1 animals. As reported by Goodchild and McMeniman ( 1994 ), supplementation of poor quality forage with good protein feed has increased the availability of nitrogen in the rumen, thereby improving the rate of degradation and utilization of the feed. The average total CP intake of goats in the present study is in line with the recommended requirements of 65–70 g/day CP for goats weighing 20–25 kg (Kearl 1982 ). This indicates that the ration used in the present study can support better performance in goats. The estimated daily energy intakes were in the range of 4.8–5.9 MJ ME, which are within the recommended range of 3.25–6.47 MJ (Devendra and Burns 1983 ). Growth performance The difference in growth performance of goats fed different concentrate levels in the present study reflects the variations in feed conversion efficiency. The extra 1 kg weight gain of L2 over L1 goats can be due to their increased DM and protein intakes. The average daily weight gain recorded in the present study is within the range of 29–51 g reported for Small East African goats fed low and high levels of concentrate (Hango et al. 2007 ). Daily weight gains of 33–49 g/day were reported for HH goats fed different levels of leftover khat ( Catha edulis ) (Wallie et al. 2012 ), which are very close to the present values. Higher growth rates were also reported for Somali (Mengistu et al. 2007 ; Solomon and Simret 2008 ) and Sidama goats (Solomon et al. 2008 ) supplemented with concentrate and fed hay as basal diet. Body linear dimensions such as heart girth, height at wither, and body lengths are reported to be closely related to the size or weight of an animal (Vargas et al. 2007 ). In present study, only height at wither was found to be significantly higher for Bati and HH goats than SS goats, probably due to the fact that these goats are naturally taller than SS goats (FARM-Africa 1996 ). However, supplementation of goats with low to moderate quality basal diets did not affect body length and heart girth. The fact that SS goats had shorter height (p < 0.05) but comparable body length and heart girth with that of Bati goats indicates that SS goats have compact body than Bati goats. This is also confirmed by the result that SS goats tended to have relatively higher DP than Bati goats (Table 4 ). Mahgoub and Lu ( 1998 ) reported that Dhofari goat with smaller body size had higher carcass muscularity than Batina goat of larger body size. Carcass characteristics The carcass weight recorded in the present study is within the carcass weight of 6–9 kg reported for other Ethiopian goats (Hailu et al. 2005 ; Ameha et al. 2007 ). The effect of diet on carcass weight was clearly seen. The difference in carcass weight between supplement groups may be attributed to higher feed intake and, consequently, greater nutrient availability to promote weight gain and tissue development in goats fed L2 diet. Mushi et al. ( 2009a ) also reported higher weights and carcass yields in goats fed higher energy levels. The dressing percentage, expressed as percentage of full and empty body weight, were 42.4 and 52.8 %, respectively. The values are within the range of 39.5–41.8 and 53.3–56.6 % reported for Ethiopian (Ameha et al. 2007 ) and Omani goat breeds (Kadim et al. 2003 ). The reason for variation in DP among genotypes in the present study is because of greater difference in proportion of non-carcass components such as GIT. Dhanda et al. ( 1999 ) and Addisu et al. ( 2002 ) also reported a significant effect of genotype on DP and attributed this difference to variations in weight of digestive tract contents. On the other hand, the higher DP of L2 goat compared to that of L1 goat is likely associated to better development of muscle and fat tissue due to their increased DM and CP intakes (Safari et al. 2011 ). According to De Boer et al. ( 1974 ) linear carcass measurements are indices indirectly help to determine carcass conformation and affected by genotype, sex and feeding regimes. HH and SS goats had relatively wider (p < 0.05) rib-eye area than Bati goats implying that they have more desirable carcass. According to the report of Wolf et al. ( 1980 ), larger rib-eye area is associated with higher production of lean in the carcass and higher lean to bone ratio. The proportion of the legs (32–33 %) calculated in the present study is within 28–33 % reported for well-conformed goat breeds (Sen et al. 2004 ; Webb et al. 2005 ). The reason for SS goats to have equal proportions of primal cuts compared to that of Bati goat is because they deposited more muscle tissue relative to their body size. Mahgoub and Lu ( 1998 ) noted that small body sized goats had a higher carcass muscularity than large body sized ones. Non-carcass characteristics Information on yield of non-carcass components of animals is important especially in developing countries where they are more valuable for the households (Mushi et al. 2009b ). The presence of variations in internal fat deposition among different breeds of goat has been reported earlier by Mahgoub and Lu ( 1998 ) and Kadim et al. ( 2003 ). On average, about 0.32 kg total non-carcass fat was recorded in the present study, which is lower than 0.57 kg reported for other Ethiopian indigenous goats (Ameha et al. 2007 ). Among the three goat types, SS goats can be regarded as physiologically early maturing animals as they have higher deposition of internal fats, and also better composition of chemical fat in the muscle as indicated by Tadesse et al. ( 2015 ). According to Ameha et al. ( 2007 ), different animals can have different physiological maturities as a result of difference in chemical fat composition and total non-carcass fat contents. Higher proportions of total and edible offals were obtained from L2 than L1 goats, which is in agreement with the results of Solomon et al. ( 2008 ) and Solomon and Simret ( 2008 ). The total edible offals (2.8–3.2 kg) reported for HH (Asnakew and Berhan 2007 ) and Sidama goats (Tadesse et al. 2013 ) are also similar with finding of the present study. The fact that SS goats had lighter total offals in the present study indicates that this breed can produce more proportion of usable carcass components relative to their body size than Bati and HH goats. Goats fed L2 had heavier liver than those fed L1, may be due to their higher nutrient intake and, consequently, greater deposition of reserve substances such as glycogen as noted by Mushi et al. ( 2009b ). Because of their bigger body size, thicker skins and dense hair cover, HH and Bati goats produced heavier (p < 0.05) skin than SS goats. Similar observation has been noted by Kadim et al. ( 2003 ) on skins obtained from different Omani goat breeds. Conclusion The study demonstrated the effect of genotypes and levels of concentrate on growth rate and slaughter characteristics of selected Ethiopian indigenous goats. Concentrate feeding at the rate of 1.5 % than 1 % BW provided adequate nutrients to support maintenance and growth rates of up to 51 g/day. It also resulted in heavier final body and carcass weights despite lack of influence on most of the body and carcass linear measurements. Among the three genotypes, HH goats had better yield and carcass weight with more developed muscles. The study further revealed that SS goats, though smaller in size, are capable of producing comparable carcass compared to Bati goats. This indicates the potential of SS goats to produce more carcass yield relative to their body size/weight. In general, the study indicates the potential of Ethiopian indigenous goat for meat production through supplementation of concentrate at the rate of 1.5 % body weight. Authors' information DT is assistant professor in Tropical Animal Production in Department of Animal Sciences, Debre Berhan University, Ethiopia. MU and GA are associate professor of animal nutrition in the School of Animal and Range Sciences, Haramaya University, Ethiopia. YM is currently working in the International Livestock Research Institute as Regional Coordinator and Senior Expert, Addis Ababa, Ethiopia. Acknowledgements This study was funded by Swedish International Development Agency (Sida) through Haramaya University. The authors wish to thank Sida and Haramaya University for the financial and material support. Competing interests The authors declare that they have no competing interests.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094474/

Removal and retention of viral aerosols by a novel alumina nanofiber filter

Nanomaterial, due to its unique physical, chemical and biological properties compared to its bulk counterparts, has the potential to provide a product superior to its bulk predecessor. In this study, a novel alumina nanofiber filter was assessed for its removal and retention capability for MS2 aerosol. Its physical removal efficiency in the 10–400 nm range was 94.35%, while its viable removal efficiency was 98.87%, which was slightly lower than three conventional HEPA filters tested. However, its pressure drop was much lower than HEPA filters, yielding a higher filter quality than HEPA filters. The average extracted fraction from the nanofiber filter was 8.64×10 −2 ±7.00×10 −2 , which is three orders lower than other HEPA filters, demonstrating that the viruses were effectively retained in the nanofiber filter. Furthermore, the performance of the nanofiber filter showed no dependence on relative humidity. In conclusion, this novel alumina nanofiber filter presents advantageous potential for removal and retention of viral aerosol agents. 1 Introduction Bioaerosols are aerosols of biological origins such as viruses, bacteria, fungi, spores, pollen and allergens ( Hinds, 1999 ). They can cause adverse health and welfare effects, including allergy, asthma, respiratory illnesses, crop damage and animals infection. The spread of airborne pathogens such as severe acute respiratory syndrome (SARS) and avian flu as well as the potential of intentional release of biological agents by terrorists such as the Anthrax in 2001 have raised the public's concerns about bioaerosols and protection measures ( Kortepeter & Parker, 1999 ; Rengasamy, Zhuang, & BerryAnn, 2004 ; Lee et al., 2008 ). Filtration is one of the most commonly applied methods for aerosol removal because it is simple and economical. However, there are two main issues associated with filtration: (1) High aerosol removal efficiency is achieved only at the cost of high pressure drop, which is translated into high energy consumption for collective protection or high breathing resistance for individual protection. How to increase efficiency while maintaining low pressure drop is a critical challenge to filter development. (2) Collected biological agents may creep through the filter and reaerosolize. The reaerosolization may impose hazards to the person intended to protect. How to effectively retain the biological agents loaded on the filter, hence, is an important consideration ( Ratnesar-Shumate et al., 2008 ; Rengasamy et al., 2004 ). Nanofibrous media have the potential to solve the above issues due to their high surface area and ability to incorporate special functional groups at the nanoscale level. ( Barhate & Ramakrishna, 2007 ). A large surface-area-to-volume ratio of nanofiber can increase the probability of aerosol deposition on the fiber surface by diffusion and improve the filter efficiency ( Ahn et al., 2006 ; Huang, Zhang, Kotaki, & Ramakrishna, 2003 ). Their smaller meshes can significantly increase the removal efficiency of submicrometer particles by direct interception ( Podgórski, Balazy, & Gradoń, 2006 ). Due to the slip on the nanofiber surface, the drag force on the nanofiber is smaller than in a non-slip flow, which translates into a lower pressure drop than conventional filters ( Kosmider & Scott, 2002 ). Most microorganisms have an electronegative surface due to their carboxyl and phosphate groups ( Mozes et al., 1987 ; Valegård, Liljas, Fridborg, & Unge, 1990 ). Thus, an electropositive surface has the potential to have enhanced bioaerosol removal. This electrostatic attraction has been well demonstrated in aqueous environment. For example, iron oxyhydroxide floc particles have been investigated for adsorbing negatively charged MS2 viruses in water ( Zhu, Clifford, & Chellam, 2005 ). Coating of sand and granular activated carbon with iron aluminum hydroxide increased the zeta potential and therefore improved virus removal ( Scott et al., 2002 ). On the same basis, nanofibrous media can also be fabricated with electropositive surface to attract microorganisms. A novel alumina nanofiber filter has been developed recently that has demonstrated ability to remove microbiological agents in aqueous environment using electrostatic attraction ( Tepper & Kaledin, 2005 ). Although there has been no assessment in the aerosol phase yet, its feature is expected to have enhanced bioaerosol collection and retention efficiency that utilizes the above addressed advantages: large surface-area-to-volume ratio, lower pressure drop and higher surface charge density. The objective of this study was therefore to evaluate the performance of the alumina nanofilter for removal and retention of viral aerosols. Experiments were conducted to assess the filter's physical removal efficiency (PRE), viable removal efficiency (VRE), extracted fraction (EF) and filter quality ( q F ). Three conventional filters (two glass fiber filters and one PTFE filter) were also tested for comparison. In addition, the nanofiber filter was examined in three different relative humidities (RHs) to determine its response to different environmental conditions. 2 Experimental method 2.1 Test agent MS2, an Escherichia coli bacteriophage, (ATCC ® 15597-B1™) was chosen as the biological test agent. It has a single-stranded RNA genome and an approximate diameter of 28 nm ( Golmohammadi, Valegard, Fridborg, & Liljas, 1993 ; Prescott, Harley, & Klein, 2002 ). MS2 is hydrophilic and electronegative in water ( Valegård et al., 1990 ). It infects only male E. coli bacteria by injection of its RNA and A-protein. Due to the similar physical characteristics, it has been frequently used as a surrogate for mammalian viruses ( Aranha-Creado & Brandwein, 1999 ). Freeze-dried MS2 bacteriophage was suspended with filtered deionized (DI) water to the concentration of 10 8 –10 9 PFL/mL, which was the virus stock suspension. 2.2 Test filters The test nanofiber filter consists of a single layer of alumina nanofiber grafted to a microglass fiber backbone. SEM and TEM images of fresh nanofiber filter are shown in Figs. 1 a and c. The diameter of the alumina nanofiber is approximately 2–4 nm and its surface area is 450–600 m 2 /g ( Tepper & Kaledin, 2005 ). Its composition has been identified as boehmite (AlOOH) by X-ray diffraction patterns and the zeta potential is +50 mV at pH 7. The filter is 800 μm thick with 92% porosity. The pore size for water filtration is 2 μm while for air use is 28 μm to allow for higher permeability. Its composition, synthesis and characteristics are defined as sample AF16 in Tepper and Kaledin (2007) . This structure is different from other nanofibers tested for air filtration; their nanofiber diameters are in the 80–400 nm range ( Ahn et al., 2006 ; Kosmider & Scott, 2002 ; Yun et al., 2007 ). Fig. 1 SEM images of nanofiber filter: (a) blank filter and (b) after experiment; TEM images of nanofiber filter: (c) blank filter and (d) after experiment. Three commercially available HEPA filters were also compared in the study: a 790-μm thick glass fiber filter (A) with resin binder, 1.0 μm pore size and 90% porosity (Millipore, AP1504700); a 78-g/m 2 basis weight HEPA glass fiber filter (B) that has 99.982% DOP collection efficiency (Lydall, grade 4450-HS); a 152-μm thick PTFE membrane filter (polytetrafluoroethylene with polytetrafluoroethylene support, Zefluor membrane) with a 2.0-μm pore size (Pall, P5PJ047). 2.3 Experimental system Fig. 2 shows a schematic drawing of the experimental system. The bioaerosol was generated by a six-jet Collison nebulizer (Model CN25, BGI Inc., Waltham, MA) and passed through a dilution drier to remove the moisture. The virus concentration in the nebulizer was around 10 7 PFU/mL and was prepared by diluting 0.1 mL of virus stock suspension in 50 mL of sterile DI water. The corresponding MS2 aerosol concentration was approximately 4000 PFU/L. The temperature and humidity of the air stream were measured by a thermometer and a RH meter (Model HX94C, OMEGA Engineering Inc., Stamford, CT), respectively. The airflow was then divided into two parallel streams. One stream that contained the test filter was designed for experimental collection while the other, which had no test filter, served as the control. The bioaerosol was collected by a biosampler (SKC Inc., Eighty-Four, PA) that had 15 mL of phosphate buffer solution (PBS) as collection liquid. MS2 collected in the biosampler was then enumerated following the procedures in Adams (1959) and plates were incubated at 37 °C for 18 h. The experiment was conducted at 22±2 °C and 50±5% RH for 30-min intervals, and the face velocity of filtration was 26.0 cm/s. In addition, the nanofiber filter was tested in three RHs (35%, 50% and 75%) to determine its response to different environmental conditions. RH was regulated by controlling the ratio of dry cylinder air to the nebulizer flow. The pressure drop of the test filter was measured using a pressure gauge (2010AV C, Dwyer Instruments, Michigan City, IN). Fig. 2 Schematic of the bioaerosol experimental system. 2.4 Removal efficiency In this study, two types of efficiency ( η ) were determined: VRE and PRE. The efficiency was determined by comparing the penetrating viral aerosols in the experimental stream and the feed viral aerosols in the control stream. VRE was determined from the number of plaque-forming units (PFUs) on the petri dish for each collection stream following Eq. (1) : (1) η ( VRE or PRE ) = 1 - N p / N c where N c is the number of viral aerosols entering the filter and N p is the number of viral aerosols penetrating the filter. Regarding PRE, the particle size distributions (PSDs) of the aerosols entering and penetrating the test filters were measured by a scanning mobility particle sizer (SMPS, Model #3936, TSI) and the PRE was calculated according to Eq. (1) based on the SMPS measurements. It should be noted that the freeze-dried MS2 from ATCC contains non-MS2 content, e.g. protein. Hence, the SMPS was measuring particles containing MS2 virons rather than purely MS2 virons. 2.5 Extracted fraction After 2 h of the experiment for removal efficiency, the test filter was removed from the filter holder and subjected to vortex mixing (Model # M16715, Barnstead) to extract the MS2 collected on the filter. After various vortexing times (0, 1, 3 and 5 min), the infectivity of MS2 in the vortexing solution was analyzed and expressed as EF, as defined by Eq. (2) : (2) EF = N Ext / N Coll where N Ext is the MS2 count extracted from the filter and N Coll is the MS2 count of control collection (MS2 collected by the biosampler without nanofiber filter) minus the count of experimental collection (penetrating MS2 collected by the biosampler with nanofiber filter). 2.1 Test agent MS2, an Escherichia coli bacteriophage, (ATCC ® 15597-B1™) was chosen as the biological test agent. It has a single-stranded RNA genome and an approximate diameter of 28 nm ( Golmohammadi, Valegard, Fridborg, & Liljas, 1993 ; Prescott, Harley, & Klein, 2002 ). MS2 is hydrophilic and electronegative in water ( Valegård et al., 1990 ). It infects only male E. coli bacteria by injection of its RNA and A-protein. Due to the similar physical characteristics, it has been frequently used as a surrogate for mammalian viruses ( Aranha-Creado & Brandwein, 1999 ). Freeze-dried MS2 bacteriophage was suspended with filtered deionized (DI) water to the concentration of 10 8 –10 9 PFL/mL, which was the virus stock suspension. 2.2 Test filters The test nanofiber filter consists of a single layer of alumina nanofiber grafted to a microglass fiber backbone. SEM and TEM images of fresh nanofiber filter are shown in Figs. 1 a and c. The diameter of the alumina nanofiber is approximately 2–4 nm and its surface area is 450–600 m 2 /g ( Tepper & Kaledin, 2005 ). Its composition has been identified as boehmite (AlOOH) by X-ray diffraction patterns and the zeta potential is +50 mV at pH 7. The filter is 800 μm thick with 92% porosity. The pore size for water filtration is 2 μm while for air use is 28 μm to allow for higher permeability. Its composition, synthesis and characteristics are defined as sample AF16 in Tepper and Kaledin (2007) . This structure is different from other nanofibers tested for air filtration; their nanofiber diameters are in the 80–400 nm range ( Ahn et al., 2006 ; Kosmider & Scott, 2002 ; Yun et al., 2007 ). Fig. 1 SEM images of nanofiber filter: (a) blank filter and (b) after experiment; TEM images of nanofiber filter: (c) blank filter and (d) after experiment. Three commercially available HEPA filters were also compared in the study: a 790-μm thick glass fiber filter (A) with resin binder, 1.0 μm pore size and 90% porosity (Millipore, AP1504700); a 78-g/m 2 basis weight HEPA glass fiber filter (B) that has 99.982% DOP collection efficiency (Lydall, grade 4450-HS); a 152-μm thick PTFE membrane filter (polytetrafluoroethylene with polytetrafluoroethylene support, Zefluor membrane) with a 2.0-μm pore size (Pall, P5PJ047). 2.3 Experimental system Fig. 2 shows a schematic drawing of the experimental system. The bioaerosol was generated by a six-jet Collison nebulizer (Model CN25, BGI Inc., Waltham, MA) and passed through a dilution drier to remove the moisture. The virus concentration in the nebulizer was around 10 7 PFU/mL and was prepared by diluting 0.1 mL of virus stock suspension in 50 mL of sterile DI water. The corresponding MS2 aerosol concentration was approximately 4000 PFU/L. The temperature and humidity of the air stream were measured by a thermometer and a RH meter (Model HX94C, OMEGA Engineering Inc., Stamford, CT), respectively. The airflow was then divided into two parallel streams. One stream that contained the test filter was designed for experimental collection while the other, which had no test filter, served as the control. The bioaerosol was collected by a biosampler (SKC Inc., Eighty-Four, PA) that had 15 mL of phosphate buffer solution (PBS) as collection liquid. MS2 collected in the biosampler was then enumerated following the procedures in Adams (1959) and plates were incubated at 37 °C for 18 h. The experiment was conducted at 22±2 °C and 50±5% RH for 30-min intervals, and the face velocity of filtration was 26.0 cm/s. In addition, the nanofiber filter was tested in three RHs (35%, 50% and 75%) to determine its response to different environmental conditions. RH was regulated by controlling the ratio of dry cylinder air to the nebulizer flow. The pressure drop of the test filter was measured using a pressure gauge (2010AV C, Dwyer Instruments, Michigan City, IN). Fig. 2 Schematic of the bioaerosol experimental system. 2.4 Removal efficiency In this study, two types of efficiency ( η ) were determined: VRE and PRE. The efficiency was determined by comparing the penetrating viral aerosols in the experimental stream and the feed viral aerosols in the control stream. VRE was determined from the number of plaque-forming units (PFUs) on the petri dish for each collection stream following Eq. (1) : (1) η ( VRE or PRE ) = 1 - N p / N c where N c is the number of viral aerosols entering the filter and N p is the number of viral aerosols penetrating the filter. Regarding PRE, the particle size distributions (PSDs) of the aerosols entering and penetrating the test filters were measured by a scanning mobility particle sizer (SMPS, Model #3936, TSI) and the PRE was calculated according to Eq. (1) based on the SMPS measurements. It should be noted that the freeze-dried MS2 from ATCC contains non-MS2 content, e.g. protein. Hence, the SMPS was measuring particles containing MS2 virons rather than purely MS2 virons. 2.5 Extracted fraction After 2 h of the experiment for removal efficiency, the test filter was removed from the filter holder and subjected to vortex mixing (Model # M16715, Barnstead) to extract the MS2 collected on the filter. After various vortexing times (0, 1, 3 and 5 min), the infectivity of MS2 in the vortexing solution was analyzed and expressed as EF, as defined by Eq. (2) : (2) EF = N Ext / N Coll where N Ext is the MS2 count extracted from the filter and N Coll is the MS2 count of control collection (MS2 collected by the biosampler without nanofiber filter) minus the count of experimental collection (penetrating MS2 collected by the biosampler with nanofiber filter). 3 Result and discussions 3.1 PSDs and removal efficiencies The PSDs measured by the SMPS are shown in Fig. 3 . The mode size of the feed aerosol was about 30 nm, which is similar to the size of a single MS2 virus and agrees well with prior studies using MS2 ( Burton, Grinshpun, & Reponen, 2007 ; Hogan et al., 2005 ; Richardson, Eshbaugh, Hofacre, & Gardner, 2006 ). The PSDs of penetrating aerosol from the four filters are also shown in Fig. 3 a. The most penetrating aerosol size was around 100 nm, which is smaller than the 0.3-μm standard that is commonly used in filter testing ( NIOSH, 2005 ) ( Fig. 3 b). The corresponding minimum PRE of the nanofiber filter for 100 nm was around 75%, which is lower than other HEPA filters tested. Compared to other filters tested for MS2 aerosol reported in the literature, this alumina nanofiber filter's PRE is higher than 1.0-μm and 3.0-μm pore size polycarbonate filters (68% and 27%), but slightly lower than PTFE and gelatine filters (>96%) ( Burton et al., 2007 ). Fig. 3 (a) Particle size distributions of MS2 before and after the filters and (b) PRE of MS2 in the 10–400 nm particle size range. The VREs of the nanofiber and three conventional filters are shown in Table 1 . As shown, the nanofiber filter's VRE was slightly lower than the other filters. The difference between PRE and VRE implies that the virus distribution does not follow the number size distribution of the aerosol ( Hogan et al., 2005 ), although no detailed information regarding virus distribution in aerosol is available yet. SEM and TEM images of nanofiber filters after the MS2 filtration experiment and vortexing extraction are shown in Figs. 1 b and d. Compared to those before the MS2 experiment, it is clear to observe particles trapped on the fiber surface. Table 1 Physical removal efficiency, viable removal efficiency, pressure drop, filter quality and extracted fraction for nanofiber filter, glass fiber filters A, B and PTFE filter Filters PRE a (%) VRE (%) Δ P (in H 2 O) b q F (kPa) c q F (kPa) d EF Nanofiber 94.35±3.22 98.87±0.78 2.5–2.8 4.12 6.43 8.64×10−2±7.00×10−2 Glass fiber A 99.99±0.001 99.92±0.14 15–16 2.31 1.79 162±61 PTFE 96.02±2.27 99.94±0.05 6.0–6.5 2.16 4.97 38.1±1.5 Glass fiber B 99.63±0.22 99.96±0.01 5.0–5.5 4.07 5.72 32.2±9.8 a Overall efficiency over 10–400 nm. b Measured at 26.0 cm/s face velocity. c Based on PRE. d Based on VRE. There are other reasons why the PRE and VRE are not comparable. First, the sampling particle size range is different. The SMPS only covers from 10 to 400 nm while the biosampler has no size limit. However, the biosampler has very low collection efficiency in the ultrafine range ( Hogan et al., 2005 ), even as low as 10%. On the other hand, the SMPS also has limitation of low charging efficiency and has to rely on assumed Boltzmann charge distribution for correction. 3.2 Pressure drop and filter quality The pressure drop measurements for the nanofiber filter along with three HEPA filters are displayed in Table 1 . As shown, the pressure drop of the nanofiber filter was much lower than the other three filters. Due to the slip on the nanofiber surface, the drag force on the nanofiber is smaller than in a non-slip flow, which translates into a lower pressure drop ( Kosmider & Scott, 2002 ; Podgórski et al., 2006 ). In comparison, the glass fiber filter A as an analytical filter had the highest pressure drop. Combining penetration and pressure drop, filter quality ( q F ) can be calculated by the following Eq. (3) ( Hinds, 1999 ) (3) q F = ln ( 1 / p ) / Δ P where p is penetration, and Δ P is pressure drop. A filter with better quality has less penetration for the same pressure drop compared to other filters. The results for all four filters are listed in Table 1 . As shown, based on PRE, the nanofiber filter has the best filter quality than other filters. It's 1.9 times higher than the PTFE filter. For measured VRE, the nanofiber filter still presents the best filter quality, which is 3.3 times higher than the glass fiber filter A. This is mainly contributed by its unique feature of low pressure drop. Similar improvement in filter quality by decreasing fiber diameter was reported by Podgórski et al. (2006) , who reported that their nanofiber layer was 2.6 times better than two layers of microfibrous support although it was much thinner. Yun et al. (2007) also reported similar observations. 3.3 Extracted fraction The results of EFs for all filters are listed in Table 1 . As shown, the average EF of the nanofiber filter over 1, 3, and 5 min of vortex time was 8.64×10 −2 ±7.00×10 −2 , which was significantly lower than any other compared filters. This phenomenon indicates that viruses were effectively retained in the nanofiber filter, due to electrostatic attraction between the electropositive fiber surface and electronegative MS2 particles. In contrast, the EFs of the other three filters were all greater than one. Theoretically, the EF should be less than one. However, the feed MS2 concentration determined by the biosampler collection was an underestimate because the biosampler had low collection efficiency for nanometer particles (96%) ( Burton et al., 2007 ). Fig. 3 (a) Particle size distributions of MS2 before and after the filters and (b) PRE of MS2 in the 10–400 nm particle size range. The VREs of the nanofiber and three conventional filters are shown in Table 1 . As shown, the nanofiber filter's VRE was slightly lower than the other filters. The difference between PRE and VRE implies that the virus distribution does not follow the number size distribution of the aerosol ( Hogan et al., 2005 ), although no detailed information regarding virus distribution in aerosol is available yet. SEM and TEM images of nanofiber filters after the MS2 filtration experiment and vortexing extraction are shown in Figs. 1 b and d. Compared to those before the MS2 experiment, it is clear to observe particles trapped on the fiber surface. Table 1 Physical removal efficiency, viable removal efficiency, pressure drop, filter quality and extracted fraction for nanofiber filter, glass fiber filters A, B and PTFE filter Filters PRE a (%) VRE (%) Δ P (in H 2 O) b q F (kPa) c q F (kPa) d EF Nanofiber 94.35±3.22 98.87±0.78 2.5–2.8 4.12 6.43 8.64×10−2±7.00×10−2 Glass fiber A 99.99±0.001 99.92±0.14 15–16 2.31 1.79 162±61 PTFE 96.02±2.27 99.94±0.05 6.0–6.5 2.16 4.97 38.1±1.5 Glass fiber B 99.63±0.22 99.96±0.01 5.0–5.5 4.07 5.72 32.2±9.8 a Overall efficiency over 10–400 nm. b Measured at 26.0 cm/s face velocity. c Based on PRE. d Based on VRE. There are other reasons why the PRE and VRE are not comparable. First, the sampling particle size range is different. The SMPS only covers from 10 to 400 nm while the biosampler has no size limit. However, the biosampler has very low collection efficiency in the ultrafine range ( Hogan et al., 2005 ), even as low as 10%. On the other hand, the SMPS also has limitation of low charging efficiency and has to rely on assumed Boltzmann charge distribution for correction. 3.2 Pressure drop and filter quality The pressure drop measurements for the nanofiber filter along with three HEPA filters are displayed in Table 1 . As shown, the pressure drop of the nanofiber filter was much lower than the other three filters. Due to the slip on the nanofiber surface, the drag force on the nanofiber is smaller than in a non-slip flow, which translates into a lower pressure drop ( Kosmider & Scott, 2002 ; Podgórski et al., 2006 ). In comparison, the glass fiber filter A as an analytical filter had the highest pressure drop. Combining penetration and pressure drop, filter quality ( q F ) can be calculated by the following Eq. (3) ( Hinds, 1999 ) (3) q F = ln ( 1 / p ) / Δ P where p is penetration, and Δ P is pressure drop. A filter with better quality has less penetration for the same pressure drop compared to other filters. The results for all four filters are listed in Table 1 . As shown, based on PRE, the nanofiber filter has the best filter quality than other filters. It's 1.9 times higher than the PTFE filter. For measured VRE, the nanofiber filter still presents the best filter quality, which is 3.3 times higher than the glass fiber filter A. This is mainly contributed by its unique feature of low pressure drop. Similar improvement in filter quality by decreasing fiber diameter was reported by Podgórski et al. (2006) , who reported that their nanofiber layer was 2.6 times better than two layers of microfibrous support although it was much thinner. Yun et al. (2007) also reported similar observations. 3.3 Extracted fraction The results of EFs for all filters are listed in Table 1 . As shown, the average EF of the nanofiber filter over 1, 3, and 5 min of vortex time was 8.64×10 −2 ±7.00×10 −2 , which was significantly lower than any other compared filters. This phenomenon indicates that viruses were effectively retained in the nanofiber filter, due to electrostatic attraction between the electropositive fiber surface and electronegative MS2 particles. In contrast, the EFs of the other three filters were all greater than one. Theoretically, the EF should be less than one. However, the feed MS2 concentration determined by the biosampler collection was an underestimate because the biosampler had low collection efficiency for nanometer particles (<10% for 30–100 nm; Hogan et al., 2005 ). With effective extraction, the EF therefore can be great than one. Unfortunately, there is no existing bioaerosol sampling method that allows better viable sampling and liquid impingement is the commonly accepted method ( Hogan et al., 2005 ; Wang & Brion, 2007 ). Therefore, it was used in this study. The filter's unique feature of electrostatically attracting viruses provides the capacity to retain viruses. The isoelectric point of MS2 is pH 3.9 and thus the MS2 is electronegative above this value ( Ackermann & Dubow, 1987 ; Zhu et al., 2005 ). On the other hand, the zeta potential of nanofiber media is +50 mV at pH 7 ( Tepper & Kaledin, 2007 ). To further verify the electrostatic attraction, experiments were carried out with four fresh filters added to a suspension of known MS2 concentration (∼10 7 PFU/mL) and the suspension was vortexed for up to 10 min. The results are shown in Fig. 4 . The infectivity of MS2 on the nanofiber filter decreased dramatically with an increase in vortexing time. In contrast, the infectivity of MS2 on the other three filters had a similar decay as in DI water for all vortexing times. The difference further verifies the effect of electrostatic attraction exerted by the alumina nanofiber filter. Fig. 4 The infectivity of MS2 in suspensions of known MS2 concentration without filters and with four fresh filters added. 3.4 Performance under different RHs The VRE of the nanofiber filter tested at 35%, 50% and 75% RH were 98.13±1.32%, 98.87±0.78% and 98.79±0.62%, respectively. Apparently, the performance of the nanofiber was not affected by RH change. It has been reported that high RHs decrease the removal efficiencies of electret filters ( Motyl & Lowkis, 2006 ; Moyer & Stevens, 1989 ; Raynor & Chae, 2004 ; Yang et al., 2007 ) due to the reduction of charges within the filter. For non-electret filters that rely on mechanical removal mechanisms, RH imposes no significant effect on the removal ( Huang & Yang, 2006 ; Kim, Bao, Okuyama, Shimada, & Niinuma, 2006 ). Wang and Brion (2007) showed that glass microfiber filter had similar performance at 23% and 50% RHs after the filter had reached a steady-state condition. Compared to electrets where the charge is induced during manufacture, the nanofiber's charge is intrinsic in the way the alumina is formed. Hence, the charge is not water sensitive and is retained in bulk water. 4 Conclusions This novel alumina nanofiber filter has demonstrated potential for effective removal and retention of MS2 aerosol. It presented lower PRE and VRE than conventional HEPA filters, but its pressure drop was much lower due to smaller drag force on the nanofiber surface. Hence, the nanofiber filter presented the best filter quality. The EF of nanofiber filter was three orders lower than the HEPA filters, demonstrating that viruses were effectively retained in the nanofiber filter due to electrostatic attraction. While the performance of conventional electret filters decreases at higher RHs, different RHs imposed no effects on the nanofiber filter's ability to remove aerosol.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590714/

Establishment of a National Inventory of Dangerous Pathogens in the Republic of Uganda

One of the challenges of global biosecurity is to protect and control dangerous pathogens from unauthorized access and intentional release. A practical and feasible option to protect life science institutes against theft and sabotage, and secure their biological materials against misuse, is to establish a national electronic database with a comprehensive overview of the locations of all controlled dangerous pathogens in a country. This national database could be used as an instrument to secure and account for dangerous pathogens in a country, but it could also assist in establishing a biosecurity assessing and monitoring system for laboratories that work with these controlled biological agents. The Republic of Uganda is one of the first countries, prompted by the World Health Organization's (WHO's) Joint External Evaluation (JEE), to implement a national electronic database that assembles information collected from relevant Ugandan laboratories. This Ugandan Inventory of Dangerous Pathogens is different from an institute-specific pathogen inventory system, as it is intended to store the information collected from laboratories in the country working with dangerous pathogens in 1 centralized secure location. The Uganda National Council for Science and Technology (UNCST) has coordinated the implementation of the Ugandan national inventory. The inventory was recognized by the WHO JEE as contributing to Uganda's developed capacities regarding biosafety and biosecurity. This article describes the steps in implementing Uganda's National Inventory of Dangerous Pathogens. In addition, it presents a straightforward approach that can be adapted by other countries that aim to enhance their biosecurity capacities. Methods Software The RIVM has provided secure software to the UNCST to create and manage a national inventory of dangerous pathogens in Uganda. The national inventory is survey-driven. Data can be collected through a spreadsheet, then imported and securely stored into the electronic database. The National Inventory of Dangerous Pathogens in Uganda stores information on all relevant institutes, their geographic location, the select agents these laboratories are storing and handling, their safety classification, and the contact details of the responsible biosafety officer. The database also provides options for adding additional information manually. The information stored in the databases should be considered sensitive information and protected with a high level of security. Stakeholder Consultation Before the implementation process of the national inventory, the RIVM and the public health emergency operations center conducted a stakeholder consultation, which included 4 site visits with key Ugandan institutes, assigned by the public health emergency operations center, the Infectious Disease Institute (IDI) of Makarere University, the National Public Health Laboratory Services (NPHLS), the Uganda Virus Research Institute (UVRI), and the National Animal Disease and Diagnostic Epidemiology Centre (NADDEC). The main purpose of this stakeholder consultation was to recommend to the UNCST how best to implement a national inventory of dangerous pathogens. These recommendations would ensure that the database aligns with the specific biosecurity needs of the country. These expert opinions resulted in a set of recommendations. First, the national inventory would store information from all selected institutes, with a short note that the stakeholders' list of the national inventory would need to be revised periodically. Next, selected institutes would be required to report only biological agents on the US Federal Select Agents List, 16 the 67 biological agents that have been determined to pose a severe threat to human, plant, and animal health. Laboratories should report any of those agents that have been stored longer than 2 weeks. Third, access to the National Inventory of Dangerous Pathogens would be granted only to authorized individuals from the UNCST and would be reported to the responsible authorities. Implementation Process The implementation of a National Inventory of Dangerous Pathogens can be divided into 3 stages: preparation, implementation, and maintenance. In the preparatory phase, the government of Uganda committed to the establishment of a National Inventory of Dangerous Pathogens and assigned responsibilities in the government. With a designated government focal point in Uganda, the implementation phase was initiated, and a communication plan was set up for contacting the appropriate institutes for the relevant data. The list of these institutes was compiled with the help of IDI, UVRI, UNCST, NPHLS, and NADDEC, in addition to the Biosafety and Biosecurity Association Uganda, and included approximately 40 institutes being requested to provide relevant information. The decision of the prioritized pathogen list to be included in the inventory was determined to be the US Federal Select Agents List, and the information was gathered and inserted into the dedicated software. In the maintenance phase, the focal point is responsible for informing the appropriate Ugandan authorities (ie, the ministry of health, as part of the team for the JEE, and the Uganda representative at the BWC) about the number and location of institutes storing dangerous pathogens, as well as the variety of dangerous pathogens present in the Republic of Uganda and plans for annual updates of the inventory. In all of these stages, communication, ownership, and data-collection activities lie with the Uganda focal point, and no sensitive information was shared with or handled by anyone not approved by the focal point. The database will be owned and controlled by the government of Uganda. Although institutional data on working with high-risk pathogens may not be sensitive information, the consolidated national data could be considered sensitive information and should therefore be stored safely and securely according to Ugandan procedures and relevant official information confidentiality laws. The public health emergency operations center of the ministry of health in Uganda initially served as the focal point for organizing, coordinating, supporting, and managing all aspects of public health emergency response efforts. Subsequently, ownership of the national point was handed over from the ministry of health to the UNCST, which would carry out the laboratory mapping activities, host and maintain inventory, and report to the relevant ministries. Two professionals from the UNCST were active on this project, which included an IT specialist and a biosafety and biosecurity expert, which is sufficient for the maintenance phase during the annual updates. Support from the Netherlands included a program coordinator, a program officer, and an additional subject matter expert. There were 6 in-person meetings in Uganda: 1 meeting with the ministry of health director general for commitment, 1 meeting for determining the responsible government entity, 1 dedicated to site visits to the 4 large Ugandan institutes working on human and zoonotic pathogens, 1 meeting dedicated to IT infrastructure, 1 dedicated to communication strategies, and 1 evaluative meeting. Software The RIVM has provided secure software to the UNCST to create and manage a national inventory of dangerous pathogens in Uganda. The national inventory is survey-driven. Data can be collected through a spreadsheet, then imported and securely stored into the electronic database. The National Inventory of Dangerous Pathogens in Uganda stores information on all relevant institutes, their geographic location, the select agents these laboratories are storing and handling, their safety classification, and the contact details of the responsible biosafety officer. The database also provides options for adding additional information manually. The information stored in the databases should be considered sensitive information and protected with a high level of security. Stakeholder Consultation Before the implementation process of the national inventory, the RIVM and the public health emergency operations center conducted a stakeholder consultation, which included 4 site visits with key Ugandan institutes, assigned by the public health emergency operations center, the Infectious Disease Institute (IDI) of Makarere University, the National Public Health Laboratory Services (NPHLS), the Uganda Virus Research Institute (UVRI), and the National Animal Disease and Diagnostic Epidemiology Centre (NADDEC). The main purpose of this stakeholder consultation was to recommend to the UNCST how best to implement a national inventory of dangerous pathogens. These recommendations would ensure that the database aligns with the specific biosecurity needs of the country. These expert opinions resulted in a set of recommendations. First, the national inventory would store information from all selected institutes, with a short note that the stakeholders' list of the national inventory would need to be revised periodically. Next, selected institutes would be required to report only biological agents on the US Federal Select Agents List, 16 the 67 biological agents that have been determined to pose a severe threat to human, plant, and animal health. Laboratories should report any of those agents that have been stored longer than 2 weeks. Third, access to the National Inventory of Dangerous Pathogens would be granted only to authorized individuals from the UNCST and would be reported to the responsible authorities. Implementation Process The implementation of a National Inventory of Dangerous Pathogens can be divided into 3 stages: preparation, implementation, and maintenance. In the preparatory phase, the government of Uganda committed to the establishment of a National Inventory of Dangerous Pathogens and assigned responsibilities in the government. With a designated government focal point in Uganda, the implementation phase was initiated, and a communication plan was set up for contacting the appropriate institutes for the relevant data. The list of these institutes was compiled with the help of IDI, UVRI, UNCST, NPHLS, and NADDEC, in addition to the Biosafety and Biosecurity Association Uganda, and included approximately 40 institutes being requested to provide relevant information. The decision of the prioritized pathogen list to be included in the inventory was determined to be the US Federal Select Agents List, and the information was gathered and inserted into the dedicated software. In the maintenance phase, the focal point is responsible for informing the appropriate Ugandan authorities (ie, the ministry of health, as part of the team for the JEE, and the Uganda representative at the BWC) about the number and location of institutes storing dangerous pathogens, as well as the variety of dangerous pathogens present in the Republic of Uganda and plans for annual updates of the inventory. In all of these stages, communication, ownership, and data-collection activities lie with the Uganda focal point, and no sensitive information was shared with or handled by anyone not approved by the focal point. The database will be owned and controlled by the government of Uganda. Although institutional data on working with high-risk pathogens may not be sensitive information, the consolidated national data could be considered sensitive information and should therefore be stored safely and securely according to Ugandan procedures and relevant official information confidentiality laws. The public health emergency operations center of the ministry of health in Uganda initially served as the focal point for organizing, coordinating, supporting, and managing all aspects of public health emergency response efforts. Subsequently, ownership of the national point was handed over from the ministry of health to the UNCST, which would carry out the laboratory mapping activities, host and maintain inventory, and report to the relevant ministries. Two professionals from the UNCST were active on this project, which included an IT specialist and a biosafety and biosecurity expert, which is sufficient for the maintenance phase during the annual updates. Support from the Netherlands included a program coordinator, a program officer, and an additional subject matter expert. There were 6 in-person meetings in Uganda: 1 meeting with the ministry of health director general for commitment, 1 meeting for determining the responsible government entity, 1 dedicated to site visits to the 4 large Ugandan institutes working on human and zoonotic pathogens, 1 meeting dedicated to IT infrastructure, 1 dedicated to communication strategies, and 1 evaluative meeting. Results and Discussion Finalizing the National Inventory The implementation process took approximately 1 year and included the government and institutional commitment process, identifying all relevant laboratories in the country, allocating responsibilities regarding the implementation process, assigning access rights to the information, setting up the communication plan, arranging the dedicated and secure IT infrastructure at a central location, and finally collecting, registering, and processing the requested electronic information from Ugandan institutes. The National Inventory of Dangerous Pathogens in Uganda stores electronic data on approximately 40 institutes in the country and their geographic locations, the select agents these laboratories are storing and their safety classification, and the contact details of the responsible biosafety officer. It is important to note that not all institutes were necessarily storing dangerous pathogens, but it was considered relevant to store information on all institutes and include these in subsequent annual updates. Finally, this resulted in an operational and accurate Ugandan National Inventory of Dangerous Pathogens. Since a National Inventory of Dangerous Pathogens contains sensitive information, this case study therefore provides no concrete information on the contents of this inventory. In 2017, the WHO JEE scored the biosafety and biosecurity capacities of the Republic of Uganda as "developed capacity" (score 3 out of 5), 17 which was a substantial increase in the score from the 2015 GHSA pilot assessment in Uganda. Although a number of recommendations in the area of biosafety and biosecurity still must be fulfilled, the initial inventory was specifically mentioned as one of the strengths and best practices in the whole of government biosafety and biosecurity system. 17 By including the National Inventory in the JEE process, the government of Uganda showed continued commitment to this activity. Challenges Encountered Several challenges were encountered during the various stages of implementation. For example, in the preparatory phase, there was initially some confusion about the aims of the National Inventory of Dangerous Pathogens, which was occasionally considered to be an actual (physical) specimen collection. This required an elaboration on the concept of the inventory regarding its purpose and the international regulations and frameworks, emphasizing that the inventory is an electronic resource only. Some institutes were initially reluctant to disclose information on high-risk pathogens. This highlighted the need to raise awareness about the purpose of the inventory, its relevance to national biosecurity, and what is expected from institutes concerning data sharing. Communication on the commitment by the government of Uganda contributed to more compliance. One recommendation was that prior to the first official communication, a workshop with representatives of all institutes would have been helpful in raising awareness and providing tailored information. An additional challenge is that the BWC and other international treaties rarely specify what constitutes dangerous pathogens, and therefore no protocols exist on which pathogens should be monitored and controlled. Decisions on such issues can be challenging when a large number of stakeholders are involved. For this reason, it was decided to include organisms from the US Federal Select Agent List, with the possibility to design and implement a national prioritized pathogen list in the Ugandan context at a later stage. Although the various international regulations concerning biosecurity provide little guidance on how precisely to set up a practical system to account for and secure biological agents and toxins, there is some level of flexibility regarding the relevant elements to be included in the electronic database. Still, it was important to decide early in the process what elements pertinent to Uganda should be included. Finalizing the National Inventory The implementation process took approximately 1 year and included the government and institutional commitment process, identifying all relevant laboratories in the country, allocating responsibilities regarding the implementation process, assigning access rights to the information, setting up the communication plan, arranging the dedicated and secure IT infrastructure at a central location, and finally collecting, registering, and processing the requested electronic information from Ugandan institutes. The National Inventory of Dangerous Pathogens in Uganda stores electronic data on approximately 40 institutes in the country and their geographic locations, the select agents these laboratories are storing and their safety classification, and the contact details of the responsible biosafety officer. It is important to note that not all institutes were necessarily storing dangerous pathogens, but it was considered relevant to store information on all institutes and include these in subsequent annual updates. Finally, this resulted in an operational and accurate Ugandan National Inventory of Dangerous Pathogens. Since a National Inventory of Dangerous Pathogens contains sensitive information, this case study therefore provides no concrete information on the contents of this inventory. In 2017, the WHO JEE scored the biosafety and biosecurity capacities of the Republic of Uganda as "developed capacity" (score 3 out of 5), 17 which was a substantial increase in the score from the 2015 GHSA pilot assessment in Uganda. Although a number of recommendations in the area of biosafety and biosecurity still must be fulfilled, the initial inventory was specifically mentioned as one of the strengths and best practices in the whole of government biosafety and biosecurity system. 17 By including the National Inventory in the JEE process, the government of Uganda showed continued commitment to this activity. Challenges Encountered Several challenges were encountered during the various stages of implementation. For example, in the preparatory phase, there was initially some confusion about the aims of the National Inventory of Dangerous Pathogens, which was occasionally considered to be an actual (physical) specimen collection. This required an elaboration on the concept of the inventory regarding its purpose and the international regulations and frameworks, emphasizing that the inventory is an electronic resource only. Some institutes were initially reluctant to disclose information on high-risk pathogens. This highlighted the need to raise awareness about the purpose of the inventory, its relevance to national biosecurity, and what is expected from institutes concerning data sharing. Communication on the commitment by the government of Uganda contributed to more compliance. One recommendation was that prior to the first official communication, a workshop with representatives of all institutes would have been helpful in raising awareness and providing tailored information. An additional challenge is that the BWC and other international treaties rarely specify what constitutes dangerous pathogens, and therefore no protocols exist on which pathogens should be monitored and controlled. Decisions on such issues can be challenging when a large number of stakeholders are involved. For this reason, it was decided to include organisms from the US Federal Select Agent List, with the possibility to design and implement a national prioritized pathogen list in the Ugandan context at a later stage. Although the various international regulations concerning biosecurity provide little guidance on how precisely to set up a practical system to account for and secure biological agents and toxins, there is some level of flexibility regarding the relevant elements to be included in the electronic database. Still, it was important to decide early in the process what elements pertinent to Uganda should be included. Conclusions In 2016, the government of Uganda successfully implemented a National Inventory of Dangerous Pathogens. This activity has been formally recognized in the WHO JEE of 2017 as contributing to the Ugandan operational capacity regarding biosafety and biosecurity. In 2018, the UNCST organized a follow-up activity: a national stakeholders meeting to discuss the national biosecurity framework and to simultaneously update the National Inventory of Dangerous Pathogens, thereby ensuring sustainable implementation. A National Inventory of Dangerous Pathogens could contribute to several national biosafety and biosecurity issues simultaneously. First, an accurate inventory is in line with international regulations (eg, the BWC) concerning nonproliferation commitments. Second, a national inventory of dangerous pathogens could provide meaningful information for establishing a national policy framework on biosafety and biosecurity, based on the actual occurrence of pathogens in-country. Third, emergency response plans for both intentional and accidental biological calamities in laboratories can be more appropriately tailored when the government, and by extension first responders, has access to accurate information concerning the presence of dangerous pathogens in facilities. Additionally, an existing national inventory could aid in the preparations of emergency response plans—for example, by identifying national reference laboratories for infectious disease outbreaks. Numerous WHO JEEs have been conducted, and the biosafety and biosecurity recommendation frequently noted is the need to set up and implement a national pathogen inventory system. 18 , 19 The fact that numerous countries still do not have a meaningful system in place to account for dangerous pathogens indicates that such inventories constitute a significant biosecurity gap worldwide. This need also coincides with the fundamental biosecurity priority of the Global Partnership against the Spread of Weapons and Materials of Mass Destruction: that states should secure and account for materials that represent biological proliferation risks. Currently, a guidance document on how to implement a national inventory of dangerous pathogens for other interested countries is still lacking. However, as the Uganda experience demonstrates, the establishment of such an inventory is feasible and practical, but requires tailored, country-specific protocols conscientiously implemented.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2974426/

Technical Advance: caspase-1 activation and IL-1β release correlate with the degree of lysosome damage, as illustrated by a novel imaging method to quantify phagolysosome damage

A novel imaging method quantifies phagolysosome damage.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3799554/

Exploring the membrane topology of prohormone convertase 1 in AtT20 Cells: in situ analysis by immunofluorescence microscopy

Prohormone convertase 1 (PC1) was previously characterized as a partially transmembrane protein in purified chromaffin granules of bovine adrenal medulla1. This was challenged with experiments on transfected PC1 in COS1 cells, a non-endocrine cell line2. To address this issue, we undertook to analyze its extraction properties in vitro and its immunocytochemical localization in situ in AtT20 cells, an endocrine cell line that expresses PC1. Most of the 87 kDa form of PC1 was resistant to carbonate extraction suggesting that it had properties of a transmembrane protein. Under semi-permeabilized conditions whereby only the plasma membrane was permeabilized, the carboxy-terminus of PC1 was specifically immunostained whereas the amino-terminus was not. These results indicate that the amino-terminus of PC1 was within the lumen of the Golgi and granules, and some of the C-terminus was exposed to the cytosol. Thus, endogenous PC1 can assume a transmembrane orientation in situ in AtT20 cells. 1. Introduction The proprotein convertases (PCs) belong to a family of endoproteinases that cleave proproteins specifically at basic residue cleavage sites 3 . The first mammalian member of this group, furin, was identified by sequence homology 4 , 5 to the yeast prohormone convertase, Kex2, which was the first eukaryotic enzyme to be described as a prohormone convertase 6 , 7 . Both Kex2 and furin are transmembrane proteins 5 , 7 – 9 . Other mammalian enzymes, homologous to furin were subsequently cloned, of which PC1 (also described as PC3 or SPC3) and PC2 were found to be exclusively expressed in (neuro)endocrine tissue 10 – 14 , suggesting their function to be specific for the maturation of peptide hormones and neuropeptides. Both enzymes do not contain classical amino acid sequences that would predict them to have a transmembrane domain. PC1 is expressed as a pre-pro-protein of ~92 kDa in mass. After removal of the signal peptide, the pro-protein undergoes autocatalytic conversion in the ER to an 87 kDa form 15 – 18 . This form of PC1 can subsequently be converted to a 64–66 kDa form 19 , 20 which is the predominant form found in dense-core granules of the bovine pituitary 21 . The conversion from 87 kDa to 64 kDa is the result of the removal of the carboxyl terminus in a late compartment of the secretory pathway 22 . We and others have investigated the function of the C-terminus of PC1, since it does not appear to be involved in catalysis per se and is distinct from the P-domain of PC1 which is involved in the stability and pH and calcium dependence of PC1 activity 23 . Initial studies revealed that the C-terminus of PC1 is involved in the efficient trafficking of PC1 to the regulated secretory pathway (RSP) 24 – 26 , giving rise to the identification of three alpha-helical amphipathic sequences important in this function. Recent studies by Dikeakos et al. 27 , 28 , have characterized by NMR the extreme C-terminal sequence and identified important residues within the sequence involved in binding to membrane patches which were important for sorting. Previously, we showed by immuno-labeling and classical extraction studies, that in intact purified bovine chromaffin granules, PC1 behaved in part like a transmembrane protein 1 . We identified, immunologically and functionally, the putative transmembrane (TM) sequence (aa617–638), and showed that when fused with the soluble extracellular domain of the IL2 receptor alpha-subunit (Tac), it could direct this protein to secretory granules of the RSP 1 . Indeed, deletion studies identified this sequence as necessary for PC1 sorting to the RSP 29 . We speculated about how PC1 might assume a TM orientation and the consequences of having a cytosolic domain. However, Stettler et al. provided evidence that transfected PC1 is not synthesized as a TM protein in COS1 cells 30 . In our current study we address in a model endocrine cell line, AtT20 cells, which expresses PC1 endogenously, whether PC1 has properties consistent with that of a TM protein. 2. Materials and methods 2.1 Cell culture AtT20 and COS7 cells were grown in high glucose DMEM containing 10% fetal bovine serum, 1X penicillin/streptomycin and 50 μg/ml normocin (Invitrogen, San Diego, CA) in an incubator maintained at 37°C and 5% CO 2 . For sodium carbonate extraction, AtT20 cells were rinsed 3 times with ice cold phosphate buffered saline (PBS), scraped and collected in PBS, sedimented by centrifugation (3,000 rpm for 3 min) and resuspended in a smaller volume of PBS. The cell suspension was dispensed into equal aliquots and stored at -30°C until used. 2.2 Sodium carbonate extraction A frozen aliquot of AtT20 cells was thawed on ice. One hundred μl of the cell suspension was saved immediately and 100 μl were placed into two airfuge tubes for centrifugation at 24 psi for 10 min, i.e. >100,000 × g (Airfuge, Beckman, Fullerton, CA). To block the non-specific binding of proteins to the plastic, the airfuge tubes were previously incubated on ice with 10% BSA for 30 min, after which they were rinsed 3 times with PBS. After centrifugation of the samples, the supernatants were removed and saved, and the pellets were resuspended in an additional 100 μl PBS each. The samples were centrifuged again and the resulting supernatants were combined with their original supernatants. To collect and save the pellet from the PBS extraction, the pellet from one tube was resuspended with 100 μl PBS and saved. The empty tube was rinsed once with 100 μl PBS and this was combined with the resuspended pellet. The other pellet was resuspended by vigorous pipetting with 100 μl of 0.1 M sodium carbonate, pH 11.5, and allowed to incubate on ice for 30 min. The sample was then centrifuged at 24 psi for 10 min and the resulting supernatant saved. The tube was rinsed carefully with 100 μl of fresh carbonate solution and this was added to the supernatant. The pellet was collected in PBS in the same way as was done for the first pellet. Thirty μl 1 M Tris/Cl, pH 7.4, 90 μl 4X SDS sample buffer and 36 μl 10X sample reducing agent were added to each of the extracted samples (containing 200 μl). To the original 100 μl aliquot of the starting material, half these volumes were added to maintain equivalent dilutions. Ten μl of the starting material and 20 μl of each extracted samples were analyzed by Western blot after SDS-PAGE through a 4–12% NuPAGE gel using the Bis-Tris buffer system and electro-blotting to nitrocellulose. Three transmembrane proteins; transferrin receptor, synaptotagmin 1 and aquaporin 1, and 3 non-transmembrane proteins; chromogranin A (β-granin), p115 and Grasp65 were analyzed. The blots were also probed for PC1 (N-terminal specific, ABR Inc., Golden, CO) in order to determine its pattern of extraction and to compare it to the patterns of the known transmembrane and non-transmembrane proteins listed above. Visualization of the proteins was by detection of secondary antibodies labeled with fluorophores that emit in the infra-red region of the spectrum using the Odyssey Infrared Imaging System (Licor Biosciences). 2.3 Generation and immunopurification of PC1 C-Terminal antibodies For the detection of the C-terminus of PC1, a new antibody was generated against the peptide DSEDSLYSDYVDVFYN, which is present within the C-terminus of PC1 (amino acid numbering D 714 -N 729 ). A cysteine residue was incorporated at the amino terminal to facilitate the coupling of the peptide to keyhole limpet hemocyanin (KLH). The synthesis of the peptide, coupling to KLH and generation of immune sera was performed under contract by Covance Research (Princeton, NJ). The antibodies were immunopurified as follows. Three mg of peptide (without KLH), dissolved in DMSO, were coupled to Affi-Gel 15 beads (Biorad, Hercules, CA) according to the manufacturer's protocol. The beads were loaded into a column (~1.5 ml bed volume) and prepared for affinity chromatography. Five ml of the PC1 C-terminal antiserum (#5450) were mixed with 5 ml PBS containing 0.1% Tween 20 (PBST) and added to the column. The flow through sample was re-applied 3 times after which the column was washed with 30 ml PBST. The bound antibodies were eluted with 0.9 ml aliquots of 0.1 M Glycine, pH 2.9 into eppendorf tubes containing 100 μl 1 M Tris/Cl buffer, pH 7.5. Analysis by SDS-PAGE under reducing conditions and Coomassie Blue staining of the eluted fractions verified the presence of 50 kDa and 25 kDa immunoglobulin bands at an apparent purity estimated at >95% (data not shown). The purified IgGs were pooled and concentrated by centrifugation through 50 kDa molecular mass cutoff membranes (Pall Filtron, Northborough, MA). The buffer was also replaced with PBS/0.1% sodium azide by diafiltration through the same membranes. The resultant sample of immuno-purified IgGs (165 μg/ml) was stored at 4°C. These IgGs were used for the immunoprecipitation (IP) and immunocytochemistry (ICC) experiments. 2.4 Characterization of the purified IgGs by immunoprecipitation of PC1 from AtT20 cells To demonstrate the specificity of the new C-terminal PC1 purified IgGs, an immunoprecipitation was performed using radio-labeled proteins from AtT20 cells. The cells were labeled for 24 h with a mixture of [ 35 S]-Met/[ 35 S]-Cys (100 μCi/ml). Following this, the cells were rinsed 3 times with ice cold PBS and then harvested in 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 2 mM EDTA (TNE buffer) containing 0.1% Triton X-100 and freshly prepared phenyl-methylsulfonyl fluoride (PMSF, 1 mM). The homogenate was centrifuged at 13,000 rpm for 10 min to sediment insoluble cellular material and a second extraction was performed on this pellet. The supernatants from both extractions were combined and incubated at 60°C for 20 min after addition of SDS to a final concentration of 0.1%. Following centrifugation of the sample (13,000 rpm, 10 min), Triton X-100 was added to the supernatant to a final concentration of 1%. The sample was pre-cleared by addition of protein A-sepharose beads (50 μl of a 50% slurry, 30 min, 4°C). After centrifugation to remove the beads, the supernatant was incubated with 1 μg of PC1 C-terminal immuno-purified IgGs or 1 μg of PC1 N-terminal immuno-purified IgGs for 18 h at 4°C. Antibody:antigen complexes were precipitated with 30 μl of protein-A sepharose beads and were washed extensively. The beads were resuspended in 1X tris-glycine SDS sample buffer containing β-mercaptoethanol, boiled for 10 min to elute the proteins and then analyzed by autoradiography after SDS-PAGE and electroblotting onto PVDF membrane. An additional gel was run later for the analysis of the IP supernatant. In that case, a 4–12% NuPAGE gel was used and the proteins transferred to nitrocellulose for autoradiography. 2.5 PC1 topology by immunocytochemistry It was demonstrated previously in purified bovine adrenal chromaffin granules that some PC1 could adopt a transmembrane orientation 1 . This suggested that some of the C-terminus of PC1 is localized on the cytosolic side of secretory granules in the cell. In order to investigate this, in situ , we employed a procedure that has previously been characterized 31 and one which we had been investigating independently. This procedure utilizes the observation that fixation of cells with para -formaldehyde (PFA) in PBS selectively permeabilizes the plasma membrane and allows access by immunoglobulins to the cytosolic space. Thus, cytosolic epitopes would be accessible for immunofluorescence microscopy in PFA/PBS fixed cells. On the other hand, proteins within the lumen of organelles, such as those found within the secretory pathway would not be accessible 31 . Using this procedure it is therefore possible to demonstrate the topology of a transmembrane protein within cells in situ if domain specific antibodies were available. AtT20 cells were grown in two-chambered glass slides, rinsed 3 times with room temperature (RT) PBS and then fixed in 2% PFA/PBS for 30 min at RT. One set of chambers was permeabilized by 0.25% Triton X-100/PBS for 5 min at RT while the other set received only PBS. After blocking with 1% bovine serum albumen (BSA)/PBS for 2 h at RT, the cells were then incubated for 16–20 h at 4°C with primary antiserum diluted as indicated in 1% BSA/PBS. Primary antibodies used were as follows; mouse anti-transferrin receptor (1:1,000, cytoplasmic epitope) (Invitrogen, Carlsbad, CA), mouse anti-p115 (1:1,000) (BD Biosciences, San Jose CA), rabbit anti-GRASP65 (1:2,000) (Proteus Biosciences, Ramona, CA), mouse anti-ACTH (1:1,000) (Abcam, Cambridge, MA), rabbit anti-PC1 (10 μg/ml, N-terminal specific) (Affinity Bioreagents Inc., Golden, CO) and rabbit anti-chromogranin A (1:10,000) 32 . The rabbit anti-PC1 (C-terminal specific) immuno-purified antibody, which was generated in our laboratory (see above), was used at a concentration of 1.6 μg/ml. This antibody was also used in combination with the mouse anti-p115 in a double labeling experiment. To demonstrate specificity, the C-terminal specific PC1 purified antibodies were pre-absorbed with the immunogenic peptide (1 μg/ml) and also used. After extensive washing with PBS, primary antibodies were detected with Alexa dye-conjugated secondary antibodies; goat anti-rabbit-568 (1:1,000) or goat anti-mouse-488 (1:1,000) from Molecular Probes (Invitrogen, Carlsbad, CA). All pictures were captured on an LSM 510 inverted scanning confocal microscope in the NICHD Microscopy and Imaging Core facility. For each antigen, power settings were optimized for the Triton X-100 (TX-100) treated cells until a clear, strong picture was obtained. These settings were then used to detect the same antigen in the non TX-100 treated cells, so that a direct comparison could be made between the staining intensities of the same antigen under the two conditions. Prohormone convertase 1 cDNA 29 , encoded in the mammalian expression vector, pcDNA3.1, was transfected into COS7 cells using Lipofectamine 2000 according to the manufacturer (Invitrogen). Forty-eight h after transfection, the cells were processed for ICC under TX-100 treated and untreated conditions and images captured as described above. 2.1 Cell culture AtT20 and COS7 cells were grown in high glucose DMEM containing 10% fetal bovine serum, 1X penicillin/streptomycin and 50 μg/ml normocin (Invitrogen, San Diego, CA) in an incubator maintained at 37°C and 5% CO 2 . For sodium carbonate extraction, AtT20 cells were rinsed 3 times with ice cold phosphate buffered saline (PBS), scraped and collected in PBS, sedimented by centrifugation (3,000 rpm for 3 min) and resuspended in a smaller volume of PBS. The cell suspension was dispensed into equal aliquots and stored at -30°C until used. 2.2 Sodium carbonate extraction A frozen aliquot of AtT20 cells was thawed on ice. One hundred μl of the cell suspension was saved immediately and 100 μl were placed into two airfuge tubes for centrifugation at 24 psi for 10 min, i.e. >100,000 × g (Airfuge, Beckman, Fullerton, CA). To block the non-specific binding of proteins to the plastic, the airfuge tubes were previously incubated on ice with 10% BSA for 30 min, after which they were rinsed 3 times with PBS. After centrifugation of the samples, the supernatants were removed and saved, and the pellets were resuspended in an additional 100 μl PBS each. The samples were centrifuged again and the resulting supernatants were combined with their original supernatants. To collect and save the pellet from the PBS extraction, the pellet from one tube was resuspended with 100 μl PBS and saved. The empty tube was rinsed once with 100 μl PBS and this was combined with the resuspended pellet. The other pellet was resuspended by vigorous pipetting with 100 μl of 0.1 M sodium carbonate, pH 11.5, and allowed to incubate on ice for 30 min. The sample was then centrifuged at 24 psi for 10 min and the resulting supernatant saved. The tube was rinsed carefully with 100 μl of fresh carbonate solution and this was added to the supernatant. The pellet was collected in PBS in the same way as was done for the first pellet. Thirty μl 1 M Tris/Cl, pH 7.4, 90 μl 4X SDS sample buffer and 36 μl 10X sample reducing agent were added to each of the extracted samples (containing 200 μl). To the original 100 μl aliquot of the starting material, half these volumes were added to maintain equivalent dilutions. Ten μl of the starting material and 20 μl of each extracted samples were analyzed by Western blot after SDS-PAGE through a 4–12% NuPAGE gel using the Bis-Tris buffer system and electro-blotting to nitrocellulose. Three transmembrane proteins; transferrin receptor, synaptotagmin 1 and aquaporin 1, and 3 non-transmembrane proteins; chromogranin A (β-granin), p115 and Grasp65 were analyzed. The blots were also probed for PC1 (N-terminal specific, ABR Inc., Golden, CO) in order to determine its pattern of extraction and to compare it to the patterns of the known transmembrane and non-transmembrane proteins listed above. Visualization of the proteins was by detection of secondary antibodies labeled with fluorophores that emit in the infra-red region of the spectrum using the Odyssey Infrared Imaging System (Licor Biosciences). 2.3 Generation and immunopurification of PC1 C-Terminal antibodies For the detection of the C-terminus of PC1, a new antibody was generated against the peptide DSEDSLYSDYVDVFYN, which is present within the C-terminus of PC1 (amino acid numbering D 714 -N 729 ). A cysteine residue was incorporated at the amino terminal to facilitate the coupling of the peptide to keyhole limpet hemocyanin (KLH). The synthesis of the peptide, coupling to KLH and generation of immune sera was performed under contract by Covance Research (Princeton, NJ). The antibodies were immunopurified as follows. Three mg of peptide (without KLH), dissolved in DMSO, were coupled to Affi-Gel 15 beads (Biorad, Hercules, CA) according to the manufacturer's protocol. The beads were loaded into a column (~1.5 ml bed volume) and prepared for affinity chromatography. Five ml of the PC1 C-terminal antiserum (#5450) were mixed with 5 ml PBS containing 0.1% Tween 20 (PBST) and added to the column. The flow through sample was re-applied 3 times after which the column was washed with 30 ml PBST. The bound antibodies were eluted with 0.9 ml aliquots of 0.1 M Glycine, pH 2.9 into eppendorf tubes containing 100 μl 1 M Tris/Cl buffer, pH 7.5. Analysis by SDS-PAGE under reducing conditions and Coomassie Blue staining of the eluted fractions verified the presence of 50 kDa and 25 kDa immunoglobulin bands at an apparent purity estimated at >95% (data not shown). The purified IgGs were pooled and concentrated by centrifugation through 50 kDa molecular mass cutoff membranes (Pall Filtron, Northborough, MA). The buffer was also replaced with PBS/0.1% sodium azide by diafiltration through the same membranes. The resultant sample of immuno-purified IgGs (165 μg/ml) was stored at 4°C. These IgGs were used for the immunoprecipitation (IP) and immunocytochemistry (ICC) experiments. 2.4 Characterization of the purified IgGs by immunoprecipitation of PC1 from AtT20 cells To demonstrate the specificity of the new C-terminal PC1 purified IgGs, an immunoprecipitation was performed using radio-labeled proteins from AtT20 cells. The cells were labeled for 24 h with a mixture of [ 35 S]-Met/[ 35 S]-Cys (100 μCi/ml). Following this, the cells were rinsed 3 times with ice cold PBS and then harvested in 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 2 mM EDTA (TNE buffer) containing 0.1% Triton X-100 and freshly prepared phenyl-methylsulfonyl fluoride (PMSF, 1 mM). The homogenate was centrifuged at 13,000 rpm for 10 min to sediment insoluble cellular material and a second extraction was performed on this pellet. The supernatants from both extractions were combined and incubated at 60°C for 20 min after addition of SDS to a final concentration of 0.1%. Following centrifugation of the sample (13,000 rpm, 10 min), Triton X-100 was added to the supernatant to a final concentration of 1%. The sample was pre-cleared by addition of protein A-sepharose beads (50 μl of a 50% slurry, 30 min, 4°C). After centrifugation to remove the beads, the supernatant was incubated with 1 μg of PC1 C-terminal immuno-purified IgGs or 1 μg of PC1 N-terminal immuno-purified IgGs for 18 h at 4°C. Antibody:antigen complexes were precipitated with 30 μl of protein-A sepharose beads and were washed extensively. The beads were resuspended in 1X tris-glycine SDS sample buffer containing β-mercaptoethanol, boiled for 10 min to elute the proteins and then analyzed by autoradiography after SDS-PAGE and electroblotting onto PVDF membrane. An additional gel was run later for the analysis of the IP supernatant. In that case, a 4–12% NuPAGE gel was used and the proteins transferred to nitrocellulose for autoradiography. 2.5 PC1 topology by immunocytochemistry It was demonstrated previously in purified bovine adrenal chromaffin granules that some PC1 could adopt a transmembrane orientation 1 . This suggested that some of the C-terminus of PC1 is localized on the cytosolic side of secretory granules in the cell. In order to investigate this, in situ , we employed a procedure that has previously been characterized 31 and one which we had been investigating independently. This procedure utilizes the observation that fixation of cells with para -formaldehyde (PFA) in PBS selectively permeabilizes the plasma membrane and allows access by immunoglobulins to the cytosolic space. Thus, cytosolic epitopes would be accessible for immunofluorescence microscopy in PFA/PBS fixed cells. On the other hand, proteins within the lumen of organelles, such as those found within the secretory pathway would not be accessible 31 . Using this procedure it is therefore possible to demonstrate the topology of a transmembrane protein within cells in situ if domain specific antibodies were available. AtT20 cells were grown in two-chambered glass slides, rinsed 3 times with room temperature (RT) PBS and then fixed in 2% PFA/PBS for 30 min at RT. One set of chambers was permeabilized by 0.25% Triton X-100/PBS for 5 min at RT while the other set received only PBS. After blocking with 1% bovine serum albumen (BSA)/PBS for 2 h at RT, the cells were then incubated for 16–20 h at 4°C with primary antiserum diluted as indicated in 1% BSA/PBS. Primary antibodies used were as follows; mouse anti-transferrin receptor (1:1,000, cytoplasmic epitope) (Invitrogen, Carlsbad, CA), mouse anti-p115 (1:1,000) (BD Biosciences, San Jose CA), rabbit anti-GRASP65 (1:2,000) (Proteus Biosciences, Ramona, CA), mouse anti-ACTH (1:1,000) (Abcam, Cambridge, MA), rabbit anti-PC1 (10 μg/ml, N-terminal specific) (Affinity Bioreagents Inc., Golden, CO) and rabbit anti-chromogranin A (1:10,000) 32 . The rabbit anti-PC1 (C-terminal specific) immuno-purified antibody, which was generated in our laboratory (see above), was used at a concentration of 1.6 μg/ml. This antibody was also used in combination with the mouse anti-p115 in a double labeling experiment. To demonstrate specificity, the C-terminal specific PC1 purified antibodies were pre-absorbed with the immunogenic peptide (1 μg/ml) and also used. After extensive washing with PBS, primary antibodies were detected with Alexa dye-conjugated secondary antibodies; goat anti-rabbit-568 (1:1,000) or goat anti-mouse-488 (1:1,000) from Molecular Probes (Invitrogen, Carlsbad, CA). All pictures were captured on an LSM 510 inverted scanning confocal microscope in the NICHD Microscopy and Imaging Core facility. For each antigen, power settings were optimized for the Triton X-100 (TX-100) treated cells until a clear, strong picture was obtained. These settings were then used to detect the same antigen in the non TX-100 treated cells, so that a direct comparison could be made between the staining intensities of the same antigen under the two conditions. Prohormone convertase 1 cDNA 29 , encoded in the mammalian expression vector, pcDNA3.1, was transfected into COS7 cells using Lipofectamine 2000 according to the manufacturer (Invitrogen). Forty-eight h after transfection, the cells were processed for ICC under TX-100 treated and untreated conditions and images captured as described above. 3. Results 3.1 Sodium carbonate extraction Three transmembrane proteins; transferrin receptor, aquaporin 1 and synaptotagmin 1, were studied as a set of control proteins for the classical procedure of alkaline sodium carbonate extraction. All 3 of these proteins were predominantly recovered in the sodium carbonate pellet indicative of their resistance to extraction by this procedure ( Figure 1 ). Three non-transmembrane proteins were also studied as another set of control proteins for this procedure. These proteins, β-actin, chromogranin A (or β-granin when processed) and p115, a protein peripherally associated with the cytoplasmic side of the Golgi 33 , were recovered in the PBS supernatant ( Figure 1 ). Residual levels of these proteins that were recovered in the PBS pellet were subsequently extracted in the sodium carbonate supernatant, demonstrating, as non-transmembrane proteins, their susceptibility to extraction by this procedure. These 6 proteins were established as positive and negative controls for the carbonate extraction procedure of AtT20 cells and all volumes were maintained equivalently so that a direct relative quantification of the proteins recovered at each step could be obtained when compared to the starting material. The distribution of PC1 was also analyzed using an N-terminal specific PC1 antibody. It was found that the 64 kDa form of PC1 was predominantly recovered in the PBS supernatant whereas the 87 kDa form was recovered in the PBS pellet. The 87 kDa form was subsequently recovered predominantly in the sodium carbonate pellet along with a small amount of the 64 kDa form ( Figure 1 ). Figure 1. Carbonate extraction of AtT20 cells. AtT20 cells were subjected to extraction by PBS followed by 0.1 M sodium carbonate, pH 11.5. Equivalent volumes from each step were analyzed by Western blot. Three TM proteins (transferrin receptor (TfR), synaptotagmin 1 (Syt-1) and aquaporin 1 (AQP-1) and 3 non-TM proteins (β-actin, β-granin and p115) were analyzed as controls. The 3 TM proteins were recovered in the sodium carbonate pellet while the 3 non-TM proteins were predominantly recovered in the PBS supernantant. The distribution of PC1 was also analyzed. The 64 kDa form was predominantly recovered in the PBS supernatant while the 87 kDa form (and a small amount of the 64 kDa form) was predominantly recovered in the carbonate pellet. This suggested that the 87 kDa form and some of the 64 kDa form of PC1 have properties consistent with a TM protein. T, total; S, supernatant; P, pellet. 3.2 Immunoprecipitation of PC1 from AtT20 cells Under steady state conditions, 2 forms of PC1 are found in AtT20 cells, an 87 kDa form and a 64 kDa form, both of which have an identical N-terminus. Immunoprecipitation by the N-terminal specific IgGs resulted in the capture of both these forms ( Figure 2 , N-term). When the C-terminal specific IgGs were used, one major band was captured consistent with being the 87 kDa PC1 form as it co-migrated with the 87 kDa form captured by the N-terminal specific IgGs ( Figure 2 , C-term). A faint band, with an apparent molecular mass of ~20 kDa based on the molecular mass standards (SeeBlue Plus 2, Invitrogen) was also seen. This band was considered likely to be the processed carboxyl terminus of PC1 since it was only present in the C-terminal specific IP lane and it has the same molecular mass as a previously expressed form of the C-terminal domain of mouse PC1 34 . Western blot analysis of a subsequent IP of unlabeled AtT20 cells (both carried out with the PC1 C-terminal IgGs), failed to show such a PC1 immunoreactive protein (data not shown), indicating that it's levels were too low for Western blot detection (compared to the radiolabeled band) or was possibly a protein that co-immunoprecipitated with the 87 kDa form of PC1. Figure 2. Immunoprecipitation of PC1 from AtT20 cells. To demonstrate the specificity of the immuno-purified PC1 C-terminal specific IgGs, an immunoprecipitation (IP) was performed on radiolabeled AtT20 cells. From a multitude of labeled proteins (Sup lane), one major band at 87 kDa was immunoprecipitated with these IgGs (C-term lane). As a control, immunoprecipitation with N-terminal specific PC1 IgGs yielded the two expected bands of 87 kDa and 64 kDa PC1 (N-term lane). A faint band at ~20 kDa was deemed non-specific as it was not immuno-reactive with the C-terminal specific IgGs in a Western blot of a subsequent IP of unlabeled AtT20 cell lysate (data not shown). 3.3 PC1 topology by immunocytochemistry To demonstrate that PFA fixation selectively permeabilizes the plasma membrane, we performed ICC on TX-100 treated and non-treated AtT20 cells and analyzed the staining pattern of 6 endogenous proteins; 3 with epitopes localized in the cytosol and 3 proteins localized within the lumen of organelles belonging to the regulated secretory pathway (RSP) which includes the Golgi and secretory granules. For all 3 lumenal proteins, CgA, ACTH and the N-terminus of PC1, strong staining of the Golgi and tips of the processes were observed only in the TX-100 treated cells consistent with their presence in the RSP ( Figure 3A-C ). In the absence of TX-100, no staining of these proteins could be detected ( Figure 3D-F ), demonstrating the requirement for the detergent to expose these proteins to the antibodies by permeabilizing the membranes of the organelles. For the 3 proteins with cytosolic epitopes, p115, Grasp 65 and transferrin receptor, strong immuno-staining was observed whether TX-100 was used or not ( Figure 4 ). Thus PFA fixation allows accessibility of the IgGs to the cytosol, where they can bind their antigens, but not to the lumen of the organelles of the RSP. Figure 3. Immunocytochemical analysis of RSP luminal proteins in AtT20 cells. AtT20 cells were chemically fixed with 2% PFA/PBS and then treated with and without the detergent, Triton X-100. Three luminal proteins belonging to the RSP (Chromogranin A, ACTH and the N-terminus of PC1) were stained by indirect immunofluorescence. For all three proteins, the Golgi (arrows) and the tips of the processes (arrow heads) were specifically stained when Triton X-100 was used ( A – C ). No staining was seen when Triton X-100 was not used ( D – F ). This staining pattern is consistent with the presence of these proteins within the organelles of the RSP and demonstrates that PFA fixation does not cause an access of the antibodies to these compartments. Bar 20 μm Figure 4. Immunocytochemical analysis of cytosolic proteins in AtT20 cells. AtT20 cells were chemically fixed with 2% PFA/PBS and then treated with and without the detergent, Triton X-100. Three cytosolic proteins (p115, Grasp65 and N-terminus of the transferrin receptor) were stained by indirect immunofluorescence. For p115 ( A , D ) and Grasp65 ( B , E ), staining of the Golgi area was observed and for the transferrin receptor ( C , F ), staining of the plasma membrane/endosomes were observed, whether the cells were treated with Triton X-100 or not. This demonstrated that the antibodies had access to the cytosol even with only PFA fixation. Bar 20 μm Using this procedure with the C-terminal specific immuno-purified PC1 antibodies, a pattern of staining for PC1 was observed in the TX-100 treated cells that was similar to that of CgA, ACTH and the N-terminal specific PC1 antibodies, i.e. strong staining of the Golgi and a punctate pattern in the processes ( Figure 5 , top panel, PC1). The staining pattern exhibited by these purified antibodies is consistent with the localization of PC1 in the Golgi, as evidenced by its colocalization with p115 ( Figure 5 , top panel, p115 and Merged) and secretory granules of the RSP. No staining could be seen when immuno-purified C-terminal PC1 IgGs were used that had been pre-absorbed by the antigenic peptide ( Figure 5 , Absorption control). In the absence of TX-100, however, while staining with the N-terminal specific PC1 antibodies was negative ( Figure 3F ); staining of the Golgi and processes was observed in the untreated cells with the C-terminal specific IgGs ( Figure 5 , lower panel, PC1). This pattern of staining indicated that the C-terminus of PC1 is present in the cytosol and the N-terminus of PC1 is in the lumen of the Golgi and secretory granules, indicating that at least some PC1 is in a transmembrane orientation in situ and supports the results of the extraction experiments ( Figure 1 ). Figure 5. Immunocytochemical analysis of PC1 C-terminus in AtT20 cells. AtT20 cells were chemically fixed with 2% PFA/PBS and then treated with and without the detergent, Triton X-100. PC1 was stained with C-terminal specific immunopurifed IgGs by indirect immunofluorescence. In the Triton X-100 treated cells, the staining pattern of the C-terminus of PC1 was similar to that of the N-terminus of PC1 described in Figure 3 , in that Golgi staining (arrows) was observed (top panel). In the Triton X-100 untreated cells, however, a reduced but similar staining pattern was observed to that of the Triton X-100 treated cells (lower panel). This demonstrates that some of the C-terminus of PC1 was localized in the cytosol. Bar 20 μm. The topology of PC1 transfected into non-endocrine COS7 cells was assessed also by this procedure. After fixation by PFA, the C-terminus of PC1 was strongly stained by the C-terminus specific purified IgGs only after permeabilization with TX-100 ( Figure 6A ). This result indicated that PC1 did not assume a TM orientation in COS7 cells consistent with the results of Stettler et al. in COS1 cells 30 . Figure 6. Immunocytochemical analysis of PC1 C-terminus in COS7 cells. COS7 cells, expressing transfected full length PC1, were chemically fixed with 2% PFA/PBS and then treated with and without the detergent, Triton X-100. PC1 was stained with C-terminal specific immunopurifed IgGs by indirect immunofluorescence. In the Triton X-100 treated cells, a strong staining pattern of the C-terminus of PC1 was observed in the transfected cells consistent with a distribution in the reticular network of the ER and in the Golgi. ( A ) Only a low level of background staining was observed for the TX-100 untreated cells ( B ). Bar 20 μM. 3.1 Sodium carbonate extraction Three transmembrane proteins; transferrin receptor, aquaporin 1 and synaptotagmin 1, were studied as a set of control proteins for the classical procedure of alkaline sodium carbonate extraction. All 3 of these proteins were predominantly recovered in the sodium carbonate pellet indicative of their resistance to extraction by this procedure ( Figure 1 ). Three non-transmembrane proteins were also studied as another set of control proteins for this procedure. These proteins, β-actin, chromogranin A (or β-granin when processed) and p115, a protein peripherally associated with the cytoplasmic side of the Golgi 33 , were recovered in the PBS supernatant ( Figure 1 ). Residual levels of these proteins that were recovered in the PBS pellet were subsequently extracted in the sodium carbonate supernatant, demonstrating, as non-transmembrane proteins, their susceptibility to extraction by this procedure. These 6 proteins were established as positive and negative controls for the carbonate extraction procedure of AtT20 cells and all volumes were maintained equivalently so that a direct relative quantification of the proteins recovered at each step could be obtained when compared to the starting material. The distribution of PC1 was also analyzed using an N-terminal specific PC1 antibody. It was found that the 64 kDa form of PC1 was predominantly recovered in the PBS supernatant whereas the 87 kDa form was recovered in the PBS pellet. The 87 kDa form was subsequently recovered predominantly in the sodium carbonate pellet along with a small amount of the 64 kDa form ( Figure 1 ). Figure 1. Carbonate extraction of AtT20 cells. AtT20 cells were subjected to extraction by PBS followed by 0.1 M sodium carbonate, pH 11.5. Equivalent volumes from each step were analyzed by Western blot. Three TM proteins (transferrin receptor (TfR), synaptotagmin 1 (Syt-1) and aquaporin 1 (AQP-1) and 3 non-TM proteins (β-actin, β-granin and p115) were analyzed as controls. The 3 TM proteins were recovered in the sodium carbonate pellet while the 3 non-TM proteins were predominantly recovered in the PBS supernantant. The distribution of PC1 was also analyzed. The 64 kDa form was predominantly recovered in the PBS supernatant while the 87 kDa form (and a small amount of the 64 kDa form) was predominantly recovered in the carbonate pellet. This suggested that the 87 kDa form and some of the 64 kDa form of PC1 have properties consistent with a TM protein. T, total; S, supernatant; P, pellet. 3.2 Immunoprecipitation of PC1 from AtT20 cells Under steady state conditions, 2 forms of PC1 are found in AtT20 cells, an 87 kDa form and a 64 kDa form, both of which have an identical N-terminus. Immunoprecipitation by the N-terminal specific IgGs resulted in the capture of both these forms ( Figure 2 , N-term). When the C-terminal specific IgGs were used, one major band was captured consistent with being the 87 kDa PC1 form as it co-migrated with the 87 kDa form captured by the N-terminal specific IgGs ( Figure 2 , C-term). A faint band, with an apparent molecular mass of ~20 kDa based on the molecular mass standards (SeeBlue Plus 2, Invitrogen) was also seen. This band was considered likely to be the processed carboxyl terminus of PC1 since it was only present in the C-terminal specific IP lane and it has the same molecular mass as a previously expressed form of the C-terminal domain of mouse PC1 34 . Western blot analysis of a subsequent IP of unlabeled AtT20 cells (both carried out with the PC1 C-terminal IgGs), failed to show such a PC1 immunoreactive protein (data not shown), indicating that it's levels were too low for Western blot detection (compared to the radiolabeled band) or was possibly a protein that co-immunoprecipitated with the 87 kDa form of PC1. Figure 2. Immunoprecipitation of PC1 from AtT20 cells. To demonstrate the specificity of the immuno-purified PC1 C-terminal specific IgGs, an immunoprecipitation (IP) was performed on radiolabeled AtT20 cells. From a multitude of labeled proteins (Sup lane), one major band at 87 kDa was immunoprecipitated with these IgGs (C-term lane). As a control, immunoprecipitation with N-terminal specific PC1 IgGs yielded the two expected bands of 87 kDa and 64 kDa PC1 (N-term lane). A faint band at ~20 kDa was deemed non-specific as it was not immuno-reactive with the C-terminal specific IgGs in a Western blot of a subsequent IP of unlabeled AtT20 cell lysate (data not shown). 3.3 PC1 topology by immunocytochemistry To demonstrate that PFA fixation selectively permeabilizes the plasma membrane, we performed ICC on TX-100 treated and non-treated AtT20 cells and analyzed the staining pattern of 6 endogenous proteins; 3 with epitopes localized in the cytosol and 3 proteins localized within the lumen of organelles belonging to the regulated secretory pathway (RSP) which includes the Golgi and secretory granules. For all 3 lumenal proteins, CgA, ACTH and the N-terminus of PC1, strong staining of the Golgi and tips of the processes were observed only in the TX-100 treated cells consistent with their presence in the RSP ( Figure 3A-C ). In the absence of TX-100, no staining of these proteins could be detected ( Figure 3D-F ), demonstrating the requirement for the detergent to expose these proteins to the antibodies by permeabilizing the membranes of the organelles. For the 3 proteins with cytosolic epitopes, p115, Grasp 65 and transferrin receptor, strong immuno-staining was observed whether TX-100 was used or not ( Figure 4 ). Thus PFA fixation allows accessibility of the IgGs to the cytosol, where they can bind their antigens, but not to the lumen of the organelles of the RSP. Figure 3. Immunocytochemical analysis of RSP luminal proteins in AtT20 cells. AtT20 cells were chemically fixed with 2% PFA/PBS and then treated with and without the detergent, Triton X-100. Three luminal proteins belonging to the RSP (Chromogranin A, ACTH and the N-terminus of PC1) were stained by indirect immunofluorescence. For all three proteins, the Golgi (arrows) and the tips of the processes (arrow heads) were specifically stained when Triton X-100 was used ( A – C ). No staining was seen when Triton X-100 was not used ( D – F ). This staining pattern is consistent with the presence of these proteins within the organelles of the RSP and demonstrates that PFA fixation does not cause an access of the antibodies to these compartments. Bar 20 μm Figure 4. Immunocytochemical analysis of cytosolic proteins in AtT20 cells. AtT20 cells were chemically fixed with 2% PFA/PBS and then treated with and without the detergent, Triton X-100. Three cytosolic proteins (p115, Grasp65 and N-terminus of the transferrin receptor) were stained by indirect immunofluorescence. For p115 ( A , D ) and Grasp65 ( B , E ), staining of the Golgi area was observed and for the transferrin receptor ( C , F ), staining of the plasma membrane/endosomes were observed, whether the cells were treated with Triton X-100 or not. This demonstrated that the antibodies had access to the cytosol even with only PFA fixation. Bar 20 μm Using this procedure with the C-terminal specific immuno-purified PC1 antibodies, a pattern of staining for PC1 was observed in the TX-100 treated cells that was similar to that of CgA, ACTH and the N-terminal specific PC1 antibodies, i.e. strong staining of the Golgi and a punctate pattern in the processes ( Figure 5 , top panel, PC1). The staining pattern exhibited by these purified antibodies is consistent with the localization of PC1 in the Golgi, as evidenced by its colocalization with p115 ( Figure 5 , top panel, p115 and Merged) and secretory granules of the RSP. No staining could be seen when immuno-purified C-terminal PC1 IgGs were used that had been pre-absorbed by the antigenic peptide ( Figure 5 , Absorption control). In the absence of TX-100, however, while staining with the N-terminal specific PC1 antibodies was negative ( Figure 3F ); staining of the Golgi and processes was observed in the untreated cells with the C-terminal specific IgGs ( Figure 5 , lower panel, PC1). This pattern of staining indicated that the C-terminus of PC1 is present in the cytosol and the N-terminus of PC1 is in the lumen of the Golgi and secretory granules, indicating that at least some PC1 is in a transmembrane orientation in situ and supports the results of the extraction experiments ( Figure 1 ). Figure 5. Immunocytochemical analysis of PC1 C-terminus in AtT20 cells. AtT20 cells were chemically fixed with 2% PFA/PBS and then treated with and without the detergent, Triton X-100. PC1 was stained with C-terminal specific immunopurifed IgGs by indirect immunofluorescence. In the Triton X-100 treated cells, the staining pattern of the C-terminus of PC1 was similar to that of the N-terminus of PC1 described in Figure 3 , in that Golgi staining (arrows) was observed (top panel). In the Triton X-100 untreated cells, however, a reduced but similar staining pattern was observed to that of the Triton X-100 treated cells (lower panel). This demonstrates that some of the C-terminus of PC1 was localized in the cytosol. Bar 20 μm. The topology of PC1 transfected into non-endocrine COS7 cells was assessed also by this procedure. After fixation by PFA, the C-terminus of PC1 was strongly stained by the C-terminus specific purified IgGs only after permeabilization with TX-100 ( Figure 6A ). This result indicated that PC1 did not assume a TM orientation in COS7 cells consistent with the results of Stettler et al. in COS1 cells 30 . Figure 6. Immunocytochemical analysis of PC1 C-terminus in COS7 cells. COS7 cells, expressing transfected full length PC1, were chemically fixed with 2% PFA/PBS and then treated with and without the detergent, Triton X-100. PC1 was stained with C-terminal specific immunopurifed IgGs by indirect immunofluorescence. In the Triton X-100 treated cells, a strong staining pattern of the C-terminus of PC1 was observed in the transfected cells consistent with a distribution in the reticular network of the ER and in the Golgi. ( A ) Only a low level of background staining was observed for the TX-100 untreated cells ( B ). Bar 20 μM. 4. Discussion Prohormone convertase 1 (PC1) is sorted to the regulated secretory pathway (RSP) of (neuro)endocrine cells where it functions to cleave prohormones and proneuropeptides into smaller peptides that ultimately function in important biological processes. How PC1 is sorted to the RSP has been actively studied and several proposals have been put forward. A commonality among these ideas is the belief that association of the C-terminal tail of PC1 with components of the trans-Golgi network (TGN) membrane, where sorting to the RSP is believed to be initiated, must occur, although binding via the prodomain has also been implicated 35 . In light of the extraction and/or binding studies by Hill et al. 21 , Arnaoutova et al. 1 and Jutras et al. 25 , it is considered that such binding or membrane association is quite strong. Without evidence of amino acid sequences that predict a classical transmembrane (TM) domain similar to furin and Kex2, and with the previously identified membrane binding amphipathic α-helices within the C-terminus 25 , it is reasonable to expect that binding would be to the luminal side of the TGN membrane in a non-TM manner and that such binding would be necessary for sorting to the RSP. However, we have previously studied the membrane association properties of carboxypeptidase E (CPE) that contains an amphipathic α-helix at its C-terminus 36 , 37 and demonstrated that it can assume a transmembrane topology in a lipid raft dependent manner. Indeed, live cell imaging demonstrated a role of its cytoplasmic tail in peptide hormone granule transport via interaction with dynactin and the microtubule dependent motor proteins, kinesin and cytoplasmic dynein 38 , 39 . Hence, based on these and other observations of the novel TM behavior of CPE and PC2 via their C-termini 37 , 40 , we speculated that lipid raft-association and TM orientation of PC1 might occur through a similar C-terminal domain that was identified in PC1. Thus, in our previous work we tested this idea principally in intact purified, bovine adrenal medulla chromaffin granules and showed the presence of the C-terminus of PC1 on the outside of these granules. In such a case the cytosolic C-terminus would be quite long (~114 aa). To explain how this might occur, we speculated that, since insertion through the membrane in the Golgi or post-Golgi compartment would be energetically unlikely, PC1 might be synthesized as a TM protein 1 . Our speculations, however, have been challenged by Stettler et al. who showed by various methods that transfected PC1 is not synthesized as a TM protein in COS1 cells 30 . The results of Stettler et al. were important for two reasons. One, it provided information about the initial synthesis of PC1, albeit in a non-endocrine cell, and two; it questioned whether PC1 was a TM protein at all, because the data by Arnaoutova et al. which demonstrated it to be a TM protein in intact purified granules, was discounted by Stettler et al. simply as the result of contamination. While this is a valid point, we do not consider it to be probable because, had there been even a small amount of contamination, chromogranin A, the most abundant protein in chromaffin granules (47% of soluble granule content, 7% of total adrenal medulla content 41 , would, like PC1, also have been biotinylated when the purified granules were used in the biotinylation experiment. The observation that PC1 was biotinylated and CgA was not provided very strong evidence that PC1 was there in a transmembrane orientation rather than by contamination from ruptured organelles. Regardless of this explanation and to readdress the issue of PC1 topology, we undertook to analyze endogenously expressed PC1 in a model endocrine cell line where both forms are known to exist at steady state. Initial extractions by sodium carbonate, pH 11.5, a classical procedure for the characterization of TM proteins initially described by Fujiki et al. 42 , suggested that the 87 kDa form (and some of the 64 kDa form) of PC1 had properties of a TM protein because its partitioning behavior was similar to three known TM proteins ( Figure 1 ). These results are consistent with the previously published data on PC1 from bovine chromaffin granules 1 and indicated to us that PC1 in AtT20 cells had similar properties to PC1 found in chromaffin cells from bovine adrenal medulla. While resistance to alkaline sodium carbonate extraction is not definitive proof of TM orientation, it is considered to be strong evidence for such a conclusion. To investigate this further we studied PC1 topology in AtT20 cells under steady state conditions in situ by immunofluorescence confocal microscopy. This simple but powerful procedure is based on the observation that fixing cells for immunocytochemistry (ICC) with para -formaldehyde (PFA) in PBS, permeabilizes the plasma membrane sufficiently to allow immunoglobulins (IgGs) into the cytosol 31 . However, since PFA/PBS does not have the same effect on membranes of internal organelles, we can determine the topology of organellar proteins if domain specific IgGs are available. To demonstrate the validity of this technique in AtT20 cells, we performed ICC on PFA fixed cells with and without detergent permeabilization and analyzed the staining of 3 known cytosolic proteins (Grasp65, p115 and the N-terminus of the transferrin receptor) and 3 known luminal proteins belonging to the regulated secretory pathway (RSP) (ACTH, CgA and the N-terminus of PC1). As expected, the RSP proteins were only stained when the cells were permeabilized with the detergent, Triton X-100 (TX-100) demonstrating the integrity of the membranes of the internal organelles ( Figure 3 ). The cytosolic proteins were stained whether TX-100 was used or not ( Figure 4 ) demonstrating that the antibodies had access to the cytosol even in the absence of detergent treatment. Staining of the C-terminus of PC1 with our immuno-purified IgGs gave a similar pattern of staining to that of the PC1 N-terminal specific IgGs when performed on TX-100 treated cells. In the absence of TX-100, however, reduced but specific staining with the C-terminal specific IgGs was also observed indicating that some of the C-terminus of PC1 was present in the cytosol ( Figure 5 ). Golgi staining of the C-terminus was observed (as demonstrated by its colocalization with p115) as well as punctate staining in the processes, indicative of granules, suggests that PC1 (or some of it) can be in a TM orientation in the Golgi and granules. How this happens is currently unknown. However, assuming that the results observed by Stettler et al. in the COS1 cells are similar for the synthesis of endogenous PC1 in classical (neuro)endocrine tissue/cell lines, we are now directed to re-consider the possibility that an insertion event might be taking place after synthesis in the ER. Although the sequence identified as the TM domain (aa619–638) 1 , 29 , 43 does not have classical TM characteristics, it is conceivable that one or several factors that are still unknown may facilitate or stabilize PC1 in such an insertion/orientation, thus reducing the free energy necessary for such an event. An example of a "helper" protein exists for the diphtheria toxin where insertion into model membranes as a TM protein only occurs in the presence of molten globule-like proteins 44 . In addition, while it is known that C-terminal tail-anchored proteins (TA proteins, e.g. cytochrome b5) are TM proteins, the mechanism by which membrane insertion of these cytosolic proteins occurs is still unknown as it is independent of the Sec61 translocon 45 . It is probable that not all of the PC1 becomes TM, in which case an equilibrium may exist between luminal/peripheral and TM partitioning and that this equilibrium may depend on levels of endogenous factors as indicated above. Indeed a more intense signal of the PC1 C-terminal signal in the granules was observed in the TX-100 treated cells ( Figure 5 , top panel versus lower panel) indicating the presence of the C-terminus within the lumen of the granules presumably as a cleaved product. The concept of "helper" proteins is also supported by our observations that when we transfected PC1 into COS7 cells ( Figure 6 ) or PC12 cells (a model neuroendocrine cell line) (data not shown) and performed the ICC experiment we could only observe specific PC1 C-terminal staining when TX-100 was used indicating that in transfected cells the PC1 did not measurably adopt a TM orientation. This suggested to us that TM insertion is a saturable process that appears to require components that are limiting in (neuro)endocrine cells or not present in non-endocrine cells. Studies are underway to identify such components.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886983/

Mechanisms of Effector-Mediated Immunity Revealed by the Accidental Human Pathogen Legionella pneumophila

Many Gram-negative bacterial pathogens employ translocated virulence factors, termed effector proteins, to facilitate their parasitism of host cells and evade host anti-microbial defenses. However, eukaryotes have evolved to detect effector-mediated virulence strategies through a phenomenon termed effector-triggered immunity (ETI). Although ETI was discovered in plants, a growing body of literature demonstrates that metazoans also utilize effector-mediated immunity to detect and clear bacterial pathogens. This mini review is focused on mechanisms of effector-mediated immune responses by the accidental human pathogen Legionella pneumophila . We highlight recent advancements in the field and discuss the future prospects of harnessing effectors for the development of novel therapeutics, a critical need due to the prevalence and rapid spread of antibiotic resistance. Effector-Mediated Immunity Enhances Host Defense Against Bacterial Pathogens The evolutionary arms race between host and pathogen has necessitated the use of several complementary innate immune pathways to detect and eradicate pathogens. Initial pathogen recognition occurs through engagement of pathogen associated molecular patterns (PAMPs) by host pattern recognition receptors (PRRs) ( Janeway, 1989 ). PRRs include toll-like-receptors (TLRs), located on either the plasma membrane or endosomal membranes ( Medzhitov and Janeway, 2000 ; Massis and Zamboni, 2011 ). PRR recognition of PAMPs activates signaling cascades that culminate in production of pro-inflammatory cytokines that contribute to controlling infection ( Janeway and Medzhitov, 2002 ). Inflammasomes are multimeric intracellular protein complexes that activate inflammatory caspases in response to cellular damage or pathogen infection [reviewed in ( Martinon et al., 2002 )]. Bacterial pathogens have evolved diverse repertoires of virulence factors to promote their survival within hosts by acquisition of host-derived nutrients and avoidance of host defenses. Bacterial effectors are directly injected into host cells through specialized secretion systems and functions within the host cells to facilitate pathogen survival in close association with host cells ( Cambronne and Roy, 2006 ; Galán, 2009 ). Both intracellular and extracellular pathogens utilize effector proteins, emphasizing their importance in the virulence strategies of diverse bacterial pathogens. Multiple effector-mediated virulence processes are similar between seemingly diverse bacterial pathogens. Multicellular eukaryotes are able to detect bacterial effectors and/or their virulence processes via effector-triggered immunity (ETI) ( Stuart et al., 2013 ; Rajamuthiah and Mylonakis, 2014 ; Fischer et al., 2019 ). ETI was first described in immune defense against pathogens in plants as "gene-for-gene resistance" where resistance (R) genes in plants recognize bacterial effectors (Avr) within the plant cell and trigger an immune response ( Flor, 1971 ; Chisholm et al., 2006 ). However, animals also detect pathogen infection through ETI and effector-mediated responses [reviewed in ( Fischer et al., 2019 )]. Plant ETI results from either direct recognition of the effector itself or sensing of intracellular effector activity, whereas only the latter has been observed in metazoans ( Stuart et al., 2013 ). In plants, several models have been described of how resistant strains directly or indirectly detect pathogen effectors. The "receptor-ligand model" describes direct recognition of bacterial effectors whereby a host R protein binds and inactivates a bacterial Avr effector ( Stuart et al., 2013 ). The Pseudomonas syringae effector AvrPto blocks host pathogen recognition through subversion of receptor-mediated signaling. In resistant plants, AvrPto is directly inactivated by the R protein, Pto ( Xiang et al., 2008 ). The "guard hypothesis," "decoy model," and "bait-and-switch model" are indirect models through which resistant plants detect pathogen effector function ( Dangl and Jones, 2001 ; Stuart et al., 2013 ). In animal cells, effector function is detected indirectly though cell-autonomous sensing of homeostatic perturbations elicited by the effectors to the benefit of the pathogen ( Colaço and Moita, 2016 ). Examples include cellular detection of effector-mediated translation inhibition, inhibition of Rho GTPases, and pore formation ( Fischer et al., 2019 ). This mini review is focused specifically on mechanisms of effector-mediated and -triggered host defense against the accidental human pathogen Legionella pneumophila. Use of the "Accidental" Pathogen L. pneumophila as a Model to Understand Effector-Mediated Immune Responses Several mechanisms of effector-mediated immunity have been uncovered by studying the accidental human pathogen Legionella pneumophila . Legionella spp. are Gram-negative intracellular bacteria that are ubiquitous in aquatic and soil environments, where they parasitize free-living protozoa ( Rowbotham, 1980 ; Fliermans et al., 1981 ; Barbaree et al., 1986 ). Anthropomorphic fresh-water environments such as cooling towers, water fountains and any system that allows for aerosolization of water droplets, have potential to be the source of Legionella infection, collectively termed legionellosis ( Barbaree et al., 1986 ). Inhalation or aspiration of L. pneumophila can result in an inflammatory pneumonia called Legionnaires' disease, which is fatal in ~10% of cases ( Soda et al., 2017 ). Legionnaires' disease primarily affects elderly and immunocompromised individuals and was named for the initial outbreak, which occurred at the 1976 American Legion Convention in Philadelphia ( Fraser et al., 1977 ; McDade et al., 1980 ). In immunocompetent individuals, L. pneumophila can cause a mild self-limiting flu-like illness called Pontiac Fever ( Glick et al., 1978 ). Legionellosis is a consequence of L. pneumophila replication within alveolar macrophages ( Nash et al., 1984 ; Friedman et al., 2002 ); however, the infection is readily cleared by innate immune responses in vivo , owing in part to orchestrated production of pro-inflammatory cytokines ( Shin, 2012 ; Liu et al., 2020 ). The opportunistic colonization of built freshwater environments, rarity of person-to-person transmission and susceptibility to innate immune responses has led to description of L. pneumophila as an "accidental pathogen" ( Borges et al., 2016 ; Boamah et al., 2017 ). Virulence strategies evolved by L. pneumophila to parasitize free-living protozoa have conferred the ability to replicate within mammalian macrophages ( Park et al., 2020 ). Upon phagocytosis, L. pneumophila rapidly remodels its vacuole to prevent lysosomal degradation and establish an intracellular replicative niche called the Legionella containing vacuole (LCV) ( Horwitz, 1983 ). For biogenesis of LCV and intracellular replication, L. pneumophila employs over three hundred individual effector proteins translocated into host cells by a Dot/Icm type IVB secretion system (T4SS) ( Berger and Isberg, 1993 ; Zhu et al., 2011 ; Ensminger, 2016 ). L. pneumophila encodes the largest arsenal of translocated effector proteins identified to date, due to its broad and diverse tropism for free-living protozoa ( Park et al., 2020 ). Armed with these effectors, L. pneumophila proliferates to high numbers within host phagocytes. Effectors are essential for biogenesis of the LCV and intracellular replication through facilitating nutrient acquisition and prevention of lysosomal degradation. However, several L. pneumophila effectors that perform these essential functions paradoxically amplify pro-inflammatory immune responses in macrophages. Thus, L. pneumophila has become a useful model pathogen to delineate mechanisms of effector-mediated immune detection and clearance. Legionella have also served as a valuable model to study molecular basis of inflammasome activation; however, this aspect of Legionella biology has been reviewed previously and will not be discussed here ( Mascarenhas and Zamboni, 2017 ). Below, we discuss mechanisms of effector-mediated immune defense against L. pneumophila and the potential for effector-mediated immunity to be harnessed for development of novel therapeutics to combat infectious diseases. L. pneumophila Effector-Mediated Translation Inhibition Enhances Macrophage Inflammatory Responses Effector-mediated host protein translation inhibition, a virulence strategy employed by multiple pathogens, enhances inflammatory signaling in L. pneumophila infected macrophages ( Fontana et al., 2011 ; Barry et al., 2013 ). To replicate intracellularly, L. pneumophila is reliant on host-derived amino acids ( George et al., 1980 ; Bruckert et al., 2013 ; Price et al., 2014 ; Schunder et al., 2014 ). Since free amino acid levels are tightly regulated in eukaryotic cells, L. pneumophila utilizes several effectors to facilitate acquisition of amino acids from host cells. The effectors Lgt1-3, SidI, SidL, LegK4, and RavX collectively inhibit host protein translation [recently reviewed in ( Belyi, 2020 )]. The mechanisms by which RavX, SidL, and SidI inhibit translation have not been fully elucidated. However, Lgt1-3 glycosylation of the host translation elongation factor eEF1A on a conserved Ser residue inhibits host polypeptide elongation ( Belyi et al., 2006 ; Belyi et al., 2008 ) and LegK4 impairs polypeptide refolding through phosphorylation of host Hsp90 ( Moss et al., 2019 ). SidI interacts with eEF1A and eEF1Bγ; however, this interaction is not sufficient for translation inhibition ( Shen et al., 2009 ; Joseph et al., 2020 ). The collective activity of this redundant family of effectors enhances the inflammatory response to L. pneumophila ( Shin and Roy, 2008 ; Barry et al., 2013 ). Effector-mediated protein translation inhibition synergizes with PAMP-mediated signaling to enhance inflammation in L. pneumophila –infected macrophages. Fontana and colleagues originally discovered that activity of the effectors Lgt1-3, SidI, and SidL induced selective mitogen activated protein kinase (MAPK)-mediated upregulation of interleukin (IL)-1α in L. pneumophila –infected macrophages ( Fontana et al., 2011 ; Fontana et al., 2012 ). MAP kinase signaling cascades culminate in activation of the dimeric activating protein (AP-1) transcription factor—made up of Jun and Fos—which facilitates pro-inflammatory gene expression ( Fujioka et al., 2004 ; Hess et al., 2004 ; Alonso et al., 2018 ; Gazon et al., 2018 ). Interestingly, complementation of a L. pneumophila mutant lacking lgt1-3 , sidL , and sidI with just lgt-3 is sufficient to restore MAPK activation during infection ( Figure 1 ) ( Fontana et al., 2012 ). Translation inhibition results in selective upregulation of IL-1α, which is critical for host defense against L. pneumophila ( Barry et al., 2013 ; Copenhaver et al., 2015 ; Mascarenhas et al., 2015 ). The selective upregulation of Il1a is a consequence of mRNA superinduction, a phenomenon whereby increased de novo transcription of specific genes overcomes bacterial blockade of protein translation and initiates a pro-inflammatory response ( Barry et al., 2017 ). This selective production of IL-1α by infected macrophages results in amplification of pro-inflammatory cytokine production by uninfected translation-competent bystander cells ( Copenhaver et al., 2015 ; Liu et al., 2020 ) (see below). Translation inhibition also occurs via an effector-independent mechanism, which may be a consequence of metabolic reprogramming ( Barry et al., 2017 ; Price et al., 2020 ) (see below). However, effector-mediated restriction of host protein translation, which liberates amino acids for use by L. pneumophila ( De Leon et al., 2017 ), contributes to a highly orchestrated pro-inflammatory response in accidental hosts and is an example of canonical ETI. Figure 1 Schematic representation of L. pneumophila effector-mediated host defense in macrophages. From the LCV, L. pneumophila (purple) translocates hundreds of individual effector proteins (red squares/rectangles) into the host cytosol through the Dot/Icm T4SS (orange). Multiple effectors inhibit host translation elongation (RavX, SidI, SidL, LegK4, and Lgt1-3), which results in activation of MAPK signaling and pro-inflammatory cytokine expression [AP-1 (Jun, Fos)]. The activity of Lgt1-3 also activates the mTORC1 complex, which results in downregulation of pro-inflammatory genes. However, in macrophages, mTOR signaling is attenuated by detection of pathogen-derived molecules. Activation of NF-κB downstream of PRR (TLRs shown) engagement is enhanced by LegS2, LnaB, and LegK1, the latter of which phosphorylates IκBα. L. pneumophila replication within macrophages is also impaired by LegA9 and LegC4, the latter of which augments cytokine-mediated restriction. Finally, LamA, a recently characterized L. pneumophila effector, degrades cellular glycogen, leading to increased aerobic glycolysis and proinflammatory cytokine production. For clarity, the SidE family of effectors and the role of IL-1 production by infected macrophages are not shown. Question marks indicate unknowns. See text for additional details. L. pneumophila Effector-Mediated Translation Inhibition Impacts Mechanistic Target of Rapamycin (mTOR) Signaling Modulation of host protein translation also impacts activity of the mechanistic target of rapamycin (mTOR). mTOR is central to many cellular processes and regulates host amino acid metabolism, where availability and dearth of amino acids results in activation or inactivation of mTOR signaling, respectively [ Figure 1 ; reviewed in ( Condon and Sabatini, 2019 )]. Several viral pathogens and protozoan parasites, such as Leishmania , have evolved to directly target this pathway and its processes for their own benefit ( Buchkovich et al., 2008 ; Jaramillo et al., 2011 ; Leroux et al., 2018 ). Recent work has revealed the central, albeit complex, role of mTOR in L. pneumophila pathogenesis and host defense. Ivanov and Roy initially reported that macrophages detect cytosolic "pathogen signatures," which results in suppression of mTOR and selective production of pro-inflammatory cytokines and independently of translocated effectors ( Ivanov and Roy, 2013 ). Concomitantly, L. pneumophila virulence is attenuated in the lungs of mice with mTOR-deficient macrophages. However, subsequent studies uncovered a role for effectors in mTOR regulation during L. pneumophila infection ( Abshire et al., 2016 ; De Leon et al., 2017 ). The mTORC1 complex (a multiprotein complex containing mTOR) is both suppressed and activated by distinct families of L. pneumophila effectors ( De Leon et al., 2017 ). Translation inhibition, through the activity of the Lgt effector family (see above), and consequent increases in free amino acids, activates mTORC1 in macrophages. However, mTORC1 is suppressed through ubiquitination and suppression of Rag GTPases by the SidE effector family (SidE/SdeABC), which also inhibit host protein translation ( De Leon et al., 2017 ). Thus, the SidE family of effectors may prevent mTORC1 sensing amino acids that are liberated downstream of Lgt1-3 activity. In macrophages, mTOR activation by the Lgts is downstream of potent translation inhibition. As discussed above, inhibition of protein translation in macrophages results in selective production of a subset of pro-inflammatory mediators, such as IL-1α. Thus, inhibition of mTOR would contribute to selective production of cytokines that orchestrate a robust inflammatory response in the lung through engagement of bystander cells (see below) ( Ivanov and Roy, 2013 ; Copenhaver et al., 2015 ; Barry et al., 2017 ; Liu et al., 2020 ). Together, these studies collectively emphasize the central and complex role of mTOR in Legionella pathogenesis and the inflammatory response elicited in accidental hosts. An Effector-Mediated Strategy for Replication in Amoebae Leads to Pro-Inflammatory Macrophage Restriction L. pneumophila is ubiquitous in freshwater environments where it parasitizes and replicates within unicellular eukaryotes, including amoebae ( Molmeret et al., 2005 ; Albert-Weissenberger et al., 2006 ). When environmental conditions are not optimal for growth and survival, amoeboid trophozoites undergo encystation, a condition where the amoeba turns into a metabolically inactive cyst containing cellulose rich cell wall that is resistant to hostile environmental conditions ( Moon and Kong, 2012 ; Aqeel et al., 2013 ). Although L. pneumophila survives in amoebal cysts, encystation is restrictive to intracellular replication ( Kilvington and Price, 1990 ; Bouyer et al., 2007 ). Prior to encystation, amoebae accumulate glycogen, which is used for biogenesis of the characteristic cellulose-rich cell wall ( Weisman et al., 1970 ; Fouque et al., 2012 ; Moon and Kong, 2012 ; Schaap and Schilde, 2018 ). To maintain amoebae as replication-permissive trophozoites, L. pneumophila utilizes the effector LamA, an amylase that catalyzes glycogenolysis to limit glycogen accumulation in infection amoebae ( Price et al., 2020 ). LamA alone is not required for L. pneumophila replication in the natural hose Acanthamoeba polyphaga , likely due to functional redundancy with other effectors ( Ghosh and O'Connor, 2017 ; Park et al., 2020 ). However, other natural host amoebae were not examined in this study. Thus, it is tempting to speculate that LamA activity is individually important in other species, such as A. castellanii , in which encystation is highly restrictive to L. pneumophila ( Weisman et al., 1970 ; Bouyer et al., 2007 ). LamA-mediated metabolic reprogramming is deleterious to L. pneumophila in accidental hosts ( Price et al., 2020 ). Excess cellular glucose results in increased aerobic glycolysis in both A. polyphaga and human monocyte-derived macrophages (hMDM) ( Price et al., 2020 ). Aerobic glycolysis in macrophages promotes their activation, M1 polarization and secretion of pro-inflammatory cytokines ( Langston et al., 2017 ). Thus, LamA activity enhances secretion of pro-inflammatory cytokines from hMDMs during infection [ Figure 1 ( Price et al., 2020 )] and impairs L. pneumophila in hMDM through IFN-γ–mediated indolamine-2,3-dioxygenase (IDO) activity ( Murray et al., 1989 ; Price et al., 2020 ). This result is intriguing since translation inhibition during L. pneumophila infection limits production of most cytokines (see above). The authors propose that the amount of IFN-γ produced is sufficient for IDO activation; however, it would be interesting to determine if IFN-γ activation is indeed required for LamA-mediated restriction. In the mouse lung, IL-1α production is severely decreased following infection with a lamA mutant compared to wild-type bacteria. However, the lamA mutant strain presumably still translocates effector translation inhibitors (see above). Thus, macrophage metabolic reprogramming may contribute to effector-independent translation inhibition and concomitant inflammation ( Barry et al., 2017 ). Moreover, LamA-mediated macrophage activation is a direct consequence of its enzymatic activity, distinguishing this response from canonical ETI. Effector-Mediated Augmentation of Cytokine-Mediated Restriction of L. pneumophila Pro-inflammatory cytokines activate resting macrophages and are critical for restriction of L. pneumophila in mammalian hosts ( Archer and Roy, 2006 ). The effector LegC4 was initially identified in a high-throughput forward genetic screen for individual effectors that impact L. pneumophila virulence in amoeba and mammalian infection models ( Shames et al., 2017 ; Rolando and Buchrieser, 2018 ). This screen identified LegC4 as conferring fitness disadvantage on L. pneumophila relative to the isogenic parental strain in a mouse model of Legionnaires' disease but not BMMs ex vivo ( Shames et al., 2017 ; Ngwaga et al., 2019 ). Interestingly, LegC4 is individually important for L. pneumophila replication in the natural host, A. castellanii ( Shames et al., 2017 ). Further investigation revealed that LegC4 is deleterious to L. pneumophila specifically within cytokine-activated macrophages ( Ngwaga et al., 2019 ) ( Figure 1 ). In cultured mouse BMMs, LegC4-mediated restriction is contingent on autocrine and paracrine TNF receptor 1(TNFR1)-mediated signaling. However, loss of TNFR1 is insufficient to rescue LegC4-mediated replication defects in the mouse lung, likely due to LegC4-mediated exacerbation of IFN-γ-mediated restriction ( Ngwaga et al., 2019 ). LegC4 additionally enhances secretion of several pro-inflammatory cytokines, including IL-12, IL-6, and TNF-α, from L. pneumophila –infected BMMs despite global translation inhibition ( Shames et al., 2017 ; Ngwaga et al., 2019 ). Whether LegC4-mediated increases in cytokine production from L. pneumophila –infected macrophages is due to enhanced transcription or translation is unknown. Revealing the influence of LegC4 on production of IL-1α, TNF-α, and IFN-γ in the lung will provide a foundation for understanding the mechanism of LegC4-mediated restriction. Interestingly, LegC4 is also augments cytokine-mediated restriction of L. longbeachae , and the second leading cause of Legionnaires' disease globally ( Gobin et al., 2009 ). L. longbeachae is lethal to mice and is reliant on a Dot/Icm T4SS for intracellular replication; however, L. longbeachae does not encode a homolog of legC4 ( Cazalet et al., 2010 ; Wood et al., 2015 ; Massis et al., 2016 ). LegC4 is sufficient to attenuate L. longbeachae replication within BMMs activated with either TNF-α or IFN-γ ( Ngwaga et al., 2019 ), demonstrating that LegC4-mediated restriction is not specific to L. pneumophila . LegC4—like LamA—enhances L. pneumophila virulence in a natural host, but its activity in macrophages is deleterious. The mechanism by which LegC4 impacts L. pneumophila fitness in natural and accidental hosts, respectively, and its potential to enhance cytokine-mediated restriction of other intracellular pathogens are currently under investigation in our lab. Effector-Mediated Activation of Inflammatory Gene Expression and Autophagy Many bacterial pathogens actively attenuate inflammatory signaling by restricting activation of the NF-κB transcription factor ( Brodsky and Medzhitov, 2009 ). However, NF-κB is activated in mammalian cells infected with L. pneumophila . Within L. pneumophila –infected cells, NF-κB activation occurs in two waves; effector-independent TLR-dependent activation when bacteria first make contact with host cells and effector-mediated activation after several hours of infection ( Losick and Isberg, 2006 ; Asrat et al., 2014 ). Several L. pneumophila effectors contribute to NF-κB activation in mammalian cells ( Figure 1 ). LegK1 is a eukaryotic-like serine/threonine kinase that phosphorylates the inhibitor of κBα (IκBα), which results in nuclear localization of NF-κB and consequent upregulation of pro-inflammatory and pro-survival gene ( Ge et al., 2009 ; Rahman and McFadden, 2011 ). However, LegK1-mediated NF-κB activation occurs only upon ectopic expression of legK1 in epithelial cells ( Ge et al., 2009 ). LnaB enhances NF-κB-mediated gene expression by an unknown mechanism following ectopic expression and during L. pneumophila infection of epithelial cells ( Losick et al., 2010 ). LegK1 does not contribute to NF-κB activity in L. pneumophila –infected epithelial cells ( Losick et al., 2010 ), which raises the possibility that this phenotype is a consequence of dose-dependent effect, or mislocalization due to ectopic expression. Neither lnaB nor legK1 are required for L. pneumophila replication in mouse macrophages individually or in combination ( Losick et al., 2010 ). Since amoebae lack NF-κB signaling components, direct effector-mediated activation of this pathway is perplexing. It is possible that IκBα phosphorylation by LegK1 is due promiscuous enzymatic activity and/or presence highly conserved target motifs. Identification of LegK1 substrates in amoebae would shed light on this possibility. Effector-mediated NF-κB activation enhances L. pneumophila survival in macrophages through prevention of premature apoptosis but also results in expression of pro-inflammatory cytokines, including IL-1α ( Losick and Isberg, 2006 ). NF-κB plays a multifaceted role in L. pneumophila infection of accidental hosts, but the evolutionary basis for its activation has yet to be elucidated. Autophagy is central to cell-autonomous restriction of intracellular bacterial pathogens in phagotrophs. L. pneumophila has evolved several effectors capable of regulating host autophagy and two, LegS2 and LegA9, are deleterious to L. pneumophila in accidental hosts ( Khweek et al., 2016 ; Rolando et al., 2016 ; Sherwood and Roy, 2016 ). LegS2 is a mitochondria-targeted sphingosine-1-phosphate lyase that restricts L. pneumophila replication in macrophages, suppresses autophagy, and enhances NF-κB activation ( Degtyar et al., 2009 ; Khweek et al., 2016 ; Rolando et al., 2016 ). Suppression of starvation-induced autophagy by LegS2 is facilitated by modulation of host sphingosine metabolism ( Rolando et al., 2016 ), but whether amplification of NF-κB is linked to LegS2-mediated sphingosine metabolism and suppression of autophagy is unknown. LegA9 enhances L. pneumophila macrophage clearance by upregulating autophagy in BMMs ( Khweek et al., 2013 ); however, further investigation is required to define the mechanism by which LegA9 augments L. pneumophila macrophage clearance. Consequences of Effector-Mediated Immunity in a Mouse Model of Legionnaires' Disease L. pneumophila replicates robustly in macrophages derived from permissive mice but is efficiently cleared from the lung just days after infection. Restriction of L. pneumophila in the mouse lung is due to a rapid and robust pro-inflammatory response orchestrated through engagement of multiple cell types ( Blanchard et al., 1987 ; Blanchard et al., 1989 ; Brieland et al., 1998 ; Archer and Roy, 2006 ). L. pneumophila –infected alveolar macrophages are poor producers of TNF-α, IL-6, and IL-12 in vivo due to effector-mediated translation inhibition ( Copenhaver et al., 2014 ; Copenhaver et al., 2015 ). However, selective upregulation of IL-1α by infected translation-impaired cells ultimately results in pro-inflammatory cytokine production by uninfected bystander cells, namely Ly6C hi monocytes and neutrophils ( Copenhaver et al., 2015 ; Barry et al., 2017 ; Casson et al., 2017 ). A central role for IL-1α in immune defense against L. pneumophila has been well established and is contingent on MyD88-mediated signaling ( Barry et al., 2013 ; Asrat et al., 2014 ; Copenhaver et al., 2015 ; Mascarenhas et al., 2015 ). However, the mechanism by which IL-1α facilitates bacterial clearance in vivo was only recently uncovered. IL-1α produced by infected alveolar macrophages engages IL-1R on alveolar epithelial cells, which in turn secrete granulocyte colony stimulating factor (GM-CSF) ( Liu et al., 2020 ). Consequent GM-CSF signaling in inflammatory monocytes upregulates aerobic glycolysis leading to pro-inflammatory cytokine production ( Liu et al., 2020 ). This work exemplifies how effector-driven virulence mechanisms, such as translation inhibition, trigger a highly orchestrated inflammatory response to L. pneumophila in the lung. Harnessing Effector-Mediated Immunity to Combat Bacterial Infection Antimicrobial resistance comprises a major global public health challenge. Thus, to control the emergence and spread of antimicrobial-resistant pathogens, innovative therapeutic strategies are desperately needed. Anti-virulence therapy a promising alternative approach to combat resistant pathogens via targeting virulence pathways of the pathogen ( Rasko and Sperandio, 2010 ; Martínez et al., 2019 ). As pathogen-centric therapeutics are susceptible to evolution of resistance, host-centric therapeutics are an attractive alternative to control bacterial infection. Information gleaned from effector-mediated immune response and the effectors themselves have potential to combat infection by regulated amplification of host immunity. Bacterial products, including effectors, modulate host immunity. Bacterial PAMPs are promising innate immunologicals, but whether immune-activating effectors can be harnessed directly or indirectly to combat infectious diseases has not been investigated. CpG oligodeoxynucleotides (ODN), a TLR9 agonist, have been used as vaccine adjuvants and can amplify immune responses to multiple bacterial, parasitic and viral pathogens, including Leishmania major , Mycobacterium tuberculosis , Francisella tularensis and, more recently, SARS-CoV-2, the etiological agent of COVID-19 ( Zimmermann et al., 1998 ; Elkins et al., 1999 ; Juffermans et al., 2002 ; Scheiermann and Klinman, 2014 ; Oberemok et al., 2020 ). Moreover, multiple effectors from well-adapted human pathogens that dampen host immunity have attracted attention as potential drug candidates for the treatment of inflammatory diseases ( Rüter and Hardwidge, 2014 ). Use of effectors that amplify immunity directly into host cells as possible host-specific therapeutics has not been evaluated. Effector-mediated subversion of host homeostasis triggers ETI but perturbation of host cellular processes poses a major challenge. Global inhibition of translation as a means to enhance anti-microbial immunity would be impractical. However, based on insight from the response to ETI, treatment with IL-1α could initiate an early and robust immune response in the lung against diverse pathogens (see above). The effector LegC4 is an intriguing candidate for host-specific therapeutics based on its ability to amplify cytokine-mediated pathogen restriction. However, further investigation is required to determine the mechanisms of LegC4 function within immune cells and if immune-activating effectors can be harnessed as host-specific therapeutics. To exert their functions, effectors require access to the cytosol of host cells. Current use of effectors as therapeutics is accomplished by autonomous translocation into host cells via fusion to cell-penetrating peptides ( Rüter and Schmidt, 2017 ). However, the N-terminal domain of anthrax lethal factor (LFn), when co-delivered with the anthrax protective antigen, facilitates translocation of cargo protein directly into mammalian cells ( Rabideau and Pentelute, 2016 ). LFn fusion has been successfully utilized to deliver bacterial proteins into host cells and mice ( Kofoed and Vance, 2011 ; Shi et al., 2015 ). In addition, nanoparticles can be used for specific delivery of nucleic acids for orthologous expression of effectors within target cells or direct delivery of protein cargo ( Avila et al., 2016 ; Rincon-Restrepo et al., 2017 ). Thus, potential exists for bacterial effectors to function as therapeutic agents. Effector based therapeutics offer several advantages over conventional biologics such as autonomous translocation, enhanced specificity, efficacy at low concentrations, targeted and topical applications, comparatively fewer side effects, cost-effective and stability at variable pH and temperatures. Moreover, further understanding of novel effectors, mechanisms of effector-mediated immunity, and the development of selective delivery mechanisms offers potential for improved combinatorial therapeutics in the future. Conclusions In addition to sensing pathogens through PRRs, the mammalian immune system has developed additional mechanisms to detect the activity of virulence factors secreted by pathogens. Effector-mediated immunity facilitates detection and/or enhanced clearance of pathogens. This additional mechanism to detect pathogens is important because pathogenic microorganisms have evolved ways to avoid, modulate and hide from the first line of immune defense offered by triggering of PRRs. As an accidental human pathogen, L. pneumophila continues to serve as a useful model used to study innate immune mechanisms without the complications of evasion strategies used by mammalian-adapted pathogens. L. pneumophila has provided valuable insight into mechanisms of innate immune defense against intracellular bacterial pathogens, including how effector-mediated virulence strategies trigger inflammation. Further investigation of L. pneumophila effector function will undoubtedly reveal yet additional mechanisms by which cells of the innate immune system restrict intracellular pathogens. Exploiting effector-mediated immunity to elicit pathogen-centric immunotherapeutics may provide additional treatment or prevention strategies against antimicrobial resistant pathogens. Author Contributions SS conceived the idea for the manuscript. TN, DC, and SS wrote the paper. All authors contributed to the article and approved the submitted version. Funding The Shames Lab is supported by an NIH NIGMS COBRE Research Project Grant (P20GM130448; SS), an NIH Kansas-INBRE Postdoctoral Fellowship (P20GM103418; DC), U.S. Department of Agriculture Research Service project (#3020-43440-001-00D; SS) and institutional start-up funds from Kansas State University (SS). Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581486/

Colicin N Binds to the Periphery of Its Receptor and Translocator, Outer Membrane Protein F

Summary Colicins kill Escherichia coli after translocation across the outer membrane. Colicin N displays an unusually simple translocation pathway, using the outer membrane protein F (OmpF) as both receptor and translocator. Studies of this binary complex may therefore reveal a significant component of the translocation pathway. Here we show that, in 2D crystals, colicin is found outside the porin trimer, suggesting that translocation may occur at the protein-lipid interface. The major lipid of the outer leaflet interface is lipopolysaccharide (LPS). It is further shown that colicin N binding displaces OmpF-bound LPS. The N-terminal helix of the pore-forming domain, which is not required for pore formation, rearranges and binds to OmpF. Colicin N also binds artificial OmpF dimers, indicating that trimeric symmetry plays no part in the interaction. The data indicate that colicin is closely associated with the OmpF-lipid interface, providing evidence that this peripheral pathway may play a role in colicin transmembrane transport. Introduction Protein translocation across membranes is a ubiquitous feature of biology and was once thought to require a water-filled pore to allow polar protein molecules across the hydrophobic bilayer. However, several models have been proposed recently whereby lipids play a critical role in the translocation pathway ( Hessa et al., 2005; Rapaport, 2005; Slatin et al., 2002 ). Probably the most fundamental process is represented by the protein secretion apparatus known as Sec61 in eukaryotes, SecYEG in bacteria, and SecYEβ in archaea. In this example, unfolded polypeptides are translocated before folding ( Robson and Collinson, 2006 ). Translocation of unfolded polypeptides reduces the minimum diameter of the pore required to shield polar polypeptide regions from the low dielectric constant of the membrane interior. Nevertheless, this pore must also deal with the insertion of hydrophobic helices of integral membrane proteins into the lipid bilayer ( Rapoport et al., 2004 ). It appears to achieve this by a transient lateral opening of the pore, and, recently, strong evidence was obtained for the sorting of hydrophobic and amphipathic segments at a protein-lipid interface ( Hessa et al., 2005 ). Mitochondrial proteins are largely nuclear encoded and translocate across the outer membrane from the cytoplasm ( Mokranjac and Neupert, 2005 ). This is accomplished by the TOM (translocase outer membrane) and TIM (translocase inner membrane) complexes ( Rapaport, 2005 ). The β-barrel TOM complex provides a pore to deliver proteins across the outer membrane. Outer membrane β-barrel proteins are imported via the TOM pore into the intermembrane space and then inserted into the outer membrane by the SAM (sorting and assembly machinery) or TOB (topogenesis outer membrane β-barrel) complex ( Paschen et al., 2005 ). This final step is similar to that in Gram-negative bacteria and involves at least one homologous protein, Omp85 ( Gentle et al., 2005 ). Mitochondria also import hydrophobic helical proteins into their outer membrane and do this in a TOM-dependent manner. Examples include those with single transmembrane stands, such as signal anchor proteins ( Habib et al., 2003 ), and apoptosis regulators, such as Bcl ( Rapaport, 2005 ), but possibly also multiple membrane-spanning proteins, such as liver carnitine palmitoyltransferase ( Cohen et al., 2001 ), and viral proteins ( Valentin et al., 2005 ). Because of the rigid β-barrel structure of the TOM pore, a mechanism for sideways release as in Sec is unlikely ( Habib et al., 2003; Horie et al., 2003 ). Thus, it has been proposed that they insert via the protein-lipid interface at the periphery of the β-barrel Tom40 and possibly between several Tom40 dimers ( Rapaport, 2005 ). The only helical proteins known to reverse translocate across the Gram-negative bacterial outer membrane are toxic bacteriocins, such as the colicins of Escherichia coli. Colicins are 40–80 kDa proteins that kill cells closely related to the producer by translocating a large (15–25 kDa) toxic domain across the protective outer membrane. This domain is either a pore former or nuclease. The outer membrane normally acts as a molecular sieve permeable only to solutes smaller than 600 Da. Although large protein export pathways exist ( Economou et al., 2006 ) and one colicin (E1) ( James et al., 1996 ) does require TolC, ( Koronakis et al., 2000 ) through which hemolysin toxins are exported ( Holland et al., 2005 ), there is no evidence of a general link between colicins and dedicated protein export systems. Because OmpF or a close homolog, such as PhoE or OmpC, is absolutely required for translocation of a number of colicins ( Bourdineaud et al., 1990; Evans et al., 1996a; Fourel et al., 1990 ), their role in translocation has been discussed intensively ( Bainbridge et al., 1998; Cao and Klebba, 2002; Kurisu et al., 2003; Vetter et al., 1998; Zakharov et al., 2004 ). Although OmpF is the translocator for most Tol-dependent ( Lazdunski et al., 1998 ) colicins, most of which first bind a high-affinity receptor, such as BtuB ( Cascales et al., 2007; Housden et al., 2005 ), Colicin N binds only to OmpF, which plays the role of both receptor and translocator ( El-Kouhen et al., 1993 ). This simple complex may thus reveal how this protein acts as the general translocation route for many different colicins ( Vetter et al., 1998 ). Experiments have clearly shown the blockage of OmpF ion channels by colicin domains ( Stora et al., 1999; Zakharov et al., 2004, 2006 ) and the binding of colicin T domains to OmpF by isothermal titration calorimetry ( Evans et al., 1996a; Housden et al., 2005 ). Nevertheless, we do not yet have conclusive evidence for the admittedly attractive and simple idea of a protein pore pathway through OmpF ( Sharma et al., 2007; Vetter et al., 1998; Zakharov et al., 2004, 2006 ). It is well known that colicins unfold during translocation ( Benedetti et al., 1992; Duché et al., 1994 ), but even elongated peptides exceed the diameter of the OmpF pore ( Bainbridge et al., 1998; Cowan et al., 1992 ). Interestingly, TonB-dependent colicins ( Lazdunski et al., 1998 ) seem only to require a high-affinity receptor ( Buchanan et al., 2007 ). Here, we describe the results of a combined biochemical and electron microscopy (EM) structural study indicating that colicin N binds to the outer surface of its receptor and translocator OmpF, displacing OmpF-bound LPS. The first helix of the pore-forming domain rearranges to allow binding to OmpF, which need not be in a trimeric conformation. Such an interaction with the periphery of OmpF thus raises the intriguing possibility that, as suggested for mitochondrial protein import, some part of the transmembrane translocation may occur at the protein-lipid interface. Results OmpF-Colicin N Complexes Form Ordered 2D Crystals Isolated complexes of colicin N with OmpF can be observed in negatively stained samples but, although they are clearly different from OmpF alone, they are currently of insufficient quality to contribute to a structural study (see the Supplemental Data available with this article online). The 2D crystallization of OmpF has been described elsewhere, and the dependence of lipid-to-protein ratio (LPR) on lattice structure was demonstrated ( Dorset et al., 1983; Hasler et al., 1998 ). As a result of the difficulties in repeating and maintaining a precise LPR throughout detergent removal, several LPRs were evaluated (i.e., 0.8, 1.0, and 1.2). Vesicle structures of varying sizes were seen with all LPRs and appeared within 24 hr of dialysis. An LPR of 1.2 gave the best results with respect to size and crystal order. At lower LPRs, smaller vesicles were predominant, containing little or no ordered lattice. The crystals form in large vesicles (up to 5 μm in diameter) that collapse to form multiple-layers of 2D crystals, most of which were in register with each other. A construct consisting of the colicin N pore-forming and receptor binding domains (colN-RP) was used to form crystals of the complex, to avoid the influence of the unstructured translocation domain. Colicin N-RP/OmpF 2D crystals formed within 24 hr of dialysis in a two-fold molar excess of colN-RP. After washing to remove free protein, the crystals were analyzed for their protein content by SDS-PAGE. At each LPR (0.6, 0.8, 1.0, 1.2, and 1.4), both colN-RP and OmpF were present ( Figure 1 A). These crystals were similar to the OmpF-only crystals, with a diffraction pattern ( Figure 1 B) that confirms a hexagonal lattice and p 3 symmetry consistent with previous OmpF crystals produced at these LPRs ( Dorset et al., 1983; Hoenger et al., 1990 ). The best quality crystals were again seen at an LPR of 1.2. At other LPRs, the vesicles were smaller, with little or no ordered lattice. Image processing of the colN-RP/OmpF crystals gave a unit cell of a = b = 97.1 ± 0.6 à ( Figure 1 C), slightly larger than that of the OmpF crystal (a = b = 93.6 ± 0.8 à ) ( Figure 1 D). Four separate images of each crystal type were merged in p 3 symmetry to a resolution of 25 à . The resulting OmpF map is consistent with previously published data ( Dorset et al., 1983; Hoenger et al., 1990 ). Colicin N Is Located at the Periphery of OmpF Trimers Comparison of the superposed, merged, and scaled projection maps from Figure 1 revealed some subtle differences between the two structures ( Figure 2 A). Because the crystallization methods were the same (i.e., the detergent and its concentration, lipid type and the LPR, buffer, and dialysis times), we conclude that the reproducible differences between the two structures in a series of samples are a result of colN-RP binding. A difference map was calculated by subtracting the merged and scaled OmpF map from that of the colN-RP/OmpF map in Fourier space to show features solely resulting from the presence of colN-RP ( Figure 2 B). This map reveals significant density extending from the external face of the OmpF barrel within the cleft between monomer-subunit interfaces of OmpF. This density almost certainly arises from bound colicin N-RP, which must interact with OmpF having a considerable proportion of the protein lying at the periphery of the OmpF envelope, possibly interacting with surrounding LPS. LPS Electron Density Is Removed by Colicin N Areas of density at the outer edges of each monomer in the OmpF map are missing in the complex map ( Figure 2 D, blue circles). Disappearance of this density is manifest in the difference map by a slight negative density at the same location. This location has been proposed as an LPS-binding site on the basis of the 2D crystallization of purified OmpF-LPS complexes ( Figure 2 E; Hoenger et al., 1990 ), and it is likely that this loss of electron density indicates a possible displacement of LPS upon colN binding. Previous work on the outer membrane protein FhuA has identified a conserved LPS-binding motif ( Ferguson et al., 2000 ). Interaction of 11 charged or polar residues with the negatively charged phosphate groups of the lipid A inner core and the diglucosamine were found and proposed to be responsible for the tight binding of LPS to FhuA. ( Ferguson et al., 2000 ; Figure 2 F). Of these 11 amino acids, four were found to be conserved between known LPS-binding proteins, which were identified using a structural search of the PDB ( Ferguson et al., 2000 ). Colicins and outer membrane proteins, including OmpF, were also highlighted by the search (K. Diederichs, personal communication). By use of these data, a possible OmpF LPS-binding site is shown in Figure 2 G comprising the conserved lysine and arginine residues of the LPS-binding motif. A similar site has been modeled onto the LPS-dependent outer membrane protease OmpT ( Vandeputte-Rutten et al., 2001 ). The area indicated in Figure 2 G shows good correlation with the areas of extra density found in the OmpF projection map ( Figure 2 D blue circles) and those found by Hoenger et al. ( Hoenger et al., 1990 ) ( Figure 2 E). As a result of additional ion exchange purification steps, peripheral LPS molecules were not present in the detergent-solubilized OmpF X-ray structure ( Cowan et al., 1992 ). Colicin N Displaces LPS from OmpF Without extensive ion exchange chromatography, LPS copurifies with OmpF, and it has also been shown to be critical in the assembly of outer membrane proteins in general ( Bulieris et al., 2003; de Cock et al., 1999; Fourel et al., 1994 ). LPS associated with OmpF results in the formation of a "ladder/smear" upon SDS-PAGE because of differing numbers of LPS molecules associated with OmpF trimers ( Holzenburg et al., 1989 ). It has been shown by free flow electrophoresis that four forms can be isolated— lb LPS (no loosely bound LPS), ∗lb LPS (1 molecule of loosely bound LPS per trimer), ∗∗lb LPS (2 molecules of loosely bound LPS per trimer), and +lb LPS (8 molecules of loosely bound LPS per trimer). Each form had a defined homogenous mass measurable by SDS-PAGE and analytical ultracentrifugation. 2D crystals formed with +lb LPS (as here) showed no effect of LPS upon the 2D lattice ( Holzenburg et al., 1989 ). To demonstrate this further we used newly available, refolded trimeric OmpF (RF OmpF). This OmpF has been isolated from inclusion bodies and refolded in vitro to produce a fully folded, fully functional LPS-free trimeric OmpF ( Visudtiphole et al., 2005 ). Figure 3 A shows formation of the characteristic ladder on SDS-PAGE due to OmpF-associated LPS in both +lb LPS (WT) OmpF purified from the outer membrane of E. coli BE3000 ( Figure 3 A, lane WT OmpF) and refolded trimeric OmpF with the addition of exogenous LPS from E. coli 0111:B4 ( Figure 3 A, lane RF OmpF+ LPS). These are both compared to the pure RF OmpF without LPS, which shows a single clear band ( Figure 3 A, lane RF OmpF). The slight difference seen in the migration patterns of WT OmpF and RF OmpF+LPS may be due to the use of a smooth LPS in the RF OmpF samples ( Diedrich et al., 1990 ). Smooth LPS molecules contain the full oligosaccharide core and O antigen units and are therefore larger than those derived from rough strains (such as E. coli BE3000) and have been shown to bind preferentially to OmpF ( Borneleit et al., 1989; Diedrich et al., 1990 ). WT OmpF/colN complex formation ( Derouiche et al., 1996; Dover et al., 2000 ) results in the loss of the ladder effect, suggesting that LPS is displaced during complex formation ( Figure 3 B). Not only does complex formation appear to displace LPS, but it also results in dissociation of higher order OmpF structures/aggregates ( Figure 3 B). This effect is seen with all P-domain/OmpF complexes and also TolAII/OmpF complexes observable on SDS-PAGE ( Derouiche et al., 1996; Dover et al., 2000 ). To determine whether the disappearance of the ladder on SDS-PAGE is due to removal of LPS, we used the anti-LPS antibody WN1 222-5 ( Di Padova et al., 1993 ). No LPS could be detected in the complex formed by WT OmpF and colN or in RF OmpF, but a strong signal was observed in WT-OmpF alone ( Figure 3 C). To ensure that only the complex was present in the western blot, an excess of colN was used. Structural homology searches have revealed a possible LPS-binding site on colicin N ( Ferguson et al., 2000 ), so we used fluorescently labeled LPS to detect whether LPS displaced from OmpF was bound by free colN. In an SDS-PAGE experiment where FITC-LPS was preincubated with RF OmpF, there was no fluorescence at the level of the free excess colN-RP. This experiment was inconclusive regarding LPS displacement from the complex, because free FITC-LPS migrated the same distance as OmpF (data not shown). Previously, the main role of LPS in colicin action was thought to be in the ability of long O antigen chains to inhibit both colicin and phage action on E. coli ( Lakey et al., 1994; van der Ley et al., 1986 ) and possible interactions with Tol proteins ( Cascales et al., 2007 ). Because the LPS is bound to the outer surface of the OmpF trimer, the current data indicate a clearer interaction of colicin N with this surface than has been previously proposed. The significant density from the EM study shows the colicin to be situated at the interface between two monomers in the trimer, but it has also been shown to bind dimeric OmpF that arises as a contaminant in normal preparations ( Dover et al., 2000 ). Here, we made use of refolded dimeric OmpF, and our results confirmed ( Figure 3 D) that it also forms complexes with colicin N on SDS-PAGE. The dimer is asymmetric and is likely to form a structure resembling a trimer with a subunit missing so that the intermonomer interface is likely to remain ( Visudtiphole et al., 2005 ). Thus, the binding site does not require a trimer but since we lack a folded monomer preparation this experiment cannot be taken to its natural conclusion. The First Helix of the Pore-Forming Domain Is Involved in Complex Formation It was shown previously that the colicin P-domain and TolA-II (periplasmic domain) bind competitively to the OmpF trimer ( Derouiche et al., 1996; Dover et al., 2000 ). TolA-II is a helical protein composed of 11 mer tandem repeats ( Levengood et al., 1991 ), so it is straightforward to compare with likely sequences in colicin N. The most similar region is part of the N-terminal helix of the P-domain (ColN184–199). To test its involvement in complex formation, two disulfide bond mutants were designed that hold opposite ends of this helix in the conformation observed in the X-ray structure ( Figure 4 ) (PDB code: 1A84 ) ( Vetter et al., 1998 ). The mutant N191C-A288C, which binds the helix-1 (H1), was predicted by the program SSBOND ( Hazes and Dijkstra, 1988 ) as having the correct geometry for a disulfide. However, in the absence of a useful prediction by SSBOND for the other end of H1, we chose Y213C-V352C, which links H1 to the tip of hydrophobic helix formed by H8/H9 with less favorable geometry ( Figure 4 ). Each mutant showed shifts on SDS-PAGE upon oxidation, indicating disulfide formation ( Supplemental Data ), and was mixed with OmpF under both oxidizing and reducing conditions. For both cases, the formation of the disulfide bond inhibits complex formation, with N191C-A288C being more inhibitory than Y213C-V352C ( Figure 4 ). Both mutants behaved as wild-type in the reduced state. Toxicity was tested on live cells in a fluorescent membrane depolarization assay ( Bainbridge et al., 1998 ), and both mutants were inactive in the oxidized (disulfide) state. The addition of DTT allowed the mutants to regain their killing activity and, therefore, also confirms that the mutant Y213C-V352C does form a stable disulfide bond ( Supplemental Data ). Thus, conformational change of this region is required for complex formation with OmpF and toxicity. To further indicate the role of this region in complex formation, the entire P-domain and just the sequence K185-A195 were added to the C terminus of glutathione-S-transferase (GST) ( Sharrocks, 1994 ). GST does not bind to OmpF in the SDS-PAGE assay, and an anti-GST western blot was used to detect interaction of the fusion proteins with trimeric OmpF. The GST-P-domain construct binds strongly, but the GST-colicin N (185–195) fusion (GST-H1) was easily proteolyzed. Nevertheless, the blot shows a clear binding imparted by this ten residue sequence ( Figure 4 ). OmpF-Colicin N Complexes Form Ordered 2D Crystals Isolated complexes of colicin N with OmpF can be observed in negatively stained samples but, although they are clearly different from OmpF alone, they are currently of insufficient quality to contribute to a structural study (see the Supplemental Data available with this article online). The 2D crystallization of OmpF has been described elsewhere, and the dependence of lipid-to-protein ratio (LPR) on lattice structure was demonstrated ( Dorset et al., 1983; Hasler et al., 1998 ). As a result of the difficulties in repeating and maintaining a precise LPR throughout detergent removal, several LPRs were evaluated (i.e., 0.8, 1.0, and 1.2). Vesicle structures of varying sizes were seen with all LPRs and appeared within 24 hr of dialysis. An LPR of 1.2 gave the best results with respect to size and crystal order. At lower LPRs, smaller vesicles were predominant, containing little or no ordered lattice. The crystals form in large vesicles (up to 5 μm in diameter) that collapse to form multiple-layers of 2D crystals, most of which were in register with each other. A construct consisting of the colicin N pore-forming and receptor binding domains (colN-RP) was used to form crystals of the complex, to avoid the influence of the unstructured translocation domain. Colicin N-RP/OmpF 2D crystals formed within 24 hr of dialysis in a two-fold molar excess of colN-RP. After washing to remove free protein, the crystals were analyzed for their protein content by SDS-PAGE. At each LPR (0.6, 0.8, 1.0, 1.2, and 1.4), both colN-RP and OmpF were present ( Figure 1 A). These crystals were similar to the OmpF-only crystals, with a diffraction pattern ( Figure 1 B) that confirms a hexagonal lattice and p 3 symmetry consistent with previous OmpF crystals produced at these LPRs ( Dorset et al., 1983; Hoenger et al., 1990 ). The best quality crystals were again seen at an LPR of 1.2. At other LPRs, the vesicles were smaller, with little or no ordered lattice. Image processing of the colN-RP/OmpF crystals gave a unit cell of a = b = 97.1 ± 0.6 à ( Figure 1 C), slightly larger than that of the OmpF crystal (a = b = 93.6 ± 0.8 à ) ( Figure 1 D). Four separate images of each crystal type were merged in p 3 symmetry to a resolution of 25 à . The resulting OmpF map is consistent with previously published data ( Dorset et al., 1983; Hoenger et al., 1990 ). Colicin N Is Located at the Periphery of OmpF Trimers Comparison of the superposed, merged, and scaled projection maps from Figure 1 revealed some subtle differences between the two structures ( Figure 2 A). Because the crystallization methods were the same (i.e., the detergent and its concentration, lipid type and the LPR, buffer, and dialysis times), we conclude that the reproducible differences between the two structures in a series of samples are a result of colN-RP binding. A difference map was calculated by subtracting the merged and scaled OmpF map from that of the colN-RP/OmpF map in Fourier space to show features solely resulting from the presence of colN-RP ( Figure 2 B). This map reveals significant density extending from the external face of the OmpF barrel within the cleft between monomer-subunit interfaces of OmpF. This density almost certainly arises from bound colicin N-RP, which must interact with OmpF having a considerable proportion of the protein lying at the periphery of the OmpF envelope, possibly interacting with surrounding LPS. LPS Electron Density Is Removed by Colicin N Areas of density at the outer edges of each monomer in the OmpF map are missing in the complex map ( Figure 2 D, blue circles). Disappearance of this density is manifest in the difference map by a slight negative density at the same location. This location has been proposed as an LPS-binding site on the basis of the 2D crystallization of purified OmpF-LPS complexes ( Figure 2 E; Hoenger et al., 1990 ), and it is likely that this loss of electron density indicates a possible displacement of LPS upon colN binding. Previous work on the outer membrane protein FhuA has identified a conserved LPS-binding motif ( Ferguson et al., 2000 ). Interaction of 11 charged or polar residues with the negatively charged phosphate groups of the lipid A inner core and the diglucosamine were found and proposed to be responsible for the tight binding of LPS to FhuA. ( Ferguson et al., 2000 ; Figure 2 F). Of these 11 amino acids, four were found to be conserved between known LPS-binding proteins, which were identified using a structural search of the PDB ( Ferguson et al., 2000 ). Colicins and outer membrane proteins, including OmpF, were also highlighted by the search (K. Diederichs, personal communication). By use of these data, a possible OmpF LPS-binding site is shown in Figure 2 G comprising the conserved lysine and arginine residues of the LPS-binding motif. A similar site has been modeled onto the LPS-dependent outer membrane protease OmpT ( Vandeputte-Rutten et al., 2001 ). The area indicated in Figure 2 G shows good correlation with the areas of extra density found in the OmpF projection map ( Figure 2 D blue circles) and those found by Hoenger et al. ( Hoenger et al., 1990 ) ( Figure 2 E). As a result of additional ion exchange purification steps, peripheral LPS molecules were not present in the detergent-solubilized OmpF X-ray structure ( Cowan et al., 1992 ). Colicin N Displaces LPS from OmpF Without extensive ion exchange chromatography, LPS copurifies with OmpF, and it has also been shown to be critical in the assembly of outer membrane proteins in general ( Bulieris et al., 2003; de Cock et al., 1999; Fourel et al., 1994 ). LPS associated with OmpF results in the formation of a "ladder/smear" upon SDS-PAGE because of differing numbers of LPS molecules associated with OmpF trimers ( Holzenburg et al., 1989 ). It has been shown by free flow electrophoresis that four forms can be isolated— lb LPS (no loosely bound LPS), ∗lb LPS (1 molecule of loosely bound LPS per trimer), ∗∗lb LPS (2 molecules of loosely bound LPS per trimer), and +lb LPS (8 molecules of loosely bound LPS per trimer). Each form had a defined homogenous mass measurable by SDS-PAGE and analytical ultracentrifugation. 2D crystals formed with +lb LPS (as here) showed no effect of LPS upon the 2D lattice ( Holzenburg et al., 1989 ). To demonstrate this further we used newly available, refolded trimeric OmpF (RF OmpF). This OmpF has been isolated from inclusion bodies and refolded in vitro to produce a fully folded, fully functional LPS-free trimeric OmpF ( Visudtiphole et al., 2005 ). Figure 3 A shows formation of the characteristic ladder on SDS-PAGE due to OmpF-associated LPS in both +lb LPS (WT) OmpF purified from the outer membrane of E. coli BE3000 ( Figure 3 A, lane WT OmpF) and refolded trimeric OmpF with the addition of exogenous LPS from E. coli 0111:B4 ( Figure 3 A, lane RF OmpF+ LPS). These are both compared to the pure RF OmpF without LPS, which shows a single clear band ( Figure 3 A, lane RF OmpF). The slight difference seen in the migration patterns of WT OmpF and RF OmpF+LPS may be due to the use of a smooth LPS in the RF OmpF samples ( Diedrich et al., 1990 ). Smooth LPS molecules contain the full oligosaccharide core and O antigen units and are therefore larger than those derived from rough strains (such as E. coli BE3000) and have been shown to bind preferentially to OmpF ( Borneleit et al., 1989; Diedrich et al., 1990 ). WT OmpF/colN complex formation ( Derouiche et al., 1996; Dover et al., 2000 ) results in the loss of the ladder effect, suggesting that LPS is displaced during complex formation ( Figure 3 B). Not only does complex formation appear to displace LPS, but it also results in dissociation of higher order OmpF structures/aggregates ( Figure 3 B). This effect is seen with all P-domain/OmpF complexes and also TolAII/OmpF complexes observable on SDS-PAGE ( Derouiche et al., 1996; Dover et al., 2000 ). To determine whether the disappearance of the ladder on SDS-PAGE is due to removal of LPS, we used the anti-LPS antibody WN1 222-5 ( Di Padova et al., 1993 ). No LPS could be detected in the complex formed by WT OmpF and colN or in RF OmpF, but a strong signal was observed in WT-OmpF alone ( Figure 3 C). To ensure that only the complex was present in the western blot, an excess of colN was used. Structural homology searches have revealed a possible LPS-binding site on colicin N ( Ferguson et al., 2000 ), so we used fluorescently labeled LPS to detect whether LPS displaced from OmpF was bound by free colN. In an SDS-PAGE experiment where FITC-LPS was preincubated with RF OmpF, there was no fluorescence at the level of the free excess colN-RP. This experiment was inconclusive regarding LPS displacement from the complex, because free FITC-LPS migrated the same distance as OmpF (data not shown). Previously, the main role of LPS in colicin action was thought to be in the ability of long O antigen chains to inhibit both colicin and phage action on E. coli ( Lakey et al., 1994; van der Ley et al., 1986 ) and possible interactions with Tol proteins ( Cascales et al., 2007 ). Because the LPS is bound to the outer surface of the OmpF trimer, the current data indicate a clearer interaction of colicin N with this surface than has been previously proposed. The significant density from the EM study shows the colicin to be situated at the interface between two monomers in the trimer, but it has also been shown to bind dimeric OmpF that arises as a contaminant in normal preparations ( Dover et al., 2000 ). Here, we made use of refolded dimeric OmpF, and our results confirmed ( Figure 3 D) that it also forms complexes with colicin N on SDS-PAGE. The dimer is asymmetric and is likely to form a structure resembling a trimer with a subunit missing so that the intermonomer interface is likely to remain ( Visudtiphole et al., 2005 ). Thus, the binding site does not require a trimer but since we lack a folded monomer preparation this experiment cannot be taken to its natural conclusion. The First Helix of the Pore-Forming Domain Is Involved in Complex Formation It was shown previously that the colicin P-domain and TolA-II (periplasmic domain) bind competitively to the OmpF trimer ( Derouiche et al., 1996; Dover et al., 2000 ). TolA-II is a helical protein composed of 11 mer tandem repeats ( Levengood et al., 1991 ), so it is straightforward to compare with likely sequences in colicin N. The most similar region is part of the N-terminal helix of the P-domain (ColN184–199). To test its involvement in complex formation, two disulfide bond mutants were designed that hold opposite ends of this helix in the conformation observed in the X-ray structure ( Figure 4 ) (PDB code: 1A84 ) ( Vetter et al., 1998 ). The mutant N191C-A288C, which binds the helix-1 (H1), was predicted by the program SSBOND ( Hazes and Dijkstra, 1988 ) as having the correct geometry for a disulfide. However, in the absence of a useful prediction by SSBOND for the other end of H1, we chose Y213C-V352C, which links H1 to the tip of hydrophobic helix formed by H8/H9 with less favorable geometry ( Figure 4 ). Each mutant showed shifts on SDS-PAGE upon oxidation, indicating disulfide formation ( Supplemental Data ), and was mixed with OmpF under both oxidizing and reducing conditions. For both cases, the formation of the disulfide bond inhibits complex formation, with N191C-A288C being more inhibitory than Y213C-V352C ( Figure 4 ). Both mutants behaved as wild-type in the reduced state. Toxicity was tested on live cells in a fluorescent membrane depolarization assay ( Bainbridge et al., 1998 ), and both mutants were inactive in the oxidized (disulfide) state. The addition of DTT allowed the mutants to regain their killing activity and, therefore, also confirms that the mutant Y213C-V352C does form a stable disulfide bond ( Supplemental Data ). Thus, conformational change of this region is required for complex formation with OmpF and toxicity. To further indicate the role of this region in complex formation, the entire P-domain and just the sequence K185-A195 were added to the C terminus of glutathione-S-transferase (GST) ( Sharrocks, 1994 ). GST does not bind to OmpF in the SDS-PAGE assay, and an anti-GST western blot was used to detect interaction of the fusion proteins with trimeric OmpF. The GST-P-domain construct binds strongly, but the GST-colicin N (185–195) fusion (GST-H1) was easily proteolyzed. Nevertheless, the blot shows a clear binding imparted by this ten residue sequence ( Figure 4 ). Discussion Several groups of toxins are known to act by translocating proteins across membranes ( Parker and Feil, 2005 ). In some examples, such as anthrax or cholera, a defined protein pore is created to insert a toxic subunit into the cytoplasm, but in diphtheria toxin, the translocon that transports the 270 residue catalytic domain is much less well defined. Colicin Ia has been shown to transport arbitrary cargo proteins, engineered onto its N terminus, across the lipid bilayer. This general transport system uses voltage to perform the seemingly impossible task of translocating folded charged proteins through a low dielectric barrier ( Slatin et al., 2002 ). Furthermore, it has been proposed that combined protein-lipid or toroidal pores are formed by colicins in the inner membrane of E. coli ( Sobko et al., 2004, 2006 ), by E. coli Hemolysin E ( Tzokov et al., 2006 ) and by the eukaryotic channel-forming toxin Equinatoxin ( Anderluh et al., 2003; Barlic et al., 2004 ). Thus, recent proposals for the involvement of lipid ( Hessa et al., 2005; Rapaport, 2005 ), once considered "the last refuge of the intellectually bankrupt" ( Qiu et al., 1996 ), have begun to suggest further alternatives to the protein-only model for transmembrane translocation pathways. The translocation of Tol, but not Ton-dependent ( Buchanan et al., 2007 ), colicins into Gram-negative cells requires either a trimeric porin (OmpF, OmpC, or PhoE) ( Evans et al., 1996a ) or TolC ( Lazzaroni et al., 2002 ) and, thus, parasitizes host proteins not designed for protein import. The absolute requirement for these proteins leaves no doubt as to their central role in providing a pathway across the outer membrane. Isothermal titration calorimetry (ITC) measurements of colicin N binding to OmpF, OmpC, and PhoE showed that all three bound colicin with similar affinity, even though OmpF-bearing cells were much more sensitive. The difference in toxicity must therefore be due to differences in translocation. OmpF binds colicin N with a much larger enthalpic component, which is compensated by a significant entropic penalty; thus, efficient colicin translocation by OmpF correlates with unique colicin N-binding thermodynamics. Such binding is observed only when using full-length colicin N ( Evans et al., 1996a, 1996b ), and it has recently been demonstrated by ITC that the flexible translocation domain of colicin E9 binds specifically to OmpF ( Housden et al., 2005 ). Because this domain also binds a periplasmic receptor (TolB), it is likely that it interacts with OmpF on its periplasmic face ( Housden et al., 2005 ). Thus, complexes of pore-forming colicins with OmpF can require interactions with all three domains—translocation ( Evans et al., 1996a ), receptor ( Evans et al., 1996b ), and pore forming ( Dover et al., 2000 ). Ion channel measurements in artificial lipid membranes also reveal OmpF interactions with the R domain ( Stora et al., 1999 ) and T domain ( Zakharov et al., 2004 ) of colicin N by observation of transient blocking of the pore. The blocking by T domain occurs on one side of OmpF, but whether this is the extracellular ( Zakharov et al., 2004 ) or periplasmic side ( Danelon et al., 2003 ), as is likely from the biology ( Housden et al., 2005 ), is not clear. Mutations in OmpF that affect colicin N binding are on the outer loops (E285,G285) or in the pore lumen (G119D) ( Fourel et al., 1993; Jeanteur et al., 1994 ), and it is the latter, deep inside the pore, that conflicts most with a possible exterior route for protein translocation. However, this mutation is a true receptor-binding mutant whose effects are overcome under low-salt receptor bypass conditions where the role of OmpF is purely a translocator ( Jeanteur et al., 1994 ). The narrow "eyelet" region of the OmpF pore is probably too small to accommodate a polypeptide, and OmpF unfolding would need to provide a suitable pore size such as that found in the anthrax toxin ( Krantz et al., 2005 ). Disulfide bond mutants, which prevent localized eyelet unfolding, have no effect upon translocation and thus argue against the pore route ( Bainbridge et al., 1998 ), although there are arguments supporting the classical model ( Cao and Klebba, 2002 ). Studies using OmpF/OmpC chimeras show that translocation of colicin N by OmpF is dependent on residues 143–262 ( Fourel et al., 1990 ), which form the outer wall of the β-barrel ( Supplemental Data ), are separated from the pore by the invaginated loop3 and coincide with the proposed LPS-binding site ( Figure 2 F and Supplemental Data ). Importantly, both colicin N and C termini of colicin need to gain access to the periplasmic space through the outer membrane barrier for toxicity to occur. The evidence here is that the unfolded C-terminal domain inserts in clefts at the periphery of OmpF with direct binding by its first helix. The remaining helices are sufficient to span the periplasm and form a functional toxic pore ( Baty et al., 1990 ; Figure 5 ). It is not clear where the N-terminal translocation domain fits in the current proposal. Finally, because colicin activity relies on the Tol-Pal complex, which has recently been shown to be part of the cell division machinery ( Gerding et al., 2007 ), the OmpF employed by colicins may be newly synthesized. The relevance to the majority of colicins, which also bind to a high-affinity receptor, is best appreciated by examination of the X-ray crystal structure of the receptor complex of colicin E3 ( Kurisu et al., 2003 ) and of the detailed model for OmpF recruitment provided by work on colicin E9 ( Housden et al., 2005 ). The initial receptor-bound structure may thus present the N-terminal disordered domain for OmpF binding and the C-terminal toxic domain for translocation in a format comparable to that shown here. In conclusion, we have revealed by electron crystallography the first, to our knowledge, visualization of a colicin within a membrane translocon. By such direct imaging and indirect biochemical methods, we show that colicin N makes intimate contact with the exterior of its translocator, displacing tightly bound lipid as it does so. Furthermore, we measured the interaction of helix-1 with OmpF, which was predicted in most models of group A colicin translocation ( Cascales et al., 2007; Vetter et al., 1998 ). These discoveries argue strongly for the transmembrane translocation of colicins at the protein-lipid interface. Together with recently published evidence for protein translocation at other protein-lipid interfaces ( Hessa et al., 2005; Rapaport, 2005 ), our data question the general assumption that protein translocation across membranes occurs exclusively though protein pores. Experimental Procedures Protein Expression and Purification The Colicin N-RP construct was created using Quick Change mutagenesis to "loop-out" the translocation domain (residues 1–81) of the full-length gene. The mutagenic primer contained a 5′ region complementary to the MCS of the pET8c and the 3′ complementary half of the colicin N receptor-binding domain (the underlined half being complementary to the start of the receptor-binding domain [5′-CATCACCATCACTCGAGC AGTGCTAAGGTTGGAGAG- 3′]). The Quick Change product thus lacked the translocation domain. All colicin constructs were expressed using the modified pET8c vector giving N-terminal six histidine tag ( Politou et al., 1994 ). Expressed protein from E. coli BL21 pLysE was then purified using Ni-NTA affinity resin ( Fridd et al., 2002 ). WT OmpF was extracted from the outer membrane of E. coli BE3000, as described elsewhere ( Lakey et al., 1985 ). Refolded trimeric and dimeric OmpF was purified from inclusion bodies, as described in Visudtiphole et al., 2005 . Preparation of OmpF/Colicin N Complexes in Detergent for Negative-Stain Electron Microscopy Each complex was formed in a 2-fold molar excess of colicin in the presence of SDS (0.1% w/v) and was incubated for 30 min at 37°C. These complexes were applied to glow-discharged, carbon-coated grids and stained with uranyl acetate (2% w/v). Micrographs were recorded at 100 kV on a Philips CM100 EM onto Kodak Electron Image Film, SO163. Preparation of 2D Crystals for Negative-Stain Electron Microscopy Formation of the 2D crystals followed the method developed by Dorset et al. (1983) . WT OmpF purified in SDS from the outer membrane was buffer exchanged into 2D crystallization buffer (20 mM HEPES [pH 7.0], 10 mM MgCl 2 , 100 mM NaCl, 0.2 mM EDTA, 0.2 mM DTT, and 3 mM NaN 3 ) supplemented with octyl-POE (1.0% v/v). Where required, colN-RP was added at a molar ratio of 1:2 (monomeric OmpF:colN-RP) and was incubated for 30 min at 37°C. To this, DMPC (Avanti Polar Lipids Inc., Alabaster, AL; 20 mM Tris [pH 7.5] and 1% [v/v] octyl-POE) was added at the relevant LPR (w/w). After mixing, the samples were incubated for a further 30 min at 37°C. Dialysis of the mixture using a 3,500 MWCO Float A Lyzer (Spectrum Laboratories Ltd., Rancho Dominguez, CA) was performed at 37°C against 2D-crystallization buffer for at least 20 hr (all buffers were pre-equilibrated at 37°). After 50% of the dialysis buffer was replaced with fresh 2D-crystallization buffer, dialysis continued for a further 20 hr at 37°C. A further 50% of the dialysis buffer was then changed for Nano-pure water and dialyzed for a further 4 hr. This step was repeated three times. The sample was then incubated on ice for 10 min before being centrifuged at 2000× g for 5 min. Resuspension of the sample into equal volumes of Nano-pure water was followed by centrifugation at 2000× g for 5 min. This step was repeated, and SDS-PAGE was used to determine the presence of protein in the final crystals. Samples were negatively stained with uranyl formate (0.75% w/v). Image Processing Micrographs were recorded at 100 kV on a Philips CM100 electron microscope equipped with a 1024 × 1024 CCD camera. Images of crystals that showed good diffraction were processed to a resolution of 25 à , as described elsewhere ( Crowther et al., 1996; Henderson et al., 1986 ). Symmetry analysis was performed using ALLSPACE ( Valpuesta et al., 1994 ), and four separate images of each type of crystal merged in p 3 symmetry. Difference maps were calculated by subtraction of the Fourier terms after first scaling amplitudes to yield equal total amplitude for each data set ( Kubalek et al., 1987 ). SDS-PAGE Gel Shift Assay All gel shift assays were performed on 12% (w/v) SDS-PAGE, as described elsewhere ( Dover et al., 2000 ). Complex formation was achieved by incubating samples at 37°C for 30 min in the presence of SDS (0.1% w/v). Samples were analyzed without heat denaturation. Protein Expression and Purification The Colicin N-RP construct was created using Quick Change mutagenesis to "loop-out" the translocation domain (residues 1–81) of the full-length gene. The mutagenic primer contained a 5′ region complementary to the MCS of the pET8c and the 3′ complementary half of the colicin N receptor-binding domain (the underlined half being complementary to the start of the receptor-binding domain [5′-CATCACCATCACTCGAGC AGTGCTAAGGTTGGAGAG- 3′]). The Quick Change product thus lacked the translocation domain. All colicin constructs were expressed using the modified pET8c vector giving N-terminal six histidine tag ( Politou et al., 1994 ). Expressed protein from E. coli BL21 pLysE was then purified using Ni-NTA affinity resin ( Fridd et al., 2002 ). WT OmpF was extracted from the outer membrane of E. coli BE3000, as described elsewhere ( Lakey et al., 1985 ). Refolded trimeric and dimeric OmpF was purified from inclusion bodies, as described in Visudtiphole et al., 2005 . Preparation of OmpF/Colicin N Complexes in Detergent for Negative-Stain Electron Microscopy Each complex was formed in a 2-fold molar excess of colicin in the presence of SDS (0.1% w/v) and was incubated for 30 min at 37°C. These complexes were applied to glow-discharged, carbon-coated grids and stained with uranyl acetate (2% w/v). Micrographs were recorded at 100 kV on a Philips CM100 EM onto Kodak Electron Image Film, SO163. Preparation of 2D Crystals for Negative-Stain Electron Microscopy Formation of the 2D crystals followed the method developed by Dorset et al. (1983) . WT OmpF purified in SDS from the outer membrane was buffer exchanged into 2D crystallization buffer (20 mM HEPES [pH 7.0], 10 mM MgCl 2 , 100 mM NaCl, 0.2 mM EDTA, 0.2 mM DTT, and 3 mM NaN 3 ) supplemented with octyl-POE (1.0% v/v). Where required, colN-RP was added at a molar ratio of 1:2 (monomeric OmpF:colN-RP) and was incubated for 30 min at 37°C. To this, DMPC (Avanti Polar Lipids Inc., Alabaster, AL; 20 mM Tris [pH 7.5] and 1% [v/v] octyl-POE) was added at the relevant LPR (w/w). After mixing, the samples were incubated for a further 30 min at 37°C. Dialysis of the mixture using a 3,500 MWCO Float A Lyzer (Spectrum Laboratories Ltd., Rancho Dominguez, CA) was performed at 37°C against 2D-crystallization buffer for at least 20 hr (all buffers were pre-equilibrated at 37°). After 50% of the dialysis buffer was replaced with fresh 2D-crystallization buffer, dialysis continued for a further 20 hr at 37°C. A further 50% of the dialysis buffer was then changed for Nano-pure water and dialyzed for a further 4 hr. This step was repeated three times. The sample was then incubated on ice for 10 min before being centrifuged at 2000× g for 5 min. Resuspension of the sample into equal volumes of Nano-pure water was followed by centrifugation at 2000× g for 5 min. This step was repeated, and SDS-PAGE was used to determine the presence of protein in the final crystals. Samples were negatively stained with uranyl formate (0.75% w/v). Image Processing Micrographs were recorded at 100 kV on a Philips CM100 electron microscope equipped with a 1024 × 1024 CCD camera. Images of crystals that showed good diffraction were processed to a resolution of 25 à , as described elsewhere ( Crowther et al., 1996; Henderson et al., 1986 ). Symmetry analysis was performed using ALLSPACE ( Valpuesta et al., 1994 ), and four separate images of each type of crystal merged in p 3 symmetry. Difference maps were calculated by subtraction of the Fourier terms after first scaling amplitudes to yield equal total amplitude for each data set ( Kubalek et al., 1987 ). SDS-PAGE Gel Shift Assay All gel shift assays were performed on 12% (w/v) SDS-PAGE, as described elsewhere ( Dover et al., 2000 ). Complex formation was achieved by incubating samples at 37°C for 30 min in the presence of SDS (0.1% w/v). Samples were analyzed without heat denaturation. Supplemental Data Document S1. Five Figures and Supplemental References

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2772353/

Impact of Spore Biology on the Rate of Kill and Suppression of Resistance in Bacillus anthracis ▿

Bacillus anthracis is complex because of its spore form. The spore is invulnerable to antibiotic action. It also has an impact on the emergence of resistance. We employed the hollow-fiber infection model to study the impacts of different doses and schedules of moxifloxacin on the total-organism population, the spore population, and the subpopulations of vegetative- and spore-phase organisms that were resistant to moxifloxacin. We then generated a mathematical model of the impact of moxifloxacin, administered by continuous infusion or once daily, on vegetative- and spore-phase organisms. The ratio of the rate constant for vegetative-phase cells going to spore phase ( K vs ) to the rate constant for spore-phase cells going to vegetative phase ( K sv ) determines the rate of organism clearance. The continuous-infusion drug profile is more easily sensed as a threat; the K vs / K sv ratio increases at lower drug exposures (possibly related to quorum sensing). This movement to spore phase protects the organism but makes the emergence of resistance less likely. Suppression of resistance requires a higher level of drug exposure with once-daily administration than with a continuous infusion, a difference that is related to vegetative-to-spore (and back) transitioning. Spore biology has a major impact on drug therapy and resistance suppression. These findings explain why all drugs of different classes have approximately the same rate of organism clearance for Bacillus anthracis .

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2583550/

Identification of a Surrogate Marker for Infection in the African Green Monkey Model of Inhalation Anthrax ▿

In 2001, a bioterrorism attack involving Bacillus anthracis spore-laced letters resulted in 22 cases of inhalation anthrax, with five fatalities. This incident identified gaps in our health care system and precipitated a renewed interest in identifying both therapeutics and rapid diagnostic assays. To address those gaps, well-characterized animal models that resemble the human disease are needed. In addition, a rapid assay for a reliable diagnostic marker is key to the success of these efforts. In this study, we exposed African green monkeys to B. anthracis spores; examined clinical signs and physiological parameters, including fever, heart rate, complete blood count, and bacteremia; and evaluated the PCR assay and electrochemiluminescence (ECL) immunoassay for the biomarkers protective antigen and capsule. The results demonstrated that although there were neither objective clinical nor physiological signs that consistently identified either infection or the onset of clinical anthrax disease, the African green monkey is a suitable animal model exhibiting a disease course similar to that observed in the rhesus model and humans. We also demonstrated that detection of the biomarkers protective antigen and capsule correlated with bacterial loads in the blood of these nonhuman primates. The ECL immunoassay described here is simple and sensitive enough to provide results in one to two hours, making this assay a viable option for use in the diagnosis of anthrax, leading to timely initiation of treatment, which is a key component of B. anthracis therapeutic development.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8590609/

CircANTXR1 Contributes to the Malignant Progression of Hepatocellular Carcinoma by Promoting Proliferation and Metastasis

Background Circular RNA (circRNA) is a key regulator for the malignant progression of cancer. However, the role of circRNA anthrax toxin receptor 1 (circANTXR1) in hepatocellular carcinoma (HCC) is still unclear. Methods Quantitative real-time PCR was performed to detect RNA expression. Cell proliferation, migration and invasion were determined using MTT assay, EdU staining, colony formation assay, wound healing assay and transwell assay. The protein levels of metastasis markers, x-ray repair cross complementing 5 (XRCC5) and exosome markers were examined using Western blot analysis. Xenograft tumor models were built to investigate the role of circANTXR1 in HCC tumorigenesis. The relationship between microRNA (miR)-532-5p and circANTXR1 or XRCC5 was confirmed by dual-luciferase reporter assay and RNA pull-down assay. The identification of exosomes were performed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Results CircANTXR1 was a stable and highly expressed circRNA in HCC. Silenced circANTXR1 inhibited the proliferation, migration and invasion of HCC cells in vitro, and suppressed HCC tumor growth in vivo. MiR-532-5p could be sponged by circANTXR1, and its inhibitor could reverse the inhibition of circANTXR1 silencing on HCC cells progression. In addition, we discovered that XRCC5 was a target of miR-532-5p. Furthermore, XRCC5 overexpression could reverse the suppressive effect of miR-532-5p overexpression on HCC cell proliferation, migration and invasion. Exosome was involved in the transport of circANTXR1 in HCC cells. Exosome circANTXR1 might be a potential serum biomarker for HCC patients. Conclusion CircANTXR1 promotes the progression of HCC through the miR-532-5p/XRCC5 axis, which might be a potential serum biomarker and therapeutic target of HCC. Background Circular RNA (circRNA) is a key regulator for the malignant progression of cancer. However, the role of circRNA anthrax toxin receptor 1 (circANTXR1) in hepatocellular carcinoma (HCC) is still unclear. Methods Quantitative real-time PCR was performed to detect RNA expression. Cell proliferation, migration and invasion were determined using MTT assay, EdU staining, colony formation assay, wound healing assay and transwell assay. The protein levels of metastasis markers, x-ray repair cross complementing 5 (XRCC5) and exosome markers were examined using Western blot analysis. Xenograft tumor models were built to investigate the role of circANTXR1 in HCC tumorigenesis. The relationship between microRNA (miR)-532-5p and circANTXR1 or XRCC5 was confirmed by dual-luciferase reporter assay and RNA pull-down assay. The identification of exosomes were performed using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Results CircANTXR1 was a stable and highly expressed circRNA in HCC. Silenced circANTXR1 inhibited the proliferation, migration and invasion of HCC cells in vitro, and suppressed HCC tumor growth in vivo. MiR-532-5p could be sponged by circANTXR1, and its inhibitor could reverse the inhibition of circANTXR1 silencing on HCC cells progression. In addition, we discovered that XRCC5 was a target of miR-532-5p. Furthermore, XRCC5 overexpression could reverse the suppressive effect of miR-532-5p overexpression on HCC cell proliferation, migration and invasion. Exosome was involved in the transport of circANTXR1 in HCC cells. Exosome circANTXR1 might be a potential serum biomarker for HCC patients. Conclusion CircANTXR1 promotes the progression of HCC through the miR-532-5p/XRCC5 axis, which might be a potential serum biomarker and therapeutic target of HCC. Introduction Hepatocellular carcinoma (HCC) refers to malignant tumors that occur from liver cells, and is the common pathological type of primary liver cancer. 1 , 2 HCC has the characteristics of high mortality, high invasiveness and easy recurrence. 3 , 4 Although a lot of efforts have been made, the prognosis of HCC is still not optimistic, and the number of patients is increasing year by year. 5 , 6 Therefore, it is necessary to understand the underlying mechanism of HCC development in order to determine more accurate and reliable biomarkers for HCC diagnosis and treatment. Circular RNAs (circRNAs) are a class of RNAs with regulatory functions, which have a closed circular structure and exist in large quantities in eukaryotic transcriptome. 7 , 8 Most of the circRNAs are composed of exon sequences, which are conserved in different species and have specific expression in tissues. 9 , 10 Most circRNAs function as microRNA (miRNA) sponges, which can interact with miRNAs to regulate target gene expression. 11 The high stability of circRNAs gives it obvious advantages in becoming a novel clinical diagnostic marker for human diseases including cancer. 12 , 13 Currently, many circRNAs have been found to participate in regulating HCC malignant progression, such as circ-5692, 14 circ_104075, 15 and circ_0001955. 16 Here, we screened the differentially expressed circRNAs in HCC tissues and normal tissues using GEO database, and showed that circ_0055033 (derived from anthrax toxin receptor 1 (circANTXR1) gene, also called circANTXR1) was remarkably upregulated in HCC tissues. Nevertheless, circANTXR1 role in HCC progression remains unclear. Exosomes are nanometer-sized (30–150 nm) extracellular vesicles, which are widely present and distributed in various body fluids. 17 Exosomes carry a variety of important signal molecules, which are closely related to the occurrence of various diseases. 18 Exosomes are a key medium of communication, and the proteins or RNA contained in them can trigger phenotypic changes in recipient cells. 19 Studies have shown that circRNA is relatively enriched and stable in exosomes, and exosomal circRNA may serve as a potential molecular target for disease diagnosis. 20 Therefore, the identification of potential exosomal circRNA is essential for the early diagnosis of cancer. Our study aims to investigate the role of circANTXR1 in HCC proliferation and metastasis, and further reveal its underlying molecular mechanism through the hypothesis of circRNA/miRNA/mRNA axis. In addition, we also extracted exosomes from HCC cells and patients' serum to confirm the potential of exosomes circANTXR1 as a diagnosis and treatment of HCC. Materials and Methods Samples Collection A total of 70 patients with HCC were recruited from The Affiliated Cancer Hospital of Zhengzhou University, and their peripheral blood, tumor tissues and paracancerous normal tissues were collected. The blood was centrifuged, and serum was collected and stored at −80°C for later use. Fifty healthy control subjects were recruited from our hospital for routine physical examination, and their peripheral blood was collected to extract serum. The research has been carried out in accordance with the World Medical Association Declaration of Helsinki, All the personnel signed the informed consent. Our research was approved from The Affiliated Cancer Hospital of Zhengzhou University. Cell Culture and Transfection HCC cells (HuH-7 and HCCLM3) and human liver epithelial cell line (THLE-2) were bought from Biovector NTCC (Beijing, China). HCCLM3 and HuH-7 cells were cultured in DMEM (Hyclone, Logan, UT, USA), while THLE-2 cells were grown in BEGM Bullet Kit (Lonza, Walkersville, MD, USA) at 37°C with 5% CO 2 . All the mediums were additionally supplemented with 10% FBS (Hyclone) and 1% double antibiotics (Invitrogen, Carlsbad, CA, USA). Cell transfection was carried out using Lipofectamine 3000 (Invitrogen). All oligonucleotides and vectors were synthesized from RiboBio (Guangzhou, China), including circANTXR1 small interference RNA, lentiviral short hairpin RNA and overexpression vector (si-circANTXR1#1/#2/#3, sh-circANTXR1 and oe-circANTXR1) or scrambled controls (si-NC, sh-NC and vector), miR-532-5p mimic and inhibitor (miR-532-5p and anti-miR-532-5p) or scrambled controls (miR-NC and anti-miR-NC), pcDNA XRCC5 overexpression vector (pcDNA-XRCC5) and scrambled control (pcDNA-NC). Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted using RNA simple (Tiangen, Beijing, China), and cDNA was synthesized with First Strand cDNA Synthesis Kit (Beyotime, Shanghai, China) or TaqMan Advanced miRNA cDNA Synthesis Kit (ABI, Foster City, CA, USA). PCR operation was performed using SYBR Premix Kit (Takara, Dalian, China). Relative expression was analyzed with 2 −ΔΔCT method with normalization to GAPDH or U6. Primer sequences were shown in Table 1 . Table 1 The Primer Sequences Used for qRT-PCR Gene Forward Sequence (5ʹ-3ʹ) Reverse Sequence (5ʹ-3ʹ) circANTXR1 TTTGAAGAAGTCCTGCATCG AGAGCCTGAAAGCCGTCAT ANTXR1 ACAGTTGGCTCACAAATTCATCA TCACTGGCCCTTTCAAATCCT miR-3681-5p TCGGCAGGTAGTCCATGATGCACT CTCAACTGGTGTCGTGGA miR-532-5p GGGCATGCCTTGAGTGTAG CAGTGCGTGTCGTGGAGT XRCC5 GTGCGGTCGGGGAATAAGG GGGGATTCTATACCAGGAATGGA GAPDH AGCCACATCGCTCAGACAC GCCCAATACGACCAAATCC U6 CTCGCTTCGGCAGCACA AACGCTTCACGAATTTGCGT Identification of circRNA Random primers and oligo (dT) 18 primers were used to determine whether circANTXR1 had poly-A tail, and RNase R assay was utilized to assess circANTXR1 stability. Briefly, circANTXR1 and linear ANTXR1 were amplified by random primers and oligo (dT) 18 primers, and then qRT-PCR was performed to measure RNA expression. In RNase R assay, the RNA isolated from HuH-7 and HCCLM3 cells was incubated with RNase R (Geneseed, Guangzhou, China). After that, circANTXR1 and linear ANTXR1 expression was determined using qRT-PCR. MTT Assay HuH-7 and HCCLM3 cells were collected and reseeded into 96-well-plates. The timing was started after the cells were attached to the well. Four treatment points were set: 0, 24, 48 and 72 h. At the indicated time points, MTT solution (Invitrogen) was added into cells for 4 h. Later, cells were hatched with DMSO solution (Solarbio, Beijing, China) for 10 min, and the optical density (OD) value was assessed using microplate reader at 570 nm. EdU Staining Basing on the instructions of EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime), HuH-7 and HCCLM3 cells were incubated with EdU staining and DAPI staining. Using a fluorescent microscope (200 ×), the cell fluorescent was visualized and the EdU positive cells (%) were counted. Colony Formation Assay Transfected HuH-7 and HCCLM3 cells were inoculated into 6-well plates to culture for 2 weeks. The colonies were fixed with 4% paraformaldehyde (Beyotime) and stained with crystal violet (Beyotime). Then, the colonies were imaged and its number was counted under a microscope. Wound Healing Assay HuH-7 and HCCLM3 cells were plated in 6-well plates (5 × 10 5 cells per well). When the cells reached 90% confluences, a 200 μL pipette tip was used to create a wound in the cell layer. After the cells were incubated with serum-free medium for 24 h, the wound area at 0 h and 24 h was photographed under a microscope (40 ×). The wound healing rate (%) was calculated according to the formula: (the wound area at 0 h - The wound area at 24 h)/ the wound area at 0 h. Transwell Assay HuH-7 and HCCLM3 cells were resuspended with serum-free medium and seeded into the upper of transwell chambers (BD Bioscience, San Jose, CA, USA). The lower chamber was filled with serum medium. The only difference between the detection of cell migration and invasion was that in the cell invasion assay, the upper of transwell chambers was pre-coated with a Matrigel (BD Biosciences). Twenty four h later, cells were fixed and stained, and the numbers of migrated and invaded cells were calculated under a microscope at 100 ×. Western Blot (WB) Analysis Total protein was obtained using RIPA lysis buffer (Sangon, Shanghai, China), and the BCA method (Beyotime) was used to quantify the protein. The protein was separated by 10% SDS-PAGE gel and transferred to PVDF membrane (Invitrogen). After blocked with skimmed milk, the membrane was incubated with primary and secondary antibodies. The antibodies were obtained from Abcam (Cambridge, MA, USA), including anti-E-cadherin (ab40772, 1:20,000), anti-N-cadherin (ab18203, 1:1,000), anti-Vimentin (ab137321, 1:2,000), anti-XRCC5 (ab80592, 1:5,000), anti-GAPDH (ab9485, 1:2,000), anti-CD63 (ab68418, 1:1,000), anti-HSP70 (ab79852, 1:5,000), anti-TSG101 (ab30871, 1:1,000), and Goat Anti-Rabbit IgG (ab205718, 1:50,000). The ECL Western Blotting Substrate (Solarbio) was applied to visualize the protein bands. Relative protein expression was analyzed by Image J software with GAPDH as a loading control. Mice Xenograft Models All animal work was approved by The Affiliated Cancer Hospital of Zhengzhou University. Animal studies were performed in compliance with the ARRIVE guidelines and the Basel Declaration. All animals received humane care according to the National Institutes of Health (USA) guidelines. Male BALB/c nude mice (Vital, Beijing, China) were randomly divided into 4 groups (n = 6). HuH-7 and HCCLM3 cells transfected with sh-NC or sh-circANTXR1 were resuspended with PBS, and then the cell suspensions (5 × 10 6 cells/0.2 mL PBS) were subcutaneously inoculated into the right flank of mice, respectively. Tumors volume was calculated every 7 days using tumor length × width 2 /2. After 35 days, mice were sacrificed through cervical dislocation and tumor was collected for weighting. In addition, paraffin sections were prepared from tumor tissues to perform hematoxylin-eosin (HE) staining and Ki67 immunohistochemical (IHC) staining using HE staining Kit (Beyotime) and Ki67 IHC Kit (Sangon). Dual-Luciferase Reporter Assay The sequences of circANTXR1 and XRCC5 3ʹUTR containing the binding sites or mutate sites of miR-532-5p were amplified and cloned into the psiCHECK-2 vector to build the corresponding wild-type (WT) and mutated-type (MUT) vectors. HuH-7 and HCCLM3 cells were co-transfected with vector and miR-532-5p mimic or miR-NC. Relative luciferase activity (Firefly/Renilla) was measured using Dual-Lucy Assay Kit (Solarbio). RNA Pull-Down Assay Biotin-labeled miR-532-5p probe and mutate probe (bio-miR-532-5p and bio-miR-532-5p MUT) or control probe (bio-NC) were transfected into HuH-7 and HCCLM3 cells. Forty eight h later, the cell was lysed and cell lysates were hatched with streptavidin magnetic beads (Invitrogen). Then, qRT-PCR was performed to examine RNA enrichment. Exosomes Isolation and Identification The exosomes from cells and serum samples were extracted according to the instructions of MagCapture™ Exosome Isolation Kit PS (Wako, Osaka, Japan). Exosome morphology was observed under transmission electron microscopy (TEM; JEOL, Tokyo, Japan), and exosome particle size was analyzed by nanoparticle tracking analysis (NTA) using Zeta Nanoparticle Tracking Analyzer (Merkel Technologies, Yehud, Israel). To determine the success of exosome isolation, WB analysis was utilized to determine exosome markers (CD63, HSP70 and TSG101) expression. Exosome and Cell Co-Culture HuH-7 cells were transfected with si-NC, si-circANTXR1#3, vector or oe-circANTXR1 for 48 h. After that, the cell exosomes were isolated, and termed as si-NC exo, si-circANTXR1 exo, vector exo or oe-circANTXR1 exo. The isolated exosomes were co-cultured with HCCLM3 cells for 48 h. Then, HCCLM3 cells were harvested for functional experiments. Statistical Analysis Data were expressed as mean ± standard deviation. GraphPad Prism 7.0 software (GraphPad, La Jolla, CA, USA) was used for statistical analyses. Results were analyzed by one-way analysis of variance or Student's t -test. Overall survival rate was analyzed by Kaplan-Meier analysis, and correlations were determined using Pearson's correlation coefficient. P 2 and P 50 42 20 22 Tumor size 0.0081* ≤5 cm 39 25 14 >5 cm 31 10 21 TNM stage 0.0271* I–II 43 26 17 III–IV 27 9 18 HBsAg 0.6903 Negative 7 4 3 Positive 63 31 32 Note : * P 2 and P 50 42 20 22 Tumor size 0.0081* ≤5 cm 39 25 14 >5 cm 31 10 21 TNM stage 0.0271* I–II 43 26 17 III–IV 27 9 18 HBsAg 0.6903 Negative 7 4 3 Positive 63 31 32 Note : * P < 0.05 a Chi-square test. Figure 1 CircANTXR1 was a stable circRNA with high expression in HCC. ( A ) GSE78520 and GSE97332 datasets exhibited the expression of circANTXR1 in HCC tumor tissues and normal tissues. ( B ) The information of circANTXR1 analyzed by circBase software were shown. ( C ) QRT-PCR was used to detect the circANTXR1 expression in HCC tumor tissues and paracancerous normal tissues. ( D ) The comparison of circANTXR1 expression between HCC tumor tissues (T) and paracancerous normal tissues (N) was shown. ( E ) CircANTXR1 expression in HCC cells (HuH-7 and HCCLM3) and THLE-2 cells was measured by qRT-PCR. ( F ) Kaplan-Meier analysis was used to analyze the correlation between circANTXR1 expression and overall survival rate of HCC patients. ( G and H ) Random primers and oligo (dT) 18 primers were used to explore whether circANTXR1 had poly-A tails. ( I and J ) RNase R assay was performed to assess the stability of circANTXR1. * P < 0.05. CircANTXR1 Silencing Inhibited the Proliferation, Migration and Invasion of HCC Cells To investigate the function of circANTXR1 in HCC, we silenced circANTXR1 expression in HCC cells using the siRNAs of circANTXR1. Our results showed that the 3 siRNAs of circANTXR1 could significantly inhibit the expression of circANTXR1 in HuH-7 and HCCLM3 cells, among which si-circANTXR1#3 had the best effect ( Figure 2A ). Therefore, si-circANTXR1#3 was used in the functional experiments. MTT assay results showed that the viability of HuH-7 and HCCLM3 cells could be suppressed by circANTXR1 knockdown ( Figure 2B and C ). Moreover, silenced circANTXR1 also reduced the EdU positive cells and the number of colonies in HuH-7 and HCCLM3 cells ( Figure 2D and E ). After knockdown of circANTXR1 in HuH-7 and HCCLM3 cells, the wound healing rate and the numbers of migrated and invaded cells were markedly inhibited ( Figure 2F – I ). By detecting the protein expression of metastasis markers (E-cadherin, N-cadherin and Vimentin), we found that downregulation of circANTXR1 could promote E-cadherin expression, while decrease N-cadherin and Vimentin expression in HuH-7 and HCCLM3 cells ( Figure 2J and K ). All data showed that circANTXR1 could facilitate HCC proliferation and metastasis. Figure 2 CircANTXR1 silencing inhibited the proliferation, migration and invasion of HCC cells. ( A ) The transfection efficiency of 3 siRNAs for circANTXR1 was confirmed by detecting circANTXR1 expression using qRT-PCR. ( B – K ) HuH-7 and HCCLM3 cells were transfected with si-NC or si-circANTXR1#3. MTT assay ( B and C ), EdU staining ( D ), colony formation assay ( E ), wound healing assay ( F and G ) and transwell assay ( H and I ) were used to measure cell viability, EdU positive cells, colony numbers, wound healing rate and the numbers of migrated and invaded cells. ( J and K ) The protein levels of E-cadherin, N-cadherin and Vimentin were determined by WB analysis. * P < 0.05. Knockdown of circANTXR1 Reduced the Tumorigenesis of HCC To further confirm the role of circANTXR1 in HCC, we constructed the xenograft models using HuH-7 and HCCLM3 cells transfected with sh-circANTXR1 or sh-NC. By analyzing the tumor volume curve, we found that the tumor volume of the sh-circANTXR1 group was significantly lower than that of the sh-NC group ( Figure 3A ). Also, the tumor weight of the sh-circANTXR1 group also was smaller than the sh-NC group ( Figure 3B ). The tumor pictures for each group were shown in Figure 3C . Moreover, we confirmed that circANTXR1 expression was significantly downregulated in the tumor tissues of the sh-circANTXR1 group ( Figure 3D ). In addition, HE staining was performed on the tumor tissues of each group, and IHC staining results also showed that Ki67 positive cells in the sh-circANTXR1 group was remarkably reduced ( Figure 3E ). These results revealed that circANTXR1 indeed played an active role in the tumor growth of HCC. Figure 3 Knockdown of circANTXR1 reduced the tumorigenesis of HCC. HuH-7 and HCCLM3 cells transfected with sh-NC or sh-circANTXR1 were injected into nude mice. ( A ) Tumor volume was measured every 7 days until 35 days. ( B ) Tumor weight in each group was detected after 35 days. ( C ) The tumor picture of each group was shown. ( D ) The expression of circANTXR1 in each group was examined by qRT-PCR. ( E ) The HE staining pictures and Ki67 IHC staining pictures were exhibited. * P < 0.05. CircANTXR1 Acted as a Sponge of miR-532-5p In order to determine the targeted miRNA of circANTXR1, we used starbase ( http://starbase.sysu.edu.cn/ ) and circbank ( http://www.circbank.cn/ ) software to jointly predict miRNAs that could interact with circANTXR1, and found that 2 miRNAs (miR-532-5p and miR-3681-5p) had complementary binding sites with circANTXR1 ( Figure 4A ). Subsequently, we examined the expression of miR-532-5p and miR-3681-5p in si-circANTXR1#3-transfected HuH-7 and HCCLM3 cells and found that only the expression of miR-532-5p was significantly increased ( Figure 4B and C ). Therefore, miR-532-5p was selected as the target miRNA of circANTXR1 for our study. The binding sites and mutate sites between circANTXR1 and miR-532-5p were shown in Figure 4D . After confirming that miR-532-5p mimic could increase miR-532-5p expression in HuH-7 and HCCLM3 cells ( Figure 4E ), we transfected with circANTXR1 WT/MUT vector and miR-532-5p mimic into HuH-7 and HCCLM3 cells. Our data indicated that miR-532-5p mimic markedly inhibited the luciferase activity of circANTXR1 WT vector, while not effect on that of the circANTXR1 MUT vector ( Figure 4F and G ). Meanwhile, the enrichment of circANTXR1 also was increased in the bio-miR-532-5p probe rather than the bio-miR-532-5p MUT probe ( Figure 4H ). Additionally, miR-532-5p was lowly expressed in HCC tumor tissues and cells ( Figure 4I and J ), and its expression was negatively correlated with circANTXR1 expression in HCC tumor tissues ( Figure 4K ). Therefore, we confirmed that circANTXR1 could sponge miR-532-5p in HCC. Figure 4 CircANTXR1 acted as a sponge of miR-532-5p. ( A ) Venn Diagram showed the targeted miRNAs of circANTXR1 using starbase and circbank software. ( B and C ) In HuH-7 and HCCLM3 cells transfected with si-NC or si-circANTXR1#3, the expression of miR-3681-5p and miR-532-5p was measured by qRT-PCR. ( D ) The binding sites and mutate sites between circANTXR1 and miR-532-5p were shown. ( E ) The transfection efficiency of miR-532-5p mimic was confirmed by measuring miR-532-5p expression using qRT-PCR. Dual-luciferase reporter assay ( F and G ) and RNA pull-down assay ( H ) were utilized to verify the interaction between circANTXR1 and miR-532-5p. ( I and J ) The expression of miR-532-5p in HCC tumor tissues and cells was evaluated by qRT-PCR. ( K ) Pearson's correlation coefficient was used to assess the correlation between circANTXR1 and miR-532-5p. * P < 0.05. Inhibition of miR-532-5p Reversed the Regulation of circANTXR1 Knockdown on HCC Progression The anti-miR-532-5p was constructed and it was confirmed that anti-miR-532-5p indeed reduced miR-532-5p expression in HuH-7 and HCCLM3 cells ( Figure 5A ). To explore whether circANTXR1 regulated HCC progression by sponging miR-532-5p, we co-transfected with si-circANTXR1#3 and anti-miR-532-5p into HuH-7 and HCCLM3 cells. Function analysis results suggested that the suppressive effect of circANTXR1 silencing on cell viability, the number of colonies and EdU positive cells could be abolished by miR-532-5p inhibitor ( Figure 5B – E ). Also, miR-532-5p inhibitor reversed the inhibition of circANTXR1 knockdown on the wound healing rate and the numbers of migrated and invaded cells ( Figure 5F – H ). Moreover, the regulation of circANTXR1 knockdown on the protein levels of E-cadherin, N-cadherin and Vimentin also could be reversed by miR-532-5p inhibitor ( Figure 5I and J ). Hence, our data showed that circANTXR1 sponged miR-532-5p to mediate HCC progression. Figure 5 Inhibition of miR-532-5p reversed the regulation of circANTXR1 knockdown on HCC progression. ( A ) QRT-PCR was performed to measure miR-532-5p expression to assess the transfection efficiency of miR-532-5p inhibitor. ( B – J ) HuH-7 and HCCLM3 cells were transfected with si-NC, si-circANTXR1#3, si-circANTXR1#3 + anti-miR-NC or si-circANTXR1#3 + anti-miR-532-5p. Cell viability, colony numbers, EdU positive cells, wound healing rate and the numbers of migrated and invaded cells were determined using MTT assay ( B and C ), colony formation assay ( D ), EdU staining ( E ), wound healing assay ( F ) and transwell assay ( G and H ). ( I and J ) WB analysis was performed to detect the protein levels of E-cadherin, N-cadherin and Vimentin. * P < 0.05. XRCC5 Could Be Targeted by miR-532-5p Using the starbase tool, we found that the 3ʹUTR of XRCC5 had binding sites for miR-532-5p ( Figure 6A ). Further analysis revealed that the luciferase activity of XRCC5 3ʹUTR WT vector rather than the corresponding MUT vector could be reduced by miR-532-5p mimic ( Figure 6B and C ), and XRCC5 enrichment also was higher in the bio-miR-532-5p probe compared to the bio-miR-532-5p-MUT probe ( Figure 6D ). Also, the mRNA and protein expression of XRCC5 could be inhibited by miR-532-5p mimic ( Figure 6E and F ). In HCC tumor tissues and cells, we discovered that XRCC5 expression was significantly higher than in corresponding controls at the mRNA level and protein level ( Figure 6G – J ). And XRCC5 mRNA expression in HCC tumor tissues also was negatively correlated with miR-532-5p expression ( Figure 6K ). Furthermore, we found that circANTXR1 silencing could decrease XRCC5 mRNA and protein expression, while these effects could be reversed by miR-532-5p inhibitor ( Figure 6L and M ). Correlation analysis revealed that there was a positive correlation between XRCC5 expression and circANTXR1 expression in HCC tumor tissues ( Figure 6N ). These results suggested that circANTXR1 positively regulated XRCC5 by sponging miR-532-5p. Figure 6 XRCC5 could be targeted by miR-532-5p. ( A ) The binding sites and mutate sites between XRCC5 3ʹUTR and miR-532-5p were exhibited. The interaction between XRCC5 and miR-532-5p was confirmed by dual-luciferase reporter assay ( B and C ) and RNA pull-down assay ( D ). ( E and F ) In HuH-7 and HCCLM3 cells transfected with miR-NC or miR-532-5p mimic, XRCC5 mRNA and protein expression was measured by qRT-PCR and WB analysis. ( G – J ) QRT-PCR and WB analysis were used to determine XRCC5 mRNA and protein expression in HCC tumor tissues and cells. 6H showing 3 representative bands for each group in the Western blot image. ( K ) The correlation between XRCC5 and miR-532-5p in HCC tumor tissues was assessed using Pearson's correlation coefficient. ( L and M ) The mRNA and protein expression of XRCC5 was examined by qRT-PCR and WB analysis in HuH-7 and HCCLM3 cells transfected with si-NC, si-circANTXR1#3, si-circANTXR1#3 + anti-miR-NC or si-circANTXR1#3 + anti-miR-532-5p. ( N ) Pearson's correlation coefficient was performed to evaluate the correlation between XRCC5 and circANTXR1. * P < 0.05. Overexpressed XRCC5 Partially Reversed the Inhibition of miR-532-5p on HCC Progression After that, the pcDNA XRCC5 overexpression vector was built and its transfection efficiency was confirmed by detecting XRCC5 expression in HuH-7 and HCCLM3 cells after transfection ( Figure 7A ). In HuH-7 and HCCLM3 cells transfected with miR-532-5p mimic and pcDNA-XRCC5, we found that miR-532-5p overexpression could repress cell viability, the number of colonies and the EdU positive cells, while pcDNA-XRCC5 could reverse these effects ( Figure 7B – E ). Overexpression of XRCC5 also could abolish the inhibitory effect of miR-532-5p on the wound healing rate and the numbers of migrated and invaded cells ( Figure 7F – H ). Besides, miR-532-5p promoted E-cadherin protein level and inhibited N-cadherin and Vimentin protein levels. However, these effects also could be overturned by XRCC5 overexpression ( Figure 7I and J ). Hence, our data illumined that miR-532-5p targeted XRCC5 to suppress HCC progression. Figure 7 Overexpressed XRCC5 partially reversed the inhibition of miR-532-5p on HCC progression. ( A ) WB analysis was used to detect XRCC5 protein expression to evaluate the transfection efficiency of pcDNA-XRCC5. ( B – J ) HuH-7 and HCCLM3 cells were transfected with miR-NC, miR-532-5p, miR-532-5p+ pcDNA-NC or miR-532-5p + pcDNA-XRCC5. MTT assay ( B and C ), colony formation assay ( D ), EdU staining ( E ), wound healing assay ( F ) and transwell assay ( G and H ) were performed to determine cell viability, colony numbers, EdU positive cells, wound healing rate and the numbers of migrated and invaded cells. ( I and J ) The protein levels of E-cadherin, N-cadherin and Vimentin were assessed by WB analysis. * P < 0.05. Exosome Mediated the Intercellular Transmission of circANTXR1 in HCC Cells After extracted exosome from HuH-7 cells, the morphology of exosomes was observed under TEM ( Figure 8A ). NTA analysis showed that the particle size of isolated exosomes was mostly about 100 nm ( Figure 8B ). The expression of exosome markers (CD63, HSP70, and TSG101) was detected in the isolated exosomes from HuH-7 and HCCLM3 cells, which confirmed that the extraction of exosomes from cells was successful ( Figure 8C ). Through measuring circANTXR1 expression, we confirmed that circANTXR1 was markedly upregulated in the exosomes isolated from HuH-7 cells compared to HCCLM3 cells ( Figure 8D ). Therefore, HuH-7 cells were used to extract exosome. After HuH-7 cells were transfected with si-circANTXR1#3 or oe-circANTXR1, the cell exosomes were isolated. By detecting circANTXR1 expression in exosome, we confirmed that circANTXR1 expression was decreased in si-circANTXR1 exo and was increased in oe-circANTXR1 exo ( Supplementary Figure 2A ). Then, both exosomes were co-cultured with HCCLM3 cells for 48 h. Our data revealed that silenced exosome circANTXR1 could decrease circANTXR1 expression, and overexpressed exosome circANTXR1 markedly enhanced circANTXR1 expression in HCCLM3 cells ( Figure 8E ). Additionally, si-circANTXR1 exo treatment also promoted miR-532-5p expression and inhibited XRCC5 mRNA and protein expression, while overexpressed exosome circANTXR1 had an opposite regulation ( Supplementary Figure 2B – D ). Function analysis results showed that knockdown of exosome circANTXR1 could inhibit EdU positive cells, the number of colonies, the wound healing rate and the numbers of migrated and invaded cells, while overexpression of exosome circANTXR1 had an opposite effect ( Figure 8F – J ). Also, the treatment of si-circANTXR1 exo also promoted E-cadherin expression and restrained N-cadherin and Vimentin expression in HCCLM3 cells ( Figure 8K ). In addition, we found that E-cadherin expression was reduced, while N-cadherin and Vimentin expression was increased in HCCLM3 cells co-cultured with oe-circANTXR1 exo ( Figure 8K ). These results confirmed that circANTXR1 mainly existed in exosomes and that exosome circANTXR1 mediated intercellular communication. Figure 8 Exosome mediated the intercellular transmission of circANTXR1 in HCC cells. ( A ) Exosomes isolated from HuH-7 cells were observed under TEM. ( B ) NTA was used to analyze the particle size of exosomes. ( C ) The CD63, HSP70 and TSG101 proteins in exosomes were observed using WB analysis. ( D ) The expression of circANTXR1 was measured by qRT-PCR in exosomes isolated from HuH-7 and HCCLM3 cells. ( E – K ) HuH-7 cells transfected with si-NC, si-circANTXR1#3, vector or oe-circANTXR1. The exosomes (si-NC exo, si-circANTXR1 exo, vector exo or oe-circANTXR1 exo) isolated from transfected HuH-7 cells were co-cultured with HCCLM3 cells for 48 h. ( E ) The expression of circANTXR1 in HCCLM3 cells was measured by qRT-PCR. EdU staining ( F ), colony formation assay ( G ), wound healing assay ( H ) and transwell assay ( I and J ) were used to detect EdU positive cells, colony numbers, wound healing rate and the numbers of migrated and invaded cells. ( K ) WB analysis was utilized for measuring the protein levels of E-cadherin, N-cadherin and Vimentin. * P < 0.05. Exosome circANTXR1 Could Serve as a Potential Serum Biomarker for HCC Patients In addition, we isolated exosomes from HCC patients, and TEM analysis showed the morphology of exosomes ( Figure 9A ). Using the NTA analysis, we confirmed that the particle size of exosomes was mostly about 120 nm ( Figure 9B ). By detecting the expression of exosome markers CD63, HSP70 and TSG101, we determined that the isolation of exosomes was successful ( Figure 9C ). We analyzed the expression of circANTXR1 in serum exosomes from HCC patients and determined that circANTXR1 was significantly overexpressed compared with that of in healthy control ( Figure 9D ). ROC curve analysis indicated that the area under the ROC curve (AUC) was 0.76, suggesting that the serum exosome circANTXR1 level had clinical diagnostic significance for HCC patients ( Figure 9E ). These results suggested that serum exosome circANTXR1 might be a diagnostic biomarker for HCC. Figure 9 Exosome circANTXR1 could serve as a potential serum biomarker for HCC patients. ( A ) Exosomes isolated from the serum of HCC patients were observed under TEM. ( B ) The particle size of exosomes was analyzed by NTA. ( C ) The CD63, HSP70 and TSG101 proteins in exosomes from HCC patients were detected by WB analysis. ( D ) The expression of circANTXR1 in the exosomes from the serum of healthy control and HCC patients was measured by qRT-PCR. ( E ) ROC curve analysis was used to evaluate the clinical diagnostic significance of serum exosome circANTXR1 in HCC patients. * P < 0.05. Discussion Differential expression of circRNAs often indicates that they have different roles in disease. In HCC-related studies, circTP63 was found to be upregulated in HCC, and was considered to be a tumor promoter to accelerate HCC proliferation and metastasis via the miR-155-5p/ZBTB18 network. 21 On the contrary, Ma et al reported that circ_0014717 was downregulated in HCC, which could suppress HCC growth and metastasis by reducing BTG2 through sponging miR-668-3p. 22 Here, we screened a new circRNA, circANTXR1. Our analysis showed that the high expression of circANTXR1 was related to the poor prognosis of HCC patients. Functional analysis results indicated that circANTXR1 knockdown hindered HCC cell proliferation and metastasis in vitro, and reduced HCC tumorigenic ability in vivo. These results confirmed the positive role of circANTXR1 in HCC progression, suggesting that circANTXR1 might be a potential therapeutic target for HCC. Studies have suggests that circRNA is a natural miRNA sponge. In this, we proposed that circANTXR1 might be a sponge for miR-532-5p. In previous studies, miR-532-5p was found to be abnormally expressed in many cancers and to have different effects in different cancers. Increasing evidence showed that miR-532-5p could suppress cancer progression by inhibiting proliferation and metastasis, including lung cancer, 23 bladder cancer, 24 and colorectal cancer. 25 On the contrary, Huang et al suggested that miR-532-5p could promote breast cancer proliferation and migration, and it might play an pro-cancer role in breast cancer. 26 In HCC, many researches verified that miR-532-5p was underexpressed in HCC, and it could restrain HCC proliferation, migration and invasion to inhibit cancer development. 27–30 Similar to these results, our study also revealed that miR-532-5p might play a tumor suppressor role in HCC by inhibiting HCC cell proliferation and metastasis. The reversal effect of anti-miR-532-5p on si-circANTXR1-mediated HCC progression showed that circANTXR1 regulated HCC progression via sponging miR-532-5p. This was an exciting discovery for us. XRCC5 is a DNA double-strand break repair gene, and its abnormal expression is closely related to cancer development. 31 High XRCC5 expression had been confirmed to promote HCC proliferation and metastasis, and could predict patients' poor prognosis. 32 , 33 Our data revealed that miR-532-5p could target XRCC5, an oncogene. The expression of XRCC5 was not only negatively regulated by miR-532-5p, but also positively correlated with circANTXR1 expression. Furthermore, overexpressed XRCC5 abolished miR-532-5p-inhibited HCC proliferation and metastasis, suggesting that miR-532-5p indeed targeted XRCC5 to repress HCC progression. These results confirmed the existence of circANTXR1/miR-532-5p/XRCC5 axis and improved the mechanism of circANTXR1 regulating HCC malignant progression. At present, the role of exosomes in cancer has received more and more attention, and exosomal circRNAs have shown great advantages in the treatment and diagnosis of cancer. 20 In this, we found that circANTXR1 was expressed in exosomes from HCC cells, and the overexpressed exosome circANTXR1 promoted HCCLM3 cell proliferation and metastasis. These results confirmed that exosomes were involved in the intercellular transport of circANTXR1 to affect HCC cell biological functions. In the serum exosomes from patients with HCC, we confirmed that circANTXR1 was significantly overexpressed. Further ROC analysis confirmed the clinical significance of serum exosome circANTXR1 levels in HCC patients. In summary, our results suggested that circANTXR1 was a novel circRNA that mediated HCC progression. Our study revealed that circANTXR1 facilitated HCC proliferation and metastasis via upregulating XRCC5 by sponging miR-532-5p. These results showed that targeted inhibition of circANTXR1 might be an effective strategy for HCC treatment. Importantly, our results also indicated that exosomal circANTXR1 could be used as an indicator for early diagnosis of HCC. Disclosure The authors declare that they have no conflicts of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842784/

Preparation of a Bacteriophage T4-based Prokaryotic-eukaryotic Hybrid Viral Vector for Delivery of Large Cargos of Genes and Proteins into Human Cells

A viral vector that can safely and efficiently deliver large and diverse molecular cargos into cells is the holy grail of curing many human diseases. Adeno-associated virus (AAV) has been extensively used but has a very small capacity. The prokaryotic virus T4 has a large capacity but lacks natural mechanisms to enter mammalian cells. Here, we created a hybrid vector by combining T4 and AAV into one nanoparticle that possesses the advantages of both. The small 25 nm AAV particles are attached to the large 120 nm x 86 nm T4 head through avidin-biotin cross-bridges using the phage decoration proteins Soc (small outer capsid protein) and Hoc (highly antigenic outer capsid protein). AAV thus "piggy-backed" on T4 capsid, by virtue of its natural ability to enter many types of human cells efficiently acts as a "driver" to deliver large cargos associated with the T4 head. This unique T4-AAV hybrid vector approach could pave the way for the development of novel therapeutics in the future.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071550/

A Computational Investigation of In Vivo Cytosolic Protein Delivery for Cancer Therapy

The ability to specifically block or degrade cytosolic targets using therapeutic proteins would bring tremendous therapeutic opportunities in cancer therapy. Over the last few years, significant progress has been made with respect to tissue targeting, cytosolic delivery, and catalytic inactivation of targets, placing this aim within reach. Here, we developed a mathematical model specifically built for the evaluation of approaches towards cytosolic protein delivery, involving all steps from systemic administration to translocation into the cytosol and target engagement. Focusing on solid cancer tissues, we utilized the model to investigate the effects of microvascular permeability, receptor affinity, the cellular density of targeted receptors, as well as the mode of activity (blocking/degradation) on therapeutic potential. Our analyses provide guidance for the rational optimization of protein design for enhanced activity and highlight the importance of tuning the receptor affinity as a function of receptor density as well as the receptor internalization rate. Furthermore, we provide quantitative insights into how enzymatic cargoes can enhance the distribution, extent, and duration of therapeutic activity, already at very low catalytic rates. Our results illustrate that with current protein engineering approaches, the goal of delivery of cytosolic delivery of proteins for therapeutic effects is well within reach. 1. Introduction In comparison to the rapid growth of the arsenal of protein-based therapies targeting extracellular receptors, the development of therapies in which proteins address an intracellular target lags far behind. Only three have been approved so far: the recombinant immunotoxins denileukin diftitox (Ontak), tagraxofusp, and moxetumomab pasudotox [ 1 , 2 , 3 ]. All act by delivering catalytic protein domains that block protein synthesis. These isolated examples made it into the clinic because they combine an extremely high potency with relatively well-accessible targets in T cell lymphomas and B cell leukemias. This particular set of properties is shared with many more similar agents currently in clinical development [ 4 ]. In contrast, few therapeutic proteins acting inside the cell are far in development for solid tumors or other diseases in which cells in a tissue need to be reached [ 4 ]. Beyond recombinant immunotoxins, there are ample potential applications for cytosolically delivered proteins that can be divided into those that (temporarily) add a function and those that block a pathway as inhibitors. Examples for the former include the delivery of enzymes [ 5 ] or proteins that reprogram or genetically modify cell populations in vivo [ 6 , 7 ]. The latter can be achieved either via direct protein inhibition or, as has attracted more attention recently, through enzymatic target modification, which, for instance, was achieved for RAS oncoproteins [ 8 ]. It can, in principle, also occur via targeted protein degradation [ 9 ], although in vivo targeted protein degradation using engineered proteins has not yet been reported. Blocking type applications can be especially powerful for cancer therapies or senescent cell removal, where blocking a pathway can drive cells that rely on this pathway for survival into apoptosis. Through binding of large interaction surfaces, proteins can easily mediate levels of specificity and modes of activity that are very difficult if not impossible to achieve with small molecules [ 10 ], thus massively expanding the druggable genome [ 11 ]. During the last years, there has been a steady development of approaches to deliver proteins into the cytosol of cells cultured in 2D in vitro systems [ 12 , 13 , 14 , 15 ]. However, there has been little success to translate these results into activity in vivo. Due to its challenges, access of therapeutic proteins to intracellular targets has been referred to as "high-hanging fruit": highly desirable, but difficult to achieve [ 16 ]. This lack of progress may be attributed to the fact that in vivo protein delivery to cells that reside outside of the bloodstream presents several additional challenges. These challenges include extravasation in the organ of interest, adequate penetration into the tissue, and accumulation at the target cells [ 17 ]. For cytosolic delivery, as opposed to endosomal delivery, there additionally needs to be a moiety that enables the protein to escape endosomes; for instance, an endosomal escape peptide or a bacterial translocation domain [ 18 ]. Currently, there are several excellent computational models available that can be used to simulate the delivery of agents that bind to extracellular targets or to tumor tissues in general [ 19 , 20 ]. In contrast, in-depth modeling approaches to understand and define the requirements to yield effective cytosolic protein delivery for therapeutic applications have, to the best of our knowledge, not yet been reported. Here, we present a mathematical model specifically built for the evaluation of protein engineering approaches directed at cytosolic protein delivery in in vivo solid tumor tissues. We employ this model to investigate how microvascular permeability, molecular size, affinity for a cellular target receptor, density of targeted receptors, and finally, mode of action, influence the drug activity. We focused our investigations on solid tumors, while reflecting on the consequences for protein-based therapeutics in various therapeutic areas. Our results demonstrate that for cytosolic protein delivery for a given area of application, in vivo activity can be rationally optimized through the proper tuning of parameters that are readily controllable through protein engineering and/or proper dosing regimes. 2. Materials and Methods A technical explanation of the model, outcome parameters, as well as the rationale for the choice of input parameters and model assumptions is provided as a Supplementary Materials section. 3. Results 3.1. Modeling Protein Delivery To generate a quantitative understanding of the requirements for effective in vivo cytosolic protein delivery, we developed a mathematical model incorporating key elements of cellular targeting, entry, and intracellular activity. The model was, in part, inspired by work from Thurber et al., in which antibody delivery in vivo was described within a Krogh geometry consisting of two concentric cylinders; the inner cylinder represented a capillary, and the outer represented the surrounding tissue ( Figure 1 ) [ 19 ]. The model permits the in silico evaluation of all physical and biological phenomena that we reasoned would significantly influence cytosolic protein delivery and its therapeutic effects. Those parameters that relate to the engineered protein are size, affinity, intracellular binding, plasma half-life, half-life in the cytosol, as well as the effects of enzymatic target modification on the levels of functional cytosolic target proteins. Those parameters that relate more to the targeted tissue are vascular permeability, interstitial diffusivity, cellular internalization rates, and cytosolic delivery efficiency ( Figure 1 ). An extensive description of how the model was designed and the rationale for the choice of all parameters is provided in the Supplementary Materials section. As a representative tissue, we focused on solid tumors of the breast overexpressing the tumor marker epithelial cell adhesion molecule (EpCAM), while in some instances, we used normal skeletal muscle tissue for comparison. We made a distinction between tumors that exhibited convection, i.e., those with functional lymph vessels, and those that did not, and therefore exhibited little or no fluid flow and where macromolecule transport is driven by diffusion. Results are, in most cases, described as the degree of inhibition as a function of time (i.e., inhibition of a cytosolic target protein), maximum inhibition (i.e., single time-point across the entire tissue), or as an inhibitory effect (effect integrated over time and entire tissue). A technical explanation of how these terms were calculated is given in the Supplementary Materials . 3.2. Simulation of Delivery and Therapeutic Effects In Vivo We initially simulated delivery of a protein with a molecular weight of 70 kDa binding to the tumor marker EpCAM. Binding properties were taken from the designed ankyrin repeat protein (DARPin) Ec1, which binds EpCAM with an affinity (K d ) of 68 pM [ 21 ]. We assumed a tumor tissue containing a cell density of 2.9 × 10 8 cells/mL, representing a tumor with a high cellularity, and a receptor density of 5.4 × 10 5 receptors/cell, which we previously determined for EpCAM on MCF-7 cells [ 22 ]. As indicated above, we thus considered the tissue as a homogenous medium in which cellular structures are not explicitly defined. We simulated a starting protein plasma concentration of 1 μM, a concentration that can be realistically reached upon intravenous administration of a therapeutic protein [ 23 ] ( Figure 2 A). A sharp decline in the delivered protein concentration away from the capillary was observed, with the majority of protein being confined to the first 20 μm of tissue. When an EpCAM binder is combined with a moiety or vector that mediates efficient cytosolic delivery, e.g., an endosomal escape peptide or a bacterial toxin-derived translocation domain as we reported previously [ 24 ] (see Supplementary Table S2 for detailed information on assumptions), the simulated degree of target inhibition (see Supplements for a technical explanation) varies across different distances from the capillary ( Figure 2 B) and directly reflects protein delivery ( Figure 2 A), with maximum inhibition in the tissue reached at around 24 h ( Supplementary Video S1 ). We then evaluated the maximum cytosolic delivery and inhibition with a targeted protein ( Figure 2 C,D) or a targeted peptide ( Figure 2 E,F) in tumors with and without convection and compared it with delivery in normal tissue (skeletal muscle was chosen as a representative tissue because of the availability of experimental values of needed parameters in the literature). The relevant difference between both types of compounds is size, and we modelled a molecular weight (MW) of 3.5 kDa for the peptide, which is the MW of the calcitonin peptide hormone. Calcitonin is a medically used peptide for which half-lives have been determined in vivo in humans [ 25 ] ( Supplementary Table S3 ). Employing a targeted peptide resulted in more rapid clearance but also faster penetration due to a higher interstitial diffusivity. We also included peptides because for this class of compounds, several strategies for cytosolic delivery have been described. For both proteins and peptides, cytosolic delivery and inhibition in tumors vastly surpassed that achieved in muscle tissue in terms of tissue reached, mostly due to the enhanced permeability of leaky blood vessels. Convection additionally contributed substantially to an enhanced depth of penetration for proteins ( Figure 2 C). While targeted proteins were delivered in high concentrations to the tissue immediately adjacent to the capillary but rapidly decreased in concentration further away, peptides permeated the entire tissue more evenly by comparison, although in lower levels ( Figure 2 C,E). 3.3. Effect of Receptor Affinity on Peptide and Protein Delivery The binding-site barrier is a phenomenon where high-affinity targeted agents bind tightly to receptors in the first cell layers encountered and only travel further into the tissue upon receptor saturation. The affinity for receptors, the receptor density, and the receptor internalization rate are well-established factors with respect to the binding-site barrier [ 19 , 28 , 29 ], although quantitative insights into the impact of the binding-site barrier on tissue penetration under different conditions are lacking. One approach to minimize the negative consequences of the binding-site barrier is to modulate the affinity of a targeted agent towards its cellular receptor. Importantly, a lower affinity may benefit tissue penetration. First, we determined the optimum receptor affinity for tumor targeting with or without convection for a fixed receptor density for both targeted proteins and peptides ( Figure 3 ), as determined by maximal delivery to the tissue. Maximal delivery was defined as the situation when the average concentration across the tissue of the free target protein was at its lowest. Convection facilitates the rapid transport of macromolecules from the immediate proximity of the endothelium deeper into the tissue, and its effect on protein delivery was therefore of particular interest. We found that higher affinities for targeted proteins were needed for the optimal delivery in tumors without convection. The presence of convection in tumors greatly reduced the optimal affinity and enhanced the overall therapeutic effect ( Figure 3 A). For peptide delivery, on the other hand, almost identical optimal affinities were observed in the presence or absence of convection, and delivery only mildly increased in the presence of convection. When comparing peptides vs. proteins, peptides produced superior delivery in tumors without convection due to the higher permeability and diffusivity, while proteins surpass peptides in tumors with convection due to their longer plasma half-life (see also Figure 2 C,E). The steeper decline in delivered molecules observed for proteins vs. peptides indicates a more pronounced binding-site barrier for slower-diffusing proteins. Targeted proteins can easily be engineered to be highly selective and have affinities that are considered optimal by our modeling approach ( Figure 3 A). However, for peptides, the ability to obtain high affinities appears to be a limiting factor for their efficacy ( Figure 3 B), although peptides with very high affinities have been reported [ 30 ], including peptides that bind with subnanomolar K d values to vascular endothelial growth factor (VEGF) receptor 2 and c-MET, or hapten peptides that bind to single chain variable fragments with affinities as high as 2.3 nM [ 31 ]. Interestingly, for targeted proteins, the optimal affinities for targeting tumors with and without convection, of 16.4 nM and 625 pM, respectively, for these simulated parameters, appear to be readily achievable, as demonstrated by the superimposition of the frequency distribution of protein affinities reported in the literature and compiled in the kinetics database KOFFI [ 30 ]. When investigating the effect of receptor density for our default conditions (i.e., using a proteinaceous EpCAM binder with an affinity of 68 pM), we noted a very strong dependency of the therapeutic effect on the receptor density ( Supplementary Figure S3 ). This mirrors the effects of modulating receptor affinity (see Figure 3 A) and reflects the characteristics of the binding-site barrier. Therapeutic effects were highest at around 1–2 × 10 5 receptors/cell ( Supplementary Figure S3 ). 3.4. Interplay Between Receptor Affinity, Receptor Density and Internalization Rate At present, we are not aware of reports that relate the optimal affinity to receptor density and the receptor internalization rate. Ultimately, this knowledge may guide the choice of suitable receptors for targeting in the development of cancer therapies. Next to target affinity and receptor density, the internalization rate of cellular receptors plays a prominent role in determining the properties of the binding-site barrier [ 29 ]. This is illustrated by determining the affinity that yields the greatest therapeutic effect (i.e., optimum affinity) as a function of receptor density for receptors that differ in internalization rates, either in the presence or absence of convection ( Figure 4 A). Although internalization, recycling rates, and even trafficking routes for receptors can differ as a function of the ligand utilized for targeting (e.g., natural ligands vs. engineered binders), as well as the epitope addressed on a specific receptor, our simulations indicated that receptors which generally internalize faster, e.g., epidermal growth factor receptor (EGFR), necessitate a lower optimal affinity for maximal tissue delivery. Again, convection decreased the optimal affinity for delivery for all internalization rates and also increased the internalization rate-dependent differences ( Figure 4 ). In our model, we simulated steady state levels of receptors by matching the rate of internalization with the rate of recycling, thus mimicking the delivery of non-toxic proteins (for details, see the Supplementary Materials ). A fraction of targeted agents was delivered to the cytosol in the model, with the rest being degraded. Even in the presence of a moiety that mediates endosomal escape, degradation in the endolysosomal system is a common fate of endocytosed proteins, and only a fraction of internalized protein reaches the cytosol [ 18 ]. Remarkably, for a binder exhibiting the optimal affinity, the same overall magnitude of delivery and inhibition can be achieved largely irrespective of the receptor density and internalization rate ( Figure 4 B). This implies that targeting should not only focus on those receptors which are present at very high levels and for which very high affinity binders are available, which would increase the options for productive targeted drug delivery. Nonetheless, specificity and potential off-target effects should always be taken into consideration. Notably, for diffusion-based delivery, maximal delivery is always lower, emphasizing the relevance of convection in targeted delivery to tumors. 3.5. Effect of Cold Dosing, Targeted Protein Degradation and Degradation-Resistant Proteins on the Binding-Site Barrier As illustrated by our analyses so far, protein targeting deep into tissues faces several dilemmas. Small size, on the one hand, favors penetration, but on the other hand, leads to faster clearance. A high receptor affinity promotes effective cellular targeting, although limits penetration due the rapid capture of extravasated proteins by the first layer of cells, also referred to as the binding-site barrier. As an approach to overcome the binding-site barrier, cold dosing, where the therapeutic moiety is only present on a fraction of administered targeted agents, has been investigated for a long time. Mostly, this has been performed in the context of radiolabeled antibody [ 34 , 35 ] but, more recently, also in the context of antibody–drug conjugates [ 36 , 37 ]. In addition, there are multiple protein engineering approaches aimed to enhance intracellular delivery and/or subsequent therapeutic effects, some of which are only recently coming to the fore for engineered proteins. The therapeutic potency can be increased without affecting delivery itself, through either delivery of (target protein-inactivating) enzymes [ 8 , 38 ], targeted protein degradation [ 39 ], or by enhancing the stability of the delivered agent against proteasomal degradation [ 40 , 41 ]. In our simulations, catalytic inactivators represent both enzymes that inactivate target proteins and protein-based agents that induce target protein degradation, because for both types of activities, the functional outcome is the same. Given our assumptions (see Supplementary Materials ), catalytic agents strongly outperform binders whose effect is limited to inhibition by direct blockade of the protein—protein interaction, in particular at lower concentrations ( Figure 5 A) or when the target protein concentration is high ( Figure 5 B). For instance, at a target protein level of 1.0 × 10 5 molecules/cell and using catalytic inactivators, a very strong and pronounced inhibition can be observed as compared to inhibition by binding only ( Figure 5 B). Irrespective of the presence of a binding-site barrier, the high activity upon cytosolic delivery of even small amounts of catalytic inactivators results in much stronger therapeutic effects throughout the tissue ( Figure 5 A,B). Assuming identical delivery properties and stabilities in the cytosol, the effects are much longer-lasting, because after removal of the inactivator, the pool of target proteins first has to be replenished by translation ( Figure 5 B). Even with relatively low estimates of rates of inactivation (low k cat values), the effects are remarkably potent ( Figure 5 A). Enhanced effects of catalytic cargoes can be further increased by repeated dosing approaches. For a scenario with application of a dose resulting in a 200 nM concentration of an enzymatic cargo in the plasma approximately every 4.5 days, greater therapeutic effects are produced as compared to the twice-daily administration of binders to a plasma concentration of 1 μM ( Figure 5 C). In a clinical setting, the lower plasma exposure required for catalytic agents ( Figure 5 D) might have considerable benefits with respect to eliciting non-specific side effects as well as immunogenic reactions [ 42 ]. We subsequently investigated the effect of cold dosing and analyzed the influence of the time interval between warm and cold dose in tumors exhibiting convection and those that do not. Cold dosing enhanced the overall inhibitory (i.e., therapeutic) effect, with stronger effects in tumors exhibiting convection and in the presence of shorter time intervals ( Figure 5 E). Of note is that for binders that show optimal receptor affinities, cold dosing tends to have little effect (see below; Figure 5 G). As alluded to before, an alternative approach to increase the activity of proteins delivered to the cytosol is to enhance the stability against degradation, for instance, by removing lysine residues on the protein surface, through which the canonical pathway of ubiquitination and proteasomal degradation occurs [ 43 ]. Alternatively, the (partial) use of D-amino acids can improve stability [ 40 , 41 ], because protein stretches composed of D-amino acids are resistant to proteolytic degradation. A higher cytosolic stability was simulated as a longer cytosolic half-life of binders in our model ( Figure 5 F). Our findings indicate that a moderate enhancement of the cytosolic half-life (4×) can make the therapeutic effect much longer lasting. By contrast, a large increase in half-lives (40×/100×) shows limited additional effects due to the constant synthesis of new target proteins. Conversely, shortening the half-life cuts down the duration of the therapeutic effect, although the maximum level of inhibition (i.e., at a single time-point) is surprisingly unaffected even when a 10-fold shorter cytosolic half-life is assumed ( Figure 5 F). When comparing strategies side-by-side, an optimization of receptor affinity was more powerful than cold dosing with respect to maximizing effects throughout the tissue ( Figure 5 G). When investigating the maximum inhibition instead of the inhibitory effect achieved by these different strategies, the efficacy of a cold dose in tumors with convection is strong when the affinity is not optimal, but has no positive effects when an optimal K d is modelled ( Supplementary Figure S5 ). Extending the cytosolic half-life is, within the conditions tested, ineffective with respect to increasing the level of maximum inhibition. 3.1. Modeling Protein Delivery To generate a quantitative understanding of the requirements for effective in vivo cytosolic protein delivery, we developed a mathematical model incorporating key elements of cellular targeting, entry, and intracellular activity. The model was, in part, inspired by work from Thurber et al., in which antibody delivery in vivo was described within a Krogh geometry consisting of two concentric cylinders; the inner cylinder represented a capillary, and the outer represented the surrounding tissue ( Figure 1 ) [ 19 ]. The model permits the in silico evaluation of all physical and biological phenomena that we reasoned would significantly influence cytosolic protein delivery and its therapeutic effects. Those parameters that relate to the engineered protein are size, affinity, intracellular binding, plasma half-life, half-life in the cytosol, as well as the effects of enzymatic target modification on the levels of functional cytosolic target proteins. Those parameters that relate more to the targeted tissue are vascular permeability, interstitial diffusivity, cellular internalization rates, and cytosolic delivery efficiency ( Figure 1 ). An extensive description of how the model was designed and the rationale for the choice of all parameters is provided in the Supplementary Materials section. As a representative tissue, we focused on solid tumors of the breast overexpressing the tumor marker epithelial cell adhesion molecule (EpCAM), while in some instances, we used normal skeletal muscle tissue for comparison. We made a distinction between tumors that exhibited convection, i.e., those with functional lymph vessels, and those that did not, and therefore exhibited little or no fluid flow and where macromolecule transport is driven by diffusion. Results are, in most cases, described as the degree of inhibition as a function of time (i.e., inhibition of a cytosolic target protein), maximum inhibition (i.e., single time-point across the entire tissue), or as an inhibitory effect (effect integrated over time and entire tissue). A technical explanation of how these terms were calculated is given in the Supplementary Materials . 3.2. Simulation of Delivery and Therapeutic Effects In Vivo We initially simulated delivery of a protein with a molecular weight of 70 kDa binding to the tumor marker EpCAM. Binding properties were taken from the designed ankyrin repeat protein (DARPin) Ec1, which binds EpCAM with an affinity (K d ) of 68 pM [ 21 ]. We assumed a tumor tissue containing a cell density of 2.9 × 10 8 cells/mL, representing a tumor with a high cellularity, and a receptor density of 5.4 × 10 5 receptors/cell, which we previously determined for EpCAM on MCF-7 cells [ 22 ]. As indicated above, we thus considered the tissue as a homogenous medium in which cellular structures are not explicitly defined. We simulated a starting protein plasma concentration of 1 μM, a concentration that can be realistically reached upon intravenous administration of a therapeutic protein [ 23 ] ( Figure 2 A). A sharp decline in the delivered protein concentration away from the capillary was observed, with the majority of protein being confined to the first 20 μm of tissue. When an EpCAM binder is combined with a moiety or vector that mediates efficient cytosolic delivery, e.g., an endosomal escape peptide or a bacterial toxin-derived translocation domain as we reported previously [ 24 ] (see Supplementary Table S2 for detailed information on assumptions), the simulated degree of target inhibition (see Supplements for a technical explanation) varies across different distances from the capillary ( Figure 2 B) and directly reflects protein delivery ( Figure 2 A), with maximum inhibition in the tissue reached at around 24 h ( Supplementary Video S1 ). We then evaluated the maximum cytosolic delivery and inhibition with a targeted protein ( Figure 2 C,D) or a targeted peptide ( Figure 2 E,F) in tumors with and without convection and compared it with delivery in normal tissue (skeletal muscle was chosen as a representative tissue because of the availability of experimental values of needed parameters in the literature). The relevant difference between both types of compounds is size, and we modelled a molecular weight (MW) of 3.5 kDa for the peptide, which is the MW of the calcitonin peptide hormone. Calcitonin is a medically used peptide for which half-lives have been determined in vivo in humans [ 25 ] ( Supplementary Table S3 ). Employing a targeted peptide resulted in more rapid clearance but also faster penetration due to a higher interstitial diffusivity. We also included peptides because for this class of compounds, several strategies for cytosolic delivery have been described. For both proteins and peptides, cytosolic delivery and inhibition in tumors vastly surpassed that achieved in muscle tissue in terms of tissue reached, mostly due to the enhanced permeability of leaky blood vessels. Convection additionally contributed substantially to an enhanced depth of penetration for proteins ( Figure 2 C). While targeted proteins were delivered in high concentrations to the tissue immediately adjacent to the capillary but rapidly decreased in concentration further away, peptides permeated the entire tissue more evenly by comparison, although in lower levels ( Figure 2 C,E). 3.3. Effect of Receptor Affinity on Peptide and Protein Delivery The binding-site barrier is a phenomenon where high-affinity targeted agents bind tightly to receptors in the first cell layers encountered and only travel further into the tissue upon receptor saturation. The affinity for receptors, the receptor density, and the receptor internalization rate are well-established factors with respect to the binding-site barrier [ 19 , 28 , 29 ], although quantitative insights into the impact of the binding-site barrier on tissue penetration under different conditions are lacking. One approach to minimize the negative consequences of the binding-site barrier is to modulate the affinity of a targeted agent towards its cellular receptor. Importantly, a lower affinity may benefit tissue penetration. First, we determined the optimum receptor affinity for tumor targeting with or without convection for a fixed receptor density for both targeted proteins and peptides ( Figure 3 ), as determined by maximal delivery to the tissue. Maximal delivery was defined as the situation when the average concentration across the tissue of the free target protein was at its lowest. Convection facilitates the rapid transport of macromolecules from the immediate proximity of the endothelium deeper into the tissue, and its effect on protein delivery was therefore of particular interest. We found that higher affinities for targeted proteins were needed for the optimal delivery in tumors without convection. The presence of convection in tumors greatly reduced the optimal affinity and enhanced the overall therapeutic effect ( Figure 3 A). For peptide delivery, on the other hand, almost identical optimal affinities were observed in the presence or absence of convection, and delivery only mildly increased in the presence of convection. When comparing peptides vs. proteins, peptides produced superior delivery in tumors without convection due to the higher permeability and diffusivity, while proteins surpass peptides in tumors with convection due to their longer plasma half-life (see also Figure 2 C,E). The steeper decline in delivered molecules observed for proteins vs. peptides indicates a more pronounced binding-site barrier for slower-diffusing proteins. Targeted proteins can easily be engineered to be highly selective and have affinities that are considered optimal by our modeling approach ( Figure 3 A). However, for peptides, the ability to obtain high affinities appears to be a limiting factor for their efficacy ( Figure 3 B), although peptides with very high affinities have been reported [ 30 ], including peptides that bind with subnanomolar K d values to vascular endothelial growth factor (VEGF) receptor 2 and c-MET, or hapten peptides that bind to single chain variable fragments with affinities as high as 2.3 nM [ 31 ]. Interestingly, for targeted proteins, the optimal affinities for targeting tumors with and without convection, of 16.4 nM and 625 pM, respectively, for these simulated parameters, appear to be readily achievable, as demonstrated by the superimposition of the frequency distribution of protein affinities reported in the literature and compiled in the kinetics database KOFFI [ 30 ]. When investigating the effect of receptor density for our default conditions (i.e., using a proteinaceous EpCAM binder with an affinity of 68 pM), we noted a very strong dependency of the therapeutic effect on the receptor density ( Supplementary Figure S3 ). This mirrors the effects of modulating receptor affinity (see Figure 3 A) and reflects the characteristics of the binding-site barrier. Therapeutic effects were highest at around 1–2 × 10 5 receptors/cell ( Supplementary Figure S3 ). 3.4. Interplay Between Receptor Affinity, Receptor Density and Internalization Rate At present, we are not aware of reports that relate the optimal affinity to receptor density and the receptor internalization rate. Ultimately, this knowledge may guide the choice of suitable receptors for targeting in the development of cancer therapies. Next to target affinity and receptor density, the internalization rate of cellular receptors plays a prominent role in determining the properties of the binding-site barrier [ 29 ]. This is illustrated by determining the affinity that yields the greatest therapeutic effect (i.e., optimum affinity) as a function of receptor density for receptors that differ in internalization rates, either in the presence or absence of convection ( Figure 4 A). Although internalization, recycling rates, and even trafficking routes for receptors can differ as a function of the ligand utilized for targeting (e.g., natural ligands vs. engineered binders), as well as the epitope addressed on a specific receptor, our simulations indicated that receptors which generally internalize faster, e.g., epidermal growth factor receptor (EGFR), necessitate a lower optimal affinity for maximal tissue delivery. Again, convection decreased the optimal affinity for delivery for all internalization rates and also increased the internalization rate-dependent differences ( Figure 4 ). In our model, we simulated steady state levels of receptors by matching the rate of internalization with the rate of recycling, thus mimicking the delivery of non-toxic proteins (for details, see the Supplementary Materials ). A fraction of targeted agents was delivered to the cytosol in the model, with the rest being degraded. Even in the presence of a moiety that mediates endosomal escape, degradation in the endolysosomal system is a common fate of endocytosed proteins, and only a fraction of internalized protein reaches the cytosol [ 18 ]. Remarkably, for a binder exhibiting the optimal affinity, the same overall magnitude of delivery and inhibition can be achieved largely irrespective of the receptor density and internalization rate ( Figure 4 B). This implies that targeting should not only focus on those receptors which are present at very high levels and for which very high affinity binders are available, which would increase the options for productive targeted drug delivery. Nonetheless, specificity and potential off-target effects should always be taken into consideration. Notably, for diffusion-based delivery, maximal delivery is always lower, emphasizing the relevance of convection in targeted delivery to tumors. 3.5. Effect of Cold Dosing, Targeted Protein Degradation and Degradation-Resistant Proteins on the Binding-Site Barrier As illustrated by our analyses so far, protein targeting deep into tissues faces several dilemmas. Small size, on the one hand, favors penetration, but on the other hand, leads to faster clearance. A high receptor affinity promotes effective cellular targeting, although limits penetration due the rapid capture of extravasated proteins by the first layer of cells, also referred to as the binding-site barrier. As an approach to overcome the binding-site barrier, cold dosing, where the therapeutic moiety is only present on a fraction of administered targeted agents, has been investigated for a long time. Mostly, this has been performed in the context of radiolabeled antibody [ 34 , 35 ] but, more recently, also in the context of antibody–drug conjugates [ 36 , 37 ]. In addition, there are multiple protein engineering approaches aimed to enhance intracellular delivery and/or subsequent therapeutic effects, some of which are only recently coming to the fore for engineered proteins. The therapeutic potency can be increased without affecting delivery itself, through either delivery of (target protein-inactivating) enzymes [ 8 , 38 ], targeted protein degradation [ 39 ], or by enhancing the stability of the delivered agent against proteasomal degradation [ 40 , 41 ]. In our simulations, catalytic inactivators represent both enzymes that inactivate target proteins and protein-based agents that induce target protein degradation, because for both types of activities, the functional outcome is the same. Given our assumptions (see Supplementary Materials ), catalytic agents strongly outperform binders whose effect is limited to inhibition by direct blockade of the protein—protein interaction, in particular at lower concentrations ( Figure 5 A) or when the target protein concentration is high ( Figure 5 B). For instance, at a target protein level of 1.0 × 10 5 molecules/cell and using catalytic inactivators, a very strong and pronounced inhibition can be observed as compared to inhibition by binding only ( Figure 5 B). Irrespective of the presence of a binding-site barrier, the high activity upon cytosolic delivery of even small amounts of catalytic inactivators results in much stronger therapeutic effects throughout the tissue ( Figure 5 A,B). Assuming identical delivery properties and stabilities in the cytosol, the effects are much longer-lasting, because after removal of the inactivator, the pool of target proteins first has to be replenished by translation ( Figure 5 B). Even with relatively low estimates of rates of inactivation (low k cat values), the effects are remarkably potent ( Figure 5 A). Enhanced effects of catalytic cargoes can be further increased by repeated dosing approaches. For a scenario with application of a dose resulting in a 200 nM concentration of an enzymatic cargo in the plasma approximately every 4.5 days, greater therapeutic effects are produced as compared to the twice-daily administration of binders to a plasma concentration of 1 μM ( Figure 5 C). In a clinical setting, the lower plasma exposure required for catalytic agents ( Figure 5 D) might have considerable benefits with respect to eliciting non-specific side effects as well as immunogenic reactions [ 42 ]. We subsequently investigated the effect of cold dosing and analyzed the influence of the time interval between warm and cold dose in tumors exhibiting convection and those that do not. Cold dosing enhanced the overall inhibitory (i.e., therapeutic) effect, with stronger effects in tumors exhibiting convection and in the presence of shorter time intervals ( Figure 5 E). Of note is that for binders that show optimal receptor affinities, cold dosing tends to have little effect (see below; Figure 5 G). As alluded to before, an alternative approach to increase the activity of proteins delivered to the cytosol is to enhance the stability against degradation, for instance, by removing lysine residues on the protein surface, through which the canonical pathway of ubiquitination and proteasomal degradation occurs [ 43 ]. Alternatively, the (partial) use of D-amino acids can improve stability [ 40 , 41 ], because protein stretches composed of D-amino acids are resistant to proteolytic degradation. A higher cytosolic stability was simulated as a longer cytosolic half-life of binders in our model ( Figure 5 F). Our findings indicate that a moderate enhancement of the cytosolic half-life (4×) can make the therapeutic effect much longer lasting. By contrast, a large increase in half-lives (40×/100×) shows limited additional effects due to the constant synthesis of new target proteins. Conversely, shortening the half-life cuts down the duration of the therapeutic effect, although the maximum level of inhibition (i.e., at a single time-point) is surprisingly unaffected even when a 10-fold shorter cytosolic half-life is assumed ( Figure 5 F). When comparing strategies side-by-side, an optimization of receptor affinity was more powerful than cold dosing with respect to maximizing effects throughout the tissue ( Figure 5 G). When investigating the maximum inhibition instead of the inhibitory effect achieved by these different strategies, the efficacy of a cold dose in tumors with convection is strong when the affinity is not optimal, but has no positive effects when an optimal K d is modelled ( Supplementary Figure S5 ). Extending the cytosolic half-life is, within the conditions tested, ineffective with respect to increasing the level of maximum inhibition. 4. Discussion The ability to deliver proteins efficiently into the cytosol of specific cells in vivo would enable numerous novel therapeutic opportunities [ 16 ], including the interference with signaling pathways in cancer and senescent cells, the restoration of missing functions in genetic diseases, and potentially even the ability to reprogram cells in vivo to restore or redirect their identity [ 44 ]. Here, we built a mathematical model for investigating the challenges of protein delivery in vivo and to evaluate the merit of engineering approaches that are geared towards overcoming these challenges. As we demonstrate, the results provide clear guidelines for protein engineers on how to design proteins more rationally towards specific applications. While our quantitative analyses are focused on cancer targeting in vivo, the findings are also qualitatively pertinent to other disease areas. Furthermore, variants of the model can be employed to study protein transport and activity in microfluidic models mimicking the tumor microenvironment with various degrees of complexity, and we are actively pursuing this line of research. As a starting point, it is useful to consider that approaches that report cytosolic protein delivery in 2D systems in vitro often report values of cytosolic concentrations for the delivered protein that are comparatively high (mid- or high-nanomolar range) [ 18 , 24 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 ], which is well over the average level of molecules of a specific protein in a cell, which is around 2000–8000 molecules/cell (~2–10 nM) [ 27 ]. Hence, addressing cytosolic targets in vitro is already possible for some applications. However, similarly to challenges associated with a homogenous delivery to targeted agents to extracellular receptors [ 55 ], delivery challenges and a rapid degradation in the cytosol imply that reaching these levels and concomitant biological effects in vivo remains very difficult. The binding-site barrier is an often-mentioned factor that limits effective tissue penetration [ 28 , 56 ]. While it has long been known that reducing the affinity of a binder for a specific receptor may facilitate tumor penetration [ 57 ], our simulations indicate that tuning the receptor affinity for a particular receptor density, receptor internalization rate, and the presence or absence of convection can be very powerful in increasing targeting. For our default scenario (agent binding EpCAM with 68 pM), we observed a 16% increase in maximum inhibition in tumors without convection and a 118% increase in tumors with convection. The 16% increase in tumors without convection is modest because our starting affinity was fairly close to the optimal affinity already (which was 625 pM), although in general, improvements in tissue delivery by optimizing affinity surpass those achieved by cold dosing approaches. Two notable outcomes of our simulations are (i) that the optimal affinity for delivery is much lower in tumor tissue exhibiting convection compared to its counterpart without convection, with optimal affinities differing between one and three orders of magnitude, depending on the receptor internalization rate and receptor density ( Figure 4 ); and (ii) that within a large range of receptor internalization rates and receptor densities, equal overall therapeutic effects can be accomplished, provided the affinity for the receptor has been optimized ( Figure 4 ). With contemporary screening and protein engineering approaches, the tuning of affinities is a feasible undertaking. Nevertheless, in practice, a balance needs to be found between the optimal affinity for a specific set of conditions (e.g., type of receptor, receptor density, convection) and the degree of heterogeneity in the tumor—often, tumors are characterized by regions that differ in extracellular matrix (ECM) densities, in the presence of convective flow [ 58 , 59 ], and in levels of receptor expression. As a consequence, heterogeneous delivery is often observed in antibody-treated tumors [ 60 ], and this heterogeneity is even more pronounced when utilizing antibody–drug conjugates that already have dose-limiting toxicities at low doses [ 36 ]. Our analyses on the interplay between the various factors that govern cellular delivery (receptor density, internalization, affinity, convection) will aid the understanding of the impact of heterogeneity on delivery. While tumor heterogeneity is an inherent and challenging aspect of tumor targeting that complicates finding one optimal solution, by covering broad ranges of values in our simulations, the decision to address a receptor with higher or lower affinity can be made more rational. We focused our investigations on the binding and internalization of low- to medium-sized proteins that reach the target cells through active targeting. For that reason, our model does not apply to delivery by the enhanced permeability and retention (EPR) effect, which describes the passive accumulation of large entities, e.g., nanoparticles, in tumor tissue due to a high local microvascular permeability and a poor lymphatic clearance [ 61 ]. However, for delivery by the EPR effect, the incorporation of modalities for active targeting has only limited added value [ 62 ]. Our analyses demonstrate the promise of two strategies to mitigate the effect of the binding-site barrier: optimizing the affinity towards the cell surface receptor and enzymatic target modulation or targeted protein degradation ( Figure 5 G). Optimizing the affinity towards cell surface receptors was identified as an approach that could facilitate overcoming the binding-site barrier in a straightforward manner. Importantly, affinity optimization is not synonymous with achieving as high an affinity as possible. The findings hold for both tumors exhibiting convection and those that do not. Cytosolic delivery of enzymes that inactivate oncogenes was recently demonstrated through the diphtheria toxin-mediated delivery of an enzyme that inactivates wild-type and mutant RAS [ 8 ]. The k cat of the RAS-cleaving enzyme that was used was 2.35 min −1 [ 63 ], and is on the high end of the range of catalytic rates that were simulated by us ( Figure 5 A), emphasizing that this particular approach is indeed very powerful. While tumor growth inhibition in vivo was achieved, challenges associated with full tumor penetration were identified that contributed to suboptimal therapeutic effects. Targeted protein degradation, as a more generic approach for intracellular protein depletion, also exhibits great potency and is rapidly moving towards clinical application. Although current agents to trigger targeted protein degradation are mostly small molecule-based rather than protein-based [ 64 , 65 , 66 ], a monobody binder targeting the Lck tyrosine kinase fused to von Hippel-Lindau (VHL), a substrate receptor of the Cullin2-E3 ubiquitin ligase complex, has recently been shown to mediate targeted protein degradation in vitro upon cytosolic delivery [ 39 ]. Notwithstanding its potential, the high potency of targeted protein degradation or enzymatic inactivation means that off-target delivery could prove more toxic at lower concentrations (see Figure 5 A,B), which emphasizes the necessity to place great importance on the specificity of the targeting approach. A better quantitative understanding of in vivo protein delivery has the potential to act synergistically with a better quantitative knowledge of how many proteins are necessary to exert a specific effect. This can range from targeted protein degradation of highly overexpressed anti-apoptotic proteins in cancer cells or senescent cells, e.g., members of the BCL-2 protein family [ 67 , 68 ], to the delivery of transcription factors over several days for in vivo reprogramming efforts, as was recently accomplished using an mRNA-based approach [ 69 ]. Given the immunogenic risks associated with long-term therapy, application areas beyond cancer in which transient administration is sufficient are particularly promising, especially those where partial effectivity already gives rise to substantial therapeutic effect. For example, by interfering with signaling pathways that mediate the survival of senescent cells, a short-term protein-based approach to interfere with these pathways may remove a fraction of the senescent cells, which is already expected to yield multiple health-related benefits [ 70 ]. Limitations and Future Perspectives A limitation in our model is that surface charge of the delivered proteins has not been taken into account. Surface charge has been shown to affect both the permeability and interstitial diffusivity of macromolecules and nanoparticles. and this effect has been attributed to interactions with the negatively charged basement membranes and ECM, respectively [ 71 , 72 , 73 ]. Our reliance on data from neutral (i.e., non-charged) dextrans for some parameters, for reasons of availability, means that the extrapolation towards proteins with very different charge characteristics has to be performed with caution. However, upon the availability of better-suited data for proteins with specific charges, these data can be easily implemented in our model. For our analyses, we chose a shorter plasma half-life for the therapeutic proteins than what is common for IgG antibodies, because for cytosolic delivery, full-length antibodies are an unlikely choice and antibodies also show only poor tissue penetration [ 16 ]. Nevertheless, for non-IgG proteins, plasma half-lives can also now be engineered to a large extent; for example, through the inclusion of Fc segments [ 74 ] or by fusion to albumin-binding domains to exploit FcRn receptors [ 75 , 76 ]. The presented model has been employed here to extract general principles but should be refined towards protein engineering approaches for specific applications. This can be achieved through the implementation of more detailed and context-relevant experimental data obtained from in vivo studies and studies with microfluidic models. Experimental studies can refine the model and provide better estimates of plasma and intracellular half-life, the approach utilized for mediating endosomal escape, interstitial diffusivities, surface receptor internalization as a function of the targeting agent, ubiquitination rates, and microvascular permeabilities. When these values are unknown, a broad range of values can be tested to investigate the sensitivity towards there parameters. This would help to understand the relevance of measuring and/or optimizing these values. At present, the model also does not recapitulate the complexity of tumor vasculature in vivo. However, because the aim of the model is to understand extravasation from the vasculature, tissue penetration, and entry into cells in the direct vicinity of a capillary, we do not consider this simplification a significant limitation. Finally, through incorporating specific rates of transcytosis through receptors such as transferrin or insulin receptors, which are often used for delivery across the blood–brain barrier [ 77 , 78 ], our model can also be employed to simulate delivery to the brain or other tissues in which transcytosis is the main mode of transport. In conclusion, we have shown that the "high-hanging fruit" of proteins that exert their therapeutic effect inside cells is well within reach of the protein engineering approaches that are currently being pursued. Nevertheless, the proper engineering of their characteristics will be crucial, and models such as the one presented here will guide this way. Limitations and Future Perspectives A limitation in our model is that surface charge of the delivered proteins has not been taken into account. Surface charge has been shown to affect both the permeability and interstitial diffusivity of macromolecules and nanoparticles. and this effect has been attributed to interactions with the negatively charged basement membranes and ECM, respectively [ 71 , 72 , 73 ]. Our reliance on data from neutral (i.e., non-charged) dextrans for some parameters, for reasons of availability, means that the extrapolation towards proteins with very different charge characteristics has to be performed with caution. However, upon the availability of better-suited data for proteins with specific charges, these data can be easily implemented in our model. For our analyses, we chose a shorter plasma half-life for the therapeutic proteins than what is common for IgG antibodies, because for cytosolic delivery, full-length antibodies are an unlikely choice and antibodies also show only poor tissue penetration [ 16 ]. Nevertheless, for non-IgG proteins, plasma half-lives can also now be engineered to a large extent; for example, through the inclusion of Fc segments [ 74 ] or by fusion to albumin-binding domains to exploit FcRn receptors [ 75 , 76 ]. The presented model has been employed here to extract general principles but should be refined towards protein engineering approaches for specific applications. This can be achieved through the implementation of more detailed and context-relevant experimental data obtained from in vivo studies and studies with microfluidic models. Experimental studies can refine the model and provide better estimates of plasma and intracellular half-life, the approach utilized for mediating endosomal escape, interstitial diffusivities, surface receptor internalization as a function of the targeting agent, ubiquitination rates, and microvascular permeabilities. When these values are unknown, a broad range of values can be tested to investigate the sensitivity towards there parameters. This would help to understand the relevance of measuring and/or optimizing these values. At present, the model also does not recapitulate the complexity of tumor vasculature in vivo. However, because the aim of the model is to understand extravasation from the vasculature, tissue penetration, and entry into cells in the direct vicinity of a capillary, we do not consider this simplification a significant limitation. Finally, through incorporating specific rates of transcytosis through receptors such as transferrin or insulin receptors, which are often used for delivery across the blood–brain barrier [ 77 , 78 ], our model can also be employed to simulate delivery to the brain or other tissues in which transcytosis is the main mode of transport. In conclusion, we have shown that the "high-hanging fruit" of proteins that exert their therapeutic effect inside cells is well within reach of the protein engineering approaches that are currently being pursued. Nevertheless, the proper engineering of their characteristics will be crucial, and models such as the one presented here will guide this way.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100532/

The dichotomy of pathogens and allergens in vaccination approaches

Traditional prophylactic vaccination to prevent illness is the primary objective of many research activities worldwide. The golden age of vaccination began with an approach called variolation in ancient China and the evolution of vaccines still continues today with modern developments such as the production of Gardasil TM against HPV and cervical cancer. The historical aspect of how different forms of vaccination have changed the face of medicine and communities is important as it dictates our future approaches on both a local and global scale. From the eradication of smallpox to the use of an experimental vaccine to save a species, this review will explore these successes in infectious disease vaccination and also discuss a few significant failures which have hampered our efforts to eradicate certain diseases. The second part of the review will explore designing a prophylactic vaccine for the growing global health concern that is allergy. Allergies are an emerging global health burden. Of particular concern is the rise of food allergies in developed countries where 1 in 10 children is currently affected. The formation of an allergic response results from the recognition of a foreign component by our immune system that is usually encountered on a regular basis. This may be a dust-mite or a prawn but this inappropriate immune response can result in a life-time of food avoidance and lifestyle restrictions. These foreign components are very similar to antigens derived from infectious pathogens. The question arises: should the allergy community be focussing on protective measures rather than ongoing therapeutic interventions to deal with these chronic inflammatory conditions? We will explore the difficulties and benefits of prophylactic vaccination against various allergens by means of genetic technology that will dictate how vaccination against allergens could be utilized in the near future. INTRODUCTION Globally, the burden of disease and infection is diverse and inescapable. It is a shared affliction for humanity and one that is constantly moderated by better hygiene, enhanced education, and improved vaccines and therapeutic interventions. In terms of healthcare, it is always more beneficial to prevent a disease or infection from occurring than to treat and cure it. The development of vaccines is dependent on the knowledge of: what pathogen causes the disease; how it establishes itself in the host; how the host's innate and cell-mediated immunity responds to pathogens; and how it maintains ongoing protection after the disease using antibodies. Whilst there are many successful vaccines currently available, there are still no registered vaccines for some globally prevalent infectious diseases such as malaria and human immunodeficiency virus (HIV). Although we have made enormous progress in medicine over the last 300 years since the practice of vaccination first began, there are still diseases that are killing millions of people globally which desperately require a vaccine. Furthermore, there is a multitude of autoimmune conditions such as food allergies which may benefit from a traditional prophylactic vaccination approach. This review will explore the progression of traditional vaccines from empirical vaccines to the more recent novel vaccines and how recent advancements could change the field of allergy research. A BRIEF HISTORY OF VACCINATION The first crude attempt at disease control was the procedure of variolation where the inoculated person stood a good chance at surviving both the procedure and later exposure to the pathogen. Variolation consisted of directly transferring the infection from a sick person to a healthy person, through direct contact or by infectious matter such as pus, saliva or blood ( Dinc and Ulman, 2007 ). This form of vaccination is believed to have begun in either ancient China or India, but was only brought to the UK by the wife of a British diplomat, Lady Wortley-Montagu in 1721 ( Dinc and Ulman, 2007 ). Lady Wortley-Montagu had observed that harem girls in Constantinople had pox-free faces which were attributed to them being variolated; hence she had her son variolated in Istanbul in 1718 to save him from experiencing what she had as a young adult – smallpox. Later she also variolated her daughter in London; however, this was only after she had confirmed that it did not result in death or disease in eleven orphans and six convicted murderers from Newgate Prison ( Dinc and Ulman, 2007 ). Lady Wortley-Montagu was so impressed that she implored her surgeon, Dr. Charles Maitland, to learn the technique and demonstrate it to the Royal British Court ( Stewart and Devlin, 2006 ). After this demonstration, 200 upper-class members of British society, including members of the royal family, underwent the procedure, and in 1729 a further 897 more inoculations were performed with only 17 deaths post-procedure which is infinitely fewer than smallpox mortality at the time ( Dinc and Ulman, 2007 ). Even though Lady Wortley-Montagu was severely criticized for bringing the procedure to Britain, it was slowly implemented throughout the UK over the following years, and in 1757 a young boy named Edward Jenner would be variolated against smallpox ( Dinc and Ulman, 2007 ). This ultimately saved countless lives from smallpox; the most devastating disease of the time. However, there were two issues with variolation: (1) it could impair the patient or even kill them if the dosage was incorrect or if they were not physically fit enough to withstand the infection, and (2) whilst the patient would be protected from further infections, they would become contagious during the active infection ( Bazin, 2003 ). Although variolation was popular in the cities, in the English countryside there were many rumors that if you contracted cowpox you were protected from the deadly smallpox. Subsequently, a farmer named Benjamin Jesty in Yetminster, England, inoculated his wife and two sons with cowpox in the hope of surviving a smallpox epidemic ( Pead, 2003 , 2006 ). Even though his wife became very ill, she and the whole family survived and went on to survive many smallpox epidemics in the area. This transpired a full 20 years before scientist Jenner began his experiment with a boy called James Phipps ( Pead, 2003 ); however, Jesty was recognized for his contribution in 1805 by a published statement and a portrait commissioned by the Original Vaccine Pock Institution, London ( Pead, 2006 ). It is believed that Jenner was also aware of the rumors of cowpox protecting against smallpox, and that this was the inspiration for his experiment, resulting in him being the first to document that a person infected with cowpox would survive subsequent exposure to smallpox ( Stewart and Devlin, 2006 ). This technique evolved into using cow inoculums as the vaccine, which did provide immunity to smallpox although not to the same degree as natural disease or variolation. This discovery was heralded as the new age of vaccines and instigated new research into other common diseases. A couple of centuries later, medicine would again make another considerable leap forward with the separate works of scientists Louis Pasteur and Robert Koch, and the publication of germ theory. The most famous of these works would be Pasteur's and his attenuation of the bacteria Pasteurella multocida, which causes fowl cholera, by exposing the cultures to air and room temperature for extended periods of time ( Bordenave, 2003 ). He demonstrated that whilst the bacteria were avirulent, they provided full protection from the virulent strain of the bacteria, which was a revolutionary idea at the time. Pasteur also went on to attenuate the rabies virus by passage through rabbits ( Bazin, 2003 ). Koch, on the other hand, would discover the bacterial agents of anthrax, tuberculosis and cholera whilst also compiling postulates with fellow scientist Jacob Henle that would transform the world of microbiology ( Kaufmann and Schaible, 2005 ). All of these discoveries led to the development of immunology and non-empirical vaccines. The first whole cell vaccine was produced by Salmon and Smith in 1886 and was based on a Salmonella strain that was killed by heat and injected into pigeons to provide immunity ( Bazin, 2003 ). Around the same time, others were investigating bacterial components and methods to purify them. This was the beginning of traditional vaccine methodology. During this era there were many great innovations in the field of immunology and vaccinology, such as the discovery of toxins and the consequent inactivation of toxins by heat and formalin, killed vaccines, adjuvants, sub-unit or acellular vaccines, tissue culture and live attenuated vaccines. With the establishment of molecular biology and genetic engineering in the late 1950s, a new era began where vaccine development no longer needed to be empirical and bacterial components could be produced artificially or even in vivo by unrelated vectors. VACCINES IN THE MODERN ERA What makes a good vaccine? The traditional definition of a vaccine is one that protects against a particular (or group of) infectious agent(s); however, these days there are many vaccines that could be designated as therapeutic agents against diseases such as cancer ( Bergman et al., 2006 ), although the goal is still to prevent illness. In this review we will focus on prophylactic vaccines. The global market for vaccines is estimated to be around US$8 billion per year whilst the cost to develop each vaccine from concept to commercialism is around US$300–800 million ( Plotkin, 2005 ). The reason for the high expenditure is that each vaccine has to be rigorously tested before commercial release and the average time it takes to fully develop a vaccine is between 15 and 20 years ( Arntzen et al., 2005 ). A successful vaccine is measured by its effectiveness, its spectrum of protection, the duration of immunity and the strength of immunological memory that it induces. Secondary considerations of a good vaccine are its stability, ease of administration and storage, achievable mass production and its toxicity. Biotechnology is a rapidly developing area which allows continued improvement into the exploration of antigens suitability as vaccine candidates. Choosing the right antigen is a core decision in the development of a vaccine candidate as some antigens that are immunogenic in vivo may not elicit long term protection. The same antigen may also vary in structure and sequence between strains, limiting its usefulness. Some antigens are also hard to express and purify on a large scale which is required for mass production ( Mora et al., 2003 ). This is where novel vaccine methodology hopes to improve how vaccines are made and administered; this will be examined subsequently. Routes of administration The oldest technique for vaccination is that of subcutaneous delivery via scarification and one of the newest techniques is intramuscular injection ( Bazin, 2003 ). Whilst the intramuscular route of vaccination is quite standard today in developed countries, it is an inconvenient method of application as it requires sterile needles and syringes, and usually a medical physician to administer it. This is the major drawback of vaccines that rely on intramuscular injections to be effective. In one study, a viral vectored vaccine was found to elicit stronger systemic and detectable mucosal responses via a single intramuscular injection than if it was applied via the oral route. The oral route proved to stimulate suboptimal T-cell responses and did induce a higher level of mucosal antibody than the intramuscular route ( Lin et al., 2007 ). Nasal and oral administration routes of a vaccine are more desirable than intramuscular as they are non-invasive, painless, not required to be sterile and do not require a physician for administration. This final point is most important as it is one of the reasons that third world countries have the lowest level of immunizations in the world ( Costantino et al., 2007 ). Nasal immunization would place the vaccine in contact with the large surface of the nasal mucosa which consists of the nasal-associated lymphoid tissue (NALT), which can lead to both humoral and cellular immune responses ( Zuercher et al., 2002 ; Costantino et al., 2007 ). The most well-known nasal vaccine is FluMist ® ; a live cold-adapted influenza virus. It can be given as one or two doses from a syringe sprayer, is licensed for use in the USA for persons aged 5–50 and has shown high efficacy from its inception ( Plotkin, 2005 ; Costantino et al., 2007 ). However, one of the detriments of a nasal vaccine is that an unpleasant taste and nasal discomfort can occur often discouraging repeated use ( Atmar et al., 2007 ). Oral administration is a practical method of application if it can be achieved without diminishing the effectiveness of the vaccine, and immunity can be achieved with a single dose. The objective of oral vaccines is to mimic a natural infection and provide mucosal immunity. Orally delivered vaccines can induce suboptimal T-cell responses with high levels of mucosal antibody than the intramuscular route; however, the vaccine must be very stable as it will have to survive the acidic environment of the stomach before it reaches the M cells of the intestinal wall where it can be processed by antigen-presenting cells (APCs; Lin et al., 2007 ). Adjuvants Adjuvants are defined as compounds that influence the immune system into mounting a Th1 or Th2 response and whilst doing so, greatly enhances the magnitude of immune response against the antigen ( Marciani, 2003 ). They are an important aspect of vaccines due to their tendency to make an ineffective antigen become effective. It is vital that adjuvants have the following properties: a non-toxic nature or have minimal toxicity at the dosage to elicit effective adjuvanticity; able to stimulate a strong humoral and/or T-cell immune response; provide good immunological memory or long-term protection; not induce autoimmunity; are non-mutagenic, non-carcinogenic, non-teratogenic, and non-pyrogenic; and be stable under broad ranges of storage time, temperature and pH levels ( Marciani, 2003 ). The most popular adjuvants are aluminum-based and were first described and published in 1926 ( Glenny et al., 1926 ). There are three adjuvants that are currently licensed for human use: aluminum hydroxide, also known as alum ( Davies and Flower, 2007 ); monophosphoryl lipid A (MPL) and AS03 consisting of D , L -alpha-tocopherol (vitamin E), squalene and polysorbate 80 ( U.S. Food and Drug Administration, 2014 ). It is well established that aluminum adjuvants stimulate the production of IgE and a Th2 immune profile, yet for some diseases this would not be adequate protection against pathogens as a Th1 response would be required ( Lindblad, 2004 ). MPL and AS03 have demonstrated clinical efficacy when used in a HPV and influenza vaccine, respectively ( Mohan et al., 2013 ). Adjuvants are used to lengthen the dissemination time of the antigen from the site of injection which allows the antigen to be released over a prolonged period improving the effectiveness of the vaccine. This feature is called the depot effect and is traditionally associated with aluminum-based adjuvants ( Lindblad, 2004 ; Mohan et al., 2013 ); however, recently the depot effect has been questioned as reducing the dissemination time of the antigen does not alter the magnitude of the immune response ( Hutchison et al., 2012 ) along with other evidence suggesting other modes of action (reviewed in De De Gregorio et al., 2013 ). Other methods in which adjuvants improve the immune response are to form complexes with the antigen and to target the vaccine towards specific receptors. For example, the use of mannose in the adjuvant is recognized by pattern recognition receptors (PRRs) that initiate endocytosis and antigen processing ( Stahl and Ezekowitz, 1998 ). The use of pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and CpG-DNA, and of synthetic low-MW imidazoquinolines in adjuvants, all trigger innate immune responses that lead to a Th1 or Th2 response in the vaccinated person ( Marciani, 2003 ). Other adjuvants consist of cytokines ( Cheng et al., 2007 ) and glycolipids ( Singh et al., 1999 ; Ko et al., 2005 ) and other immunomodulators ( Morrow et al., 2004 ) that bind to highly specific receptors on T cells which activate them. TRADITIONAL VACCINE METHODOLOGY The early development of vaccines focused on using killed organisms, inactivated toxins or modified organisms, but currently there are many different approaches to vaccine development, which will be examined subsequently. As these approaches were empirical in design, these types of vaccines, whilst being successful, are now viewed as being traditional vaccines. These can be divided into three different types: (a) killed vaccines; (b) attenuated vaccines; and (c) sub-unit vaccines. Killed vaccines A killed or "inactivated" vaccine is developed by the pathogen being grown and then being made inactive by means of heat, chemical or radiation treatment and was the basis of most vaccines until the 1980s. This results in the pathogen being unable to cause disease whilst providing the immune system with stimulation via its normal antigenic epitopes on its cell surface. One major disadvantage of this approach is that, whilst these vaccines are immunogenic, they do not replicate in vivo infectivity limiting the spectrum of the immunity acquired as the agent is incapable of going through its normal antigenic variation over the course of the infection. This results in decreased immunity and a requirement of booster shots to maintain immunity ( Moylett and Hanson, 2003 ). Another disadvantage of this vaccine type is that during the inactivation process the antigenic epitopes can be modified resulting in a less efficacious vaccine ( Tano et al., 2007 ). Despite these limitations, killed vaccines are commonly used today with the typhoid, Salk poliomyelitis, and seasonal influenza vaccines still being administered ( Bazin, 2003 ; Palese, 2006 ). Attenuated vaccines Among the more efficacious of the traditional vaccines are the attenuated ones. In this case, a pathogen is subjected to altered growth conditions, is passage through a host or is genetically modified to eliminate its virulence, yet retaining its ability to replicate albeit at a greatly reduced rate. These vaccines are more successful at eliciting a robust lifelong immunity than other traditional vaccines. This can be attributed to their ability to cause an asymptomatic infection which stimulates both the humoral and cellular branches of the immune system. However, this ability to replicate carries the greatest risk as the vaccine can persist in immune-compromised persons or the elderly due to limited immune responses. A benefit to these vaccines is they express their own immunogenic antigens which stimulate the immune system strongly thereby negating the need for an adjuvant to be used ( Loessner et al., 2008 ). The most commonly used attenuated vaccine is the MMR vaccine which protects children worldwide against measles, mumps and rubella and with subsequent boosters provides lifelong immunity ( Vandermeulen et al., 2007 ). Attenuated vaccines have further progressed into carrier vaccines where they can deliver heterologous antigens ( Bachtiar et al., 2003 ; Lotter et al., 2008 ; Schoen et al., 2008 ). For live carrier vaccines that deliver multiple heterologous antigens, there is a risk that the host immune system will dampen the immune response to the heterologous antigens by misdirecting the immune response against the carrier ( Berzofsky et al., 2004 ). However, if the immunity induced is cell-mediated the response can be enhanced by pre-existing immunity to the carrier strain ( Saxena et al., 2013 ). Subunit vaccines Traditionally, it was thought that the only way to protect against a disease was to use the whole organism to vaccinate the host. However, it was elucidated that specific parts of the organisms, when purified or isolated, demonstrated immunogenic properties. These components could be the capsule, the flagella or even an outer membrane protein of the cell wall. These types of vaccines are known as subunit vaccines or acellular vaccines. These vaccines are not able to cause the disease and in comparison to whole cell killed vaccines they are not as efficacious. This is both an advantage as they are safe for immune-compromised patients and a disadvantage as they do not elicit long-term immunity and will often require multiple vaccinations to maintain immunity ( Schmitt et al., 2008 ). An advantage of this type of vaccine is that it can be engineered to protect against various strains of the organism. An example of a successful subunit vaccine is the Haemophilus influenzae type b (HiB) conjugate vaccine which consists of a polysaccharide-protein conjugate. This vaccine has eliminated or significantly reduced this disease in children in regions of South America ( Ribeiro et al., 2007 ; Franco-Paredes et al., 2008 ) and Africa ( Adegbola et al., 2005 ; Muganga et al., 2007 ) where it was once endemic. In the UK, the success of this vaccination program was compromised by a highly publicized paper (which has now been retracted) that linked autism to early childhood vaccination which lead to a rise in HiB infections as parents chose not to vaccinate; however, subsequent booster campaigns by the NHS has seen a reduction in infection rates again ( Ladhani et al., 2008 ). A recent meta-analysis covering studies involving over 1.2 million children has discredited any link between vaccinations or vaccine components thimerosal or mercury to the development of autism or autism spectrum disorders ( Taylor et al., 2014 ). DNA VACCINES – A NEXT GENERATION EXAMPLE There are multiple novel types of vaccines that are currently under development, such as bacterial ghosts ( Szostak et al., 1996 ; Jawale and Lee, 2014 ) and nanovaccines ( Cho et al., 2014 ). However, one that holds great promise and has had documented successes is DNA vaccines. DNA vaccines differ from traditional vaccines as they do not consist of a protein or a cell component but only the DNA that encodes an immunogenic antigen within a plasmid vector. The plasmid can be administered by injection, gene gun, electroporation, or aerosol delivery, upon which the host's immune cells, usually dendritic cells, will sample the plasmid and express the encoded antigens. These antigens are then degraded by the cell into peptides and presented via MHC class I and class II molecules depending on the mode of administration and the cell type. From this, both antibody and cellular responses can be induced ( Forde, 2005 ). The first reported use of a plasmid DNA vaccine outside of trial or experimental conditions was in 2003 and was a desperate attempt to save an endangered species from extinction ( Bouchie, 2003 ). The vaccine was for the highly endangered California condors against the lethal West Nile virus. West Nile virus had emerged in New York in 1999 and spread to 41 out of the 50 US states killing birds from 138 species in a matter of years. It was believed that if the virus spread to California, the remaining 200 or so condors would face extinction. The US Centers for Disease Control and Prevention (CDC) expedited the manufacture of an experimental vaccine and permitted the condors to be vaccinated with it ( Bouchie, 2003 ). The DNA vaccine expressed West Nile virus pre-membrane/membrane and envelope proteins. The vaccinated condors were monitored and it was observed that the DNA vaccination stimulated protective immunity in adults, nestlings and newly hatched chicks. Following two intramuscular vaccinations, the condors demonstrated excellent neutralizing antibodies 60 days post-vaccination with a continued increase until approximately 1 year post-vaccination. It was also noted that the birds did not show any unusual behaviors, health changes or side effects post-vaccination ( Chang et al., 2007 ). This vaccine has also demonstrated efficacy in other bird species such as the American robins ( Turdus migratorius ) ( Kilpatrick et al., 2010 ) and the fish crows ( Corvus ossifragus ; Turell et al., 2003 ). The first two DNA vaccines for veterinary use were granted US approval in 2005 for West Nile virus vaccine for horses and haematopoietic necrosis vaccine for farm-reared Atlantic salmon ( Chalmers, 2006 ). Even though there are no currently approved DNA vaccines for human use, as of May, 2014 there are 128 open trials listed on Clinicaltrials.Gov (2014) that involve DNA-based vaccines and therapies GLOBAL VACCINE SUCCESS The global eradication of smallpox is, to date; the most successful vaccination campaign in history. Smallpox has existed for many thousands of years and spread through the world following the migration of humans to new settlements ( Barquet and Domingo, 1997 ). As mentioned previously, Edward Jenner is famously credited with developing a smallpox vaccination using the cow-pox virus (vaccinia virus) and published many observations on both the successful and adverse events ( Jenner, 1809 ) associated with his vaccination protocol. Small pox was an indiscriminate disease that is caused by two virus variants Variola major and Variola minor and was responsible for 300–500 million deaths before its eradication ( Theves et al., 2014 ). The smallpox vaccine that was developed by Jenner produces both neutralizing antibodies and cell mediated responses that are protective against other members of the Orthopoxvirus genus ( Barquet and Domingo, 1997 ). After years of vaccination success but with deaths from smallpox still common, the World Health Assembly, the executive body of the [ World Health Organization (WHO), 2013 ] set a target to eradicate smallpox. This was only achievable as humans are the only reservoir for the virus and the vaccine had demonstrated high efficacy ( Fenner et al., 1988 ). In the late 1960s, the efforts of the WHO were strengthened with more funding and new surveillance protocols. The last natural occurrence of smallpox occurred in Somalia, where cook Ali Maow Maalin developed the rash on October 26th 1977, but tragically it was not the last global smallpox death [ World Health Organization (WHO), 1980 ]. Medical photographer Janet Parker became the last person to die of smallpox in the world when she was accidently exposed to it in her workplace at the University of Birmingham and unfortunately a lapse in obtaining her booster vaccination led to her being susceptible at the time of exposure ( Barquet and Domingo, 1997 ). Eradication of smallpox was declared on May 8, 1980 by the WHO when the Final Report of Global Commission for Certification of Smallpox Eradication was published [ World Health Organization (WHO), 1980 ]. As of 2014, two depositories of smallpox still exist at the CDC in the USA and the State Research Center of Virology and Biotechnology VECTOR in Koltsovo, Russia. The destruction of these viral stocks has been delayed and debated since the declaration of eradication occurred in, 1980. Discovery of smallpox victims during building excavations often fuels these debates although no viable virus has been recovered from these corpses, so the risk of a modern smallpox outbreak is improbable ( Reardon, 2014 ; Theves et al., 2014 ). The WHO is again debating the existence of these stocks in May, 2014 ( Reardon, 2014 ). Another successful vaccine that has been implemented globally is those against poliomyelitis – the Salk, and Sabin vaccines. There are three different poliovirus serotypes and all of them can lead to serious disability in children, even death by acute flaccid paralysis [ World Health Organization (WHO), 2014a ]. Due to its moderate mortality rates, its long-term severe disability consequences and like smallpox, humans being the only natural reservoir for the virus, the World Health Assembly set a target of eradication by the year 2000. This project is known as the Global Polio Eradication Initiative. Poliovirus Type 2 infection has not been observed since 1999 in India and Type 3 since 2012. However, in 2014, poliovirus Type 1 is still endemic in regions of Nigeria, Pakistan, and Afghanistan [ World Health Organization (WHO), 2014a ]. The reasons behind these persisting endemics will be discussed later. There are two vaccines, an oral live attenuated vaccine known as the Sabin vaccine and the inactivated poliovirus vaccine also known as the Salk vaccine [ World Health Organization (WHO), 2014a ]. The Sabin vaccine was derived from passages of the poliovirus strains through rats and mice and then through cell cultures more than 50 times resulting in an attenuated forms of the virus types that all induced good antibody levels ( Sabin, 1957 ; Baicus, 2012 ). In 1972, Sabin donated his vaccine strains to the WHO which increased the number of vaccine recipients from 5 to 80% [ Baicus, 2012 ; World Health Organization (WHO), 2014a ]. The Sabin vaccine is no longer in use in the USA or UK as the only poliomyelitis cases reported in the populations were vaccine-associated paralytic poliomyelitis where the vaccine strain has caused an outbreak but it is still used in some developing countries due to its ease of administration and cost (US$0.14 a dose vs US$2–3 a dose for Salk vaccine; Willyard, 2014 ). There are now plans to eliminate the Sabin vaccine entirely in the 124 countries that still use it by 2015 ( Willyard, 2014 ). The Salk vaccine is grown in monkey kidney cells and inactivated with formalin ( Salk et al., 1954 ) and was introduced in the USA in 1955 and by 1961, the incidence of poliomyelitis had decreased from 13.9 cases per 100,000 in 1954 to 0.8 cases per 100,000 in 1961 [ Baicus, 2012 ; World Health Organization (WHO), 2014a ]. Besides preventing deaths, the main benefit to come from polio vaccination is the cost savings to the healthcare system which is estimated at US$40–50 billion for the period between 1988 and 2035 in the USA alone [ World Health Organization (WHO), 2014a ]. Most countries that have been certified polio-free still have rare isolated cases which have come from travelers importing the virus from endemic areas, for example in Australia had one such case in 2007 ( Paterson and Durrheim, 2013 ). However, the Global Polio Eradication Initiative has a new timeline for eradication and with a new strategy of phasing out the Sabin vaccines, hopefully the world will be certified polio-free in 2018 ( Willyard, 2014 ). A more recent vaccine accomplishment is the pneumococcal conjugate vaccine (PCV) against Streptococcus pneumoniae (pneumococcus) infections which include acute otitis media, sinusitis, pneumonia and invasive pneumococcal diseases such as meningitis and sepsis. The first conjugate vaccine was a heptavalent vaccine which protects against seven different serotypes of pneumococcus and it was licensed in the USA in 2000 ( Black et al., 2000 ; Lee et al., 2014 ). Since that time, 10- ( Domingues et al., 2014 ), 13- ( Spijkerman et al., 2013 ), and 23-valent ( Grabenstein and Manoff, 2012 ) vaccines have been licensed with all producing strong immunity against a broad spectrum of strains. In the USA, all age groups from children under 5 years to adults over 65 years had dramatic reductions in incidence of pneumococcal infections over a seven year period after the PCV was available ( Pilishvili et al., 2010 ). It is predicted that if the heptavalent PCV was implemented in China it would prevent 4222 cases of invasive pneumococcal disease, 4,061,524 cases of otitis media and 472,527 cases of pneumonia, as well as preventing an additional 2682 deaths from pneumococcal disease; however, the implementation cost would be estimated at US$6.44 billion ( Che et al., 2014 ). The current overall cost of pneumococcal disease in the unvaccinated population in China is estimated to be US$3.5 billion ( Che et al., 2014 ). Following the introduction of PCV in the USA, an estimated 211,000 serious pneumococcal infections and 13,000 deaths were prevented in the period of 2000–2008 ( Pilishvili et al., 2010 ). The influence of this vaccine on public health is in its early stages and has already had impacts on child mortality in over 88 countries that have included various PCV on their recommended immunization schedule ( Whitney et al., 2014 ). There are other vaccines that have been successfully implemented in the past decade. The most recent and highly publicized vaccine is the quadrivalent human papillomavirus vaccine against cervical cancer, marketed as Gardasil ® , which prevents the premalignant disease that leads to cervical cancers and fulfills all the above criteria of being a successful vaccine ( Zhou et al., 1991 ; Govan, 2008 ). Initially the cost of Gardasil ® was extremely prohibitive at US$120 per dose with three doses required; however, in collaboration with GAVI Alliance, the cost from the supplier has dropped to US4.50 per dose which increases its affordability and likelihood of being implemented in developing countries ( Anon, 2013 ). As the cost of the vaccine decreases and more people are immunized this vaccine which has been included in over 30 countries immunization schedules, in conjunction with regular Pap screening, may lead to a long-term reduction in cervical cancer incidence ( Harper et al., 2010 ; Ribeiro-Muller and Muller, 2014 ). VACCINE FAILURES AND CHALLENGES Historically there have been more vaccine failures than successes and unfortunately those failures can be publicized and instill fear in the general public long after the event. One such failure is one that occurred early in the rollout of the Salk polio vaccine is known as the Cutter incident. In April, 1955 a few weeks after Salk's polio vaccine had been declared safe and efficacious, there were reports from California that five children had become paralyzed after receiving the vaccine ( Offit, 2005 ). These vaccines were traced to Cutter which was one of the five pharmaceutical companies that were granted a license to produce the vaccine in the USA ( Nathanson and Langmuir, 1995 ). It was found that two production batches failed the deactivation steps; so live virulent poliovirus was found in 120,000 doses of the vaccine. Of the children vaccinated from this pool, 40,000 developed abortive polio, 51 suffered from permanent paralysis and five died ( Nathanson and Langmuir, 1995 ). Unfortunately this was not the end of the tragedy, a polio outbreak followed where a further 113 people in close contact with the vaccinated children were infected and subsequently paralyzed, and a further five deaths ( Nathanson and Langmuir, 1995 ; Offit, 2005 ). This incident halted the implementation of the polio vaccine program and severely affected public confidence in the vaccination, not only in the USA but as far reaching as New Zealand ( Day, 2009 ), Germany, the UK and Sweden ( Axelsson, 2012 ) and in the end, it caused the USA to recommend Sabin's vaccine in the long term which, barring manufacturing failures, proved to be the more risky of the two vaccines as it could revert to full virulence and cause outbreaks of vaccine-associated paralytic poliomyelitis ( Offit, 2005 ; Fitzpatrick, 2006 ). Following its emergence in 1981, HIV infections and its subsequent disease acquired immunodeficiency syndrome (AIDS) has become a global pandemic with millions of deaths and over 34 million people living with HIV ( De Cock et al., 2012 ). According to the [ World Health Organization (WHO, 2014c ) ] and the Joint United Nations Programme on Hiv/Aids (2013) the pandemic appears to have peaked as AIDS-related deaths have decreased by 25% in the past decade as well as new infections decreasing by 20% since 2006. This is the combined effect of the development of anti-retroviral drugs, and better education about the transmission of this disease. However, a vaccine is desperately needed to prevent new infections and to stop this pandemic from affecting future generations. Multiple HIV vaccines have been tested in clinical trials with limited success ( Johnson et al., 2013 ). In the last decade, the most prominent vaccine trial failures was that of the Merck STEP phase II test of concept and efficacy trial for an Adenovirus5 (Ad5) vaccine. It showed that the MRKAd5 HIV-1 gag/pol/nef vaccine was highly immunogenic and elicited a higher magnitude of HIV-specific CD8+ T cells than any of the other HIV candidate vaccines over the past 15 years but it did not prevent HIV infection or reduce viral loads in infected patients ( Buchbinder et al., 2008 ). In fact, more disturbingly, there was an increase in the number of HIV-1 infections in male recipients of the vaccine compared to the controls ( McElrath et al., 2008 ). This trial was immediately ceased when the independent data and safety monitoring board determined that the study could not demonstrate efficacy ( Buchbinder et al., 2008 ). One of the reasons behind the failure of the Merck STEP clinical trial was the pre-existing neutralizing antibodies against Ad5. A recent study confirmed that the international epidemiology of pre-existing immunity to different adenovirus types can severely compromise its efficacy as only 14.8% of the 1904 participants were seronegative for neutralizing antibodies against Ad5 ( Mast et al., 2010 ). This indicates that naturally acquired infections from virulent forms of the vaccine vectors can limit their usefulness in the same species. However, choosing a virus from a different species for which no prior exposure is possible but may sound too risky to be accepted by the general population. It was also found that whilst the group of men that became more susceptible to HIV infection post-vaccination were seropositive against the Ad5 vector, they were also uncircumcised and had sexual relations with the same sex implying that pre-existing immunity may not be the sole factor that caused this vaccine failure ( Gray et al., 2010 ; Duerr et al., 2012 ). Whilst this phase II trial failure was a major setback for the HIV research community, it raised fundamental questions about the pathogenesis of HIV and also gave insight into immunological mechanisms that were previously unexplored ( Johnson et al., 2013 ; Fauci et al., 2014 ). The search for a HIV vaccine is ongoing and as of May 2014, there are 92 open HIV vaccine trials according to Clinicaltrials.Gov (2014) . Another infectious disease that is under surveillance by health departments worldwide is a double-stranded RNA virus called rotavirus. Rotavirus causes acute enteritis resulting in severe, dehydrating diarrhea in infants and young children and is very transmissible through close contact [ Bishop et al., 1976 ; World Health Organization (WHO), 2013 ]. In the pre-vaccination era, rotavirus caused 111 million cases of illness with 25 million medical visits, 2 million hospitalizations and between 352,000 and 592,000 rotavirus gastroenteritis-associated deaths worldwide annually with most of these occurring in low income countries ( Parashar et al., 2003 ). The first rotavirus vaccine was RotaShield which was developed by Wyeth-Lederle Vaccines and Pediatrics, Philadelphia, as an oral vaccine and showed high efficacy at 80% protection from severe illness; hence it was recommended for all infants in the USA once it was approved by the Food and Drug Administration (FDA) on August 31, 1998 ( American Academy of Pediatrics, 1998 ). Over the eleven month period after the vaccine was approved until July 7, 1999, 15 cases of intussusception, a type of intestinal blockage requiring surgical intervention, were reported and linked to the vaccination. In consultation with the FDA, Wyeth-Lederle Vaccines withdrew Rotashield from the market on October 15, 1999. Before this withdrawal, the cases of confirmed intussusception had risen to 101 ( Delage, 2000 ) and fortuitously, because there were no deaths caused by this vaccine, physician trust in vaccine safety measures were not compromised by this withdrawal ( McPhillips et al., 2001 ). In 2006, two new oral rotavirus vaccines were released onto the market: Rotarix ® – a live monovalent attenuated human strain by GlaxoSmithKline Biologicals ( Vesikari et al., 2004 ; Keating, 2006b ) and RotaTeq ® – a live pentavalent human-bovine reassortant vaccine by Merck & Co. Inc. ( Clark et al., 2004 ; Keating, 2006a ). After 6 years of use, a Cochrane Review found that both of these vaccines are efficacious with no increased risk of adverse side effects such as intussusception ( Soares-Weiser et al., 2012 ). However, in 2013, a small increase in risk was confirmed when the data was analyzed comparing the risk of intussusception in the post-vaccine period with other periods ( Haber et al., 2013 ; Quinn et al., 2014 ). A year later, the vaccines are still on the market albeit with an intussusception warning even though there is an estimated up to sixfold increase with the use of these two rotavirus vaccines. So far the Vaccine Safety Datalink has reported that Rotarix ® has had 66 intussusception cases in 200,000 doses, whilst RotaTeq ® had eight cases for 1.3 million doses administered with most occurring within 7 days after the first dose [ World Health Organization (WHO), 2014b ]. Currently the risk of intussusception is estimated to be 1–2 per 100,000 infants vaccinated [ World Health Organization (WHO), 2013 ]. However, the general view is that there are great benefits to vaccination against rotavirus as the infant mortality rates in countries that have added this to their vaccination schedule have significantly decreased ( Buttery et al., 2014 ) and this is reflected in the WHO's Global Advisory Committee on Vaccine Safety in their weekly epidemiological record [ World Health Organization (WHO), 2014b ] stated this in regards to the new intussusception risk: "the findings remain reassuring that the risk of intussusception following current rotavirus vaccines remains small compared to the benefits of preventing the impact of severe diarrhea." Surveillance of such adverse effects requires long-term study in order to make sound decisions about the appropriateness of the vaccine. There may come a time where the relative risk is too high and the vaccine is withdrawn like Rotashield which had a rate of intussusception of 1 in 10,000 infant doses [ World Health Organization (WHO), 2013 ], even though it provided strong immunological protection. This is one of the hardest aspects in vaccine development to plan for and may lead to public distrust in future vaccines, if it is not done expediently when those risks increases. POLITICAL AND GLOBAL ASPECTS OF VACCINE USAGE When a vaccine is designed, it is assumed that if it proves effective it will be used in various countries around the world to vaccinate the population; however, this is not always the case. Within each country there are government agencies, industry and community health advocates, and outside agencies such as the WHO that will make recommendations for vaccination strategies. Often this process will result in a successful vaccination strategy such as the global eradication of smallpox ( Stewart and Devlin, 2006 ), but it can also lead to confusion and scepticism in the chosen strategy. One such example was the choice of pertussis vaccine for a national vaccination campaign in the Netherlands. Originally, the Dutch government chose to use a whole cell vaccine based on the Bordetella pertussis bacterium; however, after speculation that the vaccine could cause brain damage, alternative vaccines were sought. At this time, acellular vaccines comprising three to five bacterial components were being used by many countries in Europe as they were comparable in protection to the whole cell vaccines and demonstrated minimal side effects ( Blume and Zanders, 2006 ). Over the course of 7 years, the debate over the new vaccine became very convoluted as many government agencies, drug companies, and consumer groups presented opposing studies and evidence. There was also external pressure from neighboring countries and global non-profit groups including the WHO and United Nations Children's Fund (UNICEF) for the Dutch government to make a decision. Concurrently, many parents had lost faith in the old vaccine strategy; hence an epidemic of pertussis ensued. To combat the growing epidemic the Dutch government chose an acellular vaccine which was used in primary vaccinations in 2005; however, the Health Minister advised that this decision was not based on recommendations and evidence provided by the Dutch Health Council, but on the need to appease parents and re-establish their confidence in the vaccine strategy ( Blume and Zanders, 2006 ). By contrast, in areas where any disease is endemic and the health system is overwhelmed, often the vaccination strategy proposed by governing bodies will be accepted by the population and acquiesced as mandatory ( Chalmers, 2006 ). Unfortunately this has not worked in areas such as Pakistan, Nigeria, and Afghanistan where the eradication of polio has failed due in part to misinformation, violence, politics, and mistrust about vaccination. There is a distinct divide in these populations between vaccine acceptors and non-acceptors which is based in the abundance of misinformation about the vaccine, religious beliefs and the emotional fear about the agenda; however, if there is an outbreak many non-acceptors will accept the vaccination as the fear of disease outweighs the perceived risks ( Murele et al., 2014 ). Socio-cultural, educational and perceptual factors are particularly strong in these regions and in some cases targeting male authority figures could improve vaccination uptake ( Murele et al., 2014 ); however, in other regions maternal education and empowerment has been suggested as a strong motivator in vaccine acceptance ( Larson et al., 2014 ). Violence is another contributing factor to this program's failure particularly when there are fatal attacks on vaccination workers in Pakistan and Nigeria ( Abimbola et al., 2013 ). In Afghanistan, both the Taliban regime and the militant Islamist terrorist group Al Qaeda support the Global Polio Eradication Initiative; however, factions within these groups can disrupt it as they view it as a Westernization issue, rather than a health one ( Abimbola et al., 2013 ). In Nigeria and Pakistan, militants can gain international media attention by attacking polio health workers ( Riaz and Rehman, 2013 ) and spreading propaganda that immunization programs are actually covert sterilization campaigns to reduce the Muslim population, which puts more fear into the local communities than the disease itself ( Abimbola et al., 2013 ; Willyard, 2014 ). All of the aforementioned issues affect the successful eradication of infectious diseases with well documented epidemiology and pathology. However, there exist conditions and disorders where the mechanisms of development and ongoing chronic pathology are yet to be fully ascertained. One such condition causing concern among health professionals globally is allergy. VACCINES IN THE MODERN ERA What makes a good vaccine? The traditional definition of a vaccine is one that protects against a particular (or group of) infectious agent(s); however, these days there are many vaccines that could be designated as therapeutic agents against diseases such as cancer ( Bergman et al., 2006 ), although the goal is still to prevent illness. In this review we will focus on prophylactic vaccines. The global market for vaccines is estimated to be around US$8 billion per year whilst the cost to develop each vaccine from concept to commercialism is around US$300–800 million ( Plotkin, 2005 ). The reason for the high expenditure is that each vaccine has to be rigorously tested before commercial release and the average time it takes to fully develop a vaccine is between 15 and 20 years ( Arntzen et al., 2005 ). A successful vaccine is measured by its effectiveness, its spectrum of protection, the duration of immunity and the strength of immunological memory that it induces. Secondary considerations of a good vaccine are its stability, ease of administration and storage, achievable mass production and its toxicity. Biotechnology is a rapidly developing area which allows continued improvement into the exploration of antigens suitability as vaccine candidates. Choosing the right antigen is a core decision in the development of a vaccine candidate as some antigens that are immunogenic in vivo may not elicit long term protection. The same antigen may also vary in structure and sequence between strains, limiting its usefulness. Some antigens are also hard to express and purify on a large scale which is required for mass production ( Mora et al., 2003 ). This is where novel vaccine methodology hopes to improve how vaccines are made and administered; this will be examined subsequently. Routes of administration The oldest technique for vaccination is that of subcutaneous delivery via scarification and one of the newest techniques is intramuscular injection ( Bazin, 2003 ). Whilst the intramuscular route of vaccination is quite standard today in developed countries, it is an inconvenient method of application as it requires sterile needles and syringes, and usually a medical physician to administer it. This is the major drawback of vaccines that rely on intramuscular injections to be effective. In one study, a viral vectored vaccine was found to elicit stronger systemic and detectable mucosal responses via a single intramuscular injection than if it was applied via the oral route. The oral route proved to stimulate suboptimal T-cell responses and did induce a higher level of mucosal antibody than the intramuscular route ( Lin et al., 2007 ). Nasal and oral administration routes of a vaccine are more desirable than intramuscular as they are non-invasive, painless, not required to be sterile and do not require a physician for administration. This final point is most important as it is one of the reasons that third world countries have the lowest level of immunizations in the world ( Costantino et al., 2007 ). Nasal immunization would place the vaccine in contact with the large surface of the nasal mucosa which consists of the nasal-associated lymphoid tissue (NALT), which can lead to both humoral and cellular immune responses ( Zuercher et al., 2002 ; Costantino et al., 2007 ). The most well-known nasal vaccine is FluMist ® ; a live cold-adapted influenza virus. It can be given as one or two doses from a syringe sprayer, is licensed for use in the USA for persons aged 5–50 and has shown high efficacy from its inception ( Plotkin, 2005 ; Costantino et al., 2007 ). However, one of the detriments of a nasal vaccine is that an unpleasant taste and nasal discomfort can occur often discouraging repeated use ( Atmar et al., 2007 ). Oral administration is a practical method of application if it can be achieved without diminishing the effectiveness of the vaccine, and immunity can be achieved with a single dose. The objective of oral vaccines is to mimic a natural infection and provide mucosal immunity. Orally delivered vaccines can induce suboptimal T-cell responses with high levels of mucosal antibody than the intramuscular route; however, the vaccine must be very stable as it will have to survive the acidic environment of the stomach before it reaches the M cells of the intestinal wall where it can be processed by antigen-presenting cells (APCs; Lin et al., 2007 ). Adjuvants Adjuvants are defined as compounds that influence the immune system into mounting a Th1 or Th2 response and whilst doing so, greatly enhances the magnitude of immune response against the antigen ( Marciani, 2003 ). They are an important aspect of vaccines due to their tendency to make an ineffective antigen become effective. It is vital that adjuvants have the following properties: a non-toxic nature or have minimal toxicity at the dosage to elicit effective adjuvanticity; able to stimulate a strong humoral and/or T-cell immune response; provide good immunological memory or long-term protection; not induce autoimmunity; are non-mutagenic, non-carcinogenic, non-teratogenic, and non-pyrogenic; and be stable under broad ranges of storage time, temperature and pH levels ( Marciani, 2003 ). The most popular adjuvants are aluminum-based and were first described and published in 1926 ( Glenny et al., 1926 ). There are three adjuvants that are currently licensed for human use: aluminum hydroxide, also known as alum ( Davies and Flower, 2007 ); monophosphoryl lipid A (MPL) and AS03 consisting of D , L -alpha-tocopherol (vitamin E), squalene and polysorbate 80 ( U.S. Food and Drug Administration, 2014 ). It is well established that aluminum adjuvants stimulate the production of IgE and a Th2 immune profile, yet for some diseases this would not be adequate protection against pathogens as a Th1 response would be required ( Lindblad, 2004 ). MPL and AS03 have demonstrated clinical efficacy when used in a HPV and influenza vaccine, respectively ( Mohan et al., 2013 ). Adjuvants are used to lengthen the dissemination time of the antigen from the site of injection which allows the antigen to be released over a prolonged period improving the effectiveness of the vaccine. This feature is called the depot effect and is traditionally associated with aluminum-based adjuvants ( Lindblad, 2004 ; Mohan et al., 2013 ); however, recently the depot effect has been questioned as reducing the dissemination time of the antigen does not alter the magnitude of the immune response ( Hutchison et al., 2012 ) along with other evidence suggesting other modes of action (reviewed in De De Gregorio et al., 2013 ). Other methods in which adjuvants improve the immune response are to form complexes with the antigen and to target the vaccine towards specific receptors. For example, the use of mannose in the adjuvant is recognized by pattern recognition receptors (PRRs) that initiate endocytosis and antigen processing ( Stahl and Ezekowitz, 1998 ). The use of pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and CpG-DNA, and of synthetic low-MW imidazoquinolines in adjuvants, all trigger innate immune responses that lead to a Th1 or Th2 response in the vaccinated person ( Marciani, 2003 ). Other adjuvants consist of cytokines ( Cheng et al., 2007 ) and glycolipids ( Singh et al., 1999 ; Ko et al., 2005 ) and other immunomodulators ( Morrow et al., 2004 ) that bind to highly specific receptors on T cells which activate them. What makes a good vaccine? The traditional definition of a vaccine is one that protects against a particular (or group of) infectious agent(s); however, these days there are many vaccines that could be designated as therapeutic agents against diseases such as cancer ( Bergman et al., 2006 ), although the goal is still to prevent illness. In this review we will focus on prophylactic vaccines. The global market for vaccines is estimated to be around US$8 billion per year whilst the cost to develop each vaccine from concept to commercialism is around US$300–800 million ( Plotkin, 2005 ). The reason for the high expenditure is that each vaccine has to be rigorously tested before commercial release and the average time it takes to fully develop a vaccine is between 15 and 20 years ( Arntzen et al., 2005 ). A successful vaccine is measured by its effectiveness, its spectrum of protection, the duration of immunity and the strength of immunological memory that it induces. Secondary considerations of a good vaccine are its stability, ease of administration and storage, achievable mass production and its toxicity. Biotechnology is a rapidly developing area which allows continued improvement into the exploration of antigens suitability as vaccine candidates. Choosing the right antigen is a core decision in the development of a vaccine candidate as some antigens that are immunogenic in vivo may not elicit long term protection. The same antigen may also vary in structure and sequence between strains, limiting its usefulness. Some antigens are also hard to express and purify on a large scale which is required for mass production ( Mora et al., 2003 ). This is where novel vaccine methodology hopes to improve how vaccines are made and administered; this will be examined subsequently. Routes of administration The oldest technique for vaccination is that of subcutaneous delivery via scarification and one of the newest techniques is intramuscular injection ( Bazin, 2003 ). Whilst the intramuscular route of vaccination is quite standard today in developed countries, it is an inconvenient method of application as it requires sterile needles and syringes, and usually a medical physician to administer it. This is the major drawback of vaccines that rely on intramuscular injections to be effective. In one study, a viral vectored vaccine was found to elicit stronger systemic and detectable mucosal responses via a single intramuscular injection than if it was applied via the oral route. The oral route proved to stimulate suboptimal T-cell responses and did induce a higher level of mucosal antibody than the intramuscular route ( Lin et al., 2007 ). Nasal and oral administration routes of a vaccine are more desirable than intramuscular as they are non-invasive, painless, not required to be sterile and do not require a physician for administration. This final point is most important as it is one of the reasons that third world countries have the lowest level of immunizations in the world ( Costantino et al., 2007 ). Nasal immunization would place the vaccine in contact with the large surface of the nasal mucosa which consists of the nasal-associated lymphoid tissue (NALT), which can lead to both humoral and cellular immune responses ( Zuercher et al., 2002 ; Costantino et al., 2007 ). The most well-known nasal vaccine is FluMist ® ; a live cold-adapted influenza virus. It can be given as one or two doses from a syringe sprayer, is licensed for use in the USA for persons aged 5–50 and has shown high efficacy from its inception ( Plotkin, 2005 ; Costantino et al., 2007 ). However, one of the detriments of a nasal vaccine is that an unpleasant taste and nasal discomfort can occur often discouraging repeated use ( Atmar et al., 2007 ). Oral administration is a practical method of application if it can be achieved without diminishing the effectiveness of the vaccine, and immunity can be achieved with a single dose. The objective of oral vaccines is to mimic a natural infection and provide mucosal immunity. Orally delivered vaccines can induce suboptimal T-cell responses with high levels of mucosal antibody than the intramuscular route; however, the vaccine must be very stable as it will have to survive the acidic environment of the stomach before it reaches the M cells of the intestinal wall where it can be processed by antigen-presenting cells (APCs; Lin et al., 2007 ). Adjuvants Adjuvants are defined as compounds that influence the immune system into mounting a Th1 or Th2 response and whilst doing so, greatly enhances the magnitude of immune response against the antigen ( Marciani, 2003 ). They are an important aspect of vaccines due to their tendency to make an ineffective antigen become effective. It is vital that adjuvants have the following properties: a non-toxic nature or have minimal toxicity at the dosage to elicit effective adjuvanticity; able to stimulate a strong humoral and/or T-cell immune response; provide good immunological memory or long-term protection; not induce autoimmunity; are non-mutagenic, non-carcinogenic, non-teratogenic, and non-pyrogenic; and be stable under broad ranges of storage time, temperature and pH levels ( Marciani, 2003 ). The most popular adjuvants are aluminum-based and were first described and published in 1926 ( Glenny et al., 1926 ). There are three adjuvants that are currently licensed for human use: aluminum hydroxide, also known as alum ( Davies and Flower, 2007 ); monophosphoryl lipid A (MPL) and AS03 consisting of D , L -alpha-tocopherol (vitamin E), squalene and polysorbate 80 ( U.S. Food and Drug Administration, 2014 ). It is well established that aluminum adjuvants stimulate the production of IgE and a Th2 immune profile, yet for some diseases this would not be adequate protection against pathogens as a Th1 response would be required ( Lindblad, 2004 ). MPL and AS03 have demonstrated clinical efficacy when used in a HPV and influenza vaccine, respectively ( Mohan et al., 2013 ). Adjuvants are used to lengthen the dissemination time of the antigen from the site of injection which allows the antigen to be released over a prolonged period improving the effectiveness of the vaccine. This feature is called the depot effect and is traditionally associated with aluminum-based adjuvants ( Lindblad, 2004 ; Mohan et al., 2013 ); however, recently the depot effect has been questioned as reducing the dissemination time of the antigen does not alter the magnitude of the immune response ( Hutchison et al., 2012 ) along with other evidence suggesting other modes of action (reviewed in De De Gregorio et al., 2013 ). Other methods in which adjuvants improve the immune response are to form complexes with the antigen and to target the vaccine towards specific receptors. For example, the use of mannose in the adjuvant is recognized by pattern recognition receptors (PRRs) that initiate endocytosis and antigen processing ( Stahl and Ezekowitz, 1998 ). The use of pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and CpG-DNA, and of synthetic low-MW imidazoquinolines in adjuvants, all trigger innate immune responses that lead to a Th1 or Th2 response in the vaccinated person ( Marciani, 2003 ). Other adjuvants consist of cytokines ( Cheng et al., 2007 ) and glycolipids ( Singh et al., 1999 ; Ko et al., 2005 ) and other immunomodulators ( Morrow et al., 2004 ) that bind to highly specific receptors on T cells which activate them. TRADITIONAL VACCINE METHODOLOGY The early development of vaccines focused on using killed organisms, inactivated toxins or modified organisms, but currently there are many different approaches to vaccine development, which will be examined subsequently. As these approaches were empirical in design, these types of vaccines, whilst being successful, are now viewed as being traditional vaccines. These can be divided into three different types: (a) killed vaccines; (b) attenuated vaccines; and (c) sub-unit vaccines. Killed vaccines A killed or "inactivated" vaccine is developed by the pathogen being grown and then being made inactive by means of heat, chemical or radiation treatment and was the basis of most vaccines until the 1980s. This results in the pathogen being unable to cause disease whilst providing the immune system with stimulation via its normal antigenic epitopes on its cell surface. One major disadvantage of this approach is that, whilst these vaccines are immunogenic, they do not replicate in vivo infectivity limiting the spectrum of the immunity acquired as the agent is incapable of going through its normal antigenic variation over the course of the infection. This results in decreased immunity and a requirement of booster shots to maintain immunity ( Moylett and Hanson, 2003 ). Another disadvantage of this vaccine type is that during the inactivation process the antigenic epitopes can be modified resulting in a less efficacious vaccine ( Tano et al., 2007 ). Despite these limitations, killed vaccines are commonly used today with the typhoid, Salk poliomyelitis, and seasonal influenza vaccines still being administered ( Bazin, 2003 ; Palese, 2006 ). Attenuated vaccines Among the more efficacious of the traditional vaccines are the attenuated ones. In this case, a pathogen is subjected to altered growth conditions, is passage through a host or is genetically modified to eliminate its virulence, yet retaining its ability to replicate albeit at a greatly reduced rate. These vaccines are more successful at eliciting a robust lifelong immunity than other traditional vaccines. This can be attributed to their ability to cause an asymptomatic infection which stimulates both the humoral and cellular branches of the immune system. However, this ability to replicate carries the greatest risk as the vaccine can persist in immune-compromised persons or the elderly due to limited immune responses. A benefit to these vaccines is they express their own immunogenic antigens which stimulate the immune system strongly thereby negating the need for an adjuvant to be used ( Loessner et al., 2008 ). The most commonly used attenuated vaccine is the MMR vaccine which protects children worldwide against measles, mumps and rubella and with subsequent boosters provides lifelong immunity ( Vandermeulen et al., 2007 ). Attenuated vaccines have further progressed into carrier vaccines where they can deliver heterologous antigens ( Bachtiar et al., 2003 ; Lotter et al., 2008 ; Schoen et al., 2008 ). For live carrier vaccines that deliver multiple heterologous antigens, there is a risk that the host immune system will dampen the immune response to the heterologous antigens by misdirecting the immune response against the carrier ( Berzofsky et al., 2004 ). However, if the immunity induced is cell-mediated the response can be enhanced by pre-existing immunity to the carrier strain ( Saxena et al., 2013 ). Subunit vaccines Traditionally, it was thought that the only way to protect against a disease was to use the whole organism to vaccinate the host. However, it was elucidated that specific parts of the organisms, when purified or isolated, demonstrated immunogenic properties. These components could be the capsule, the flagella or even an outer membrane protein of the cell wall. These types of vaccines are known as subunit vaccines or acellular vaccines. These vaccines are not able to cause the disease and in comparison to whole cell killed vaccines they are not as efficacious. This is both an advantage as they are safe for immune-compromised patients and a disadvantage as they do not elicit long-term immunity and will often require multiple vaccinations to maintain immunity ( Schmitt et al., 2008 ). An advantage of this type of vaccine is that it can be engineered to protect against various strains of the organism. An example of a successful subunit vaccine is the Haemophilus influenzae type b (HiB) conjugate vaccine which consists of a polysaccharide-protein conjugate. This vaccine has eliminated or significantly reduced this disease in children in regions of South America ( Ribeiro et al., 2007 ; Franco-Paredes et al., 2008 ) and Africa ( Adegbola et al., 2005 ; Muganga et al., 2007 ) where it was once endemic. In the UK, the success of this vaccination program was compromised by a highly publicized paper (which has now been retracted) that linked autism to early childhood vaccination which lead to a rise in HiB infections as parents chose not to vaccinate; however, subsequent booster campaigns by the NHS has seen a reduction in infection rates again ( Ladhani et al., 2008 ). A recent meta-analysis covering studies involving over 1.2 million children has discredited any link between vaccinations or vaccine components thimerosal or mercury to the development of autism or autism spectrum disorders ( Taylor et al., 2014 ). Killed vaccines A killed or "inactivated" vaccine is developed by the pathogen being grown and then being made inactive by means of heat, chemical or radiation treatment and was the basis of most vaccines until the 1980s. This results in the pathogen being unable to cause disease whilst providing the immune system with stimulation via its normal antigenic epitopes on its cell surface. One major disadvantage of this approach is that, whilst these vaccines are immunogenic, they do not replicate in vivo infectivity limiting the spectrum of the immunity acquired as the agent is incapable of going through its normal antigenic variation over the course of the infection. This results in decreased immunity and a requirement of booster shots to maintain immunity ( Moylett and Hanson, 2003 ). Another disadvantage of this vaccine type is that during the inactivation process the antigenic epitopes can be modified resulting in a less efficacious vaccine ( Tano et al., 2007 ). Despite these limitations, killed vaccines are commonly used today with the typhoid, Salk poliomyelitis, and seasonal influenza vaccines still being administered ( Bazin, 2003 ; Palese, 2006 ). Attenuated vaccines Among the more efficacious of the traditional vaccines are the attenuated ones. In this case, a pathogen is subjected to altered growth conditions, is passage through a host or is genetically modified to eliminate its virulence, yet retaining its ability to replicate albeit at a greatly reduced rate. These vaccines are more successful at eliciting a robust lifelong immunity than other traditional vaccines. This can be attributed to their ability to cause an asymptomatic infection which stimulates both the humoral and cellular branches of the immune system. However, this ability to replicate carries the greatest risk as the vaccine can persist in immune-compromised persons or the elderly due to limited immune responses. A benefit to these vaccines is they express their own immunogenic antigens which stimulate the immune system strongly thereby negating the need for an adjuvant to be used ( Loessner et al., 2008 ). The most commonly used attenuated vaccine is the MMR vaccine which protects children worldwide against measles, mumps and rubella and with subsequent boosters provides lifelong immunity ( Vandermeulen et al., 2007 ). Attenuated vaccines have further progressed into carrier vaccines where they can deliver heterologous antigens ( Bachtiar et al., 2003 ; Lotter et al., 2008 ; Schoen et al., 2008 ). For live carrier vaccines that deliver multiple heterologous antigens, there is a risk that the host immune system will dampen the immune response to the heterologous antigens by misdirecting the immune response against the carrier ( Berzofsky et al., 2004 ). However, if the immunity induced is cell-mediated the response can be enhanced by pre-existing immunity to the carrier strain ( Saxena et al., 2013 ). Subunit vaccines Traditionally, it was thought that the only way to protect against a disease was to use the whole organism to vaccinate the host. However, it was elucidated that specific parts of the organisms, when purified or isolated, demonstrated immunogenic properties. These components could be the capsule, the flagella or even an outer membrane protein of the cell wall. These types of vaccines are known as subunit vaccines or acellular vaccines. These vaccines are not able to cause the disease and in comparison to whole cell killed vaccines they are not as efficacious. This is both an advantage as they are safe for immune-compromised patients and a disadvantage as they do not elicit long-term immunity and will often require multiple vaccinations to maintain immunity ( Schmitt et al., 2008 ). An advantage of this type of vaccine is that it can be engineered to protect against various strains of the organism. An example of a successful subunit vaccine is the Haemophilus influenzae type b (HiB) conjugate vaccine which consists of a polysaccharide-protein conjugate. This vaccine has eliminated or significantly reduced this disease in children in regions of South America ( Ribeiro et al., 2007 ; Franco-Paredes et al., 2008 ) and Africa ( Adegbola et al., 2005 ; Muganga et al., 2007 ) where it was once endemic. In the UK, the success of this vaccination program was compromised by a highly publicized paper (which has now been retracted) that linked autism to early childhood vaccination which lead to a rise in HiB infections as parents chose not to vaccinate; however, subsequent booster campaigns by the NHS has seen a reduction in infection rates again ( Ladhani et al., 2008 ). A recent meta-analysis covering studies involving over 1.2 million children has discredited any link between vaccinations or vaccine components thimerosal or mercury to the development of autism or autism spectrum disorders ( Taylor et al., 2014 ). DNA VACCINES – A NEXT GENERATION EXAMPLE There are multiple novel types of vaccines that are currently under development, such as bacterial ghosts ( Szostak et al., 1996 ; Jawale and Lee, 2014 ) and nanovaccines ( Cho et al., 2014 ). However, one that holds great promise and has had documented successes is DNA vaccines. DNA vaccines differ from traditional vaccines as they do not consist of a protein or a cell component but only the DNA that encodes an immunogenic antigen within a plasmid vector. The plasmid can be administered by injection, gene gun, electroporation, or aerosol delivery, upon which the host's immune cells, usually dendritic cells, will sample the plasmid and express the encoded antigens. These antigens are then degraded by the cell into peptides and presented via MHC class I and class II molecules depending on the mode of administration and the cell type. From this, both antibody and cellular responses can be induced ( Forde, 2005 ). The first reported use of a plasmid DNA vaccine outside of trial or experimental conditions was in 2003 and was a desperate attempt to save an endangered species from extinction ( Bouchie, 2003 ). The vaccine was for the highly endangered California condors against the lethal West Nile virus. West Nile virus had emerged in New York in 1999 and spread to 41 out of the 50 US states killing birds from 138 species in a matter of years. It was believed that if the virus spread to California, the remaining 200 or so condors would face extinction. The US Centers for Disease Control and Prevention (CDC) expedited the manufacture of an experimental vaccine and permitted the condors to be vaccinated with it ( Bouchie, 2003 ). The DNA vaccine expressed West Nile virus pre-membrane/membrane and envelope proteins. The vaccinated condors were monitored and it was observed that the DNA vaccination stimulated protective immunity in adults, nestlings and newly hatched chicks. Following two intramuscular vaccinations, the condors demonstrated excellent neutralizing antibodies 60 days post-vaccination with a continued increase until approximately 1 year post-vaccination. It was also noted that the birds did not show any unusual behaviors, health changes or side effects post-vaccination ( Chang et al., 2007 ). This vaccine has also demonstrated efficacy in other bird species such as the American robins ( Turdus migratorius ) ( Kilpatrick et al., 2010 ) and the fish crows ( Corvus ossifragus ; Turell et al., 2003 ). The first two DNA vaccines for veterinary use were granted US approval in 2005 for West Nile virus vaccine for horses and haematopoietic necrosis vaccine for farm-reared Atlantic salmon ( Chalmers, 2006 ). Even though there are no currently approved DNA vaccines for human use, as of May, 2014 there are 128 open trials listed on Clinicaltrials.Gov (2014) that involve DNA-based vaccines and therapies GLOBAL VACCINE SUCCESS The global eradication of smallpox is, to date; the most successful vaccination campaign in history. Smallpox has existed for many thousands of years and spread through the world following the migration of humans to new settlements ( Barquet and Domingo, 1997 ). As mentioned previously, Edward Jenner is famously credited with developing a smallpox vaccination using the cow-pox virus (vaccinia virus) and published many observations on both the successful and adverse events ( Jenner, 1809 ) associated with his vaccination protocol. Small pox was an indiscriminate disease that is caused by two virus variants Variola major and Variola minor and was responsible for 300–500 million deaths before its eradication ( Theves et al., 2014 ). The smallpox vaccine that was developed by Jenner produces both neutralizing antibodies and cell mediated responses that are protective against other members of the Orthopoxvirus genus ( Barquet and Domingo, 1997 ). After years of vaccination success but with deaths from smallpox still common, the World Health Assembly, the executive body of the [ World Health Organization (WHO), 2013 ] set a target to eradicate smallpox. This was only achievable as humans are the only reservoir for the virus and the vaccine had demonstrated high efficacy ( Fenner et al., 1988 ). In the late 1960s, the efforts of the WHO were strengthened with more funding and new surveillance protocols. The last natural occurrence of smallpox occurred in Somalia, where cook Ali Maow Maalin developed the rash on October 26th 1977, but tragically it was not the last global smallpox death [ World Health Organization (WHO), 1980 ]. Medical photographer Janet Parker became the last person to die of smallpox in the world when she was accidently exposed to it in her workplace at the University of Birmingham and unfortunately a lapse in obtaining her booster vaccination led to her being susceptible at the time of exposure ( Barquet and Domingo, 1997 ). Eradication of smallpox was declared on May 8, 1980 by the WHO when the Final Report of Global Commission for Certification of Smallpox Eradication was published [ World Health Organization (WHO), 1980 ]. As of 2014, two depositories of smallpox still exist at the CDC in the USA and the State Research Center of Virology and Biotechnology VECTOR in Koltsovo, Russia. The destruction of these viral stocks has been delayed and debated since the declaration of eradication occurred in, 1980. Discovery of smallpox victims during building excavations often fuels these debates although no viable virus has been recovered from these corpses, so the risk of a modern smallpox outbreak is improbable ( Reardon, 2014 ; Theves et al., 2014 ). The WHO is again debating the existence of these stocks in May, 2014 ( Reardon, 2014 ). Another successful vaccine that has been implemented globally is those against poliomyelitis – the Salk, and Sabin vaccines. There are three different poliovirus serotypes and all of them can lead to serious disability in children, even death by acute flaccid paralysis [ World Health Organization (WHO), 2014a ]. Due to its moderate mortality rates, its long-term severe disability consequences and like smallpox, humans being the only natural reservoir for the virus, the World Health Assembly set a target of eradication by the year 2000. This project is known as the Global Polio Eradication Initiative. Poliovirus Type 2 infection has not been observed since 1999 in India and Type 3 since 2012. However, in 2014, poliovirus Type 1 is still endemic in regions of Nigeria, Pakistan, and Afghanistan [ World Health Organization (WHO), 2014a ]. The reasons behind these persisting endemics will be discussed later. There are two vaccines, an oral live attenuated vaccine known as the Sabin vaccine and the inactivated poliovirus vaccine also known as the Salk vaccine [ World Health Organization (WHO), 2014a ]. The Sabin vaccine was derived from passages of the poliovirus strains through rats and mice and then through cell cultures more than 50 times resulting in an attenuated forms of the virus types that all induced good antibody levels ( Sabin, 1957 ; Baicus, 2012 ). In 1972, Sabin donated his vaccine strains to the WHO which increased the number of vaccine recipients from 5 to 80% [ Baicus, 2012 ; World Health Organization (WHO), 2014a ]. The Sabin vaccine is no longer in use in the USA or UK as the only poliomyelitis cases reported in the populations were vaccine-associated paralytic poliomyelitis where the vaccine strain has caused an outbreak but it is still used in some developing countries due to its ease of administration and cost (US$0.14 a dose vs US$2–3 a dose for Salk vaccine; Willyard, 2014 ). There are now plans to eliminate the Sabin vaccine entirely in the 124 countries that still use it by 2015 ( Willyard, 2014 ). The Salk vaccine is grown in monkey kidney cells and inactivated with formalin ( Salk et al., 1954 ) and was introduced in the USA in 1955 and by 1961, the incidence of poliomyelitis had decreased from 13.9 cases per 100,000 in 1954 to 0.8 cases per 100,000 in 1961 [ Baicus, 2012 ; World Health Organization (WHO), 2014a ]. Besides preventing deaths, the main benefit to come from polio vaccination is the cost savings to the healthcare system which is estimated at US$40–50 billion for the period between 1988 and 2035 in the USA alone [ World Health Organization (WHO), 2014a ]. Most countries that have been certified polio-free still have rare isolated cases which have come from travelers importing the virus from endemic areas, for example in Australia had one such case in 2007 ( Paterson and Durrheim, 2013 ). However, the Global Polio Eradication Initiative has a new timeline for eradication and with a new strategy of phasing out the Sabin vaccines, hopefully the world will be certified polio-free in 2018 ( Willyard, 2014 ). A more recent vaccine accomplishment is the pneumococcal conjugate vaccine (PCV) against Streptococcus pneumoniae (pneumococcus) infections which include acute otitis media, sinusitis, pneumonia and invasive pneumococcal diseases such as meningitis and sepsis. The first conjugate vaccine was a heptavalent vaccine which protects against seven different serotypes of pneumococcus and it was licensed in the USA in 2000 ( Black et al., 2000 ; Lee et al., 2014 ). Since that time, 10- ( Domingues et al., 2014 ), 13- ( Spijkerman et al., 2013 ), and 23-valent ( Grabenstein and Manoff, 2012 ) vaccines have been licensed with all producing strong immunity against a broad spectrum of strains. In the USA, all age groups from children under 5 years to adults over 65 years had dramatic reductions in incidence of pneumococcal infections over a seven year period after the PCV was available ( Pilishvili et al., 2010 ). It is predicted that if the heptavalent PCV was implemented in China it would prevent 4222 cases of invasive pneumococcal disease, 4,061,524 cases of otitis media and 472,527 cases of pneumonia, as well as preventing an additional 2682 deaths from pneumococcal disease; however, the implementation cost would be estimated at US$6.44 billion ( Che et al., 2014 ). The current overall cost of pneumococcal disease in the unvaccinated population in China is estimated to be US$3.5 billion ( Che et al., 2014 ). Following the introduction of PCV in the USA, an estimated 211,000 serious pneumococcal infections and 13,000 deaths were prevented in the period of 2000–2008 ( Pilishvili et al., 2010 ). The influence of this vaccine on public health is in its early stages and has already had impacts on child mortality in over 88 countries that have included various PCV on their recommended immunization schedule ( Whitney et al., 2014 ). There are other vaccines that have been successfully implemented in the past decade. The most recent and highly publicized vaccine is the quadrivalent human papillomavirus vaccine against cervical cancer, marketed as Gardasil ® , which prevents the premalignant disease that leads to cervical cancers and fulfills all the above criteria of being a successful vaccine ( Zhou et al., 1991 ; Govan, 2008 ). Initially the cost of Gardasil ® was extremely prohibitive at US$120 per dose with three doses required; however, in collaboration with GAVI Alliance, the cost from the supplier has dropped to US4.50 per dose which increases its affordability and likelihood of being implemented in developing countries ( Anon, 2013 ). As the cost of the vaccine decreases and more people are immunized this vaccine which has been included in over 30 countries immunization schedules, in conjunction with regular Pap screening, may lead to a long-term reduction in cervical cancer incidence ( Harper et al., 2010 ; Ribeiro-Muller and Muller, 2014 ). VACCINE FAILURES AND CHALLENGES Historically there have been more vaccine failures than successes and unfortunately those failures can be publicized and instill fear in the general public long after the event. One such failure is one that occurred early in the rollout of the Salk polio vaccine is known as the Cutter incident. In April, 1955 a few weeks after Salk's polio vaccine had been declared safe and efficacious, there were reports from California that five children had become paralyzed after receiving the vaccine ( Offit, 2005 ). These vaccines were traced to Cutter which was one of the five pharmaceutical companies that were granted a license to produce the vaccine in the USA ( Nathanson and Langmuir, 1995 ). It was found that two production batches failed the deactivation steps; so live virulent poliovirus was found in 120,000 doses of the vaccine. Of the children vaccinated from this pool, 40,000 developed abortive polio, 51 suffered from permanent paralysis and five died ( Nathanson and Langmuir, 1995 ). Unfortunately this was not the end of the tragedy, a polio outbreak followed where a further 113 people in close contact with the vaccinated children were infected and subsequently paralyzed, and a further five deaths ( Nathanson and Langmuir, 1995 ; Offit, 2005 ). This incident halted the implementation of the polio vaccine program and severely affected public confidence in the vaccination, not only in the USA but as far reaching as New Zealand ( Day, 2009 ), Germany, the UK and Sweden ( Axelsson, 2012 ) and in the end, it caused the USA to recommend Sabin's vaccine in the long term which, barring manufacturing failures, proved to be the more risky of the two vaccines as it could revert to full virulence and cause outbreaks of vaccine-associated paralytic poliomyelitis ( Offit, 2005 ; Fitzpatrick, 2006 ). Following its emergence in 1981, HIV infections and its subsequent disease acquired immunodeficiency syndrome (AIDS) has become a global pandemic with millions of deaths and over 34 million people living with HIV ( De Cock et al., 2012 ). According to the [ World Health Organization (WHO, 2014c ) ] and the Joint United Nations Programme on Hiv/Aids (2013) the pandemic appears to have peaked as AIDS-related deaths have decreased by 25% in the past decade as well as new infections decreasing by 20% since 2006. This is the combined effect of the development of anti-retroviral drugs, and better education about the transmission of this disease. However, a vaccine is desperately needed to prevent new infections and to stop this pandemic from affecting future generations. Multiple HIV vaccines have been tested in clinical trials with limited success ( Johnson et al., 2013 ). In the last decade, the most prominent vaccine trial failures was that of the Merck STEP phase II test of concept and efficacy trial for an Adenovirus5 (Ad5) vaccine. It showed that the MRKAd5 HIV-1 gag/pol/nef vaccine was highly immunogenic and elicited a higher magnitude of HIV-specific CD8+ T cells than any of the other HIV candidate vaccines over the past 15 years but it did not prevent HIV infection or reduce viral loads in infected patients ( Buchbinder et al., 2008 ). In fact, more disturbingly, there was an increase in the number of HIV-1 infections in male recipients of the vaccine compared to the controls ( McElrath et al., 2008 ). This trial was immediately ceased when the independent data and safety monitoring board determined that the study could not demonstrate efficacy ( Buchbinder et al., 2008 ). One of the reasons behind the failure of the Merck STEP clinical trial was the pre-existing neutralizing antibodies against Ad5. A recent study confirmed that the international epidemiology of pre-existing immunity to different adenovirus types can severely compromise its efficacy as only 14.8% of the 1904 participants were seronegative for neutralizing antibodies against Ad5 ( Mast et al., 2010 ). This indicates that naturally acquired infections from virulent forms of the vaccine vectors can limit their usefulness in the same species. However, choosing a virus from a different species for which no prior exposure is possible but may sound too risky to be accepted by the general population. It was also found that whilst the group of men that became more susceptible to HIV infection post-vaccination were seropositive against the Ad5 vector, they were also uncircumcised and had sexual relations with the same sex implying that pre-existing immunity may not be the sole factor that caused this vaccine failure ( Gray et al., 2010 ; Duerr et al., 2012 ). Whilst this phase II trial failure was a major setback for the HIV research community, it raised fundamental questions about the pathogenesis of HIV and also gave insight into immunological mechanisms that were previously unexplored ( Johnson et al., 2013 ; Fauci et al., 2014 ). The search for a HIV vaccine is ongoing and as of May 2014, there are 92 open HIV vaccine trials according to Clinicaltrials.Gov (2014) . Another infectious disease that is under surveillance by health departments worldwide is a double-stranded RNA virus called rotavirus. Rotavirus causes acute enteritis resulting in severe, dehydrating diarrhea in infants and young children and is very transmissible through close contact [ Bishop et al., 1976 ; World Health Organization (WHO), 2013 ]. In the pre-vaccination era, rotavirus caused 111 million cases of illness with 25 million medical visits, 2 million hospitalizations and between 352,000 and 592,000 rotavirus gastroenteritis-associated deaths worldwide annually with most of these occurring in low income countries ( Parashar et al., 2003 ). The first rotavirus vaccine was RotaShield which was developed by Wyeth-Lederle Vaccines and Pediatrics, Philadelphia, as an oral vaccine and showed high efficacy at 80% protection from severe illness; hence it was recommended for all infants in the USA once it was approved by the Food and Drug Administration (FDA) on August 31, 1998 ( American Academy of Pediatrics, 1998 ). Over the eleven month period after the vaccine was approved until July 7, 1999, 15 cases of intussusception, a type of intestinal blockage requiring surgical intervention, were reported and linked to the vaccination. In consultation with the FDA, Wyeth-Lederle Vaccines withdrew Rotashield from the market on October 15, 1999. Before this withdrawal, the cases of confirmed intussusception had risen to 101 ( Delage, 2000 ) and fortuitously, because there were no deaths caused by this vaccine, physician trust in vaccine safety measures were not compromised by this withdrawal ( McPhillips et al., 2001 ). In 2006, two new oral rotavirus vaccines were released onto the market: Rotarix ® – a live monovalent attenuated human strain by GlaxoSmithKline Biologicals ( Vesikari et al., 2004 ; Keating, 2006b ) and RotaTeq ® – a live pentavalent human-bovine reassortant vaccine by Merck & Co. Inc. ( Clark et al., 2004 ; Keating, 2006a ). After 6 years of use, a Cochrane Review found that both of these vaccines are efficacious with no increased risk of adverse side effects such as intussusception ( Soares-Weiser et al., 2012 ). However, in 2013, a small increase in risk was confirmed when the data was analyzed comparing the risk of intussusception in the post-vaccine period with other periods ( Haber et al., 2013 ; Quinn et al., 2014 ). A year later, the vaccines are still on the market albeit with an intussusception warning even though there is an estimated up to sixfold increase with the use of these two rotavirus vaccines. So far the Vaccine Safety Datalink has reported that Rotarix ® has had 66 intussusception cases in 200,000 doses, whilst RotaTeq ® had eight cases for 1.3 million doses administered with most occurring within 7 days after the first dose [ World Health Organization (WHO), 2014b ]. Currently the risk of intussusception is estimated to be 1–2 per 100,000 infants vaccinated [ World Health Organization (WHO), 2013 ]. However, the general view is that there are great benefits to vaccination against rotavirus as the infant mortality rates in countries that have added this to their vaccination schedule have significantly decreased ( Buttery et al., 2014 ) and this is reflected in the WHO's Global Advisory Committee on Vaccine Safety in their weekly epidemiological record [ World Health Organization (WHO), 2014b ] stated this in regards to the new intussusception risk: "the findings remain reassuring that the risk of intussusception following current rotavirus vaccines remains small compared to the benefits of preventing the impact of severe diarrhea." Surveillance of such adverse effects requires long-term study in order to make sound decisions about the appropriateness of the vaccine. There may come a time where the relative risk is too high and the vaccine is withdrawn like Rotashield which had a rate of intussusception of 1 in 10,000 infant doses [ World Health Organization (WHO), 2013 ], even though it provided strong immunological protection. This is one of the hardest aspects in vaccine development to plan for and may lead to public distrust in future vaccines, if it is not done expediently when those risks increases. POLITICAL AND GLOBAL ASPECTS OF VACCINE USAGE When a vaccine is designed, it is assumed that if it proves effective it will be used in various countries around the world to vaccinate the population; however, this is not always the case. Within each country there are government agencies, industry and community health advocates, and outside agencies such as the WHO that will make recommendations for vaccination strategies. Often this process will result in a successful vaccination strategy such as the global eradication of smallpox ( Stewart and Devlin, 2006 ), but it can also lead to confusion and scepticism in the chosen strategy. One such example was the choice of pertussis vaccine for a national vaccination campaign in the Netherlands. Originally, the Dutch government chose to use a whole cell vaccine based on the Bordetella pertussis bacterium; however, after speculation that the vaccine could cause brain damage, alternative vaccines were sought. At this time, acellular vaccines comprising three to five bacterial components were being used by many countries in Europe as they were comparable in protection to the whole cell vaccines and demonstrated minimal side effects ( Blume and Zanders, 2006 ). Over the course of 7 years, the debate over the new vaccine became very convoluted as many government agencies, drug companies, and consumer groups presented opposing studies and evidence. There was also external pressure from neighboring countries and global non-profit groups including the WHO and United Nations Children's Fund (UNICEF) for the Dutch government to make a decision. Concurrently, many parents had lost faith in the old vaccine strategy; hence an epidemic of pertussis ensued. To combat the growing epidemic the Dutch government chose an acellular vaccine which was used in primary vaccinations in 2005; however, the Health Minister advised that this decision was not based on recommendations and evidence provided by the Dutch Health Council, but on the need to appease parents and re-establish their confidence in the vaccine strategy ( Blume and Zanders, 2006 ). By contrast, in areas where any disease is endemic and the health system is overwhelmed, often the vaccination strategy proposed by governing bodies will be accepted by the population and acquiesced as mandatory ( Chalmers, 2006 ). Unfortunately this has not worked in areas such as Pakistan, Nigeria, and Afghanistan where the eradication of polio has failed due in part to misinformation, violence, politics, and mistrust about vaccination. There is a distinct divide in these populations between vaccine acceptors and non-acceptors which is based in the abundance of misinformation about the vaccine, religious beliefs and the emotional fear about the agenda; however, if there is an outbreak many non-acceptors will accept the vaccination as the fear of disease outweighs the perceived risks ( Murele et al., 2014 ). Socio-cultural, educational and perceptual factors are particularly strong in these regions and in some cases targeting male authority figures could improve vaccination uptake ( Murele et al., 2014 ); however, in other regions maternal education and empowerment has been suggested as a strong motivator in vaccine acceptance ( Larson et al., 2014 ). Violence is another contributing factor to this program's failure particularly when there are fatal attacks on vaccination workers in Pakistan and Nigeria ( Abimbola et al., 2013 ). In Afghanistan, both the Taliban regime and the militant Islamist terrorist group Al Qaeda support the Global Polio Eradication Initiative; however, factions within these groups can disrupt it as they view it as a Westernization issue, rather than a health one ( Abimbola et al., 2013 ). In Nigeria and Pakistan, militants can gain international media attention by attacking polio health workers ( Riaz and Rehman, 2013 ) and spreading propaganda that immunization programs are actually covert sterilization campaigns to reduce the Muslim population, which puts more fear into the local communities than the disease itself ( Abimbola et al., 2013 ; Willyard, 2014 ). All of the aforementioned issues affect the successful eradication of infectious diseases with well documented epidemiology and pathology. However, there exist conditions and disorders where the mechanisms of development and ongoing chronic pathology are yet to be fully ascertained. One such condition causing concern among health professionals globally is allergy. ALLERGY AND VACCINE POTENTIAL Allergy is a hypersensitivity disease characterized by the production of IgE antibodies against antigenic components (i.e., allergens) that can enter the body via the respiratory and gastrointestinal tract, the skin, an insect sting or injection of a drug ( Sicherer and Sampson, 2014 ). The clinical reactions experienced by sensitized patients vary in different target organs and include rhinitis, urticaria, and allergic asthma to life-threatening anaphylactic shock ( Sampson, 2003 , 2004 ). The acute symptoms of allergy are usually due to the release of inflammatory mediators by tissue-bound mast cells and circulation basophils. These inflammatory mediators include histamine, platelet-activating factor, leukotrienes, mast cell proteases, and a range of cytokines. Mediators are released when allergen binds to IgE antibody attached to FεRI receptors on the cell surface, causing degranulation. Studies show a skewing towards a Th2 response, with elevated levels of IL-4, IL-5, and IL-13, while tolerant individuals usually have higher levels of the Th1 cytokines IFN-gamma and TNF-alpha, and the regulatory cytokine IL-10 ( Andre et al., 1996 ; Noma et al., 1996 ; Schade et al., 2003 ; Turcanu et al., 2003 ; Tiemessen et al., 2004 ). The class switch to produce IgE antibody occurs during primary sensitization in allergic patients and seems to be driven by IL-4, which is a direct product of Th2 cells and other effector cells of the allergic immune response. The activation of allergen-specific T cells is achieved by the presentation of allergens via APCs, including dendritic cells ( Grainger et al., 2014 ; Nagai et al., 2014 ). As the prevalence and potential fatality of this disease has increased, so have the efforts to find effective therapies and prophylaxis also intensified ( Valenta et al., 2010 ). Specific immunotherapy (SIT) is effective for desensitization against inhalant allergens; however, it is not advised as a therapy against food allergy because of the high risk of adverse side-effects ( Sabato et al., 2014 ). Oral administration of antigens usually leads to tolerance, and has been effective in decreasing allergic sensitization to antibiotics and other medications ( Stevenson, 2000 , 2003 ). Obviously native food allergens cannot be administered in this way, but it may be possible for hypoallergenic or CpG-conjugated derivatives. Microencapsulation provides a promising way of delivering allergens without degradation in the stomach ( Litwin et al., 1996 ), thereby inducing oral tolerance, and has already been applied in clinical trials ( TePas et al., 2004 ). Conjugation or co-administration of recombinant allergens with Th1-inducing heat-killed bacteria has yielded good protective results in mice ( Li et al., 2003a , b ) and allergic dogs ( Frick et al., 2005 ). Various approaches have been attempted to develop safe and effective DNA vaccines and are discussed in the following section. DNA VACCINES AND ALLERGY DNA vaccines, as demonstrated in the California condors, can induce protective immune responses against infectious diseases. Plasmid DNA injected intramuscularly, intraperitoneally or with a gene gun results in transcription and translation of encoded genes and elicits an antibody response in the host ( Tang et al., 1992 ; Ulmer et al., 1993 ; Hsu et al., 1996b ). This method of immunization preferentially induces a Th1 immune response and suppression of IgE ( Raz et al., 1996 ; Yoshida et al., 2000 ). These effects appear to be mediated by both CD8+ and CD4+ cells ( Hsu et al., 1996a ; Lee et al., 1997 ; Peng et al., 2002 ), and plasmid DNA requires immunostimulatory sequences such as CpG for optimal immunogenicity ( Sato et al., 1996 ; Adel-Patient et al., 2001 ; Jilek et al., 2001 ; Hartl et al., 2004 ). Unmethylated CpG motifs either in bacterial DNA or as synthetic oligodeoxynucleotides (CpG-ODN) are recognized by the mammalian immune system via toll-like receptor 9 (and possibly other PRRs) and trigger a Th1 response ( Hartmann and Krieg, 1999 ; Stacey et al., 2000 ; Bauer et al., 2001 ). Experiments in murine models of allergic asthma, rhino sinusitis, and conjunctivitis show that administration of CpG-ODN alone prevents symptoms and reduces already established disease by reducing Th2 immune responses and IgE ( Kline et al., 1998 , 1999 ; Magone et al., 2000 ; Serebrisky et al., 2000 ). Allergen/CpG-ODN conjugates have been shown to be less allergenic and more immunogenic than native allergen ( Tighe et al., 2000 ; Horner et al., 2002 ). The major allergen from ragweed, Amb a 1, linked to an immunostimulatory DNA sequence promoted Th1 cytokine expression and down regulated Th2 expression in vitro ( Simons et al., 2004 ), reversed established airway hyperreactivity in a murine model of asthma ( Marshall et al., 2001 ; Santeliz et al., 2002 ) and yielded promising results in Phase II clinical trials ( Tulic et al., 2004 ). Genetic immunization to specific allergens using plasmid DNA offers a powerful solution to the major problems associated with protein immunization, such as cross-linking of IgE antibody on effector cells or even de novo synthesis of IgE antibodies to the immunized protein itself. However, genetic vaccination may lead to an uncontrolled synthesis of allergens in the vaccinated host ( Slater et al., 1998 ) and has been a major hurdle for application in allergic patients. Three approaches are currently used to prevent this from occurring: (a) cutting the allergen-coding gene into fragments, lacking the antigenic determinant but containing the original T cell epitope repertoire, (b) the use of hypoallergenic protein derivatives, or (c) fusing allergen with proteins that promote immune responses. Several allergens have been tested in DNA vaccination approaches using murine models, including Ara h 2 (peanut), bovine beta-lactoglobulin (cow's milk), Cry j 1 (Japanese cedar), phospholipase A2 (bee venom), Der f 11 and Der p 1 (dust-mite), and Bet v 1 and Phl p 2 (grass; Roy et al., 1999 ; Toda et al., 2000 ; Adel-Patient et al., 2001 ; Jilek et al., 2001 ; Kwon et al., 2001 ; Peng et al., 2002 ; Hochreiter et al., 2003 ; Ludwig-Portugall et al., 2004 ). Most studies observed elicitation of a Th1 response and increased IL-10 production. Mice vaccinated against phospholipase A2 were protected against fatal anaphylaxis following allergen challenge ( Jilek et al., 2001 ), while mice receiving an oral DNA vaccine containing the peanut allergen Ara h 2 ( Roy et al., 1999 ) experienced significantly less severe and delayed allergic reactions upon subsequent sensitization and challenge. However, prophylactic effects, while promising, are not sufficient to aid patients who have existing food allergy. In mice pre-sensitized to phospholipase A (bee venom), therapeutic gene vaccination prevented only 30% of mice from anaphylaxis ( Jilek et al., 2001 ). In addition to direct DNA vaccination, these approaches provide the option of co-delivering genes or adjuvant molecules with immunomodulatory properties together with the antigen sequence ( Hartl et al., 2004 ; Mutschlechner et al., 2009 ). Allergen–allergen hybrid molecules may combine different allergens from one complex allergen source or use allergens from different sources as demonstrated for grass pollen ( Linhart et al., 2005 ; Wallner et al., 2009 ). Furthermore, hybrid molecules using only T cell epitopes have been successfully used ( Linhart et al., 2008 ). Vaccination of mice with a plasmid containing the cDNA for OVA fused to the cDNA of IL-18 (Allergen–cytokine fusion protein), a potent Th1 inducer, reversed established airway hyperreactivity, while a plasmid containing OVA alone had only a prophylactic effect ( Maecker et al., 2001 ). Ubiquitination of allergens represents another routine approach for destroying IgE-binding epitopes on proteins to produce hypoallergenic DNA vaccines. This approach has been applied for the production of a DNA-based vaccine encoding an ubiquitinated version of Linhart v 1, the major allergen from birch pollen ( Bauer et al., 2006 ). It was demonstrated in a murine study that this vaccine did not produce any detectable antibody response, but T cell reactivity was preserved as well as allergic reactions prevented. In summary, several novel therapeutic and prophylactic therapies against allergy are currently under investigation ( Nieuwenhuizen and Lopata, 2005 ; Flicker et al., 2013 ; Weiss et al., 2013 ). Genetic immunization has proven a powerful method to induce anti-allergic immune responses. The underlying functional principle described seems to be based on the recruitment of allergen-specific Th1 cells, CD8+ cells and the establishment of a Th1 cytokine milieu. This response can be protective by preventing the development of a Th2-biased response towards allergens, as well as balance an ongoing Th2-type response in a more therapeutic application. More studies are needed to increase our understanding of the pathophysiology and immunological mechanisms of allergy, and to characterize the molecular structure and epitopes of allergens, to develop safer and more effective ways of combating this debilitating and potentially life-threatening disease. DNA VACCINES AND ALLERGY DNA vaccines, as demonstrated in the California condors, can induce protective immune responses against infectious diseases. Plasmid DNA injected intramuscularly, intraperitoneally or with a gene gun results in transcription and translation of encoded genes and elicits an antibody response in the host ( Tang et al., 1992 ; Ulmer et al., 1993 ; Hsu et al., 1996b ). This method of immunization preferentially induces a Th1 immune response and suppression of IgE ( Raz et al., 1996 ; Yoshida et al., 2000 ). These effects appear to be mediated by both CD8+ and CD4+ cells ( Hsu et al., 1996a ; Lee et al., 1997 ; Peng et al., 2002 ), and plasmid DNA requires immunostimulatory sequences such as CpG for optimal immunogenicity ( Sato et al., 1996 ; Adel-Patient et al., 2001 ; Jilek et al., 2001 ; Hartl et al., 2004 ). Unmethylated CpG motifs either in bacterial DNA or as synthetic oligodeoxynucleotides (CpG-ODN) are recognized by the mammalian immune system via toll-like receptor 9 (and possibly other PRRs) and trigger a Th1 response ( Hartmann and Krieg, 1999 ; Stacey et al., 2000 ; Bauer et al., 2001 ). Experiments in murine models of allergic asthma, rhino sinusitis, and conjunctivitis show that administration of CpG-ODN alone prevents symptoms and reduces already established disease by reducing Th2 immune responses and IgE ( Kline et al., 1998 , 1999 ; Magone et al., 2000 ; Serebrisky et al., 2000 ). Allergen/CpG-ODN conjugates have been shown to be less allergenic and more immunogenic than native allergen ( Tighe et al., 2000 ; Horner et al., 2002 ). The major allergen from ragweed, Amb a 1, linked to an immunostimulatory DNA sequence promoted Th1 cytokine expression and down regulated Th2 expression in vitro ( Simons et al., 2004 ), reversed established airway hyperreactivity in a murine model of asthma ( Marshall et al., 2001 ; Santeliz et al., 2002 ) and yielded promising results in Phase II clinical trials ( Tulic et al., 2004 ). Genetic immunization to specific allergens using plasmid DNA offers a powerful solution to the major problems associated with protein immunization, such as cross-linking of IgE antibody on effector cells or even de novo synthesis of IgE antibodies to the immunized protein itself. However, genetic vaccination may lead to an uncontrolled synthesis of allergens in the vaccinated host ( Slater et al., 1998 ) and has been a major hurdle for application in allergic patients. Three approaches are currently used to prevent this from occurring: (a) cutting the allergen-coding gene into fragments, lacking the antigenic determinant but containing the original T cell epitope repertoire, (b) the use of hypoallergenic protein derivatives, or (c) fusing allergen with proteins that promote immune responses. Several allergens have been tested in DNA vaccination approaches using murine models, including Ara h 2 (peanut), bovine beta-lactoglobulin (cow's milk), Cry j 1 (Japanese cedar), phospholipase A2 (bee venom), Der f 11 and Der p 1 (dust-mite), and Bet v 1 and Phl p 2 (grass; Roy et al., 1999 ; Toda et al., 2000 ; Adel-Patient et al., 2001 ; Jilek et al., 2001 ; Kwon et al., 2001 ; Peng et al., 2002 ; Hochreiter et al., 2003 ; Ludwig-Portugall et al., 2004 ). Most studies observed elicitation of a Th1 response and increased IL-10 production. Mice vaccinated against phospholipase A2 were protected against fatal anaphylaxis following allergen challenge ( Jilek et al., 2001 ), while mice receiving an oral DNA vaccine containing the peanut allergen Ara h 2 ( Roy et al., 1999 ) experienced significantly less severe and delayed allergic reactions upon subsequent sensitization and challenge. However, prophylactic effects, while promising, are not sufficient to aid patients who have existing food allergy. In mice pre-sensitized to phospholipase A (bee venom), therapeutic gene vaccination prevented only 30% of mice from anaphylaxis ( Jilek et al., 2001 ). In addition to direct DNA vaccination, these approaches provide the option of co-delivering genes or adjuvant molecules with immunomodulatory properties together with the antigen sequence ( Hartl et al., 2004 ; Mutschlechner et al., 2009 ). Allergen–allergen hybrid molecules may combine different allergens from one complex allergen source or use allergens from different sources as demonstrated for grass pollen ( Linhart et al., 2005 ; Wallner et al., 2009 ). Furthermore, hybrid molecules using only T cell epitopes have been successfully used ( Linhart et al., 2008 ). Vaccination of mice with a plasmid containing the cDNA for OVA fused to the cDNA of IL-18 (Allergen–cytokine fusion protein), a potent Th1 inducer, reversed established airway hyperreactivity, while a plasmid containing OVA alone had only a prophylactic effect ( Maecker et al., 2001 ). Ubiquitination of allergens represents another routine approach for destroying IgE-binding epitopes on proteins to produce hypoallergenic DNA vaccines. This approach has been applied for the production of a DNA-based vaccine encoding an ubiquitinated version of Linhart v 1, the major allergen from birch pollen ( Bauer et al., 2006 ). It was demonstrated in a murine study that this vaccine did not produce any detectable antibody response, but T cell reactivity was preserved as well as allergic reactions prevented. In summary, several novel therapeutic and prophylactic therapies against allergy are currently under investigation ( Nieuwenhuizen and Lopata, 2005 ; Flicker et al., 2013 ; Weiss et al., 2013 ). Genetic immunization has proven a powerful method to induce anti-allergic immune responses. The underlying functional principle described seems to be based on the recruitment of allergen-specific Th1 cells, CD8+ cells and the establishment of a Th1 cytokine milieu. This response can be protective by preventing the development of a Th2-biased response towards allergens, as well as balance an ongoing Th2-type response in a more therapeutic application. More studies are needed to increase our understanding of the pathophysiology and immunological mechanisms of allergy, and to characterize the molecular structure and epitopes of allergens, to develop safer and more effective ways of combating this debilitating and potentially life-threatening disease. CONCLUSION The advent of vaccination changed global society and our everyday lives dramatically, especially in conjunction with improved healthcare, infrastructure and technology. Over the last century with increasing knowledge of the immune system and infectious diseases, infant mortality associated with infectious diseases dropped, in developed countries debilitating illnesses like polio disappeared from public view, and the youth of today did not experience the threat or fear of death via infectious diseases. However, some diseases such as HIV and malaria are yet to have efficacious vaccines developed and successfully complete Phase III clinical trials. So the fight continues against these known enemies and with each failure, we learn more. The list of global health threats consists of many incurable infectious diseases; immunological disorders such as allergy should be added to that list. Currently, therapeutic interventions are adequate, but with population and allergy prevalence increasing there is a strong need for a prophylactic vaccine. Although the establishment of allergy is not fully elucidated, researchers should be mining the already long history of infectious disease vaccines to create new avenues of allergen vaccine development. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3982875/

The View from the Trenches Part 1: Emergency Medical Response Plans and the Need for EPR Screening

Few natural disasters or intentional acts of war or terrorism have the potential for such severe impact upon a population and infrastructure as the intentional detonation of a nuclear device within a major U.S. city. In stark contrast to other disasters or even a "dirty bomb," hundreds of thousands will be affected and potentially exposed to a clinically significant dose of ionizing radiation. This will result in immediate deaths and injuries and subsequently the development of Acute Radiation Syndrome (ARS). Additionally, millions more who are unlikely to develop ARS will seek medical evaluation and treatment, overwhelming the capacity of an already compromised medical system. In this paper, we propose that in vivo electron paramagnetic resonance (EPR) dosimetry be utilized to screen large numbers of potentially exposed victims, and that this screening process be incorporated into the medical-surge framework that is currently being implemented across the nation for other catastrophic public health emergencies. The National Incident Management System (NIMS), the National Response Framework (NRF), the Target Capabilities list (TCL), Homeland Security Presidential Directives (HSPD), as well as additional guidance from multiple federal agencies provides a solid framework for this response. The effective screening of potentially exposed victims directly following a nuclear attack could potentially decrease the number of patients seeking immediate medical care by greater than 90%.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7417294/

Notes on Brachymenium in Guyana with a new species from Mt. Ayanganna

Abstract A relative of the African species described by Brotherus as Bryum perspinidens , has been discovered in Guyana with erect capsules and a short inner peristome. The Guyana material is recognized as a new species, and both species are placed in the genus Brachymenium . The characteristics that distinquish the genus are discussed with reference to the Guyana specimens of Brachymenium speciosum . Citation Robinson H, Golinski GK (2020) Notes on Brachymenium in Guyana with a new species from Mt. Ayanganna. PhytoKeys 154: 11–17. https://doi.org/10.3897/phytokeys.154.39105 Introduction Study of bryophyte collections obtained during the Smithsonian Biological Diversity of the Guianas project, has revealed a number of interesting species. Among these are two two collections of a bryaceous moss with capsules identifiable as a Brachymenium Schwaegr., Spec. Musc. Suppl. 2(1): 131. 1824, with a leaf that superficially matches the illustration of Bryum perspinidens Broth. in the Brotherus treatments in the two editions (1904 and 1925) of Engler and Prantl. The only problems were that the Brotherus illustration was of an African species named as a Bryum . The spiniform teeth of the leaf margins were nevertheless similar, and a relationship seemed to be involved. As for the generic placement, the Brotherus (1897) species was described from sterile material so that the placement in Bryum Hedw. lacked any real evidence. The relationship of Guyana Highland species to African species fits a pattern noted by Robinson (1965) . In addition, there is ample material from Guyana of another species of Brachymenium , B. speciosum that is newly discussed and illustrated. Methods Specimens in this study were obtained during the Smithsonian Biological Diversity of the Guianas Program conducted over a period of years from 1985 to 2014 ( Kelloff et al. 2019 ). The particular specimens of the new species involved in this study were collected during a separately funded trip conducted by M.D. Clark in 2001 that collected on Mt. Ayanganna. The bryophytes were deposited at the US National Herbarium awaiting identification. They have been in storage since that time. A note with the specimens indicates that when they arrived in the US they were irradiated during the Anthrax scare of 2001. Results The South American material includes one species that seems to be distinct from others from the Western Hemisphere (see for example Allen 2002 ) and from the related African species. Taxon classification Plantae Bryales Bryaceae Brachymenium ayangannensis H.Rob. & G.K.Golinski sp. nov. 559AD855-EF85-57AE-9984-DA2994EE2C2F Figure 1 Type. Guyana. Region: Potaro–Siparuni. Mt. Ayanganna, east face, plateau above second of four escarpments. 1380 m, 05°22.550'N, 059°58.350'W . Scrub forest on sandstone and peat, with Clusia , Pagamea and Sphagnum . Epiphyte; sporophytes green. 17 June 2001. H.D. Clarke 9299 , with R. Williams , C. Perry , E. Tripp & J. Kelly (US). 10.3897/phytokeys.154.39105.figure1 B9CA91A5-4CDE-5904-8594-2AA3D07A54A5 Figure 1. Brachymenium ayangannensis H.Rob. & G.K.Golinski. A Leafy stem showing distorted leaves B distal part of leaves, showing well-developed pale margin and spiniform teeth on both margin and long-acuminate tip C remoistened erect capsule showing partially detached short-rostrate operculum D B. perspinidens (Broth.) H.Rob. & Golinski, distal part of leaf of holotype (from H) E tip of capsule mounted in Hoyer's solution showing short exostome and lack of well-developed endostome. The photographs were taken using a Leica DM4B Compound microscope, using a 5× objective. https://binary.pensoft.net/fig/438302 Description. Stems up to 3 cm tall, leaves not closely spaced, rather firm in structure but contorted when dry and resistant to wetting. Costa percurrent into a long slender acumination, median cells narrowly oval, with firm walls showing slight porosity, mostly 80–100 μm long and ca. 30 μm wide, without shorter quadrate cells at base, margin with numerous rows of linear pale cells forming a strong border, border with numerous cells projecting as spiniform teeth, such spiniform teeth extending onto apical acumination. Synoicous? Seta pale yellowish-red, ca. 17 mm long, smooth. Capsules erect, ca. 2 mm long, with short hypophysis, operculum short-rostrate, higher than wide. Outer peristome teeth reddish, rudimentary, ca. 80 μm long, inner peristome a low pale membrane ca. 70 μm without projecting segments or cilia. Calyptra not seen. Spores ca. 10 μm in size. Additional material. Guyana. Region: Potaro–Siparuni. Mt. Ayanganna, east face, area near camp at base of fourth of four escarpments. Elev. 1545 m, 05°23.083'N, 059°58.550'W . Dense forest on sandstone and peat, with Euterpe , Clusia , and Brocchinia . Sporophytes green. On tree limb. H.D. Clarke 9551 with R. Williams , C. Perry , E. Tripp & J. Kelly (US). The peristome teeth of the new species have proven extremely fragile, possibly because of the radiation treatment. The spiniform teeth of the leaf margin are distinctive, but the manner in which they occur on the acuminate apical extension is reminiscent of the illustration by Brotherus (1904 : 557 fig. C; 1925: 367, figs C, D). This illustration has led to the comparison, but it proves to be somewhat inaccurate compared to the more recent illustration made from the type by Ochi (1972) The African species is well illustrated by Ochi (1972) , but the type from Helsinki has been borrowed not because of doubts of relationship so much as to insure that the two species are not the same. The principle difference is the absence of spinose teeth extending on to the apical acumination of the leaf. Nevertheless, there is no doubt the two are close, and the African species was placed in Bryum only because there was no sporophyte to indicate otherwise. A important point derived from the Ochi study is that none of the species in typical Bryum have spinose marginal teeth, all with such teeth are in what is now in the Brachymenium , Rhodobryum relationship. On the basis of the evident relationship between the African and Guyana species, the following transfer of the African species is provided. Taxon classification Plantae Bryales Bryaceae Brachymenium perspinidens (Broth.) H.Rob. & G.K.Golinski comb. nov. 673E4191-B2F7-52F6-B9C3-CE439A8FCF10 Figure 1D Bryum perspinidens Broth., Bot. Jahrb. Syst. 24: 246. 1897. Britische Ostafrika, Seengebiet: Ru– Nssóro, 3300–3600 m (Uganda: Ruwenzori, heather forest 10–12000'), Scott Elliot 266 , Sterile. Rhodobryum perspinidens (Broth.) Pócs, in Bizot & Pócs, Acta Bot. Acad. Sci. Hungaricae 25: 257. 1979 [1980]. With record of species from Tanzania, also sterile. Ochi (1972) indicated the species was rather an oddity in Bryum Hedw. subgenus Rhodobryum Schimp. in which he placed it. Notes. Placement of the new species in Brachymenium is based on the capsules being erect with an inner peristome being a low membrane lacking segments or cilia, the traditional distinctions of the genus. Recent DNA studies ( Pedersen et al. 2003 , Pedersen and Hedenas 2005 ; Cox and Hedderson 2003 ) indicate that species that have been placed in the genus Brachymenium are mostly in basal branches of the Bryaceae while Bryum species are more derived. According to such studies, the genus Brachymenium is more entangled phyletically with the genus Rhodobryum (Schimp.) Hampe, Linnaea 38: 663. 1874, a later established genus and Osculatia De Not., Mem. Reale Accad. Sci. Torino, ser 2, 18: 445. 1859 ( Robinson 1965 ; Ochyra et al. 2018 ). A survey of the illustrations in Brotherus (1904 , 1925 ) shows an additional trend in Brachymenium that is lacking in typical Bryum , conical to rostrate opercula such is seen in the new species. In fact, within the present definition of Brachymenium , fully rostrate opercula occur in another species recently collected in Guyana. Taxon classification Plantae Bryales Bryaceae Brachymenium speciosum (Hook. & Wils.) Steere. 17399882-F918-5D65-968F-86F1F8CCAEAA Figure 2 Notes. The latter species has been collected on a mountain near Ayanganna as indicated below. Mt. Wokomung, Little Ayanganna, upper slopes of highest point of Mount Wokomunga massif. 5°5'8"N, 59°50'32"W . elev. 1525 m. Tepui scrub forest on sandstone and peat, with Schefflera , Clusia and Guadua . 5 July 2003, H.D. Clarke 10550 , with R. Williams , C. Perry , J. Kelly , D. Gittens , S. Stern ; Guiana. Mt. Wokomung, Little Ayanganna, upper slopes of highest point of Mount Wokomung massif. 5°5'8"N, 59°50'32"W . elev. 1525 m. Tepui scrub forest on sandstone and peat, with Schefflera , Clusia and Guadua , elev. 1525 m. 5 July 2003, H.D. Clarke 10575 , with R. Williams , C. Perry , J. Kelly , D. Gittens , S. Stern. Guiana. Mt. Wokomung, Little Ayanganna, upper slopes of highest point of Mount Wokomung massif. 5°4'53.1"N, 59°50'26.1"W . elev. 1525 m. Tepui bog on sandstone and peat, with Brocchinia , Bonnetia and Rapatea , elev. 1660 m. 6 July 2003, H.D. Clarke 10576 , with R. Williams , C. Perry , J. Kelly , D. Gittens , S. Stern. Mt. Wokomung, area above third of four escarpments, 1 km NE of Mt. Wokomung, 5°4'30"N, 59°51'15"W . elev. 1490 m. dense forest on laterite, with Clusia , Euterpe and Licania , elev. 1490 m. 8 July 2003, H.D. Clarke 10802 , with R. Williams , C. Perry , J. Kelly , D. Gittens , S. Stern. The material shows the additional feature of the species, the multistratose leaf margin with teeth on the margin and upper and lower surfaces. The species is otherwise reported from Suriname, Ecuador, and supposedly described from Jamaica (Maracaibo, Venezuela?)( Allen 2013 ; Steere 1948 ). 10.3897/phytokeys.154.39105.figure2 A773665D-5A78-5BAA-B8C7-A58E5EA80620 Figure 2. Brachymenium speciosum (Hook. & Wils.) Steere. A Part of leafy stem showing distorted leaves B tip of leaf showing thickened margin with teeth on margin and upper and lower surfaces, some of these appearing as double teeth C capsule showing rostrate operculum D peristome teeth mounted in Hoyer's solution, showing elongate exostome teeth erect on one half and reflexed on other half, the latter showing endostome lacking cilia and segments. The photographs were taken using a Leica DM4B Compound microscope, 5× objective. https://binary.pensoft.net/fig/438303 Taxon classification Plantae Bryales Bryaceae Brachymenium ayangannensis H.Rob. & G.K.Golinski sp. nov. 559AD855-EF85-57AE-9984-DA2994EE2C2F Figure 1 Type. Guyana. Region: Potaro–Siparuni. Mt. Ayanganna, east face, plateau above second of four escarpments. 1380 m, 05°22.550'N, 059°58.350'W . Scrub forest on sandstone and peat, with Clusia , Pagamea and Sphagnum . Epiphyte; sporophytes green. 17 June 2001. H.D. Clarke 9299 , with R. Williams , C. Perry , E. Tripp & J. Kelly (US). 10.3897/phytokeys.154.39105.figure1 B9CA91A5-4CDE-5904-8594-2AA3D07A54A5 Figure 1. Brachymenium ayangannensis H.Rob. & G.K.Golinski. A Leafy stem showing distorted leaves B distal part of leaves, showing well-developed pale margin and spiniform teeth on both margin and long-acuminate tip C remoistened erect capsule showing partially detached short-rostrate operculum D B. perspinidens (Broth.) H.Rob. & Golinski, distal part of leaf of holotype (from H) E tip of capsule mounted in Hoyer's solution showing short exostome and lack of well-developed endostome. The photographs were taken using a Leica DM4B Compound microscope, using a 5× objective. https://binary.pensoft.net/fig/438302 Description. Stems up to 3 cm tall, leaves not closely spaced, rather firm in structure but contorted when dry and resistant to wetting. Costa percurrent into a long slender acumination, median cells narrowly oval, with firm walls showing slight porosity, mostly 80–100 μm long and ca. 30 μm wide, without shorter quadrate cells at base, margin with numerous rows of linear pale cells forming a strong border, border with numerous cells projecting as spiniform teeth, such spiniform teeth extending onto apical acumination. Synoicous? Seta pale yellowish-red, ca. 17 mm long, smooth. Capsules erect, ca. 2 mm long, with short hypophysis, operculum short-rostrate, higher than wide. Outer peristome teeth reddish, rudimentary, ca. 80 μm long, inner peristome a low pale membrane ca. 70 μm without projecting segments or cilia. Calyptra not seen. Spores ca. 10 μm in size. Additional material. Guyana. Region: Potaro–Siparuni. Mt. Ayanganna, east face, area near camp at base of fourth of four escarpments. Elev. 1545 m, 05°23.083'N, 059°58.550'W . Dense forest on sandstone and peat, with Euterpe , Clusia , and Brocchinia . Sporophytes green. On tree limb. H.D. Clarke 9551 with R. Williams , C. Perry , E. Tripp & J. Kelly (US). The peristome teeth of the new species have proven extremely fragile, possibly because of the radiation treatment. The spiniform teeth of the leaf margin are distinctive, but the manner in which they occur on the acuminate apical extension is reminiscent of the illustration by Brotherus (1904 : 557 fig. C; 1925: 367, figs C, D). This illustration has led to the comparison, but it proves to be somewhat inaccurate compared to the more recent illustration made from the type by Ochi (1972) The African species is well illustrated by Ochi (1972) , but the type from Helsinki has been borrowed not because of doubts of relationship so much as to insure that the two species are not the same. The principle difference is the absence of spinose teeth extending on to the apical acumination of the leaf. Nevertheless, there is no doubt the two are close, and the African species was placed in Bryum only because there was no sporophyte to indicate otherwise. A important point derived from the Ochi study is that none of the species in typical Bryum have spinose marginal teeth, all with such teeth are in what is now in the Brachymenium , Rhodobryum relationship. On the basis of the evident relationship between the African and Guyana species, the following transfer of the African species is provided. Type. Guyana. Region: Potaro–Siparuni. Mt. Ayanganna, east face, plateau above second of four escarpments. 1380 m, 05°22.550'N, 059°58.350'W . Scrub forest on sandstone and peat, with Clusia , Pagamea and Sphagnum . Epiphyte; sporophytes green. 17 June 2001. H.D. Clarke 9299 , with R. Williams , C. Perry , E. Tripp & J. Kelly (US). 10.3897/phytokeys.154.39105.figure1 B9CA91A5-4CDE-5904-8594-2AA3D07A54A5 Figure 1. Brachymenium ayangannensis H.Rob. & G.K.Golinski. A Leafy stem showing distorted leaves B distal part of leaves, showing well-developed pale margin and spiniform teeth on both margin and long-acuminate tip C remoistened erect capsule showing partially detached short-rostrate operculum D B. perspinidens (Broth.) H.Rob. & Golinski, distal part of leaf of holotype (from H) E tip of capsule mounted in Hoyer's solution showing short exostome and lack of well-developed endostome. The photographs were taken using a Leica DM4B Compound microscope, using a 5× objective. https://binary.pensoft.net/fig/438302 Description. Stems up to 3 cm tall, leaves not closely spaced, rather firm in structure but contorted when dry and resistant to wetting. Costa percurrent into a long slender acumination, median cells narrowly oval, with firm walls showing slight porosity, mostly 80–100 μm long and ca. 30 μm wide, without shorter quadrate cells at base, margin with numerous rows of linear pale cells forming a strong border, border with numerous cells projecting as spiniform teeth, such spiniform teeth extending onto apical acumination. Synoicous? Seta pale yellowish-red, ca. 17 mm long, smooth. Capsules erect, ca. 2 mm long, with short hypophysis, operculum short-rostrate, higher than wide. Outer peristome teeth reddish, rudimentary, ca. 80 μm long, inner peristome a low pale membrane ca. 70 μm without projecting segments or cilia. Calyptra not seen. Spores ca. 10 μm in size. Additional material. Guyana. Region: Potaro–Siparuni. Mt. Ayanganna, east face, area near camp at base of fourth of four escarpments. Elev. 1545 m, 05°23.083'N, 059°58.550'W . Dense forest on sandstone and peat, with Euterpe , Clusia , and Brocchinia . Sporophytes green. On tree limb. H.D. Clarke 9551 with R. Williams , C. Perry , E. Tripp & J. Kelly (US). The peristome teeth of the new species have proven extremely fragile, possibly because of the radiation treatment. The spiniform teeth of the leaf margin are distinctive, but the manner in which they occur on the acuminate apical extension is reminiscent of the illustration by Brotherus (1904 : 557 fig. C; 1925: 367, figs C, D). This illustration has led to the comparison, but it proves to be somewhat inaccurate compared to the more recent illustration made from the type by Ochi (1972) The African species is well illustrated by Ochi (1972) , but the type from Helsinki has been borrowed not because of doubts of relationship so much as to insure that the two species are not the same. The principle difference is the absence of spinose teeth extending on to the apical acumination of the leaf. Nevertheless, there is no doubt the two are close, and the African species was placed in Bryum only because there was no sporophyte to indicate otherwise. A important point derived from the Ochi study is that none of the species in typical Bryum have spinose marginal teeth, all with such teeth are in what is now in the Brachymenium , Rhodobryum relationship. On the basis of the evident relationship between the African and Guyana species, the following transfer of the African species is provided. Taxon classification Plantae Bryales Bryaceae Brachymenium perspinidens (Broth.) H.Rob. & G.K.Golinski comb. nov. 673E4191-B2F7-52F6-B9C3-CE439A8FCF10 Figure 1D Bryum perspinidens Broth., Bot. Jahrb. Syst. 24: 246. 1897. Britische Ostafrika, Seengebiet: Ru– Nssóro, 3300–3600 m (Uganda: Ruwenzori, heather forest 10–12000'), Scott Elliot 266 , Sterile. Rhodobryum perspinidens (Broth.) Pócs, in Bizot & Pócs, Acta Bot. Acad. Sci. Hungaricae 25: 257. 1979 [1980]. With record of species from Tanzania, also sterile. Ochi (1972) indicated the species was rather an oddity in Bryum Hedw. subgenus Rhodobryum Schimp. in which he placed it. Notes. Placement of the new species in Brachymenium is based on the capsules being erect with an inner peristome being a low membrane lacking segments or cilia, the traditional distinctions of the genus. Recent DNA studies ( Pedersen et al. 2003 , Pedersen and Hedenas 2005 ; Cox and Hedderson 2003 ) indicate that species that have been placed in the genus Brachymenium are mostly in basal branches of the Bryaceae while Bryum species are more derived. According to such studies, the genus Brachymenium is more entangled phyletically with the genus Rhodobryum (Schimp.) Hampe, Linnaea 38: 663. 1874, a later established genus and Osculatia De Not., Mem. Reale Accad. Sci. Torino, ser 2, 18: 445. 1859 ( Robinson 1965 ; Ochyra et al. 2018 ). A survey of the illustrations in Brotherus (1904 , 1925 ) shows an additional trend in Brachymenium that is lacking in typical Bryum , conical to rostrate opercula such is seen in the new species. In fact, within the present definition of Brachymenium , fully rostrate opercula occur in another species recently collected in Guyana. Notes. Placement of the new species in Brachymenium is based on the capsules being erect with an inner peristome being a low membrane lacking segments or cilia, the traditional distinctions of the genus. Recent DNA studies ( Pedersen et al. 2003 , Pedersen and Hedenas 2005 ; Cox and Hedderson 2003 ) indicate that species that have been placed in the genus Brachymenium are mostly in basal branches of the Bryaceae while Bryum species are more derived. According to such studies, the genus Brachymenium is more entangled phyletically with the genus Rhodobryum (Schimp.) Hampe, Linnaea 38: 663. 1874, a later established genus and Osculatia De Not., Mem. Reale Accad. Sci. Torino, ser 2, 18: 445. 1859 ( Robinson 1965 ; Ochyra et al. 2018 ). A survey of the illustrations in Brotherus (1904 , 1925 ) shows an additional trend in Brachymenium that is lacking in typical Bryum , conical to rostrate opercula such is seen in the new species. In fact, within the present definition of Brachymenium , fully rostrate opercula occur in another species recently collected in Guyana. Taxon classification Plantae Bryales Bryaceae Brachymenium speciosum (Hook. & Wils.) Steere. 17399882-F918-5D65-968F-86F1F8CCAEAA Figure 2 Notes. The latter species has been collected on a mountain near Ayanganna as indicated below. Mt. Wokomung, Little Ayanganna, upper slopes of highest point of Mount Wokomunga massif. 5°5'8"N, 59°50'32"W . elev. 1525 m. Tepui scrub forest on sandstone and peat, with Schefflera , Clusia and Guadua . 5 July 2003, H.D. Clarke 10550 , with R. Williams , C. Perry , J. Kelly , D. Gittens , S. Stern ; Guiana. Mt. Wokomung, Little Ayanganna, upper slopes of highest point of Mount Wokomung massif. 5°5'8"N, 59°50'32"W . elev. 1525 m. Tepui scrub forest on sandstone and peat, with Schefflera , Clusia and Guadua , elev. 1525 m. 5 July 2003, H.D. Clarke 10575 , with R. Williams , C. Perry , J. Kelly , D. Gittens , S. Stern. Guiana. Mt. Wokomung, Little Ayanganna, upper slopes of highest point of Mount Wokomung massif. 5°4'53.1"N, 59°50'26.1"W . elev. 1525 m. Tepui bog on sandstone and peat, with Brocchinia , Bonnetia and Rapatea , elev. 1660 m. 6 July 2003, H.D. Clarke 10576 , with R. Williams , C. Perry , J. Kelly , D. Gittens , S. Stern. Mt. Wokomung, area above third of four escarpments, 1 km NE of Mt. Wokomung, 5°4'30"N, 59°51'15"W . elev. 1490 m. dense forest on laterite, with Clusia , Euterpe and Licania , elev. 1490 m. 8 July 2003, H.D. Clarke 10802 , with R. Williams , C. Perry , J. Kelly , D. Gittens , S. Stern. The material shows the additional feature of the species, the multistratose leaf margin with teeth on the margin and upper and lower surfaces. The species is otherwise reported from Suriname, Ecuador, and supposedly described from Jamaica (Maracaibo, Venezuela?)( Allen 2013 ; Steere 1948 ). 10.3897/phytokeys.154.39105.figure2 A773665D-5A78-5BAA-B8C7-A58E5EA80620 Figure 2. Brachymenium speciosum (Hook. & Wils.) Steere. A Part of leafy stem showing distorted leaves B tip of leaf showing thickened margin with teeth on margin and upper and lower surfaces, some of these appearing as double teeth C capsule showing rostrate operculum D peristome teeth mounted in Hoyer's solution, showing elongate exostome teeth erect on one half and reflexed on other half, the latter showing endostome lacking cilia and segments. The photographs were taken using a Leica DM4B Compound microscope, 5× objective. https://binary.pensoft.net/fig/438303 Notes. The latter species has been collected on a mountain near Ayanganna as indicated below. Mt. Wokomung, Little Ayanganna, upper slopes of highest point of Mount Wokomunga massif. 5°5'8"N, 59°50'32"W . elev. 1525 m. Tepui scrub forest on sandstone and peat, with Schefflera , Clusia and Guadua . 5 July 2003, H.D. Clarke 10550 , with R. Williams , C. Perry , J. Kelly , D. Gittens , S. Stern ; Guiana. Mt. Wokomung, Little Ayanganna, upper slopes of highest point of Mount Wokomung massif. 5°5'8"N, 59°50'32"W . elev. 1525 m. Tepui scrub forest on sandstone and peat, with Schefflera , Clusia and Guadua , elev. 1525 m. 5 July 2003, H.D. Clarke 10575 , with R. Williams , C. Perry , J. Kelly , D. Gittens , S. Stern. Guiana. Mt. Wokomung, Little Ayanganna, upper slopes of highest point of Mount Wokomung massif. 5°4'53.1"N, 59°50'26.1"W . elev. 1525 m. Tepui bog on sandstone and peat, with Brocchinia , Bonnetia and Rapatea , elev. 1660 m. 6 July 2003, H.D. Clarke 10576 , with R. Williams , C. Perry , J. Kelly , D. Gittens , S. Stern. Mt. Wokomung, area above third of four escarpments, 1 km NE of Mt. Wokomung, 5°4'30"N, 59°51'15"W . elev. 1490 m. dense forest on laterite, with Clusia , Euterpe and Licania , elev. 1490 m. 8 July 2003, H.D. Clarke 10802 , with R. Williams , C. Perry , J. Kelly , D. Gittens , S. Stern. The material shows the additional feature of the species, the multistratose leaf margin with teeth on the margin and upper and lower surfaces. The species is otherwise reported from Suriname, Ecuador, and supposedly described from Jamaica (Maracaibo, Venezuela?)( Allen 2013 ; Steere 1948 ). 10.3897/phytokeys.154.39105.figure2 A773665D-5A78-5BAA-B8C7-A58E5EA80620 Figure 2. Brachymenium speciosum (Hook. & Wils.) Steere. A Part of leafy stem showing distorted leaves B tip of leaf showing thickened margin with teeth on margin and upper and lower surfaces, some of these appearing as double teeth C capsule showing rostrate operculum D peristome teeth mounted in Hoyer's solution, showing elongate exostome teeth erect on one half and reflexed on other half, the latter showing endostome lacking cilia and segments. The photographs were taken using a Leica DM4B Compound microscope, 5× objective. https://binary.pensoft.net/fig/438303 Supplementary Material XML Treatment for Brachymenium ayangannensis XML Treatment for Brachymenium perspinidens XML Treatment for Brachymenium speciosum

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8858844/

Pyroptosis: A Developing Foreland of Ovarian Cancer Treatment

Ovarian cancer (OVCA) has the second highest mortality among all gynecological cancers worldwide due to its complexity and difficulty in early-stage diagnosis and a lack of targeted therapy. Modern strategies of OVCA treatment involve debulking surgery combined with chemotherapy. Nonetheless, the current treatment is far from satisfactory sometimes and therefore the demand for novel therapeutic measures needs to be settled. Pyroptosis is a notable form of programmed cell death characterized by influx of sodium with water, swelling of cells, and finally osmotic lysis, which is distinctive from numerous classes of programmed cell death. So far, four major pathways underlying mechanisms of pyroptosis have been identified and pyroptosis is indicated to be connected with a variety of disorders including cancerous diseases. Interestingly enough, pyroptosis plays an important role in ovarian cancer with regard to long non-coding RNAs and several regulatory molecules, as is shown by previously published reports. In this review, we summarized major pathways of pyroptosis and the current research foundations of pyroptosis and ovarian cancer, anticipating enriching the thoughts for the treatment of ovarian cancer. What is more, some problems yet unsolved in this field were also raised to hopefully propose several potential threads of OVCA treatment and research directions in future. Introduction Among all gynecological cancers, ovarian cancer (OVCA) does not represent the largest portion of new cases, but it is the cancer type with the second highest mortality worldwide ( 1 , 2 ). Although the incidence has almost been stable for several years, OVCA is still estimated as the fifth cancer death reason for American women in 2021 due to its complexity and difficulty in early-stage diagnosis and a lack of targeted therapy ( 3 ). Moreover, the ovarian cancer patients usually show no evident symptoms at the early stage. Even in advanced OVCA patients, some certain symptoms including back pain, fatigue, abdominal pain, bloating, constipation, and urinary symptoms cannot guarantee an accurate diagnosis, nor can the exploratory laparotomy ( 4 , 5 ). Based on histopathological characteristics, ovarian cancers can be divided into three main types including epithelial, germ cell, and sex-cord-stromal types ( 6 , 7 ). Surgery is undoubtedly the foundation of treating ovarian cancer. However, it is far from satisfactory and the traditional treatment of advanced ovarian cancer has become the combination of surgery and chemotherapy ( 7 – 9 ). Accordingly, many novel drugs selectively acting on specific targets such as prexasertib specifically inhibiting cell cycle checkpoint kinase (Chk) 1/2 have been developed for certain classifications of OVCA ( 10 ). Nevertheless, prexasertib acting as a Chk 1/2 inhibitor is now under investigation for the treatment of high-grade serous OVCA, whereas its promising efficacy has been preliminarily evidenced only in phase 1 studies on account of its moderate hematological toxicity ( 11 ). Therefore, larger confirmatory studies are required to evaluate these new drugs and innovative methods of treating other types of OVCA are needed as well. Programmed cell death (PCD) is an essential biological process in all multicellular organisms, underlying many physiological progressions involving growth and development, anti-infection, and survival in extreme condition ( 12 , 13 ), etc. Moreover, diseases comprising neoplasm, autoimmune diseases, infection, etc., could emerge when PCD is interrupted. Several famous forms of PCD have been well acknowledged so far, encompassing apoptosis, autophagy, necroptosis, ferroptosis, and pyroptosis ( 14 ). Apoptosis is characterized by cytoplasmic shrinkage, nuclear condensation, and the maintenance of completeness of membranes and organelles. Many molecules are involved in apoptosis, and the key initiators are caspase-2, -8, -9, and -10 while the main executioners are caspase-3, -6, and -7 ( 13 , 15 , 16 ). Autophagy is distinguished by the formation of autophagosomes, with the indispensable autophagy-related proteins. Moreover, caspase-2, -3, -6 and -8 are found to work as regulators ( 16 – 18 ). Necroptosis, a programmed cell death similar to necrosis, is realized by the activation of receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase domain-like pseudokinase (MLKL) pathway and the downregulation of caspase-8 simultaneously ( 14 ). As another newfound PCD, the physiological roles of ferroptosis remain intangible but it shows great potential in tumors. Therefore, it is a promising area of cancer treatment ( 18 , 19 ). More recently, pyroptosis, an inflammatory PCD, is made up of two Greek roots "pyro" and 'ptosis', which is presumed to happen in response to infection and is reported to be triggered by inflammasomes customarily. After the discovery of pyroptosis in the field of infection, the scope of research was gradually extended and pyroptosis has been revealed to be of vital importance in many other diseases, including metabolic diseases ( 20 ), cardiovascular diseases ( 21 ), neurological diseases ( 22 ). As inflammation is evidently one of the hallmarks of cancers ( 23 ), a strong association might exist between pyroptosis and malignant diseases. Importantly, in recent years, some chemotherapeutic agents have been found to stimulate the formation of inflammasomes, hinting that there may be a correlation between cancer treatment and pyroptosis ( 24 , 25 ). Generally speaking, with activation of caspase-1, -4 (in human), -5 (in human), and -11 (in mice) and cleavage of gasdermins (GSDMs), plasma membrane pores subsequently form as a result of N-termini of GSDMs and cause membrane perforation, cell swelling, plasma membrane lysis, chromatin fragmentation, and release of intracellular proinflammatory contents, which distinguishes pyroptosis from apoptosis biochemically and morphologically ( 14 , 17 , 26 , 27 ). Moreover, great strides have been made in detecting the underlying mechanisms of pyroptosis, broadening our understanding of cancers and providing new threads of cancer management. Hereof, in this review, we mainly summarized some cardinal mechanisms of pyroptosis and discussed the relationship between pyroptosis and ovarian cancer with an emphasis on the current study foundations, hopefully to provide some potential perspectives in OVCA treatment. Main Mechanisms of Pyroptosis: Setting the Cells on Fire The Gasdermin Family The gasdermin family is a cluster of proteins encoded by GSDM family genes, including GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and PJVK. All the members share a similar structure containing a C-terminal repressor domain (RD) and an N-terminal pore-forming domain (PFD). Besides, there exists a linker region in all GSDMs except for PJVK. Significantly, the N-terminus and C-terminus are highly conserved in the GSDM family, while the linker regions are diverse ( 28 ), resulting in cleavage by different caspases or granzymes. Once the cleavage occurs, RD and PFD fall apart, and hence PFD could come into play. Then the PFD binds to membrane phospholipids and generates pores ( 29 ). The GSDM family possesses extensive functions and is widely expressed in human, although regrettably, a lot of detailed mechanisms are still unknown. Moreover, pyroptosis, as yet, is proved to be associated with GSDMB, GSDMD, and GSDME ( 30 ). GSDMA, related to mitochondrial homeostasis ( 31 ) and an increased apoptosis-inducing activity in human mucus-secreting pit cells, is found to be inhibited in gastric cancers ( 32 ). The biological functions of GSDMC and PJVK remain unknown, but it is reported that the expression level of GSDMC is positively correlated with the metastatic ability of melanoma cells ( 33 ), indicating the possible relationship between GSDMC and tumorigenesis. The Canonical Pathway As pyroptosis was first coined in 2001, it is mostly concerned with inflammation ( 34 ) and largely depends on the assembly of a crucial component, the inflammasome complex, which is composed of pattern-recognition receptors (PRRs), procaspase-1, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) ( Figure 1 ). The activation of canonical inflammasomes mostly appears in macrophages and dendritic cells ( 35 ). Figure 1 Four prestigious pathways indicated in mechanisms of pyroptosis. Of note is that the canonical pathway is composed of inflammasomes, caspase-1, and GSDMD. Moreover, the inflammasome complex consists of PRRs (NLRP1, NLRP3, NLRC4, and AIM2), procaspase-1, and ASC, with the last one being dispensable in NLRC4 inflammasome. Different PRRs constitute corresponding types of inflammasomes and recognize different types of PAMPs or DAMPs. After recognition of PAMPs or DAMPs, the assembled inflammasomes activate caspase-1, thus cleaving GSDMD. The gasdermin pore formed by N-terminus of GSDMD results in pyroptosis characterized by outlet for IL-1β and IL-18, influx of sodium with water, swelling of cells, and finally osmotic lysis. In the non-canonical pathway, LPS derived from gram-negative bacteria could trigger pyroptosis through activating caspase-4, -5, and -11 to cleave GSDMD. Besides, the activated caspase-11 could also inspire the activation of the NLRP3 inflammasome. As for the caspase 3/8-dependent pathway, activated RIPK1 by inhibition of TAK1 helps caspase-8 to cut GSDMD and to mediate pyroptosis while the activated caspase-3 by chemotherapeutic drugs could split GSDME, leading to pyroptosis as well. In the granzyme A/B-dependent pathway, Gzm B released by CAR T cells could induce GSDME-modulated pyroptosis by both direct cleavage of GSDME and indirect cleavage of GSDME via activation of caspase-3, while cytotoxic lymphocyte-released Gzm A cleaves GSDMB to induce pyroptosis. PRRs of canonical inflammasomes often cover NLRP1, NLRP3, and NLRC4, absent in melanoma 2 (AIM2), with these four proteins constituting four corresponding types of inflammasomes. The first three belong to the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, with NLRP possessing a pyrin domain (PYD) and NLRC possessing an N-terminal caspase recruitment domain (CARD) ( 36 ). AIM2 is endowed with a PYD and a DNA-binding HIN-200 domain ( 37 ), and the latter decides the connection between AIM2 and endogenous or pathogen-derived DNA ( 38 ). PYD and CARD of these inflammasome receptors contribute to recognition of certain pathogen-associated molecular patterns (PAMPs) and damaged-associated molecular patterns (DAMPs) ( 36 , 39 ). For example, the NLRP1 inflammasome mediates the recognition of lethal toxin from Bacillus anthracis , muramyl dipeptide, and Salmonella ( 40 – 42 ), whereas the NLRP3 inflammasome recognizes multiple stimuli, including PAMPs such as Sendai virus, influenza, and bacterial pore-forming toxins, as well as DAMPs such as extracellular ATP, hyaluronan, and glucose ( 35 , 43 – 47 ). Additionally, the NLRC4 inflammasome recognizes PAMPs including flagellin and muramyl dipeptide ( 48 , 49 ), while the AIM2 inflammasome only recognizes endogenous or pathogen-derived double-stranded DNA (dsDNA) ( 38 ). PAMPs and DAMPs are activated to recruit inflammasome adaptors ASC after recognition by PRRs. PYD and CARD are contained in ASC as well, similar to that of PRRs and participating in a homotypic interaction. The PYD–PYD interaction helps PRRs to summon ASC, and in the meantime CARD of ASC is indispensable for recruiting procaspase-1 into the inflammasome complex via CARD–CARD interaction ( 50 ). Apart from recruiting procaspase-1, ASC is indispensable in the maturation of IL-1β ( 51 ). Besides, NLRP1B and NLRC4 probably recruit procaspase-1 directly as they have CARD themselves ( 52 ). Moreover, the self-cleavage of procaspase-1 could give rise to caspase-1 activation primarily in macrophages and dendritic cells ( 53 – 55 ) ( Figure 1 ). Caspase-1, also referred to as interleukin-1-beta-converting enzyme, is another pivotal core in this pathway, distinguishing pyroptosis from apoptosis ( 56 ). It was first described as an inflammatory cysteine protease by Thornberry et al. in 1995 ( 57 ). After being recruited to inflammasomes, the concentration of regional caspase-1 monomers increases and consequently the dimerization might be accelerated ( 58 ), since the dimeric form of caspase-1 has protease activity. In caspase-1, there exists a CARD domain linker between the CARD domain and C-terminus, along with an interdomain linker inside the C-terminus which separates it into a larger subunit (p20) and a smaller one (p10) ( 59 ). As these two linkers could be self-cleaved by caspase-1 at diverse sites ( 60 ), the p20 subunit and p10 subunit are separated to reunite the active tetramer which is composed of two p20 subunits and two p10 subunits ( 61 ). Also, following research revealed that active caspase-1 could transform precursors of IL-1β and IL-18 into mature forms ( 62 ), while cleaving GSDMD into two termini as well ( 53 ). Then, the N-terminus of GSDMD, PFD, could generate a gasdermin pore in the plasma membrane when the inhibitory RD is cleaved apart. These pores bring about the outlet for IL-1β and IL-18, the influx of sodium with water, the swelling of cells, and finally the osmotic lysis ( 29 , 63 – 65 ) ( Figure 1 ). Intriguingly, in gastric cancer cells, the expression of GSDMD is downregulated according to a previously published article, which results in abnormal proliferation of cancer cells ( 66 ), indicating that elevating the expression of GSDMD might inhibit the progression of gastric cancer. Non-Canonical Pathway Unlike that of the canonical pathway, the non-canonical pathway requires caspase-4 and -5 in human and ortholog caspase-11 in mice ( 67 , 68 ). In the 1990s, the study by Li found that caspase-1 knockout mice showed high resistance to the injection of lipopolysaccharide (LPS) ( 69 ). Moreover, following articles described possible mechanisms. It was found that caspase-11 is expressed in a great quantity due to the stimulation of LPS ( 70 ). This expression causes the induction of pyroptosis in macrophages, which possibly depends on the ATP-mediated P2X7 signaling pathway according to Yang et al. They observed the instantly fast release of extracellular ATP after transfection of LPS in bone marrow-derived macrophages, mediated by the cleavage of pannexin-1 depending on caspase-11 ( 71 ). ATP finally triggered the activation of P2X7, leading to its opening with ion movement, formation of larger pores on the membrane, and following pyroptosis ( 72 , 73 ). Besides, the stimulation of LPS results in potassium's efflux, in which pannexin-1 is indispensable. Caspase-11 somehow could activate NLRP3 inflammasome mentioned in the canonical pathway, for the efflux of potassium plays a critical part in this procession ( 67 , 71 , 74 ). The direct combination of LPS and orthologs of caspase-11, caspase-4, and caspase-5 could induce the activation of caspases themselves ( 68 , 75 , 76 ). All these activated caspases engender the cleavage of GSDMD resembling that of caspase-1 and ensuing pyroptosis as mentioned above ( 53 , 77 , 78 ) ( Figure 1 ). In a study conducted by Yokoyama et al., it was revealed that secretoglobin 3A2 was capable of inhibiting growth of human non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cells in the mouse metastasis model by means of the caspase-4-mediated non-canonical pyroptosis pathway ( 79 ). According to a study analyzing the caspase-1, -4, and -5 gene mutations in cancers, it is indicated that inhibition of caspase-5 probably contributes to carcinogenesis in microsatellite instability-positive tumor entities ( 80 ). Terlizzi et al. also found that in patients with NSCLC, the circulating level of caspase-4 is raised compared with those without ( 81 ). With further diligent work, their recent study clearly declared that caspase-4 is highly expressed in NSCLC compared to normal lung tissues, while caspase-11 motivates the development of lung cancer in mice. Notably, this high expression of caspase-4 is associated with a poor survival rate in NSCLC patients ( 82 ). Caspase 3/8-Dependent Pathway In 2017, Feng and colleagues firstly demonstrated the novel function of caspase-3 in pyroptosis, breaking the stereotype that pyroptosis could be induced only by inflammatory caspases. In their experiment, chemotherapy drugs could mediate the caspase-3-governed cleavage of GSDME, exposing its gasdermin N-terminal domain and executing pyroptosis as well ( Figure 1 ). Moreover, TNF-induced apoptosis was also found to be switched to pyroptosis by GSDME1 ( 83 ). Their results were later reconfirmed in various sorts of cancers, including gastric cancer ( 84 ), lung cancer ( 85 ), and colon cancer ( 86 ). Besides, in murine macrophages, it was indicated that when the traditional canonical NLRP3-inflammasome pathway is blocked, its activators like ATP could induce pyroptosis through the caspase-3/GSDME pathway, a switch between apoptosis and pyroptosis in cancers ( 87 ), instead of the caspase-1/GSDMD pathway ( 88 ). Briefly, the switch between pyroptosis and apoptosis is primarily determined by the expression level of GSDME, and both the PCD pathways are caspase-dependent. When GSDME is highly expressed, active caspase-3 cleaves it in two termini with the N-terminal domains punching holes on the cell membrane and causing pyroptosis. Conversely, apoptosis will occur if there is a low expression level of GSDME. However, more studies are needed to reconfirm the mechanisms underlying this switch ( 87 ). Only 1 year later in 2018, two back-to-back studies revealed that inhibition of TGF-β-activated kinase-1 (TAK1) by Yersinia YopJ has the ability to provoke pyroptotic cell death in murine macrophages during Yersinia infection ( 89 , 90 ). They uniformly agreed that during the aforementioned process, TAK1 blockade by Yersinia bacteria could lead to activation of RIPK1, together with the subsequent activation of caspase-8, and caspase-8 could chop GSDMD, finally unleashing IL-1β as a result of the pores formed by N-termini of GSDMDs ( 89 , 90 ) ( Figure 1 ). This process was then reassured by Schwarzer et al. in intestinal epithelial cells in a gut inflammation model ( 91 ). Moreover, intriguingly, in two recent works, caspase-8 was regarded as the pivot of the apoptosis–necroptosis–pyroptosis network ( 92 , 93 ), exhibiting its shining role in cell death. Granzyme A/B-Dependent Pathway So far, five subtypes of human granzymes (Gzms) have been described in natural killer cells and cytotoxic T lymphocytes whereas eleven subtypes of murine granzymes are now known to us ( 94 ). Among all, Gzm A and B are of vital importance, which also function in cell death, inflammation, infection, and tumor immunity ( 95 ). Over the years, much attention has been given to Gzm A and B in cell death, where their roles in either caspase-dependent or caspase-independent cell death are well explained. Moreover, perforin, a 67-kDa protein guarding the entrance of granzymes, is widely expressed in immune cells and could induce cell apoptosis in synergy with granzymes ( 96 ). In January of last year, Liu et al. described their conclusion that chimeric antigen receptor (CAR) T cells stimulate caspase-3 to cut GSDME through unleashing granzyme B, the function of which is to cleave and activate caspase-3 in cooperation with perforin, and thus pyroptosis happens in target cells ( 97 ). Shortly afterward, Zhang et al. reported that Gzm B could split GSDME without the existence of caspase-3. In other words, Gzm B could induce GSDME-modulated target tumor cell pyroptosis by both direct cleavage of GSDME and indirect cleavage of GSDME via activation of caspase-3 ( 98 ) ( Figure 1 ). Additionally in the same year, it was demonstrated that other than Gzm B, Gzm A also takes effect as a pyroptosis executioner. In GSDMB-positive cells, natural killer cells and cytotoxic T lymphocytes cause cell death through pyroptosis. What is more, cytotoxic lymphocytes are confirmed to release Gzm A, which then specifically cuts GSDMB through the interdomain with the help of perforin as well, resulting in pyroptosis ( Figure 1 ). Furthermore, this remarkable pathway could successfully promote tumor clearance in mice ( 99 ), providing a new paradigm for pyroptosis and cancer treatment. The Gasdermin Family The gasdermin family is a cluster of proteins encoded by GSDM family genes, including GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and PJVK. All the members share a similar structure containing a C-terminal repressor domain (RD) and an N-terminal pore-forming domain (PFD). Besides, there exists a linker region in all GSDMs except for PJVK. Significantly, the N-terminus and C-terminus are highly conserved in the GSDM family, while the linker regions are diverse ( 28 ), resulting in cleavage by different caspases or granzymes. Once the cleavage occurs, RD and PFD fall apart, and hence PFD could come into play. Then the PFD binds to membrane phospholipids and generates pores ( 29 ). The GSDM family possesses extensive functions and is widely expressed in human, although regrettably, a lot of detailed mechanisms are still unknown. Moreover, pyroptosis, as yet, is proved to be associated with GSDMB, GSDMD, and GSDME ( 30 ). GSDMA, related to mitochondrial homeostasis ( 31 ) and an increased apoptosis-inducing activity in human mucus-secreting pit cells, is found to be inhibited in gastric cancers ( 32 ). The biological functions of GSDMC and PJVK remain unknown, but it is reported that the expression level of GSDMC is positively correlated with the metastatic ability of melanoma cells ( 33 ), indicating the possible relationship between GSDMC and tumorigenesis. The Canonical Pathway As pyroptosis was first coined in 2001, it is mostly concerned with inflammation ( 34 ) and largely depends on the assembly of a crucial component, the inflammasome complex, which is composed of pattern-recognition receptors (PRRs), procaspase-1, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) ( Figure 1 ). The activation of canonical inflammasomes mostly appears in macrophages and dendritic cells ( 35 ). Figure 1 Four prestigious pathways indicated in mechanisms of pyroptosis. Of note is that the canonical pathway is composed of inflammasomes, caspase-1, and GSDMD. Moreover, the inflammasome complex consists of PRRs (NLRP1, NLRP3, NLRC4, and AIM2), procaspase-1, and ASC, with the last one being dispensable in NLRC4 inflammasome. Different PRRs constitute corresponding types of inflammasomes and recognize different types of PAMPs or DAMPs. After recognition of PAMPs or DAMPs, the assembled inflammasomes activate caspase-1, thus cleaving GSDMD. The gasdermin pore formed by N-terminus of GSDMD results in pyroptosis characterized by outlet for IL-1β and IL-18, influx of sodium with water, swelling of cells, and finally osmotic lysis. In the non-canonical pathway, LPS derived from gram-negative bacteria could trigger pyroptosis through activating caspase-4, -5, and -11 to cleave GSDMD. Besides, the activated caspase-11 could also inspire the activation of the NLRP3 inflammasome. As for the caspase 3/8-dependent pathway, activated RIPK1 by inhibition of TAK1 helps caspase-8 to cut GSDMD and to mediate pyroptosis while the activated caspase-3 by chemotherapeutic drugs could split GSDME, leading to pyroptosis as well. In the granzyme A/B-dependent pathway, Gzm B released by CAR T cells could induce GSDME-modulated pyroptosis by both direct cleavage of GSDME and indirect cleavage of GSDME via activation of caspase-3, while cytotoxic lymphocyte-released Gzm A cleaves GSDMB to induce pyroptosis. PRRs of canonical inflammasomes often cover NLRP1, NLRP3, and NLRC4, absent in melanoma 2 (AIM2), with these four proteins constituting four corresponding types of inflammasomes. The first three belong to the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, with NLRP possessing a pyrin domain (PYD) and NLRC possessing an N-terminal caspase recruitment domain (CARD) ( 36 ). AIM2 is endowed with a PYD and a DNA-binding HIN-200 domain ( 37 ), and the latter decides the connection between AIM2 and endogenous or pathogen-derived DNA ( 38 ). PYD and CARD of these inflammasome receptors contribute to recognition of certain pathogen-associated molecular patterns (PAMPs) and damaged-associated molecular patterns (DAMPs) ( 36 , 39 ). For example, the NLRP1 inflammasome mediates the recognition of lethal toxin from Bacillus anthracis , muramyl dipeptide, and Salmonella ( 40 – 42 ), whereas the NLRP3 inflammasome recognizes multiple stimuli, including PAMPs such as Sendai virus, influenza, and bacterial pore-forming toxins, as well as DAMPs such as extracellular ATP, hyaluronan, and glucose ( 35 , 43 – 47 ). Additionally, the NLRC4 inflammasome recognizes PAMPs including flagellin and muramyl dipeptide ( 48 , 49 ), while the AIM2 inflammasome only recognizes endogenous or pathogen-derived double-stranded DNA (dsDNA) ( 38 ). PAMPs and DAMPs are activated to recruit inflammasome adaptors ASC after recognition by PRRs. PYD and CARD are contained in ASC as well, similar to that of PRRs and participating in a homotypic interaction. The PYD–PYD interaction helps PRRs to summon ASC, and in the meantime CARD of ASC is indispensable for recruiting procaspase-1 into the inflammasome complex via CARD–CARD interaction ( 50 ). Apart from recruiting procaspase-1, ASC is indispensable in the maturation of IL-1β ( 51 ). Besides, NLRP1B and NLRC4 probably recruit procaspase-1 directly as they have CARD themselves ( 52 ). Moreover, the self-cleavage of procaspase-1 could give rise to caspase-1 activation primarily in macrophages and dendritic cells ( 53 – 55 ) ( Figure 1 ). Caspase-1, also referred to as interleukin-1-beta-converting enzyme, is another pivotal core in this pathway, distinguishing pyroptosis from apoptosis ( 56 ). It was first described as an inflammatory cysteine protease by Thornberry et al. in 1995 ( 57 ). After being recruited to inflammasomes, the concentration of regional caspase-1 monomers increases and consequently the dimerization might be accelerated ( 58 ), since the dimeric form of caspase-1 has protease activity. In caspase-1, there exists a CARD domain linker between the CARD domain and C-terminus, along with an interdomain linker inside the C-terminus which separates it into a larger subunit (p20) and a smaller one (p10) ( 59 ). As these two linkers could be self-cleaved by caspase-1 at diverse sites ( 60 ), the p20 subunit and p10 subunit are separated to reunite the active tetramer which is composed of two p20 subunits and two p10 subunits ( 61 ). Also, following research revealed that active caspase-1 could transform precursors of IL-1β and IL-18 into mature forms ( 62 ), while cleaving GSDMD into two termini as well ( 53 ). Then, the N-terminus of GSDMD, PFD, could generate a gasdermin pore in the plasma membrane when the inhibitory RD is cleaved apart. These pores bring about the outlet for IL-1β and IL-18, the influx of sodium with water, the swelling of cells, and finally the osmotic lysis ( 29 , 63 – 65 ) ( Figure 1 ). Intriguingly, in gastric cancer cells, the expression of GSDMD is downregulated according to a previously published article, which results in abnormal proliferation of cancer cells ( 66 ), indicating that elevating the expression of GSDMD might inhibit the progression of gastric cancer. Non-Canonical Pathway Unlike that of the canonical pathway, the non-canonical pathway requires caspase-4 and -5 in human and ortholog caspase-11 in mice ( 67 , 68 ). In the 1990s, the study by Li found that caspase-1 knockout mice showed high resistance to the injection of lipopolysaccharide (LPS) ( 69 ). Moreover, following articles described possible mechanisms. It was found that caspase-11 is expressed in a great quantity due to the stimulation of LPS ( 70 ). This expression causes the induction of pyroptosis in macrophages, which possibly depends on the ATP-mediated P2X7 signaling pathway according to Yang et al. They observed the instantly fast release of extracellular ATP after transfection of LPS in bone marrow-derived macrophages, mediated by the cleavage of pannexin-1 depending on caspase-11 ( 71 ). ATP finally triggered the activation of P2X7, leading to its opening with ion movement, formation of larger pores on the membrane, and following pyroptosis ( 72 , 73 ). Besides, the stimulation of LPS results in potassium's efflux, in which pannexin-1 is indispensable. Caspase-11 somehow could activate NLRP3 inflammasome mentioned in the canonical pathway, for the efflux of potassium plays a critical part in this procession ( 67 , 71 , 74 ). The direct combination of LPS and orthologs of caspase-11, caspase-4, and caspase-5 could induce the activation of caspases themselves ( 68 , 75 , 76 ). All these activated caspases engender the cleavage of GSDMD resembling that of caspase-1 and ensuing pyroptosis as mentioned above ( 53 , 77 , 78 ) ( Figure 1 ). In a study conducted by Yokoyama et al., it was revealed that secretoglobin 3A2 was capable of inhibiting growth of human non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cells in the mouse metastasis model by means of the caspase-4-mediated non-canonical pyroptosis pathway ( 79 ). According to a study analyzing the caspase-1, -4, and -5 gene mutations in cancers, it is indicated that inhibition of caspase-5 probably contributes to carcinogenesis in microsatellite instability-positive tumor entities ( 80 ). Terlizzi et al. also found that in patients with NSCLC, the circulating level of caspase-4 is raised compared with those without ( 81 ). With further diligent work, their recent study clearly declared that caspase-4 is highly expressed in NSCLC compared to normal lung tissues, while caspase-11 motivates the development of lung cancer in mice. Notably, this high expression of caspase-4 is associated with a poor survival rate in NSCLC patients ( 82 ). Caspase 3/8-Dependent Pathway In 2017, Feng and colleagues firstly demonstrated the novel function of caspase-3 in pyroptosis, breaking the stereotype that pyroptosis could be induced only by inflammatory caspases. In their experiment, chemotherapy drugs could mediate the caspase-3-governed cleavage of GSDME, exposing its gasdermin N-terminal domain and executing pyroptosis as well ( Figure 1 ). Moreover, TNF-induced apoptosis was also found to be switched to pyroptosis by GSDME1 ( 83 ). Their results were later reconfirmed in various sorts of cancers, including gastric cancer ( 84 ), lung cancer ( 85 ), and colon cancer ( 86 ). Besides, in murine macrophages, it was indicated that when the traditional canonical NLRP3-inflammasome pathway is blocked, its activators like ATP could induce pyroptosis through the caspase-3/GSDME pathway, a switch between apoptosis and pyroptosis in cancers ( 87 ), instead of the caspase-1/GSDMD pathway ( 88 ). Briefly, the switch between pyroptosis and apoptosis is primarily determined by the expression level of GSDME, and both the PCD pathways are caspase-dependent. When GSDME is highly expressed, active caspase-3 cleaves it in two termini with the N-terminal domains punching holes on the cell membrane and causing pyroptosis. Conversely, apoptosis will occur if there is a low expression level of GSDME. However, more studies are needed to reconfirm the mechanisms underlying this switch ( 87 ). Only 1 year later in 2018, two back-to-back studies revealed that inhibition of TGF-β-activated kinase-1 (TAK1) by Yersinia YopJ has the ability to provoke pyroptotic cell death in murine macrophages during Yersinia infection ( 89 , 90 ). They uniformly agreed that during the aforementioned process, TAK1 blockade by Yersinia bacteria could lead to activation of RIPK1, together with the subsequent activation of caspase-8, and caspase-8 could chop GSDMD, finally unleashing IL-1β as a result of the pores formed by N-termini of GSDMDs ( 89 , 90 ) ( Figure 1 ). This process was then reassured by Schwarzer et al. in intestinal epithelial cells in a gut inflammation model ( 91 ). Moreover, intriguingly, in two recent works, caspase-8 was regarded as the pivot of the apoptosis–necroptosis–pyroptosis network ( 92 , 93 ), exhibiting its shining role in cell death. Granzyme A/B-Dependent Pathway So far, five subtypes of human granzymes (Gzms) have been described in natural killer cells and cytotoxic T lymphocytes whereas eleven subtypes of murine granzymes are now known to us ( 94 ). Among all, Gzm A and B are of vital importance, which also function in cell death, inflammation, infection, and tumor immunity ( 95 ). Over the years, much attention has been given to Gzm A and B in cell death, where their roles in either caspase-dependent or caspase-independent cell death are well explained. Moreover, perforin, a 67-kDa protein guarding the entrance of granzymes, is widely expressed in immune cells and could induce cell apoptosis in synergy with granzymes ( 96 ). In January of last year, Liu et al. described their conclusion that chimeric antigen receptor (CAR) T cells stimulate caspase-3 to cut GSDME through unleashing granzyme B, the function of which is to cleave and activate caspase-3 in cooperation with perforin, and thus pyroptosis happens in target cells ( 97 ). Shortly afterward, Zhang et al. reported that Gzm B could split GSDME without the existence of caspase-3. In other words, Gzm B could induce GSDME-modulated target tumor cell pyroptosis by both direct cleavage of GSDME and indirect cleavage of GSDME via activation of caspase-3 ( 98 ) ( Figure 1 ). Additionally in the same year, it was demonstrated that other than Gzm B, Gzm A also takes effect as a pyroptosis executioner. In GSDMB-positive cells, natural killer cells and cytotoxic T lymphocytes cause cell death through pyroptosis. What is more, cytotoxic lymphocytes are confirmed to release Gzm A, which then specifically cuts GSDMB through the interdomain with the help of perforin as well, resulting in pyroptosis ( Figure 1 ). Furthermore, this remarkable pathway could successfully promote tumor clearance in mice ( 99 ), providing a new paradigm for pyroptosis and cancer treatment. Current Research Foundations of Pyroptosis and Ovarian Cancer Genes That Might Regulate Pyroptosis in OVCA With more studies focusing on pyroptosis and ovarian cancer, it was not so long ago that Berkel et al. published their paper comparing differential expression and copy number variations of certain GSDM family members in normal ovarian tissues with those of malignant serous ovarian tissues ( 100 ). They firstly pointed out that the expression of GSDME is downregulated whereas GSDMD and GSDMC are expressed at a high level in serous OVCA, which is associated with a poor prognosis of TP53 -mutated OVCA patients. Likewise, as executioners of GSDMs, the expression of caspase-1, -3, -4, -5, and -8 is decreased at the mRNA level in serous ovarian cancer. Also, the copy number variation events happen more frequently in genes encoding GSDMD and GSDMC, in accordance with their expression. Additionally, various histological subtypes of epithelial ovarian cancer express GSDMB and GSDME differently ( 100 ) ( Table 1 ) . Table 1 Current research foundations of pyroptosis and ovarian cancer. Year Authors Research object Ovarian cancer cell lines Gate molecules Signaling pathways Additional information 2021 Berkel et al. ( 100 ) Differential expression and copy number variations of GSDMs / / / In epithelial ovarian cancer, the expression of GSDMB is increased in mucinous histotype compared to endometrioid and serous histotypes. Also, the expression of GSDMD is elevated in clear cell and serous histotypes compared to endometrioid histotype. 2021 Ye et al. ( 101 ) Pyroptosis-related genes / / / The 13 downregulated genes include PRKACA, GSDMB, SCAF11, PJVK, CASP9, NOD1, PLCG1, NLRP1, GSDME, ELANE, TIRAP, CASP4, and GSDMD while the 18 upregulated genes are GPX4, NLRP7, NLRP2, CASP3, CASP6, TNF, IL1B, IL18, CASP8, NLRP6, GSDMA, GSDMC, PYCARD, CASP5, AIM2, NOD2, NLRC4, and NLRP3. 2018 Li et al. ( 102 ) LncRNA GAS5 SKOV3, OVCAR-3, A2780, and 3AO GSDMD The canonical pathway Depletion of lncRNA GAS5 promotes viability of OVCA cells, while the overexpression of lncRNA GAS5 inhibits proliferation and colony formation in OVCA cells. 2021 Tan et al. ( 103 ) LncRNA HOTTIP CAOV-3, A2780, SKOV3, and OVCAR3 GSDMD ASK1/JNK signaling pathway LncRNA HOTTIP is upregulated in ovarian cancer tissues, and microRNA-148a-3p was a downstream target gene of HOTTIP, exerting negative effects on the regulatory functions of HOTTIP. 2020 Liang et al. ( 104 ) Osthole A2780 and OVCAR3 GSDME / Osthole could mediate GSDME-dependent pyroptosis while suppressing cell death by mitochondria-mediated apoptosis and causing cell autophagy in OVCA. 2020 Zhang et al. ( 105 ) Nobiletin A2780 and OVCAR3 GSDMD, GSDME / Nobiletin could inhibit cell proliferation, induce apoptosis via DNA damage in a dose-dependent way, and mediate pyroptosis through induction of autophagy in OVCA cells. 2019 Qiao et al. ( 106 ) α-NETA Ho8910, Ho8910PM, Hey, SKOV3, and A2780 GSDMD GSDMD/caspase-4 pathway α-NETA treatment causes epithelial ovarian cancer cell membrane blistering and cytoplasm leakage, typical manifestations of cells undergoing pyroptosis, which could be arrested by β-arrestin-2. Secondly yet importantly, not long ago Qi and colleagues identified 31 differentially expressed genes (DEGs) that might regulate pyroptosis between OVCA and normal ovarian tissues, based on which the OVCA cases were classified. Among the 31 DEGs, 13 genes were downregulated while the remaining 18 genes were enriched in the tumor tissues. Moreover, a total of 7 DEGs including 3 downregulated (PLCG1, ELANE, and PJVK) and 4 upregulated (AIM2, CASP3, CASP6, and GSDMA) genes were retained for generating a prognostic model and a risk model because of their significant p -values, where 3 genes (PLCG1, ELANE, and GSDMA) were shown to be risk factors, while the other 4 genes (AIM2, PJVK, CASP3, and CASP6) were protective in the TCGA cohort. Thereafter, prognostic value was evaluated and pyroptosis-related genes were ascertained to play a key role in tumor immunity and predicting the prognosis of OVCA ( 101 ) ( Table 1 ) . LncRNAs and Pyroptosis in OVCA Alternatively, two studies revealed that two long non-coding RNAs (lncRNAs), lncRNA growth arrest-specific transcript 5 (GAS5) and lncRNA HOXA transcript at the distal tip (HOTTIP), could regulate the pyroptosis process in OVCA, serving as a good cop and a bad cop, respectively ( 102 , 103 ). Li et al. determined the positive effect of lncRNA GAS5 on pyroptosis in OVCA. Not only did they determine the repressed expression of lncRNA GAS5 in ovarian cancer tissues, but also they used lncRNA GAS5 overexpression and depletion models to identify that lncRNA GAS5 triggers the formation of inflammasome, thus leading to pyroptosis both in vivo and in vitro ( 102 ). The work done by Tan et al. was more complicated, with several downstream effectors discovered. In ovarian cancer tissues and cell lines, lncRNA HOTTIP is upregulated, the knockdown of which could lead to pyroptosis, hampering the progression of OVCA. Mechanistically, silencing lncRNA HOTTIP brings about upregulation of its downstream target gene microRNA (miRNA)-148a-3p, low AKT2 expression, positive modulation of the ASK1/JNK signaling pathway, and elevated formation of NLRP1-inflammasome ( 103 ) ( Figure 2 , Table 1 ) . In view of the broad research prospects of pyroptosis in OVCA, more potential lncRNAs that could modulate pyroptosis are yet to be unearthed. Figure 2 Potential mechanisms underlying pyroptosis in ovarian cancer cells and current study foundations. Notably, two lncRNAs, GAS5 and HOTTIP, play an important role in the regulation of inflammasomes. The inhibited expression of lncRNA GAS5 in ovarian cancer could trigger the formation of inflammasome while lncRNA HOTTIP is highly expressed in ovarian cancer, the knockdown of which leads to upregulation of ASK1/JNK signaling, elevated formation of NLRP1-inflammasome, and pyroptosis. Moreover, three novel small molecules including osthole, nobiletin, and α-NETA are reported to regulate the pyroptosis process in ovarian cancer cells. Osthole and nobiletin are of high similarity since they both have an effect on ROS production, MMP, and LC3-related autophagy. However, osthole could mediate GSDME-dependent pyroptosis while nobiletin could mediate pyroptosis through GSDMD- and GSDME-dependent ways. Moreover, α-NETA treatment causes epithelial ovarian cancer cell death through pyroptosis, with dramatically augmented level of GSDMD and caspase-4. Several Regulatory Molecules of Pyroptosis in OVCA Meanwhile, some reports showed that apart from lncRNAs, pyroptosis in OVCA could also be induced by various molecules comprising osthole, nobiletin, and 2-(alpha-naphthoyl)ethyltrimethylammonium iodide (α-NETA) ( 104 – 106 ). Osthole, a natural compound found in several medicinal plants such as Cnidium monnieri and Angelica pubescens , is reported to show potential anticancer, antioxidant, antimicrobial, and anti-inflammatory activities ( 107 , 108 ). Similarly, nobiletin is another plant-derived natural compound targeting various oncogene and onco-suppressor pathways, thus showing great anticancer activity ( 109 , 110 ). Moreover, α-NETA is a stable, non-competitive, slowly reversible choline acetylcholine transferase inhibitor ( 106 ). Recently, Liang et al. have found that osthole could mediate GSDME-dependent pyroptosis while eliciting reactive oxygen species (ROS) generation, decreasing mitochondrial membrane potential (MMP), and inducing LC3-mediated autophagy. In their study, the level of cleavage of GSDME was raised by osthole, exerting tremendous influence on the occurrence of pyroptosis ( 104 ). Remarkably, and perhaps not coincidentally, Zhang et al. uncovered the new identity of nobiletin as the pyroptosis trigger in OVCA in the same year. Highly similar to osthole, nobiletin could also stimulate ROS production, decrease MMP, and promote the evocation of classical autophagy in connection with LC3. Besides, nobiletin was verified to evoke the pyroptosis process in an autophagy-related, ROS-mediated, GSDMD- and GSDME-dependent way, slightly different from that of osthole ( 105 ). What is more, a later published paper further convinced that α-NETA treatment causes epithelial ovarian cancer cell death through pyroptosis, with a dramatically augmented level of GSDMD, caspase-4, LC3B, and IL-18 secretion ( 106 ) ( Figure 2 , Table 1 ) . Characteristics of Pyroptosis in OVCA and Other Types of Cancer Cells The pyroptosis process happens not only in OVCA cells but also in many other types of tumor cells. For example, in NSCLC patients, GSDMD is highly expressed, the same as that in malignant serous ovarian tissues, and indicates a poor prognosis as well. Moreover, in digestive system carcinomas, caspase-1 is demonstrated to be low-expressed in hepatocellular carcinoma and colorectal cancer ( 111 ). Surprisingly in colorectal cancer, lncRNA RP1-85F18.6 is reported to promote proliferation and invasion as well as suppress pyroptosis ( 112 ) whereas lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) could mediate ionizing radiation-induced pyroptosis relying on upregulation of GSDME expression ( 113 ). Besides, as a platinum antitumor agent, lobaplatin could remarkably elevate the level of ROS in CRC cells and phosphorylate JNK. Then activated JNK could cause mitochondrial damage and release of cytochrome C, promoting caspase-3 and -9 cleavage and GSDME-dependent pyroptosis, which shows a moderate overlap between OVCA and CRC ( 86 ). Genes That Might Regulate Pyroptosis in OVCA With more studies focusing on pyroptosis and ovarian cancer, it was not so long ago that Berkel et al. published their paper comparing differential expression and copy number variations of certain GSDM family members in normal ovarian tissues with those of malignant serous ovarian tissues ( 100 ). They firstly pointed out that the expression of GSDME is downregulated whereas GSDMD and GSDMC are expressed at a high level in serous OVCA, which is associated with a poor prognosis of TP53 -mutated OVCA patients. Likewise, as executioners of GSDMs, the expression of caspase-1, -3, -4, -5, and -8 is decreased at the mRNA level in serous ovarian cancer. Also, the copy number variation events happen more frequently in genes encoding GSDMD and GSDMC, in accordance with their expression. Additionally, various histological subtypes of epithelial ovarian cancer express GSDMB and GSDME differently ( 100 ) ( Table 1 ) . Table 1 Current research foundations of pyroptosis and ovarian cancer. Year Authors Research object Ovarian cancer cell lines Gate molecules Signaling pathways Additional information 2021 Berkel et al. ( 100 ) Differential expression and copy number variations of GSDMs / / / In epithelial ovarian cancer, the expression of GSDMB is increased in mucinous histotype compared to endometrioid and serous histotypes. Also, the expression of GSDMD is elevated in clear cell and serous histotypes compared to endometrioid histotype. 2021 Ye et al. ( 101 ) Pyroptosis-related genes / / / The 13 downregulated genes include PRKACA, GSDMB, SCAF11, PJVK, CASP9, NOD1, PLCG1, NLRP1, GSDME, ELANE, TIRAP, CASP4, and GSDMD while the 18 upregulated genes are GPX4, NLRP7, NLRP2, CASP3, CASP6, TNF, IL1B, IL18, CASP8, NLRP6, GSDMA, GSDMC, PYCARD, CASP5, AIM2, NOD2, NLRC4, and NLRP3. 2018 Li et al. ( 102 ) LncRNA GAS5 SKOV3, OVCAR-3, A2780, and 3AO GSDMD The canonical pathway Depletion of lncRNA GAS5 promotes viability of OVCA cells, while the overexpression of lncRNA GAS5 inhibits proliferation and colony formation in OVCA cells. 2021 Tan et al. ( 103 ) LncRNA HOTTIP CAOV-3, A2780, SKOV3, and OVCAR3 GSDMD ASK1/JNK signaling pathway LncRNA HOTTIP is upregulated in ovarian cancer tissues, and microRNA-148a-3p was a downstream target gene of HOTTIP, exerting negative effects on the regulatory functions of HOTTIP. 2020 Liang et al. ( 104 ) Osthole A2780 and OVCAR3 GSDME / Osthole could mediate GSDME-dependent pyroptosis while suppressing cell death by mitochondria-mediated apoptosis and causing cell autophagy in OVCA. 2020 Zhang et al. ( 105 ) Nobiletin A2780 and OVCAR3 GSDMD, GSDME / Nobiletin could inhibit cell proliferation, induce apoptosis via DNA damage in a dose-dependent way, and mediate pyroptosis through induction of autophagy in OVCA cells. 2019 Qiao et al. ( 106 ) α-NETA Ho8910, Ho8910PM, Hey, SKOV3, and A2780 GSDMD GSDMD/caspase-4 pathway α-NETA treatment causes epithelial ovarian cancer cell membrane blistering and cytoplasm leakage, typical manifestations of cells undergoing pyroptosis, which could be arrested by β-arrestin-2. Secondly yet importantly, not long ago Qi and colleagues identified 31 differentially expressed genes (DEGs) that might regulate pyroptosis between OVCA and normal ovarian tissues, based on which the OVCA cases were classified. Among the 31 DEGs, 13 genes were downregulated while the remaining 18 genes were enriched in the tumor tissues. Moreover, a total of 7 DEGs including 3 downregulated (PLCG1, ELANE, and PJVK) and 4 upregulated (AIM2, CASP3, CASP6, and GSDMA) genes were retained for generating a prognostic model and a risk model because of their significant p -values, where 3 genes (PLCG1, ELANE, and GSDMA) were shown to be risk factors, while the other 4 genes (AIM2, PJVK, CASP3, and CASP6) were protective in the TCGA cohort. Thereafter, prognostic value was evaluated and pyroptosis-related genes were ascertained to play a key role in tumor immunity and predicting the prognosis of OVCA ( 101 ) ( Table 1 ) . LncRNAs and Pyroptosis in OVCA Alternatively, two studies revealed that two long non-coding RNAs (lncRNAs), lncRNA growth arrest-specific transcript 5 (GAS5) and lncRNA HOXA transcript at the distal tip (HOTTIP), could regulate the pyroptosis process in OVCA, serving as a good cop and a bad cop, respectively ( 102 , 103 ). Li et al. determined the positive effect of lncRNA GAS5 on pyroptosis in OVCA. Not only did they determine the repressed expression of lncRNA GAS5 in ovarian cancer tissues, but also they used lncRNA GAS5 overexpression and depletion models to identify that lncRNA GAS5 triggers the formation of inflammasome, thus leading to pyroptosis both in vivo and in vitro ( 102 ). The work done by Tan et al. was more complicated, with several downstream effectors discovered. In ovarian cancer tissues and cell lines, lncRNA HOTTIP is upregulated, the knockdown of which could lead to pyroptosis, hampering the progression of OVCA. Mechanistically, silencing lncRNA HOTTIP brings about upregulation of its downstream target gene microRNA (miRNA)-148a-3p, low AKT2 expression, positive modulation of the ASK1/JNK signaling pathway, and elevated formation of NLRP1-inflammasome ( 103 ) ( Figure 2 , Table 1 ) . In view of the broad research prospects of pyroptosis in OVCA, more potential lncRNAs that could modulate pyroptosis are yet to be unearthed. Figure 2 Potential mechanisms underlying pyroptosis in ovarian cancer cells and current study foundations. Notably, two lncRNAs, GAS5 and HOTTIP, play an important role in the regulation of inflammasomes. The inhibited expression of lncRNA GAS5 in ovarian cancer could trigger the formation of inflammasome while lncRNA HOTTIP is highly expressed in ovarian cancer, the knockdown of which leads to upregulation of ASK1/JNK signaling, elevated formation of NLRP1-inflammasome, and pyroptosis. Moreover, three novel small molecules including osthole, nobiletin, and α-NETA are reported to regulate the pyroptosis process in ovarian cancer cells. Osthole and nobiletin are of high similarity since they both have an effect on ROS production, MMP, and LC3-related autophagy. However, osthole could mediate GSDME-dependent pyroptosis while nobiletin could mediate pyroptosis through GSDMD- and GSDME-dependent ways. Moreover, α-NETA treatment causes epithelial ovarian cancer cell death through pyroptosis, with dramatically augmented level of GSDMD and caspase-4. Several Regulatory Molecules of Pyroptosis in OVCA Meanwhile, some reports showed that apart from lncRNAs, pyroptosis in OVCA could also be induced by various molecules comprising osthole, nobiletin, and 2-(alpha-naphthoyl)ethyltrimethylammonium iodide (α-NETA) ( 104 – 106 ). Osthole, a natural compound found in several medicinal plants such as Cnidium monnieri and Angelica pubescens , is reported to show potential anticancer, antioxidant, antimicrobial, and anti-inflammatory activities ( 107 , 108 ). Similarly, nobiletin is another plant-derived natural compound targeting various oncogene and onco-suppressor pathways, thus showing great anticancer activity ( 109 , 110 ). Moreover, α-NETA is a stable, non-competitive, slowly reversible choline acetylcholine transferase inhibitor ( 106 ). Recently, Liang et al. have found that osthole could mediate GSDME-dependent pyroptosis while eliciting reactive oxygen species (ROS) generation, decreasing mitochondrial membrane potential (MMP), and inducing LC3-mediated autophagy. In their study, the level of cleavage of GSDME was raised by osthole, exerting tremendous influence on the occurrence of pyroptosis ( 104 ). Remarkably, and perhaps not coincidentally, Zhang et al. uncovered the new identity of nobiletin as the pyroptosis trigger in OVCA in the same year. Highly similar to osthole, nobiletin could also stimulate ROS production, decrease MMP, and promote the evocation of classical autophagy in connection with LC3. Besides, nobiletin was verified to evoke the pyroptosis process in an autophagy-related, ROS-mediated, GSDMD- and GSDME-dependent way, slightly different from that of osthole ( 105 ). What is more, a later published paper further convinced that α-NETA treatment causes epithelial ovarian cancer cell death through pyroptosis, with a dramatically augmented level of GSDMD, caspase-4, LC3B, and IL-18 secretion ( 106 ) ( Figure 2 , Table 1 ) . Characteristics of Pyroptosis in OVCA and Other Types of Cancer Cells The pyroptosis process happens not only in OVCA cells but also in many other types of tumor cells. For example, in NSCLC patients, GSDMD is highly expressed, the same as that in malignant serous ovarian tissues, and indicates a poor prognosis as well. Moreover, in digestive system carcinomas, caspase-1 is demonstrated to be low-expressed in hepatocellular carcinoma and colorectal cancer ( 111 ). Surprisingly in colorectal cancer, lncRNA RP1-85F18.6 is reported to promote proliferation and invasion as well as suppress pyroptosis ( 112 ) whereas lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) could mediate ionizing radiation-induced pyroptosis relying on upregulation of GSDME expression ( 113 ). Besides, as a platinum antitumor agent, lobaplatin could remarkably elevate the level of ROS in CRC cells and phosphorylate JNK. Then activated JNK could cause mitochondrial damage and release of cytochrome C, promoting caspase-3 and -9 cleavage and GSDME-dependent pyroptosis, which shows a moderate overlap between OVCA and CRC ( 86 ). Discussion Taken together, as a notable style of lately identified programmed cell death, pyroptosis displays a significant role in multitudinous diseases embodying cancerous ailments ( 84 , 86 , 104 ), infectious diseases ( 90 , 114 ), neurological diseases ( 115 , 116 ) and cardiovascular events ( 117 , 118 ). Among them, nevertheless, carcinomas are emerging as one of the auspicious prospects. Moreover, as is conspicuously stated above, compelling evidence denotes a close relation between pyroptosis and ovarian cancer. With four major pathways of pyroptosis being discovered one after another, the gasdermin family becomes the kernel of pyroptosis induction, and caspases that have the capacity to mediate pyroptosis are no longer confined to inflammatory ones. Therefore, questions are gradually starting to surface. Are the existing pathways complete mechanisms of pyroptosis? We now know that caspases triggering pyroptosis, for example caspase-3 and -8, could also participate in apoptosis. Particularly, caspase-8 serves as hub of the apoptosis–necroptosis–pyroptosis network, whose bigger potential needs to be tapped. So is there a chance that other apoptosis-related caspases, such as caspase-2, -6, -7, -9, and -10, could also function in pyroptosis? For this reason, a grand network involving apoptosis-related caspases and yet undetected further GSDMs is worth looking forward to. What is more, since mounting studies demonstrated an association between pyroptosis and tumor immunotherapy, it might be possible to treat cancer patients with immunotherapy assisted by pyroptosis-inducing nanoparticles ( 119 – 121 ) in the future. It was reported that one of those nanoparticles could mediate tumor cell pyroptosis in a mouse colon carcinoma model, and the pyroptotic tumor cells could release DAMPs, thus initiating adaptive immunity and boosting the efficacy of immune checkpoint inhibitors (ICIs) ( 120 ). However, the safety of those nanoparticles should be taken into consideration when applied. Additionally, it was also reported that ICIs could kill resistant tumors only in the context of the concomitant induction of pyroptosis ( 122 ), highlighting the importance of the combination of pyroptosis inducers and ICIs in treating ICI-resistant tumors. Nevertheless, since the occurrence of pyroptosis brings about the release of inflammatory components, which might promote the development of tumors ( 123 , 124 ), pyroptosis, as a double-edged sword, should be carefully harnessed, either shutting a door or opening a window for a great deal of cancer patients. Aside from the aforementioned issues and back to OVCA, pyroptosis in cancer treatment and cancer patients is another thing to be addressed. Since distinct chemotherapy drugs are of benefit with respect to ovarian cancer via stimulation of pyroptosis, along with generation of ROS and decrease of mitochondrial membrane potential, many other precisely targeting pyroptosis medications intended for diverse specific subtypes of ovarian cancer are urgently needed to be developed, as well as more in vivo experiments. Besides, the possibility of treating OVCA patients with immunotherapy in conjunction with pyroptosis is worth exploring. Moreover, as mechanisms of pyroptosis in OVCA are still poorly studied, whether unsuspected mechanisms could solve problems related to drug resistance, progression, or recurrence in OVCA patients is yet unknown. Moreover, there might be a subtle correlation of pyroptosis with ferroptosis and mitochondrial autophagy, which awaits further elucidation. So is it possible to treat OVCA patients with medications that could mediate ovarian cancer cell death through induction of pyroptosis, ferroptosis, necroptosis, and autophagy so as to kill target cells to the greatest degree? Now that a few lncRNAs are reported to regulate pyroptosis in OVCA, chances are that ovarian cancer could be treated at a genetic level. Back to patients themselves, when the pyroptosis progress occurs, a variety of immune components partake including cytotoxic lymphocytes, CAR T cells, IL-1β, and IL-18. Cytotoxic lymphocytes could kill tumor cells by transferring granzymes into target cells. During this process, GSDMB activated by Gzm A or GSDME activated by Gzm B and caspase-3 induces pyroptosis, which probably reinforces the cytotoxicity ( 125 ). CAR T cells are supposed to experience a similar course to launch attack, and Gzm B plays a significant role in activating GSDME and caspase-3, as well as inducing pyroptosis. Besides, due to a high affinity between CAR T cells and their ligands, it is more efficient for those cells to induce pyroptosis ( 126 ). Moreover, although the cytokines could be properly utilized to assist in fighting against malignancies, for cancer patients, the possibly forthcoming inflammatory cytokine storm under infectious conditions might make things worse. Besides, the newly discovered pyroptosis-related DEGs between OVCA and normal tissues, along with the prognostic and risk models derived from DEGs, might play a critical role in predicting the prognosis of OVCA patients in the future. Last but not least, there are many FDA-approved drugs in clinical practice that could induce pyroptosis ( 122 ). These drugs involve antidiabetes drug metformin, anticancer drugs paclitaxel and doxorubicin, and nutrients anthocyanin and DHA, which show great antitumor activity. In particular, paclitaxel and doxorubicin exhibit enormous potential owing to their dual effects including treating cancers and inducing pyroptosis, but cancer cells could still quickly develop resistance against them, which remains an unsolved but interesting problem. In a study focusing on nasopharyngeal carcinoma (NPC), it was discovered that caspase-1 inhibition and GSDMD knockout could induce a Taxol-resistant phenotype in vitro and in vivo and that autophagy could negatively regulate the canonical pathway of pyroptosis in NPC cells ( 127 ). Additionally, it was also found that the knockdown of USP47, a cysteine protease, could increase doxorubicin-induced pyroptosis in CRC while the ectopic expression of USP47 leads to doxorubicin resistance in CRC cells ( 128 ). Thus, we speculate the fact that patients taking particular drugs with dual effects experience drug resistance or tumor relapse might possibly result from the fine regulation of the intricate PCD pathway network. Moreover, although the induction of pyroptosis by these drugs might not directly follow the aforementioned four pathways, the preclinical studies did bring hope to us. Consequently, developing drugs targeting pyroptosis in tumor cells is a promising area. Furthermore, clinical trials regarding pyroptosis do exist, with one focusing on diabetes ( 129 ) and the other on leukemia ( 130 ). In B cell acute lymphoblastic leukemia patients, B leukemic cell pyroptosis was stimulated through the Gzm B pathway triggered by CAR T cells. However, target cell pyroptosis stimulates macrophages to cause cytokine release syndrome ( 130 ), which might be detrimental to patients and become a flaw in pyroptosis, limiting its development. Similarly in patients with type 2 diabetes, alleviation of diabetes via inhibiting pyroptosis was observed ( 129 ), further confirming the negative inflammatory process during pyroptosis. Therefore, it is inevitable to take the concomitant inflammatory process during pyroptosis into account. By and large, the continuous exploration centering upon pyroptosis and ovarian cancer provides clinicians with more choices from a genetic level to a chemotherapeutic or an immunotherapeutic level, enriching the thoughts for the treatment of ovarian cancer. Despite some problems to be settled, the significant and promising prospect of pyroptosis is worthy of the wait. Author Contributions TL, MH, and LL had the idea for the article. TL, MH, ML, and LL were the major contributors in the drafting of the work. CQ, LC, TZ, and JQ critically revised the work. All authors contributed to the article and approved the submitted version. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's Note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Abbreviations OVCA, ovarian cancer; Chk, cell cycle checkpoint kinase; PCD, programmed cell death; RIPK3, receptor-interacting protein kinase 3; MLKL, mixed lineage kinase domain-like pseudokinase; GSDM, gasdermin; RD, repressor domain; PFD, pore-forming domain; PRR, pattern-recognition receptor; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; AIM2, absent in melanoma 2; NOD, nucleotide-binding oligomerization domain; NLR, NOD-like receptor; PYD, pyrin domain; CARD, caspase recruitment domain; PAMP, pathogen-associated molecular pattern; DAMP, damaged-associated molecular pattern; dsDNA, double-stranded DNA; IL-1β, interleukin-1β; IL-18, interleukin-18; LPS, lipopolysaccharide; P2X7, purinergic receptor P2X, ligand-gated ion channel, 7; NSCLC, non-small cell lung cancer; CRC, colorectal cancer; TNF, tumor necrosis factor; TAK1, TGF-β activated kinase-1; Gzm, granzyme; CAR, chimeric antigen receptor; DEG, differentially expressed gene; lncRNA, long non-coding RNA; GAS5, growth arrest-specific transcript 5; HOTTIP, HOXA transcript at the distal tip; miRNA, microRNA; ASK1, apoptosis signal-regulating kinase1; JNK, c-Jun N-terminal kinase; α-NETA, 2-(alpha-naphthoyl)ethyltrimethylammonium iodide; ROS, reactive oxygen species; MMP, mitochondrial membrane potential; ICI, immune checkpoint inhibitor; NPC, nasopharyngeal carcinoma.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3509669/

Advances and Future Challenges in Recombinant Adenoviral Vectored H5N1 Influenza Vaccines

The emergence of a highly pathogenic avian influenza virus H5N1 has increased the potential for a new pandemic to occur. This event highlights the necessity for developing a new generation of influenza vaccines to counteract influenza disease. These vaccines must be manufactured for mass immunization of humans in a timely manner. Poultry should be included in this policy, since persistent infected flocks are the major source of avian influenza for human infections. Recombinant adenoviral vectored H5N1 vaccines are an attractive alternative to the currently licensed influenza vaccines. This class of vaccines induces a broadly protective immunity against antigenically distinct H5N1, can be manufactured rapidly, and may allow mass immunization of human and poultry. Recombinant adenoviral vectors derived from both human and non-human adenoviruses are currently being investigated and appear promising both in nonclinical and clinical studies. This review will highlight the current status of various adenoviral vectored H5N1 vaccines and will outline novel approaches for the future. 1. Introduction Influenza is a contagious acute respiratory disease that remains a serious public-health problem today [ 1 , 2 ] and results in substantial economic burden every year [ 3 ], even though most influenza virus infections are self-limited. There are three type influenza viruses (A, B, and C). All of them can infect humans, but influenza A viruses are the most virulent types of responsible for pandemics [ 4 ]. Influenza A viruses can be divided into subtypes based on antigenic differences in their surface glycoprotein hemagglutinin (HA) and neuraminidase (NA). Currently, 17 HA and 9 NA subtypes of influenza A viruses have been described [ 5 , 6 ]. The genome of influenza A viruses comprise eight single–stranded, negative-sense RNA segments that encode eleven proteins. These viruses are continuously evolving through accumulated mutations (antigenic drift) and genetic reassortment (antigenic shift) that results in the emergence of new strains. An influenza pandemic may take place when a new strain of influenza A virus carrying novel HA and/or NA genes enters the general population with little or no immunity against it. Compelling evidence suggests that avian influenza viruses (AIV) have contributed with genetic material to pandemic influenza strains that hit the world in 1918 ('Spanish flu'), 1957 ('Asian flu'), 1968 ('Hong Kong flu'), and 2009 ('Mexican flu') [ 7 , 8 , 9 ]. Currently circulating highly pathogenic avian influenza (HPAI) H5N1 virus poses another potential pandemic threat to humans [ 10 ]. This virus emerged in 1996 and the first confirmed direct transmission of AIV to humans without an intermediate host was in 1997 during a poultry outbreak in Hong Kong [ 11 , 12 ]. After re-emergence in 2003, the H5N1 virus strains have spread from Asia to Europe and Africa and has caused severe disease in poultry and wild birds in multiple countries [ 13 ]. These viruses crossed the species barriers infecting mammals, including domestic cats, Owston's civets, leopards, tigers, dogs, stone martens, pigs, plateau pika, and humans [ 13 ]. As of August 2012, there have been a total of 608 confirmed human cases of H5N1 infection, with 359 deaths (59% mortality rate) in 15 countries since 2003, as reported the World Health Organization [ 14 ]. Although up to now H5N1 viruses have not yet shown efficient transmissibility among humans, the transmissions of H5N1 viruses from human to human have been documented in several countries [ 15 ]. Two recent laboratory-engineered mammalian-transmissible H5N1 virus strains [ 16 , 17 ] and additional surveillance data [ 18 ] clearly indicate that HPAI H5N1 virus can evolve into a strain capable of human-to-human transmission, which may induce a global disaster because the human population, overall, has no immunity against this virus. In addition, a human–to–human transmissible HPAI H5N1 virus has the potential of being utilized as a bioterrorist weapon [ 19 ]. 2. The Need for Better H5N1 Pandemic Vaccines To mitigate the spread of this contagious virus and reduce the degree of pathogenicity in infected hosts, there is a critical need for an effective H5N1 pandemic preparedness plan [ 20 ], that likely requires a combination of pharmaceutical prophylaxis, treatment (vaccines and antiviral drugs) and non-pharmaceutical interventions (general personal hygiene and reduction of nonessential contacts) [ 21 , 22 ]. H5N1 pandemic vaccine development is considered to be the cornerstone of pandemic influenza control and prevention. Traditional inactivated or live attenuated vaccines are somewhat effective in protecting people against seasonal influenza by targeting HA. However, it is difficult to produce sufficient amounts of effective H5N1 pandemic vaccines in a timely manner using the conventional egg-based system because: (i) It takes at least four months to produce the first vaccine after the identification of a new potential strain [ 23 ]; (ii) H5N1 viruses are highly lethal to personnel [ 14 ], requiring biosafety level 3 containment facilities for vaccine production; (iii) H5N1 viruses do not replicate well in chicken embryos, resulting in low yields of the H5N1 vaccine virus per egg [ 24 ]. In addition, the supply of eggs for vaccine production might be compromised during an H5N1 pandemic when many chickens are infected or culled. In general, both inactivated and live attenuated H5N1 vaccines are only mildly immunogenic in humans, requiring high doses of antigen, multiple cycles of vaccination, and/or the use of adjuvants [ 13 , 25 , 26 ]. Furthermore, the live attenuated influenza vaccine (LAIV) is only licensed for healthy people from 2 through 49 years of age and excludes high-risk groups [ 27 ]. Overall, the platforms that are licensed for the existing seasonal influenza vaccines are not optimal for an H5N1 pandemic scenario as experienced in the 2009 H1N1 influenza pandemic [ 28 ]. Thus, it is urgent to explore alternative pandemic influenza vaccine strategies capable of preventing and controlling H5N1 infection in a timely manner. Several egg-independent vaccine strategies, such as mammalian cell-based vaccines, recombinant protein-based vaccines, virus-like particle (VLP)-based vaccines, DNA vaccines, bacterial vectored vaccines, and viral vectored vaccines, have been extensively studied as alternative approaches [ 28 , 29 , 30 ]. Included in the list of alternative strategies are the recombinant adenoviral (rAd) vectored H5N1 vaccines, which are promising candidates that induce rapid and long-term cross-protective immunity against continuously evolving H5N1 viruses [ 31 , 32 , 33 , 34 , 35 ]. 3. Recombinant Adenoviral Vectors for Vaccination Since the first isolation of adenovirus (Ad) from human adenoid tissue culture nearly 60 years ago, numerous adenoviruses (Ads) have been isolated from a variety of animal species and humans [ 36 , 37 ]. Based on serological properties and genome DNA sequences, human Ads have been classified into 53 distinct serotypes and grouped into 7 subgroups (A–G) [ 38 ], with the majority causing only mild clinical symptoms such as colds or gastroenteritis [ 39 ]. Ads are non-enveloped icosahedral DNA viruses of about 70 to 100 nm in diameter [ 40 ]. Ad viral genome, a linear double-stranded DNA of about 33 to 38 kb, is flanked by two inverted terminal repeats (ITRs) and its packaging signal sequence (ψ) locates adjacent the left ITR [ 41 ]. This genome is tightly associated with viral core proteins and packaged in a naked icosahedral capsid which composes three major structural proteins, hexon, penton, and a knobbed fiber, along with a number of other minor proteins [ 42 ]. Based on the transcription, which occur either before or after replication of viral DNA, the genome can be divided into early (E1a, E1b, E2a, E2b, E3, and E4) or late (L1-L5) regions [ 41 , 43 ]. In 1977, Graham and colleagues developed a system for the production of replication defective rAd vectors in a helper free environment. This system was based on the human embryonic kidney (HEK) 293 packaging cell line, which provided E1 gene products in trans [ 44 ]. Since then, rAd vectors have been extensively investigated for their applications in vaccine development. There are numerous noteworthy reasons for utilizing a rAd as a vaccine delivery vector: (i) the safety of rAd based vaccines has been demonstrated in nonclinical and clinical studies against a number of infectious diseases; (ii) the techniques are well established for rAd vaccine construction and large-scale production in well characterized suspension cells ( i.e. PER.C6) in a very cost effective manner [ 32 , 45 ]; (iii) novel formulations have allowed rAd vectored vaccines to be stored in liquid buffer [ 46 ] or as lyophilized dry power [ 47 ] at 4 o C for at least one year and newly developed thermostabilization techniques may allow the storage of rAd vectors at room temperature at up to 45 o C for 6 months with minimal declining infectivity [ 48 ]; (iv) unlike unnatural adjuvants (manmade molecules and/or administration to an inappropriate site) that may induce unpredictable consequences [ 49 ], rAd vectored vaccines can provide 'self-adjuvanting' activity [ 50 ] by activating innate immunity [ 51 ], which may lower regulatory and commercialization hurdles; and (v) rAd vectored vaccines can be administered by various routes [ 52 , 53 , 54 , 55 , 56 ] because they can infect a wide variety of cell types and tissues in both dividing and non-dividing cells, including antigen-presenting cells [ 31 ]. Both replication-competent and replication-defective rAd vectors have been used as vaccine carriers in early studies [ 57 , 58 , 59 , 60 ]. Due to the fact that human Ad serotype 5 (Ad5) is the best characterized of all Ads in virus-cell interaction, DNA replication and transcription, protein expression, and viral assembly [ 41 ], it is thus not surprising that the majority of currently used rAd vectors are derived from Ad5. Three generations of rAd vectors based on Ad5 have been created and tested. The first-generation rAd vectors are E1 or E1/E3 deleted vectors which are generated and propagated in packaging cells, such as HEK 293 and PER.C6 cells, that supply the products of the deleted genes in trans. The disadvantage with the HEK 293 cells is that replication-competent adenoviruses (RCA) can be generated within rAd vector productions through homologous recombination between identical sequences framing the E1 locus displayed by HEK 293 cells and the rAd vector backbone [ 61 ]. The presence of RCA in rAd vaccine stocks raises the possibilities of undesired Ad infection, amplification, and mobilization. It also triggers the host immune response and gives rise to inflammation and tissue damage [ 62 , 63 ]. RCA-free rAd vectors can be constructed and produced by using the PER.C6 cell line and its matched plasmids [ 32 , 64 ]. PER.C6 is one of the most comprehensively documented mammalian cell lines, with an excellent safety record and a current biologics master file on record with the FDA [ 65 ]. The standard operating procedures for large-scale production of RCA-free first-generation rAd vectors by using PER.C6 cells for clinical trials are well established [ 32 , 66 , 67 ]. In an attempt to reduce vector toxicity, increase their cloning capacity, and prolong transgene expression in vivo , several investigators designed second-generation rAd vectors by further deletion of some or all of E2 and/or E4 genes in the E1 or E1/E3 deleted vector backbone [ 68 , 69 , 70 , 71 ]. The third-generation rAd, which are helper-dependent adenoviral vectors (HDAd), lacks all or nearly all viral coding sequences except necessary cis-acting elements: the packaging signal sequences (ψ) and two ITRs [ 72 ]. Although the second or third-generation rAd vectors might have the advantage of evading any pre-existing anti-vector immunity and inducing better immune responses [ 73 , 74 ], they remain difficult to produce and purify for clinical application. One potential problem with rAd vectors derived from Ad5 is that the majority human populations have pre-existing immunity to Ad5 as a result of natural exposure [ 75 , 76 ] that can severely reduce the potency of injected Ad5 vectored vaccines [ 77 , 78 , 79 ]. Several strategies to circumvent this potential drawback have been studied including the development of rare serotype human rAd vectors, non-human rAd vectors, and molecular engineered Ad5 vectors. A number of novel rAd vectors based on the rare serotype human Ads ( i.e. Ad11, Ad26, Ad35, Ad48, Ad49, and Ad50) [ 80 , 81 , 82 , 83 , 84 ] and non-human rAd vectors such as chimpanzee Ads [ 85 , 86 , 87 ], bovine Ad 3 [ 88 ], canine Ad 2 [ 89 ], and porcine Ad 3 [ 90 ] have been examined as alternative vectors for evading pre-existing Ad5 immunity. However, studies have clearly showed the transgene product-specific antibody responses induced by the vectors derived from both the rare human serotype viruses and chimpanzees viruses are markedly lower than those induced by Ad5 based vectors [ 83 , 91 , 92 ]. Moreover, the pre-existing Ad5-specific CD4+ T and CD8+ T cells may still have a negative impact on the potency of a chimpanzee rAd vectored vaccine [ 93 , 94 ]. Given the impressive immune responses generated by Ad5 based vectors in naïve animals, several investigators have constructed molecular engineered Ad5 vectors to minimize the effect of the anti-Ad5 immunity [ 95 , 96 ]. Despite the substantial progress in molecular engineering of rAd vectors that has been made since the initial studies, these vectors have been proven difficult to construct and produce. In addition, the safety of the rare serotype human rAd vectors, non-human rAd vectors, and molecular engineered rAd vectors needs to be investigated before their use in humans [ 97 ]. 4. Adenoviral Vectored Nasal Vaccines Can Bypass Pre-Existing Immunity Immunization with rAd vectored vaccines by different routes and doses can have a significant effect on the type and strength of the induced immune responses [ 98 , 99 , 100 , 101 ]. Similar to traditional vaccines, rAd vectored vaccines are usually delivered parenterally to stimulate humoral and cellular immune responses. However, the efficacy of the parenterally delivered rAd vectored vaccines may have interference by the presence of pre-existing Ad5 immunity. In contrast, administration of rAd vectored vaccines via alternative routes might overcome pre-existing immunity against the Ad5 vector [ 53 , 55 , 102 , 103 , 104 , 105 ]. There are evidences that intranasal immunizations with rAd vectored vaccines can overcome the effect of pre-existing immunity to Ad5 vectors. They also induce sufficient immune response against encoded antigens and provide protection against challenge of pathogens in mice, rabbits, and non-human primates [ 106 , 107 , 108 , 109 , 110 , 111 ]. These events could be caused due to high-efficiency gene delivery into cells in the superficial layer along the mucosal barrier, together with the potent antigen presentation associated with this immunocompetent interface tissue. Although intranasal inoculation of rAd vectors in mice or rats could lead to some viral dissemination to the olfactory bulb and the central nervous system [ 112 , 113 , 114 , 115 ], no cytopathic effect was detected in the central nervous system structures [ 112 ]. After intranasal delivery of a rAd vectored anthrax vaccine in rabbits, the rAd vector DNA was transiently detected in trachea, olfactory bulb, and lymph node. However, no Ad vector DNA was found in liver, blood, and brain, but the majority of DNA was detected in the lung and could persist there for up to a month (Vaxin unpublished data). Intranasal administration of a rAd vectored vaccine had no undesirable systemic effects in rabbits, cynomolgus macaques or humans [ 53 ] (Vaxin unpublished data). The natural tropism of Ad5 vectors for the respiratory tract makes them useful for the purpose of intranasal vaccination against pathogens ( e.g . influenza virus), that preferentially initiate infection at the mucosal site. Thus, intranasal immunization of Ad vectored vaccines through the use of a nasal spray could be advantageous since immunization becomes simple, practical, economical, and well suited for mass vaccination campaigns. 5. Adenoviral Vectored H5N1 Vaccines 5.1. Multifaceted Immune Responses Comparative data have demonstrated that rAd vectored vaccines induced better humoral and cellular immune responses than recombinant protein vaccines, plasmid-based DNA vaccines, and other recombinant vector systems currently available [ 83 , 116 , 117 ]. The effectiveness of rAd vectored influenza vaccines has been intensively evaluated in a series of efforts focused on the development of vaccines against various influenza virus subtypes, particularly in HPAI H5N1 vaccines. In 2006, Gao et al. reported that a rAd vector encoding HA gene from A/Vietnam/1194/04 elicited robust humoral and/or cellular immune responses in vaccinated mice and chickens. Immunized animals were completely protected from a lethal challenges with H5N1 (A/Vietnam/1194/04) [ 118 ]. They also described that mice immunized with a rAd vector containing HA2 domain from A/Vietnam/1194/04 generated negligible humoral neutralizing response but strong cellular immunity. Also, animals were partially protected when challenged with a lethal dose of A/Vietnam/1194/04. In addition, vaccination with a rAd vector encoding HA1 domain from A/Hong Kong/156/97 could provide cross-protection against the antigenically distinct A/Vietnam/1194/04 virus [ 118 ]. At the same time, Hoelscher et al. observed that mice intramuscularly or intranasally immunized with a rAd expressing HA protein from A/Hong Kong/156/97 were effectively protected against lethal challenges with the heterologous (A/Hong Kong/483/97) and (A/Vietnam/1203/04) H5N1 influenza viruses, even without a strong humoral neutralizing response against A/Vietnam/1203/04 virus [ 119 ]. These two studies highlight that robust cellular immune responses induced by rAd based influenza vaccines have the advantage of conferring broader protection against continuously evolving H5N1 viruses. Cellular immune response also plays an important role in virus clearance and promoting early recovery during H5N1 infection [ 120 , 121 ]. In addition, a rAd vaccine encoding a HA gene is able to stimulate cross-protective immunity between different subtypes of avian influenza virus [ 52 , 122 , 123 ]. This suggests that rAd based vaccination may induce secretion of antibodies against conserved epitopes of HA molecules from different subtypes or strains [ 124 ], providing cross-protection against divergent influenza viruses. During the last ten years, the research group at Vaxin has been developing RCA-free, rAd based nasal influenza vaccines [ 32 , 53 , 125 ]. Studies from Vaxin and others have demonstrated that a single-dose intranasal administration of a rAd vectored influenza vaccine could confer protection against virulent influenza virus challenges in animals [ 126 , 127 ] (Vaxin unpublished data). Growing evidence suggests that rAd vectored nasal influenza vaccines induce greater antigen-specific IgA and IgG responses in the respiratory tract. These vaccines could also provide more virus-specific activated T-cells in the lung and better protection than intramuscularly injected rAd vaccines [ 123 , 128 , 129 ]. This is significant because mucosal immunity can potentially provide cross-protection against different strains of influenza [ 130 , 131 , 132 , 133 , 134 , 135 ]. The more relevant immune responses are found in human clinical trials where human subjects could be safely and effectively immunized with rAd vectored nasal influenza vaccines even in the presence of pre-existing anti-Ad5 immunity [ 53 ] (data of a recent rAd H5N1 nasal vaccine phase I unpublished clinical study involving 48 human). Recently and unexpectedly, Vaxin found that significant protection is afforded by a single intranasal vaccination but not intramuscular injection of either the AdNCHA1.1 vaccine (a rAd vector encoding codon-optimized HA1 domain from A/New Caledonin/20/99) or an AdE vector (a rAd vector without the transgene) against A/California/04/09 (pandemic H1N1) or A/Puerto Rico/8/34 viruses after two days administration [ 136 ]. Although the mechanism is not yet clear, this observation suggests that intranasal immunization of a rAd vector expressing HA proteins may confer seamless protection against influenza virus infection by eliciting of innate as well as adaptive immune responses. More recently, we found that the intranasal administration of an AdVNHA5 vector (a rAd vector encoding codon-optimized HA gene from A/Vietnam/1203/04) could provide similar protection against A/vitenam/1203/04 virus challenge infection in mice (Vaxin unpublished data). Similar to other rAd based vaccines [ 137 , 138 , 139 , 140 ], the rAd vectored H5N1 vaccines are able to induce rapid and long-lasting humoral and cellular protective immunity in mice [ 127 , 129 , 141 ]. This is a highly desirable attribute for an H5N1 vaccine to overcome the emergence of an H5N1 pandemic. The establishment of a complete protection could limit future infection and reduce transmission rates by providing a long lasting immunity to the herd. 5.2.Strategies for Broad Protection The HA glycoprotein is the primary influenza vaccine target that stimulates higher levels of HA inhibition (HI) and neutralizing antibodies (NAB) titers, stronger cellular immune responses, and confers better protection against homologous or heterologous H5N1 virus challenge than NA, nucleoprotein (NP), matrix protein 1 (M1) or M2 based vaccines [ 142 , 143 , 144 , 145 ]. However, a monovalent HA rAd based vaccine may not provide adequate protection against a broad range of heterologous strains of H5N1 influenza viruses, that are currently classified into more than ten antigenically unique clades on the basis of phylogenetic analysis of their HA genes [ 146 ]. Three approaches, which are not mutually exclusive, have been tested in animals to evaluate the efficacy of rAd based H5N1 vaccines against H5N1 isolates from various clades: (i) co-immunization with multivalent rAd vectors expressing HA glycoproteins or other antigens derived from different clades; (ii) a rAd vector expressing HA protein with NA protein and/or other highly conserved influenza antigens; (iii) different combination of prime-boost with rAd based H5N1 vaccines. The efficacy of a multivalent rAd based HA vaccine was first examined by immunizing mice with rAd vectors encoding HA genes from clade 1 virus (A/Vietnam/1203/04), or rAd vectors encoding HA genes from clade 2 virus (A/Indonesia/5/05), or both [ 147 ]. Serum tests showed that mice receiving clade 1 vaccine or clade 2 vaccine produced strong neutralizing antibodies against H5N1 viruses of the same clade but not against the other clade. The mice that received both vaccines generated protective immunity against H5N1 viruses from both clades, but antibody titers were relatively low [ 147 ]. In a similar study, mice immunized with both rAd vectors containing A/Hong Kong/482/97 (clade 0) HA and rAd vectors encoding A/Vietnam/1194/04 (clade 1) HA produced similar HI antibody titers against A/Vietnam/1194/04 to the mice immunized with clade 1 vaccine alone, but generated significantly higher HI titers than the mice only received clade 0 vaccine [ 148 ]. These results demonstrated that multivalent rAd based H5N1 vaccines could be mixed and delivered simultaneously. The immune response elicited by different rAd vectors suggests that some H5N1 strains may provide better protection against heterologous challenge than others. Interestingly, Hoelscher et al. showed that co-administration a rAd vector expressing HA gene with a rAd vector expressing NP gene could further enhance the efficacy of the vaccine [ 147 ]. By contrast, Petal et al. found that the immunization a rAd based HA vaccine together with a rAd vector expressing NP actually damaged the quality of the protective immune responses [ 149 ]. Future studies are needed to focus on what are the relative benefits or detriments of combinations of multivalent rAd vaccination. Several groups have tested the feasibility of a rAd H5N1 vaccine encoding multiple influenza genes to elicit heterosubtypic immunity. Holman et al. constructed two rAd vectors expressing HA, NA, and M1 genes from a clade 1 H5N1 virus A/chicken/Thailand/CH-2/04 (cAdVax-FluAv) and from the 1918 Spanish influenza strain A/South Carolina/1/18 (cAdVax-Flusp) respectively [ 150 ]. These vaccines are based on a second generation rAd platform that had been applied to several disease models [ 102 , 151 , 152 ]. The cAdVax-FluAv vaccine induced HI titers against both clade 1 (A/Vietnam/1203/04) and clade 2 (A/Indonesia/5/05) viruses, with a higher results of immune response against the more homologous clade 1 virus. The same vector induced a 100% protection in mice challenged with both clade 1 and clade 2 viruses [ 150 ]. Although the rAdVax-Flusp vaccine did not elicit HI antibodies against A/Vietnam/1203/04 and A/Indonesia/5/05 viruses, immunization with rAdVax-Flusp did provide complete protection against a lethal A/Vietnam/1203/04 virus challenge and partial protection against A/Indonesia/5/05 challenge [ 150 ]. Park et al. created a rAd H5N1 vaccine (rAdv-AI) expressing two fusion genes linked by an internal ribosome entry site (IRES) based on a traditional first-generation rAd vector [ 128 ]. One fusion gene contains three copies of M2e (2-24 amino acids of M2), the extracellular domain regions of the HA gene of A/Hong Kong/213/2003, and human CD40L. Another fusion gene carries M1 and M2 genes from A/Vietnam/1203/2004. In contrast to intramuscular immunization only eliciting systemic immunity, intranasal vaccination with the rAdv-AI vaccine was able to induce both systemic and mucosal antigen-specific immune responses [ 128 ], that may be more effective against antigenically distinct H5N1 viruses. Interestingly, mice received the rAdv-AI vaccine were completely protected against a lethal H5N2 virus challenge. However, -no anti-H5N2 neutralizing antibodies were detected [ 128 ]. More recently, Pandey et al. reported that a rAd vector encoding both HA and NP genes from A/Vietnam/1203/04 could induce protective immune response in mice, even with high levels of pre-existing Ad5 immunity [ 105 ]. The use of a rAd H5N1 vaccine encoding multiple influenza antigens may have several advantages, like: the reduction of production costs, the decrease of vaccination side effects and the induction of balanced immune responses to all antigens. In a series of studies, Epstein and her colleges demonstrated that a DNA priming-rAd boosting regimen focusing immunity on conserved influenza virus antigens (NP and/or M2) is able to stimulate a broadly protective immunity against challenge with virulent heterologous viruses, including different H5N1 strains, in both mouse and ferret models [ 129 , 153 , 154 , 155 ]. They further showed that intranasal rAd boosting could induce stronger mucosal immunity, enhance viral clearance, and confer better protection against morbidity following highly virulent H1N1 and H5N1 virus challenge. These results were superior compared to intramuscular rAd boosting or intranasal cold-adapted influenza virus boosting [ 129 ]. However, vaccines based on conserved antigens are not capable of replacing HA antigen matched vaccines because these vaccines alone cannot prevent viral infection. Therefore, only reduce the severity of disease and protect animals against low dose influenza virus challenge [ 156 ]. A recent study suggested that NP and M2 antigens may require combinatorial vaccination with HA antigens to become suitable candidates for universal influenza vaccines [ 142 ]. Using HA molecules from different subtypes of the influenza viruses, Wei et al. demonstrated that DNA priming-rAd boosting vaccination could generate a strong antigen-specific cellular immunity and high titers of broadly neutralizing antibodies, including antibodies against conserved HA2. They also protect animals against challenge with more divergent influenza strains [ 52 ], even in animals with pre-existing influenza immunity [ 157 ]. rAd vectors have been demonstrated to be very immunogenic at priming [ 117 ] and boosting [ 158 ]. In agreement with these studies, Lin et al. recently showed that a rAd encoding HA gene priming followed by a recombinant trimetric HA protein boosting elicited a robust neutralizing antibody responses against homologous and heterologous H5N1 virus strains [ 159 ]. The prime-boost immunization strategies are excellent regimes for enhancing the potency of rAd based H5N1 vaccines, but they require multiple doses over a prolonged immunization schedule, which might limit their usefulness during an H5N1 pandemic. 5.3. Human Adenovirus Serotype 4 Vectored H5N1 Vaccines In addition to Ad5 derived vectors, replicating recombinant adenovirus serotype 4 (rAd4) vector has also been studied for prevention of several disease models [ 160 , 161 ]. Recently, Alexander et al. presented nonclinical data that intranasal administration of a rAd4 encoding HA gene from A/Vietnam/1194/2004 induced HA specific HI antibodies and cellular immune responses. This adenoviral construction also protected mice against a lethal H5N1 reassortant viral challenge even in the presence of pre-existing anti-Ad4 immunity [ 162 ]. One phase I clinical trial designed to evaluate the safety and immunogenicity of an oral rAd4 H5N1 vaccine has been completed [ 163 ]. Another phase I trial in healthy adults aged 19-45 years to determine the optimal route and dose for the rAd4 H5N1 vaccine is underway [ 163 ]. 5.4. Non-Human Adenovirus Vectored H5N1 Vaccines Several non-human Ad vectors have been tested as H5N1 vaccine carriers that were primarily intended as strategies to overcome pre-existing anti-Ad5 immunity. Roy et al. tested the efficacy of a single dose of AdC7 (a chimpanzee adenovirus vector) expressing A/Puerto Rico/8/34/mount Sinai NP gene and showed that it was less effective than a Ad5 based vector in protection against A/Puerto Rico/8/34/mount Sinai, A/Vitetnam/1203/04 and A/Hong Kong/483/1997 in a mouse model [ 87 ]. Singh et al. described the effectiveness of a BAd3 (a bovine adenovirus 3 vector) containing HA gene from A/Hong Kong/156/97 in eliciting the protective immune responses against A/Hong Kong/483/97 challenges in mice with or without pre-existing anti-Ad5 immunity [ 88 ]. Patel et al. presented data with a PAV3 (a porcine adenovirus 3 vector) based H5N1 vaccine encoding A/Hanoi/30408/05 HA gene and demonstrated that it could be at least as potent as its Ad5 counterpart in mice [ 90 ]. A recombinant replication-competent canine adenovirus type 2 (cAd2) expressing HA gene from H5N1 subtype of tiger influenza virus (A/tiger/Harbin/01/2003) has also been evaluated in cats as a Felidae H5N1 vaccine [ 164 ]. 5.5. Recombinant Adenoviral Vectored H5N1 in Chicken Most H5N1 infections in humans have been preceded by outbreaks in poultry [ 165 ]. Thus, it is not surprising that mass immunization of poultry against H5N1 could reduce the risk of human exposure; hence, the decrease of H5N1 infected humans by reducing the number of susceptible poultry [ 166 ]. Current vaccination of poultry with oil emulsion, inactivated, whole-AI-virus vaccines or fowl pox-vectored AI vaccines are effective in reducing the risk of infection and disease, but injection of individual birds are labor intensive and time consuming. Improved vaccines are required in order to induce rapid and sustained broad immunity, to allow mass vaccination campaigns and DIVA (differentiation between infected and vaccinated animals) strategies. The potency of a rAd vectored H5N1 vaccine expressing HA protein of A/Vietnam/1203/04 has been demonstrated [ 101 , 118 ] and can be significantly enhanced by fusion of HA protein with chicken CD154 in chickens [ 167 ]. Additionally, chickens can be effectively immunized by in ovo administration or aerosol spray of rAd vectored AI vaccines [ 125 , 168 ]. Both types of vaccines may allow mass immunization of poultry with the rAd vectored vaccines. Furthermore, chickens immunized in ovo with a rAd vectored HA5 vaccine can be successfully vaccinated post-hatch with another rAd vectored HA7 vaccine [ 122 ], raising the possibility of protection against multiple pathogens with the same rAd vector technology. 5.1. Multifaceted Immune Responses Comparative data have demonstrated that rAd vectored vaccines induced better humoral and cellular immune responses than recombinant protein vaccines, plasmid-based DNA vaccines, and other recombinant vector systems currently available [ 83 , 116 , 117 ]. The effectiveness of rAd vectored influenza vaccines has been intensively evaluated in a series of efforts focused on the development of vaccines against various influenza virus subtypes, particularly in HPAI H5N1 vaccines. In 2006, Gao et al. reported that a rAd vector encoding HA gene from A/Vietnam/1194/04 elicited robust humoral and/or cellular immune responses in vaccinated mice and chickens. Immunized animals were completely protected from a lethal challenges with H5N1 (A/Vietnam/1194/04) [ 118 ]. They also described that mice immunized with a rAd vector containing HA2 domain from A/Vietnam/1194/04 generated negligible humoral neutralizing response but strong cellular immunity. Also, animals were partially protected when challenged with a lethal dose of A/Vietnam/1194/04. In addition, vaccination with a rAd vector encoding HA1 domain from A/Hong Kong/156/97 could provide cross-protection against the antigenically distinct A/Vietnam/1194/04 virus [ 118 ]. At the same time, Hoelscher et al. observed that mice intramuscularly or intranasally immunized with a rAd expressing HA protein from A/Hong Kong/156/97 were effectively protected against lethal challenges with the heterologous (A/Hong Kong/483/97) and (A/Vietnam/1203/04) H5N1 influenza viruses, even without a strong humoral neutralizing response against A/Vietnam/1203/04 virus [ 119 ]. These two studies highlight that robust cellular immune responses induced by rAd based influenza vaccines have the advantage of conferring broader protection against continuously evolving H5N1 viruses. Cellular immune response also plays an important role in virus clearance and promoting early recovery during H5N1 infection [ 120 , 121 ]. In addition, a rAd vaccine encoding a HA gene is able to stimulate cross-protective immunity between different subtypes of avian influenza virus [ 52 , 122 , 123 ]. This suggests that rAd based vaccination may induce secretion of antibodies against conserved epitopes of HA molecules from different subtypes or strains [ 124 ], providing cross-protection against divergent influenza viruses. During the last ten years, the research group at Vaxin has been developing RCA-free, rAd based nasal influenza vaccines [ 32 , 53 , 125 ]. Studies from Vaxin and others have demonstrated that a single-dose intranasal administration of a rAd vectored influenza vaccine could confer protection against virulent influenza virus challenges in animals [ 126 , 127 ] (Vaxin unpublished data). Growing evidence suggests that rAd vectored nasal influenza vaccines induce greater antigen-specific IgA and IgG responses in the respiratory tract. These vaccines could also provide more virus-specific activated T-cells in the lung and better protection than intramuscularly injected rAd vaccines [ 123 , 128 , 129 ]. This is significant because mucosal immunity can potentially provide cross-protection against different strains of influenza [ 130 , 131 , 132 , 133 , 134 , 135 ]. The more relevant immune responses are found in human clinical trials where human subjects could be safely and effectively immunized with rAd vectored nasal influenza vaccines even in the presence of pre-existing anti-Ad5 immunity [ 53 ] (data of a recent rAd H5N1 nasal vaccine phase I unpublished clinical study involving 48 human). Recently and unexpectedly, Vaxin found that significant protection is afforded by a single intranasal vaccination but not intramuscular injection of either the AdNCHA1.1 vaccine (a rAd vector encoding codon-optimized HA1 domain from A/New Caledonin/20/99) or an AdE vector (a rAd vector without the transgene) against A/California/04/09 (pandemic H1N1) or A/Puerto Rico/8/34 viruses after two days administration [ 136 ]. Although the mechanism is not yet clear, this observation suggests that intranasal immunization of a rAd vector expressing HA proteins may confer seamless protection against influenza virus infection by eliciting of innate as well as adaptive immune responses. More recently, we found that the intranasal administration of an AdVNHA5 vector (a rAd vector encoding codon-optimized HA gene from A/Vietnam/1203/04) could provide similar protection against A/vitenam/1203/04 virus challenge infection in mice (Vaxin unpublished data). Similar to other rAd based vaccines [ 137 , 138 , 139 , 140 ], the rAd vectored H5N1 vaccines are able to induce rapid and long-lasting humoral and cellular protective immunity in mice [ 127 , 129 , 141 ]. This is a highly desirable attribute for an H5N1 vaccine to overcome the emergence of an H5N1 pandemic. The establishment of a complete protection could limit future infection and reduce transmission rates by providing a long lasting immunity to the herd. 5.2.Strategies for Broad Protection The HA glycoprotein is the primary influenza vaccine target that stimulates higher levels of HA inhibition (HI) and neutralizing antibodies (NAB) titers, stronger cellular immune responses, and confers better protection against homologous or heterologous H5N1 virus challenge than NA, nucleoprotein (NP), matrix protein 1 (M1) or M2 based vaccines [ 142 , 143 , 144 , 145 ]. However, a monovalent HA rAd based vaccine may not provide adequate protection against a broad range of heterologous strains of H5N1 influenza viruses, that are currently classified into more than ten antigenically unique clades on the basis of phylogenetic analysis of their HA genes [ 146 ]. Three approaches, which are not mutually exclusive, have been tested in animals to evaluate the efficacy of rAd based H5N1 vaccines against H5N1 isolates from various clades: (i) co-immunization with multivalent rAd vectors expressing HA glycoproteins or other antigens derived from different clades; (ii) a rAd vector expressing HA protein with NA protein and/or other highly conserved influenza antigens; (iii) different combination of prime-boost with rAd based H5N1 vaccines. The efficacy of a multivalent rAd based HA vaccine was first examined by immunizing mice with rAd vectors encoding HA genes from clade 1 virus (A/Vietnam/1203/04), or rAd vectors encoding HA genes from clade 2 virus (A/Indonesia/5/05), or both [ 147 ]. Serum tests showed that mice receiving clade 1 vaccine or clade 2 vaccine produced strong neutralizing antibodies against H5N1 viruses of the same clade but not against the other clade. The mice that received both vaccines generated protective immunity against H5N1 viruses from both clades, but antibody titers were relatively low [ 147 ]. In a similar study, mice immunized with both rAd vectors containing A/Hong Kong/482/97 (clade 0) HA and rAd vectors encoding A/Vietnam/1194/04 (clade 1) HA produced similar HI antibody titers against A/Vietnam/1194/04 to the mice immunized with clade 1 vaccine alone, but generated significantly higher HI titers than the mice only received clade 0 vaccine [ 148 ]. These results demonstrated that multivalent rAd based H5N1 vaccines could be mixed and delivered simultaneously. The immune response elicited by different rAd vectors suggests that some H5N1 strains may provide better protection against heterologous challenge than others. Interestingly, Hoelscher et al. showed that co-administration a rAd vector expressing HA gene with a rAd vector expressing NP gene could further enhance the efficacy of the vaccine [ 147 ]. By contrast, Petal et al. found that the immunization a rAd based HA vaccine together with a rAd vector expressing NP actually damaged the quality of the protective immune responses [ 149 ]. Future studies are needed to focus on what are the relative benefits or detriments of combinations of multivalent rAd vaccination. Several groups have tested the feasibility of a rAd H5N1 vaccine encoding multiple influenza genes to elicit heterosubtypic immunity. Holman et al. constructed two rAd vectors expressing HA, NA, and M1 genes from a clade 1 H5N1 virus A/chicken/Thailand/CH-2/04 (cAdVax-FluAv) and from the 1918 Spanish influenza strain A/South Carolina/1/18 (cAdVax-Flusp) respectively [ 150 ]. These vaccines are based on a second generation rAd platform that had been applied to several disease models [ 102 , 151 , 152 ]. The cAdVax-FluAv vaccine induced HI titers against both clade 1 (A/Vietnam/1203/04) and clade 2 (A/Indonesia/5/05) viruses, with a higher results of immune response against the more homologous clade 1 virus. The same vector induced a 100% protection in mice challenged with both clade 1 and clade 2 viruses [ 150 ]. Although the rAdVax-Flusp vaccine did not elicit HI antibodies against A/Vietnam/1203/04 and A/Indonesia/5/05 viruses, immunization with rAdVax-Flusp did provide complete protection against a lethal A/Vietnam/1203/04 virus challenge and partial protection against A/Indonesia/5/05 challenge [ 150 ]. Park et al. created a rAd H5N1 vaccine (rAdv-AI) expressing two fusion genes linked by an internal ribosome entry site (IRES) based on a traditional first-generation rAd vector [ 128 ]. One fusion gene contains three copies of M2e (2-24 amino acids of M2), the extracellular domain regions of the HA gene of A/Hong Kong/213/2003, and human CD40L. Another fusion gene carries M1 and M2 genes from A/Vietnam/1203/2004. In contrast to intramuscular immunization only eliciting systemic immunity, intranasal vaccination with the rAdv-AI vaccine was able to induce both systemic and mucosal antigen-specific immune responses [ 128 ], that may be more effective against antigenically distinct H5N1 viruses. Interestingly, mice received the rAdv-AI vaccine were completely protected against a lethal H5N2 virus challenge. However, -no anti-H5N2 neutralizing antibodies were detected [ 128 ]. More recently, Pandey et al. reported that a rAd vector encoding both HA and NP genes from A/Vietnam/1203/04 could induce protective immune response in mice, even with high levels of pre-existing Ad5 immunity [ 105 ]. The use of a rAd H5N1 vaccine encoding multiple influenza antigens may have several advantages, like: the reduction of production costs, the decrease of vaccination side effects and the induction of balanced immune responses to all antigens. In a series of studies, Epstein and her colleges demonstrated that a DNA priming-rAd boosting regimen focusing immunity on conserved influenza virus antigens (NP and/or M2) is able to stimulate a broadly protective immunity against challenge with virulent heterologous viruses, including different H5N1 strains, in both mouse and ferret models [ 129 , 153 , 154 , 155 ]. They further showed that intranasal rAd boosting could induce stronger mucosal immunity, enhance viral clearance, and confer better protection against morbidity following highly virulent H1N1 and H5N1 virus challenge. These results were superior compared to intramuscular rAd boosting or intranasal cold-adapted influenza virus boosting [ 129 ]. However, vaccines based on conserved antigens are not capable of replacing HA antigen matched vaccines because these vaccines alone cannot prevent viral infection. Therefore, only reduce the severity of disease and protect animals against low dose influenza virus challenge [ 156 ]. A recent study suggested that NP and M2 antigens may require combinatorial vaccination with HA antigens to become suitable candidates for universal influenza vaccines [ 142 ]. Using HA molecules from different subtypes of the influenza viruses, Wei et al. demonstrated that DNA priming-rAd boosting vaccination could generate a strong antigen-specific cellular immunity and high titers of broadly neutralizing antibodies, including antibodies against conserved HA2. They also protect animals against challenge with more divergent influenza strains [ 52 ], even in animals with pre-existing influenza immunity [ 157 ]. rAd vectors have been demonstrated to be very immunogenic at priming [ 117 ] and boosting [ 158 ]. In agreement with these studies, Lin et al. recently showed that a rAd encoding HA gene priming followed by a recombinant trimetric HA protein boosting elicited a robust neutralizing antibody responses against homologous and heterologous H5N1 virus strains [ 159 ]. The prime-boost immunization strategies are excellent regimes for enhancing the potency of rAd based H5N1 vaccines, but they require multiple doses over a prolonged immunization schedule, which might limit their usefulness during an H5N1 pandemic. 5.3. Human Adenovirus Serotype 4 Vectored H5N1 Vaccines In addition to Ad5 derived vectors, replicating recombinant adenovirus serotype 4 (rAd4) vector has also been studied for prevention of several disease models [ 160 , 161 ]. Recently, Alexander et al. presented nonclinical data that intranasal administration of a rAd4 encoding HA gene from A/Vietnam/1194/2004 induced HA specific HI antibodies and cellular immune responses. This adenoviral construction also protected mice against a lethal H5N1 reassortant viral challenge even in the presence of pre-existing anti-Ad4 immunity [ 162 ]. One phase I clinical trial designed to evaluate the safety and immunogenicity of an oral rAd4 H5N1 vaccine has been completed [ 163 ]. Another phase I trial in healthy adults aged 19-45 years to determine the optimal route and dose for the rAd4 H5N1 vaccine is underway [ 163 ]. 5.4. Non-Human Adenovirus Vectored H5N1 Vaccines Several non-human Ad vectors have been tested as H5N1 vaccine carriers that were primarily intended as strategies to overcome pre-existing anti-Ad5 immunity. Roy et al. tested the efficacy of a single dose of AdC7 (a chimpanzee adenovirus vector) expressing A/Puerto Rico/8/34/mount Sinai NP gene and showed that it was less effective than a Ad5 based vector in protection against A/Puerto Rico/8/34/mount Sinai, A/Vitetnam/1203/04 and A/Hong Kong/483/1997 in a mouse model [ 87 ]. Singh et al. described the effectiveness of a BAd3 (a bovine adenovirus 3 vector) containing HA gene from A/Hong Kong/156/97 in eliciting the protective immune responses against A/Hong Kong/483/97 challenges in mice with or without pre-existing anti-Ad5 immunity [ 88 ]. Patel et al. presented data with a PAV3 (a porcine adenovirus 3 vector) based H5N1 vaccine encoding A/Hanoi/30408/05 HA gene and demonstrated that it could be at least as potent as its Ad5 counterpart in mice [ 90 ]. A recombinant replication-competent canine adenovirus type 2 (cAd2) expressing HA gene from H5N1 subtype of tiger influenza virus (A/tiger/Harbin/01/2003) has also been evaluated in cats as a Felidae H5N1 vaccine [ 164 ]. 5.5. Recombinant Adenoviral Vectored H5N1 in Chicken Most H5N1 infections in humans have been preceded by outbreaks in poultry [ 165 ]. Thus, it is not surprising that mass immunization of poultry against H5N1 could reduce the risk of human exposure; hence, the decrease of H5N1 infected humans by reducing the number of susceptible poultry [ 166 ]. Current vaccination of poultry with oil emulsion, inactivated, whole-AI-virus vaccines or fowl pox-vectored AI vaccines are effective in reducing the risk of infection and disease, but injection of individual birds are labor intensive and time consuming. Improved vaccines are required in order to induce rapid and sustained broad immunity, to allow mass vaccination campaigns and DIVA (differentiation between infected and vaccinated animals) strategies. The potency of a rAd vectored H5N1 vaccine expressing HA protein of A/Vietnam/1203/04 has been demonstrated [ 101 , 118 ] and can be significantly enhanced by fusion of HA protein with chicken CD154 in chickens [ 167 ]. Additionally, chickens can be effectively immunized by in ovo administration or aerosol spray of rAd vectored AI vaccines [ 125 , 168 ]. Both types of vaccines may allow mass immunization of poultry with the rAd vectored vaccines. Furthermore, chickens immunized in ovo with a rAd vectored HA5 vaccine can be successfully vaccinated post-hatch with another rAd vectored HA7 vaccine [ 122 ], raising the possibility of protection against multiple pathogens with the same rAd vector technology. 6. Future Directions There is no doubt that significant progress has been made, during the past decade, in the field of the rAd-vectored H5N1 influenza vaccines. However, these vaccines must overcome several challenges before they can be considered a suitable alternative to the currently licensed vaccines. A major drawback faced by the vaccine candidates for licensure is the lack of information associated to the correlation between immunity and protection [ 169 ]. A serum HI titer of 40 or greater is a well-established marker of immune protection for inactivated seasonal influenza vaccines intramuscularly injected. However, this may not appear to hold true for a rAd vectored H5N1 vaccine encoding heterogonous HA [ 128 , 150 ], HA1 fragment (Vaxin unpublished data), HA2 fragment [ 118 ], or conserved NP and M2 antigens [ 129 ]. Mounting evidence indicates that mucosal and T cell mediated immunity may actually more important than previously realized against a broad spectrum of H5N1 strains [ 118 , 119 , 121 , 123 , 128 , 129 ]. Therefore, the standardization of immunoassays used in the assessment of the innate and adaptive immune responses is crucial for the comparative analysis of such vaccines. Also, it is important to standardize virulent H5N1 challenge reagents and animal models for head-to-head comparisons of rAd H5N1 vaccine candidates. The cross-protection achieved by vaccination with rAd vaccines encoding HA and/or conserved antigens is very encouraging [ 52 , 87 , 129 , 147 ]. In a pandemic scenario, these vaccines may be used for emergency vaccination when an antigenically matched vaccine is not available [ 170 ]. Future nonclinical and clinical studies should be aimed to identify the optimal combination of rAd expressing HA and/or conserved NP, M1, M2 antigens for the induction of protective immunity against heterogonous strains. The development of rAd vectored H5N1 vaccines is greatly benefited from advanced synthetic DNA technologies. With improvements in surveillance and case confirmation, the modified HA or other antigen can be rapidly synthesized in order to adjust the changes in current H5N1 strains. Nonclinical studies showed that rAd or DNA vaccines encoding synthetic consensus HA genes are effective for eliciting protective immunity against mismatched virus challenges [ 140 , 171 , 172 , 173 ]. The immune responses could be substantially strengthened with codon-optimized influenza antigens inserted into rAd vectored vaccines [ 148 , 174 ]. However, our previous experience with manufacturing of rAd vaccines encoding codon-optimized HA genes showed that overexpression of HA transgene products from a number of influenza virus strains may inhibit rAd vector production in replication permissive cell lines (Vaxin unpublished data). This suggests that manufacturing process optimization will be required to maximize yields prior to scale up. Until recently, it was believed that the common presence of pre-existing Ad5 immunity in human populations could be a potential problem for the clinical use of rAd vectored vaccines. Although not studied in sufficient detail, emerging data from clinical trials suggest that this limitation can be overcome by increasing the vaccine dose [ 175 ] or by intranasal vaccination [ 53 ]. However, more clinical information is required to clarify the influence of the pre-existing immunity on the rAd vectored vaccines. Vaxin's nonclinical and Phase I clinical trial data support the overall advantages of our Ad5-vectored nasal influenza vaccine platform [ 53 ] (Vaxin unpublished data). These data are moving forward to more advanced clinical stages. Further evaluation of rAd vectored seasonal and pandemic influenza vaccines in animal models and clinical trials is needed to confirm their suitability for human use. They must include the in high-risk populations, such as the very young, the elderly, pregnant women, and immunocompromised individuals. Influenza vaccines based on egg-independent technologies have increased acceptance in recent years. Mammalian cell-based vaccines have been licensed by regulatory agencies in Europe, Asia, and Latin America [ 176 ] and close to commercialization in the USA [ 177 ]. Recombinant protein-based vaccines and VLP based vaccines can be produced in plants, insect cells, or Escherichia coli [ 178 ]. VLP based vaccines can induce both humoral and cellular immunity in nonclinical studies and have looked very promising in clinical trials [ 178 ]. rAd vectored H5N1 vaccines are considered lead-candidates among DNA based and viral vectored influenza vaccines [ 35 ]. Based on the promising results of rAd H5N1 vaccine in nonclinical and clinical studies and the increasing clinical experience with rAd vectored vaccines against various infectious pathogens, we believe that the rAd vectored nasal influenza vaccines hold great promise for the influenza pandemic preparedness.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9273079/

A participatory epidemiological and One Health approach to explore the community’s capacity to detect emerging zoonoses and surveillance network opportunities in the forest region of Guinea

The Ebola virus disease epidemic that threatened West Africa between 2013 and 2016 was of unprecedented health magnitude. After this health crisis, studies highlighted the need to introduce community-based surveillance systems and to adopt a One Health approach. This study aimed to provide preparatory insights for the definition of a community-based surveillance system for emerging zoonoses such as viral hemorrhagic fevers in Guinea. The objective was to explore the disease detection capacity and the surveillance network opportunities at the community level in two pilot areas in the forest region of Guinea, where the epidemic emerged. Based on a participatory epidemiological and One Health approach, we conducted Focus Group Discussions with human, animal and ecosystem health actors. We used a range of participatory tools, included semi-structured interviews, ranking, scoring and flow diagram, to estimate the local knowledge and perception of diseases and clinical signs and to investigate the existing health information exchange network and its related strengths and weaknesses. The results showed that there is heterogeneity in knowledge of diseases and perception of the clinical signs among actors and that there are preferred and more effective health communication channels opportunities. This preparatory study suggests that it is necessary to adapt the case definitions and the health communication channels to the different actors who can play a role in a future community-based surveillance system and provides recommendations for future surveillance activities to be carried out in West Africa. 1. Introduction The emerging zoonotic diseases constitute threats to our modern world. Their rate of incidence is increasing, driven by anthropogenic factors such as international trade, human and animal populations movements and the disruption of ecosystems due to human activities; which are no more and no less than consequences of world population growth and globalization [ 1 ]. Nowadays, at least 75% of emerging diseases affecting humans are of animal origin and most of them originate in wildlife [ 2 , 3 ]. Several zoonotic diseases emerged from wildlife over these last decades such as the Nipah epidemic in 1999 in Malaysia, the Ebola virus disease (EVD) outbreak in West Africa in 2013 and more recently the COVID-19 pandemic in 2019 [ 4 – 8 ]. The EVD outbreak that occurred in West Africa between 2013 and 2016 was on a scale never seen before, with more than 20,000 reported cases and more than 11,000 deaths [ 9 ]. Governments have been overwhelmed by this health event. The surprising nature of this health disaster also lies in its geographical distribution. The EVD used to emerge in Central and East Africa. For the first time it appeared in West Africa with an index case retrospectively identified in Guinea and dated December 2013 [ 10 ]. Then, the EVD epidemic rapidly spread to other regions of Guinea and neighboring countries. Several factors led to a late response and a difficulty in containing the epidemic in Guinea: the presence of the ecological niche of the disease, susceptible populations, an insufficient response capacity, risky behaviors conducive to human-wildlife contacts and the community mistrust [ 11 ]. Poor regions of the world face many challenges such as a lack of health infrastructures, an insufficient access to health, communication problems and a lack of resources. This situation creates difficulties for the coordination of surveillance systems and the effective use of data [ 12 ]. Nowadays, participatory epidemiology is increasingly used for active surveillance of endemic, epidemic and emerging diseases. It consists of interactive participation to collect data, analyze them and plan action [ 13 ]. Community-based surveillance "is the systematic detection and reporting of events of public health significance within community by community members" [ 14 ]. It explores the social context in which a disease occurs and the host-agent-environment interactions. The participatory surveillance system, with the help of community workers, can therefore be a solution to make monitoring possible by reducing the burden on health infrastructures and supporting data collection [ 15 ]. To avoid a future pandemic, a One Health approach is a key strategy for global health security [ 16 ]. The tripartite collaboration, included the World Health Organization (WHO), World Organization for Animal Health (OIE) and Food and Agricultural Organization (FAO), called for a One Health surveillance of diseases and recommend to coordinate and address health risk at the human-animal-ecosystems interfaces [ 17 ]. In November 2020, the United Nations Environment Programme (UNEP) joint this tripartite collaboration in a new international expert panel to address the emergence and spread of zoonotic diseases [ 18 ]. This panel defined the One Health as "an integrated, unifying approach that aims to sustainably balance and optimize the health of people, animals and ecosystems" and recognized these three sectors of health as "closely linked and inter-dependent" [ 19 ]. Several studies support the hypothesis that a One Health approach provides a positive effect for emerging infectious diseases prevention [ 20 – 22 ]. By combining knowledge on animals, humans and ecosystems, surveillance and detection strategies are strengthened [ 23 ]. The purpose of our study was to provide preparatory insights for the definition of a community-based surveillance system in Guinea for the early detection of emerging zoonoses and viral hemorrhagic fevers (VHFs) in particular; as they have a high pandemic and outbreak receptivity in this area [ 24 ]. We used a participatory epidemiological and One Health approach and conducted Focus Group Discussions (FGDs) with human, animal and ecosystem health actors to estimate local knowledge and perception of diseases and clinical signs. We also investigated the existing health information exchange network and its related strengths and weaknesses. 2. Material and methods 2.1 Ethics statement Official authorization from the National Department of Veterinary Services and at the prefectural level allowed us to work on these areas. The study was validated by the National Health Research Ethics Committee of Guinea (046/CNERS/18, 023/CNERS/19). Respondents participated freely and anonymously to the research study. The project was presented and translated before each interview so that participants could knowingly sign a consent form. 2.2. Study area The study was conducted in the prefecture of Guéckédou in the forest region of N'Zérékoré in Guinea. Based on a previous study on the socio-cultural and economic practices increasing the risk of zoonotic transmission from wildlife and the community's perception of One health surveillance and in consultation with the National Direction of Veterinary Services (Direction Nationale des Services Vétérinaires) and key resource persons, we selected the two sub-prefectures of Guendembou and Temessadou, for their close human-domestic animal-wildlife interface and for the presence of human, animal and ecosystem health actors [ 25 ]. We conducted FGDs in their respective chief towns and in the village of Mongo ( Fig 1 ). 10.1371/journal.pntd.0010462.g001 Fig 1 Map of the Republic of Guinea and location of the study areas. The study areas were located in the sub-prefectures of Guendembou and Temessadou (orange area) in the prefecture of Guéckédou (orange hatched area) in the N'Zérékoré region (green area). Focus Group Discussions were conducted in the villages of Guendembou, Temessadou and Mongo (red dots). Map created using the Free and Open Source QGIS software ( https://www.qgis.org ) and OpenStreetMap ( https://www.openstreetmap.org ) and GADM ( https://www.gadm.org ) geographic databases. 2.3 Participants We conducted FGDs with human, animal and ecosystem health actors to estimate local knowledge and perception of diseases and clinical signs and to investigate the existing health information exchange network and the potential collaborations for community-based surveillance. From the human health sector, we identified women and Community Human Health Workers (CHHWs). We decided to have non-mixed groups of women to ensure their representativeness because they are under-represented among other categories of actors. Women are involved in the care of family members and, as such, can play a role in disease detection. CHHWs are volunteers located in village and elected by the community. They are literate and specially trained in birth control, malaria treatment, awareness of diseases and surveillance. They routinely report human health information to the health center. From the animal health sector, we identified breeders and Community Animal Health Workers (CAHWs). CAHWs are volunteers located at the district level and elected by the community. They are literate and specially trained in conflict management between plant growers and animal breeders, veterinary treatment, awareness of animal diseases and surveillance. From the ecosystem health sector, we identified hunters, Community Informants (CIs) and rangers. In this study area, traditional hunting is practiced by hunters from the Kissi ethnolinguistic group. CIs are volunteers from the community located at the district level. They are literate and elected by the sub-prefectural Water and Forest Department to report on bushfires and non-compliance with hunting regulation and conservation activities. The rangers are located at the sub-prefectural level and work for the Water and Forest Department which is the most decentralized service of the Ministry of Environment. They control and manage all activities related to the environment and conservation such as fishing, hunting, deforestation, wildlife monitoring. 2.4 Data collection 2.4.1 Participatory epidemiological approach The participatory epidemiology is a bottom-up approach based on active participation of individuals in defining their own solutions tailored to their development stakes. This method is used to collect and analyze qualitative epidemiological information from local knowledge and expertise and is consistent to increase involvement of communities in the design and implementation of animal health program such as surveillance systems [ 13 , 26 ]. In this present study we investigated the perception of the disease and clinical signs to assess the case definition adaptability [ 27 ]. Participatory epidemiology is also interesting to explore the health information exchange network opportunities as it promotes local initiative and communication between the various actors [ 28 ]. Data were collected by an investigation team including a veterinarian from the National Direction of Veterinary Services, the Livestock Prefectural Director of Macenta in N'Zérékoré forest region and two French veterinary students. The investigation team was trained to conduct semi-structured interviews (SSIs), to use participatory tools and to record data in an appropriate format for analysis. For each FGD, we distributed roles among the investigation team to have one facilitator, one analyst and two note-takers. We conducted the FGDs in French or in the local language and literate persons from the sub-prefectures translated from Kissi into French. 2.4.2 Data triangulation and sampling method We applied data triangulation as main sampling criteria to ensure data reliability and saturation [ 29 – 31 ]. We expected between eight and ten participants per FGD to ensure data quality without compromising participation and facilitation. In each sub-prefecture, the head of the health center, the official veterinarian and the head of the Water and Forest Department selected the participants from the human, animal and ecosystem health sectors respectively. Participation to the FGDs was without gender restriction, except for the non-mixed groups of women, and we voluntarily included heads of breeders and heads of hunters to investigate their role in the existing animal and ecosystem health information exchange network. We conducted two sessions of FGDs in each sub-prefecture and expected as many of the participants as possible to be the same from one session to the other to foster their understanding of the research activities. 2.4.3 Data on local knowledge and perception of diseases and clinical signs Between February and March 2019, we conducted a first session of FGDs with each type of participants in both sub-prefectures to estimate local knowledge and perception of disease and clinical signs in the human, domestic animal and wildlife populations. We used several participatory tools as described by Catley et al. [ 32 ] such as SSIs, ranking and scoring. First, we asked participants to list the clinical signs, and their related diseases, they observe in the human, domestic animal or wildlife populations, and we noted them on post-it notes. Secondly, to reduce the large number of clinical signs and ensure the feasibility of the following steps, we asked them to select those they perceive as life-threatening or as a zoonotic risk. Then, we asked them to rank these selected clinical signs from the most to the least frequently observed. Finally, we asked participants to score these same and previously selected clinical signs by distributing one hundred beans proportionally to the associated threat regarding their own health; even for the signs they observe in the animal populations. 2.4.4 Data on the health information exchange network The first session of FGDs allowed us to collect qualitative data on community's response to health threats and human, animal and ecosystem health actors' roles. In April 2019, we conducted a second session of FGDs in each sub-prefecture to gather women and CHHWs, breeders and CAHWs and hunters and CIs. We used flow diagrams to explore the existing health information exchange network, by drawing the interactions between actors on a poster, and to discuss its related strengths and weaknesses and better alternatives for an efficient community-based surveillance of emerging zoonotic diseases [ 33 ]. 2.5 Data analysis All the FGDs were recorded and transcribed on Microsoft Word. We performed a thematic content analysis to extract the qualitative data from the transcripts and sort them in different broad themes on Microsoft Excel. Using a deductive approach, we identified the pre-conceived themes: stakeholders' roles, local knowledge on diseases and detection capacity, communication of health information, and One Health collaboration [ 34 ]. Data were separately analyzed for each One Health sector. 2.5.1 Qualitative analysis of data on local knowledge of diseases Data on diseases and clinical signs were reported into a Microsoft Excel table to generate diseases-clinical signs matrix. We extracted from the matrix the commonly listed diseases, the diseases under surveillance, the VHFs and the zoonotic diseases. To estimate participants' knowledge, we compared the clinical picture they used to describe the diseases under surveillance with the clinical pictures cited in the OIE and WHO descriptions and with the case definitions provided to the CAHWs and CHHWs [ 35 – 46 ]. 2.5.2 Analysis of rankings and scorings Semi-quantitative data obtained from ranking and scoring were reported into a database and processed for analysis using graphical representations on Microsoft Excel. For each group of participants, we generated scatter plots of the signs that were ranked and scored to position them according to participants' perception in terms of frequency of observation and concern regarding their own health. Then we plotted axes through the medians of the ranks and scores to generate four-way matrix: rare/frequent and health-threatening/not threatening. This method allowed to identify the clinical signs perceived as the rarest and the most health-threatening and to estimate participants' perception of the clinical signs included in the human case definitions recommended by the WHO for Ebola or Marburg virus disease surveillance and in the animal case definition recommended by the FAO for the syndromic and participatory surveillance of the Rift Valley fever (RVF) which is one of the VHFs causing disease in animals [ 35 , 47 ]. 2.5.3 Analysis of data on the health information exchange network From the flow diagrams we identified the actors who collect health information and the communication channels they use. We compiled all the communication channels drawn by the different groups of participants in a generic flow diagram. We performed a qualitative analysis of the FGDs to identify the actor's constraints, reluctance and preferences to use these communication channels. The FGDs were also an opportunity for the participants to explore better alternatives for a more efficient communication of health information. 2.1 Ethics statement Official authorization from the National Department of Veterinary Services and at the prefectural level allowed us to work on these areas. The study was validated by the National Health Research Ethics Committee of Guinea (046/CNERS/18, 023/CNERS/19). Respondents participated freely and anonymously to the research study. The project was presented and translated before each interview so that participants could knowingly sign a consent form. 2.2. Study area The study was conducted in the prefecture of Guéckédou in the forest region of N'Zérékoré in Guinea. Based on a previous study on the socio-cultural and economic practices increasing the risk of zoonotic transmission from wildlife and the community's perception of One health surveillance and in consultation with the National Direction of Veterinary Services (Direction Nationale des Services Vétérinaires) and key resource persons, we selected the two sub-prefectures of Guendembou and Temessadou, for their close human-domestic animal-wildlife interface and for the presence of human, animal and ecosystem health actors [ 25 ]. We conducted FGDs in their respective chief towns and in the village of Mongo ( Fig 1 ). 10.1371/journal.pntd.0010462.g001 Fig 1 Map of the Republic of Guinea and location of the study areas. The study areas were located in the sub-prefectures of Guendembou and Temessadou (orange area) in the prefecture of Guéckédou (orange hatched area) in the N'Zérékoré region (green area). Focus Group Discussions were conducted in the villages of Guendembou, Temessadou and Mongo (red dots). Map created using the Free and Open Source QGIS software ( https://www.qgis.org ) and OpenStreetMap ( https://www.openstreetmap.org ) and GADM ( https://www.gadm.org ) geographic databases. 2.3 Participants We conducted FGDs with human, animal and ecosystem health actors to estimate local knowledge and perception of diseases and clinical signs and to investigate the existing health information exchange network and the potential collaborations for community-based surveillance. From the human health sector, we identified women and Community Human Health Workers (CHHWs). We decided to have non-mixed groups of women to ensure their representativeness because they are under-represented among other categories of actors. Women are involved in the care of family members and, as such, can play a role in disease detection. CHHWs are volunteers located in village and elected by the community. They are literate and specially trained in birth control, malaria treatment, awareness of diseases and surveillance. They routinely report human health information to the health center. From the animal health sector, we identified breeders and Community Animal Health Workers (CAHWs). CAHWs are volunteers located at the district level and elected by the community. They are literate and specially trained in conflict management between plant growers and animal breeders, veterinary treatment, awareness of animal diseases and surveillance. From the ecosystem health sector, we identified hunters, Community Informants (CIs) and rangers. In this study area, traditional hunting is practiced by hunters from the Kissi ethnolinguistic group. CIs are volunteers from the community located at the district level. They are literate and elected by the sub-prefectural Water and Forest Department to report on bushfires and non-compliance with hunting regulation and conservation activities. The rangers are located at the sub-prefectural level and work for the Water and Forest Department which is the most decentralized service of the Ministry of Environment. They control and manage all activities related to the environment and conservation such as fishing, hunting, deforestation, wildlife monitoring. 2.4 Data collection 2.4.1 Participatory epidemiological approach The participatory epidemiology is a bottom-up approach based on active participation of individuals in defining their own solutions tailored to their development stakes. This method is used to collect and analyze qualitative epidemiological information from local knowledge and expertise and is consistent to increase involvement of communities in the design and implementation of animal health program such as surveillance systems [ 13 , 26 ]. In this present study we investigated the perception of the disease and clinical signs to assess the case definition adaptability [ 27 ]. Participatory epidemiology is also interesting to explore the health information exchange network opportunities as it promotes local initiative and communication between the various actors [ 28 ]. Data were collected by an investigation team including a veterinarian from the National Direction of Veterinary Services, the Livestock Prefectural Director of Macenta in N'Zérékoré forest region and two French veterinary students. The investigation team was trained to conduct semi-structured interviews (SSIs), to use participatory tools and to record data in an appropriate format for analysis. For each FGD, we distributed roles among the investigation team to have one facilitator, one analyst and two note-takers. We conducted the FGDs in French or in the local language and literate persons from the sub-prefectures translated from Kissi into French. 2.4.2 Data triangulation and sampling method We applied data triangulation as main sampling criteria to ensure data reliability and saturation [ 29 – 31 ]. We expected between eight and ten participants per FGD to ensure data quality without compromising participation and facilitation. In each sub-prefecture, the head of the health center, the official veterinarian and the head of the Water and Forest Department selected the participants from the human, animal and ecosystem health sectors respectively. Participation to the FGDs was without gender restriction, except for the non-mixed groups of women, and we voluntarily included heads of breeders and heads of hunters to investigate their role in the existing animal and ecosystem health information exchange network. We conducted two sessions of FGDs in each sub-prefecture and expected as many of the participants as possible to be the same from one session to the other to foster their understanding of the research activities. 2.4.3 Data on local knowledge and perception of diseases and clinical signs Between February and March 2019, we conducted a first session of FGDs with each type of participants in both sub-prefectures to estimate local knowledge and perception of disease and clinical signs in the human, domestic animal and wildlife populations. We used several participatory tools as described by Catley et al. [ 32 ] such as SSIs, ranking and scoring. First, we asked participants to list the clinical signs, and their related diseases, they observe in the human, domestic animal or wildlife populations, and we noted them on post-it notes. Secondly, to reduce the large number of clinical signs and ensure the feasibility of the following steps, we asked them to select those they perceive as life-threatening or as a zoonotic risk. Then, we asked them to rank these selected clinical signs from the most to the least frequently observed. Finally, we asked participants to score these same and previously selected clinical signs by distributing one hundred beans proportionally to the associated threat regarding their own health; even for the signs they observe in the animal populations. 2.4.4 Data on the health information exchange network The first session of FGDs allowed us to collect qualitative data on community's response to health threats and human, animal and ecosystem health actors' roles. In April 2019, we conducted a second session of FGDs in each sub-prefecture to gather women and CHHWs, breeders and CAHWs and hunters and CIs. We used flow diagrams to explore the existing health information exchange network, by drawing the interactions between actors on a poster, and to discuss its related strengths and weaknesses and better alternatives for an efficient community-based surveillance of emerging zoonotic diseases [ 33 ]. 2.4.1 Participatory epidemiological approach The participatory epidemiology is a bottom-up approach based on active participation of individuals in defining their own solutions tailored to their development stakes. This method is used to collect and analyze qualitative epidemiological information from local knowledge and expertise and is consistent to increase involvement of communities in the design and implementation of animal health program such as surveillance systems [ 13 , 26 ]. In this present study we investigated the perception of the disease and clinical signs to assess the case definition adaptability [ 27 ]. Participatory epidemiology is also interesting to explore the health information exchange network opportunities as it promotes local initiative and communication between the various actors [ 28 ]. Data were collected by an investigation team including a veterinarian from the National Direction of Veterinary Services, the Livestock Prefectural Director of Macenta in N'Zérékoré forest region and two French veterinary students. The investigation team was trained to conduct semi-structured interviews (SSIs), to use participatory tools and to record data in an appropriate format for analysis. For each FGD, we distributed roles among the investigation team to have one facilitator, one analyst and two note-takers. We conducted the FGDs in French or in the local language and literate persons from the sub-prefectures translated from Kissi into French. 2.4.2 Data triangulation and sampling method We applied data triangulation as main sampling criteria to ensure data reliability and saturation [ 29 – 31 ]. We expected between eight and ten participants per FGD to ensure data quality without compromising participation and facilitation. In each sub-prefecture, the head of the health center, the official veterinarian and the head of the Water and Forest Department selected the participants from the human, animal and ecosystem health sectors respectively. Participation to the FGDs was without gender restriction, except for the non-mixed groups of women, and we voluntarily included heads of breeders and heads of hunters to investigate their role in the existing animal and ecosystem health information exchange network. We conducted two sessions of FGDs in each sub-prefecture and expected as many of the participants as possible to be the same from one session to the other to foster their understanding of the research activities. 2.4.3 Data on local knowledge and perception of diseases and clinical signs Between February and March 2019, we conducted a first session of FGDs with each type of participants in both sub-prefectures to estimate local knowledge and perception of disease and clinical signs in the human, domestic animal and wildlife populations. We used several participatory tools as described by Catley et al. [ 32 ] such as SSIs, ranking and scoring. First, we asked participants to list the clinical signs, and their related diseases, they observe in the human, domestic animal or wildlife populations, and we noted them on post-it notes. Secondly, to reduce the large number of clinical signs and ensure the feasibility of the following steps, we asked them to select those they perceive as life-threatening or as a zoonotic risk. Then, we asked them to rank these selected clinical signs from the most to the least frequently observed. Finally, we asked participants to score these same and previously selected clinical signs by distributing one hundred beans proportionally to the associated threat regarding their own health; even for the signs they observe in the animal populations. 2.4.4 Data on the health information exchange network The first session of FGDs allowed us to collect qualitative data on community's response to health threats and human, animal and ecosystem health actors' roles. In April 2019, we conducted a second session of FGDs in each sub-prefecture to gather women and CHHWs, breeders and CAHWs and hunters and CIs. We used flow diagrams to explore the existing health information exchange network, by drawing the interactions between actors on a poster, and to discuss its related strengths and weaknesses and better alternatives for an efficient community-based surveillance of emerging zoonotic diseases [ 33 ]. 2.5 Data analysis All the FGDs were recorded and transcribed on Microsoft Word. We performed a thematic content analysis to extract the qualitative data from the transcripts and sort them in different broad themes on Microsoft Excel. Using a deductive approach, we identified the pre-conceived themes: stakeholders' roles, local knowledge on diseases and detection capacity, communication of health information, and One Health collaboration [ 34 ]. Data were separately analyzed for each One Health sector. 2.5.1 Qualitative analysis of data on local knowledge of diseases Data on diseases and clinical signs were reported into a Microsoft Excel table to generate diseases-clinical signs matrix. We extracted from the matrix the commonly listed diseases, the diseases under surveillance, the VHFs and the zoonotic diseases. To estimate participants' knowledge, we compared the clinical picture they used to describe the diseases under surveillance with the clinical pictures cited in the OIE and WHO descriptions and with the case definitions provided to the CAHWs and CHHWs [ 35 – 46 ]. 2.5.2 Analysis of rankings and scorings Semi-quantitative data obtained from ranking and scoring were reported into a database and processed for analysis using graphical representations on Microsoft Excel. For each group of participants, we generated scatter plots of the signs that were ranked and scored to position them according to participants' perception in terms of frequency of observation and concern regarding their own health. Then we plotted axes through the medians of the ranks and scores to generate four-way matrix: rare/frequent and health-threatening/not threatening. This method allowed to identify the clinical signs perceived as the rarest and the most health-threatening and to estimate participants' perception of the clinical signs included in the human case definitions recommended by the WHO for Ebola or Marburg virus disease surveillance and in the animal case definition recommended by the FAO for the syndromic and participatory surveillance of the Rift Valley fever (RVF) which is one of the VHFs causing disease in animals [ 35 , 47 ]. 2.5.3 Analysis of data on the health information exchange network From the flow diagrams we identified the actors who collect health information and the communication channels they use. We compiled all the communication channels drawn by the different groups of participants in a generic flow diagram. We performed a qualitative analysis of the FGDs to identify the actor's constraints, reluctance and preferences to use these communication channels. The FGDs were also an opportunity for the participants to explore better alternatives for a more efficient communication of health information. 2.5.1 Qualitative analysis of data on local knowledge of diseases Data on diseases and clinical signs were reported into a Microsoft Excel table to generate diseases-clinical signs matrix. We extracted from the matrix the commonly listed diseases, the diseases under surveillance, the VHFs and the zoonotic diseases. To estimate participants' knowledge, we compared the clinical picture they used to describe the diseases under surveillance with the clinical pictures cited in the OIE and WHO descriptions and with the case definitions provided to the CAHWs and CHHWs [ 35 – 46 ]. 2.5.2 Analysis of rankings and scorings Semi-quantitative data obtained from ranking and scoring were reported into a database and processed for analysis using graphical representations on Microsoft Excel. For each group of participants, we generated scatter plots of the signs that were ranked and scored to position them according to participants' perception in terms of frequency of observation and concern regarding their own health. Then we plotted axes through the medians of the ranks and scores to generate four-way matrix: rare/frequent and health-threatening/not threatening. This method allowed to identify the clinical signs perceived as the rarest and the most health-threatening and to estimate participants' perception of the clinical signs included in the human case definitions recommended by the WHO for Ebola or Marburg virus disease surveillance and in the animal case definition recommended by the FAO for the syndromic and participatory surveillance of the Rift Valley fever (RVF) which is one of the VHFs causing disease in animals [ 35 , 47 ]. 2.5.3 Analysis of data on the health information exchange network From the flow diagrams we identified the actors who collect health information and the communication channels they use. We compiled all the communication channels drawn by the different groups of participants in a generic flow diagram. We performed a qualitative analysis of the FGDs to identify the actor's constraints, reluctance and preferences to use these communication channels. The FGDs were also an opportunity for the participants to explore better alternatives for a more efficient communication of health information. 3. Results 3.1 Participation in the FGDs For the first session of FGDs, we conducted twelve collective SSIs, six in the sub-prefecture of Guendembou and six others in the sub-prefecture of Temessadou. We recorded one-hundred-twelve participations. We identified the CIs after a first fieldwork in Guendembou and we included them in subsequent FGDs. For the second session, we conducted six FGDs, three in Guendembou and three in Temessadou. We recorded a fifty-two participation rate. The rangers were not included in this second session of FGDs because we preferred to have actors as close to the community as possible to complete information ( Table 1 ). The average number of participants per FGDs was of 9.3 for the first session and of 8.7 for the second one. 10.1371/journal.pntd.0010462.t001 Table 1 Number and type of participants per Focus Group Discussions. Focus Group Discussions First session Second session Participants Area 1 Area 2 Total Area 1 Area 2 Total Human health Women 14 9 23 6 5 11 CHHWs 9 11 20 5 5 10 Animal health Breeders 10 7 17 3 6 9 CAHWs 8 12 20 1 5 6 Ecosystem health Hunters 11 12 23 8 5 13 CIs Unidentified 2 2 0 3 3 Rangers 4 3 7 Not included - Total 56 56 112 23 29 52 Area 1, sub-prefecture of Guendembou; Area 2, sub-prefecture of Temessadou; CHHWs, Community Human Health Workers; CAHWs, Community Animal Health Workers; CIs, Community Informants. 3.2 Capacity of the communities to detect human diseases 3.2.1 Local knowledge about human diseases The disease-clinical signs matrix showed knowledge heterogeneity among groups of participants on diseases and their associated clinical pictures (Matrix A in S1 Matrix ). Both groups of CHHWs cited seven of the eight diseases under surveillance for which they received a technical sheet with case definitions. Among these diseases under surveillance, they commonly mentioned the VHFs such EVD and yellow fever and CHHWs from Temessadou mentioned the Lassa fever as well. Women from Guendembou mentioned four of the eight diseases under surveillance for which they were sensitized through awareness campaign. Among these four diseases they mentioned two VHFs such as EVD and another one they named "nasal hemorrhagic disease". In comparison, women from Temessadou did not mentioned any disease under surveillance and VHFs. None of the groups mentioned the five other diseases under surveillance for which CHHWs did not receive technical sheet with case definitions ( Table 2 ). 10.1371/journal.pntd.0010462.t002 Table 2 Clinical pictures associated with human diseases known by women and Community Human Health Workers. Group of participants CHHWs Women Area 1 Area 2 Area 1 Area 2 Diseases under surveillance VHFs EVD 0 Hematemesis ✓ 0 - Lassa fever - Hematemesis ✓ ✓ - - NHF - - Bleeding ✓ - Headache ✓ Yellow fever Red eyes ✓ Yellow eyes ✓ ✓ - - Yellow urine ✓ Yellow urine ✓ Anorexia ✓ Bloating Breathing difficulties Weight loss Not VHFs Measles Pimples ✓ ✓ Pimples ✓ ✓ Pimples ✓ ✓ - Fever ✓ ✓ Yellow eyes Meningitis Neck stiffness ✓ ✓ Neck stiffness ✓ ✓ - - Cholera Diarrhea ✓ ✓ Diarrhea ✓ ✓ Diarrhea ✓ ✓ - Deep-set eyes Vomiting Vomiting Poliomyelitis Paralysis ✓ ✓ Paralysis ✓ ✓ - - Neonatal tetanus Convulsions ✓ - - - Diseases not under surveillance Common Malaria Coma Coma Convulsions Convulsions Convulsions Fever Fever Fever Fever Vomiting Vomiting Vomiting Headache Headache Headache Vertigo Vertigo Loss of consciousness Nausea Diarrhea Pallor Tuberculosis Cough Hematemesis Cough - Weight loss Breathing difficulties Fatigue Anemia Palmar pallor Pallor Pallor - White eyes Dehydration Child malnutrition Bloating Growth delay - - Edema Weight loss Tension - - 0 Paralysis Asthma - - Breathing difficulties Breathing difficulties Cough Fatigue Opimo - - Headache Headache Area 1, sub-prefecture of Guendembou; Area 2, sub-prefecture of Temessadou; CHHWs, Community Human Health Workers; VHFs, viral hemorrhagic fevers; EVD, Ebola virus disease; NHF, nasal hemorrhagic fever; Opimo, intracranial vascular hypertension; green tick symbol, clinical signs in case definitions provided to the Community Human Health Workers; orange tick symbol, clinical signs in WHO descriptions of diseases; yellow underlined text is the clinical signs commonly cited by at least two groups of participants. The VHFs are poorly described. CHHWs from Temessadou associated hematemesis with Lassa fever and EVD but did not associate fever, other signs of hemorrhage or mortality which are including in the VHFs case definition. CHHWs and women from Guendembou did not associate any signs with EVD. Women from Guendembou associated bleeding and headache to the "nasal hemorrhagic disease". Among the signs included in the specific yellow fever case definition, only CHHWs from Temessadou associated the yellow eye sign. None of CHHWs groups associated fever and yellow skin but they associated other signs included in the WHO definition such yellow urine. The other diseases under surveillance are better and more homogeneously described, except by women from Temessadou. The groups that mentioned these diseases associated at least one of the signs including in case definition, except for neonatal tetanus. CHHWs from both sub-prefectures did not associate fever to measles and bulging fontanelle in children and fever to meningitis. Seven diseases which are not under surveillance were listed by at least two groups of participants. The groups associated at least one common clinical sign. Malaria is the only disease mentioned and similarly described by all groups of participants. Except the group of women from Guendembou, all the groups described the disease with five to seven signs. This disease is subject to a specific health program and is frequently observed in the community, as mentioned by a CHHW from Temessadou: "Malaria is very common: vomiting, fever, headache". Women from Guendembou also identified diseases they suffer from or they observe in the community: "All of those [women] sitting here are all sick. Tension, filariasis, malaria are spread in this area.". 3.2.2 Ranking and scoring of clinical signs in human health CHHWs from Guendembou listed twenty clinical signs or symptoms, selected seventeen of them as health-threatening, and ranked and scored these seventeen signs. CHHWs from Temessadou listed thirty-three signs, selected twenty-seven of them as health-threatening, they only ranked the eighteen most frequently observed signs but did not rank the nine signs identified as the rarest. They added four signs during the scoring (appendicitis, epilepsy, hernia, hydrocele) and scored thirty-one signs in total. Women from Guendembou listed twenty-two signs, selected eleven of them as health-threatening and ranked these eleven signs. They added one sign (joint pain) during the scoring and scored twelve signs in total. Women from Mongo, sub-prefecture of Temessadou, listed thirty-six signs and selected twenty-five of them as health-threatening. They did not rank these clinical signs but divided them into two categories: nineteen frequently observed signs and six rarely observed signs. They scored these twenty-five signs. Most of the VHFs clinical signs are perceived as frequent and health-threatening but the specific hemorrhagic signs are heterogeneously ranked and scored by the groups of participants. Among all the hemorrhagic signs mentioned, and which are of interest for the detection of VHFs, the ones which are perceived as rare are: bleeding by women from the two areas, red eyes by CHHWs from Guendembou, hematemesis and hematuria by CHHWs from Temessadou; and the ones which are perceived as threatening are: bleeding by women from Guendembou, bloody stools and hematemesis by women and CHHWs from Temessadou. A CHHW and a woman from Temessadou particularly said: "If you vomit blood, you will die.". Bleeding, hematemesis and hematuria are the only hemorrhagic signs perceived as both rare and threatening. Other non-pathognomonic signs which are of interest for the detection of VHFs, particularly during outbreak, were ranked and scored. Most of them are perceived as frequent, except, anorexia by CHHWs from Guendembou and vomiting and diarrhea by women from Guendembou. The ones which are perceived as health-threatening are: headache, vomiting, fever, breathing difficulties by the groups that scored them. Diarrhea is perceived as health-threatening by all the groups, except women from Guendembou. Abdominal pain is perceived as health-threatening by CHHWs and women from Temessadou and as not threatening by women from Guendembou. Muscle pain is not perceived as threatening. Anorexia is perceived as threatening by CHHWs from Guendembou and as not threatening by CHHWs from Temessadou. Fatigue is perceived as threatening for women from Guendembou and not for the CHHWs from Temessadou ( Fig 2 ). 10.1371/journal.pntd.0010462.g002 Fig 2 Perception of the signs ranked and scored according to rarity and concern by Community Human Health Workers and women from the community. On the x-axis: rank associated with the frequency of observation of the sign, from the least to the most frequent. On the y-axis: score associated with the threat for human health, from the least to the most health threatening. Axes are cut at their median. Red dots represent hemorrhagic signs: 1. Bleeding 2. Hematemesis 3. Bloody stools 4. Hematuria 5. Red eyes and dark yellow urine. Orange dots represent signs included in the WHO case definitions for viral hemorrhagic fever surveillance: 6. Anorexia 7. Diarrhea 8. Vomiting 9. Headache 10. Fever 11. Breathing difficulties 12. Fatigue 13. Abdominal pain 14. Muscle pain. The blue dots represent all other signs that are ranked and scored as not fitting these categories: 15. Paralysis 16. Bloating 17. Edema 18. Pimples 19. Loss of consciousness / coma 20. Neck stiffness 21. Palmar pallor 22. Cough 23. Convulsions 24. Weight loss 25. Deep-set eyes 26. Yellow eyes 27. Yellow urine 28. Pimples located on one side of the body 29. Mutilation 30. Weight gain 31. Polyuria 32. Malnutrition 33. Pyuria 34. Pallor 35. Rectal prolapse 36. Wound 37. Epilepsy 38. Loss of speech 39. Vertigo 40. Gbassama 41. Taeniasis 42. Abortion 43. Constipation 44. Opimo 45. Breast pain 46. Swelling 47. Chest pain 48. Toothache 49. Absence of menstrual period. Women from Temessadou did not assign a rank to the signs mentioned but classified them into two categories: rare and frequent. 3.3 Capacity of the communities to detect animal diseases 3.3.1 Local knowledge about animal diseases The disease-clinical signs matrix showed knowledge heterogeneity among groups of participants on diseases and their associated clinical pictures (Matrix B in S1 Matrix ). Most of the diseases mentioned by CAHWs and breeders are observed in livestock. Some of them worry them because of the zoonotic risk, as mentioned by a breeder from Guendembou: "Scabies is also a worrying disease. The consumption of contaminated meat is a serious risk to human health.". Other diseases worry them because of the economic loss they generate, as a breeder from Temessadou mentioned: "Animal diseases affect our moral because of their negative economic impact.". CAHWs from Guendembou mentioned four of the five diseases under surveillance for which they received technical sheets with case definitions. CAHWs from Temessadou mentioned three of them. Breeders from Guendembou and from Temessadou mentioned two of the diseases under surveillance for which they were sensitized. Among the zoonotic diseases under surveillance, all the groups mentioned anthrax whereas this disease is not prevalent in the study area as mentioned by a breeder from Guendembou: "We have never observed case of anthrax but we know this disease.". They did not mention the hepatic lesion used in the case definition but they associated other signs provided in the OIE definition such as bleeding and mortality by the CAHWs and the breeders respectively. CAHWs from both sub-prefectures commonly mentioned rabies. They associated the clinical picture provided in the case definition but also associated other signs which are not described in the OIE technical disease card. Rabies is prevalent in our study area as mentioned by a CAHW from Guendembou: "Rabies affects dogs and is very common in our country.". CAHWs from Temessadou and breeders from Guendembou mentioned avian influenza. They did not associate the nasal discharge sign cited in the case definition but breeders from Guendembou associated mortality. CAHWs from Guendembou and breeders from Temessadou mentioned other diseases under surveillance which are not zoonotic. They commonly mentioned the New Castle disease and CAHWs from Guendembou mentioned the PPR as well. They did not associate signs to the PPR. They described the New Castle disease with signs not included in case definition, except dirty hind legs. Some of these signs are part of the OIE clinical picture such falling wings, fatigue and mortality. CAHWs from Guendembou mentioned two additional zoonotic diseases which are not under surveillance such scabies and tuberculosis. The breeders from Guendembou also mentioned scabies but did not associate clinical signs. Three other diseases which are not under surveillance and not zoonotic were listed by at least two groups of participants. Hair loss and diarrhea were commonly used to define eczema and parasitosis respectively ( Table 3 ). 10.1371/journal.pntd.0010462.t003 Table 3 Clinical pictures associated with animal diseases known by Community Animal Health Workers and breeders. Group of participants CAHWs Breeders Area 1 Area 2 Area 1 Area 2 Diseases under surveillance Zoonotic Rabies Aggressiveness ✓ ✓ Aggressiveness ✓ ✓ - - Photophobia Salivation ✓ ✓ Animal turning on itself Animal turning on itself Hydrophobia Anthrax Bleeding ✓ Bleeding ✓ Mortality ✓ Mortality ✓ Bloating ✓ Bloody stools ✓ Black meat Black blood Fall ✓ Tremors ✓ Salivation Black body part Avian influenza - 0 Mortality ✓ - Not zoonotic PPR 0 - - - New Castle disease Dirty hind legs ✓ - - Falling wings ✓ Fatigue ✓ Nasal discharge Mortality ✓ Salivation Salivation Tearing Diseases not under surveillance Zoonotic Scabies Hair loss - 0 - Presence of lice or ticks Wound Tuberculosis Cough - - - Common Foot rot Hoof wound - 0 Excessive hoof growth Lameness Eczema - Hair loss - Hair loss Crusts Hard skin Itching Parasitosis - Diarrhea Diarrhea - Bloating Area 1, sub-prefecture of Guendembou; Area 2, sub-prefecture of Temessadou; CAHWs, Community Animal Health Workers; green tick symbol, clinical signs in case definitions provided to the Community Animal Health Workers; orange tick symbol, clinical signs in OIE descriptions of diseases; yellow underlined text is the clinical signs commonly cited by at least two groups of participants. 3.3.2 Ranking and scoring of clinical signs in animal health CAHWs from Guendembou listed twenty-five signs, selected ten of them as threatening for their own lives, ranked and scored these ten signs. CAHWs from Temessadou listed twenty-nine signs, selected sixteen of them as threatening for their own lives, ranked and scored these sixteen signs. Breeders from Guendembou listed seventeen signs, selected fifteen of them as threatening for their own lives, ranked and scored these fifteen signs. Breeders from Temessadou, listed twenty-eight signs and selected fourteen of them as threatening for their own lives, ranked and scored these fourteen signs. The Fig 3 showed a heterogeneous perception of the clinical signs among groups. Abortion and mortality, which are included in case definition for the surveillance of RVF are perceived differently among groups of participants. Abortion is perceived as frequent by CAHWs and breeders from Temessadou, as rare by breeders from Guendembou and as health-threatening by these three groups. We learned that brucellosis prevailed in this region and cause abortion in livestock. Mortality is perceived as rare by all groups except by CAHWs from Temessadou and as threatening for CAHWs and breeders from Temessadou and not threatening by CAHWs and breeders from Guendembou. Other non-pathognomonic signs of RVF were mentioned, ranked and scored. They are all perceived as rare except nasal discharge by CAHWs and breeders from Guendembou. Hemorrhagic signs of interest such as bleeding and bloody stools are perceived as threatening and oral bleeding as not threatening. Fatigue is perceived as threatening by breeders from Guendembou and as not threatening by CAHWs from Temessadou. Anorexia is perceived as not threatening by breeders from Guendembou and Temessadou. 10.1371/journal.pntd.0010462.g003 Fig 3 Perception of the signs ranked and scored according to rarity and concern by Community Animal Health Workers and breeders. On the x-axis: rank associated with the frequency of observation of the sign, from least to most frequent. On the y-axis: score associated with the concern for human health, from the least to the most threatening. Axes are cut at their median. Red dots represent signs included in case definitions for Rift Valley Fever surveillance: 1. Mortality 2. Abortion. Orange dots represent other non-pathognomonic signs of Rift Valley Fever: 3. Bleeding and black blood 4. Nasal discharge 5. Oral bleeding 6. Bloody stools 7. Fatigue 8. Anorexia 9. Bleeding. The blue dots represent all other signs that are ranked and scored as not fitting these categories: 10. Aggressiveness 11. Animal turning on itself, photophobia and hydrophobia 12. Falling wings and tearing in poultry 13. Hair loss 14. Salivation 15. Cough 16. Diarrhea 17. Black body part 18. Vulvar wound 19. Presence of worms in the flesh 20. Tremors and fall 21. Presence of flies 22. Bloating 23. Presence of ticks 24. Presence of venom in eyes 25. Malformation 26. Weight loss 27. Scabies 28. Cold 29. Fatigue in poultry populations 30. Presence of abdominal fat 31. Black meat 32. Hair loss, hard skin, itching and pimples 33. Cough and white lungs. 3.4 Capacity of the communities to detect wildlife diseases 3.4.1 Local knowledge about wildlife diseases Forest rangers, hunters and CIs from both sub-prefectures did not mention any disease which affects wildlife populations but since the EVD outbreak they understood the zoonotic risk, as a ranger of Temessadou mentioned: "This is when Ebola emerged from the monkeys, from the bats, that we understood the zoonotic risk. Before we didn't know. They constitute vital animal proteins but now we are very afraid to consume them.". They defined a sick animal as a living animal that seems tired and presents an unusual behavior or a dead animal for no apparent reason. Hunters and CIs from Temessadou identified clinical signs or lesions on carcasses that they perceived as an indicator of disease occurrence but they did not associate these signs with a disease but rather with animal species (palm rat, mouse, bat, doe, agouti, etc.). 3.4.2 Ranking and scoring of clinical signs in wildlife Only hunters from Temessadou listed twenty-one signs and selected all of them as threatening for their own lives. They ranked the eight most frequently observed signs but did not rank the thirteen signs identified as the rarest. They scored these twenty-one signs. Among signs of RVF they mentioned nasal discharge, fatigue and yellow organs which are perceived as rare and only nasal discharge is perceived as threatening ( Fig 4 ). They did not list mortality but they said that it is very rare to see several dead animals in the same area ("We have never seen an epidemic that kills many wild animals."). The most threatening signs are the ones perceived as frequent. Some of these signs are visible at the autopsy such as presence of worms in liver, presence of water in the body, black blood, presence of red worms in stomach. 10.1371/journal.pntd.0010462.g004 Fig 4 Perception of the signs ranked and scored according to rarity and concern by hunters from Temessadou. On the x-axis: rank associated with the frequency of observation of the sign, from least to most frequent. On the y-axis: score associated with the concern for human health conferred by these signs, from the least to the most threatening. Axes are cut at their median. Orange dots represent non-pathognomonic signs of Rift Valley Fever: 1. Nasal discharge 2. Fatigue 3. Yellow organs. Blue dots represent all signs that are ranked and scored: 4. Presence of water in the body 5. Black blood 6. Tearing 7. Immobility 8. Adhesion of organs 9. Absence of blood 10. Atrophied organs 11. Facial hypertrophy 12. Malformation 13. Hypotrophy of gall bladder 14. Presence of worms in liver 15. Presence of external maggots 16. Presence of red worms in stomach 17. Cough 18. Red wounds 19. Weight loss 20. Hair loss 21. Diarrhea. 3.5 Health information exchange network The Fig 5 shows that there are several opportunities to communicate health information from the village level to the sub-prefectural relevant actors in each health sector. 10.1371/journal.pntd.0010462.g005 Fig 5 The community health information exchange network including human, animal and ecosystem health actors. Blue boxes: human health actors; green boxes: ecosystem health actors; red boxes: animal health actors; blue arrows: human health communication channels identified by actors from Guendembou (light blue) or Temessadou (dark blue); green arrows: ecosystem health communication channels identified by actors from Guendembou (light green) or Temessadou (dark green); orange arrows: animal health communication channels identified by actors from Guendembou; purple arrows: health communication channels commonly identified by actors from Guendembou and Temessadou; dashed arrows: health communication channels identified but not triangulated by actors; circle: actors from both sub-prefectures (purple) or ecosystem health actors from Guendembou (light green) or from Temessadou (dark green) who communicate health information to local authorities. 3.5.1 Human health communication channels In both sub-prefectures, when someone shows signs of disease, community members can inform the CHHW or directly inform or consult the health center. Women specifically consult matrons in case of women's health issues or for birth. In Temessadou, community members also inform their community or consult the hospital, through ignorance of CHHW's role, or traditional healers, mainly for financial reasons. According to CHHWs, the traditional healers and matrons may withhold information as mentioned by CHHWs: "The communication between matrons and CHHWs is only for birth issue. Otherwise, matrons generally do not transmit information to the CHHWs.", "Healers only communicate with CHHWs when they do not manage to cure patients". CHHWs from Guendembou and Temessadou refer patients to the health post or health center and inform the health center if there is a suspected case. In general, the community members trust the CHHW that they elected, but there is still some reluctance to inform him, as mentioned by a CHHW from Temessadou: "When a CHHW comes to provide free care to a person with malaria, people in village still mistrust the CHHW and say that CHHWs administer drugs that kills them.". 3.5.2 Animal health communication channels In both sub-prefectures, breeders generally inform the official veterinarian or the head of breeders. Breeders can inform the CAHW but some of them are still reluctant towards CAHWs as mentioned: "There is reluctance. Even if a breeder sees his animal in a dilapidated state, he does not care. For him we ca not treat the animal.". Some breeders do not want the veterinarian to be informed because of financial reasons as mentioned by a CAHW: "Sometimes the breeders do not have money and they are afraid to not be able to pay the veterinarian.". In Temessadou breeders can also inform the animal's owner or the head of breeders at the village level. CAHWs from Gendembou and Temessadou always inform the official veterinarian. 3.5.3 Ecosystem health communication channels At the community level, there is no surveillance system to report suspected case in the wildlife population. Nevertheless, the ecosystem health actors want to participate in the surveillance and there are existing communication channels to share information on species conservation, hunting management and forestry activities. In both sub-prefectures, hunters can directly inform the Water and Forest Department but they prefer communicate with their representative in the village because of the economic loss due to the carcass confiscation by rangers. For the same reason, hunters from Temessadou are reluctant to communicate with the CIs who inform the Water and Forest Department. Hunters and CIs from Temessadou suggested to inform the official veterinarian if there is a suspected case in the wildlife population whereas hunters from Guendembou suggested to inform the health center. 3.5.4 Strengths and weaknesses of the health information exchange network The presence of community workers in the three health sectors at the village or district levels is a real asset for a One Health surveillance system. However, they are not systematically informed because of reluctance or ignorance of their role whereas CHHWs and CAHWs are elected by their community. In addition, some community workers do not have telephone or the means to pay telephone credit or to travel the great distance (up to eighteen km) that separates them from the relevant sub-prefectural institutions. These constraints can delay the communication of the health information. CHHWs and CAHWs reported the difficulty to sensitize the community with case definitions and supports for awareness campaigns which are not translated into the Kissi local language. CHHWs required more in-depth training and on more diseases to better detect them. The official veterinarian of Temessadou is overburdened because there are no other veterinarians in this large sub-prefecture and the Fig 5 shows that he could be more solicited by ecosystem health actors for surveillance tasks. CAHWs suggested to train five to six voluntary CAHWs and to officially authorize them to perform specific acts to relieve the official veterinarian. Hunters, CIs and rangers want to participate to a community-based surveillance system but with training on these diseases and on the biosecurity precautions and with protective equipment if they have to handle carcasses. In both sub-prefectures, community workers, community members and breeders compulsory inform local authorities when there is a case of disease. This is an interesting parallel communication channel as the local authorities share the information with the relevant institutions. In return, they are notified if the case is confirmed and provide feedback to their community. 3.1 Participation in the FGDs For the first session of FGDs, we conducted twelve collective SSIs, six in the sub-prefecture of Guendembou and six others in the sub-prefecture of Temessadou. We recorded one-hundred-twelve participations. We identified the CIs after a first fieldwork in Guendembou and we included them in subsequent FGDs. For the second session, we conducted six FGDs, three in Guendembou and three in Temessadou. We recorded a fifty-two participation rate. The rangers were not included in this second session of FGDs because we preferred to have actors as close to the community as possible to complete information ( Table 1 ). The average number of participants per FGDs was of 9.3 for the first session and of 8.7 for the second one. 10.1371/journal.pntd.0010462.t001 Table 1 Number and type of participants per Focus Group Discussions. Focus Group Discussions First session Second session Participants Area 1 Area 2 Total Area 1 Area 2 Total Human health Women 14 9 23 6 5 11 CHHWs 9 11 20 5 5 10 Animal health Breeders 10 7 17 3 6 9 CAHWs 8 12 20 1 5 6 Ecosystem health Hunters 11 12 23 8 5 13 CIs Unidentified 2 2 0 3 3 Rangers 4 3 7 Not included - Total 56 56 112 23 29 52 Area 1, sub-prefecture of Guendembou; Area 2, sub-prefecture of Temessadou; CHHWs, Community Human Health Workers; CAHWs, Community Animal Health Workers; CIs, Community Informants. 3.2 Capacity of the communities to detect human diseases 3.2.1 Local knowledge about human diseases The disease-clinical signs matrix showed knowledge heterogeneity among groups of participants on diseases and their associated clinical pictures (Matrix A in S1 Matrix ). Both groups of CHHWs cited seven of the eight diseases under surveillance for which they received a technical sheet with case definitions. Among these diseases under surveillance, they commonly mentioned the VHFs such EVD and yellow fever and CHHWs from Temessadou mentioned the Lassa fever as well. Women from Guendembou mentioned four of the eight diseases under surveillance for which they were sensitized through awareness campaign. Among these four diseases they mentioned two VHFs such as EVD and another one they named "nasal hemorrhagic disease". In comparison, women from Temessadou did not mentioned any disease under surveillance and VHFs. None of the groups mentioned the five other diseases under surveillance for which CHHWs did not receive technical sheet with case definitions ( Table 2 ). 10.1371/journal.pntd.0010462.t002 Table 2 Clinical pictures associated with human diseases known by women and Community Human Health Workers. Group of participants CHHWs Women Area 1 Area 2 Area 1 Area 2 Diseases under surveillance VHFs EVD 0 Hematemesis ✓ 0 - Lassa fever - Hematemesis ✓ ✓ - - NHF - - Bleeding ✓ - Headache ✓ Yellow fever Red eyes ✓ Yellow eyes ✓ ✓ - - Yellow urine ✓ Yellow urine ✓ Anorexia ✓ Bloating Breathing difficulties Weight loss Not VHFs Measles Pimples ✓ ✓ Pimples ✓ ✓ Pimples ✓ ✓ - Fever ✓ ✓ Yellow eyes Meningitis Neck stiffness ✓ ✓ Neck stiffness ✓ ✓ - - Cholera Diarrhea ✓ ✓ Diarrhea ✓ ✓ Diarrhea ✓ ✓ - Deep-set eyes Vomiting Vomiting Poliomyelitis Paralysis ✓ ✓ Paralysis ✓ ✓ - - Neonatal tetanus Convulsions ✓ - - - Diseases not under surveillance Common Malaria Coma Coma Convulsions Convulsions Convulsions Fever Fever Fever Fever Vomiting Vomiting Vomiting Headache Headache Headache Vertigo Vertigo Loss of consciousness Nausea Diarrhea Pallor Tuberculosis Cough Hematemesis Cough - Weight loss Breathing difficulties Fatigue Anemia Palmar pallor Pallor Pallor - White eyes Dehydration Child malnutrition Bloating Growth delay - - Edema Weight loss Tension - - 0 Paralysis Asthma - - Breathing difficulties Breathing difficulties Cough Fatigue Opimo - - Headache Headache Area 1, sub-prefecture of Guendembou; Area 2, sub-prefecture of Temessadou; CHHWs, Community Human Health Workers; VHFs, viral hemorrhagic fevers; EVD, Ebola virus disease; NHF, nasal hemorrhagic fever; Opimo, intracranial vascular hypertension; green tick symbol, clinical signs in case definitions provided to the Community Human Health Workers; orange tick symbol, clinical signs in WHO descriptions of diseases; yellow underlined text is the clinical signs commonly cited by at least two groups of participants. The VHFs are poorly described. CHHWs from Temessadou associated hematemesis with Lassa fever and EVD but did not associate fever, other signs of hemorrhage or mortality which are including in the VHFs case definition. CHHWs and women from Guendembou did not associate any signs with EVD. Women from Guendembou associated bleeding and headache to the "nasal hemorrhagic disease". Among the signs included in the specific yellow fever case definition, only CHHWs from Temessadou associated the yellow eye sign. None of CHHWs groups associated fever and yellow skin but they associated other signs included in the WHO definition such yellow urine. The other diseases under surveillance are better and more homogeneously described, except by women from Temessadou. The groups that mentioned these diseases associated at least one of the signs including in case definition, except for neonatal tetanus. CHHWs from both sub-prefectures did not associate fever to measles and bulging fontanelle in children and fever to meningitis. Seven diseases which are not under surveillance were listed by at least two groups of participants. The groups associated at least one common clinical sign. Malaria is the only disease mentioned and similarly described by all groups of participants. Except the group of women from Guendembou, all the groups described the disease with five to seven signs. This disease is subject to a specific health program and is frequently observed in the community, as mentioned by a CHHW from Temessadou: "Malaria is very common: vomiting, fever, headache". Women from Guendembou also identified diseases they suffer from or they observe in the community: "All of those [women] sitting here are all sick. Tension, filariasis, malaria are spread in this area.". 3.2.2 Ranking and scoring of clinical signs in human health CHHWs from Guendembou listed twenty clinical signs or symptoms, selected seventeen of them as health-threatening, and ranked and scored these seventeen signs. CHHWs from Temessadou listed thirty-three signs, selected twenty-seven of them as health-threatening, they only ranked the eighteen most frequently observed signs but did not rank the nine signs identified as the rarest. They added four signs during the scoring (appendicitis, epilepsy, hernia, hydrocele) and scored thirty-one signs in total. Women from Guendembou listed twenty-two signs, selected eleven of them as health-threatening and ranked these eleven signs. They added one sign (joint pain) during the scoring and scored twelve signs in total. Women from Mongo, sub-prefecture of Temessadou, listed thirty-six signs and selected twenty-five of them as health-threatening. They did not rank these clinical signs but divided them into two categories: nineteen frequently observed signs and six rarely observed signs. They scored these twenty-five signs. Most of the VHFs clinical signs are perceived as frequent and health-threatening but the specific hemorrhagic signs are heterogeneously ranked and scored by the groups of participants. Among all the hemorrhagic signs mentioned, and which are of interest for the detection of VHFs, the ones which are perceived as rare are: bleeding by women from the two areas, red eyes by CHHWs from Guendembou, hematemesis and hematuria by CHHWs from Temessadou; and the ones which are perceived as threatening are: bleeding by women from Guendembou, bloody stools and hematemesis by women and CHHWs from Temessadou. A CHHW and a woman from Temessadou particularly said: "If you vomit blood, you will die.". Bleeding, hematemesis and hematuria are the only hemorrhagic signs perceived as both rare and threatening. Other non-pathognomonic signs which are of interest for the detection of VHFs, particularly during outbreak, were ranked and scored. Most of them are perceived as frequent, except, anorexia by CHHWs from Guendembou and vomiting and diarrhea by women from Guendembou. The ones which are perceived as health-threatening are: headache, vomiting, fever, breathing difficulties by the groups that scored them. Diarrhea is perceived as health-threatening by all the groups, except women from Guendembou. Abdominal pain is perceived as health-threatening by CHHWs and women from Temessadou and as not threatening by women from Guendembou. Muscle pain is not perceived as threatening. Anorexia is perceived as threatening by CHHWs from Guendembou and as not threatening by CHHWs from Temessadou. Fatigue is perceived as threatening for women from Guendembou and not for the CHHWs from Temessadou ( Fig 2 ). 10.1371/journal.pntd.0010462.g002 Fig 2 Perception of the signs ranked and scored according to rarity and concern by Community Human Health Workers and women from the community. On the x-axis: rank associated with the frequency of observation of the sign, from the least to the most frequent. On the y-axis: score associated with the threat for human health, from the least to the most health threatening. Axes are cut at their median. Red dots represent hemorrhagic signs: 1. Bleeding 2. Hematemesis 3. Bloody stools 4. Hematuria 5. Red eyes and dark yellow urine. Orange dots represent signs included in the WHO case definitions for viral hemorrhagic fever surveillance: 6. Anorexia 7. Diarrhea 8. Vomiting 9. Headache 10. Fever 11. Breathing difficulties 12. Fatigue 13. Abdominal pain 14. Muscle pain. The blue dots represent all other signs that are ranked and scored as not fitting these categories: 15. Paralysis 16. Bloating 17. Edema 18. Pimples 19. Loss of consciousness / coma 20. Neck stiffness 21. Palmar pallor 22. Cough 23. Convulsions 24. Weight loss 25. Deep-set eyes 26. Yellow eyes 27. Yellow urine 28. Pimples located on one side of the body 29. Mutilation 30. Weight gain 31. Polyuria 32. Malnutrition 33. Pyuria 34. Pallor 35. Rectal prolapse 36. Wound 37. Epilepsy 38. Loss of speech 39. Vertigo 40. Gbassama 41. Taeniasis 42. Abortion 43. Constipation 44. Opimo 45. Breast pain 46. Swelling 47. Chest pain 48. Toothache 49. Absence of menstrual period. Women from Temessadou did not assign a rank to the signs mentioned but classified them into two categories: rare and frequent. 3.2.1 Local knowledge about human diseases The disease-clinical signs matrix showed knowledge heterogeneity among groups of participants on diseases and their associated clinical pictures (Matrix A in S1 Matrix ). Both groups of CHHWs cited seven of the eight diseases under surveillance for which they received a technical sheet with case definitions. Among these diseases under surveillance, they commonly mentioned the VHFs such EVD and yellow fever and CHHWs from Temessadou mentioned the Lassa fever as well. Women from Guendembou mentioned four of the eight diseases under surveillance for which they were sensitized through awareness campaign. Among these four diseases they mentioned two VHFs such as EVD and another one they named "nasal hemorrhagic disease". In comparison, women from Temessadou did not mentioned any disease under surveillance and VHFs. None of the groups mentioned the five other diseases under surveillance for which CHHWs did not receive technical sheet with case definitions ( Table 2 ). 10.1371/journal.pntd.0010462.t002 Table 2 Clinical pictures associated with human diseases known by women and Community Human Health Workers. Group of participants CHHWs Women Area 1 Area 2 Area 1 Area 2 Diseases under surveillance VHFs EVD 0 Hematemesis ✓ 0 - Lassa fever - Hematemesis ✓ ✓ - - NHF - - Bleeding ✓ - Headache ✓ Yellow fever Red eyes ✓ Yellow eyes ✓ ✓ - - Yellow urine ✓ Yellow urine ✓ Anorexia ✓ Bloating Breathing difficulties Weight loss Not VHFs Measles Pimples ✓ ✓ Pimples ✓ ✓ Pimples ✓ ✓ - Fever ✓ ✓ Yellow eyes Meningitis Neck stiffness ✓ ✓ Neck stiffness ✓ ✓ - - Cholera Diarrhea ✓ ✓ Diarrhea ✓ ✓ Diarrhea ✓ ✓ - Deep-set eyes Vomiting Vomiting Poliomyelitis Paralysis ✓ ✓ Paralysis ✓ ✓ - - Neonatal tetanus Convulsions ✓ - - - Diseases not under surveillance Common Malaria Coma Coma Convulsions Convulsions Convulsions Fever Fever Fever Fever Vomiting Vomiting Vomiting Headache Headache Headache Vertigo Vertigo Loss of consciousness Nausea Diarrhea Pallor Tuberculosis Cough Hematemesis Cough - Weight loss Breathing difficulties Fatigue Anemia Palmar pallor Pallor Pallor - White eyes Dehydration Child malnutrition Bloating Growth delay - - Edema Weight loss Tension - - 0 Paralysis Asthma - - Breathing difficulties Breathing difficulties Cough Fatigue Opimo - - Headache Headache Area 1, sub-prefecture of Guendembou; Area 2, sub-prefecture of Temessadou; CHHWs, Community Human Health Workers; VHFs, viral hemorrhagic fevers; EVD, Ebola virus disease; NHF, nasal hemorrhagic fever; Opimo, intracranial vascular hypertension; green tick symbol, clinical signs in case definitions provided to the Community Human Health Workers; orange tick symbol, clinical signs in WHO descriptions of diseases; yellow underlined text is the clinical signs commonly cited by at least two groups of participants. The VHFs are poorly described. CHHWs from Temessadou associated hematemesis with Lassa fever and EVD but did not associate fever, other signs of hemorrhage or mortality which are including in the VHFs case definition. CHHWs and women from Guendembou did not associate any signs with EVD. Women from Guendembou associated bleeding and headache to the "nasal hemorrhagic disease". Among the signs included in the specific yellow fever case definition, only CHHWs from Temessadou associated the yellow eye sign. None of CHHWs groups associated fever and yellow skin but they associated other signs included in the WHO definition such yellow urine. The other diseases under surveillance are better and more homogeneously described, except by women from Temessadou. The groups that mentioned these diseases associated at least one of the signs including in case definition, except for neonatal tetanus. CHHWs from both sub-prefectures did not associate fever to measles and bulging fontanelle in children and fever to meningitis. Seven diseases which are not under surveillance were listed by at least two groups of participants. The groups associated at least one common clinical sign. Malaria is the only disease mentioned and similarly described by all groups of participants. Except the group of women from Guendembou, all the groups described the disease with five to seven signs. This disease is subject to a specific health program and is frequently observed in the community, as mentioned by a CHHW from Temessadou: "Malaria is very common: vomiting, fever, headache". Women from Guendembou also identified diseases they suffer from or they observe in the community: "All of those [women] sitting here are all sick. Tension, filariasis, malaria are spread in this area.". 3.2.2 Ranking and scoring of clinical signs in human health CHHWs from Guendembou listed twenty clinical signs or symptoms, selected seventeen of them as health-threatening, and ranked and scored these seventeen signs. CHHWs from Temessadou listed thirty-three signs, selected twenty-seven of them as health-threatening, they only ranked the eighteen most frequently observed signs but did not rank the nine signs identified as the rarest. They added four signs during the scoring (appendicitis, epilepsy, hernia, hydrocele) and scored thirty-one signs in total. Women from Guendembou listed twenty-two signs, selected eleven of them as health-threatening and ranked these eleven signs. They added one sign (joint pain) during the scoring and scored twelve signs in total. Women from Mongo, sub-prefecture of Temessadou, listed thirty-six signs and selected twenty-five of them as health-threatening. They did not rank these clinical signs but divided them into two categories: nineteen frequently observed signs and six rarely observed signs. They scored these twenty-five signs. Most of the VHFs clinical signs are perceived as frequent and health-threatening but the specific hemorrhagic signs are heterogeneously ranked and scored by the groups of participants. Among all the hemorrhagic signs mentioned, and which are of interest for the detection of VHFs, the ones which are perceived as rare are: bleeding by women from the two areas, red eyes by CHHWs from Guendembou, hematemesis and hematuria by CHHWs from Temessadou; and the ones which are perceived as threatening are: bleeding by women from Guendembou, bloody stools and hematemesis by women and CHHWs from Temessadou. A CHHW and a woman from Temessadou particularly said: "If you vomit blood, you will die.". Bleeding, hematemesis and hematuria are the only hemorrhagic signs perceived as both rare and threatening. Other non-pathognomonic signs which are of interest for the detection of VHFs, particularly during outbreak, were ranked and scored. Most of them are perceived as frequent, except, anorexia by CHHWs from Guendembou and vomiting and diarrhea by women from Guendembou. The ones which are perceived as health-threatening are: headache, vomiting, fever, breathing difficulties by the groups that scored them. Diarrhea is perceived as health-threatening by all the groups, except women from Guendembou. Abdominal pain is perceived as health-threatening by CHHWs and women from Temessadou and as not threatening by women from Guendembou. Muscle pain is not perceived as threatening. Anorexia is perceived as threatening by CHHWs from Guendembou and as not threatening by CHHWs from Temessadou. Fatigue is perceived as threatening for women from Guendembou and not for the CHHWs from Temessadou ( Fig 2 ). 10.1371/journal.pntd.0010462.g002 Fig 2 Perception of the signs ranked and scored according to rarity and concern by Community Human Health Workers and women from the community. On the x-axis: rank associated with the frequency of observation of the sign, from the least to the most frequent. On the y-axis: score associated with the threat for human health, from the least to the most health threatening. Axes are cut at their median. Red dots represent hemorrhagic signs: 1. Bleeding 2. Hematemesis 3. Bloody stools 4. Hematuria 5. Red eyes and dark yellow urine. Orange dots represent signs included in the WHO case definitions for viral hemorrhagic fever surveillance: 6. Anorexia 7. Diarrhea 8. Vomiting 9. Headache 10. Fever 11. Breathing difficulties 12. Fatigue 13. Abdominal pain 14. Muscle pain. The blue dots represent all other signs that are ranked and scored as not fitting these categories: 15. Paralysis 16. Bloating 17. Edema 18. Pimples 19. Loss of consciousness / coma 20. Neck stiffness 21. Palmar pallor 22. Cough 23. Convulsions 24. Weight loss 25. Deep-set eyes 26. Yellow eyes 27. Yellow urine 28. Pimples located on one side of the body 29. Mutilation 30. Weight gain 31. Polyuria 32. Malnutrition 33. Pyuria 34. Pallor 35. Rectal prolapse 36. Wound 37. Epilepsy 38. Loss of speech 39. Vertigo 40. Gbassama 41. Taeniasis 42. Abortion 43. Constipation 44. Opimo 45. Breast pain 46. Swelling 47. Chest pain 48. Toothache 49. Absence of menstrual period. Women from Temessadou did not assign a rank to the signs mentioned but classified them into two categories: rare and frequent. 3.3 Capacity of the communities to detect animal diseases 3.3.1 Local knowledge about animal diseases The disease-clinical signs matrix showed knowledge heterogeneity among groups of participants on diseases and their associated clinical pictures (Matrix B in S1 Matrix ). Most of the diseases mentioned by CAHWs and breeders are observed in livestock. Some of them worry them because of the zoonotic risk, as mentioned by a breeder from Guendembou: "Scabies is also a worrying disease. The consumption of contaminated meat is a serious risk to human health.". Other diseases worry them because of the economic loss they generate, as a breeder from Temessadou mentioned: "Animal diseases affect our moral because of their negative economic impact.". CAHWs from Guendembou mentioned four of the five diseases under surveillance for which they received technical sheets with case definitions. CAHWs from Temessadou mentioned three of them. Breeders from Guendembou and from Temessadou mentioned two of the diseases under surveillance for which they were sensitized. Among the zoonotic diseases under surveillance, all the groups mentioned anthrax whereas this disease is not prevalent in the study area as mentioned by a breeder from Guendembou: "We have never observed case of anthrax but we know this disease.". They did not mention the hepatic lesion used in the case definition but they associated other signs provided in the OIE definition such as bleeding and mortality by the CAHWs and the breeders respectively. CAHWs from both sub-prefectures commonly mentioned rabies. They associated the clinical picture provided in the case definition but also associated other signs which are not described in the OIE technical disease card. Rabies is prevalent in our study area as mentioned by a CAHW from Guendembou: "Rabies affects dogs and is very common in our country.". CAHWs from Temessadou and breeders from Guendembou mentioned avian influenza. They did not associate the nasal discharge sign cited in the case definition but breeders from Guendembou associated mortality. CAHWs from Guendembou and breeders from Temessadou mentioned other diseases under surveillance which are not zoonotic. They commonly mentioned the New Castle disease and CAHWs from Guendembou mentioned the PPR as well. They did not associate signs to the PPR. They described the New Castle disease with signs not included in case definition, except dirty hind legs. Some of these signs are part of the OIE clinical picture such falling wings, fatigue and mortality. CAHWs from Guendembou mentioned two additional zoonotic diseases which are not under surveillance such scabies and tuberculosis. The breeders from Guendembou also mentioned scabies but did not associate clinical signs. Three other diseases which are not under surveillance and not zoonotic were listed by at least two groups of participants. Hair loss and diarrhea were commonly used to define eczema and parasitosis respectively ( Table 3 ). 10.1371/journal.pntd.0010462.t003 Table 3 Clinical pictures associated with animal diseases known by Community Animal Health Workers and breeders. Group of participants CAHWs Breeders Area 1 Area 2 Area 1 Area 2 Diseases under surveillance Zoonotic Rabies Aggressiveness ✓ ✓ Aggressiveness ✓ ✓ - - Photophobia Salivation ✓ ✓ Animal turning on itself Animal turning on itself Hydrophobia Anthrax Bleeding ✓ Bleeding ✓ Mortality ✓ Mortality ✓ Bloating ✓ Bloody stools ✓ Black meat Black blood Fall ✓ Tremors ✓ Salivation Black body part Avian influenza - 0 Mortality ✓ - Not zoonotic PPR 0 - - - New Castle disease Dirty hind legs ✓ - - Falling wings ✓ Fatigue ✓ Nasal discharge Mortality ✓ Salivation Salivation Tearing Diseases not under surveillance Zoonotic Scabies Hair loss - 0 - Presence of lice or ticks Wound Tuberculosis Cough - - - Common Foot rot Hoof wound - 0 Excessive hoof growth Lameness Eczema - Hair loss - Hair loss Crusts Hard skin Itching Parasitosis - Diarrhea Diarrhea - Bloating Area 1, sub-prefecture of Guendembou; Area 2, sub-prefecture of Temessadou; CAHWs, Community Animal Health Workers; green tick symbol, clinical signs in case definitions provided to the Community Animal Health Workers; orange tick symbol, clinical signs in OIE descriptions of diseases; yellow underlined text is the clinical signs commonly cited by at least two groups of participants. 3.3.2 Ranking and scoring of clinical signs in animal health CAHWs from Guendembou listed twenty-five signs, selected ten of them as threatening for their own lives, ranked and scored these ten signs. CAHWs from Temessadou listed twenty-nine signs, selected sixteen of them as threatening for their own lives, ranked and scored these sixteen signs. Breeders from Guendembou listed seventeen signs, selected fifteen of them as threatening for their own lives, ranked and scored these fifteen signs. Breeders from Temessadou, listed twenty-eight signs and selected fourteen of them as threatening for their own lives, ranked and scored these fourteen signs. The Fig 3 showed a heterogeneous perception of the clinical signs among groups. Abortion and mortality, which are included in case definition for the surveillance of RVF are perceived differently among groups of participants. Abortion is perceived as frequent by CAHWs and breeders from Temessadou, as rare by breeders from Guendembou and as health-threatening by these three groups. We learned that brucellosis prevailed in this region and cause abortion in livestock. Mortality is perceived as rare by all groups except by CAHWs from Temessadou and as threatening for CAHWs and breeders from Temessadou and not threatening by CAHWs and breeders from Guendembou. Other non-pathognomonic signs of RVF were mentioned, ranked and scored. They are all perceived as rare except nasal discharge by CAHWs and breeders from Guendembou. Hemorrhagic signs of interest such as bleeding and bloody stools are perceived as threatening and oral bleeding as not threatening. Fatigue is perceived as threatening by breeders from Guendembou and as not threatening by CAHWs from Temessadou. Anorexia is perceived as not threatening by breeders from Guendembou and Temessadou. 10.1371/journal.pntd.0010462.g003 Fig 3 Perception of the signs ranked and scored according to rarity and concern by Community Animal Health Workers and breeders. On the x-axis: rank associated with the frequency of observation of the sign, from least to most frequent. On the y-axis: score associated with the concern for human health, from the least to the most threatening. Axes are cut at their median. Red dots represent signs included in case definitions for Rift Valley Fever surveillance: 1. Mortality 2. Abortion. Orange dots represent other non-pathognomonic signs of Rift Valley Fever: 3. Bleeding and black blood 4. Nasal discharge 5. Oral bleeding 6. Bloody stools 7. Fatigue 8. Anorexia 9. Bleeding. The blue dots represent all other signs that are ranked and scored as not fitting these categories: 10. Aggressiveness 11. Animal turning on itself, photophobia and hydrophobia 12. Falling wings and tearing in poultry 13. Hair loss 14. Salivation 15. Cough 16. Diarrhea 17. Black body part 18. Vulvar wound 19. Presence of worms in the flesh 20. Tremors and fall 21. Presence of flies 22. Bloating 23. Presence of ticks 24. Presence of venom in eyes 25. Malformation 26. Weight loss 27. Scabies 28. Cold 29. Fatigue in poultry populations 30. Presence of abdominal fat 31. Black meat 32. Hair loss, hard skin, itching and pimples 33. Cough and white lungs. 3.3.1 Local knowledge about animal diseases The disease-clinical signs matrix showed knowledge heterogeneity among groups of participants on diseases and their associated clinical pictures (Matrix B in S1 Matrix ). Most of the diseases mentioned by CAHWs and breeders are observed in livestock. Some of them worry them because of the zoonotic risk, as mentioned by a breeder from Guendembou: "Scabies is also a worrying disease. The consumption of contaminated meat is a serious risk to human health.". Other diseases worry them because of the economic loss they generate, as a breeder from Temessadou mentioned: "Animal diseases affect our moral because of their negative economic impact.". CAHWs from Guendembou mentioned four of the five diseases under surveillance for which they received technical sheets with case definitions. CAHWs from Temessadou mentioned three of them. Breeders from Guendembou and from Temessadou mentioned two of the diseases under surveillance for which they were sensitized. Among the zoonotic diseases under surveillance, all the groups mentioned anthrax whereas this disease is not prevalent in the study area as mentioned by a breeder from Guendembou: "We have never observed case of anthrax but we know this disease.". They did not mention the hepatic lesion used in the case definition but they associated other signs provided in the OIE definition such as bleeding and mortality by the CAHWs and the breeders respectively. CAHWs from both sub-prefectures commonly mentioned rabies. They associated the clinical picture provided in the case definition but also associated other signs which are not described in the OIE technical disease card. Rabies is prevalent in our study area as mentioned by a CAHW from Guendembou: "Rabies affects dogs and is very common in our country.". CAHWs from Temessadou and breeders from Guendembou mentioned avian influenza. They did not associate the nasal discharge sign cited in the case definition but breeders from Guendembou associated mortality. CAHWs from Guendembou and breeders from Temessadou mentioned other diseases under surveillance which are not zoonotic. They commonly mentioned the New Castle disease and CAHWs from Guendembou mentioned the PPR as well. They did not associate signs to the PPR. They described the New Castle disease with signs not included in case definition, except dirty hind legs. Some of these signs are part of the OIE clinical picture such falling wings, fatigue and mortality. CAHWs from Guendembou mentioned two additional zoonotic diseases which are not under surveillance such scabies and tuberculosis. The breeders from Guendembou also mentioned scabies but did not associate clinical signs. Three other diseases which are not under surveillance and not zoonotic were listed by at least two groups of participants. Hair loss and diarrhea were commonly used to define eczema and parasitosis respectively ( Table 3 ). 10.1371/journal.pntd.0010462.t003 Table 3 Clinical pictures associated with animal diseases known by Community Animal Health Workers and breeders. Group of participants CAHWs Breeders Area 1 Area 2 Area 1 Area 2 Diseases under surveillance Zoonotic Rabies Aggressiveness ✓ ✓ Aggressiveness ✓ ✓ - - Photophobia Salivation ✓ ✓ Animal turning on itself Animal turning on itself Hydrophobia Anthrax Bleeding ✓ Bleeding ✓ Mortality ✓ Mortality ✓ Bloating ✓ Bloody stools ✓ Black meat Black blood Fall ✓ Tremors ✓ Salivation Black body part Avian influenza - 0 Mortality ✓ - Not zoonotic PPR 0 - - - New Castle disease Dirty hind legs ✓ - - Falling wings ✓ Fatigue ✓ Nasal discharge Mortality ✓ Salivation Salivation Tearing Diseases not under surveillance Zoonotic Scabies Hair loss - 0 - Presence of lice or ticks Wound Tuberculosis Cough - - - Common Foot rot Hoof wound - 0 Excessive hoof growth Lameness Eczema - Hair loss - Hair loss Crusts Hard skin Itching Parasitosis - Diarrhea Diarrhea - Bloating Area 1, sub-prefecture of Guendembou; Area 2, sub-prefecture of Temessadou; CAHWs, Community Animal Health Workers; green tick symbol, clinical signs in case definitions provided to the Community Animal Health Workers; orange tick symbol, clinical signs in OIE descriptions of diseases; yellow underlined text is the clinical signs commonly cited by at least two groups of participants. 3.3.2 Ranking and scoring of clinical signs in animal health CAHWs from Guendembou listed twenty-five signs, selected ten of them as threatening for their own lives, ranked and scored these ten signs. CAHWs from Temessadou listed twenty-nine signs, selected sixteen of them as threatening for their own lives, ranked and scored these sixteen signs. Breeders from Guendembou listed seventeen signs, selected fifteen of them as threatening for their own lives, ranked and scored these fifteen signs. Breeders from Temessadou, listed twenty-eight signs and selected fourteen of them as threatening for their own lives, ranked and scored these fourteen signs. The Fig 3 showed a heterogeneous perception of the clinical signs among groups. Abortion and mortality, which are included in case definition for the surveillance of RVF are perceived differently among groups of participants. Abortion is perceived as frequent by CAHWs and breeders from Temessadou, as rare by breeders from Guendembou and as health-threatening by these three groups. We learned that brucellosis prevailed in this region and cause abortion in livestock. Mortality is perceived as rare by all groups except by CAHWs from Temessadou and as threatening for CAHWs and breeders from Temessadou and not threatening by CAHWs and breeders from Guendembou. Other non-pathognomonic signs of RVF were mentioned, ranked and scored. They are all perceived as rare except nasal discharge by CAHWs and breeders from Guendembou. Hemorrhagic signs of interest such as bleeding and bloody stools are perceived as threatening and oral bleeding as not threatening. Fatigue is perceived as threatening by breeders from Guendembou and as not threatening by CAHWs from Temessadou. Anorexia is perceived as not threatening by breeders from Guendembou and Temessadou. 10.1371/journal.pntd.0010462.g003 Fig 3 Perception of the signs ranked and scored according to rarity and concern by Community Animal Health Workers and breeders. On the x-axis: rank associated with the frequency of observation of the sign, from least to most frequent. On the y-axis: score associated with the concern for human health, from the least to the most threatening. Axes are cut at their median. Red dots represent signs included in case definitions for Rift Valley Fever surveillance: 1. Mortality 2. Abortion. Orange dots represent other non-pathognomonic signs of Rift Valley Fever: 3. Bleeding and black blood 4. Nasal discharge 5. Oral bleeding 6. Bloody stools 7. Fatigue 8. Anorexia 9. Bleeding. The blue dots represent all other signs that are ranked and scored as not fitting these categories: 10. Aggressiveness 11. Animal turning on itself, photophobia and hydrophobia 12. Falling wings and tearing in poultry 13. Hair loss 14. Salivation 15. Cough 16. Diarrhea 17. Black body part 18. Vulvar wound 19. Presence of worms in the flesh 20. Tremors and fall 21. Presence of flies 22. Bloating 23. Presence of ticks 24. Presence of venom in eyes 25. Malformation 26. Weight loss 27. Scabies 28. Cold 29. Fatigue in poultry populations 30. Presence of abdominal fat 31. Black meat 32. Hair loss, hard skin, itching and pimples 33. Cough and white lungs. 3.4 Capacity of the communities to detect wildlife diseases 3.4.1 Local knowledge about wildlife diseases Forest rangers, hunters and CIs from both sub-prefectures did not mention any disease which affects wildlife populations but since the EVD outbreak they understood the zoonotic risk, as a ranger of Temessadou mentioned: "This is when Ebola emerged from the monkeys, from the bats, that we understood the zoonotic risk. Before we didn't know. They constitute vital animal proteins but now we are very afraid to consume them.". They defined a sick animal as a living animal that seems tired and presents an unusual behavior or a dead animal for no apparent reason. Hunters and CIs from Temessadou identified clinical signs or lesions on carcasses that they perceived as an indicator of disease occurrence but they did not associate these signs with a disease but rather with animal species (palm rat, mouse, bat, doe, agouti, etc.). 3.4.2 Ranking and scoring of clinical signs in wildlife Only hunters from Temessadou listed twenty-one signs and selected all of them as threatening for their own lives. They ranked the eight most frequently observed signs but did not rank the thirteen signs identified as the rarest. They scored these twenty-one signs. Among signs of RVF they mentioned nasal discharge, fatigue and yellow organs which are perceived as rare and only nasal discharge is perceived as threatening ( Fig 4 ). They did not list mortality but they said that it is very rare to see several dead animals in the same area ("We have never seen an epidemic that kills many wild animals."). The most threatening signs are the ones perceived as frequent. Some of these signs are visible at the autopsy such as presence of worms in liver, presence of water in the body, black blood, presence of red worms in stomach. 10.1371/journal.pntd.0010462.g004 Fig 4 Perception of the signs ranked and scored according to rarity and concern by hunters from Temessadou. On the x-axis: rank associated with the frequency of observation of the sign, from least to most frequent. On the y-axis: score associated with the concern for human health conferred by these signs, from the least to the most threatening. Axes are cut at their median. Orange dots represent non-pathognomonic signs of Rift Valley Fever: 1. Nasal discharge 2. Fatigue 3. Yellow organs. Blue dots represent all signs that are ranked and scored: 4. Presence of water in the body 5. Black blood 6. Tearing 7. Immobility 8. Adhesion of organs 9. Absence of blood 10. Atrophied organs 11. Facial hypertrophy 12. Malformation 13. Hypotrophy of gall bladder 14. Presence of worms in liver 15. Presence of external maggots 16. Presence of red worms in stomach 17. Cough 18. Red wounds 19. Weight loss 20. Hair loss 21. Diarrhea. 3.4.1 Local knowledge about wildlife diseases Forest rangers, hunters and CIs from both sub-prefectures did not mention any disease which affects wildlife populations but since the EVD outbreak they understood the zoonotic risk, as a ranger of Temessadou mentioned: "This is when Ebola emerged from the monkeys, from the bats, that we understood the zoonotic risk. Before we didn't know. They constitute vital animal proteins but now we are very afraid to consume them.". They defined a sick animal as a living animal that seems tired and presents an unusual behavior or a dead animal for no apparent reason. Hunters and CIs from Temessadou identified clinical signs or lesions on carcasses that they perceived as an indicator of disease occurrence but they did not associate these signs with a disease but rather with animal species (palm rat, mouse, bat, doe, agouti, etc.). 3.4.2 Ranking and scoring of clinical signs in wildlife Only hunters from Temessadou listed twenty-one signs and selected all of them as threatening for their own lives. They ranked the eight most frequently observed signs but did not rank the thirteen signs identified as the rarest. They scored these twenty-one signs. Among signs of RVF they mentioned nasal discharge, fatigue and yellow organs which are perceived as rare and only nasal discharge is perceived as threatening ( Fig 4 ). They did not list mortality but they said that it is very rare to see several dead animals in the same area ("We have never seen an epidemic that kills many wild animals."). The most threatening signs are the ones perceived as frequent. Some of these signs are visible at the autopsy such as presence of worms in liver, presence of water in the body, black blood, presence of red worms in stomach. 10.1371/journal.pntd.0010462.g004 Fig 4 Perception of the signs ranked and scored according to rarity and concern by hunters from Temessadou. On the x-axis: rank associated with the frequency of observation of the sign, from least to most frequent. On the y-axis: score associated with the concern for human health conferred by these signs, from the least to the most threatening. Axes are cut at their median. Orange dots represent non-pathognomonic signs of Rift Valley Fever: 1. Nasal discharge 2. Fatigue 3. Yellow organs. Blue dots represent all signs that are ranked and scored: 4. Presence of water in the body 5. Black blood 6. Tearing 7. Immobility 8. Adhesion of organs 9. Absence of blood 10. Atrophied organs 11. Facial hypertrophy 12. Malformation 13. Hypotrophy of gall bladder 14. Presence of worms in liver 15. Presence of external maggots 16. Presence of red worms in stomach 17. Cough 18. Red wounds 19. Weight loss 20. Hair loss 21. Diarrhea. 3.5 Health information exchange network The Fig 5 shows that there are several opportunities to communicate health information from the village level to the sub-prefectural relevant actors in each health sector. 10.1371/journal.pntd.0010462.g005 Fig 5 The community health information exchange network including human, animal and ecosystem health actors. Blue boxes: human health actors; green boxes: ecosystem health actors; red boxes: animal health actors; blue arrows: human health communication channels identified by actors from Guendembou (light blue) or Temessadou (dark blue); green arrows: ecosystem health communication channels identified by actors from Guendembou (light green) or Temessadou (dark green); orange arrows: animal health communication channels identified by actors from Guendembou; purple arrows: health communication channels commonly identified by actors from Guendembou and Temessadou; dashed arrows: health communication channels identified but not triangulated by actors; circle: actors from both sub-prefectures (purple) or ecosystem health actors from Guendembou (light green) or from Temessadou (dark green) who communicate health information to local authorities. 3.5.1 Human health communication channels In both sub-prefectures, when someone shows signs of disease, community members can inform the CHHW or directly inform or consult the health center. Women specifically consult matrons in case of women's health issues or for birth. In Temessadou, community members also inform their community or consult the hospital, through ignorance of CHHW's role, or traditional healers, mainly for financial reasons. According to CHHWs, the traditional healers and matrons may withhold information as mentioned by CHHWs: "The communication between matrons and CHHWs is only for birth issue. Otherwise, matrons generally do not transmit information to the CHHWs.", "Healers only communicate with CHHWs when they do not manage to cure patients". CHHWs from Guendembou and Temessadou refer patients to the health post or health center and inform the health center if there is a suspected case. In general, the community members trust the CHHW that they elected, but there is still some reluctance to inform him, as mentioned by a CHHW from Temessadou: "When a CHHW comes to provide free care to a person with malaria, people in village still mistrust the CHHW and say that CHHWs administer drugs that kills them.". 3.5.2 Animal health communication channels In both sub-prefectures, breeders generally inform the official veterinarian or the head of breeders. Breeders can inform the CAHW but some of them are still reluctant towards CAHWs as mentioned: "There is reluctance. Even if a breeder sees his animal in a dilapidated state, he does not care. For him we ca not treat the animal.". Some breeders do not want the veterinarian to be informed because of financial reasons as mentioned by a CAHW: "Sometimes the breeders do not have money and they are afraid to not be able to pay the veterinarian.". In Temessadou breeders can also inform the animal's owner or the head of breeders at the village level. CAHWs from Gendembou and Temessadou always inform the official veterinarian. 3.5.3 Ecosystem health communication channels At the community level, there is no surveillance system to report suspected case in the wildlife population. Nevertheless, the ecosystem health actors want to participate in the surveillance and there are existing communication channels to share information on species conservation, hunting management and forestry activities. In both sub-prefectures, hunters can directly inform the Water and Forest Department but they prefer communicate with their representative in the village because of the economic loss due to the carcass confiscation by rangers. For the same reason, hunters from Temessadou are reluctant to communicate with the CIs who inform the Water and Forest Department. Hunters and CIs from Temessadou suggested to inform the official veterinarian if there is a suspected case in the wildlife population whereas hunters from Guendembou suggested to inform the health center. 3.5.4 Strengths and weaknesses of the health information exchange network The presence of community workers in the three health sectors at the village or district levels is a real asset for a One Health surveillance system. However, they are not systematically informed because of reluctance or ignorance of their role whereas CHHWs and CAHWs are elected by their community. In addition, some community workers do not have telephone or the means to pay telephone credit or to travel the great distance (up to eighteen km) that separates them from the relevant sub-prefectural institutions. These constraints can delay the communication of the health information. CHHWs and CAHWs reported the difficulty to sensitize the community with case definitions and supports for awareness campaigns which are not translated into the Kissi local language. CHHWs required more in-depth training and on more diseases to better detect them. The official veterinarian of Temessadou is overburdened because there are no other veterinarians in this large sub-prefecture and the Fig 5 shows that he could be more solicited by ecosystem health actors for surveillance tasks. CAHWs suggested to train five to six voluntary CAHWs and to officially authorize them to perform specific acts to relieve the official veterinarian. Hunters, CIs and rangers want to participate to a community-based surveillance system but with training on these diseases and on the biosecurity precautions and with protective equipment if they have to handle carcasses. In both sub-prefectures, community workers, community members and breeders compulsory inform local authorities when there is a case of disease. This is an interesting parallel communication channel as the local authorities share the information with the relevant institutions. In return, they are notified if the case is confirmed and provide feedback to their community. 3.5.1 Human health communication channels In both sub-prefectures, when someone shows signs of disease, community members can inform the CHHW or directly inform or consult the health center. Women specifically consult matrons in case of women's health issues or for birth. In Temessadou, community members also inform their community or consult the hospital, through ignorance of CHHW's role, or traditional healers, mainly for financial reasons. According to CHHWs, the traditional healers and matrons may withhold information as mentioned by CHHWs: "The communication between matrons and CHHWs is only for birth issue. Otherwise, matrons generally do not transmit information to the CHHWs.", "Healers only communicate with CHHWs when they do not manage to cure patients". CHHWs from Guendembou and Temessadou refer patients to the health post or health center and inform the health center if there is a suspected case. In general, the community members trust the CHHW that they elected, but there is still some reluctance to inform him, as mentioned by a CHHW from Temessadou: "When a CHHW comes to provide free care to a person with malaria, people in village still mistrust the CHHW and say that CHHWs administer drugs that kills them.". 3.5.2 Animal health communication channels In both sub-prefectures, breeders generally inform the official veterinarian or the head of breeders. Breeders can inform the CAHW but some of them are still reluctant towards CAHWs as mentioned: "There is reluctance. Even if a breeder sees his animal in a dilapidated state, he does not care. For him we ca not treat the animal.". Some breeders do not want the veterinarian to be informed because of financial reasons as mentioned by a CAHW: "Sometimes the breeders do not have money and they are afraid to not be able to pay the veterinarian.". In Temessadou breeders can also inform the animal's owner or the head of breeders at the village level. CAHWs from Gendembou and Temessadou always inform the official veterinarian. 3.5.3 Ecosystem health communication channels At the community level, there is no surveillance system to report suspected case in the wildlife population. Nevertheless, the ecosystem health actors want to participate in the surveillance and there are existing communication channels to share information on species conservation, hunting management and forestry activities. In both sub-prefectures, hunters can directly inform the Water and Forest Department but they prefer communicate with their representative in the village because of the economic loss due to the carcass confiscation by rangers. For the same reason, hunters from Temessadou are reluctant to communicate with the CIs who inform the Water and Forest Department. Hunters and CIs from Temessadou suggested to inform the official veterinarian if there is a suspected case in the wildlife population whereas hunters from Guendembou suggested to inform the health center. 3.5.4 Strengths and weaknesses of the health information exchange network The presence of community workers in the three health sectors at the village or district levels is a real asset for a One Health surveillance system. However, they are not systematically informed because of reluctance or ignorance of their role whereas CHHWs and CAHWs are elected by their community. In addition, some community workers do not have telephone or the means to pay telephone credit or to travel the great distance (up to eighteen km) that separates them from the relevant sub-prefectural institutions. These constraints can delay the communication of the health information. CHHWs and CAHWs reported the difficulty to sensitize the community with case definitions and supports for awareness campaigns which are not translated into the Kissi local language. CHHWs required more in-depth training and on more diseases to better detect them. The official veterinarian of Temessadou is overburdened because there are no other veterinarians in this large sub-prefecture and the Fig 5 shows that he could be more solicited by ecosystem health actors for surveillance tasks. CAHWs suggested to train five to six voluntary CAHWs and to officially authorize them to perform specific acts to relieve the official veterinarian. Hunters, CIs and rangers want to participate to a community-based surveillance system but with training on these diseases and on the biosecurity precautions and with protective equipment if they have to handle carcasses. In both sub-prefectures, community workers, community members and breeders compulsory inform local authorities when there is a case of disease. This is an interesting parallel communication channel as the local authorities share the information with the relevant institutions. In return, they are notified if the case is confirmed and provide feedback to their community. 4. Discussion The resurgence of the EVD outbreak from January to June 2021 in Guinea showed improved detection and control capacity compared to the first epidemic in 2014–2016 [ 48 ]. This improvement is surely due to the numerous efforts in detection, diagnosis and outbreak response that have been made in the country. However, there are still gaps in the system and efforts still need to be deployed [ 49 ]. Our study provides useful insights for strengthening surveillance capacities at a community level, while opting for a tripartite vision of health. We propose recommendations for case definitions and health information exchange network as components of a future community-based surveillance system of emerging zoonotic diseases such as VHFs in Guinea [ 50 ]. We believe that an ex ante participatory epidemiology approach improves the performance of the future surveillance system by including contextual factors and local knowledge and empowers communities to define their own solutions to their issues and to take actions [ 32 ]. Their participation to this study may increase the acceptability, the feasibility and the sustainability of the future community-based surveillance system. The participatory and One Health approach allowed the integration of actors from the different health sectors (human health, animal health, ecosystem health) that need to be involved in the surveillance of zoonotic diseases such VHFs [ 51 ]. The inclusion of the ecosystem health sector in disease surveillance systems is relatively new and we identified potential actors and communication channels [ 52 ]. We think that the context of our study area is conducive to the design of a One Health community-based surveillance system because of the actors' motivation to collaborate, the presence of stakeholders from the three health sectors including community workers [ 53 ]. In other contexts, where these conditions are not met, it would be more difficult to implement such a participatory system. 4.1 Capacity of the communities to detect diseases Appropriate case definitions are crucial to ensure the efficiency of the surveillance system, especially for early detection of emerging diseases [ 54 ]. A high sensitivity of case definitions will lead to a larger proportion of true positive in the surveillance system, while a low specificity will burden the surveillance system with false positives and will require more investigation resources. If the objective of surveillance is the early detection of emerging zoonotic diseases as serious as the VHFs, a high sensitivity and positive predictive value of the case definitions is desirable [ 50 , 55 , 56 ]. In low-income countries such as Guinea, which have limited resources for diagnosis and case confirmation, it is also important to avoid false positives as much as possible to not burden the surveillance system. We investigated actors' knowledge on the diseases to estimate their detection capacity. We also explored actors' perception of clinical signs to estimate their reaction when they observe one of them and to identify potential signs to include in case definitions. We hypothesized that signs that might constitute an alert, and thus be relevant for inclusion, should be perceived as both rare and health-threatening. The rarity criterion is consistent with a syndromic and community-based surveillance system that aims to early detect emerging diseases [ 57 , 58 ]. We selected the human health threat criterion as an indicator of trigger event for health information communication. Our results showed that VHFs are poorly described and their associated clinical signs are differently perceived by all human health actors. The controversial aspect of VHFs may have influenced the discussion because it became a taboo issue after the EVD outbreak which caused a tense social and political climate in West Africa [ 11 , 59 ]. This health crisis weakened governments and altered relations between institutions and communities. Communities may be reluctant to report cases for fear of restrictions applied during the epidemic [ 60 ]. We did not clearly identify signs of VHFs that are perceived as both rare and health-threatening. Most of them are perceived as frequent and threatening and are not specific to VHFs. Some participants reported to be more threatened by the clinical signs they suffer from or observe in their community. The hemorrhagic signs that are more specific of VHFs are differently perceived among groups. Bleeding is perceived as rare but not threatening by women in both sub-prefectures and red eyes, hematemesis, hematuria and bloody stools are perceived as rare and/or threatening by CHHWs. We suggest to sensitize all the actors to the VHFs' threat and on the importance to detect them because most of the specific hemorrhagic signs are not perceived as an alert. It could be possible to increase the sensitivity of the case definition by including the unspecific VHFs clinical signs which are perceived as a threat. Except the VHFs, CHHWs showed a better and more homogeneous knowledge of diseases under surveillance than women. None of the interviewed groups perfectly mentioned the clinical pictures included in case definitions but we believe that the training and sensitization of community human health actors had an effect because none of the groups mentioned the diseases under surveillance for which the CHHWs did not have technical sheet with case definitions. Nevertheless, our findings showed that these efforts are insufficient. We suggest to improve training of the CHHWs on diseases under surveillance so that they can all detect them and potentially with more specific case definitions. Awareness efforts should be improved and particularly in Temessadou. We suggest to explore the success key factors of the health program addressing the malaria disease which is well known by communities. Our results showed that CAHWs have a better knowledge on diseases under surveillance than breeders. We believe that the sensitization and training improved knowledge because the actors better know zoonotic diseases under surveillance than the ones not under surveillance. Nevertheless, sensitization and training efforts should be increased because none of the interviewed groups perfectly mentioned the clinical pictures included in case definitions and some diseases are not mentioned. Our results showed that the clinical signs associated to RVF, that might be interesting to include in case definitions for the surveillance of VHFs in domestic animal populations, are differently perceived among groups. We suggest to create sensitive case definitions including clinical signs perceived as rare or as a threat for human health such as mortality and abortion which are proposed by the FAO for syndromic and participatory surveillance of RVF [ 47 ]. We suggest to increase sensitization of animal health actors on the zoonotic risk related to VHFs because they are concerned by the clinical signs they associate with a zoonotic disease ("Everything that is harmful for animal health is harmful for human health", "We are afraid of diseases transmitted from animals to humans."). Contrary to the human and animal health compartments, the ecosystem sector is not involved in health surveillance at the community level. Knowledge of ecosystem health actors on diseases is almost non-existent but they are able to associate clinical signs to species and to observe abnormalities such lesions on carcasses or one or more animals sick or dead with no apparent cause in the same area. There is no sign perceived both as a threat for human health and rare but all the hemorrhagic signs are perceived as rare. We suggest to include these rare events in a standardized and specific case definition and to train all the ecosystem health actors to zoonotic diseases. 4.2 Health information exchange network We investigated the surveillance network opportunities at the community level. The participants identified the existing communication channels and discussed the strengths and weaknesses of the current information exchange network, their preferences and better alternatives. The results highlighted the fact that community actors face different constraints and communicate with various stakeholders. This situation may delay the report of a suspected case to the relevant institutions and therefore the confirmatory diagnosis and outbreak control actions [ 51 , 61 ]. Our study showed interesting communication channels opportunities in the current health information exchange network to bypass potential barriers to health report. The CHHWs and the CAHWs are a strong link in the information chain because they are geographically close to the community and trained to the surveillance of disease. Therefore, community members and breeders from both sub-prefectures who inform the CHHWs and the CAHWs overcome the lack of transport means that could demotivate them to inform the health center and the official veterinarian but they also potentially reduce the burden on these sub-prefectural actors with not suspected case as the community workers better know the case definitions. Some community members and breeders do not inform the community workers, despite the fact they elected them, because they do not know their role or by reluctance or because they prefer to inform other actors such as matrons, traditional healers and heads of breeders. Promoting the role of community workers and involving other actors who collect health information in the surveillance system may be levers to activate. In the ecosystem health sector any actor is trained to the health surveillance but we identified communication channels opportunities. Because of reluctance hunters prefer to inform the village head of hunters rather than the CIs who are in their immediate area and trained to communicate with Water and Forest Department. The election of the CIs by the community, the promotion of their role and involving heads of hunters in the surveillance system may encourage the health information report to the Water and Forest Department by hunters while avoiding them to travel a great distance and to overwhelm the health center and the official veterinarian with ecosystem health information. One Health collaborations between the health center, the Water and Forest Department and the official veterinarian may be interesting to cross data and detect more efficiently the emergence of a zoonotic disease. They could also share means to confirm cases and jointly notify the existing prefectural One Health unit at the prefectural level rather than notifying each prefectural institution separately [ 53 ]. We believe that this study on health communication channels was an essential basis for the future community-based surveillance system. The use of participatory approach created a forum for sharing opinions among actors and may encourage them to collectively design and use adapted health communication channels [ 62 ]. 4.3 Bias and limitations of the study The major limitation of our study is the low sample size and the resulting potential lack of representativeness of our results. These selection and representativeness bias to our study were due to various reasons [ 63 ]. We did not have time to conduct a large number of FGDs and therefore to collect sufficient data for relevant statistical analysis and generalization of results. The participation rate for some FGDs was low and some categories of actors were not interviewed such as traditional healers. We expected to be as representative as possible but the participants convened by our key resource persons in the villages may have be among the most influential of the village. Despite their hierarchical position, we deliberately included heads of breeders and hunters in the FGDs to understand their potential role in the surveillance related to data collection and communication. Women were under-represented and participated less than men during some FGDs. We anticipated this social bias related to gender by conducting non-mixed FGDs. We noted that some activities and occupations are exclusively practiced by men (hunter, breeder) or women (matron). Because of road conditions, we did not visit the most remote villages and therefore we interviewed an unrepresentative sample of the sub-prefectural population. Most of the participants came from the chief towns of the sub-prefectures, where we conducted the FGDs, and neighboring villages. Local knowledge and health communication channels may be different in remote villages as they are further from the human, animal and ecosystem health sub-prefectural institutions and community workers. There is a translation bias to our study as the interviews were conducted in the Kissi language that none of the investigators understood and there may have been approximations or omissions of the information by the translator [ 64 ]. We wrote clinical signs and flow diagrams in the Kissi language and we anticipated the fact that some participants were illiterate by using visual tools such clinical signs cards. Due to these constraints, we were not always able to facilitate the participatory tools as originally planned and applied the principle of flexibility to adapt the participatory process to the participants. The investigation team was trained on participatory approaches to avoid professional bias involving interpretation of local knowledge on diseases as veterinarians. 4.4 Recommendations At the end of this study, we recommend to conduct further FGDs with each category of actors we identified as having a potential role in the collection of health information to adapt the case definitions to their knowledge and perception of diseases and clinical signs. Additional FGDs with all the actors are needed to co-design a community health information exchange network and to formalize the One Health collaborations. The implementation of the future community-based surveillance system requires actors training and awareness on case definitions and VHFs; particularly in the ecosystem health sector. Collaboration with the central authorities is essential to ensure the health communication from the community level to the competent institutions for outbreak control. Once implemented we recommend an evaluation of the community-based surveillance system to adjust its components, such as case definitions and health communication channels, to communities' knowledge and capacities over time. This study could be extended to other regions of Guinea or West Africa that aim to implement a One Health community-based surveillance system of emerging zoonotic diseases. 4.1 Capacity of the communities to detect diseases Appropriate case definitions are crucial to ensure the efficiency of the surveillance system, especially for early detection of emerging diseases [ 54 ]. A high sensitivity of case definitions will lead to a larger proportion of true positive in the surveillance system, while a low specificity will burden the surveillance system with false positives and will require more investigation resources. If the objective of surveillance is the early detection of emerging zoonotic diseases as serious as the VHFs, a high sensitivity and positive predictive value of the case definitions is desirable [ 50 , 55 , 56 ]. In low-income countries such as Guinea, which have limited resources for diagnosis and case confirmation, it is also important to avoid false positives as much as possible to not burden the surveillance system. We investigated actors' knowledge on the diseases to estimate their detection capacity. We also explored actors' perception of clinical signs to estimate their reaction when they observe one of them and to identify potential signs to include in case definitions. We hypothesized that signs that might constitute an alert, and thus be relevant for inclusion, should be perceived as both rare and health-threatening. The rarity criterion is consistent with a syndromic and community-based surveillance system that aims to early detect emerging diseases [ 57 , 58 ]. We selected the human health threat criterion as an indicator of trigger event for health information communication. Our results showed that VHFs are poorly described and their associated clinical signs are differently perceived by all human health actors. The controversial aspect of VHFs may have influenced the discussion because it became a taboo issue after the EVD outbreak which caused a tense social and political climate in West Africa [ 11 , 59 ]. This health crisis weakened governments and altered relations between institutions and communities. Communities may be reluctant to report cases for fear of restrictions applied during the epidemic [ 60 ]. We did not clearly identify signs of VHFs that are perceived as both rare and health-threatening. Most of them are perceived as frequent and threatening and are not specific to VHFs. Some participants reported to be more threatened by the clinical signs they suffer from or observe in their community. The hemorrhagic signs that are more specific of VHFs are differently perceived among groups. Bleeding is perceived as rare but not threatening by women in both sub-prefectures and red eyes, hematemesis, hematuria and bloody stools are perceived as rare and/or threatening by CHHWs. We suggest to sensitize all the actors to the VHFs' threat and on the importance to detect them because most of the specific hemorrhagic signs are not perceived as an alert. It could be possible to increase the sensitivity of the case definition by including the unspecific VHFs clinical signs which are perceived as a threat. Except the VHFs, CHHWs showed a better and more homogeneous knowledge of diseases under surveillance than women. None of the interviewed groups perfectly mentioned the clinical pictures included in case definitions but we believe that the training and sensitization of community human health actors had an effect because none of the groups mentioned the diseases under surveillance for which the CHHWs did not have technical sheet with case definitions. Nevertheless, our findings showed that these efforts are insufficient. We suggest to improve training of the CHHWs on diseases under surveillance so that they can all detect them and potentially with more specific case definitions. Awareness efforts should be improved and particularly in Temessadou. We suggest to explore the success key factors of the health program addressing the malaria disease which is well known by communities. Our results showed that CAHWs have a better knowledge on diseases under surveillance than breeders. We believe that the sensitization and training improved knowledge because the actors better know zoonotic diseases under surveillance than the ones not under surveillance. Nevertheless, sensitization and training efforts should be increased because none of the interviewed groups perfectly mentioned the clinical pictures included in case definitions and some diseases are not mentioned. Our results showed that the clinical signs associated to RVF, that might be interesting to include in case definitions for the surveillance of VHFs in domestic animal populations, are differently perceived among groups. We suggest to create sensitive case definitions including clinical signs perceived as rare or as a threat for human health such as mortality and abortion which are proposed by the FAO for syndromic and participatory surveillance of RVF [ 47 ]. We suggest to increase sensitization of animal health actors on the zoonotic risk related to VHFs because they are concerned by the clinical signs they associate with a zoonotic disease ("Everything that is harmful for animal health is harmful for human health", "We are afraid of diseases transmitted from animals to humans."). Contrary to the human and animal health compartments, the ecosystem sector is not involved in health surveillance at the community level. Knowledge of ecosystem health actors on diseases is almost non-existent but they are able to associate clinical signs to species and to observe abnormalities such lesions on carcasses or one or more animals sick or dead with no apparent cause in the same area. There is no sign perceived both as a threat for human health and rare but all the hemorrhagic signs are perceived as rare. We suggest to include these rare events in a standardized and specific case definition and to train all the ecosystem health actors to zoonotic diseases. 4.2 Health information exchange network We investigated the surveillance network opportunities at the community level. The participants identified the existing communication channels and discussed the strengths and weaknesses of the current information exchange network, their preferences and better alternatives. The results highlighted the fact that community actors face different constraints and communicate with various stakeholders. This situation may delay the report of a suspected case to the relevant institutions and therefore the confirmatory diagnosis and outbreak control actions [ 51 , 61 ]. Our study showed interesting communication channels opportunities in the current health information exchange network to bypass potential barriers to health report. The CHHWs and the CAHWs are a strong link in the information chain because they are geographically close to the community and trained to the surveillance of disease. Therefore, community members and breeders from both sub-prefectures who inform the CHHWs and the CAHWs overcome the lack of transport means that could demotivate them to inform the health center and the official veterinarian but they also potentially reduce the burden on these sub-prefectural actors with not suspected case as the community workers better know the case definitions. Some community members and breeders do not inform the community workers, despite the fact they elected them, because they do not know their role or by reluctance or because they prefer to inform other actors such as matrons, traditional healers and heads of breeders. Promoting the role of community workers and involving other actors who collect health information in the surveillance system may be levers to activate. In the ecosystem health sector any actor is trained to the health surveillance but we identified communication channels opportunities. Because of reluctance hunters prefer to inform the village head of hunters rather than the CIs who are in their immediate area and trained to communicate with Water and Forest Department. The election of the CIs by the community, the promotion of their role and involving heads of hunters in the surveillance system may encourage the health information report to the Water and Forest Department by hunters while avoiding them to travel a great distance and to overwhelm the health center and the official veterinarian with ecosystem health information. One Health collaborations between the health center, the Water and Forest Department and the official veterinarian may be interesting to cross data and detect more efficiently the emergence of a zoonotic disease. They could also share means to confirm cases and jointly notify the existing prefectural One Health unit at the prefectural level rather than notifying each prefectural institution separately [ 53 ]. We believe that this study on health communication channels was an essential basis for the future community-based surveillance system. The use of participatory approach created a forum for sharing opinions among actors and may encourage them to collectively design and use adapted health communication channels [ 62 ]. 4.3 Bias and limitations of the study The major limitation of our study is the low sample size and the resulting potential lack of representativeness of our results. These selection and representativeness bias to our study were due to various reasons [ 63 ]. We did not have time to conduct a large number of FGDs and therefore to collect sufficient data for relevant statistical analysis and generalization of results. The participation rate for some FGDs was low and some categories of actors were not interviewed such as traditional healers. We expected to be as representative as possible but the participants convened by our key resource persons in the villages may have be among the most influential of the village. Despite their hierarchical position, we deliberately included heads of breeders and hunters in the FGDs to understand their potential role in the surveillance related to data collection and communication. Women were under-represented and participated less than men during some FGDs. We anticipated this social bias related to gender by conducting non-mixed FGDs. We noted that some activities and occupations are exclusively practiced by men (hunter, breeder) or women (matron). Because of road conditions, we did not visit the most remote villages and therefore we interviewed an unrepresentative sample of the sub-prefectural population. Most of the participants came from the chief towns of the sub-prefectures, where we conducted the FGDs, and neighboring villages. Local knowledge and health communication channels may be different in remote villages as they are further from the human, animal and ecosystem health sub-prefectural institutions and community workers. There is a translation bias to our study as the interviews were conducted in the Kissi language that none of the investigators understood and there may have been approximations or omissions of the information by the translator [ 64 ]. We wrote clinical signs and flow diagrams in the Kissi language and we anticipated the fact that some participants were illiterate by using visual tools such clinical signs cards. Due to these constraints, we were not always able to facilitate the participatory tools as originally planned and applied the principle of flexibility to adapt the participatory process to the participants. The investigation team was trained on participatory approaches to avoid professional bias involving interpretation of local knowledge on diseases as veterinarians. 4.4 Recommendations At the end of this study, we recommend to conduct further FGDs with each category of actors we identified as having a potential role in the collection of health information to adapt the case definitions to their knowledge and perception of diseases and clinical signs. Additional FGDs with all the actors are needed to co-design a community health information exchange network and to formalize the One Health collaborations. The implementation of the future community-based surveillance system requires actors training and awareness on case definitions and VHFs; particularly in the ecosystem health sector. Collaboration with the central authorities is essential to ensure the health communication from the community level to the competent institutions for outbreak control. Once implemented we recommend an evaluation of the community-based surveillance system to adjust its components, such as case definitions and health communication channels, to communities' knowledge and capacities over time. This study could be extended to other regions of Guinea or West Africa that aim to implement a One Health community-based surveillance system of emerging zoonotic diseases. Supporting information S1 Matrix Diseases-clinical signs matrix. Matrix A in S1 Matrix , Human diseases-clinical signs matrix; Matrix B in S1 Matrix , Animal diseases-clinical signs matrix. Guenin, Marie Jeanne, 2022, "Plos NTD research article—A participatory epidemiological and One Health approach for surveillance opportunities", https : //doi . org/10 . 18167/DVN1/DBYZXU , CIRAD Dataverse, V1. (XLSX) Click here for additional data file. S2 Matrix Scoring and ranking of clinical signs. Guenin, Marie Jeanne, 2022, "Plos NTD research article—A participatory epidemiological and One Health approach for surveillance opportunities", https : //doi . org/10 . 18167/DVN1/DBYZXU , CIRAD Dataverse, V1. (XLSX) Click here for additional data file. S1 Picture Flow diagrams of the information health exchange network. This picture file includes the pictures of flow diagrams representing the health information exchange networks in human health realized by groups of women and community human health workers in Guendembou (Picture A) and Temessadou (Picture B), by breeders and community animal health workers in Guendembou (Picture C) and Temessadou (Picture D), by hunters and community informants in Guendembou (Picture E) and Temessadou (Picture F). Guenin, Marie Jeanne, 2022, "Plos NTD research article—A participatory epidemiological and One Health approach for surveillance opportunities", https://doi.org/10.18167/DVN1/DBYZXU , CIRAD Dataverse, V2. (PNG) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4497739/

Bacterial induction of Snail1 contributes to blood-brain barrier disruption

Bacterial meningitis is a serious infection of the CNS that results when blood-borne bacteria are able to cross the blood-brain barrier (BBB). Group B Streptococcus (GBS) is the leading cause of neonatal meningitis; however, the molecular mechanisms that regulate bacterial BBB disruption and penetration are not well understood. Here, we found that infection of human brain microvascular endothelial cells (hBMECs) with GBS and other meningeal pathogens results in the induction of host transcriptional repressor Snail1, which impedes expression of tight junction genes. Moreover, GBS infection also induced Snail1 expression in murine and zebrafish models. Tight junction components ZO-1, claudin 5, and occludin were decreased at both the transcript and protein levels in hBMECs following GBS infection, and this repression was dependent on Snail1 induction. Bacteria-independent Snail1 expression was sufficient to facilitate tight junction disruption, promoting BBB permeability to allow bacterial passage. GBS induction of Snail1 expression was dependent on the ERK1/2/MAPK signaling cascade and bacterial cell wall components. Finally, overexpression of a dominant-negative Snail1 homolog in zebrafish elevated transcription of tight junction protein–encoding genes and increased zebrafish survival in response to GBS challenge. Taken together, our data support a Snail1-dependent mechanism of BBB disruption and penetration by meningeal pathogens.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5328221/

How often people google for vaccination: Qualitative and quantitative insights from a systematic search of the web-based activities using Google Trends

ABSTRACT Nowadays, more and more people surf the Internet seeking health-related information. Information and communication technologies (ICTs) can represent an important opportunities in the field of Public Health and vaccinology. The aim of our current research was to investigate a) how often people search the Internet for vaccination-related information, b) if this search is spontaneous or induced by media, and c) which kind of information is in particular searched. We used Google Trends (GT) for monitoring the interest for preventable infections and related vaccines. When looking for vaccine preventable infectious diseases, vaccine was not a popular topic, with some valuable exceptions, including the vaccine against Human Papillomavirus (HPV). Vaccines-related queries represented approximately one third of the volumes regarding preventable infections, greatly differing among the vaccines. However, the interest for vaccines is increasing throughout time: in particular, users seek information about possible vaccine-related side-effects. The five most searched vaccines are those against 1) influenza; 2) meningitis; 3) diphtheria, pertussis (whooping cough), and tetanus; 4) yellow fever; and 5) chickenpox. ICTs can have a positive influence on parental vaccine-related knowledge, attitudes, beliefs and vaccination willingness. GT can be used for monitoring the interest for vaccinations and the main information searched.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4319067/

Cancerous inhibitor of PP2A is targeted by natural compound celastrol for degradation in non-small-cell lung cancer

Summary Celastrol binds CIP2A and enhances CIP2A–CHIP interaction, leading to ubiquitination/degradation of CIP2A and inhibition of lung cancer cells in vitro and in vivo . Celastrol potentiates cisplatin's efficacy by suppressing the CIP2A–Akt pathway, and therefore CIP2A inhibitors may represent novel therapeutics for cancer.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663889/

Anthrax lethal toxin rapidly reduces c-Jun levels by inhibiting c-Jun gene transcription and promoting c-Jun protein degradation

Anthrax is a life-threatening disease caused by infection with Bacillus anthracis , which expresses lethal factor and the receptor-binding protective antigen. These two proteins combine to form anthrax lethal toxin (LT), whose proximal targets are mitogen-activated kinase kinases (MKKs). However, the downstream mediators of LT toxicity remain elusive. Here we report that LT exposure rapidly reduces the levels of c-Jun, a key regulator of cell proliferation and survival. Blockade of proteasome-dependent protein degradation with the 26S proteasome inhibitor MG132 largely restored c-Jun protein levels, suggesting that LT promotes degradation of c-Jun protein. Using the MKK1/2 inhibitor U0126, we further show that MKK1/2–Erk1/2 pathway inactivation similarly reduces c-Jun protein, which was also restored by MG132 pre-exposure. Interestingly, c-Jun protein rebounded to normal levels 4 h following U0126 exposure but not after LT exposure. The restoration of c-Jun in U0126-exposed cells was associated with increased c-Jun mRNA levels and was blocked by inactivation of the JNK1/2 signaling pathway. These results indicate that LT reduces c-Jun both by promoting c-Jun protein degradation via inactivation of MKK1/2–Erk1/2 signaling and by blocking c-Jun gene transcription via inactivation of MKK4–JNK1/2 signaling. In line with the known functions of c-Jun, LT also inhibited cell proliferation. Ectopic expression of LT-resistant MKK2 and MKK4 variants partially restored Erk1/2 and JNK1/2 signaling in LT-exposed cells, enabling the cells to maintain relatively normal c-Jun protein levels and cell proliferation. Taken together, these findings indicate that LT reduces c-Jun protein levels via two distinct mechanisms, thereby inhibiting critical cell functions, including cellular proliferation.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6999370/

Monitoring bioactive and total antibody concentrations for continuous process control by surface plasmon resonance spectroscopy

Abstract Monoclonal antibodies have become an increasingly important part of fundamental research and medical applications. To meet the high market demand for monoclonal antibodies in the biopharmaceutical sector, industrial manufacturing needs to be achieved by large scale, highly productive and consistent production processes. These are subject to international guidelines and have to be monitored intensely due to high safety standards for medical applications. Surface plasmon resonance spectroscopy — a fast, real‐time, and label‐free bio‐sensing method — represents an interesting alternative to the quantification of monoclonal antibody concentrations by enzyme‐linked immunosorbent assay during monoclonal antibody production. For the application of monitoring bioactive and total monoclonal antibody concentrations in cell culture samples, a surface plasmon resonance assay using a target‐monoclonal antibody model system was developed. In order to ensure the subsequent detection of bioactive monoclonal antibody concentrations, suitable immobilization strategies of the target were identified. A significant decrease of the limit of detection was achieved by using an adapted affinity method compared to the commonly used amine coupling. Furthermore, the system showed limit of detection in the low ng/mL range similar to control quantifications by enzyme‐linked immunosorbent assay. Moreover, the comparison of total to bioactive monoclonal antibody concentrations allows analysis of antibody production efficiency. The development of an alternative quantification system to monitor monoclonal antibody production was accomplished using surface plasmon resonance with the advantage of low analyte volume, shorter assay time, and biosensor reusability by target‐layer regeneration. The established method provides the basis for the technical development of a surface plasmon resonance‐based system for continuous process monitoring.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8708306/

The Effects of Bariatric Surgery and Gastrectomy on the Absorption of Drugs, Vitamins, and Mineral Elements

Bariatric surgery, which is an effective treatment for obesity, and gastrectomy, which is the primary treatment method for gastric cancer, alter the anatomy and physiology of the digestive system. Weight loss and changes in the gastrointestinal tract may affect the pharmacokinetic parameters of oral medications. Both bariatric and cancer patients use drugs chronically or temporarily. It is important to know how surgery affects their pharmacokinetics to ensure an effective and safe therapy. The Cochrane, PubMed, and Scopus databases were searched independently by two authors. The search strategy included controlled vocabulary and keywords. Studies show that bariatric surgery and gastrectomy most often reduce the time to maximum plasma concentration ( t max ) and decrease the maximum plasma concentration (C max ) in comparison with the values of these parameters measured in healthy volunteers. Vitamin and mineral deficiencies are also observed. The effect depends on the type of surgery and the properties of the drug. It is recommended to use the drugs that have been tested on these groups of patients as it is possible to monitor them. 1. Introduction Bariatric surgery is an increasingly common obesity treatment applied to patients when other treatments have not been successful. It has proved to successfully reduce the weight of patients with a body mass index (BMI) greater than 40 kg/m 2 or 35 kg/m 2 in the presence of comorbidities (type 2 diabetes or cardiovascular diseases). According to the World Health Organization (WHO), in 2016 33% of adults were overweight or obese [ 1 ]. Bariatric procedures are classified as restrictive, malabsorptive, or restrictive and malabsorptive. Restrictive procedures reduce the amount of food that can be stored in the stomach but do not interfere with normal digestion, often resulting in a small gastric pouch with a narrow mouth. Examples of restrictive procedures are: (laparoscopic) sleeve gastrectomy (L)SG, (laparoscopic) adjustable gastric banding (L)AGB, and vertical banded gastroplasty (VBG). Malabsorptive procedures such as biliopancreatic diversion (BPD) consist of the shortening of the gastrointestinal tract to limit the possible extent of absorption. The following procedures combine both categories: Roux-en-Y gastric bypass (RYGB), mini gastric bypass (MGB), biliopancreatic diversion with duodenal switch (BPD-DS), single anastomosis duodeno-ileal bypass (SADI), and single anastomosis gastric-ileal bypass (SAGI) [ 1 , 2 ]. Figure 1 shows selected types of the abovementioned bariatric procedures. The type of bariatric surgery affects changes in the body functions. Although the exact mechanisms of action are unknown, the effects of some bariatric procedures appear to be purely anatomical and can cause significant weight loss without significantly changing metabolic pathways. Other treatments change the anatomy of the digestive tract in a way that changes certain physiological parameters. These treatments decrease orexigenicity and increase the count of anorexogenic hormones, so physical hunger is suppressed despite progressive weight loss [ 3 ]. RYGB is the most common type of bariatric surgery [ 4 ]. It consists of creating a gastric sac with a capacity of 20–30 mL by sewing the upper part of the stomach and then restoring the continuity of the gastrointestinal tract by creating a Roux-en-Y branch of the gastrointestinal tract in the jejunum, which requires gastrointestinal and gastrojejunal anastomosis. This surgical procedure results in a weight loss of 57–67%. Weight loss is caused by both diet restriction and decreased absorption due to short-circuiting and hormonal changes [ 5 ]. In SG, 70–80% of the outer stomach is removed from the body and only a narrow gastric tube is left. While SG promotes some reduction in consumption, it also involves metabolic mechanisms of action, including increases in PYY and GLP-1, as well as an increased feeling of fullness. It also causes a permanent decrease in ghrelin levels as a result of resection of the cell mass responsible for its secretion [ 3 ]. SG results in a weight loss of 55–65% [ 5 ]. In 2016, SG was the most common primary surgery in the world (54%), followed by RYGBP (30%) [ 6 ]. Laparoscopic adjustable gastric banding (LAGB) was the most common technique of bariatric surgery for several years until its indications gradually decreased in favor of other interventions. LAGB consists of wrapping the upper part of the stomach with an adjustable band. Subcutaneous injection of physiological serum through a small port enables adjustment of the band. This intervention is purely restrictive and leads patients to change their eating behavior by inducing early satiety. LAGB allows patients to lose 40–54% of their excess weight. However, the effectiveness of this procedure declines over time as patients adjust their eating habits. Biliopancreatic diversion with duodenal switch (BPD/DS) differs from RYGB in the size of the stomach (a small 20 mL bag for RYGB; a longitudinal 150 mL bag for BPD/DS) and, more importantly, the resulting common digestive canal (100 cm for BPD/DS; about 400 cm for RYGB), which makes BPD/DS a much larger component of malabsorption. Due to many malabsorption disorders, this procedure is applied to a very small percentage of patients undergoing bariatric surgery [ 5 ]. Anatomical and physiological changes in the gastrointestinal tract occurring after bariatric surgery may change various factors and result in reduced bioavailability of drugs. The absorption of a drug strongly depends on its physicochemical properties (solubility, lipophilicity, particle size and polarity) and the physiology of the gastrointestinal tract. Bariatric surgery procedures result in bypassing part of the intestine which is rich in metabolising enzymes. This may affect the oral bioavailability of some drugs. After absorption, drugs undergo intestinal and hepatic metabolism, which is an important factor limiting their oral bioavailability. RYGBP bypasses the proximal part of the intestine, which is rich in metabolising enzymes. This bypass places drugs directly in the more distal part of the intestine, which is less metabolic, and thus results in higher oral bioavailability. The dominant drug-metabolising enzymes are cytochrome P450 (CYP) enzymes, the most numerous of which is the CYP3A subfamily. In addition to its extensive expression in the liver, the CYP3A subfamily is widely expressed in the duodenum and proximal jejunum. At least 50% of the drugs available on the market are metabolized by CYP3A. CYP3A enzymes have been reported to constitute 80% of the total P450 content in the proximal small intestine. In consequence, the gastrointestinal rearrangement after bariatric surgery, especially RYGBP, greatly affects the oral bioavailability of CYP3A substrates. Other enzymes found in the small intestine are CYP2C9, CYP2C19, and CYP2D6, as well as UDP-glucuronosyltransferases (UGTs). Drugs penetrate the intestinal mucosa by passive diffusion or active transport, depending on their solubility and lipophilicity. Transport proteins found in the gastrointestinal tract facilitate active transport. Therefore, they may also affect both the absorption and the intestinal metabolism of substrate drugs. Many different drug transporters, including P-glycoprotein (P-gp), are expressed in the gastrointestinal tract. Gastrointestinal rearrangement after bariatric surgery may affect the pharmacokinetics of drugs [ 6 ]. The Biopharmaceutical Classification System (BCS) distinguishes four classes of drugs according to their permeability and solubility. This system might help to predict some of the effects of bariatric surgery on various drugs on the basis of their physicochemical properties. Insufficient knowledge on this subject causes pharmacological problems in patients after bariatric surgery. The aim of our review was to present changes in the pharmacokinetics and pharmacodynamics of selected drug groups in patients after bariatric surgery and gastric resection [ 6 ]. Moreover, the patients classified for this surgery are often characterized by extreme obesity and it is essential to consider changes in drug pharmacokinetics typical for obesity. They result in increased lean body mass (fat body mass in obese people is also increased), accelerated gastric emptying, altered activity of enzymes involved in drug metabolism, and enhanced glomerular filtration rate [ 6 ]. Additionally, considering obese patients, a few studies have revealed metabolic differences in visceral adipose tissue (VAT) between obese and non-obese individuals, which could be the next essential aspect in pharmacotherapy of patients after gastrectomy [ 7 , 8 ]. Dysfunctional VAT has pro-inflammatory features and promotes cardiovascular disease and type 2 diabetes mellitus. Adipose tissue secretes adipokines, for example, which are known mediators of various metabolic processes [ 9 ]. These aspects confirm that obesity is a complex disease, also because of related health concerns. To prevent excessive weight regain and improve comorbidities (e.g., diabetes, hypertension) in bariatric patients after surgery, more frequent long-term medical follow-up visits and regular monitoring are recommended [ 10 ]. Unfortunately, approximately 20–30% of bariatric patients do not achieve successful weight outcomes, because of many factors such as food tolerance, patient knowledge, and also type of surgery [ 11 ]. We also note an interesting study by Bellia et al. It was observed that 25OHD levels were higher in metabolically healthy obese patients than in insulin-resistant obese patients. An interesting fact was highlighted: the higher the 25OHD value, the lower the risk of insulin resistance [ 12 ]. The aim of our review was to present changes in the pharmacokinetics and pharmacodynamics of selected drug groups in patients after bariatric surgery and gastric resection [ 13 ]. Each year, about one million cases of gastric cancer are diagnosed worldwide. The mortality rate in this group of patients is high, i.e., about 70–75% [ 6 , 14 ]. Gastric cancer patients require various forms of gastrectomy (surgical removal of part or the whole stomach) [ 15 ]. Surgery is the only treatment option [ 14 ]. Currently, three gastrectomy procedures are available: proximal gastrectomy, distal gastrectomy, and total gastrectomy. Total gastrectomy with lymph node resection is the standard procedure for treating gastric cancer. However, in the case of limited gastric cancer, it is possible to use a different gastric resection procedure [ 6 ]. There are several methods of reconstructing the gastrointestinal tract after total gastrectomy: Roux-en-Y reconstruction, jejunal interposition, jejunal interposition with a pouch. Roux-en-Y reconstruction consists of esophageal jejunostomy of the remaining esophagus into the jejunum and jejunojejunostomy between the initial part of the left jejunum and the first loop of the jejunum. Reconstruction can be done with or without a pouch [ 16 ]. Figure 2 shows the methods of reconstruction of the gastrointestinal tract after total gastrectomy. Reconstructions after distal gastrectomy include: Billroth I reconstruction, Billroth II reconstruction, and Roux-en-Y reconstruction. Billroth I includes a gastroduodenal anastomosis. Billroth II includes gastrojejunostomy of the remaining stomach to the first jejunal loop. Roux-en-Y includes gastrojejunostomy of the remaining stomach to an excluded jejunal limb and end-to-side jejunojejunostomy between the excluded jejunum to the first jejunal loop [ 10 ]. Figure 3 shows the methods of reconstruction of the gastrointestinal tract after distal gastrectomy. Reconstruction schemes after proximal gastric resection are currently being tested. Reconstruction after proximal gastrectomy was initially performed as direct esophagogastrostomy, but this procedure involves a high rate of gastric reflux. To prevent the occurrence of a gastric reflux, different approaches have been tested, e.g., combining esophagogastrostomy with fundoplication, jejunal interposition with and without a pouch, double tract reconstruction, and ileocolic interposition [ 16 ]. Figure 4 shows the methods of reconstruction of the gastrointestinal tract after proximal gastrectomy. Various forms of gastrectomy may significantly change the pharmacokinetics of orally taken drugs [ 17 ]. The lack of stomach results in mechanometabolic and deficiency metabolic disorders. The former group of disorders includes postprandial syndrome and alkaline esophagitis due to regurgitation. The latter group of disorders includes anemia, osteoporosis and/or osteomalacia, and weight loss [ 18 ]. The consequences of gastric surgery, such as reduced gastric volume, reduced secretion of gastric, pancreatic and biliary juices, accelerated gastric emptying, and impaired fat absorption, may affect the pharmacokinetics of drugs [ 6 , 15 ]. The increase in gastric pH after gastrectomy may limit the absorption of acidic drugs [ 18 ]. Gastrectomy may change the rate and range of drug absorption by altering the time of gastric emptying into the small intestine. In consequence, the following parameters may change: area under the plasma concentration time curve (AUC), maximum drug concentration in blood (C max ), time to maximum drug concentration in blood ( t max ), absorption rate constant (k a ), bioavailability (F), and biological half-life ( t 0.5 ), which determine the therapeutic effect of the drug, its efficacy and result in treatment-induced toxicity [ 14 ]. Of course, it should be highlighted that differences between benign disease and cancer patients are relevant. A 2018 meta-analysis showed that obese patients undergoing surgery for malignancy were at increased risk of major complications, whereas obese patients undergoing surgery for benign indications were at decreased risk compared to normal weight patients [ 19 ]. 2. Materials and Methods The search strategy included controlled vocabulary and keywords. The Cochrane, PubMed, and Scopus databases were searched independently by two authors. The main search concept was to combine 'gastrectomy', 'gastric bypass', 'bariatric surgery' with related terms such as 'pharmacokinetic', 'absorption', 'changes', and 'bioavailability'. The inclusion criterion was the data included in the studies related to the groups of drugs selected by the authors. Due to the small number of studies in recent years, the time criterion was not applied. Table 1 shows the steps for including articles in the review. 2.1. Antibiotics Rocha et al., conducted a study on patients ( n = 8) before and two months after the RYGB procedure to investigate changes in the pharmacokinetics of amoxicillin (AMX) [ 4 ]. AMX is the most common antibiotic, used since the 1970s, with good absorption (85–90%), especially in the duodenum and jejunum [ 20 ]. The drug has a non-linear absorption profile, so it means that the process rate is saturable [ 21 ]. Rocha et al., conducted research on obese subjects who received a single dose of amoxicillin in a 500 mg capsule. After the surgery, the mean weight loss was 17.03 ± 5.51 kg, and the mean body mass index (BMI) decreased from 46.21 ± 2.82 to 38.82 ± 3.32 kg/m 2 . The mean amoxicillin area under the plasma concentration versus time curve from time zero to the time of the last quantifiable concentration (AUC 0–last ) increased significantly (2.03 vs. 7.21 μg∙h/mL; p = 0.0224); the peak plasma concentration (C max ) also increased significantly (0.62 vs. 1.77 μg/mL; p = 0.0279) after bariatric surgery. There was no correlation between amoxicillin absorption, BMI, and weight loss percentage. The changes observed in the pharmacokinetics of amoxicillin suggest that the obese subjects enrolled in this study had significant increases in the systemic amoxicillin exposure after the RYGB surgery. However, despite this increasee, this exposure was lower than that of the non-obese volunteers, whose AUC 0–last values ranged from 12.44 to 12.05 μg∙h/mL, whereas their C max ranged from 4.94 to 5.31 μg/mL after a single oral administration of 500 mg amoxicillin capsules [ 4 ]. This may be related to body mass. Mellon et al., observed that the amoxicillin C max decreased significantly with weight. Considering the target PK/PD value for beta-lactams fT > MIC ≥ 40%, the standard dosage of co-amoxiclav (1000/125 mg every 8 h) should be efficacious for obese adults [ 22 ], but Soares et al., suggested that amoxicillin treatment would fail if standard therapeutic regimens were applied because of a significantly higher volume of distribution in this group of patients [ 23 ]. In another study, the bioavailability of an oral AMX tablet and suspension was tested on patients who had undergone an RYGB surgery 3 months to 10 years before. The patients received an 875 mg AMX tablet or 800 mg AMX suspension. Twenty people with a body mass index of 29.88 ± 4.36 kg/m 2 were qualified for the study. The C max of AMX in the plasma of tablets and suspensions (normalized to 875 mg) was 7.42 ± 2.99 mg/L and 8.73 ± 3.26 mg/L (90% CI = 70.71–99.11), and the area under the plasma concentration versus time curve from time zero to infinity (AUC 0–∞ ) was 23.10 ± 7.41 mg⋠h/L and 27.59 ± 8.32 mg⋠h/L, respectively (Cl = 71.25–97.32). The values of these parameters were compared with the results noted in healthy subjects, as described in available literature. The healthy subjects received 875 mg AMX tablets (alone or in combination with clavulanic acid). The AUC 0–∞ and C max values increased from 43.80 to 51.29 mg⋠h/L and from 12.13 to 15.30 mg/L [ 24 ]. Padwal et al., conducted a study on the pharmacokinetics of azithromycin, which is a macrolide antibiotic with a broad spectrum of activity against various aerobic and anaerobic bacteria [ 25 ]. Azithromycin is preferentially absorbed in the duodenum and upper jejunum. The oral bioavailability of azithromycin in healthy subjects amounts to about 37% [ 26 ]. A total of 14 women who were at least 3 months post RYGB surgery and 14 healthy women (the control group) with matched body mass index (BMI) (mean age 44 years and BMI 36.4 kg/m 2 ) were administered a single dose of two 250 mg azithromycin tablets. The AUC 0–24 of the patients who had undergone the RYGB procedure was reduced by 32% (1.41 vs. 2.07 mg⋠h/L; p = 0.008), whereas the dose-normalized AUC 0–24 was reduced by 33% (0.27 vs. 0.40 kg⋠h/L; p = 0.009). The azithromycin C max of the patients after the RYGB surgery amounted to 0.260 mg/L, as compared with 0.363 mg/L in the control group ( p = 0.08) and it was reached after 2.14 h and 2.36 h ( p = 0.75), respectively. These results show that there is a possibility of early treatment failure. Therefore, modified dosage and closer clinical monitoring of gastric bypass patients receiving azithromycin should be considered [ 27 ]. The PK/PD relationship for azithromycin is AUC/MIC. Therefore, a lower AUC in patients after gastrectomy may cause treatment failure. Ciprofloxacin is a fluorinated quinolone antibiotic with high activity against a wide spectrum of Gram-positive and Gram-negative bacteria. Clinical trials with an orally administered ciprofloxacin preparation proved the effectiveness of this drug in the treatment of urinary tract infections, sexually transmitted infections, skin, bone and joint infections, prostatitis, typhoid fever, gastrointestinal infections, lower respiratory tract infections, anthrax, plague, and salmonellosis. Significant quantities of ciprofloxacin are absorbed after its oral administration. The drug is mainly absorbed in the upper part of the intestinal tract (duodenum, jejunum). The absolute bioavailability is about 70% [ 28 ]. Rivas et al., conducted a study on the pharmacokinetics of ciprofloxacin after its single administration to patients after an RYGB surgery. The study involved overweight and obese patients aged 18–60 years. The assessment was performed once in the control group and three times in the group of overweight and obese patients (first before the surgery and then one and six months after the surgery). The subjects received a single oral dose of 500 mg of ciprofloxacin at each visit. Taking the postoperative change in body weight into account, the parameters were adjusted according to the dose (mg)/body weight (kg). The ciprofloxacin C max decreased significantly one month after the surgery (1840.9 ± 485.2 vs. 1459.6 ± 354.8 ng/mL; p = 0.032), but not after six months (1840.9 ± 485.2 vs. 1589.6 ± 32.8 ng/mL; p = 0.116). The C max measured after sixth months was lower than the C max in the control group (2160.4 ± 408.6 vs. 1589.6 ± 321.8 ng/mL; p 40, G(−) = 100–125) [ 29 ]. In conclusion, by the sixth month, the effect on the C max and AUC 0–∞ had disappeared due to weight loss. There is no need to modify the doses of ciprofloxacin in these patients [ 30 ]. As results from the abovementioned studies, the values of pharmacokinetic parameters may be influenced by the time after bariatric surgery. Another conclusion concerns the form of the drug. It is important to note that the suspension and the tablet affect the pharmacokinetic parameters differently in patients after RYGB surgery. 2.2. Analgesic Drugs Acetaminophen is an analgesic drug of choice for patients after gastrectomy, even in oral formulations. This drug is mainly absorbed by passive transport in the small intestine [ 31 ]. The oral route of administration of acetaminophen increases the risk of postoperative nausea and vomiting, as compared with the intravenous route, but the efficacy of both routes of administration is comparable [ 32 ]. Porat et al., conducted a clinical, crossover study on the pharmacokinetics of paracetamol in obese patients enrolled for LSG. The patients received randomly 500 mg of paracetamol in a caplet or in syrup. The other form of the drug was administered after 1–2 weeks. The study was repeated 4–6 months after the surgery. The mean weight loss was 26 kg. The researchers observed that the AUC and C max were higher after these few weeks than before the surgery. The bioavailability of paracetamol increased twice and it was higher when administered as a liquid. The t 1/2 was longer after LSG. The changes in the pharmacokinetic parameters were associated with the patients' loss of weight. The bioavailability of acetaminophen in obese patients was much lower [ 17 ]. This may have been caused by an increase in the metabolic pathways (including glucuronidation) in these patients [ 33 ]. In addition, LSG accelerated gastric emptying, so the t max in the syrup group was shorter after the surgery [ 17 ]. The pharmacokinetics of paracetamol were also investigated in patients after total gastrectomy. Szałek et al., conducted a study comparing the pharmacokinetic parameters after the administration of two generic products. A group of 30 people after gastrectomy with Roux-en-Y reconstruction was divided into two groups. The participants received two tablets containing 500 mg of paracetamol each. The C max and AUC in both groups were lower than in healthy subjects. The t max and t 1/2 were similar to the values of these parameters measured in the volunteers without gastrectomy. The results suggest that total gastrectomy reduces the absorption of this drug [ 34 ]. Tramadol is another analgesic drug. It is indicated for the treatment of chronic and postoperative pain, renal and biliary colic, and trauma. Tramadol is a weak opioid. It is often used in combination with paracetamol at a dose of 37.5 mg tramadol and 325 mg paracetamol. This combination is in the form of conventional or effervescent tablets [ 35 ]. When administered orally, its absorption in the upper small intestine amounts to 95–100% [ 36 ]. The pharmacokinetics of these two forms of paracetamol and tramadol were investigated in patients after gastrectomy with Roux-en-Y reconstruction. A total of 26 patients were divided into two groups. The first group received two film-coated tablets, whereas the other group received two effervescent tablets. Each tablet contained 37.5 mg of tramadol and 325 mg of paracetamol. The C max of paracetamol administered orally as a conventional tablet and the C max of tramadol administered orally as an effervescent tablet were significantly lower than in healthy subjects. The t max of paracetamol administered in the form of effervescent tablets to the gastrectomy patients and the t max of tramadol in both groups were shorter. According to the researchers, this may have been caused by the shorter gastric emptying time. According to Szałek et al., conventional tablets are a better choice for patients after gastrectomy [ 35 ]. Ketoprofen belongs to the group of non-steroidal anti-inflammatory drugs. The indications for the use of this drug are postoperative pain, cancer, and rheumatoid arthritis. Ketoprofen is absorbed by passive diffusion in the stomach. It occurs in an undissociated form. Porażka et al., investigated the effect of gastrectomy on the pharmacokinetics of ketoprofen administered orally to two groups of patients. One group ( n = 15) consisted of patients after total stomach resection, whereas the other group ( n = 5) included patients after partial resection. All the participants received one film-coated tablet containing 100 mg of ketoprofen. The C max of the patients after total stomach resection was significantly lower than in the group of the patients after partial resection. According to the researchers, this may have been caused by reduced tablet disintegration and slower mixing of the gastric contents due to the smaller size of the stomach. Faster gastric emptying, higher gastric pH and a smaller absorption area may result in lower C max and t max in gastrectomy patients. The patients had higher V d (volume of distribution), most likely due to hypoalbuminaemia, which is a common symptom of gastric cancer. Gastrectomy patients may require higher doses of ketoprofen for effective pain relief [ 6 ]. Morphine is the most commonly used opioid to treat moderate to severe pain. After oral administration of the drug, its absorption from the gastrointestinal tract amounts to almost 100%. Morphine is a substrate of P-glycoprotein [ 37 ]. It is absorbed mainly in the upper part of the small intestine and, to a lesser extent, in the stomach. The absolute bioavailability of morphine is low (20–30%) due to the first pass effect [ 38 ]. A study was conducted on 30 patients to determine the effect of RYGB on the pharmacokinetics of this drug. Each patient received an oral dose of 30 mg of liquid morphine at each of three visits (7–30 days before the surgery, 7–15 days after the surgery, and 6 months after the surgery). The t max decreased, whereas the C max increased significantly. The AUC also increased. The study showed that RYGB significantly increased the rate of morphine absorption. The increase in the C max and AUC may also have been caused by reduced first pass metabolism and weight loss, because non-obese patients have less glucuronidation than obese ones. According to researchers, after RYGB patients should receive lower doses of morphine in the form of a solution before the surgery to reduce the risk of side effects. Sublingual, intranasal or gingival application of fentanyl can be an alternative to immediate-release forms of morphine [ 39 ]. Post-operative pain is also treated with oxycodone. This semi-synthetic opioid is stronger than morphine [ 40 ]. It is absorbed mainly in the small intestine [ 41 ]. Szałek et al., conducted a study on the pharmacokinetics of oxycodone in patients after total gastrectomy. A total of 24 patients received prolonged-release tablets containing 10 mg of oxycodone. The mean C max and systemic exposure of oxycodone in the men were higher than in the women. The t max of the patients after resection was slightly shorter than that of the healthy patients. This effect may have been caused by the shorter gastric emptying time. The C max was similar in both groups. The study showed that total gastrectomy did not affect the pharmacokinetics of oxycodone [ 40 ]. 2.3. Antidepressants About 30–50% of patients after bariatric surgery use psychotropic drugs, mostly antidepressants [ 42 ]. It is important to determine changes in the pharmacokinetics of these drugs after surgery to ensure the safety and effectiveness of therapy [ 43 ]. Escitalopram belongs to the group of selective serotonin reuptake inhibitors (SSRIs). It increases synaptic signalling. It is used to treat major depression and generalized anxiety disorder. It is rapidly absorbed when administered orally. Marzinke et al., conducted a study on the pharmacokinetics of escitalopram in patients after Roux-en-Y gastric bypass surgery. There were four obese patients who used 10 or 20 mg escitalopram once a day. Samples were taken two weeks before the surgery and two and six weeks after the surgery. The serum levels of the drug decreased after surgery. At the third visit, the drug levels were even lower than at the second visit. Obese patients have elevated levels of C-reactive protein (CRP), which indicates current inflammation. This may result in decreased activity of CYP 450 enzymes and a higher concentration of escitalopram before the bariatric surgery. Another reason may be altered absorption after the surgery [ 42 ]. Sertraline also belongs to the SSRI group. Apart from depression, it is also used to treat social phobia, obsessive-compulsive disorders, post-traumatic stress disorder, and panic disorder [ 44 ]. It is mainly absorbed in the duodenum. Roerig et al., conducted a study on five RYGB patients (9–15 months after the surgery) and five non-surgical patients as the control group. The aim of the study was to determine changes in the pharmacokinetics of sertraline after the bariatric surgery. All participants received a single 100 mg dose of sertraline. The AUC 0–10.5 and C max were significantly lower in the postoperative group. The t max did not differ significantly between the two groups [ 43 ]. Depression is also treated with duloxetine. This drug is also used to treat anxiety disorders and neuropathic pain. Roerig et al., conducted a study to determine the effect of Roux-en-Y gastric bypass on the pharmacokinetics of duloxetine. Ten patients who had undergone RYGB 9–15 months before and 10 volunteers from a control group received a single dose of 60 mg of duloxetine. The postoperative patients had significantly lower AUC 0–∞ and shorter t max than the control group. The differences in the C max and half-life were not clinically significant. The researchers speculated that the absorption of duloxetine was reduced as a result of surgery and the loss of the absorptive surface of the duodenum [ 45 ]. Venlafaxine is a norepinephrine reuptake inhibitor. It is used to treat depression, social anxiety disorder, generalized anxiety disorder, and panic disorder. It is available as an immediate-release and extended-release drug [ 46 ]. Ten RYGB patients were enrolled in a prospective study of venlafaxine pharmacokinetics. At least one week before and 3–4 months after the surgery, the participants received one 75 mg capsule of ER venlafaxine. The AUC 0–24 , C max , and t max values measured before and after the RYGB did not differ significantly. According to Krieger et al., gastric bypass surgery does not significantly affect the amount and time of venlafaxine absorption [ 47 ]. Vortioxetine is a multimodal serotonin modulator used to treat depression. It acts on the 5-HT1A, 5-HT1B, 5-HT3, and 5-HT7 receptors and inhibits the reuptake of serotonin. Vandenberghe et al., presented a case report of a patient who underwent RYGB and used vortioxetine regularly. The blood level of the drug was determined 126 and 200 days before surgery. The drug was administered at a dose of 10 mg/day. The vortioxetine levels were also measured 91 days after surgery. The concentration was more than twice lower than before the surgery. The dose was increased to 20 mg/day. On days 224 and 308 after the surgery, the concentration was similar to that in the preoperative period. Researchers recommend therapeutic drug monitoring and crushing tablets or using a liquid form of the drug in the case of poor absorption [ 48 ]. 2.4. Anticoagulant Drugs Rivaroxaban belongs to the group of direct oral anticoagulants (DOAC). It is an inhibitor of factor Xa. It is used to prevent venous thromboembolism [ 49 ]. Rivaroxaban is absorbed in the upper gastrointestinal tract. The drug is characterized by high oral bioavailability [ 50 ]. Kröll et al., conducted a study comparing the pharmacokinetic and pharmacodynamic parameters of rivaroxaban administered one day before and three days after bariatric surgery. Six sleeve gastrectomy patients and six Roux-en-Y gastric bypass patients participated in the study. All of them received 10 mg of rivaroxaban each time. A slight increase in the AUC was observed after both types of surgery. The C max was higher after SG and lower after RYGB than before the surgery. The t max increased in the patients after RYGB. According to the researchers, the bariatric surgery did not significantly affect the pharmacokinetic and pharmacodynamic parameters [ 49 ]. The influence of weight loss after bariatric surgery on the pharmacokinetics and pharmacodynamics of rivaroxaban was investigated on six post-SG and six post-RYGB patients, who received a single 10 mg dose of rivaroxaban 6–8 months after the surgery. The results were compared with the values measured before the surgery. Six months after the surgery the mean weight loss was over 34 kg. The postoperative t max was slightly longer than before the surgery. The C max was lower after the RYGB surgery, whereas the C max of the patients after SG was similar to the value measured before the surgery. Kröll et al., observed that weight loss and bariatric surgery did not significantly affect the pharmacokinetics and pharmacodynamics of rivaroxaban. The researchers assumed that changes in the body weight did not affect these parameters due to the high degree of plasma protein binding of this drug and its low volume of distribution [ 50 ]. Dabigatran is another drug from the DOAC group. It is used to treat and prevent venous thromboembolism and to prevent stroke and systemic embolism [ 51 ]. The drug is absorbed in the lower stomach and duodenum. Grainger et al., measured the pharmacokinetic and pharmacodynamic parameters (including C max ) of dabigatran in patients ( n = 9) after laparoscopic Roux-en-Y gastric bypass who regularly used this drug and compared them with the results from phase II studies. The drug concentration decreased significantly. According to the researchers, this may indicate impaired or delayed absorption [ 52 ]. Rottenstreich et al., observed that the C max of dabigatran in BS patients regularly using this drug was within the expected range [ 53 ]. Similarly to rivaroxaban, apixaban is also a direct factor Xa inhibitor. It is indicated to reduce the risk of stroke and thromboprophylaxis and to treat deep vein thrombosis and pulmonary embolism. Apixaban is mainly absorbed in the small intestine. Its bioavailability amounts to about 50% [ 54 ]. The study assessing the effect of BS on the level of DOAC included nine patients who took apixaban chronically. The blood levels of this drug were measured and compared with the values for the general population. The peak apixaban level was within the expected range [ 55 ]. Bitar et al., reported a failure of apixaban anticoagulant therapy in a patient who had undergone bariatric surgery four years before. The researchers suggested that the surgery may have contributed to the subtherapeutic level of the drug [ 55 ]. Warfarin is an antagonist of vitamin K. It reduces the activity of coagulation factors (II, VII, IX, X) and therefore it is used to prevent and treat thromboembolic disorders. Its efficacy is measured with the International Normalized Ratio (INR). Warfarin is absorbed in the proximal duodenum. Steffen et al., conducted a retrospective study to collect data on warfarin dosage in patients after RYGB surgery. The results measured six months before and after the surgery were analysed. The mean weekly doses before and after the surgery differed significantly. After the surgery the dose was reduced by approximately 25%. It was necessary to apply lower doses after than before the surgery to maintain the INR level. The cause of the changes in the warfarin parameters was not fully explained. According to the researchers, they may have been caused by changes in the consumption and storage of vitamin K, as well as changes in the bacterial flora [ 56 ]. Vitamin K antagonists are believed to be a better choice than DOAC for patients after BS, as they are easy to monitor and to make dose adjustments [ 53 ]. 2.5. Immunosuppressants Adequate modulation of the immune system after transplantation is essential for patient survival and prevents rejection of the transplanted organ. A three-drug regimen of corticosteroid, mycophenolate mofetil, and tacrolimus is the most common. Cyclosporine and sirolimus are also used in immunosuppressive therapy [ 57 ]. Only reliable absorption from the gastrointestinal tract can ensure adequate exposure and efficacy of these drugs [ 58 ]. Mycophenolate mofetil is mainly absorbed in the proximal gastrointestinal tract. It is a prodrug activated during first pass metabolism. Tacrolimus is available as an immediate-release (IR-TAC) and extended-release drug (ER-TAC). The bioavailability of both forms is relatively low. IR-TAC is absorbed from the duodenum to the colon, whereas ER-TAC is absorbed more distally in the gastrointestinal tract [ 59 ]. Ciclosporin is a drug with a narrow therapeutic index. As the absorption of the drug is highly variable, it is important to monitor its concentration. Absorption is incomplete and slow. It occurs mainly in the upper intestine [ 60 ]. Sirolimus, like tacrolimus, is mainly absorbed in the duodenum [ 61 ]. Its bioavailability is low (approximately 25%) [ 62 ]. Chan et al., conducted a prospective study on changes in the pharmacokinetics of immunosuppressants. Twelve patients with end-stage renal disease were involved. The pharmacokinetic parameters after administration of a single dose of tacrolimus, extended-release tacrolimus, mycophenolate mofetil, and enteric-coated mycophenolate sodium (EC-MPS) were measured two months before and 9–12 months after LSG. First, 3 mg of IR-TAC and 720 mg of EC-MPS were administered orally. Two weeks later, the patients received orally 6 mg of ER-TAC and 1000 mg of mycophenolate mofetil (MFF). The median excess body weight loss (EBWL) was 26.5 kg. The study revealed a significant increase in the exposure to all four drugs after LSG. The exposure to tacrolimus was reduced in obese patients. After weight loss, the AUC and C max of both tacrolimus forms were significantly higher. The t max and t 1/2 did not change significantly. The researchers speculated that the increased exposure to tacrolimus was caused by a decrease in the P-glycoprotein expression. They also suggested that accelerated gastric emptying after the surgery resulted in earlier delivery of tacrolimus to the proximal intestine, which also increased exposure to this drug. After LSG, the apparent total plasma clearance (Cl/F) of both forms of mycophenolate mofetil decreased by about 60%. According to the researchers, the UGT activity decreased due to weight loss. The MFF t 1/2 was several times longer after LSG. The t max of EC-MPS decreased by over 30%. It is recommended to monitor immunosuppression in patients after LSG [ 63 ]. The pharmacokinetics of immunosuppressants were also investigated on patients after Roux-en-Y gastric bypass (approximately 2 months to 8 years after surgery). The study involved four dialysis patients and two kidney transplant patients. All the dialysis patients received orally one dose of sirolimus (6 mg), two doses of MMF (1000 mg), and two doses of tacrolimus (4 mg) within 24 h. The patients after transplantation remained on their maintenance regimen. The pharmacokinetic parameters were compared with the results of other studies. The comparison of the AUC:dose ratio of the patients under study with the ratio of healthy volunteers showed that after gastric bypass surgery, a higher dose of sirolimus was necessary to achieve the same exposure as in healthy subjects. The patients with a gastric bypass had much lower AUC 0–12 and AUC 0–∞ than the healthy volunteers. As with sirolimus, the study showed that patients after bariatric surgery require a higher dose of tacrolimus. According to the researchers, this was due to reduced absorption in the small intestine. The C max and mean AUC 0–12 MPA of the patients under study were lower, which may have been caused by the reduced absorption area [ 61 ]. Chen et al., presented a case study of a patient after total gastrectomy with Roux-en-Y reconstruction with end-stage renal disease. The gastrectomy was done 5 years earlier. The patient had pharmacokinetic tests before transplantation to select the most appropriate immunosuppressive regimen. Five different treatment regimens, including EC-MPS, MMF, cyclosporine, tacrolimus, and sirolimus were tested. The regimens were tested sequentially and steady-state concentrations were obtained each time. The measured values were compared with the reference group without gastrectomy. The C max of ciclosporin and the C max and AUC of tacrolimus were higher than in the patients without gastrectomy. The patient 5 years after gastrectomy was characterized by good absorption of the drugs under study. The rate and extent of sirolimus and MPA absorption from EC-MPS was similar to the reference group. The MPA from MFF was absorbed worse than from EC-MPS [ 58 ]. A case of a patient who underwent liver transplantation and sleeve gastrectomy at the same time was also described. The patient received tacrolimus and everolimus. The appropriate level of immunosuppression was maintained. No drug absorption problems were observed. According to Tariciotti et al., it is beneficial for the patient to carry out these two treatments at the same time [ 64 ]. 2.6. Thyroid Hormones Gadiraju S. et al., conducted a meta-analysis of the effect of the type of bariatric surgery on the dosage of levothyroxine. Levothyroxine is a synthetic thyroid hormone used to treat diseases of the thyroid gland resulting from thyroxine deficiency. The drug is mainly absorbed in the jejunum and ileum. This fact suggests a higher demand for levothyroxine after jejunoileal bypass surgery. Although the jejunoileal segment of the small intestine remains intact for RYGB, SG and gastric banding, there are alternate variations to these procedures, which may result in an increased demand for levothyroxine. Probably, the small gastric sac in these procedures reduces the dissolution of levothyroxine in the stomach and results in an increased t max after RYGB. SG accelerates gastric emptying, which may contribute to the malabsorption of levothyroxine. However, most patients after RYGB and SG have a lower demand for levothyroxine. Presumably, this could be explained by the correlation between a change in the body weight and a change in levothyroxine dosage. In obesity, highly lipophilic drugs such as levothyroxine have an increased volume of distribution, which changes their pharmacokinetics. Probably, weight loss after bariatric surgery regulates the pharmacokinetics and results in a lower demand for levothyroxine. Obese patients have not only a greater mass of the adipose tissue but also a higher lean body mass, which is responsible for 20–40% of the increase in the total body weight. T4 is converted into T3 in the skeletal muscles, so it is likely that the reduction in the lean body mass after bariatric surgery results in a lower postoperative demand for levothyroxine. Lower serum leptin levels may also decrease the demand for levothyroxine in the postoperative period. Leptin regulates the expression of the thyrotropin-releasing hormone (TRH) gene and thus stimulates the production of TSH. The loss of weight causes the serum leptin and TSH levels to decrease and reduces the demand for levothyroxine. (SG (4 articles), RYGB (6 articles), biliopancreatic diversion (1 article), gastric banding 2 (article), and jejunoileal bypass (3 case reports)) [ 65 ]. 2.7. Antidiabetic Drugs More than 40% of patients with diabetes remission after gastric bypass surgery may redevelop diabetes within five years. Metformin is an oral antidiabetic, antihyperglycemic drug absorbed mainly in the upper part of the small intestine. When administered orally, it is characterized by low bioavailability of 29–60%. Padwal R. et al., studied changes in the pharmacokinetics of metformin on 16 non-diabetic post-gastric bypass patients. Surgical patients were examined ≥ 3 months after surgery. The control group consisted of 16 people selected in terms of sex and BMI (mean age 40 years and BMI 39.2 kg/m 2 ). All of them were given two 500 mg metformin tablets and then their plasma levels were measured. In comparison with the control group, the metformin AUC 0–∞ in the patients with a gastric bypass increased by 21% (13.7 vs. 11.4 μg/mL/h; p = 0.20), whereas bioavailability increased by 50% (41.8 vs. 27.8%; p = 0.007). The C max in the group of patients was 2.0 mg/mL, whereas in the control group it was 1.8 mg/mL ( p = 0.32). The results showed that the gastric bypass increased the metformin exposure, and this may cause the risk of toxicity. A change in the dosage of the drug should be considered [ 66 ]. However, Perrone et al., revealed that laparoscopic Roux-en-Y gastric bypass showed better effectiveness in type 2 diabetes mellitus resolution rate in comparison to laparoscopic sleeve gastrectomy [ 67 ]. 2.8. Loop Diuretics Furosemide is a loop diuretic drug. It is mainly absorbed in the stomach. Its peak diuretic effect occurs approximately one hour after oral administration. The bioavailability is extremely variable (10–90%). Furosemide is highly bound to plasma proteins (>95%). About 50% of furosemide is excreted in an unchanged form with urine, whereas the rest is metabolized to glucuronide by the kidneys. Patients with renal impairment exhibit a reduced response and a prolonged half-life of furosemide due to decreased urinary excretion [ 68 ]. Oral furosemide was administered to 13 RYGB patients and 14 healthy subjects (the authors do not specify the time of the examination after the surgery; the term "several" was used). The t max of furosemide was (1.8 ± 0.3 vs. 4.2 ± 1.2 h ( p = 0.006). However, there were no differences between the groups in the six-hour urine volume. The maximum plasma concentration and half-life were not different, either [ 69 ]. 2.9. Proton-Pump Inhibitors Omeprazole is a proton-pump inhibitor that effectively suppresses the secretion of gastric acid in the parietal cells. The drug is formulated as encapsulated granules to prevent its degradation in an acidic environment. Although omeprazole is well absorbed from the gastrointestinal tract, its oral bioavailability in humans is about 40–50%, which suggests its pronounced first pass metabolism. Oral omeprazole was administered to 18 RYGB recipients and 18 healthy subjects (the authors do not specify the time of the examination after the surgery; the term "several" was used). In comparison with the control group, the t max of omeprazole in the RYGB group was significantly shorter (1.1 ± 1.1 vs. 4.4 ± 1.3 h, p MIC ≥ 40%, the standard dosage of co-amoxiclav (1000/125 mg every 8 h) should be efficacious for obese adults [ 22 ], but Soares et al., suggested that amoxicillin treatment would fail if standard therapeutic regimens were applied because of a significantly higher volume of distribution in this group of patients [ 23 ]. In another study, the bioavailability of an oral AMX tablet and suspension was tested on patients who had undergone an RYGB surgery 3 months to 10 years before. The patients received an 875 mg AMX tablet or 800 mg AMX suspension. Twenty people with a body mass index of 29.88 ± 4.36 kg/m 2 were qualified for the study. The C max of AMX in the plasma of tablets and suspensions (normalized to 875 mg) was 7.42 ± 2.99 mg/L and 8.73 ± 3.26 mg/L (90% CI = 70.71–99.11), and the area under the plasma concentration versus time curve from time zero to infinity (AUC 0–∞ ) was 23.10 ± 7.41 mg⋠h/L and 27.59 ± 8.32 mg⋠h/L, respectively (Cl = 71.25–97.32). The values of these parameters were compared with the results noted in healthy subjects, as described in available literature. The healthy subjects received 875 mg AMX tablets (alone or in combination with clavulanic acid). The AUC 0–∞ and C max values increased from 43.80 to 51.29 mg⋠h/L and from 12.13 to 15.30 mg/L [ 24 ]. Padwal et al., conducted a study on the pharmacokinetics of azithromycin, which is a macrolide antibiotic with a broad spectrum of activity against various aerobic and anaerobic bacteria [ 25 ]. Azithromycin is preferentially absorbed in the duodenum and upper jejunum. The oral bioavailability of azithromycin in healthy subjects amounts to about 37% [ 26 ]. A total of 14 women who were at least 3 months post RYGB surgery and 14 healthy women (the control group) with matched body mass index (BMI) (mean age 44 years and BMI 36.4 kg/m 2 ) were administered a single dose of two 250 mg azithromycin tablets. The AUC 0–24 of the patients who had undergone the RYGB procedure was reduced by 32% (1.41 vs. 2.07 mg⋠h/L; p = 0.008), whereas the dose-normalized AUC 0–24 was reduced by 33% (0.27 vs. 0.40 kg⋠h/L; p = 0.009). The azithromycin C max of the patients after the RYGB surgery amounted to 0.260 mg/L, as compared with 0.363 mg/L in the control group ( p = 0.08) and it was reached after 2.14 h and 2.36 h ( p = 0.75), respectively. These results show that there is a possibility of early treatment failure. Therefore, modified dosage and closer clinical monitoring of gastric bypass patients receiving azithromycin should be considered [ 27 ]. The PK/PD relationship for azithromycin is AUC/MIC. Therefore, a lower AUC in patients after gastrectomy may cause treatment failure. Ciprofloxacin is a fluorinated quinolone antibiotic with high activity against a wide spectrum of Gram-positive and Gram-negative bacteria. Clinical trials with an orally administered ciprofloxacin preparation proved the effectiveness of this drug in the treatment of urinary tract infections, sexually transmitted infections, skin, bone and joint infections, prostatitis, typhoid fever, gastrointestinal infections, lower respiratory tract infections, anthrax, plague, and salmonellosis. Significant quantities of ciprofloxacin are absorbed after its oral administration. The drug is mainly absorbed in the upper part of the intestinal tract (duodenum, jejunum). The absolute bioavailability is about 70% [ 28 ]. Rivas et al., conducted a study on the pharmacokinetics of ciprofloxacin after its single administration to patients after an RYGB surgery. The study involved overweight and obese patients aged 18–60 years. The assessment was performed once in the control group and three times in the group of overweight and obese patients (first before the surgery and then one and six months after the surgery). The subjects received a single oral dose of 500 mg of ciprofloxacin at each visit. Taking the postoperative change in body weight into account, the parameters were adjusted according to the dose (mg)/body weight (kg). The ciprofloxacin C max decreased significantly one month after the surgery (1840.9 ± 485.2 vs. 1459.6 ± 354.8 ng/mL; p = 0.032), but not after six months (1840.9 ± 485.2 vs. 1589.6 ± 32.8 ng/mL; p = 0.116). The C max measured after sixth months was lower than the C max in the control group (2160.4 ± 408.6 vs. 1589.6 ± 321.8 ng/mL; p 40, G(−) = 100–125) [ 29 ]. In conclusion, by the sixth month, the effect on the C max and AUC 0–∞ had disappeared due to weight loss. There is no need to modify the doses of ciprofloxacin in these patients [ 30 ]. As results from the abovementioned studies, the values of pharmacokinetic parameters may be influenced by the time after bariatric surgery. Another conclusion concerns the form of the drug. It is important to note that the suspension and the tablet affect the pharmacokinetic parameters differently in patients after RYGB surgery. 2.2. Analgesic Drugs Acetaminophen is an analgesic drug of choice for patients after gastrectomy, even in oral formulations. This drug is mainly absorbed by passive transport in the small intestine [ 31 ]. The oral route of administration of acetaminophen increases the risk of postoperative nausea and vomiting, as compared with the intravenous route, but the efficacy of both routes of administration is comparable [ 32 ]. Porat et al., conducted a clinical, crossover study on the pharmacokinetics of paracetamol in obese patients enrolled for LSG. The patients received randomly 500 mg of paracetamol in a caplet or in syrup. The other form of the drug was administered after 1–2 weeks. The study was repeated 4–6 months after the surgery. The mean weight loss was 26 kg. The researchers observed that the AUC and C max were higher after these few weeks than before the surgery. The bioavailability of paracetamol increased twice and it was higher when administered as a liquid. The t 1/2 was longer after LSG. The changes in the pharmacokinetic parameters were associated with the patients' loss of weight. The bioavailability of acetaminophen in obese patients was much lower [ 17 ]. This may have been caused by an increase in the metabolic pathways (including glucuronidation) in these patients [ 33 ]. In addition, LSG accelerated gastric emptying, so the t max in the syrup group was shorter after the surgery [ 17 ]. The pharmacokinetics of paracetamol were also investigated in patients after total gastrectomy. Szałek et al., conducted a study comparing the pharmacokinetic parameters after the administration of two generic products. A group of 30 people after gastrectomy with Roux-en-Y reconstruction was divided into two groups. The participants received two tablets containing 500 mg of paracetamol each. The C max and AUC in both groups were lower than in healthy subjects. The t max and t 1/2 were similar to the values of these parameters measured in the volunteers without gastrectomy. The results suggest that total gastrectomy reduces the absorption of this drug [ 34 ]. Tramadol is another analgesic drug. It is indicated for the treatment of chronic and postoperative pain, renal and biliary colic, and trauma. Tramadol is a weak opioid. It is often used in combination with paracetamol at a dose of 37.5 mg tramadol and 325 mg paracetamol. This combination is in the form of conventional or effervescent tablets [ 35 ]. When administered orally, its absorption in the upper small intestine amounts to 95–100% [ 36 ]. The pharmacokinetics of these two forms of paracetamol and tramadol were investigated in patients after gastrectomy with Roux-en-Y reconstruction. A total of 26 patients were divided into two groups. The first group received two film-coated tablets, whereas the other group received two effervescent tablets. Each tablet contained 37.5 mg of tramadol and 325 mg of paracetamol. The C max of paracetamol administered orally as a conventional tablet and the C max of tramadol administered orally as an effervescent tablet were significantly lower than in healthy subjects. The t max of paracetamol administered in the form of effervescent tablets to the gastrectomy patients and the t max of tramadol in both groups were shorter. According to the researchers, this may have been caused by the shorter gastric emptying time. According to Szałek et al., conventional tablets are a better choice for patients after gastrectomy [ 35 ]. Ketoprofen belongs to the group of non-steroidal anti-inflammatory drugs. The indications for the use of this drug are postoperative pain, cancer, and rheumatoid arthritis. Ketoprofen is absorbed by passive diffusion in the stomach. It occurs in an undissociated form. Porażka et al., investigated the effect of gastrectomy on the pharmacokinetics of ketoprofen administered orally to two groups of patients. One group ( n = 15) consisted of patients after total stomach resection, whereas the other group ( n = 5) included patients after partial resection. All the participants received one film-coated tablet containing 100 mg of ketoprofen. The C max of the patients after total stomach resection was significantly lower than in the group of the patients after partial resection. According to the researchers, this may have been caused by reduced tablet disintegration and slower mixing of the gastric contents due to the smaller size of the stomach. Faster gastric emptying, higher gastric pH and a smaller absorption area may result in lower C max and t max in gastrectomy patients. The patients had higher V d (volume of distribution), most likely due to hypoalbuminaemia, which is a common symptom of gastric cancer. Gastrectomy patients may require higher doses of ketoprofen for effective pain relief [ 6 ]. Morphine is the most commonly used opioid to treat moderate to severe pain. After oral administration of the drug, its absorption from the gastrointestinal tract amounts to almost 100%. Morphine is a substrate of P-glycoprotein [ 37 ]. It is absorbed mainly in the upper part of the small intestine and, to a lesser extent, in the stomach. The absolute bioavailability of morphine is low (20–30%) due to the first pass effect [ 38 ]. A study was conducted on 30 patients to determine the effect of RYGB on the pharmacokinetics of this drug. Each patient received an oral dose of 30 mg of liquid morphine at each of three visits (7–30 days before the surgery, 7–15 days after the surgery, and 6 months after the surgery). The t max decreased, whereas the C max increased significantly. The AUC also increased. The study showed that RYGB significantly increased the rate of morphine absorption. The increase in the C max and AUC may also have been caused by reduced first pass metabolism and weight loss, because non-obese patients have less glucuronidation than obese ones. According to researchers, after RYGB patients should receive lower doses of morphine in the form of a solution before the surgery to reduce the risk of side effects. Sublingual, intranasal or gingival application of fentanyl can be an alternative to immediate-release forms of morphine [ 39 ]. Post-operative pain is also treated with oxycodone. This semi-synthetic opioid is stronger than morphine [ 40 ]. It is absorbed mainly in the small intestine [ 41 ]. Szałek et al., conducted a study on the pharmacokinetics of oxycodone in patients after total gastrectomy. A total of 24 patients received prolonged-release tablets containing 10 mg of oxycodone. The mean C max and systemic exposure of oxycodone in the men were higher than in the women. The t max of the patients after resection was slightly shorter than that of the healthy patients. This effect may have been caused by the shorter gastric emptying time. The C max was similar in both groups. The study showed that total gastrectomy did not affect the pharmacokinetics of oxycodone [ 40 ]. 2.3. Antidepressants About 30–50% of patients after bariatric surgery use psychotropic drugs, mostly antidepressants [ 42 ]. It is important to determine changes in the pharmacokinetics of these drugs after surgery to ensure the safety and effectiveness of therapy [ 43 ]. Escitalopram belongs to the group of selective serotonin reuptake inhibitors (SSRIs). It increases synaptic signalling. It is used to treat major depression and generalized anxiety disorder. It is rapidly absorbed when administered orally. Marzinke et al., conducted a study on the pharmacokinetics of escitalopram in patients after Roux-en-Y gastric bypass surgery. There were four obese patients who used 10 or 20 mg escitalopram once a day. Samples were taken two weeks before the surgery and two and six weeks after the surgery. The serum levels of the drug decreased after surgery. At the third visit, the drug levels were even lower than at the second visit. Obese patients have elevated levels of C-reactive protein (CRP), which indicates current inflammation. This may result in decreased activity of CYP 450 enzymes and a higher concentration of escitalopram before the bariatric surgery. Another reason may be altered absorption after the surgery [ 42 ]. Sertraline also belongs to the SSRI group. Apart from depression, it is also used to treat social phobia, obsessive-compulsive disorders, post-traumatic stress disorder, and panic disorder [ 44 ]. It is mainly absorbed in the duodenum. Roerig et al., conducted a study on five RYGB patients (9–15 months after the surgery) and five non-surgical patients as the control group. The aim of the study was to determine changes in the pharmacokinetics of sertraline after the bariatric surgery. All participants received a single 100 mg dose of sertraline. The AUC 0–10.5 and C max were significantly lower in the postoperative group. The t max did not differ significantly between the two groups [ 43 ]. Depression is also treated with duloxetine. This drug is also used to treat anxiety disorders and neuropathic pain. Roerig et al., conducted a study to determine the effect of Roux-en-Y gastric bypass on the pharmacokinetics of duloxetine. Ten patients who had undergone RYGB 9–15 months before and 10 volunteers from a control group received a single dose of 60 mg of duloxetine. The postoperative patients had significantly lower AUC 0–∞ and shorter t max than the control group. The differences in the C max and half-life were not clinically significant. The researchers speculated that the absorption of duloxetine was reduced as a result of surgery and the loss of the absorptive surface of the duodenum [ 45 ]. Venlafaxine is a norepinephrine reuptake inhibitor. It is used to treat depression, social anxiety disorder, generalized anxiety disorder, and panic disorder. It is available as an immediate-release and extended-release drug [ 46 ]. Ten RYGB patients were enrolled in a prospective study of venlafaxine pharmacokinetics. At least one week before and 3–4 months after the surgery, the participants received one 75 mg capsule of ER venlafaxine. The AUC 0–24 , C max , and t max values measured before and after the RYGB did not differ significantly. According to Krieger et al., gastric bypass surgery does not significantly affect the amount and time of venlafaxine absorption [ 47 ]. Vortioxetine is a multimodal serotonin modulator used to treat depression. It acts on the 5-HT1A, 5-HT1B, 5-HT3, and 5-HT7 receptors and inhibits the reuptake of serotonin. Vandenberghe et al., presented a case report of a patient who underwent RYGB and used vortioxetine regularly. The blood level of the drug was determined 126 and 200 days before surgery. The drug was administered at a dose of 10 mg/day. The vortioxetine levels were also measured 91 days after surgery. The concentration was more than twice lower than before the surgery. The dose was increased to 20 mg/day. On days 224 and 308 after the surgery, the concentration was similar to that in the preoperative period. Researchers recommend therapeutic drug monitoring and crushing tablets or using a liquid form of the drug in the case of poor absorption [ 48 ]. 2.4. Anticoagulant Drugs Rivaroxaban belongs to the group of direct oral anticoagulants (DOAC). It is an inhibitor of factor Xa. It is used to prevent venous thromboembolism [ 49 ]. Rivaroxaban is absorbed in the upper gastrointestinal tract. The drug is characterized by high oral bioavailability [ 50 ]. Kröll et al., conducted a study comparing the pharmacokinetic and pharmacodynamic parameters of rivaroxaban administered one day before and three days after bariatric surgery. Six sleeve gastrectomy patients and six Roux-en-Y gastric bypass patients participated in the study. All of them received 10 mg of rivaroxaban each time. A slight increase in the AUC was observed after both types of surgery. The C max was higher after SG and lower after RYGB than before the surgery. The t max increased in the patients after RYGB. According to the researchers, the bariatric surgery did not significantly affect the pharmacokinetic and pharmacodynamic parameters [ 49 ]. The influence of weight loss after bariatric surgery on the pharmacokinetics and pharmacodynamics of rivaroxaban was investigated on six post-SG and six post-RYGB patients, who received a single 10 mg dose of rivaroxaban 6–8 months after the surgery. The results were compared with the values measured before the surgery. Six months after the surgery the mean weight loss was over 34 kg. The postoperative t max was slightly longer than before the surgery. The C max was lower after the RYGB surgery, whereas the C max of the patients after SG was similar to the value measured before the surgery. Kröll et al., observed that weight loss and bariatric surgery did not significantly affect the pharmacokinetics and pharmacodynamics of rivaroxaban. The researchers assumed that changes in the body weight did not affect these parameters due to the high degree of plasma protein binding of this drug and its low volume of distribution [ 50 ]. Dabigatran is another drug from the DOAC group. It is used to treat and prevent venous thromboembolism and to prevent stroke and systemic embolism [ 51 ]. The drug is absorbed in the lower stomach and duodenum. Grainger et al., measured the pharmacokinetic and pharmacodynamic parameters (including C max ) of dabigatran in patients ( n = 9) after laparoscopic Roux-en-Y gastric bypass who regularly used this drug and compared them with the results from phase II studies. The drug concentration decreased significantly. According to the researchers, this may indicate impaired or delayed absorption [ 52 ]. Rottenstreich et al., observed that the C max of dabigatran in BS patients regularly using this drug was within the expected range [ 53 ]. Similarly to rivaroxaban, apixaban is also a direct factor Xa inhibitor. It is indicated to reduce the risk of stroke and thromboprophylaxis and to treat deep vein thrombosis and pulmonary embolism. Apixaban is mainly absorbed in the small intestine. Its bioavailability amounts to about 50% [ 54 ]. The study assessing the effect of BS on the level of DOAC included nine patients who took apixaban chronically. The blood levels of this drug were measured and compared with the values for the general population. The peak apixaban level was within the expected range [ 55 ]. Bitar et al., reported a failure of apixaban anticoagulant therapy in a patient who had undergone bariatric surgery four years before. The researchers suggested that the surgery may have contributed to the subtherapeutic level of the drug [ 55 ]. Warfarin is an antagonist of vitamin K. It reduces the activity of coagulation factors (II, VII, IX, X) and therefore it is used to prevent and treat thromboembolic disorders. Its efficacy is measured with the International Normalized Ratio (INR). Warfarin is absorbed in the proximal duodenum. Steffen et al., conducted a retrospective study to collect data on warfarin dosage in patients after RYGB surgery. The results measured six months before and after the surgery were analysed. The mean weekly doses before and after the surgery differed significantly. After the surgery the dose was reduced by approximately 25%. It was necessary to apply lower doses after than before the surgery to maintain the INR level. The cause of the changes in the warfarin parameters was not fully explained. According to the researchers, they may have been caused by changes in the consumption and storage of vitamin K, as well as changes in the bacterial flora [ 56 ]. Vitamin K antagonists are believed to be a better choice than DOAC for patients after BS, as they are easy to monitor and to make dose adjustments [ 53 ]. 2.5. Immunosuppressants Adequate modulation of the immune system after transplantation is essential for patient survival and prevents rejection of the transplanted organ. A three-drug regimen of corticosteroid, mycophenolate mofetil, and tacrolimus is the most common. Cyclosporine and sirolimus are also used in immunosuppressive therapy [ 57 ]. Only reliable absorption from the gastrointestinal tract can ensure adequate exposure and efficacy of these drugs [ 58 ]. Mycophenolate mofetil is mainly absorbed in the proximal gastrointestinal tract. It is a prodrug activated during first pass metabolism. Tacrolimus is available as an immediate-release (IR-TAC) and extended-release drug (ER-TAC). The bioavailability of both forms is relatively low. IR-TAC is absorbed from the duodenum to the colon, whereas ER-TAC is absorbed more distally in the gastrointestinal tract [ 59 ]. Ciclosporin is a drug with a narrow therapeutic index. As the absorption of the drug is highly variable, it is important to monitor its concentration. Absorption is incomplete and slow. It occurs mainly in the upper intestine [ 60 ]. Sirolimus, like tacrolimus, is mainly absorbed in the duodenum [ 61 ]. Its bioavailability is low (approximately 25%) [ 62 ]. Chan et al., conducted a prospective study on changes in the pharmacokinetics of immunosuppressants. Twelve patients with end-stage renal disease were involved. The pharmacokinetic parameters after administration of a single dose of tacrolimus, extended-release tacrolimus, mycophenolate mofetil, and enteric-coated mycophenolate sodium (EC-MPS) were measured two months before and 9–12 months after LSG. First, 3 mg of IR-TAC and 720 mg of EC-MPS were administered orally. Two weeks later, the patients received orally 6 mg of ER-TAC and 1000 mg of mycophenolate mofetil (MFF). The median excess body weight loss (EBWL) was 26.5 kg. The study revealed a significant increase in the exposure to all four drugs after LSG. The exposure to tacrolimus was reduced in obese patients. After weight loss, the AUC and C max of both tacrolimus forms were significantly higher. The t max and t 1/2 did not change significantly. The researchers speculated that the increased exposure to tacrolimus was caused by a decrease in the P-glycoprotein expression. They also suggested that accelerated gastric emptying after the surgery resulted in earlier delivery of tacrolimus to the proximal intestine, which also increased exposure to this drug. After LSG, the apparent total plasma clearance (Cl/F) of both forms of mycophenolate mofetil decreased by about 60%. According to the researchers, the UGT activity decreased due to weight loss. The MFF t 1/2 was several times longer after LSG. The t max of EC-MPS decreased by over 30%. It is recommended to monitor immunosuppression in patients after LSG [ 63 ]. The pharmacokinetics of immunosuppressants were also investigated on patients after Roux-en-Y gastric bypass (approximately 2 months to 8 years after surgery). The study involved four dialysis patients and two kidney transplant patients. All the dialysis patients received orally one dose of sirolimus (6 mg), two doses of MMF (1000 mg), and two doses of tacrolimus (4 mg) within 24 h. The patients after transplantation remained on their maintenance regimen. The pharmacokinetic parameters were compared with the results of other studies. The comparison of the AUC:dose ratio of the patients under study with the ratio of healthy volunteers showed that after gastric bypass surgery, a higher dose of sirolimus was necessary to achieve the same exposure as in healthy subjects. The patients with a gastric bypass had much lower AUC 0–12 and AUC 0–∞ than the healthy volunteers. As with sirolimus, the study showed that patients after bariatric surgery require a higher dose of tacrolimus. According to the researchers, this was due to reduced absorption in the small intestine. The C max and mean AUC 0–12 MPA of the patients under study were lower, which may have been caused by the reduced absorption area [ 61 ]. Chen et al., presented a case study of a patient after total gastrectomy with Roux-en-Y reconstruction with end-stage renal disease. The gastrectomy was done 5 years earlier. The patient had pharmacokinetic tests before transplantation to select the most appropriate immunosuppressive regimen. Five different treatment regimens, including EC-MPS, MMF, cyclosporine, tacrolimus, and sirolimus were tested. The regimens were tested sequentially and steady-state concentrations were obtained each time. The measured values were compared with the reference group without gastrectomy. The C max of ciclosporin and the C max and AUC of tacrolimus were higher than in the patients without gastrectomy. The patient 5 years after gastrectomy was characterized by good absorption of the drugs under study. The rate and extent of sirolimus and MPA absorption from EC-MPS was similar to the reference group. The MPA from MFF was absorbed worse than from EC-MPS [ 58 ]. A case of a patient who underwent liver transplantation and sleeve gastrectomy at the same time was also described. The patient received tacrolimus and everolimus. The appropriate level of immunosuppression was maintained. No drug absorption problems were observed. According to Tariciotti et al., it is beneficial for the patient to carry out these two treatments at the same time [ 64 ]. 2.6. Thyroid Hormones Gadiraju S. et al., conducted a meta-analysis of the effect of the type of bariatric surgery on the dosage of levothyroxine. Levothyroxine is a synthetic thyroid hormone used to treat diseases of the thyroid gland resulting from thyroxine deficiency. The drug is mainly absorbed in the jejunum and ileum. This fact suggests a higher demand for levothyroxine after jejunoileal bypass surgery. Although the jejunoileal segment of the small intestine remains intact for RYGB, SG and gastric banding, there are alternate variations to these procedures, which may result in an increased demand for levothyroxine. Probably, the small gastric sac in these procedures reduces the dissolution of levothyroxine in the stomach and results in an increased t max after RYGB. SG accelerates gastric emptying, which may contribute to the malabsorption of levothyroxine. However, most patients after RYGB and SG have a lower demand for levothyroxine. Presumably, this could be explained by the correlation between a change in the body weight and a change in levothyroxine dosage. In obesity, highly lipophilic drugs such as levothyroxine have an increased volume of distribution, which changes their pharmacokinetics. Probably, weight loss after bariatric surgery regulates the pharmacokinetics and results in a lower demand for levothyroxine. Obese patients have not only a greater mass of the adipose tissue but also a higher lean body mass, which is responsible for 20–40% of the increase in the total body weight. T4 is converted into T3 in the skeletal muscles, so it is likely that the reduction in the lean body mass after bariatric surgery results in a lower postoperative demand for levothyroxine. Lower serum leptin levels may also decrease the demand for levothyroxine in the postoperative period. Leptin regulates the expression of the thyrotropin-releasing hormone (TRH) gene and thus stimulates the production of TSH. The loss of weight causes the serum leptin and TSH levels to decrease and reduces the demand for levothyroxine. (SG (4 articles), RYGB (6 articles), biliopancreatic diversion (1 article), gastric banding 2 (article), and jejunoileal bypass (3 case reports)) [ 65 ]. 2.7. Antidiabetic Drugs More than 40% of patients with diabetes remission after gastric bypass surgery may redevelop diabetes within five years. Metformin is an oral antidiabetic, antihyperglycemic drug absorbed mainly in the upper part of the small intestine. When administered orally, it is characterized by low bioavailability of 29–60%. Padwal R. et al., studied changes in the pharmacokinetics of metformin on 16 non-diabetic post-gastric bypass patients. Surgical patients were examined ≥ 3 months after surgery. The control group consisted of 16 people selected in terms of sex and BMI (mean age 40 years and BMI 39.2 kg/m 2 ). All of them were given two 500 mg metformin tablets and then their plasma levels were measured. In comparison with the control group, the metformin AUC 0–∞ in the patients with a gastric bypass increased by 21% (13.7 vs. 11.4 μg/mL/h; p = 0.20), whereas bioavailability increased by 50% (41.8 vs. 27.8%; p = 0.007). The C max in the group of patients was 2.0 mg/mL, whereas in the control group it was 1.8 mg/mL ( p = 0.32). The results showed that the gastric bypass increased the metformin exposure, and this may cause the risk of toxicity. A change in the dosage of the drug should be considered [ 66 ]. However, Perrone et al., revealed that laparoscopic Roux-en-Y gastric bypass showed better effectiveness in type 2 diabetes mellitus resolution rate in comparison to laparoscopic sleeve gastrectomy [ 67 ]. 2.8. Loop Diuretics Furosemide is a loop diuretic drug. It is mainly absorbed in the stomach. Its peak diuretic effect occurs approximately one hour after oral administration. The bioavailability is extremely variable (10–90%). Furosemide is highly bound to plasma proteins (>95%). About 50% of furosemide is excreted in an unchanged form with urine, whereas the rest is metabolized to glucuronide by the kidneys. Patients with renal impairment exhibit a reduced response and a prolonged half-life of furosemide due to decreased urinary excretion [ 68 ]. Oral furosemide was administered to 13 RYGB patients and 14 healthy subjects (the authors do not specify the time of the examination after the surgery; the term "several" was used). The t max of furosemide was (1.8 ± 0.3 vs. 4.2 ± 1.2 h ( p = 0.006). However, there were no differences between the groups in the six-hour urine volume. The maximum plasma concentration and half-life were not different, either [ 69 ]. 2.9. Proton-Pump Inhibitors Omeprazole is a proton-pump inhibitor that effectively suppresses the secretion of gastric acid in the parietal cells. The drug is formulated as encapsulated granules to prevent its degradation in an acidic environment. Although omeprazole is well absorbed from the gastrointestinal tract, its oral bioavailability in humans is about 40–50%, which suggests its pronounced first pass metabolism. Oral omeprazole was administered to 18 RYGB recipients and 18 healthy subjects (the authors do not specify the time of the examination after the surgery; the term "several" was used). In comparison with the control group, the t max of omeprazole in the RYGB group was significantly shorter (1.1 ± 1.1 vs. 4.4 ± 1.3 h, p < 0.0001). The maximum plasma concentration, half-life, area under the curve, and oral bioavailability were not different [ 69 , 70 ]. 2.10. Vitamins Vitamin D maintains calcium homeostasis and optimizes bone mineralization. Prolonged vitamin D deficiency leads to hypocalcemia, osteopenia, and osteoporosis. During the first month after RYGB surgery there are higher 25-hydroxyvitamin D concentrations, which decrease in the following months. These observations suggest increased storage and sequestration of vitamin D by the adipose tissue with its simultaneous release during the initial weight loss. After BPD/DS there is also a progressive increase in the incidence and severity of vitamin D deficiency. Due to the increased risk of metabolic bone disease in patients after bariatric surgery, lifetime prophylaxis consisting of oral vitamin D supplementation is recommended [ 71 ]. Vitamin B12 is responsible for the proper function of the nervous system. The development of vitamin B12 deficiency in patients after bariatric surgery is mainly caused by reduced production of the intrinsic factor by a limited number of parietal cells. In consequence, there is reduced formation and absorption of the cobalamin-intrinsic factor complex. Purely restrictive surgery does not result in significant deficiency of any of the nutrients. However, about a third of patients undergoing mixed procedures such as RYGB develop vitamin B12 deficiency [ 71 ]. A comparative study of RYGB and SG showed that the risk of vitamin B12 deficiency was 3.55-times higher after RYGB than after SG [ 72 ]. Similarly, folate deficiency commonly occurs after bariatric surgery—according to reports, its incidence after RYCB is 45%. Vitamin B12 and folic acid supplementation are recommended to patients after bariatric surgery [ 71 ]. Bariatric procedures and other mixed treatments may result in malabsorption of fats due to a biliary pancreatic lesion. In consequence, there is significant deficiency of fat-soluble vitamins. The researchers observed that one year after BPD/DS, 52% of the patients under study had vitamin A deficiency, whereas 51% had vitamin K deficiency. Pre- and postoperative fat-soluble vitamin assessment and routine supplementation are recommended [ 71 ]. 2.11. Mineral Elements Due to impaired nutrient absorption and/or reduced food intake, patients after bariatric surgery develop nutritional deficiencies, which may lead to anemia and osteoporosis. Anemia occurs much more often after RYGB (45–50%) than after SG (17%). In most cases it is caused by iron deficiency. This microelement is absorbed mainly in the duodenum and proximal jejunum. Iron deficiency is mainly caused by the bypass of these parts of the gastrointestinal tract and by hypochlorhydria [ 73 ]. Enani et al., observed an incidence of iron deficiency of 24.5% after RYGB and 12.4% after SG [ 74 ]. The American Society for Metabolic and Bariatric Surgery recommended iron supplementation to all bariatric patients. The effectiveness of iron supplementation can be increased by combining iron with vitamin C or citrus fruit [ 75 ]. Reduced bone mineral density and increased bone turnover are some of the consequences of dietary restrictions and bariatric surgery. Researchers observed that the bone loss after RYGB was greater than after SG. The bone loss is mainly caused by calcium deficiency, which affects almost 10% of bariatric patients. These patients exhibit reduced calcium absorption because the main sites of absorption of this element (the duodenum and proximal jejunum) are bypassed [ 73 ]. Schafer et al., conducted a study to determine the effect of Roux-en-Y gastric bypass surgery on intestinal fractional Ca absorption (FCA) ( n = 33). Despite the recommended daily calcium intake of 1200 mg, the FCA was significantly lower after RYGB. Before the surgery it was 32.07%, whereas six months after the surgery it dropped to 6.9%. Researchers suggest that bariatric patients may require a higher dose of calcium than recommended [ 76 ]. 3. Conclusions Table 2 lists changes in the pharmacokinetic parameters observed after bariatric surgery and gastrectomy.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8277669/

Salmonella Typhimurium manipulates macrophage cholesterol homeostasis through the SseJ-mediated suppression of the host cholesterol transport protein ABCA1

Upon infection of host cells, Salmonella enterica serovar Typhimurium resides in a modified endosomal compartment referred to as the Salmonella -containing vacuole (SCV). SCV biogenesis is driven by multiple effector proteins translocated through two type III secretion systems (T3SS-1 and T3SS-2). While many host proteins targeted by these effector proteins have been characterized, the role of host lipids in SCV dynamics remains poorly understood. Previous studies have shown that S. Typhimurium infection in macrophages leads to accumulation of intracellular cholesterol, some of which concentrates in and around SCVs; however, the underlying mechanisms remain unknown. Here, we show that S. Typhimurium utilizes the T3SS-2 effector SseJ to downregulate expression of the host cholesterol transporter ABCA1 in macrophages, leading to a ~45% increase in cellular cholesterol. Mechanistically, SseJ activates a signaling cascade involving the host kinases FAK and Akt to suppress Abca1 expression. Mutational inactivation of SseJ acyltransferase activity, silencing FAK, or inhibiting Akt prevents Abca1 downregulation and the corresponding accumulation of cholesterol during infection. Importantly, RNAi-mediated silencing of ABCA1 rescued bacterial survival in FAK-deficient macrophages, suggesting that Abca1 downregulation and cholesterol accumulation are important for intracellular survival.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4958561/

Studies of DNA Aptamer OliGreen and PicoGreen Fluorescence Interactions in Buffer and Serum

Spectrofluorometric and emission peak titration and timed studies of OliGreen (OG) and PicoGreen (PG) were conducted in Tris EDTA (TE) buffer, pooled rat and fetal bovine serum with two different aptamers of 72 and 192 bases in length to determine if OG or PG were suitable for aptamer pharmacokinetic (PK) studies in sera. Results indicated that OG and PG detected the single-stranded (ss) and double- stranded (ds) stem-loop structures of the two aptamers quite well in TE with reliable standard curves having exponential character (or several linear detection regions) up to 1 μg/ml of aptamer DNA with detection limits of ~ 1 ng/ml. The intensity of OG and PG staining appeared to correlate with the number and percentage of ss and ds bases in each aptamer. OG and PG fluorescence in pooled rat serum or fetal bovine serum (FBS) did not titer as a function of DNA aptamer concentration from 1 μg/ml to 1 ng/ml. This lack of OG or PG aptamer assays in serum is contrary to most published reports of OG or PG assays for ss antisense oligonucleotides, ds PCR amplicons or other types of DNA in serum or plasma. Further studies suggested that the lack of OG and PG assay titration in serum might not be entirely due to aptamer degradation from nucleases in serum since the fluorescence signals in serum appeared relatively stable over time from 30 minutes to four hours. A hypothesis is presented which attributes the inability of OG or PG to assay aptamers in serum to a combination of high blue-green autofluorescence in serum with possible serum nuclease degradation of aptamers over time and the changing aptamer to serum protein ratio coupled to nonspecific binding of serum proteins to aptamers thereby possibly changing aptamer conformations as a function of aptamer concentration during titration experiments.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861093/

Algorithms for detecting and predicting influenza outbreaks: metanarrative review of prospective evaluations

Objectives Reliable monitoring of influenza seasons and pandemic outbreaks is essential for response planning, but compilations of reports on detection and prediction algorithm performance in influenza control practice are largely missing. The aim of this study is to perform a metanarrative review of prospective evaluations of influenza outbreak detection and prediction algorithms restricted settings where authentic surveillance data have been used. Design The study was performed as a metanarrative review. An electronic literature search was performed, papers selected and qualitative and semiquantitative content analyses were conducted. For data extraction and interpretations, researcher triangulation was used for quality assurance. Results Eight prospective evaluations were found that used authentic surveillance data: three studies evaluating detection and five studies evaluating prediction. The methodological perspectives and experiences from the evaluations were found to have been reported in narrative formats representing biodefence informatics and health policy research, respectively. The biodefence informatics narrative having an emphasis on verification of technically and mathematically sound algorithms constituted a large part of the reporting. Four evaluations were reported as health policy research narratives, thus formulated in a manner that allows the results to qualify as policy evidence. Conclusions Awareness of the narrative format in which results are reported is essential when interpreting algorithm evaluations from an infectious disease control practice perspective. Objectives Reliable monitoring of influenza seasons and pandemic outbreaks is essential for response planning, but compilations of reports on detection and prediction algorithm performance in influenza control practice are largely missing. The aim of this study is to perform a metanarrative review of prospective evaluations of influenza outbreak detection and prediction algorithms restricted settings where authentic surveillance data have been used. Design The study was performed as a metanarrative review. An electronic literature search was performed, papers selected and qualitative and semiquantitative content analyses were conducted. For data extraction and interpretations, researcher triangulation was used for quality assurance. Results Eight prospective evaluations were found that used authentic surveillance data: three studies evaluating detection and five studies evaluating prediction. The methodological perspectives and experiences from the evaluations were found to have been reported in narrative formats representing biodefence informatics and health policy research, respectively. The biodefence informatics narrative having an emphasis on verification of technically and mathematically sound algorithms constituted a large part of the reporting. Four evaluations were reported as health policy research narratives, thus formulated in a manner that allows the results to qualify as policy evidence. Conclusions Awareness of the narrative format in which results are reported is essential when interpreting algorithm evaluations from an infectious disease control practice perspective. Introduction Experiences from winter influenza seasons 1 and the pandemic pH1N1 outbreak in 2009 2 suggest that existing information systems used for detecting and predicting outbreaks and informing situational awareness show deficiencies when under heavy demand. Public health specialists seek more effective and equitable response systems, but methodological problems frequently limit the usefulness of novel approaches. 3 In these biosurveillance systems, algorithms for outbreak detection and prediction are essential components. 4 5 Regarding outbreak detection, characteristics influential for successful performance include representativeness of data and the type and specificity of the outbreak detection algorithm, while influential outbreak characteristics comprise the magnitude and shape of the signal and the timing of the outbreak. 6 After detection, mathematical models can be used to predict the progress of an outbreak and lead to the identification of thresholds that determine whether an outbreak will dissipate or develop into an epidemic. However, it has been pointed out that present prediction models have often been designed for particular situations using the data that are available and making assumptions where data are lacking. 7 8 In consequence, also biosurveillance models that have been subject to evaluation seldom produce output that fulfils standard criteria for operational readiness. 9 For instance, a recent scoping review of influenza forecasting methods assessed studies that validated models against independent data. 10 Use of independent data is vital for predictive model validation, because using the same data for model fitting and testing inflates estimates of predictive performance. 11 The review concluded that the outcomes predicted and metrics used in validations varied considerably, which limited the possibility to formulate recommendations. Building on these experiences, we set out to perform a metanarrative review of evaluations of influenza outbreak detection and prediction algorithms. To ensure that the review results can be used to inform operational readiness, we restricted the scope to settings where authentic prospective surveillance data had been used for the evaluation. Methods A metanarrative review 12 was conducted to assess publications that prospectively evaluated algorithms for the detection or short-term prediction of influenza outbreaks based on routinely collected data. A metanarrative review was conducted because it is suitable for addressing the question 'what works?', and also to elucidate a complex topic, highlighting the strengths and limitations of different research approaches to that topic. 13 Metanarrative reviews look at how particular research traditions have unfolded over time and shaped the kind of questions being asked and the methods used to answer them. They inspect the range of approaches to studying an issue, interpret and produce an account of the development of these separate 'metanarratives' and then form an overarching metanarrative summary. The principles of pragmatism (inclusion criteria are guided by what is considered to be useful to the audience), pluralism (the topic is illuminated from multiple perspectives; only research that lacks rigour is rejected), historicity (research traditions are described as they unfold over time), contestation (conflicting data are examined to generate higher order insights), reflexivity (reviewers continually reflect on the emerging findings) and peer review were applied in the analysis. 12 Four steps were taken: an electronic literature search was carried out, papers were selected, data from these papers were extracted and qualitative and semiquantitative content analyses were conducted. For data extraction and analyses, researcher triangulation (involving several researchers with different backgrounds) was used as a strategy for quality assurance. All steps were documented and managed electronically using a database. To be included in the review, an evaluation study had to apply an outbreak detection or prediction algorithm to authentic data prospectively collected to detect or predict naturally occurring influenza outbreaks among humans. Following the inclusive approach of the metanarrative review methodology, studies using clinical and laboratory diagnosis of influenza for case verification were included. 14 For the evaluations of the prediction algorithms, correlation analyses were also accepted, because interventions could have been implemented during the evaluation period. In addition, studies were required to compare syndromic data with some gold standard data from known outbreaks. All studies published from 1 January 1998 to 31 January 2016 were considered. PubMed was searched using the following search term combinations: 'influenza AND ((syndromic surveillance) OR (outbreak detection OR outbreak prediction OR real-time prediction OR real-time estimation OR real-time estimation of R))'. The database searches were conducted in February 2016. Only articles and book chapters available in the English language were selected for further analysis. To describe the characteristics of the selected papers, information was documented regarding the main objective, the publication type, whether syndromic data were used, country, algorithm applied and context of application. Information about the papers was analysed semiquantitatively by grouping papers with equal or similar characteristics and by counting the number of papers per group. In the next step, text passages, that is, sentences or paragraphs containing key terms (study aims, algorithm description and application context) were extracted and entered into the database. If necessary, sentences before and after a statement containing the key terms were added to ensure that the meaning and context were not lost. The documentation of data about the papers and the extraction of text were conducted by one reviewer and critically rechecked by a second reviewer. Next, content analysis of the extracted text was performed. The meaning of the original text was condensed. The condensed statements contained as much information as necessary to adequately represent the meaning of the text in relation to the research aim, but were as short and simple as possible to enable straightforward processing. If the original text contained several pieces of information, then a separate condensed statement was created for each piece of information. To analyse the information contained in the papers, a coding scheme was developed inductively. Also, a semantical system was developed to facilitate interpretation of algorithm performance. Values for the area under the curve (AUC) exceeding 0.90, 0.80 and 0.70, respectively, were chosen to denote very strong (outstanding), strong (excellent) and acceptable performance. 15 The same limits are used to interpret the area under the weighted receiver operating characteristic curve (AUWROC) and volume under the time-ROC surface (VUTROC) metrics. Sensitivity, specificity and positive predictive value (PPV) limits were set at 0.95, 0.90 and 0.85, respectively, when weekly data were analysed, and 0.90, 0.85 and 0.80 when daily data were analysed, denoting very strong (outstanding), strong (excellent) and acceptable discriminatory performance. To interpret the strength of correlations, limit values were modified from the Cohen scale. 16 This scale defines small, medium and large effect sizes as 0.10, 0.30 and 0.50, respectively. The limits for the present study were set at 0.90, 0.80 and 0.70 for analyses of weekly data, and 0.85, 0.75 and 0.65 for daily data, denoting very strong (outstanding), strong (excellent) and acceptable predictive performance. A summary of the sematic system is provided in table 1 . Table 1 Summary of semantic system used to interpret algorithm performance Performance Measurement Outstanding Excellent Acceptable Outbreak detection and prediction AUC, AUWROC, VUTROC 0.90 0.80 0.70 Sensitivity, specificity, PPV (weekly) 0.95 0.90 0.85 Sensitivity, specificity, PPV (daily) 0.90 0.85 0.80 Only outbreak prediction Pearson's correlation (weekly) 0.90 0.80 0.70 Pearson's correlation (daily) 0.85 0.75 0.65 AUC, area under the curve; AUWROC, area under the weighted receiver operating characteristic curve; PPV, positive predictive value; VUTROC, volume under the time-ROC surface. Condensed statements could be labelled with more than one code. The creation of the condensed statements and their coding was carried out by one reviewer and rechecked by a second reviewer. Preliminary versions were compared and agreed upon, which resulted in final versions of the condensed statements and coding. The information about the detection and prediction algorithms was summarised qualitatively in tables and analysed semiquantitatively on the basis of the coding. Next analysis phase consisted of identifying the key dimensions of algorithm evaluations, providing a narrative account of the contribution of each dimension and explaining conflicting findings. The resulting two narratives (biodefence informatics and health policy research) are presented using descriptive statistics and narratively without quantitative pooling. In the last step, a wider research team and policy leaders (n=11) with backgrounds in public health, computer science, statistics, social sciences and cognitive science were engaged in a process of testing the findings against their expectations and experience, and their feedback was used to guide further reflection and analysis. The final report was compiled following this feedback. Results The search identified eight studies reporting prospective algorithm performance based on data from naturally occurring influenza outbreaks: three studies 17–19 evaluated one or more outbreak detection algorithms and five 20–24 evaluated prediction algorithms ( figure 1 ). Figure 1 Flow chart of the paper selection process. Additional reasons for exclusion (*) included that the case definition did not comprise at least a clinical diagnosis of influenza or influenza-like illness. Regarding outbreak detection, outstanding algorithm performance was reported from a Spanish study 18 for two versions of algorithms based on hidden Markov models and Serfling regression ( table 2 ). Simple regression was reported to show poor performance in this study. The same technique displayed excellent performance on US influenza data in a study comparing algorithm performances on data from two continents, as did time-series analysis and the statistical process control method based on cumulative sum (CUSUM). 19 However, the performance of these three algorithms was found to be poor to acceptable when applied on Hong Kong data in the latter study. Table 2 Evaluation algorithms include in the metanarrative review and their absolute and relative performance Study Algorithm Modification Temporal Absolute performance Relative performance Outbreak detection Closas et al 17 Kolmogorov-Smirnov test Weekly Acceptable (sensitivity 1.00; specificity 0.88) No comparisons Martínez-Beneito et al 18 Markov model (hidden) V.1 Weekly Outstanding (AUWROC 0.97–0.98) Markov model (switching)>Markov model (hidden)>regression (Serfling)>CUSUM>regression (simple) Regression (Serfling) Outstanding (AUWROC 0.93) Markov model (switching)>Markov model (hidden)>regression (Serfling)>CUSUM>regression (simple) Markov model (hidden) V.2 Outstanding (AUWROC 0.93–0.95) Markov model (switching)>Markov model (hidden)>regression (Serfling)>CUSUM>regression (simple) Regression (simple) Poor (AUWROC 0.57) Markov model (switching)>Markov model (hidden)>regression (Serfling)>CUSUM>regression (simple) SPC (CUSUM) Poor (AUWROC 0.65–0.70) Markov model (switching)>Markov model (hidden)>regression (Serfling)>CUSUM>regression (simple) Cowling et al 19 Time series, dynamic linear model Different parameter combinations tested. W represents the assumed smoothness of the underlying system. Range: 0.025, 0.050, 0.075 or 0.100 Weekly Hong Kong: acceptable (VUTROC 0.77, sensitivity 1.00, timeliness 1.40†weeks), with fixed specificity=0.95 USA: excellent (VUTROC 0.81, sensitivity 1.00, timeliness 0.75†weeks), with fixed specificity=0.95 Hong Kong data: time series (dynamic linear model)>regression (simple)>CUSUM US data: time series (dynamic linear model)>CUSUM>regression (simple) Regression (simple) Different parameter combinations tested. m represents the number of prior weeks used to calculate the running mean and variance. Range: 3, 5, 7 or 9 Hong Kong: acceptable (VUTROC 0.75, sensitivity 1.00, timeliness 1.72†weeks), with fixed specificity=0.95 USA: excellent (VUTROC 0.81, sensitivity 0.90, timeliness 1.45†weeks), with fixed specificity=0.95 Hong Kong data: time series (dynamic linear model)>regression (simple)>CUSUMUS data: time series (dynamic linear model)>CUSUM>regression (simple) SPC (CUSUM) Different parameter combinations tested. d represents the number of weeks t separating the baseline and the index day of the outbreak. Range: 2 or 3. k represents the minimum standardised difference. Range: 1 or 2 Hong Kong: poor (VUTROC 0.56, sensitivity 0.86, timeliness 2.00†weeks), with fixed specificity=0.95 USA: excellent (VUTROC 0.90, sensitivity 0.82, timeliness 1.51†weeks), with fixed specificity=0.95 Hong Kong data: time series (dynamic linear model)>regression (simple)>CUSUMUS data: time series (dynamic linear model)>CUSUM>regression (simple) Outbreak prediction Timpka et al 20 Shewhart type Daily and weekly Pandemic outbreak: poor (AUC 0.84; PPV 0.58) on a daily basis and poor (at most acceptable) (AUC 0.78; PPV 0.79) on a weekly basis Seasonal outbreaks: outstanding (AUC 0.89; PPV 0.93) on a daily basis and excellent (AUC 0.83; PPV 1.00) on a weekly basis No comparisons Yuan et al 21 Multiple linear regression Monthly NA. Limits not defined for the adjusted metrics of residuals used (APE) No comparisons Jiang et al 22 Bayesian network Daily Outstanding (r=0.97, prediction on day 13; r=0.94, prediction on day 22) No comparisons Burkom et al 23 Regression (log-linear, non-adaptive) Non-adaptive Daily NA. Limits not defined for the adjusted metrics of residuals used (MAD, MedAPE) Ten series of case count data: Holt-Winters>regression (log-linear, adaptive)>regression (log-linear, non-adaptive) Regression (log-linear, adaptive) Adaptive Ten series of case count data: Holt-Winters>regression (log-linear, adaptive)>regression (log-linear, non-adaptive) Holt-Winters (generalised exponential smoothing) Ten series of case count data: Holt-Winters>regression (log-linear, adaptive)>regression (log-linear, non-adaptive) Viboud et al 24 Method of analogues (non-parametric time-series forecasting method) Weekly From poor (r=0.66, for 10-week-ahead prediction) to excellent (r=0.81, for 1-week-ahead prediction) Method of analogues>autoregressive model (linear)>Stone's naive method Autoregressive model (linear) From poor (r=–0.07, for 10-week-ahead prediction) to acceptable (r=0.73, for 1-week-ahead prediction) Method of analogues>autoregressive model (linear)>Stone's naive method The naive method Poor (r=–0.09, for 10-week-ahead prediction; r=0.65, for 1-week-ahead prediction) Method of analogues>autoregressive model (linear)>Stone's naive method APE, absolute percentage error; AUC, area under the curve; AUWROC, area under the weighted receiver operating characteristic curve; CUSUM, cumulative sum; MAD, median absolute residual; MedAPE, median absolute percentage error; NA, not applicable; PPV, positive predictive value; SPC, statistical process control; VUTROC, volume under the time-ROC surface. Regarding prediction algorithms, a French study predicted national-level influenza outbreaks over 18 seasons, 24 observing excellent performance for a non-parametric time-series method in 1-week-ahead predictions and poor performance in 10-week-ahead predictions. A study using county-level data from the USA 22 reported outstanding predictive performance for a Bayesian network algorithm. However, the predictions in that study were made on days 13 and 22 of one single ongoing outbreak. Another study using telenursing data from a Swedish county to predict influenza outbreaks over three seasons, including the H1N1 pandemic in 2009, showed outstanding performance for seasonal influenza outbreaks on a daily basis and excellent performance on a weekly basis. 20 However, the performance for the pandemic was poor on a daily and on a weekly basis (see online supplementary material file). 10.1136/bmjopen-2015-010683.supp1 Supplementary data An explanation of the apparent diversity of evaluation methods and findings is that the methodological perspectives and experiences from algorithm evaluations were reported in two distinct narrative formats. These narrative formats can be interpreted to represent biodefence informatics and health policy research, respectively ( table 3 ). Table 3 Summary of narrative characteristics Narrative Storyline Intended audience* Learning period dilemma Theoretical proofs Population descriptions End point measures Biodefence informatics 17 18 22 23 System verification Engineers and modellers Irregular attention Included in argument Summary Various statistical Health policy research 19–21 24 System validation Policymakers Binding attention Excluded Extensive Standard epidemiological *In addition to researchers. The biodefence informatics narrative Assessments informing construction of technically and mathematically sound algorithms for outbreak detection and prediction were reported from mathematical modelling and health informatics contexts. Research in these fields was described in a biodefence informatics narrative. The setting for this narrative is formative evaluation and justification of algorithms for detection and prediction of atypical outbreaks of infectious diseases and bioterror attacks. In other words, these studies can be said to answer the system verification question: 'Did we build the system right?' 25 The narrative is set in a context where algorithms need to be modified and assured for detection and prediction of microbiological agents with unusual or unknown characteristics, for example, novel influenza virus strains or anthrax. 26 The number of studies presented in the biodefence informatics narrative grew rapidly after the terrorist attacks in 2001. 27 Reporting of influenza algorithm performance in this narrative is characterised by presentation of statistical or technical advancements, for example, making use of increments instead of rates or introduction of methods based on Markov models. 18 As empirical data for logical reasons are scarce in biodefence settings, limited attention is in this narrative paid to the learning period dilemma. This dilemma represents a generic methodological challenge in algorithm development, that is, the statistical associations between indicative observations and the events to be predicted are determined in one time interval (the learning period) and used to predict the occurrence of corresponding events in a later interval (the evaluation period). 28 When trying to detect or predict a novel infectious agent, the learning period dilemma primarily shows unavailability of learning data for calibration of model-based algorithms. For instance, for prediction algorithms based on the reproductive number, 29 series of learning data of sufficient length for empirical determination of the serial interval cannot be made available during early outbreak stages, implying that the method cannot be used as supposed. 30 Moreover, the microbiological features of the pathogen and the environmental conditions in effect during the learning period can change after the algorithm has been defined, requiring adjustments of algorithm components and parameters to be made for preserving the predictive performance. Algorithm performance can in the biodefence informatics be narrative verified by combining prospective evaluations with formal proofs and analyses of simulated and retrospective data. Although it is commonly emphasised that the evaluation results are preliminary with regard to population outcomes, 22 the evaluation results are still included in the narrative. The health policy research narrative For evaluation study results to qualify as input to recommendations regarding infectious disease control practice, they should conform to general criteria established for health policy evidence. The analyses must be unbiased and not open for manipulation, for example, the data sources and analytic models should be described and fixed before data are accessed for analyses. 31 In the corresponding research paradigm, the use of prospective study designs is regarded as the cornerstone in the research process. 32 Correspondingly, the studies reported in the health policy research narrative answer the validation question: 'Have we built the right system for detection and prediction of influenza seasons and outbreaks?' Although the studies reported in this narrative mainly used data on clinical diagnoses and from laboratory tests, the two most recent studies also employed syndromic data: one study used data from telenursing call centres 20 and the other study used data from an internet search engine. 21 In the health policy research narrative, the foundation in real-world validation of alerts and predictions was shown, for instance, by pointing out that usually only a small number of annual infectious disease cycles of data are available for evaluations of new algorithms, leading to a constant lack of evidence-based information on which to base policy. 19 It was also shown by that space was provided for discussions regarding whether algorithms would yield worse performances when outbreak conditions change, for example, that pandemic incidences are higher than those recorded during interpandemic periods. 20 24 Moreover, evaluations presented in the health policy research narrative highlight the quantitative strength of the research evidence. For instance, in the study reporting excellent predictive performance of a non-parametric time-series method, 24 the evaluation period lasted 938†weeks and covered an entire nation. In comparison, a prospective study reported in the biodefence informatics narrative accounted for an evaluation of a Bayesian network model 22 that lasted 26†weeks and covered one US county. The biodefence informatics narrative Assessments informing construction of technically and mathematically sound algorithms for outbreak detection and prediction were reported from mathematical modelling and health informatics contexts. Research in these fields was described in a biodefence informatics narrative. The setting for this narrative is formative evaluation and justification of algorithms for detection and prediction of atypical outbreaks of infectious diseases and bioterror attacks. In other words, these studies can be said to answer the system verification question: 'Did we build the system right?' 25 The narrative is set in a context where algorithms need to be modified and assured for detection and prediction of microbiological agents with unusual or unknown characteristics, for example, novel influenza virus strains or anthrax. 26 The number of studies presented in the biodefence informatics narrative grew rapidly after the terrorist attacks in 2001. 27 Reporting of influenza algorithm performance in this narrative is characterised by presentation of statistical or technical advancements, for example, making use of increments instead of rates or introduction of methods based on Markov models. 18 As empirical data for logical reasons are scarce in biodefence settings, limited attention is in this narrative paid to the learning period dilemma. This dilemma represents a generic methodological challenge in algorithm development, that is, the statistical associations between indicative observations and the events to be predicted are determined in one time interval (the learning period) and used to predict the occurrence of corresponding events in a later interval (the evaluation period). 28 When trying to detect or predict a novel infectious agent, the learning period dilemma primarily shows unavailability of learning data for calibration of model-based algorithms. For instance, for prediction algorithms based on the reproductive number, 29 series of learning data of sufficient length for empirical determination of the serial interval cannot be made available during early outbreak stages, implying that the method cannot be used as supposed. 30 Moreover, the microbiological features of the pathogen and the environmental conditions in effect during the learning period can change after the algorithm has been defined, requiring adjustments of algorithm components and parameters to be made for preserving the predictive performance. Algorithm performance can in the biodefence informatics be narrative verified by combining prospective evaluations with formal proofs and analyses of simulated and retrospective data. Although it is commonly emphasised that the evaluation results are preliminary with regard to population outcomes, 22 the evaluation results are still included in the narrative. The health policy research narrative For evaluation study results to qualify as input to recommendations regarding infectious disease control practice, they should conform to general criteria established for health policy evidence. The analyses must be unbiased and not open for manipulation, for example, the data sources and analytic models should be described and fixed before data are accessed for analyses. 31 In the corresponding research paradigm, the use of prospective study designs is regarded as the cornerstone in the research process. 32 Correspondingly, the studies reported in the health policy research narrative answer the validation question: 'Have we built the right system for detection and prediction of influenza seasons and outbreaks?' Although the studies reported in this narrative mainly used data on clinical diagnoses and from laboratory tests, the two most recent studies also employed syndromic data: one study used data from telenursing call centres 20 and the other study used data from an internet search engine. 21 In the health policy research narrative, the foundation in real-world validation of alerts and predictions was shown, for instance, by pointing out that usually only a small number of annual infectious disease cycles of data are available for evaluations of new algorithms, leading to a constant lack of evidence-based information on which to base policy. 19 It was also shown by that space was provided for discussions regarding whether algorithms would yield worse performances when outbreak conditions change, for example, that pandemic incidences are higher than those recorded during interpandemic periods. 20 24 Moreover, evaluations presented in the health policy research narrative highlight the quantitative strength of the research evidence. For instance, in the study reporting excellent predictive performance of a non-parametric time-series method, 24 the evaluation period lasted 938†weeks and covered an entire nation. In comparison, a prospective study reported in the biodefence informatics narrative accounted for an evaluation of a Bayesian network model 22 that lasted 26†weeks and covered one US county. Discussion In a metanarrative review of studies evaluating the prospective performance of influenza outbreak detection and prediction algorithms, we found that methodological perspectives and experiences have, over time, been reported in two narratives, representing biodefence informatics and health policy discourse, respectively. Differences between the narratives are found in elements ranging from the evaluation settings and end point measures used to the structure of the argument. The biodefence informatics narrative, having an emphasis on verification of technically and mathematically sound algorithms, originates from the need to rapidly respond to evolving outbreaks of influenza pandemics and agents disseminated in bioterror attacks. Only more recently, studies presented in the biodefence informatics narrative have been directed to common public health problems, such as seasonal influenza and air pollution. 33 Although evidence-based practices have been promoted by public health agencies during the period the assessed studies were published, 34 only four prospective evaluations of influenza detection and prediction algorithms were reported as a health policy research narrative. However, despite being scarce for influenza, algorithm evaluations emphasising real-world validation of algorithm performance are relatively common for several other infectious diseases, for example, dengue fever. 35 One reason for not choosing to report evaluations of influenza detection and prediction algorithms in the health policy narrative may be that the urgent quest for knowledge in association with atypical influenza outbreaks has led to an acceptance of evaluation accounts with limited empirical grounding. These accounts agree with mathematical and engineering research practices in biodefence informatics and are thus accepted as scientific evidence within those domains. This implies that awareness of the narrative format in which evidence is reported is essential when interpreting algorithm evaluations. This study has methodological strengths and limitations that need to be taken into account when interpreting the results. A strength is that it was based on a metanarrative review. This is a relatively new method of systematic analyses of published literature, designed for topics that have been conceptualised differently and studied by different groups of researchers. 36 We found that in a historical perspective, researchers from different paradigms have evaluated algorithms for influenza outbreak detection and prediction with different means and purposes. Some researchers have conceptualised algorithm evaluations as an engineering discipline, others as a subarea of epidemiology. The intention was not to conclude recommendations for algorithm use. Instead, the aim was to summarise different perspectives on algorithm development and reporting in overarching narratives, highlighting what different researchers might learn from one another's approaches. Regarding the limitations of the review, it must be taken into consideration that the ambition was to base the narrative analysis on evaluations with relevance for operational readiness and real-world application. There is a possibility that we failed to identify some relevant evaluations due to the absence of specific indexing terms for infection disease detection and prediction methods and that we excluded studies that were not indexed in research databases. However, we believe that the probability that we missed relevant evaluations for these reasons is low. We initially identified 1084 studies out of which 116 had relevant abstracts. Following examination of the corresponding articles, the majority had to be excluded from the final review because they did not fulfil the inclusion criteria at the detailed level ( figure 1 ). One overall interpretation of this finding is that more research activity had been associated with developing detection and prediction algorithms than evaluating them and carefully reporting the results. For instance, a large number of interesting studies had to be excluded because non-prospective data were used for the evaluations, for example, the models were developed from learning data and evaluated against out-of-sample verification data from the same set using a leave-one-season-out approach. 37 38 Regarding prediction algorithms, numerous potentially interesting studies were excluded because they did not report standard evaluation metrics. One example is a prospective Japanese study of predictions conducted during the pandemic outbreak in 2009, which reported only descriptive results. 39 We found no prospective algorithm evaluations that applied an integrated outbreak detection and prediction. An Australian study applied an algorithm including detection and prediction functions, 40 but this study used simulated data for the evaluation. Nonetheless, the eligibility criteria applied in this review accepted syndromic definitions of influenza as the gold standard, that is, specified sets of symptoms not requiring laboratory confirmation for diagnosis. 41 If laboratory-confirmed diagnosis of influenza would have been included in the criteria, almost no studies would have qualified for inclusion in the review. In summary, two narratives for reporting influenza detection and prediction algorithm evaluations have been identified. In the biodefence informatics narrative, technical and mathematical verification of algorithms is described, while the health policy narrative is employed to allow conclusions to be drawn about public health policy. A main dissimilarity between the narratives is the attention paid to the learning period dilemma. This dilemma represents a generic methodological challenge in the development of biosurveillance algorithms; the statistical models used to detect or predict an influenza-related event must be determined in a preceding time interval (the learning period). This means that there is always a shortage of time when algorithms for novel infectious diseases are to be validated in real-world settings. We offer two suggestions for future research and development based on these results. First, a sequence of evaluation research phases interconnected by a translation process should be defined, starting from theoretical research on construction of new algorithms in the biodefence informatics setting and proceeding stepwise to prospective field trials performed as health policy research. In the latter setting, the evaluation study design should be registered in an international trial database, such as ClinicalTrials.gov, before the start of prospective data collection. Second, standardised and transparent reporting criteria should be formulated for all types of algorithm evaluation research. The recent development of consensus statements for evaluations of prognostic models in clinical epidemiology 42 can here be used as a reference.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10706840/

Beta-Barrel Channel Response to High Electric Fields: Functional Gating or Reversible Denaturation?

Ion channels exhibit gating behavior, fluctuating between open and closed states, with the transmembrane voltage serving as one of the essential regulators of this process. Voltage gating is a fundamental functional aspect underlying the regulation of ion-selective, mostly α-helical, channels primarily found in excitable cell membranes. In contrast, there exists another group of larger, and less selective, β-barrel channels of a different origin, which are not directly associated with cell excitability. Remarkably, these channels can also undergo closing, or "gating", induced by sufficiently strong electric fields. Once the field is removed, the channels reopen, preserving a memory of the gating process. In this study, we explored the hypothesis that the voltage-induced closure of the β-barrel channels can be seen as a form of reversible protein denaturation by the high electric fields applied in model membranes experiments—typically exceeding twenty million volts per meter—rather than a manifestation of functional gating. Here, we focused on the bacterial outer membrane channel OmpF reconstituted into planar lipid bilayers and analyzed various characteristics of the closing-opening process that support this idea. Specifically, we considered the nearly symmetric response to voltages of both polarities, the presence of multiple closed states, the stabilization of the open conformation in channel clusters, the long-term gating memory, and the Hofmeister effects in closing kinetics. Furthermore, we contemplate the evolutionary aspect of the phenomenon, proposing that the field-induced denaturation of membrane proteins might have served as a starting point for their development into amazing molecular machines such as voltage-gated channels of nerve and muscle cells. 1. Introduction In recent decades, significant efforts have been devoted to unraveling the molecular mechanisms behind membrane channel gating. The success stories in understanding the voltage gating of highly ion-selective channels, mostly α-helical, of excitable cell membranes [ 1 , 2 , 3 , 4 ] have inspired numerous investigations into the structural aspects of voltage-induced closing in a distinct class of channels: the larger, less ion-selective β-barrel channels. These channels encompass various outer membrane proteins found in Gram-negative bacteria, mitochondria, and chloroplasts, as well as exotoxins secreted by bacteria targeting mammalian cells. While the β-barrel channels are not typically associated with the excitability phenomenon, experiments involving their reconstitution into planar lipid bilayers have shown that they can also be closed by applying sufficiently high voltages to the lipid bilayer. Despite the voltage "gating" being demonstrated in β-barrel channels of diverse origins and biological functions more than four decades ago, the underlying mechanisms and potential physiological significance of this phenomenon remain unresolved [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. Furthermore, it remains unclear how much commonality exists between the voltage gating observed in the α-helical ion channels of excitable cells and that in the larger β-barrel channels. Equally uncertain is the extent of similarity in voltage gating among β-barrel channels of different origins. Notably, the physiological significance of β-barrel voltage gating, including both general and specific diffusion porins of Gram-negative bacteria and bacterial exotoxins, has been a subject of extensive debate for several decades, with Hiroshi Nikaido questioning it as early as 1988 by referring to the phenomenon as "an artifact of in vitro reconstitution" [ 14 ]. In the present study, we reexamine the fundamental aspects of voltage-induced closing in β-barrel channels, focusing on the outer membrane protein F (OmpF), which forms trimeric channels [ 15 ] in the outer membrane of the Gram-negative bacterium Escherichia coli ( E. coli ) [ 16 , 17 ]. Gram-negative bacteria are surrounded by a double-membrane envelope, where the outer membrane acts as a selective filter, providing protection against harmful components. Within this membrane, various types of β-barrel channels are found, with the OmpF trimer serving as a classic example. The OmpF trimeric structure has been determined with a resolution of 0.24 nm [ 18 ]. This protein can be isolated and reconstituted into planar lipid membranes (PLMs), allowing for examination at the single-channel level [ 6 ]. Based on our analysis of voltage-induced closing of the OmpF and data on other porins reported by our laboratory [ 19 ] and others previously [ 6 , 20 , 21 , 22 , 23 , 24 ], we propose that the "voltage gating of β-barrel channels" might represent their reversible denaturation induced by the applied electric field [ 19 ]. For a typical 100 mV transmembrane potential difference, this electric field reaches approximately 2 × 10 7 to 3 × 10 7 volts per meter. What is the magnitude of the forces acting on the channel-forming molecules in these fields? First, let us recall that a membrane with membrane-embedded proteins represents a capacitor, experiencing a compressive force between its "plates". A rough estimate indicates that at 100 mV, this force, acting on an area of 10 nm 2 (which is close, within an order of magnitude, to the solution-exposed surface of a typical β-barrel protein), is a fraction of one piconewton (pN). Importantly, this force is strong enough to be detectable in experiments on membrane voltage-induced thinning [ 25 , 26 ]. However, it is much smaller than the force of the field acting on any single charged residue within the channel-forming molecule. A quick estimate suggests that at the field strengths mentioned above, this force is an order of magnitude stronger than the compressive force, being in the range of 3 to 5 pN. Considering that at neutral pH, the OmpF molecule contains dozens of charged residues [ 27 , 28 , 29 ], this estimate indicates that a transmembrane potential difference of 100 mV exerts significant stress on the channel structure. Here, we analyze several key observations, both made in this study and reported previously, that point towards a denaturation mechanism of voltage-induced closing of OmpF. These observations include the channel's almost symmetric response to voltages of opposite polarity, the presence of multiple closed conformations, evident in the broad residual conductance distribution, the enduring memory of closing, the stabilization of open conformations in channel clusters, and the Hofmeister effect. We believe that the denaturation hypothesis provides an explanation for the slow progress in understanding the molecular mechanisms of β-barrel channel response to voltage. In many cases, voltage gating of ion-selective channels in excitable cells has been successfully linked to well-defined structural changes between the open and closed conformations of the channel-forming protein molecule. Conversely, it is widely recognized that protein denaturation, as well as the folding into functional structures, is a very complex process. This process evolves along various trajectories within a multidimensional energy landscape, often characterized by a plethora of poorly defined unfolded states, with the possible exception of small proteins [ 30 , 31 ]. 2. Results Figure 1 A illustrates the voltage response of a single trimeric OmpF channel reconstituted into a PLM. At 75 mV, the trimer exhibits a rather stable current, regardless of the polarity of the applied voltage, over a timescale of several minutes. When subjected to 150 mV of either polarity, the channel undergoes closing in discrete steps, each reducing its initial conductance by approximately one-third. This behavior is considered archetypal and has been extensively documented and reviewed in previous studies [ 6 , 8 , 9 , 13 , 32 , 33 , 34 ]. Two notable features should be highlighted: first, the closing requires a relatively large transmembrane voltage difference; second, the channel responds to both negative and positive voltages. Figure 1 B demonstrates the response of a multichannel OmpF system to the application of a slowly varying piecewise-linear periodic voltage ramp with an amplitude of ±200 mV. The response is non-Ohmic, as higher voltages lead to the closing of some channels. In Figure 1 C, we present the raw ion current data obtained from the same system at different frequencies of the ramp. Notably, at small voltages, all curves overlap, indicating that the number of channels in the membrane remains relatively constant throughout the experiment. At higher voltages, the curves diverge, exhibiting frequency-dependent hysteresis. The area of hysteresis increases as the ramp frequencies decrease from 5 to 1 mHz, meaning that at lower ramp frequencies, more channels have sufficient time to respond to the voltage. In certain parts of the records, the system displays negative differential resistance when an increase in the applied voltage results in a decrease in the current. Importantly, even in the case of the slowest ramp with a period of 1000 s (frequency of 1 mHz), the branches of the current records corresponding to increasing and decreasing voltages do not overlap. This observation suggests that the channels formed by OmpF exhibit long conformational memory, with characteristic times comparable to or significantly exceeding the 1000 s period. In the study of the voltage-dependent anion channel (VDAC) from the outer mitochondrial membrane, another classic example of a β-barrel channel [ 10 ], a widely adopted method for quantifying the voltage-induced gating was pioneered by Marco Colombini over three decades ago [ 35 ]. This method is based on the analysis of VDAC's response to slowly changing voltage ramps, similar to the experiments with the OmpF illustrated in Figure 2 . Colombini's approach focuses on the "opening" branches of the current-voltage relationship rather than the "closing" ones, as it was observed that VDAC opening generally occurs much faster than its closing. More recent studies involving the analysis of multichannel VDAC conductance kinetics in response to voltage jumps of approximately 30 mV amplitude have indeed confirmed this trend [ 36 ]. It was found that at voltage jumps of this amplitude, the opening process is approximately two orders of magnitude faster than the closing process. Specifically, the characteristic times for opening and closing were determined to be 0.8 s and 60 s, respectively. As part of the present investigation, we applied this strategy to analyze the OmpF channels, with the results shown in Figure 2 . We applied positive-polarity voltage ramps with an amplitude of 150 mV, covering a range of frequencies from 0.25 mHz to 10 mHz. Figure 2 A provides the raw data for ion current through a multichannel system at a ramp frequency of 1 mHz. While the voltage gating effect is visibly lower than that observed with the 200 mV ramp in Figure 1 , it remains measurable, as illustrated in Figure 2 B and quantified in Figure 2 C, using the hysteresis area as a metric. In line with the approach used in VDAC studies, we normalized the raw data with the following transformation [ 35 , 37 ]. (1) G n o r m V = G V − G m i n / G 0 − G m i n , where G V is the multichannel conductance measured at voltage V , G 0 is the multichannel conductance at ~0 mV where most of the channels are open, and G m i n is the minimal multichannel conductance along the hysteretic cycle. To our surprise, in contrast to the results obtained for VDAC [ 36 ], not only did the opening branches coincide, but the closing branches of the hysteresis curves measured at different ramp frequencies showed nearly complete overlap. This is demonstrated in Figure 2 D, suggesting a form of "quasi-equilibrium-like" behavior wherein the rate of voltage change remains slow enough for all ramp frequencies in the range of 0.25–5 mHz. By fitting the data to the Boltzmann distribution (2) G n o r m V = 1 + e x p n e V − V 0 / k B T − 1 , where k B , T , and e represent the Boltzmann constant, absolute temperature, and elementary charge, respectively, we determined parameters for the effective "gating charge" ( n ) and the voltage of half-effect ( V 0 ). These parameters, as shown in Figure 2 E,F, indicate the virtual independence of the ramp frequency. However, while the effective gating charges for both opening and closing processes exhibit only slight differences and are both found to be close to 1, the voltages of half-effect differ by a factor of 3. Specifically, the voltage of the half-effect for the opening branches is approximately 50 mV, whereas for the closing branches it is around 150 mV. This notable discrepancy indicates that the system possesses a long-time memory with a characteristic time significantly exceeding the largest period of the ramps used for voltage response measurements, which, in our case, is 4000 s. This observation aligns with previous reports of long relaxation times of this magnitude in other β-barrel bacterial porins [ 38 ]. It thus clearly demonstrates that the system is far from true equilibrium and, under the described experimental conditions, explores a subset of metastable states. A similar conclusion can be drawn from the conductance relaxation experiments depicted in Figure 3 . Both the processes of partial channel closing upon applying a 120 mV voltage and partial channel reopening upon returning to this voltage after holding the channels at 180 mV exhibit relatively fast relaxation components with characteristic times of about 40 and 5 s, respectively. An even faster component of the opening is seen at 100 s, upon reducing the voltage to 120 mV. However, following these fast components, there are long-term relaxations with characteristic times of many hundreds or thousands of seconds. Notably, this observation stands in stark contrast to the results published for VDAC voltage gating [ 36 ]. In Figure 3b of Ref. [ 36 ], it is shown that despite being two orders of magnitude different in their characteristic times, both closing and opening time dependences tend to converge to the same conductance. The extended memory observed in the OmpF channels, spanning hundreds or even thousands of seconds, sets their voltage sensitivity apart from that of channels of excitable membranes. Indeed, complex kinetic behavior has also been observed for the latter and has been the subject of intense discussion for over four decades, dating back to the seminal study by Francisco Bezanilla and colleagues [ 39 ]. Over time, the functional significance of this complexity in the context of excitability phenomena has been well acknowledged [ 4 , 40 ]. This complexity sparked extensive theoretical efforts, with some approaches involving multi-state discrete models [ 40 , 41 , 42 , 43 ]. Others explore diffusion across a large number of states [ 44 ] or continuous diffusion within complex energy landscapes [ 45 , 46 , 47 , 48 ]. However, it is worth noting that the recovery times observed in experiments with bona fide voltage-gated channels [ 39 , 41 , 49 ] are typically much shorter than those we report here for OmpF. The application of sufficiently high voltages to single OmpF channels induces their closure in three non-uniform increments [ 15 ] and, notably, results in a broad distribution of the "closed" state conductance [ 24 ]. This is illustrated in Figure 4 , which provides statistics of the closure amplitudes. To facilitate comparison, all current histograms in panels B, C, D, E, and F are displayed within the same current interval of 250 pA. It is evident that not only the three increments corresponding to closings of individual pores within the trimer but also the "completely" closed state ( Figure 4 F) exhibit distributions of residual current that are significantly wider than those of the fully open state ( Figure 4 B). Notably, if normalized by the mean current, the relative width of the distribution in the closed state surpasses that of the open state by two orders of magnitude. This observation implies a high degree of disorder in the protein conformations representing the closed state. We observed that the rate of the voltage-induced transition to the closed state is contingent on the ion species and follows the Hofmeister series [ 50 , 51 ] ( Figure 5 ). In our experimental approach, we applied a 150 mV potential to a single OmpF channel and measured the time spent in its fully open conformation [ 19 ]. This procedure was repeated multiple times to collect the necessary statistics. Figure 5 A provides three examples of such repetitions, while the corresponding histograms of the channel survival time in the fully open state are presented in Figure 5 B for 1 M LiCl ( left ), KCl ( middle ), and RbCl ( right ) electrolyte solutions. Figure 5 C summarizes the results obtained for several different salts, effectively demonstrating the Hofmeister effect in the rates of voltage-induced channel closing. The effect is also readily discernible in the hysteresis data plotted in Figure 5 D,E for cations, and in Figure 5 F,G for anions. Figure 5 shows both raw data ( Figure 5 D,F) and the areas encircled by the hysteresis curves ( Figure 5 E,G). Historically, the Hofmeister effect has been associated with protein stability and denaturation phenomena [ 50 , 51 , 52 ]. While the effect has been demonstrated in the context of channel-facilitated transport, to the best of our knowledge, it has not previously been linked to changes in channel conformations [ 53 , 54 , 55 ]. Although it was previously observed that not only the bathing electrolyte composition [ 19 , 56 , 57 , 58 ] but also the electrolyte concentration [ 19 , 59 , 60 ] can influence β-barrel channel gating, in the present study, we limited ourselves to 1 M salt solutions because our goal here was to demonstrate the existence of the Hofmeister effect in the voltage-induced OmpF closure. It is well-known that the voltage-induced closing of β-barrel channels depends on the composition of lipid membranes [ 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 ]. In our multichannel experiments, we have observed that the voltage sensitivity of β-barrel channels, as illustrated here with the OmpF, also depends on the manner in which the channels are organized within the membrane. Specifically, our findings reveal that when channel insertion occurs in clusters, the transition into closed conformations is impeded compared to single channels or systems with multiple non-clustered channels. This is something to be expected because of the strong protein-protein interactions revealed in the early electron microscopy studies of OmpF by Jurg Rosenbusch and colleagues [ 69 ]. Indeed, if the voltage-induced collapse of the OmpF structure involves significant changes in the geometry of the outer protein surface, such collapse could be hindered by the tight packing of channels into clusters. A possible role of channel clusterization in the β-barrel channel gating was hinted at in the studies on VDAC regulation by a pro-apoptotic protein, Bid [ 70 ], and by non-lamellar membrane lipids [ 61 ]. Figure 6 demonstrates that when OmpF insertions occur in blocks comprising two or more channels ( Figure 6 C,E), the closing process does not proceed to the nearly zero current state ( Figure 6 A) typically observed in single-channel measurements; such behavior is illustrated in Figure 4 A and Figure 5 A. As depicted in Figure 7 , most of our findings can be tentatively rationalized by illustrating a one-dimensional cross-section of a highly multidimensional energy landscape of the proposed voltage-induced denaturation of the channel structure. At zero applied transmembrane voltage ( red curve ), the molecule's lowest energy state corresponds to the folded state (F) representing the fully open channel conformation. When a large external field is imposed, for example, by the application of 150 mV of transmembrane voltage, the energy profile shifts ( blue curve ), favoring a family of (partially) unfolded states (U). Each unfolded state U exhibits significantly reduced conductance. These multiple unfolded states are denoted by the symbols i, i + 1, i + 2… It is important to note that the energy landscape undergoes a similar transformation when voltages of either polarity are applied (as exemplified by Figure 1 ), resulting in energetically favorable channel states that fall within the unfolded category. The figure illustrates only one of the many possible cross-sections of the landscape. In a different cross-section, the state i + 1 could be the most readily accessible state for a direct transition from the folded state F. This limitation is inherent in any attempt to represent the complex multidimensional energy landscape of protein folding in one or two dimensions [ 30 , 31 ]. These multiple unfolded states are metastable, separated by barriers that are typically high enough for these states to be observed within the characteristic times of experiments. However, these barriers are not excessively high, allowing for spontaneous transitions between states with different conductance levels within the array of "closed" states U. An illustrative example of such transitions is shown in Figure 4 A and Figure 5 A. According to our model, these transitions may also underlie the long memory effects demonstrated in Figure 2 and Figure 3 , enabling the channel to explore the vast space of closed states. At times, an OmpF monomer may transition to an exceptionally stable low-energy state in U. This phenomenon accounts for situations in which certain channels remain closed for several hours, even after the denaturing electric field has been removed ( Figures S1 and S2 ). Paradoxically, under these circumstances, the subsequent application of high voltages of the opposite polarity, known to induce channel closure, may aid in channel reopening [ 19 ]. If our hypothesis of reversible denaturation is correct, further investigation is needed to understand how this stress promotes the refolding of the channel to its native conformation. Overall, the OmpF memory characteristics were found to be even more complex. Over a period of extended time, the sensitivity of the OmpF closing to voltage exhibited a gradual increase in its magnitude ( Figure S1 ). It appeared that the channels undergo a form of "training" when subjected to either repeated 0–150 mV voltage ramps ( Figure S1A ) or a constant 150 mV ( Figure S1B ), resulting in an enhancement of the voltage-induced closing effect as a function of time. To investigate whether the observed enhancement results from membrane or channel aging over an extended period, we conducted experiments in which the membrane was held at 0 mV of applied voltage for many hours between three repeated 0–150 mV voltage ramp applications ( Figure S1C ). Figure S1D provides a comparison of the results obtained from these three protocols. Notably, while the hysteresis area increased by nearly 30% ( left ) and 40% ( middle ) after 6 h of ramp application ( Figure S1A ) and the voltage clamp protocol ( Figure S1B ), respectively, a modest 14% increase ( right ) in the hysteresis area was observed during the third protocol, where the membrane was held at 0 mV applied voltage for a significantly longer time interval (15 h). The interpretation of the "channel training" effect within the context of the reversible protein denaturation model is not immediately clear. The applied voltage reduces the height of the barrier for the channel to undergo unfolding ( Figure 7 ). According to the data in Figure 5 , the barrier height is also influenced by the cation and anion species following their position in the Hofmeister series. However, whether such dependence extends to the structural characteristics of the unfolded states' energy landscape remains an open question. The long relaxation times observed in Figure 2 and Figure 3 present a challenge, as they hinder reliable quantification of the true equilibrium between the folded (F) and unfolded (U) states. Remarkably, the Hofmeister effect in OmpF voltage-induced closing also exhibits an intriguing memory effect, both at the single-channel and multichannel levels ( Figure S3 ). When CsCl electrolyte solutions were replaced by LiCl without compromising membrane integrity, the channel closing kinetics displayed a gradual change in the expected direction but remained quite fast, failing to reach the level observed in LiCl solutions when used from the beginning of the experiment, including the initial channel reconstitution ( Figure S3A ). At the same time, following the solution exchange, the channel conductance aligned with that in LiCl. The same was true for the LiCl to CsCl replacement ( Figure S3B,C ). The results presented in Figure 6 strongly support the notion that the folded structure of the channel experiences stabilization when organized within channel clusters. This finding lends credence to the idea that the voltage-induced closing of the OmpF channel involves substantial structural changes, akin to what has been suggested for the voltage-induced closing of another β-barrel channel, VDAC [ 71 ]. The rationale behind this conjecture lies in the belief that if the voltage-induced closing were a result of relatively subtle changes within the barrel, such as, e.g., the motion of the L3 loop [ 72 , 73 , 74 , 75 ], screening/unscreening of charges within the channel lumen [ 23 , 76 ], or some "breakdown of the delicate ion-conducting pathway" [ 8 ], the presence of neighboring channels would not measurably interfere with the process. 3. Discussion In Table 1 , we present a compilation of empirical findings of voltage-induced closing in OmpF channels. These observations, both from our study and prior work, are divided into two categories: those supporting voltage-induced OmpF denaturation and those supporting functional gating. While most findings lean towards denaturation, we entrust the decision to the readers, allowing them to draw their own conclusion about the mechanism of large β-barrel channel voltage gating. Our study's driving motive was to find the "light at the end of the channel" by providing a plausible explanation for the stubborn mystery surrounding the voltage-induced closing of large β-barrel channels. In the present work, we explored the possibility that this phenomenon results from the denaturation of the channel-forming protein under the influence of applied electric fields, thus attempting to explain the enduring difficulties in identifying specific structures of, or even intermediates leading to, the closed states. This stands in contrast to functional voltage gating, which is a characteristic feature of ion-selective channels of excitable membranes—channels that play a crucial role in nerve pulse propagation and have been explored in an impressive bulk of celebrated research [ 1 , 2 , 3 , 4 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 ]. Our analysis, based on the data of the present and many previous studies, consistently leans towards denaturation, as argued throughout the text and summarized in Table 1 . This concept was previously hinted at by Robertson and Tielemen [ 9 ] and explored in greater detail in our conference proceedings publication [ 19 ]. Within this framework, the applied fields of more than 2 × 10 7 V/m, corresponding to a 100 mV electric potential difference across the lipid bilayer, crash the native protein structure into a set of partially unfolded, less conductive conformations. Once the denaturing field is removed, OmpF can regain its native function by refolding back to its original open-channel conformation. After considering all the pro- and contra-arguments, a reasonable question to ask is whether there exists a well-defined boundary between the phenomenon of functional voltage gating and that of voltage-induced denaturation. Our tentative answer is that this boundary is probably quite vague. One of the sources of this ambiguity stems from the possible evolutionary connection between these two phenomena. It is conceivable that, as an ancient mechanism of voltage sensitivity, nature might have employed reversible denaturation of membrane proteins in response to the transmembrane electric field. Over time, in this scenario, evolutionary pressures would have transformed these denaturation-based voltage-sensitive "proto-channels" into the perfect voltage-sensing molecular machines that exhibit distinct structures in both their open and closed states [ 1 , 2 , 83 ]. Could voltage-induced closure of OmpF function as a crucial attribute in optimizing nutrient uptake and waste product release while also serving as a defense mechanism against the infiltration of foreign molecules, like antibiotics, by controlling the number of open channels? Considering that a single E. coli cell contains approximately 10 5 copies of OmpF channels [ 85 ], alongside various other general and specific porin channels, it is evident that alteration in porin expression can influence outer membrane permeability [ 86 ]. However, could voltage-triggered protein denaturation achieve a similar outcome? Could a voltage of sufficient magnitude to induce channel closing even be attained across the outer membrane? For some E. coli outer membrane porins, such as OmpC [ 87 ], the "crucial voltage", a vague term intentionally sidestepped in the present article, was reported to be close to 200 mV, whereas other porins, e.g., the recently discovered putative β-barrel channel Triplin [ 88 ] and Vibrio cholerae OmpT exhibit voltage-induced closing at considerably lower transmembrane voltages [ 89 ]. At the same time, an increase in solution acidity [ 90 ] and the presence of polyamines, such as spermine, spermidine, and cadaverine, are cofactors known to increase the voltage sensitivity of OmpF [ 16 , 22 , 91 ]. Finding answers to these questions is not straightforward and requires further investigation and critical thinking. We should also acknowledge recent progress in our understanding, or more accurately, our increasing recognition of the complexities of membrane potential dynamics [ 92 ]. Although the Donnan potential was initially believed to have no impact on porin permeability [ 14 ], it is now recognized as a parameter that can dynamically vary based on factors like osmotic strength, external pH, and changes in cell surface charges [ 93 ]. Additionally, electrical spiking in E. coli , which is responsive to various chemical and physical influences and aligns with the rapid efflux of small molecules, has also been documented [ 94 ]. Drawing a comparison between bacteria and eukaryotes, the authors emphasized the high surface-to-volume ratio characteristic of bacteria and proposed to reconsider certain principles in neuronal electrophysiology within the bacterial context. For instance, in a bacterium with a volume of 1 fL and a cytoplasmic Na + concentration of 10 mM, there are only approximately ~10 7 ions of Na + available. This implies that a single ion channel, carrying a current of 2 pA, could deplete this ion supply in less than 1 s. It was suggested that by oscillating back and forth, the bacterium might be dynamically changing its membrane potential to various values, each of which is optimal for a distinct biological process. Finally, we hypothesize that the major channel of the outer mitochondrial membrane, VDAC [ 10 ], may have evolved to incorporate its voltage-induced denaturation as at least a partially functional feature. However, the question of whether mitochondrial outer membrane voltages alone are sufficient to gate VDAC independently of its cytosolic regulators [ 95 , 96 , 97 ] remains a topic of intense debate (see discussions in Refs. [ 98 , 99 , 100 ]). There are additional reports showing that β-barrel channels can undergo gating under the application of low, physiologically relevant voltages [ 88 , 89 ], thus suggesting gating functionality, or exhibiting a prominent asymmetry toward the applied voltage sign [ 72 , 101 , 102 , 103 , 104 ]. Over the past 25 years, researchers from our laboratories have collectively investigated eleven distinct β-barrel channel-forming proteins, encompassing five outer membrane bacterial porins, five bacterial exotoxins, and mitochondrial VDAC. All of these proteins exhibited reversible voltage-dependent closings. While certain distinctions were noted, such as in the case of the anthrax toxin which operates amidst significant endosomal voltage and pH gradients and is characterized by a pronounced asymmetry in response to the polarity of the applied voltage [ 101 ], many aspects of this process were found to be strikingly similar to other β-barrel channels. 4. Materials and Methods 4.1. Chemicals Wild-type OmpF were generously provided by Dr. Mathias Winterhalter (Jacobs University, Bremen, Germany). The protein was diluted to a concentration of 0.22, 2.2, and 22 µg/mL using 1 M KCl and 1% ( v / v ) n-octylpolyoxyethylene (octyl-POE) solution (Alexis Biochemicals, Lausen, Switzerland). The following chemical reagents were used: LiCl, NaCl, KCl, RbCl, CsCl, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), LiOH, NaOH, KOH, RbOH, CsOH (Sigma-Aldrich, St. Louis, MO, USA), "purum" hexadecane (Fluka, Buchs, Switzerland), diphytanoylphosphatidylcholine (DPhPC) in chloroform (Avanti Polar Lipids, Alabaster, AL, USA), pentane (Burdick and Jackson, Muskegon, MI, USA), and agarose (Bethesda Research Laboratory, Gaithersburg, MD, USA). The 1 M LiCl, NaCl, KCl, CsCl, and RbCl solutions were buffered at pH 7.4 by 5 mM HEPES. The pH of stock solutions was individually adjusted by adding LiOH, NaOH, KOH, RbOH, or CsOH as needed. All solutions were prepared using double-distilled water. 4.2. Channel Reconstitution "Solvent-free" planar lipid bilayer membranes were formed following the lipid monolayer opposition technique [ 105 ] from a 5 mg/mL solution of DPhPC in n-pentane on a 60 µm diameter aperture in the 15 µm thick Teflon film that separated two ( cis and trans ) compartments of the custom-made Teflon chamber. Electrolyte solutions were symmetrically added to both sides of the chamber, and measurements were performed at T = 23 ± 0.5 °C. The aperture was pretreated with a 1% solution of hexadecane in pentane and dried for 15 min prior to membrane formation. The film and the total capacitances were close to 25 and 50 pF, respectively. Single channels were reconstituted by adding 0.1–0.3 µL of 0.22 µg/mL stock solution of OmpF to the 1.5-mL aqueous phase in the cis half of the chamber while stirring at 150 mV of applied voltage for 5–10 min. In the multichannel bilayer experiments, channels were typically formed by adding 2–3 µL of a 0.22 µg/mL stock solution of OmpF prepared several weeks in advance to the cis compartment while stirring. To investigate the effect of OmpF clustering on voltage-induced closing ( Figure 6 ), we compared freshly prepared with properly stored at +4 °C eight-week-old 0.22 µg/mL OmpF solutions and used more concentrated 22 µg/mL OmpF. Steady multichannel conductance, monitored by applying a 100 mV transmembrane voltage, was achieved in about 30 min. The electric potential difference across the membrane was applied with a pair of Ag-AgCl electrodes in 2 M KCl and 1.5% agarose bridges. The potential was considered positive when it was greater on the side of OmpF addition ( cis side). Current recordings were obtained using an Axopatch 200B amplifier (Molecular Devices, San Jose, CA, USA) in the voltage-clamp mode. All experiments were carried out at a room temperature of 23.0 ± 1.5 °C. Single-channel data were filtered using a low-pass eight-pole Butterworth filter (Model 900 Frequency Active Filter, Frequency Devices, Ottawa, IL, USA) at 15 kHz, acquired with a Digidata 1322A board (Molecular Devices, San Jose, CA, USA) at a sampling frequency of 50 kHz, and analyzed using pClamp 10.7 software (Molecular Devices, San Jose, CA, USA). Multichannel data were saved with a sampling frequency of 2–5 kHz; the low-pass Bessel filter was set to 1 kHz. 4.3. Voltage Gating Measurements Five different experimental protocols were used to measure OmpF voltage-induced closing. First , hysteresis experiments [ 36 ] were conducted using periodic triangular waves applied from a Function Waveform Generator 33120A (Hewlett Packard, Palo Alto, CA, USA) with voltage changing from 200 mV to −200 mV ( Figure 1 B) or from 0 to 150 mV ( Figure 2 A) and back in the frequency range of 0.25–10 mHz. The current recordings were collected from the membranes containing 30–200 channels and averaged over several periodic triangular waves applied to the same membranes. Second , OmpF multichannel closing and opening kinetics were investigated using the previously described conductance relaxation experiments (see Figure 3 in Ref. [ 36 ]), with some modifications. The details of the "conductance relaxation to the same voltage" protocols are given in Figure 3 legends. Third , to quantify the distribution of the voltage gating event currents, we recorded the closings of all three monomers of a single OmpF trimer applying 150 mV voltage ( Figure 4 ). Fourth , the stability of single OmpF channels in response to 150 mV transmembrane voltage was studied by measuring the time needed for the closing of one (first) monomer in the trimer ( Figure 5 ). In Figure 4 and Figure 5 , after each measurement, 0 mV (or occasionally −150 mV) was applied to reopen the channel; the measurements were repeated multiple times and quantified by fitting the closing time histograms with a single exponent. Fifth , we conducted comparative multichannel PLM measurements by reconstituting OmpF from freshly diluted and eight-week-old solutions at concentrations of 0.22 and 22 µg/mL. We initially reconstituted the channels under an applied voltage of 50 mV and subsequently examined the voltage-induced closing effect at 150 mV ( Figure 6 ). 4.1. Chemicals Wild-type OmpF were generously provided by Dr. Mathias Winterhalter (Jacobs University, Bremen, Germany). The protein was diluted to a concentration of 0.22, 2.2, and 22 µg/mL using 1 M KCl and 1% ( v / v ) n-octylpolyoxyethylene (octyl-POE) solution (Alexis Biochemicals, Lausen, Switzerland). The following chemical reagents were used: LiCl, NaCl, KCl, RbCl, CsCl, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), LiOH, NaOH, KOH, RbOH, CsOH (Sigma-Aldrich, St. Louis, MO, USA), "purum" hexadecane (Fluka, Buchs, Switzerland), diphytanoylphosphatidylcholine (DPhPC) in chloroform (Avanti Polar Lipids, Alabaster, AL, USA), pentane (Burdick and Jackson, Muskegon, MI, USA), and agarose (Bethesda Research Laboratory, Gaithersburg, MD, USA). The 1 M LiCl, NaCl, KCl, CsCl, and RbCl solutions were buffered at pH 7.4 by 5 mM HEPES. The pH of stock solutions was individually adjusted by adding LiOH, NaOH, KOH, RbOH, or CsOH as needed. All solutions were prepared using double-distilled water. 4.2. Channel Reconstitution "Solvent-free" planar lipid bilayer membranes were formed following the lipid monolayer opposition technique [ 105 ] from a 5 mg/mL solution of DPhPC in n-pentane on a 60 µm diameter aperture in the 15 µm thick Teflon film that separated two ( cis and trans ) compartments of the custom-made Teflon chamber. Electrolyte solutions were symmetrically added to both sides of the chamber, and measurements were performed at T = 23 ± 0.5 °C. The aperture was pretreated with a 1% solution of hexadecane in pentane and dried for 15 min prior to membrane formation. The film and the total capacitances were close to 25 and 50 pF, respectively. Single channels were reconstituted by adding 0.1–0.3 µL of 0.22 µg/mL stock solution of OmpF to the 1.5-mL aqueous phase in the cis half of the chamber while stirring at 150 mV of applied voltage for 5–10 min. In the multichannel bilayer experiments, channels were typically formed by adding 2–3 µL of a 0.22 µg/mL stock solution of OmpF prepared several weeks in advance to the cis compartment while stirring. To investigate the effect of OmpF clustering on voltage-induced closing ( Figure 6 ), we compared freshly prepared with properly stored at +4 °C eight-week-old 0.22 µg/mL OmpF solutions and used more concentrated 22 µg/mL OmpF. Steady multichannel conductance, monitored by applying a 100 mV transmembrane voltage, was achieved in about 30 min. The electric potential difference across the membrane was applied with a pair of Ag-AgCl electrodes in 2 M KCl and 1.5% agarose bridges. The potential was considered positive when it was greater on the side of OmpF addition ( cis side). Current recordings were obtained using an Axopatch 200B amplifier (Molecular Devices, San Jose, CA, USA) in the voltage-clamp mode. All experiments were carried out at a room temperature of 23.0 ± 1.5 °C. Single-channel data were filtered using a low-pass eight-pole Butterworth filter (Model 900 Frequency Active Filter, Frequency Devices, Ottawa, IL, USA) at 15 kHz, acquired with a Digidata 1322A board (Molecular Devices, San Jose, CA, USA) at a sampling frequency of 50 kHz, and analyzed using pClamp 10.7 software (Molecular Devices, San Jose, CA, USA). Multichannel data were saved with a sampling frequency of 2–5 kHz; the low-pass Bessel filter was set to 1 kHz. 4.3. Voltage Gating Measurements Five different experimental protocols were used to measure OmpF voltage-induced closing. First , hysteresis experiments [ 36 ] were conducted using periodic triangular waves applied from a Function Waveform Generator 33120A (Hewlett Packard, Palo Alto, CA, USA) with voltage changing from 200 mV to −200 mV ( Figure 1 B) or from 0 to 150 mV ( Figure 2 A) and back in the frequency range of 0.25–10 mHz. The current recordings were collected from the membranes containing 30–200 channels and averaged over several periodic triangular waves applied to the same membranes. Second , OmpF multichannel closing and opening kinetics were investigated using the previously described conductance relaxation experiments (see Figure 3 in Ref. [ 36 ]), with some modifications. The details of the "conductance relaxation to the same voltage" protocols are given in Figure 3 legends. Third , to quantify the distribution of the voltage gating event currents, we recorded the closings of all three monomers of a single OmpF trimer applying 150 mV voltage ( Figure 4 ). Fourth , the stability of single OmpF channels in response to 150 mV transmembrane voltage was studied by measuring the time needed for the closing of one (first) monomer in the trimer ( Figure 5 ). In Figure 4 and Figure 5 , after each measurement, 0 mV (or occasionally −150 mV) was applied to reopen the channel; the measurements were repeated multiple times and quantified by fitting the closing time histograms with a single exponent. Fifth , we conducted comparative multichannel PLM measurements by reconstituting OmpF from freshly diluted and eight-week-old solutions at concentrations of 0.22 and 22 µg/mL. We initially reconstituted the channels under an applied voltage of 50 mV and subsequently examined the voltage-induced closing effect at 150 mV ( Figure 6 ).

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459463/

Ternary Fingerprints with Reference Odor for Fluctuation-Enhanced Sensing

An improved method for fluctuation-enhanced sensing (FES) is introduced. We enhanced the old binary fingerprinting method, where the fingerprint bit values were ±1, by introducing ternary fingerprint bits utilizing a reference odor. In the ternary method, the fingerprint bit values are −1, 0, and +1, where the 0 value stands for the situation where the slope of the spectrum is identical to that of the reference odor. The application of the reference odor spectrum makes the fingerprint relative to the reference. The ternary nature and the reference feature increase the information entropy of the fingerprints. The method is briefly illustrated by sensing bacterial odor in cow manure isolates. 1. Introduction: Fluctuation-Enhanced Sensing (FES) Fluctuation-enhanced sensing (FES) [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ] evolved from the observations that the random fluctuations of physical quantities potentially carry more information about the physical system than their average value. This statement is also valid for sensory signals and conductance noise of samples with non-passivated surfaces indicated an unwelcome external interference in laboratory experiments. The FES method utilizes the statistical properties of microscopic random fluctuations superimposed on the classical sensor signal to generate patterns that identify the chemical composition of odors (see Figure 1 ). The classical signal is often a DC voltage value (due to the sensor resistance response); the pattern extractor is often a spectrum analyzer generating the power density spectrum of the pre-amplified sensor noise voltage; and the pattern classifier is often a neural network, or more advanced methods as described in the text. For the non-specialist reader, we give a very brief history of FES developments below which is centered about the related patents. In 1994–1995, electrical noise for sensing chemicals was proposed by showing the variations of conductance noise spectra of conducting polymers as a function of the ambient gas composition [ 1 , 2 ]. In 1997, similar observations were made about the conductance noise spectrum of semiconductor resistors with non-passivated surfaces [ 3 ]. The first patented FES scheme [ 4 ] appeared in 1998 and it was followed by a number of patents [ 5 , 6 , 7 , 8 , 9 , 10 ] during the subsequent years. The first analytic scheme of a generic FES systems for quantitative analysis of gas mixtures with mathematical analysis about the limits and with the sensor number requirement versus the number of agents [ 4 , 11 ]. The possibility of "freezing the odor", that is, the sampling-and-hold technique [ 5 , 12 ] in a Taguchi gas sensor, was an improvement with memory and robustness of gas turbulences due to heat convection. The spectrum of surface diffusion noise on surface acoustic wave (SAW) sensors and open-gate MOS sensors was utilized for another FES technique [ 6 , 7 , 13 , 14 ]. The use of higher-order statistics and higher-order spectra was applied to enhance the extracted information from the stochastic signal component [ 8 , 15 , 16 ]. In a water-based medium, to detect and identify bacteria promptly by FES, the bacteriophage-based microscopy electrochemical cells were proposed and demonstrated [ 9 , 10 , 17 , 18 ]. The FES technique has been tested by a large body of investigations in many different systems and various conditions, see for example [ 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 ] and the present paper is a part of the ongoing research and developments to further explore and develop this method. It is important to note that the pattern generation/recognition aspects are the subject of intensive research that goes beyond the scope of our papers—see for example [ 42 , 43 , 44 , 45 ]. Obviously, such research can incorporate artificial intelligence-related technologies, too. Note, these technologies are data processing-intensive, and thus require higher energy dissipation than the simple fingerprinting ideas shown below. 2. Materials and Methods 2.1. Binary Fingerprints In principle, the generated patterns, such as the power density spectra (PDS), bispectra, etc., can directly be fed into a classifier (neural network and other machine learning/artificial intelligence tools [ 42 , 43 , 44 , 45 ]), to identify the chemical composition related to this pattern. However, machine learning tools require intensive data processing which implies a large energy dissipation. Moreover, the training of machine learning and neural networks is a tremendous task, as it requires executing a great number of measurements with a large variety of chemical compositions. Thus, much simpler and more direct approaches have also been tested with good results, for example the binary fingerprint method that extracts a bit string from the measured PDS [ 46 ] (see Figure 2 ). By generating a bit pattern characterizing the chemical environment, this bit pattern can be used as an address that directly calls the name of the chemical environment which requires only a miniscule energy dissipation. The average slope of the spectrum plotted with a log-log scale is determined by connecting the beginning and the end of the ("meaningful" part of the) PDS. Next, the same frequency band is divided into sub-bands to determine the related binary bit values. Then the local slope over these sub-bands is determined in the same way as described above. When the local slope is below the average, the bit value is −1, and otherwise it is 1 (see Figure 3 ). As soon as we have such a bit sequence, which is a binary fingerprint to characterize a chemical environment, we need only a simple interpreter to display the name of this chemical environment. Such a system can be useful in simple applications where the low power consumption is also an issue. In the present paper, we generalized the binary method to the ternary one, where each fingerprint bit had three alternative values instead of two. The new method offers additional information and ways to use comparative features with reference odors. After introducing the ternary method, we demonstrate and compare it with the binary one by generating these fingerprints with cow-manure related odor. For simplicity but without limiting generality, we are talking about PDS as a source pattern, but any other quasi-continuum patterns are suitable. 2.2. The New Method: Ternary Fingerprints In computer science, it has been well-known and demonstrated [ 47 ] that a computer with ternary bits having three different values instead of the usual binary bits of just two values is much more powerful than the binary computer version. A ternary bit has ln(3)/ln(2) times higher information entropy, which is about a 50% increase compared to a binary bit, and it has advantages in the processing, too. Moreover, for the ternary fingerprint method, we also include an enhancement by a reference agent that further increases its potential information content because various different ternary fingerprints can be generated about the same chemical environment by using alternative references. The spectrum of the reference agent serves as the reference PDS. Next, the frequency band is divided into sub-bands (similarly to the case of the binary fingerprints) to determine the related ternary bit values. Then, the local slope on these sub-bands is determined in the same way as described above. When the local slope with the agent is less than the local slope with the reference agent, the bit value is −1; when it is greater than the reference slope, the bit value is 1; and when the slopes are equal (this happens with a small probability depending on the resolution), the bit value is zero, as shown in Figure 3 and Figure 4 . 2.3. Demonstration with Bacterial Isolates from Cow Manure For the demonstration of the ternary fingerprinting method introduced above, we used a bacterial strain isolated from cow manure. The Petri plates of 58 cm 2 with the bacteria colonies were placed in a 300 cm 3 sensor chamber with the sensors attached. The microorganism used in this study was a Gram-positive toxin producing, facultative anaerobic bacteria, Bacillus cereus . Mid-log phase (OD 600 = 0.5, optical density at 600 nm) cultures of Bacillus cereus , isolated from cow manure at a dairy center in Stephenville, Texas were grown in Luria Bertani (LB) medium [ 1 ] for 4 h at 37 °C and at 150 rpm. The most abundant bacterium was isolated from different manure samples and identified as Bacillus cereus by whole genome sequencing at TIGSS (Texas A&M University Institute for Genome Sequencing and Society). One hundred microliters of the B. cereus culture were spread on Difco tryptic soy agar (TSA) plates (Becton Dickinson Co., Sparks, MD 21152, USA), and the plates were incubated overnight at 37 °C [ 48 ]. As a reference, sterile TSA plates without bacteria were also prepared. As the TSA medium itself has a strong smell, identical amounts (27 mL) of TSA medium were poured into each plastic Petri plate (VWR, Bridgeport, NJ, USA) to maintain a constant level of background odor [ 48 ]. The metal oxide (Taguchi type) sensor was a 50 nm thick SnO 2 film, sputtered on a 4 × 4 µm 2 microhotplate with a platinum heater and sensing electrodes. It was designed and manufactured by IM2NP laboratory and details were described elsewhere [ 49 ]. 2.1. Binary Fingerprints In principle, the generated patterns, such as the power density spectra (PDS), bispectra, etc., can directly be fed into a classifier (neural network and other machine learning/artificial intelligence tools [ 42 , 43 , 44 , 45 ]), to identify the chemical composition related to this pattern. However, machine learning tools require intensive data processing which implies a large energy dissipation. Moreover, the training of machine learning and neural networks is a tremendous task, as it requires executing a great number of measurements with a large variety of chemical compositions. Thus, much simpler and more direct approaches have also been tested with good results, for example the binary fingerprint method that extracts a bit string from the measured PDS [ 46 ] (see Figure 2 ). By generating a bit pattern characterizing the chemical environment, this bit pattern can be used as an address that directly calls the name of the chemical environment which requires only a miniscule energy dissipation. The average slope of the spectrum plotted with a log-log scale is determined by connecting the beginning and the end of the ("meaningful" part of the) PDS. Next, the same frequency band is divided into sub-bands to determine the related binary bit values. Then the local slope over these sub-bands is determined in the same way as described above. When the local slope is below the average, the bit value is −1, and otherwise it is 1 (see Figure 3 ). As soon as we have such a bit sequence, which is a binary fingerprint to characterize a chemical environment, we need only a simple interpreter to display the name of this chemical environment. Such a system can be useful in simple applications where the low power consumption is also an issue. In the present paper, we generalized the binary method to the ternary one, where each fingerprint bit had three alternative values instead of two. The new method offers additional information and ways to use comparative features with reference odors. After introducing the ternary method, we demonstrate and compare it with the binary one by generating these fingerprints with cow-manure related odor. For simplicity but without limiting generality, we are talking about PDS as a source pattern, but any other quasi-continuum patterns are suitable. 2.2. The New Method: Ternary Fingerprints In computer science, it has been well-known and demonstrated [ 47 ] that a computer with ternary bits having three different values instead of the usual binary bits of just two values is much more powerful than the binary computer version. A ternary bit has ln(3)/ln(2) times higher information entropy, which is about a 50% increase compared to a binary bit, and it has advantages in the processing, too. Moreover, for the ternary fingerprint method, we also include an enhancement by a reference agent that further increases its potential information content because various different ternary fingerprints can be generated about the same chemical environment by using alternative references. The spectrum of the reference agent serves as the reference PDS. Next, the frequency band is divided into sub-bands (similarly to the case of the binary fingerprints) to determine the related ternary bit values. Then, the local slope on these sub-bands is determined in the same way as described above. When the local slope with the agent is less than the local slope with the reference agent, the bit value is −1; when it is greater than the reference slope, the bit value is 1; and when the slopes are equal (this happens with a small probability depending on the resolution), the bit value is zero, as shown in Figure 3 and Figure 4 . 2.3. Demonstration with Bacterial Isolates from Cow Manure For the demonstration of the ternary fingerprinting method introduced above, we used a bacterial strain isolated from cow manure. The Petri plates of 58 cm 2 with the bacteria colonies were placed in a 300 cm 3 sensor chamber with the sensors attached. The microorganism used in this study was a Gram-positive toxin producing, facultative anaerobic bacteria, Bacillus cereus . Mid-log phase (OD 600 = 0.5, optical density at 600 nm) cultures of Bacillus cereus , isolated from cow manure at a dairy center in Stephenville, Texas were grown in Luria Bertani (LB) medium [ 1 ] for 4 h at 37 °C and at 150 rpm. The most abundant bacterium was isolated from different manure samples and identified as Bacillus cereus by whole genome sequencing at TIGSS (Texas A&M University Institute for Genome Sequencing and Society). One hundred microliters of the B. cereus culture were spread on Difco tryptic soy agar (TSA) plates (Becton Dickinson Co., Sparks, MD 21152, USA), and the plates were incubated overnight at 37 °C [ 48 ]. As a reference, sterile TSA plates without bacteria were also prepared. As the TSA medium itself has a strong smell, identical amounts (27 mL) of TSA medium were poured into each plastic Petri plate (VWR, Bridgeport, NJ, USA) to maintain a constant level of background odor [ 48 ]. The metal oxide (Taguchi type) sensor was a 50 nm thick SnO 2 film, sputtered on a 4 × 4 µm 2 microhotplate with a platinum heater and sensing electrodes. It was designed and manufactured by IM2NP laboratory and details were described elsewhere [ 49 ]. 3. Results and Discussion Figure 5 shows the measured PDS with the bacterial sample and the reference PDS which was measured using the same sensor in laboratory air. Figure 6 shows the binary and ternary fingerprints extracted from the spectra shown in Figure 5 , respectively. The reproducibility of the odor sensing systems is of great importance. Formerly binary spectra showed a good reproducibility with Escherichia coli and Bacillus anthracis (anthrax) bacterial samples [ 23 ]. Similarly, the reproducibility of the binary fingerprints for the manure isolate Bacillus cereus was satisfactory ( Figure 7 ). Therefore, with the ternary fingerprint method, a good reproducibility was also expected. We tested the reproducibility of the ternary fingerprints with our sensor system and bacteria and reference samples. Figure 8 indicates that the reproducibility results of the ternary fingerprints are satisfactory. 4. Conclusions An improved method for evaluating fluctuation-enhanced sensing (FES) results was introduced. In the ternary method, the fingerprint bit values are −1, 0, and +1, where the 0 value stands for the situation where the slope of the spectrum is identical to that of the reference odor. This step increases the information entropy of the bit pattern by 50%. The application of the reference odor spectrum makes the fingerprint relative to the reference, which further increases the information entropy of the fingerprints. Measuring bacterial odor in cow manure isolates indicates good reproducibility. Bit-based direct fingerprinting methods like this one are less intelligent than machine learning tools [ 42 , 43 , 44 , 45 ], but they have reduced energy dissipation and can be important in specific FES applications.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2364605/

Proteasome inhibition enhances the induction and impairs the maintenance of late-phase long-term potentiation

Protein degradation by the ubiquitin–proteasome pathway plays important roles in synaptic plasticity, but the molecular mechanisms by which proteolysis regulates synaptic strength are not well understood. We investigated the role of the proteasome in hippocampal late-phase long-term potentiation (L-LTP), a model for enduring synaptic plasticity. We show here that inhibition of the proteasome enhances the induction of L-LTP, but inhibits its maintenance. Proteasome inhibitor-mediated enhancement of the early part of L-LTP requires activation of NMDA receptors and the cAMP-dependent protein kinase. Augmentation of L-LTP induction by proteasome inhibition is blocked by a protein synthesis inhibitor anisomycin and is sensitive to the drug rapamycin. Our findings indicate that proteasome inhibition increases the induction of L-LTP by stabilizing locally translated proteins in dendrites. In addition, our data show that inhibition of the proteasome blocks transcription of brain-derived neurotrophic factor ( BDNF ), which is a cAMP-responsive element-binding protein (CREB)-inducible gene. Furthermore, our results demonstrate that the proteasome inhibitors block degradation of ATF4, a CREB repressor. Thus, proteasome inhibition appears to hinder CREB-mediated transcription. Our results indicate that blockade of proteasome activity obstructs the maintenance of L-LTP by interfering with transcription as well as translation required to sustain L-LTP. Thus, proteasome-mediated proteolysis has different roles during the induction and the maintenance of L-LTP.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162325/

Biosafety: Guidelines for Working with Pathogenic and Infectious Microorganisms

Abstract This unit, in conjunction with local and national guidelines and regulations (see appendix 1b ), provides the basic biosafety information needed to perform the procedures detailed in this manual. Topics discussed include routine precautions when working with biohazards, disinfectants, disposal of biohazards, biosafety levels (as established by the U.S. National Institutes of Health and the U.S. Centers for Disease Control and Prevention), animal facilities, and clinical laboratories. In addition, resources for more information are provided in the Literature Cited and Key References sections and in URLs given within the text, as well as the Internet Resources section. Curr. Protoc. Microbiol . 13:1A.1.1‐1A.1.14. © 2009 by John Wiley & Sons, Inc. Introduction One of the most important emerging technologies used by microbiologists and other life scientists and laboratory workers that handle pathogenic and infectious agents is the technology that manifests in what is collectively referred to as biosafety. Biosafety measures designed to ensure the safety of laboratory workers include the use of various primary and secondary barriers, many of which are due to the advent of new technologies in the fields of materials science and engineering. Persons carrying out the protocols in this manual may encounter potentially hazardous materials such as pathogenic and infectious biological agents, as well as toxic chemicals and carcinogenic, mutagenic, or teratogenic reagents (see unit mc01a03 ). In the case of biological agents, it has long been recognized that laboratory workers can acquire infections from the agents they manipulate, thus making the very nature of their work an occupational hazard. As an example, in 1910, Dr. Howard Taylor Ricketts acquired typhus and died while studying the disease using a primitive containment device. Since that time, there have been many reports of laboratory‐acquired infection, with many more cases probably having gone unreported. Although it would be comforting to consider laboratory‐acquired infection an artifact of a less sophisticated time in biomedical research, recent examples refute this. In 2001, for example, a public health laboratory worker in the U.S. acquired cutaneous anthrax due to an unauthorized switch in disinfectants (MMWR, 2002 ). In 2004, one of the last known deaths caused by the SARS (severe acute respiratory syndrome)‐associated coronavirus was that of the mother of a researcher who had acquired SARS in a Beijing laboratory and transmitted it to her (Normille, 2004 ). Again, the cause was likely an unauthorized or untested procedure to inactivate the virus. Also in 2004, a sample of Bacillus anthracis was presumably inactivated in a laboratory in Maryland and sent to researchers in California for use in animal studies (Enserink and Kaiser, 2004 ; MMWR, 2005 ). When the study animals died with symptoms of anthrax, it was found that the inactivation procedure had not been validated in the laboratory in Maryland and that viable B. anthracis was received by an unsuspecting laboratory. Eight laboratory workers received post‐exposure treatment; none developed anthrax. These recent events highlight the continuing possibility of the occurrence of laboratory‐acquired infections due to simple and preventable laboratory errors. New biosafety technologies and associated evolving guidelines have emerged to significantly improve ways to safely handle microbiological material. In addition, a better understanding of the risks associated with various manipulations of many agents transmissible by different routes has facilitated our ability to apply appropriate biosafety practices to specific laboratory arenas. As this knowledge base grows and new biosafety technologies emerge, evolving safety guidelines will continue to benefit laboratory workers. A combination of engineering controls, management policies, and work practices and procedures, as well as medical interventions, collectively defines these safety guidelines. Several biosafety levels, described in this unit, have been developed for microbiological and biomedical laboratories to provide increasing levels of personnel and environmental protection. unit mc01a02 provides information related to biosafety practices associated with potential agents of biocrime and biowarfare. unit mc01a03 provides guidelines for the safe use of hazardous chemicals. unit mc01a04 will discuss the safe use of radioisotopes. It is important to note that most governments regulate the use of biohazardous materials. Therefore, it is essential that they be used in strict accordance with local and national regulations (see appendix mca01b ). Cautionary notes are included in many instances throughout the manual, and some specific guidelines are provided below (and in references therein). However, we emphasize that users must proceed with the prudence and precautions associated with good laboratory practice, under the supervision of personnel responsible for implementing laboratory safety programs at their institutions. Conducting Research Safely To conduct research safely with potentially hazardous microorganisms, the researcher must (1) identify the components of the research that are hazardous (hazard identification), (2) assess the additional risks associated with manipulating the materials in experimentation (risk assessment), and (3) establish the facilities, equipment, and practices necessary to protect workers from the identified risk (risk management). The responsible party for conducting hazard identification and risk assessment and for putting risk management measures in place is generally the laboratory director. The laboratory director is further responsible for ensuring that all research personnel are aware of the risks associated with the research and are trained in the techniques established for safe work practices. Roles and responsibilities for safe conduct in research must be established for all persons involved prior to initiating research. Precautions described in this unit should be applied to the routine handling of viable pathogenic microorganisms, as well as all human‐derived materials, because they may harbor dangerous pathogens such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), cytomegalovirus (CMV), Epstein‐Barr virus (EBV), and some bacterial pathogens. In addition to concern for transmission from human‐derived material, research materials derived from animals should be evaluated for the presence of agents capable of infecting humans. An unfortunate example is the likelihood that materials derived from Old World nonhuman primates such as the macaque may contain cercopithecine herpesvirus (CHV‐1), also called herpes B virus or B‐virus. This virus causes simple cold sores in macaques but can be fatal to humans (Holmes et al., 1995 ). Other rodent‐borne diseases have been transferred to humans via cell lines that were passaged through rodent hosts (Baum et al., 1966 ). Many other pathogens that infect animals can also infect humans: these are called zoonotic agents (Hankenson et al., 2003 ). The potential hazard of a biological agent is determined by more than just the nature of the organisms. In an experimental setting, a good part of the concern comes from the manner in which biological material is handled. Experimental procedures are inherently designed to shake, poke, and probe microbes—methods that are likely to contribute to the release of an organism. Work in today's research environment often requires amplification, genetic modification, and testing in animal models. Laboratory Manipulations Many basic research activities such as centrifugation, sonication, vortexing, pipetting, etc. by their nature may generate splashes, sprays, or aerosols. Amplification of an organism creates the potential for a larger exposure if released accidentally. The equipment and process used for these methods must be designed to minimize splashes, sprays, aerosols, or other inadvertent releases. Genetic Modifications The genetic modification of microorganisms may clearly impact the level of risk. Many genetic modifications will alter the mechanisms of reproduction, replication, and/or host range or cell tropism—a risk assessment should evaluate whether this enhances or reduces virulence. Addition of genes that code for toxin production or alter antibiotic resistance must obviously be approached with a high level of caution and prudence. The introduction of genes that are known to contribute or suspected of contributing to a cancer pathway or the use of agents that could be inserted into host DNA must also be carefully evaluated for the risk they confer. Research Animals The researcher must be aware that infecting animals, intentionally or inadvertently, could add another dimension to the possible hazards. The animal may amplify the organism, especially in the case of immunodeficient animals. The shedding of the organism must be considered when establishing procedures for manipulating cages, bedding, and the animals themselves. The potential for transmission of infection to other research (or wild) animals must also be evaluated. In addition to the guidelines provided herein, experimenters can find a wealth of information about handling infectious agents in the appropriate government publications (see Literature Cited and appendix mca01b ). Laboratory Manipulations Many basic research activities such as centrifugation, sonication, vortexing, pipetting, etc. by their nature may generate splashes, sprays, or aerosols. Amplification of an organism creates the potential for a larger exposure if released accidentally. The equipment and process used for these methods must be designed to minimize splashes, sprays, aerosols, or other inadvertent releases. Genetic Modifications The genetic modification of microorganisms may clearly impact the level of risk. Many genetic modifications will alter the mechanisms of reproduction, replication, and/or host range or cell tropism—a risk assessment should evaluate whether this enhances or reduces virulence. Addition of genes that code for toxin production or alter antibiotic resistance must obviously be approached with a high level of caution and prudence. The introduction of genes that are known to contribute or suspected of contributing to a cancer pathway or the use of agents that could be inserted into host DNA must also be carefully evaluated for the risk they confer. Research Animals The researcher must be aware that infecting animals, intentionally or inadvertently, could add another dimension to the possible hazards. The animal may amplify the organism, especially in the case of immunodeficient animals. The shedding of the organism must be considered when establishing procedures for manipulating cages, bedding, and the animals themselves. The potential for transmission of infection to other research (or wild) animals must also be evaluated. In addition to the guidelines provided herein, experimenters can find a wealth of information about handling infectious agents in the appropriate government publications (see Literature Cited and appendix mca01b ). General Biosafety Guidelines Routine Precautions When Working with Biohazards The following practices are recommended for all laboratories handling potentially dangerous microorganisms, whether pathogenic or not: Limit access to work areas. Close doors during work with research materials. Decontaminate all work surfaces after each working day using an appropriate disinfectant. Decontaminate all spills of viable material immediately. See discussion under Disinfectants for Biohazards. Decontaminate all liquid or solid wastes that have come in contact with viable material. Do not pipet by mouth. Do not allow eating, drinking, smoking, or application of cosmetics in the work area. Do not store food in refrigerators that contain laboratory supplies. Wash hands with soap or detergent after handling viable materials or removing gloves, and before leaving the laboratory. Do not handle telephones, doorknobs, or other common utensils without washing hands. When handling viable materials, minimize creation of aerosols. Wear laboratory coats (preferably disposable) when in work area, but do not wear them away from the work area. Wear disposable gloves when handling viable materials. These should be disposed of as biohazardous waste. Change gloves if they are directly contaminated. Do not wear gloves away from the work area. Use sharps only when no alternatives (e.g., safety devices or non‐sharps) exist. Wear eye/face protection if splashes or sprays are anticipated. Transport materials outside of the laboratory using secondary containment. Transfer materials to other facilities according to federal and international regulations. Be familiar with written instructions for laboratory procedures and proper responses to emergencies. Report spills, exposures, illnesses, and injuries immediately. Control pest populations. Windows in the laboratory that can be opened must be equipped with screens to exclude insects. Use furniture that is easy to clean—i.e., with smooth, waterproof surfaces and as few seams as possible. Keep biohazard waste in covered containers free from leaks. Use orange or red biohazard bags (or other appropriate color in accordance with local regulations) as required by institutional procedure. Dispose of according to institutional procedure. See discussion under Disposal of Biohazards (below) for more information. Disinfectants for Biohazards Work surfaces must be disinfected prior to beginning work, after work is completed, and in the case of spills. Many types of disinfectants are available—each should be evaluated to determine if it is appropriate to inactivate the research materials being used and the surfaces to be cleaned. For example, a 1:10 dilution of commercially available household bleach is very effective against most microorganisms. However, over time, bleach can be corrosive and will pit stainless steel surfaces, thus the use of bleach should be followed by a water rinse on these surfaces. In addition, the dilution must be prepared daily due to the rapid degradation of hypochlorite ions over time. Another common disinfectant is 70% isopropyl alcohol. Although quite effective at inactivating several classes of microbes, its use on work surfaces results in nearly immediate evaporation, which does not allow for the contact time necessary required for inactivation. When using commercially available products, the label, the product information sheet, and the material safety data sheet must be reviewed to determine appropriate use. Major laboratory suppliers sell disinfectants based on quaternary ammonium compounds that are acceptable for routine biohazard decontamination (see mcaspl ). These include Roccal (Baxter), Vesphene II (Fisher), and industrial disinfectants such as concentrated Lysol. Receiving Biological Materials Only personnel trained to receive packages containing biological materials should accept and unpack packages. Arrangements must be made so that deliveries are secured as soon as they arrive. Storage of Biological Materials All refrigerators, cold rooms, freezers, or cryopreservation units housing potential pathogens and other biological materials must be labeled with the international biohazard symbol, as well as a list of contact information for those responsible for the unit and/or the materials. Access to these storage units must be limited to those who have been made aware of the potential hazard. An inventory of the materials contained within each unit should be maintained. A plan for notification of key laboratory personnel in case of power or unit failure must be developed. Experimentation Administrative Controls are procedural, administrative measures that are established well before the experimentation begins. Some administrative controls include: Applicable approvals from biosafety oversight committees or institutional offices must be obtained. Some materials may also require approval of a human subject protection or animal welfare committee. When possible, less hazardous materials should be substituted in experiments, especially at the initiation of research when unfamiliar methods are used. Laboratory procedures must be documented and recordkeeping must be implemented so that processes used during research are clearly outlined. Safe work practices and the use of personal protective equipment must be included in the procedures. Engineering Controls are generally mechanical in nature. The purpose of engineering controls is to use a mechanical means to isolate the worker from the hazard. Some engineering controls include: The availability of "safer sharps"—needles or blades that are self‐sheathing or automatically retracted—has increased recently and should replace traditional devices. The use of plastic instead of glass for vials, flasks, beakers, etc. must be a priority. Containers used to collect waste for special or normal waste handling must be sturdy, compatible with the waste to be collected, labeled appropriately, and kept closed when not in use. Biological safety cabinets (BSCs; also known as vertical laminar flow hoods or tissue culture hoods) have become a staple in biological research. These cabinets protect the experiment and those exposed to the exhaust by scrubbing the particulate‐laden air currents via high‐efficiency particulate air (HEPA) filters. A HEPA filter, by requirement, is 99.97% effective at removing particles of 0.3 µm—the particle size at which HEPA filters are the least efficient. Due to a diversity of particle‐trapping methods such as impaction, straining, diffusion, interception, and electrostatic forces, the HEPA filtration process actually has an efficiency >99.97% for removing particles both larger and smaller than 0.3 µm. For a detailed description of selection, installation, and use of this equipment, the reader is referred to appendix a in the CDC/NIH publication, Biosafety in Microbiological and Biomedical Laboratories, 5th edition. ( http://www.cdc.gov/od/ohs/biosfty/bmbl5/sections/AppendixA.pdf .) Many laboratories are equipped with single pass, directional air flow ventilation. Keeping the air currents flowing into the laboratory from the corridor keeps particulates such as microbes and gases of research chemical in the laboratory. The routine precautions listed above are Work Practices that should be familiar to and practiced by all laboratorians. The importance of frequent and routine hand washing cannot be overemphasized. Workers must wash their hands using soap and water for 10 to 30 sec prior to beginning work, any time gloves are removed or changed, and before leaving the work area at any point during the day. Soap must be provided in a dispenser near the sink so that no one need handle the outside of the container to use it. The assignment of Personal Protective Equipment (PPE) is considered to be the "last resort" for preventing and protecting against hazard exposures. When the application of administrative and engineering controls and work practices is not sufficient to protect against exposure, PPE is used to provide an additional and/or redundant layer of protection. The goal of PPE is two‐fold: (1) PPE provides a barrier to possible routes of entry on a worker's body and (2) PPE protects street clothing and shoes so that laboratory contaminants are not inadvertently taken from the laboratory into a nonresearch area, such as a worker's home. The use of protective clothing such as buttoned labcoats or back‐closing gowns and gloves are commonplace in today's research laboratory. The use of these items is appropriate even during research of minimal biosafety concern. Face protection, such as a face shield or a combination of goggles plus a surgical mask afford protection to the mucous membranes of the eyes, nose, and mouth. In certain circumstances, shoe or hair covers may be required. The use of respirators may be required in settings where airborne transmission is possible. (Note: any use of respiratory protection must follow an institutional Respiratory Protection Program. Institutional industrial hygienists or environmental health professionals must be consulted prior to using a respirator). PPE must be selected carefully for the work to be performed. For example, most gloves are suitable for creating a barrier against biological agents, but they may not be compatible with research chemicals. Some materials found in PPE may contain allergens, such as latex, that can stimulate health problems for allergic personnel. PPE must be put on (donned) in the laboratory and removed (doffed) prior to leaving the work area. Taking off PPE must be performed carefully to avoid contaminating skin or street clothes. Laboratory personnel must not launder labcoats or other reusable PPE at home. Disposal of Biohazards Regardless of the hazard posed, waste materials used in experiments with biological materials must be decontaminated prior to ultimate disposal in landfills or other legal disposal locations. Waste must be segregated into at least three categories: liquid, solid, and sharps. Liquid waste may be autoclaved or may be inactivated using a liquid disinfectant—refer to manufacturer's information for appropriate dilutions and minimum contact time. In most locations, inactivated liquid waste can be disposed into the sanitary sewer via the laboratory sink; however, it is imperative to confirm that this is acceptable for each institution. Solid waste is generally placed in autoclavable bags, which are contained in a collection container labeled with the international biohazard symbol. This waste can be autoclaved at the laboratory or collected for treatment by the institution or a contractor prior to final disposal. It must be verified that autoclaves and other treatment methods inactivate biological organisms through periodic validation tests with biological indicators. Actual records of this testing or confirmation from contractors must be kept in the laboratory or by the institution. The specific procedures for collecting and decontaminating solid waste must be confirmed for each institution. Sharps—contaminated needles, scalpels, broken glass, etc.—must be collected in puncture‐resistant sharps disposable containers available from many commercial vendors. The definition of what constitutes a sharp and how sharps disposal containers are collected must be confirmed within each institution. Emergency Preparedness and Response Even when all the previously described provisions have been addressed and implemented, emergencies or disruptions to the normal research environment will occasionally occur. In these cases, the risk management techniques described above will help limit the extent of the disruption, but some additional planning in anticipation of these events is in order. Possible disruptions include spills, exposures, injuries and illnesses; power or water loss; equipment failure; a fire in the laboratory; fire or other threat elsewhere in the facility; and the possibility of disruption or destruction due to severe weather or flooding. The response to each of these will depend on the individual and institutional circumstances of the research and are too varied and numerous to discuss here. An appropriate exercise for each laboratory director to perform is to establish procedures for "what to do when" for each of the above and other potential disruptions that might occur. In a simple example, the loss of water will preclude the ability to perform the usual and proper hand washing. Part of a plan for this disruption might include assuring that a supply of waterless hand sanitizer is available in the laboratory at all times. Spills of biological materials are generally quite simple to address. Because of the possibility that particulates or droplets will linger in the air for a while after the spill, it is best to leave the immediate area of the spill undisturbed for at least 15 min. This time allows the laboratory worker to remove and replace any contaminated PPE, to make appropriate notifications, to address any potential exposures, and/or to gather appropriate spill clean‐up materials. When ready to clean, a layer of absorbent material such as paper towels can be placed gently on top of the spill, followed by an application of an appropriately diluted disinfectant starting at the perimeter of the spill and working towards the center. When saturated by disinfectant and after the specified contact time, the spill may be wiped up with the saturated absorbent materials. The surface will require a second disinfection. All disposable clean‐up materials must be treated as normal biological waste. If a worker receives an exposure (e.g., needlestick or contamination of eyes, nose, mouth, or non‐intact skin), is injured during work with biological materials, or becomes inexplicably ill with symptoms parallel to those expected from the research agent, further medical evaluation is necessary. A relationship with an occupational health provider or other physician contracted by the institution to evaluate workplace incidents must be established by the institution or, if necessary, the laboratory director, prior to the initiation of research. Procedures and documentation for reporting and responding to these types of incidents must be established and communicated to research personnel. Transfer of Research Materials One of the idiosyncrasies of biological research is the extent to which biological materials are shared between researchers and institutions, rather than acquired from commercial sources. The responsibilities that accompany these transfers are significantly more rigorous than is generally acknowledged or implemented by the informal agreements between researchers. Increasingly, transferred materials are accompanied by material transfer agreements with provisions that, among other things, limit the use and further transfer of the materials. The shipment and receipt of diagnostic specimens (which includes most nonpathogenic research materials) and infectious substances (most cultures of pathogens) is regulated internationally by the International Air Transport Association (IATA). Nearly all express couriers follow the IATA provisions, which require that any person involved in transport, including the person who types the paperwork to accompany the shipment or who signs for the package, must be trained in the proper procedures. In addition, many countries have their own, similar provisions for ground transport. Export and import of research materials is becoming increasingly complex and nearly always requires a permit. The agencies overseeing this permitting process vary country by country. The International Biosafety Working Group (IBWG) maintains a compendium (see Internet Resources ), which provides a listing of currently identified permit requirements. Even if materials are being transferred across the hallway to a colleague's laboratory or to an internal core facility for analysis, the materials must be transferred using a secondary container that is capable of containing any spill from the primary container. Documenting the chain of custody to identify who is in control and accountable for the material at each point during the transfer is important. This is outlined in greater detail in the next unit on biosecurity ( unit mc01a02 ). Lastly, any addition or removal of materials from a laboratory's inventory must be documented and any approvals necessary for added materials must be acquired. Training All of the information discussed above must be communicated by the laboratory director to research personnel. Lack of communication of these critical elements has been seen as a key factor in recent laboratory incidents. In some cases, specific training is required by regulation and can often be obtained through the institutional biosafety professional. However, institution‐based training does not remove the obligation for the laboratory director to provide research‐specific training and continual guidance to research staff. A written training plan accompanied by records that show the training offered, who received it, and when it was provided, is a strongly recommended step for each laboratory. Routine Precautions When Working with Biohazards The following practices are recommended for all laboratories handling potentially dangerous microorganisms, whether pathogenic or not: Limit access to work areas. Close doors during work with research materials. Decontaminate all work surfaces after each working day using an appropriate disinfectant. Decontaminate all spills of viable material immediately. See discussion under Disinfectants for Biohazards. Decontaminate all liquid or solid wastes that have come in contact with viable material. Do not pipet by mouth. Do not allow eating, drinking, smoking, or application of cosmetics in the work area. Do not store food in refrigerators that contain laboratory supplies. Wash hands with soap or detergent after handling viable materials or removing gloves, and before leaving the laboratory. Do not handle telephones, doorknobs, or other common utensils without washing hands. When handling viable materials, minimize creation of aerosols. Wear laboratory coats (preferably disposable) when in work area, but do not wear them away from the work area. Wear disposable gloves when handling viable materials. These should be disposed of as biohazardous waste. Change gloves if they are directly contaminated. Do not wear gloves away from the work area. Use sharps only when no alternatives (e.g., safety devices or non‐sharps) exist. Wear eye/face protection if splashes or sprays are anticipated. Transport materials outside of the laboratory using secondary containment. Transfer materials to other facilities according to federal and international regulations. Be familiar with written instructions for laboratory procedures and proper responses to emergencies. Report spills, exposures, illnesses, and injuries immediately. Control pest populations. Windows in the laboratory that can be opened must be equipped with screens to exclude insects. Use furniture that is easy to clean—i.e., with smooth, waterproof surfaces and as few seams as possible. Keep biohazard waste in covered containers free from leaks. Use orange or red biohazard bags (or other appropriate color in accordance with local regulations) as required by institutional procedure. Dispose of according to institutional procedure. See discussion under Disposal of Biohazards (below) for more information. Disinfectants for Biohazards Work surfaces must be disinfected prior to beginning work, after work is completed, and in the case of spills. Many types of disinfectants are available—each should be evaluated to determine if it is appropriate to inactivate the research materials being used and the surfaces to be cleaned. For example, a 1:10 dilution of commercially available household bleach is very effective against most microorganisms. However, over time, bleach can be corrosive and will pit stainless steel surfaces, thus the use of bleach should be followed by a water rinse on these surfaces. In addition, the dilution must be prepared daily due to the rapid degradation of hypochlorite ions over time. Another common disinfectant is 70% isopropyl alcohol. Although quite effective at inactivating several classes of microbes, its use on work surfaces results in nearly immediate evaporation, which does not allow for the contact time necessary required for inactivation. When using commercially available products, the label, the product information sheet, and the material safety data sheet must be reviewed to determine appropriate use. Major laboratory suppliers sell disinfectants based on quaternary ammonium compounds that are acceptable for routine biohazard decontamination (see mcaspl ). These include Roccal (Baxter), Vesphene II (Fisher), and industrial disinfectants such as concentrated Lysol. Receiving Biological Materials Only personnel trained to receive packages containing biological materials should accept and unpack packages. Arrangements must be made so that deliveries are secured as soon as they arrive. Storage of Biological Materials All refrigerators, cold rooms, freezers, or cryopreservation units housing potential pathogens and other biological materials must be labeled with the international biohazard symbol, as well as a list of contact information for those responsible for the unit and/or the materials. Access to these storage units must be limited to those who have been made aware of the potential hazard. An inventory of the materials contained within each unit should be maintained. A plan for notification of key laboratory personnel in case of power or unit failure must be developed. Experimentation Administrative Controls are procedural, administrative measures that are established well before the experimentation begins. Some administrative controls include: Applicable approvals from biosafety oversight committees or institutional offices must be obtained. Some materials may also require approval of a human subject protection or animal welfare committee. When possible, less hazardous materials should be substituted in experiments, especially at the initiation of research when unfamiliar methods are used. Laboratory procedures must be documented and recordkeeping must be implemented so that processes used during research are clearly outlined. Safe work practices and the use of personal protective equipment must be included in the procedures. Engineering Controls are generally mechanical in nature. The purpose of engineering controls is to use a mechanical means to isolate the worker from the hazard. Some engineering controls include: The availability of "safer sharps"—needles or blades that are self‐sheathing or automatically retracted—has increased recently and should replace traditional devices. The use of plastic instead of glass for vials, flasks, beakers, etc. must be a priority. Containers used to collect waste for special or normal waste handling must be sturdy, compatible with the waste to be collected, labeled appropriately, and kept closed when not in use. Biological safety cabinets (BSCs; also known as vertical laminar flow hoods or tissue culture hoods) have become a staple in biological research. These cabinets protect the experiment and those exposed to the exhaust by scrubbing the particulate‐laden air currents via high‐efficiency particulate air (HEPA) filters. A HEPA filter, by requirement, is 99.97% effective at removing particles of 0.3 µm—the particle size at which HEPA filters are the least efficient. Due to a diversity of particle‐trapping methods such as impaction, straining, diffusion, interception, and electrostatic forces, the HEPA filtration process actually has an efficiency >99.97% for removing particles both larger and smaller than 0.3 µm. For a detailed description of selection, installation, and use of this equipment, the reader is referred to appendix a in the CDC/NIH publication, Biosafety in Microbiological and Biomedical Laboratories, 5th edition. ( http://www.cdc.gov/od/ohs/biosfty/bmbl5/sections/AppendixA.pdf .) Many laboratories are equipped with single pass, directional air flow ventilation. Keeping the air currents flowing into the laboratory from the corridor keeps particulates such as microbes and gases of research chemical in the laboratory. The routine precautions listed above are Work Practices that should be familiar to and practiced by all laboratorians. The importance of frequent and routine hand washing cannot be overemphasized. Workers must wash their hands using soap and water for 10 to 30 sec prior to beginning work, any time gloves are removed or changed, and before leaving the work area at any point during the day. Soap must be provided in a dispenser near the sink so that no one need handle the outside of the container to use it. The assignment of Personal Protective Equipment (PPE) is considered to be the "last resort" for preventing and protecting against hazard exposures. When the application of administrative and engineering controls and work practices is not sufficient to protect against exposure, PPE is used to provide an additional and/or redundant layer of protection. The goal of PPE is two‐fold: (1) PPE provides a barrier to possible routes of entry on a worker's body and (2) PPE protects street clothing and shoes so that laboratory contaminants are not inadvertently taken from the laboratory into a nonresearch area, such as a worker's home. The use of protective clothing such as buttoned labcoats or back‐closing gowns and gloves are commonplace in today's research laboratory. The use of these items is appropriate even during research of minimal biosafety concern. Face protection, such as a face shield or a combination of goggles plus a surgical mask afford protection to the mucous membranes of the eyes, nose, and mouth. In certain circumstances, shoe or hair covers may be required. The use of respirators may be required in settings where airborne transmission is possible. (Note: any use of respiratory protection must follow an institutional Respiratory Protection Program. Institutional industrial hygienists or environmental health professionals must be consulted prior to using a respirator). PPE must be selected carefully for the work to be performed. For example, most gloves are suitable for creating a barrier against biological agents, but they may not be compatible with research chemicals. Some materials found in PPE may contain allergens, such as latex, that can stimulate health problems for allergic personnel. PPE must be put on (donned) in the laboratory and removed (doffed) prior to leaving the work area. Taking off PPE must be performed carefully to avoid contaminating skin or street clothes. Laboratory personnel must not launder labcoats or other reusable PPE at home. Disposal of Biohazards Regardless of the hazard posed, waste materials used in experiments with biological materials must be decontaminated prior to ultimate disposal in landfills or other legal disposal locations. Waste must be segregated into at least three categories: liquid, solid, and sharps. Liquid waste may be autoclaved or may be inactivated using a liquid disinfectant—refer to manufacturer's information for appropriate dilutions and minimum contact time. In most locations, inactivated liquid waste can be disposed into the sanitary sewer via the laboratory sink; however, it is imperative to confirm that this is acceptable for each institution. Solid waste is generally placed in autoclavable bags, which are contained in a collection container labeled with the international biohazard symbol. This waste can be autoclaved at the laboratory or collected for treatment by the institution or a contractor prior to final disposal. It must be verified that autoclaves and other treatment methods inactivate biological organisms through periodic validation tests with biological indicators. Actual records of this testing or confirmation from contractors must be kept in the laboratory or by the institution. The specific procedures for collecting and decontaminating solid waste must be confirmed for each institution. Sharps—contaminated needles, scalpels, broken glass, etc.—must be collected in puncture‐resistant sharps disposable containers available from many commercial vendors. The definition of what constitutes a sharp and how sharps disposal containers are collected must be confirmed within each institution. Emergency Preparedness and Response Even when all the previously described provisions have been addressed and implemented, emergencies or disruptions to the normal research environment will occasionally occur. In these cases, the risk management techniques described above will help limit the extent of the disruption, but some additional planning in anticipation of these events is in order. Possible disruptions include spills, exposures, injuries and illnesses; power or water loss; equipment failure; a fire in the laboratory; fire or other threat elsewhere in the facility; and the possibility of disruption or destruction due to severe weather or flooding. The response to each of these will depend on the individual and institutional circumstances of the research and are too varied and numerous to discuss here. An appropriate exercise for each laboratory director to perform is to establish procedures for "what to do when" for each of the above and other potential disruptions that might occur. In a simple example, the loss of water will preclude the ability to perform the usual and proper hand washing. Part of a plan for this disruption might include assuring that a supply of waterless hand sanitizer is available in the laboratory at all times. Spills of biological materials are generally quite simple to address. Because of the possibility that particulates or droplets will linger in the air for a while after the spill, it is best to leave the immediate area of the spill undisturbed for at least 15 min. This time allows the laboratory worker to remove and replace any contaminated PPE, to make appropriate notifications, to address any potential exposures, and/or to gather appropriate spill clean‐up materials. When ready to clean, a layer of absorbent material such as paper towels can be placed gently on top of the spill, followed by an application of an appropriately diluted disinfectant starting at the perimeter of the spill and working towards the center. When saturated by disinfectant and after the specified contact time, the spill may be wiped up with the saturated absorbent materials. The surface will require a second disinfection. All disposable clean‐up materials must be treated as normal biological waste. If a worker receives an exposure (e.g., needlestick or contamination of eyes, nose, mouth, or non‐intact skin), is injured during work with biological materials, or becomes inexplicably ill with symptoms parallel to those expected from the research agent, further medical evaluation is necessary. A relationship with an occupational health provider or other physician contracted by the institution to evaluate workplace incidents must be established by the institution or, if necessary, the laboratory director, prior to the initiation of research. Procedures and documentation for reporting and responding to these types of incidents must be established and communicated to research personnel. Transfer of Research Materials One of the idiosyncrasies of biological research is the extent to which biological materials are shared between researchers and institutions, rather than acquired from commercial sources. The responsibilities that accompany these transfers are significantly more rigorous than is generally acknowledged or implemented by the informal agreements between researchers. Increasingly, transferred materials are accompanied by material transfer agreements with provisions that, among other things, limit the use and further transfer of the materials. The shipment and receipt of diagnostic specimens (which includes most nonpathogenic research materials) and infectious substances (most cultures of pathogens) is regulated internationally by the International Air Transport Association (IATA). Nearly all express couriers follow the IATA provisions, which require that any person involved in transport, including the person who types the paperwork to accompany the shipment or who signs for the package, must be trained in the proper procedures. In addition, many countries have their own, similar provisions for ground transport. Export and import of research materials is becoming increasingly complex and nearly always requires a permit. The agencies overseeing this permitting process vary country by country. The International Biosafety Working Group (IBWG) maintains a compendium (see Internet Resources ), which provides a listing of currently identified permit requirements. Even if materials are being transferred across the hallway to a colleague's laboratory or to an internal core facility for analysis, the materials must be transferred using a secondary container that is capable of containing any spill from the primary container. Documenting the chain of custody to identify who is in control and accountable for the material at each point during the transfer is important. This is outlined in greater detail in the next unit on biosecurity ( unit mc01a02 ). Lastly, any addition or removal of materials from a laboratory's inventory must be documented and any approvals necessary for added materials must be acquired. Training All of the information discussed above must be communicated by the laboratory director to research personnel. Lack of communication of these critical elements has been seen as a key factor in recent laboratory incidents. In some cases, specific training is required by regulation and can often be obtained through the institutional biosafety professional. However, institution‐based training does not remove the obligation for the laboratory director to provide research‐specific training and continual guidance to research staff. A written training plan accompanied by records that show the training offered, who received it, and when it was provided, is a strongly recommended step for each laboratory. Biosafety Levels For each biosafety level, there are specific supervisory qualifications as assurance that laboratory workers are provided appropriate role models and knowledgeable mentors. Various types of specialized equipment are used to provide primary barriers between the microorganism and the laboratory worker. These range from disposable gloves and other personnel protective equipment to complex biosafety cabinets or other containment devices. The laboratory director is specifically and primarily responsible for the safe operation of the laboratory. His/her knowledge and judgment are critical in assessing risks and appropriately applying these recommendations. The recommended biosafety level represents those conditions under which the agent can ordinarily be safely handled. Special characteristics of the agents used, the training and experience of personnel, and the nature or function of the laboratory may further influence the director in applying these recommendations. The U.S. Centers for Disease Control and Prevention (CDC; see Internet Resources ) defines four levels of biosafety, which are outlined below. Selection of an appropriate biosafety level for work with a particular agent or animal study (see Animal Facilities ) depends upon a number of factors. Some of the most important are the virulence, pathogenicity, biological stability, route of spread, and communicability of the agent; the nature or function of the laboratory; the procedures and manipulations involving the agent; the endemicity of the agent; and the availability of effective vaccines or therapeutic measures. Table 1 provides a summary of recommended biosafety levels for infectious agents. For regulations and guidelines applicable outside of the U.S., please refer to appendix mca01b and Internet Resources . Table 1 CDC Summary of Recommended Biosafety Levels for Infectious Agents a , b Biosafety level Agent characteristics Practices Safety equipment (primary barriers) c Facilities (secondary barriers) BSL‐1 Not known to consistently cause disease in healthy adults Standard microbiological practices None required Open benchtop sink BSL‐2 Associated with human disease, hazard from percutaneous injury, ingestion, mucous membrane exposure Standard microbiological practices Limited access Biohazard warning signs "Sharps" precautions Biosafety manual defining any needed waste decontamination or medical surveillance policies Class I or II biosafety cabinets (BSCs) or other physical containment devices used for all manipulations of agents that cause splashes or aerosols of infectious materials Laboratory coats and gloves Face protection as needed Open benchtop sink Autoclave BSL‐3 Indigenous or exotic agents with potential for aerosol transmission; disease may have serious or lethal consequences All BSL‐2 practices Controlled access Decontamination of all waste Decontamination of laboratory clothing before laundering Baseline serum Class I or II BSCs or other physical containment devices used for all open manipulations of agents Protective lab clothing and gloves Respiratory protection as needed Open benchtop sink Autoclave Physical separation from access corridors Self‐closing, double‐door access Exhausted air not recirculated Negative airflow into laboratory BSL‐4 Dangerous/exotic agents which pose high risk of life‐threatening disease; aerosol‐transmitted lab infections; or related agents with unknown risk of transmission All BSL‐3 practices Clothing change before entering Shower on exit All material decontaminated on exit from facility All procedures conducted in Class III BSCs, or Class I or II BSCs in combination with full‐body, air‐supplied, positive pressure personnel suit BSL‐3 plus: Separate building or isolated zone Dedicated supply and exhaust, vacuum, and decontamination systems Other requirements outlined in the text a Adapted from Biosafety in Microbiological and Biomedical Laboratories, 5th Ed., available online at http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm . b The practices, and primary and secondary barriers required for a given biosafety level include those of the all lower levels, as well as the additional required practices, equipment, and/or facilities described for the BSL in question. c See http://www.cdc.gov/od/ohs/biosfty/bmbl5/sections/AppendixA.pdf for more information concerning biological safety cabinets (BSCs). John Wiley & Sons, Inc. This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency. NOTE : The following information has been adapted from Biosafety in Microbiological and Biomedical Laboratories , 5th Ed. (BMBL, 5th Ed.), which is published jointly by the U.S. Centers for Disease Control and Prevention (CDC) and National Institutes of Health (NIH), and is available online at http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm (see Internet Resources ). Readers are strongly urged to review this publication prior to initiating any experiment outlined in this manual. Biosafety Level 1 (BSL‐1) BSL‐1 is appropriate for working with microorganisms that are not known to cause disease in healthy humans. BSL‐1 practices, safety equipment, and facility design and construction are appropriate for undergraduate and secondary educational training and teaching laboratories, and for other laboratories in which work is done with defined and characterized strains of viable microorganisms not known to consistently cause disease in healthy adult humans. Bacillus subtilis , Naegleria gruberi , infectious canine hepatitis virus, and exempt organisms under the NIH Recombinant DNA Guidelines ( http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html ) are representative of microorganisms meeting these criteria. Many agents not ordinarily associated with disease processes in humans are, however, opportunistic pathogens and may cause infection in the young, the aged, and immunodeficient or immunosuppressed individuals. Vaccine strains that have undergone multiple in vivo passages should not be considered avirulent simply because they are vaccine strains. BSL‐1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for hand washing. In this manual, when BSL‐1 conditions are appropriate to the experiments described, the following note will appear in the unit introduction. CAUTION : is a Biosafety Level 1 (BSL‐1) organism. Such organisms are not known to consistently cause disease in healthy adult humans, and are of minimal potential hazard to laboratory personnel and the environment. Standard microbiological practices should be followed when working with these organisms. See unit mc01a01 and other pertinent resources ( appendix mca01b ). Biosafety Level 2 (BSL‐2) The facility, containment devices, administrative controls (discussed below), and practices and procedures that constitute BSL‐2 are designed to maximize safe working conditions for laboratorians working with agents of moderate risk to personnel and the environment. BSL‐2 practices, equipment, and facility design and construction are applicable to clinical, diagnostic, teaching, and other laboratories in which work is done with the broad spectrum of indigenous moderate‐risk agents that are present in the community and associated with human disease of varying severity. With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided the potential for producing splashes or aerosols is low. Hepatitis B virus, HIV, the Salmonella e, and Toxoplasma spp. are representative of microorganisms assigned to this containment level. Biosafety Level 2 is also appropriate when work is done with any human‐derived blood, body fluids, tissues, or primary human cell lines where the presence of an infectious agent may be unknown. Laboratory personnel in the United States working with human‐derived materials should refer to the U.S. Occupational Safety and Health Administration (OSHA) Bloodborne Pathogen Standard (OSHA, 1991 ), available online at http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=1005 , for required precautions. For guidelines and regulations appropriate to locations outside the U.S., please refer to appendix mca01b and Internet Resources . Primary hazards to personnel working with these agents relate to accidental percutaneous or mucous membrane exposures, or ingestion of infectious materials. Extreme caution should be taken with contaminated needles or sharp instruments. Even though organisms routinely manipulated at Biosafety Level 2 are not known to be transmissible by the aerosol route, procedures with aerosol or high splash potential that may increase the risk of such personnel exposure must be conducted in primary containment equipment, or in devices such as a biological safety cabinet (BSC) or safety centrifuge cups. Personal protective equipment (PPE) should be used as appropriate, such as splash shields, face protection, gowns, and gloves. Secondary barriers such as hand washing sinks and waste decontamination facilities must be available to reduce potential environmental contamination. When BSL‐2 conditions are appropriate to the organism under investigation, the following note is included in the unit introduction. CAUTION : is a Biosafety Level 2 (BSL‐2) pathogen. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See unit mc01a01 and other pertinent resources ( appendix mca01b ) for more information. When BSL‐2 conditions are appropriate due to the use of human‐derived materials, the following note is included in the introduction. CAUTION : Follow all appropriate guidelines and regulations for the use and handling of human‐derived materials. See unit mc01a01 and other pertinent resources ( appendix mca01b ) for more information. Biosafety Level 3 (BSL‐3) BSL‐3 is suitable for work with infectious agents, which may cause serious or potentially lethal diseases as a result of exposure by the inhalation route. This may apply to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents with potential for respiratory transmission, and which may cause serious and potentially lethal infection. Mycobacterium tuberculosis , St. Louis encephalitis virus, and Coxiella burnetii are representative of the microorganisms assigned to this level. Primary hazards to personnel working with these agents relate to autoinoculation, ingestion, and exposure to infectious aerosols. At BSL‐3, more emphasis is placed on primary and secondary barriers to protect personnel in contiguous areas, the community, and the environment from exposure to potentially infectious aerosols (see Table 1 ). For example, all laboratory manipulations should be performed in a BSC or other enclosed equipment, such as a gas‐tight aerosol generation chamber. Secondary barriers for this level include controlled access to the laboratory and ventilation requirements that minimize the release of infectious aerosols from the laboratory. When BSL‐3 conditions are appropriate to the organism under investigation, the following note is included in the unit introduction. CAUTION : is a Biosafety Level 3 (BSL‐3) pathogen. Follow all appropriate guidelines for the use and handling of pathogenic microorganisms. See unit mc01a01 and other pertinent resources ( appendix mca01b ) for more information. When BSL‐3 conditions are appropriate due to the use of human‐derived materials, the following note is included in the introduction. CAUTION : Follow all appropriate guidelines and regulations for the use and handling of human‐derived materials. See unit mc01a01 and other pertinent resources ( appendix mca01b ) for more information. Biosafety Level 4 (BSL‐4) BSL‐4 practices, safety equipment, and facility design and construction are applicable for work with dangerous and exotic agents that pose a high individual risk of life‐threatening disease, which may be transmitted via the aerosol route, and for which there is no available vaccine or therapy. Agents with a close or identical antigenic relationship to Biosafety Level 4 agents also should be handled at this level. When sufficient data are obtained, work with these agents may continue at this or a lower level. Viruses such as Marburg or Congo‐Crimean hemorrhagic fever are manipulated at Biosafety Level 4. The primary hazards to personnel working with Biosafety Level 4 agents are respiratory exposure to infectious aerosols, mucous membrane or broken skin exposure to infectious droplets, and autoinoculation. All manipulations of potentially infectious diagnostic materials, isolates, and naturally or experimentally infected animals pose a high risk of exposure and infection to laboratory personnel, the community, and the environment. The laboratory worker's complete isolation from aerosolized infectious materials is accomplished primarily by working in a Class III BSC or in a full‐body, air‐supplied, positive‐pressure personnel suit. The BSL‐4 facility itself is generally a separate building or completely isolated zone with complex, specialized ventilation requirements and waste management systems to prevent release of viable agents to the environment. As of this printing, there are no experiments described in this manual, which specifically utilize BSL‐4 containment. Biosafety Level 1 (BSL‐1) BSL‐1 is appropriate for working with microorganisms that are not known to cause disease in healthy humans. BSL‐1 practices, safety equipment, and facility design and construction are appropriate for undergraduate and secondary educational training and teaching laboratories, and for other laboratories in which work is done with defined and characterized strains of viable microorganisms not known to consistently cause disease in healthy adult humans. Bacillus subtilis , Naegleria gruberi , infectious canine hepatitis virus, and exempt organisms under the NIH Recombinant DNA Guidelines ( http://www4.od.nih.gov/oba/rac/guidelines/guidelines.html ) are representative of microorganisms meeting these criteria. Many agents not ordinarily associated with disease processes in humans are, however, opportunistic pathogens and may cause infection in the young, the aged, and immunodeficient or immunosuppressed individuals. Vaccine strains that have undergone multiple in vivo passages should not be considered avirulent simply because they are vaccine strains. BSL‐1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for hand washing. In this manual, when BSL‐1 conditions are appropriate to the experiments described, the following note will appear in the unit introduction. CAUTION : is a Biosafety Level 1 (BSL‐1) organism. Such organisms are not known to consistently cause disease in healthy adult humans, and are of minimal potential hazard to laboratory personnel and the environment. Standard microbiological practices should be followed when working with these organisms. See unit mc01a01 and other pertinent resources ( appendix mca01b ). Biosafety Level 2 (BSL‐2) The facility, containment devices, administrative controls (discussed below), and practices and procedures that constitute BSL‐2 are designed to maximize safe working conditions for laboratorians working with agents of moderate risk to personnel and the environment. BSL‐2 practices, equipment, and facility design and construction are applicable to clinical, diagnostic, teaching, and other laboratories in which work is done with the broad spectrum of indigenous moderate‐risk agents that are present in the community and associated with human disease of varying severity. With good microbiological techniques, these agents can be used safely in activities conducted on the open bench, provided the potential for producing splashes or aerosols is low. Hepatitis B virus, HIV, the Salmonella e, and Toxoplasma spp. are representative of microorganisms assigned to this containment level. Biosafety Level 2 is also appropriate when work is done with any human‐derived blood, body fluids, tissues, or primary human cell lines where the presence of an infectious agent may be unknown. Laboratory personnel in the United States working with human‐derived materials should refer to the U.S. Occupational Safety and Health Administration (OSHA) Bloodborne Pathogen Standard (OSHA, 1991 ), available online at http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=1005 , for required precautions. For guidelines and regulations appropriate to locations outside the U.S., please refer to appendix mca01b and Internet Resources . Primary hazards to personnel working with these agents relate to accidental percutaneous or mucous membrane exposures, or ingestion of infectious materials. Extreme caution should be taken with contaminated needles or sharp instruments. Even though organisms routinely manipulated at Biosafety Level 2 are not known to be transmissible by the aerosol route, procedures with aerosol or high splash potential that may increase the risk of such personnel exposure must be conducted in primary containment equipment, or in devices such as a biological safety cabinet (BSC) or safety centrifuge cups. Personal protective equipment (PPE) should be used as appropriate, such as splash shields, face protection, gowns, and gloves. Secondary barriers such as hand washing sinks and waste decontamination facilities must be available to reduce potential environmental contamination. When BSL‐2 conditions are appropriate to the organism under investigation, the following note is included in the unit introduction. CAUTION : is a Biosafety Level 2 (BSL‐2) pathogen. Follow all appropriate guidelines and regulations for the use and handling of pathogenic microorganisms. See unit mc01a01 and other pertinent resources ( appendix mca01b ) for more information. When BSL‐2 conditions are appropriate due to the use of human‐derived materials, the following note is included in the introduction. CAUTION : Follow all appropriate guidelines and regulations for the use and handling of human‐derived materials. See unit mc01a01 and other pertinent resources ( appendix mca01b ) for more information. Biosafety Level 3 (BSL‐3) BSL‐3 is suitable for work with infectious agents, which may cause serious or potentially lethal diseases as a result of exposure by the inhalation route. This may apply to clinical, diagnostic, teaching, research, or production facilities in which work is done with indigenous or exotic agents with potential for respiratory transmission, and which may cause serious and potentially lethal infection. Mycobacterium tuberculosis , St. Louis encephalitis virus, and Coxiella burnetii are representative of the microorganisms assigned to this level. Primary hazards to personnel working with these agents relate to autoinoculation, ingestion, and exposure to infectious aerosols. At BSL‐3, more emphasis is placed on primary and secondary barriers to protect personnel in contiguous areas, the community, and the environment from exposure to potentially infectious aerosols (see Table 1 ). For example, all laboratory manipulations should be performed in a BSC or other enclosed equipment, such as a gas‐tight aerosol generation chamber. Secondary barriers for this level include controlled access to the laboratory and ventilation requirements that minimize the release of infectious aerosols from the laboratory. When BSL‐3 conditions are appropriate to the organism under investigation, the following note is included in the unit introduction. CAUTION : is a Biosafety Level 3 (BSL‐3) pathogen. Follow all appropriate guidelines for the use and handling of pathogenic microorganisms. See unit mc01a01 and other pertinent resources ( appendix mca01b ) for more information. When BSL‐3 conditions are appropriate due to the use of human‐derived materials, the following note is included in the introduction. CAUTION : Follow all appropriate guidelines and regulations for the use and handling of human‐derived materials. See unit mc01a01 and other pertinent resources ( appendix mca01b ) for more information. Biosafety Level 4 (BSL‐4) BSL‐4 practices, safety equipment, and facility design and construction are applicable for work with dangerous and exotic agents that pose a high individual risk of life‐threatening disease, which may be transmitted via the aerosol route, and for which there is no available vaccine or therapy. Agents with a close or identical antigenic relationship to Biosafety Level 4 agents also should be handled at this level. When sufficient data are obtained, work with these agents may continue at this or a lower level. Viruses such as Marburg or Congo‐Crimean hemorrhagic fever are manipulated at Biosafety Level 4. The primary hazards to personnel working with Biosafety Level 4 agents are respiratory exposure to infectious aerosols, mucous membrane or broken skin exposure to infectious droplets, and autoinoculation. All manipulations of potentially infectious diagnostic materials, isolates, and naturally or experimentally infected animals pose a high risk of exposure and infection to laboratory personnel, the community, and the environment. The laboratory worker's complete isolation from aerosolized infectious materials is accomplished primarily by working in a Class III BSC or in a full‐body, air‐supplied, positive‐pressure personnel suit. The BSL‐4 facility itself is generally a separate building or completely isolated zone with complex, specialized ventilation requirements and waste management systems to prevent release of viable agents to the environment. As of this printing, there are no experiments described in this manual, which specifically utilize BSL‐4 containment. Animal Facilities The CDC defines four biosafety levels for activities involving infectious disease work with experimental animals. These combinations of practices, safety equipment, and facilities are designated Animal Biosafety Levels 1 , 2 , 3 , and 4 , and provide increasing levels of protection to personnel and the environment. In this manual, when these conditions are necessary, a note is provided in the unit or protocol introduction with the following format, where x is the appropriate ABSL. CAUTION : Protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) or must conform to governmental regulations regarding the care and use of laboratory animals. This experiment requires Animal Biosafety Level x (ABSL‐ x ) conditions. Follow all appropriate guidelines for the use and handling of infected animals. See unit mc01a01 and other pertinent resources ( appendix mca01b ) for more information. For more information, refer to the Section V of the BMBL, 5th Ed., available online at http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm . Clinical Laboratories Clinical laboratories, especially those in health care facilities, receive clinical specimens with requests for a variety of diagnostic and clinical support services. Typically, the infectious nature of clinical material is unknown, and specimens are often submitted with a broad request for microbiological examination for multiple agents (e.g., sputa submitted for "routine," acid‐fast, and fungal cultures). It is the responsibility of the laboratory director to establish standard procedures in the laboratory, which realistically addresses the issue of the infective hazard of clinical specimens. Except in extraordinary circumstances (e.g., suspected hemorrhagic fever), the initial processing of clinical specimens and serological identification of isolates can be done safely at Biosafety Level 2 (see above), the recommended level for work with blood‐borne pathogens such as hepatitis B virus and HIV. The containment elements described in BSL‐2 are consistent with the OSHA standard , Occupational Exposure to Bloodborne Pathogens (Richmond, 1994 ) from the Occupational Safety and Health Administration (OSHA; see Internet Resources ). This requires the use of specific precautions with all clinical specimens of blood or other potentially infectious material (Universal or Standard Precautions; MMWR, 1988 ). Additionally, other recommendations specific for clinical laboratories may be obtained from the Clinical Laboratory Standards Institute (formerly known as the U.S. National Committee for Clinical Laboratory Standards; NCCLS, 2005 ). Biosafety Level 2 recommendations and OSHA requirements focus on the prevention of percutaneous and mucous membrane exposures to clinical material. Primary barriers such as BSCs (Class I or II; see appendix a http://www.cdc.gov/od/ohs/biosfty/bmbl5/sections/AppendixA.pdf ) should be used when performing procedures that might cause splashing, spraying, or splattering of droplets. BSCs should also be used for the initial processing of clinical specimens when the nature of the test requested or other information suggests the likely presence of an agent readily transmissible by infectious aerosols (e.g., M. tuberculosis ), or when the use of a BSC (Class II) is indicated to protect the integrity of the specimen. The segregation of clinical laboratory functions and limited or restricted access to such areas is the responsibility of the laboratory director. It is also the director's responsibility to establish standard, written procedures that address the potential hazards and the required precautions to be implemented.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2094836/

Bioterrorism in Canada: An economic assessment of prevention and postattack response

The present paper calculates the human and economic consequences of a bioterrorist attack on Canadian soil using aerosolized Bacillus anthracis and Clostridium botulinum . The study assumed that 100,000 people in a Canadian suburban neighbourhood were exposed over a 2 h period to an infectious dose of one of the agents. Using an epidemic curve based on the epidemiology and management of anthrax and botulinum poisoning, the costs of intervention and treatment after an attack were compared with the costs of preparedness before a bioterrorist attack. The results show that an investment in planning and preparedness to manage the consequences of an attack can reduce morbidity, mortality and economic costs. The sooner that an intervention program is instituted, the more significant are the health and economic benefits. The greatest benefits were realized when postattack intervention was initiated before day 3 after the event. The economic impact of a bioterrorist attack in Canada could range from $6.4 billion/100,000 exposed to B anthracis to $8.6 billion/100,000 exposed in an attack using C botulinum . Without the benefit of an effective consequence management program, predicted deaths totalled 32,875 from anthrax and 30,000 from botulinum toxin. Rapid implementation of a postattack prophylaxis program that includes the stockpiling of antibiotics, vaccines and antitoxins; training of first responders in the diagnosis, handling and treatment of pathogens; and the general enhancement of Canada's response capability would reduce both human and economic losses. The present paper calculates the human and economic consequences of a bioterrorist attack on Canadian soil using aerosolized Bacillus anthracis and Clostridium botulinum . The study assumed that 100,000 people in a Canadian suburban neighbourhood were exposed over a 2 h period to an infectious dose of one of the agents. Using an epidemic curve based on the epidemiology and management of anthrax and botulinum poisoning, the costs of intervention and treatment after an attack were compared with the costs of preparedness before a bioterrorist attack. The results show that an investment in planning and preparedness to manage the consequences of an attack can reduce morbidity, mortality and economic costs. The sooner that an intervention program is instituted, the more significant are the health and economic benefits. The greatest benefits were realized when postattack intervention was initiated before day 3 after the event. The economic impact of a bioterrorist attack in Canada could range from $6.4 billion/100,000 exposed to B anthracis to $8.6 billion/100,000 exposed in an attack using C botulinum . Without the benefit of an effective consequence management program, predicted deaths totalled 32,875 from anthrax and 30,000 from botulinum toxin. Rapid implementation of a postattack prophylaxis program that includes the stockpiling of antibiotics, vaccines and antitoxins; training of first responders in the diagnosis, handling and treatment of pathogens; and the general enhancement of Canada's response capability would reduce both human and economic losses.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2376898/

A Comparison of the Completeness and Timeliness of Automated Electronic Laboratory Reporting and Spontaneous Reporting of Notifiable Conditions

Objectives. We examined whether automated electronic laboratory reporting of notifiable-diseases results in information being delivered to public health departments more completely and quickly than is the case with spontaneous, paper-based reporting. Methods. We used data from a local public health department, hospital infection control departments, and a community-wide health information exchange to identify all potential cases of notifiable conditions that occurred in Marion County, Ind, during the first quarter of 2001. We compared traditional spontaneous reporting to the health department with automated electronic laboratory reporting through the health information exchange. Results. After reports obtained using the 2 methods had been matched, there were 4785 unique reports for 53 different conditions during the study period. Chlamydia was the most common condition, followed by hepatitis B, hepatitis C, and gonorrhea. Automated electronic laboratory reporting identified 4.4 times as many cases as traditional spontaneous, paper-based methods and identified those cases 7.9 days earlier than spontaneous reporting. Conclusions. Automated electronic laboratory reporting improves the completeness and timeliness of disease surveillance, which will enhance public health awareness and reporting efficiency.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10068770/

Semiconductor/Carbon Quantum Dot‐based Hue Recognition Strategy for Point of Need Testing: A Review

Abstract The requirement to establish novel methods for visual detection is attracting attention in many application fields of analytical chemistry, such as, healthcare, environment, agriculture, and food. The research around subjects like "point‐of‐need", "hue recognition", "paper‐based sensor", "fluorescent sensor", etc. has been always aimed at the opportunity to manufacture convenient and fast‐response devices to be used by non‐specialists. It is possible to achieve economic rationality and technical simplicity for optical sensing toward target analytes through introduction of fluorescent semiconductor/carbon quantum dot (QD) and paper‐based substrates. In this Review, the mechanisms of anthropic visual recognition and fluorescent visual assays, characteristics of semiconductor/carbon QDs and ratiometric fluorescence test paper, and strategies of semiconductor/carbon QD‐based hue recognition are described. We cover latest progress in the development and application of point‐of‐need sensors for visual detection, which is based on a semiconductor/carbon quantum dot‐based hue recognition strategy generated by ratiometric fluorescence technology. 1 Introduction Over the past decades, it has become a trend to develop point‐of‐need testing (PONT), providing simple, rapid, and cost‐effective detection methods for non‐specialists and consumers. [1] The development of PONT is seen across clinical diagnostics, food safety monitoring, and various environmental pollution detections. The appeal of versatile PONT is increased by the availability of various readout systems; one of the most common readout systems is based on optics. [2] Up to now, researchers increasingly paid attention to the combination of optics‐based PONT and visual detection for a purpose of naked‐eye detection of targets without expensive or bulky instruments. Some of the research results have already been used commercially. For example, pregnancy test kits are accessible on numerous drugstore shelves. Human visual perception is realized via the assimilation of different light (400∼780†nm) by photoreceptors in the human retina, which allows it to decipher the ambient conditions. Human capacity for discriminating distinct colors (color vision) is regulated by diverse principles in both retina and brain. [3] The human retina holds three classes of cone photoreceptors that are responsive to short, medium and long wavelengths of light respectively, which supports the visualization and division of a spectrum of colors. The capacity of mankind to distinguish discrepancies between light consist of diverse wavelengths is suitable for carrying out naked‐eye visual sensing tactics for detecting analytes. Despite that colorimetric sensors for naked‐eye identification are more common, fluorescence (FL) sensing is regarded as a very sensitive optical signal conversion pathway, accordingly enabling the fulfillment of more sensitive and selective analytical techniques. [4] The fluorescence‐based visual assays permit detecting the emergence of targets without complex and extraneous apparatus, by perceiving color change after the interaction between FL probe/sensor and corresponding targets. The variation of FL color in visual assays can be observed through color brightness in one signal among dim and bright color, or by color hue/tonality (Figure 1a ) generated from ratiometric technology which corresponds to the color modulation between two or more different fluorescent signal for naked‐eye hue recognition of targets. [5] The changes in different FL hues reflect changes in concentration of species in the respective sensors, and it is vital for easy, fast and effective visual analysis of target analyte. Moreover, not only visual qualitative and semiquantitative analysis can be implemented by naked eyes, quantitative determination can also be achieved with the assistance of smartphones installed with software program (for transforming obtained color to RGB or HSL or Lab values). [6] Figure 1 (a) Variations of color intensity (left) and color hue (right) in color wheels. (b) application of quantum dot‐based hue recognition strategy for PONT. Nowadays, the combination of ratiometric FL probe and paper‐based substrates to fabricate ratiometric FL test papers has become an ideal approach to realize PONT. It is especially attractive owing to the following advantages: i) Numerous techniques have been developed for chemical modification of papers; the introduction of different functionalities to fulfill requirements of PONT is available. [7] ii) Compared with traditional analytical instruments, test papers are cheaper, more portable and operable, and thus prefer to implement on‐site and real‐time tests. In addition, they can be prepared readily by assembling or printing FL probes into a piece of paper‐based substrates. iii) Many different assays can be performed only by naked‐eye observation. Quantitative detection can also be realized with color space model by portable devices (such as smartphones). Thanks to the integration of ratiometric technology and FL test paper, the paper‐based ratiometric FL sensor can be as an intriguing and ideal analytical platform, which promotes hue recognition‐based visual PONT without costly and sophisticated apparatus. The analytical performances including sensitivity, reproducibility and visual capability are determined by the FL properties of the sensory elements of FL test paper. So far, although many organic dyes were designed as FL probes to selectively detect target analytes, some shortcomings such as poor optical stability and easy photobleaching always limited their application. Due to the wide emission bands and narrow absorption bands, [8] the excitation light cannot simultaneously excite all sensory elements in a ratiometric FL probe. In addition, overlap of emission spectra may occur, thereby losing the ability of hue recognition toward the target analyte. To solve these problems, A variety of FL nanomaterials have been developed to construct ratiometric FL probes. Among FL nanomaterials represented by semiconductor and carbon quantum dots (QDs), [9] semiconductor QDs are very appropriate sensing labels for the fulfillment of hue recognition, due to their outstanding optical properties. These optical features are quite beneficial for constructing ratiometric fluorescent sensors: i) the tunable emission wavelengths from visible to near infrared region allow preparing visual sensor with ideal color; ii) high photoluminescence quantum yield and photochemical stability provide improved sensitivity and stability of prepared sensor; iii) narrow emission bands and wide absorption bands make it feasible concurrent excitation of several QDs of diverse emissions via utilizing one excitation wavelength. It should be pointed out that surface‐passivated carbonaceous QDs, so‐called carbon quantum" dots (CQDs) or carbon nano dots (CNDs or CDs), not only possess favorable properties of traditional semiconductor‐based QDs as mentioned above, but also have special characteristics without suffering the trouble of intrinsic toxicity or elemental scarcity. [10] In addition, CNDs are receiving increasing attention from analytical chemists, since they can be produced inexpensively and on a large scale by many approaches. However, complicated and time‐consuming procedures are usually needed for ensuring acceptable batch‐to‐batch reproducibility of CNDs. Nowadays, paper‐based sensors based on semiconductor/carbon QDs and hue recognition provide a visual tool with charming merits including flexibility, simplicity and economy. [11] Here, the phenomenon is grouped into five testing objects (Figure 1b ) involved in various PONT fields including food and drug testing, biomedicine and health protection, and environmental monitoring. In the review, how the semiconductor/carbon QD‐based probes can be combined with ratiometric FL technique and paper substrate, and subsequently applied for the hue recognition of analytes is described. In addition, the inadequacy of current research, development trends, and further opportunities in the field of semiconductor/carbon QD‐based hue recognition strategy for PONT are discussed. 2 Strategy of Semiconductor/Carbon QD‐Based Hue Recognition As displayed in Figure 2a and 2b , based on two independent probes or one probe with two different FL signals, ratiometric FL containing a response signal or dual reversible response signals can be achieved, accompanied by the evolution of color hue. [12] Obviously, because of the more obvious change of ratio of FL signal intensity, the latter strategy exhibits a more sensitive change of color hue. For example, Wang et†al. designed a dual‐emission ratiometric fluorescent probe by doping ZnS QDs with Cu 2+ and Mn 2+ ions. [13] In the presence of folic acid, because of electron transfer between QDs and folic acid, the Cu dopant emission (490†nm) was quenched while the Mn dopant emission (595†nm) was enhanced. With the change of FL intensity ratio ( I 490 / I 595 ), the probe displayed sensitive color changes from blue‐green to orange‐red. In recent years, in order to broaden the scope of hue recognition strategy for target, ratiometric FL technology with three signals has been developed (Figure 2c and 2d ). Upon analyte recognition, the reference signal remains constant while two response signals were quenched; it leads to a remarkable abundant content‐dependent color change of an orange‐yellow‐blue evolution allowing a sensitive and accurate hue recognition for target. It′s worth noting that ratiometric FL with three response signals was also developed to produce color change of a yellowish green‐pink‐blue evolution. In order to obtain good performance of hue recognition, the initial ratio of intensity of three signals and the sensitivity of different signal toward target analyte should be adjusted properly. Therefore, the mixture of three independent probes to construct ratiometric FL system with three signals seems to be appropriate strategy, and has been adopted by several research groups. [14] Figure 2 Ratiometric FL contained a response signal (a) or dual reversed response signals (b), ratiometric FL with one reference signal and two response signals (c), ratiometric FL with three signals in which one response signal has reversed change with other two signals (d). 3 Visual Sensing Applications Some reports about visual hue recognition for PONT of ions, small molecules, biomarkers and pH by using semiconductor/carbon QD‐based test paper/strip are record in several tables (Table 1 to Table 4 ) composed of detailed information. Table 1 Visual hue recognition for PONT of ions by using semiconductor/carbon QD‐based test paper. Probe Analyte λ em [nm] Type of FL signals Mechanism [a] Color effect Concentration range of visual detection [μM] LOD or discernable scale [μM] Ref GSH/DTT‐QDs/ CDs As 3+ 486/630 One reference and one response signal Aggregation‐induced quenching Peach to cyan 0–3.2 0.067 [17b] Ratiometric probe Cu 2+ 437/654 One reference and one response signal – Pink to blue 0–10 0.1 [18] r‐CDs and b‐CDs Cu 2+ 440/615 One reference and one response signal FRET Blue to orange‐red 0–0.225 0.025 [19] GSH‐f‐rCD/GQD As 3+ 410/550 One reference and one response signal Dynamic quenching Blue to green 0–1.334 – [20] CdTe QDs/ GCNNs Cu 2+ 436/572 One reference and one response signal Dynamic quenching Pink yellow to blue 0–78.68 – [21] MOF/CdTe QDs Cu 2+/ Hg 2+ 425/605 One reference and one response signal Dynamic quenching Pink to blue 0–78.68/ 0–24.93 – [22a] r‐CDs and b‐CDs Cu 2+ 443/600 One reference and one response signal Ligand metal charge transfer Orange‐red to blue 0–18 3 [23] bCDs, gQDs and rQDs Cu 2+ 440/510/600 One reference and two response signals Reduction of surface adsorbed Cu 2+ to form nonradiative surface channels Shallow pink to dark olive green to dark blue 0–0.43 0.006 [24] GQDs‐AuNCs Cu 2+ /Cd 2+ 403/565 One reference and one response signal Charge transfer Pink to blue/orange for Cu 2+ / Cd 2+ 0–100/0–1000 – [25] CDs and Au NCs Hg 2+ 450/656 One reference and one response signal – Pink to blue 0–30 – [26] Q‐dots@SiO 2 @ C‐dot Hg 2+ 453/658 One reference and one response signal – Red to blue 0–21 – [27] Si NCs and r‐CDs Hg 2+ 445/615 One reference and one response signal – Pink to orange red 0–0.105 – [28] B‐CDs and R‐CDs Hg2 + 380/620 One reference and one response signal Aggregation Blue to purple 0–0.32 0.005 [29] C‐dots‐R6G Al 3+ 410/560 One reference and one response signal FRET Blue to greenish‐ yellow 0–10000 – [30] R6G‐CD Fe 3+ 455/550 Two reversed signals FRET Blue to yellow 0–2 – [31] HBARH‐ CdTe@SiO 2 QDs hybrid sensor Fe 3+ 553/635 One reference and one response signal Metal cation coordination Red to yellow 0–30 0.5 [32] RhB‐CdTe@ SiO 2 QDs Fe 3+ 556/651 One reference and one response signals – Light blue to orange 0–0.43 1 [33] CQDs/Au NCs Ag + 472/605 One reference and one response signals Aggregation Gray orange to blue 0–50 – [34] CDs and CdTe QDs Ag + 440/628 One reference and one response signals Ion‐exchanging Red to blue 0–0.5 0.03 [35] QD‐Biopoly‐ mer‐TSPP assembly Zn 2+ 517/560 Two reversed signals Zn 2+ ‐TSPP‐ Chelation‐induced spectral modulation Yellow to bright green 0–4 – [36] CDs Pb 2+ 477/651 One reference and one response signals Static quenching Pink to cyan 0–400 – [37a] AuNCs‐QDs Pb 2+ 520/620 One reference and one response signals – Green to red 0–10 0.1 [37b] CDs and CdTe QDs UO 2 2+ 525/640 One reference and one response signals PET Red to green 0–150 0.5 [38] CDs/QDs@ ZIF‐8 Pb2 + / PO43 − 441/612 One reference and one response signals Electron transfer Red to blue/blue to red 0–60/ 0–50 – [39] CTIPS and CTe QDs F − 455/630 One reference and one response signals – Red to blue 0–300 6 [40] N, S‐CDs ClO − 478/552 One reference and one response signals – Yellow to blue 0–80 – [41] GCDs and RCDs Inorganic phosphates 475/620 One reference and one response signals Aggregation Red to green 0–50 5 [42] CDs and CdTe QDs CN − 443/611 One reference and one response signals Synergistic effect of FL recovery and enhancement Blue to pink 0–80 0.284 [43] [a] FRET: Förster resonance energy transfer, PET: photo‐induced energy transfer. Wiley‐VCH GmbH 3.1 Cations The higher concentration of metal cations like Hg 2+ and As 3+ induces various issues associated with healthiness and environment around the world. [15] Some metal ions including Pb 2+ , Hg 2+ , Cd 2+ , As 3+ and Cu 2+ can trigger different disease including osteoporosis, Alzheimer's disease, Parkinson's disease, Minamata disease, endocrine system disturbance and others. [16] The metal ions like As 3+ , Cr 3+ , Pb 2+ , and Cd 2+ give rise to severe bodily injury in spite of their trace level. However, these metal cations had been released from mining, industrial discharges, household effluent, and other garbage to pollute surroundings. Recently, the monitoring of certain metal cations in sewage and solid samples has been well implemented by the semiconductor/carbon QD‐based ratiometric FL test paper in research lab and industrial factories. Zhang and Jiang accomplished many work about color recognition‐based assay and have contributed to the development of visual test paper in color modulation strategies, detection mechanisms, and preparation methods. [17a] Zhang et†al. reported a sensitive fluorescent test paper for detecting As 3+ ions. [17b] As 3+ ions reacted with GSH/DTT‐QDs to form As−S bonds, thus induced the aggregation and FL quenching of this red QDs; meanwhile the cyan FL of CDs remained unchanged. It should be emphasized that the range of color variation between cyan and red was wider than green and red, green and blue, blue and red. So, a broad scope of sequential color hue variation for the hue recognition of As 3+ ions was achieved. These test papers were applied for the detection of As 3+ ions in water samples without pretreatments, the obtained satisfactory visual effect demonstrated its practicability. Zhang and colleagues also developed a ratiometric FL test paper using red‐emission CdTe QDs and blue‐emission CDs. [18] The test paper was applied for ratiometric detection of Cu 2+ ions; the FL color hue of the printed pattern "Copper 2+ " gradually changed from pink to blue through cyan. Although these mentioned test papers showed favorable dose‐sensitive color evolution, inferior compatibility between QDs and CDs made the preparation approach tedious. CdTe QDs possessed environmental toxicity. These issues limited the use of test papers. In order to solve these issues, Zhang put forward a ratiometric FL strategy on test paper using blue CD (b‐CD) and red CD (r‐CD) probes. (Figure 3A ). [19] The sensing mechanism of the test papers for Cu 2+ ions were described as follows: The surfaces of b‐CDs and r‐CDs had −COOH and p‐PDA ligands respectively, Cu 2+ ions will interact with b‐CDs and r‐CDs based on the surface complexing reaction. A new absorption band corresponding to the coordination of Cu 2+ with r‐CDs displayed a large spectrum overlapping with the emission of b‐CDs. Thanks to the FRET effect between b‐CDs and the Cu 2+ ‐p‐PDA complex at the surface of r‐CDs, the blue FL signal was quenched; meanwhile the red FL signal remained unchanged. The ability of this test paper to monitor Cu 2+ ions in water by the direct observation of FL colors under an excitation of 365†nm was tested; the test paper presented a sequential wide change from blue to orange‐red in the presence of increasing amounts of Cu 2+ , and had a visual LOD of 25†nM. In these studies, Zhang used an inkjet‐printing approach to prepare fluorescent test paper owing to following advantages. The uploaded amounts of probes are controllable, and the uniformity of obtained test papers can be guaranteed. Moreover, the patterns of printed probes on substrates can also be designed by computer. Figure 3 (a) Mechanism and process of ratiometric fluorescent test paper for Cu 2+ assay by a content sensitive color hue variation. Reproduced with permission from Ref.†[19] Copyright 2017, American Chemical Society. (b) Visual platform for ultrasensitive monitoring of endogenous Cu 2+ in human urine. Reproduced with permission from Ref.†[24] Copyright 2017, Elsevier B.V. (c) Major modules of the "Concentration Detection" app and (d) Smartphone based platform device for uranyl ion detection. Reproduced with permission from Ref.†[38] Copyright 2018, American Chemical Society. (e) Schematic illustration of the visual ratiometric detection system for CN − sensing and (f) hue response of prepared test paper versus CN − concentration. Reproduced with permission from Ref.†[43] Copyright 2019, Elsevier B.V. Other researchers have likewise established paper‐based sensors for hue recognition of Cu 2+ ions using QDs‐based probes. Wu and colleagues prepared a paper‐based sensor through drenching cellulose acetate membrane in probe solution. It was used for on‐site visual semi‐quantitative assay of Cu 2+ ions in red wine examples by hue recognition. [21] Wu also prepared a MOF/CdTe QDs‐based test paper for visual determination of Cu 2+ and Hg 2+ ions. [22a] Although the sensitivity and selectivity of these two test papers were not so good as other studies, on‐site and rapid determination of targets in red wine without any pretreatment procedure was realized successfully. Wang presented a simple approach to prepare fluorescent test paper with appropriate proportion of two CNDs. The strips were utilized for naked‐eye detection of Cu 2+ ions with a color change from orange‐red to blue. [23] The ratiometric FL test paper for cations has been widely created using different sensing probes, but it still remains a challenging task to prepare a test paper with multicolor variation with target dosages for definite determination. Du and Zhang reported a profuse color‐evolution‐based ratiometric FL test paper with red, green and blue three signals for visual monitoring of Cu 2+ ions in human urine. [24] As shown in Figure 3b , the tricolor probe system was obtained by mixing bCDs, gQDs and rQDs through volume ratio of 3 : 9 : 4. And the probe system‐based test papers were fabricated by inkjet‐printing approach. In the presence of Cu 2+ ions, gQDs and rQDs were simultaneously quenched whereas the FL of bCDs remains steady. Due to the change of ratio of FL intensities at three emission wavelengths, an abundant and wide color transformation (shallow pink‐light salmon‐dark orange‐olive drab‐dark olive green‐slate blue‐royal blue‐dark blue) was observed. Just by a pathway of dilution, the concentration of human urine samples can be distinguished by recognition of color hue of the test paper under a 365†nm UV lamp. The distinguished dosage scale is 6†nM in human urine, which cannot be achieved by other reported QDs‐based test papers for Cu 2+ ions. This study demonstrated that ratio FL test paper with three different signals not only had high sensitivity, but also possessed more profuse and wider color variations compared with two signals. Wang and co‐workers developed ratiometric FL nanohybrids by covalently linking prepared green QDs or Au NCs to the surface of silica nanoparticles embedded with red QDs.[ 22b , 37b ] The nanohybrid was used for on‐site visual determination of Cu 2+ and Pb 2+ ions respectively. And a probe‐based PVA‐film sensor was also constructed for sensing Pb 2+ ions. [37b] Wang and co‐workers also designed a dual‐emissive nanohybrid by hybridizing CNDs and gold nanoclusters. [26] The nanohybrid was applied for preparing test paper for the visualization of Hg 2+ ions by the distinct FL color change. The uranyl ion is regarded as an important index for nuclear industrial wastewater. It is challenging to sensitively and rapidly detect UO 2 2+ ions. For the purpose of achieving visual, rapid, on‐site, and sensitive assay of UO 2 2+ ions, Wang and colleagues designed a cellphone‐based portable platform including a 3D‐printed accessory, a smartphone installed with an app called "Concentration Detection" and a test strip (Figure 3d ). [38] Upon the addition of UO 2 2+ ions into test strip, the response signal of CdTe QDs was quenched by photo‐induced electron transfer, meanwhile, the reference signal of CNDs remained stable, thus generated significant variations in FL intensity ratio ( I 525 / I 640 ) and color of test strip. Compared with other studies, a significant advantage of this study is that after the detection process was accomplished, the colors of reference and sample were revealed synchronously in a smartphone on the result page, and the concentration of UO 2 2+ ions was computed and displayed clearly (Figure 3c and Figure†S1 in Supporting Information). Thanks to the self‐calibration capacity of ratiometric technology and designed platform device, interference such as ambient light was eliminated to a large extent; the platform for uranyl detection had been successfully applied in various real water samples. It should be noted that the interference from Cu 2+ and Ag + ions should be eliminated by the addition of sodium sulfide. Other researchers also made a lot of contributions in designing semiconductor or/and carbon QD‐based ratiometric FL test paper. For instance, Jiang and his researchers proposed a fluorescent test paper for visual detection of Hg 2+ ions by a printing process, which used CND as ink and filter paper as carrier. [29] Kim et†al. designed rhodamine‐appended CNDs‐based paper sensor for analyzing Al 3+ ions, and distinct change of color hue from blue to greenish‐yellow of the sensor can been recognized by the naked eye. [30] Wu et†al. demonstrated the employment of test paper for Fe 3+ ions sensing. [33] You et†al. illustrated a paper‐based ratiometric procedure for detection of Ag + ions, which used CdTe QDs and CNDs as probes. [35] Here, the mechanism is based on ion‐exchanging between red CdTe QDs and Ag + . On the basis of dual‐signal QD‐poly(dA)‐TSPP probes and Zn 2+ ‐chelation‐induced spectral modulation, Liu et†al. prepared a test strip with two reversed signals for on‐site naked‐eye detection of Zn 2+ ions. [36] Dong and group members constructed a fluorescent paper by doping dual‐emitting CNDs onto the cellulosic filter paper. The hue of FL color changed from pink to cyan with increasing concentration of Pb 2+ ions. [37a] On the whole, the detection mechanism of these semiconductor/carbon QD‐based test paper for cations based on FRET or electron transfer were more sensitive than other detection mechanisms such as aggregation. However, the design scheme was complicated, the distance between donor and acceptor should be regulatory, and the relative orientation of their transition dipole moments will influence the performance of detection. In addition, it was noticed that ratiometric FL test paper with two reversed signals or three signals was reported rarely: the construction of paper‐based ratiometric FL sensors with two reversed signals or three signals are demanded for improving the sensitivity and broadening color variations. 3.2 Anions The undue utilization of anions in various manufacturing industry and approaches and widespread discharge of anions into physical environment taints the natural land as well as water. This results in hazardous effects to humans and animals. Therefore, rapid, simple, on‐site visual sensors are highly demanded for sensing these anions. At present, various anions have been detected by QDs‐based FL sensors using hue recognition strategy. Zhang and Jiang designed a ratiometric fluorescent strip for sensitive, selective, naked‐eye detection of F − anions. [40] The strips used a F − ‐response organic probe and F − ‐inert CdTe QDs as signals. In the presence of different concentrations of F − , the test paper presented different FL colors with a minimum visible amount of 6†μM by naked eye. Zhang and co‐worker presented a N,S‐CD stained paper‐based test paper, which had one reference signal and one response signal for ClO − ions. [41] As the increase of concentration of ClO − anions, the FL color gradually altered from yellow to blue, which proved its potential of semi‐quantitative analysis. Zhang et†al. prepared fluorescent test strips through fixing mixed GCD/RCD/Eu 3+ buffer solutions on filter paper, of which GCD and RCD were as response and reference labels respectively. Upon the addition of inorganic phosphates, the FL color steadily varied from red to green with an observed dosage of 5†μM. [42] A standard reference card was also established for visual identification of different concentrations of inorganic phosphates on fluorescent test paper. Under a 365‐nm UV light, the trend of color changes in detection of spiked samples is similar to the card, which verified its capacity of semi‐quantitative visual detection of inorganic phosphates. CN − anions are recognized as hazardous substance in environmental and biological processes. Although many fluorescent probes had been designed for detecting CN − anions, it was a challenge to construct a CN − fluorescent assay which not only had the advantages of high sensitivity and selectivity of organic probes, but also held the good water solubility and stability of nanoprobes. In order to accomplish this challenge, our group designed a specific dual‐emission nano‐system for ultrasensitive detection of CN − ions. [43] The system used blue fluorescent CNDs as an inert reference and red fluorescent N‐acetyl‐L‐cysteine‐capped CdTe QD as a reporter. As shown in Figure 3e , the FL of N‐Acetyl‐L‐cysteine (NALC)‐capped CdTe QDs was first quenched by addition of Cu 2+ , then the introduction of carbon dots constituted a ratiometric FL detection system. Upon addition of CN − , CN − coordinated with Cu 2+ to form [Cu(CN) n ] (n−1)− complex, which caused the Cu 2+ to detach from the QDs, resulting in FL recovery. Meanwhile the CN − undergoes a nucleophilic addition reaction with the carbonyl group in the NALC, which passivated trap states and enhanced the FL of the QDs obviously. The blue emission of CDs was as a stable internal reference. Because of Cu 2+ ‐promoted complexation and addition reaction as recognition units, the presented method showed excellent selectivity and sensitivity for CN − ions. Furthermore, a fluorescent test paper fabricated with the detection system was applied for sensing CN − ions in water and cassava samples with pretreatment under 365†nm UV light. For quantitative detection, a "Color Analyzer" app installed in a phone was used to read the picture of the test strip and then transform it into hue values. Here the hue values was employed to describe the standard calibration versus various contents of CN − ions (Figure 3f ) with a LOD of 0.284†μM. On the whole, the semiconductor/carbon QDs‐based ratiometric FL test papers for visual sensing of anions are rarely reported. New detection mechanisms, patterns and materials are needed for broadening the application scope of ratiometric FL test papers for anions. And the challenge about specificity of test paper toward specific ions need to be overcome. 3.3 Small molecules Unreasonable misuse of antibiotics may cause durable and frequent ingestion of antibiotics into anatomy and injure the human body. Therefore, the remnants of antibiotics in food and the environment needs to be monitored for assuring food security and bodily health. Recently, various ratiometric FL test papers based on lanthanides‐modificatory QDs or CNDs with two reversed signals have been developed for PONT of tetracycline (TC) and oxytetracycline (OTC). For instance, Hu et†al. prepared a ratiometric fluorescent test paper for sensitive, precise and visual detection of TC, which used BCDs‐Eu/CMP‐cit as the probe. [44] The features of this test paper were described in Table 2 . Table 2 Visual hue recognition for PONT of small molecules by using semiconductor/carbon QD‐based test paper/strip. Probe Analyte λ em [nm] Type of FL signals Mechanism [a] Color effect Concentration range of visual detection [μM] LOD or discernable scale [μM] Ref BCDsEu/ CMP‐cit Tetracycline 465/620 Two reversed signals AE Bright blue/ek; to red 0–1.5 0.05 [44] BNQD‐Eu 3+ Tetracyclines 414/616 Two reversed signals IFE and PET as well as the AE Blue to red 0–20 – [45] Eu3 + /N, B‐Ti 3 C 2 MQDs Tetracycline 448/616 Two reversed signals IFE and AE Blue to red 0–20 – [46] Atta‐CDs‐Eu Tetracycline 445/617 Two reversed signals IFE and AE Blue to red 0–2 – [47] MoS 2 NPs@Gmp/ Eu‐Cit Tetracycline 430/617 Two reversed signals IFE and PET Blue to red 0–80 0.127 [48] ZnO/Eu Tetracycline 530/616 Two reversed signals AE Yellow to red 0–30 0.5 [49] SiQDs‐Cit‐Eu Tetracycline 455/617 Two reversed signals AE Blue to red 0–30 0.25 [50] La‐CDs Tetracyclines 450/515 Two reversed signals Static quenching or/and dynamic quenching Blue to green 0–1039.5 – [51] CD@AMP/ Eu NCPs Oxytetracycline 430/615 Two reversed signals IFE Blue to red 0–100 0.5 [52] BCNO QDs‐Ca 2+ Doxycycline 425/520 Two reversed signals IFE Blue to green 0–1039.5 1 [53] CDs/SiDs‐BPMA‐ Cu 2+ Ciprofloxacin 425/660 One reference and one response signals PET Slight‐blue to deep‐red 0–100 – [54] Eu‐CDs 2,6‐dipicolinic acid 475/615 One reference and one response signals AETE Blue to red 0–200 1 [55] FMn‐CDs 2,6‐dipicolinic acid 461/616 One reference and one response signals AETE Blue to red 0–200 1 [56] BCNO QDs 2,6‐dipicolinic acid 475/616 One reference and one response signals AETE Blue to red 0–700 1 [57] CDs‐Tb 2,6‐dipicolinic acid 459/495 One reference and one response signals Energy transfer Yellow to dark 0–5 0.0005 [58] hPEI‐CD‐EDTA‐ Eu 3+ 2,6‐dipicolinic acid 440/616 One reference and one response signals FRET Blue to purple 0–500 0.5 [59] CDs‐Eu 2,6‐dipicolinic acid 530/615 One reference and one response signals AE Green to red 0–50 0.067 [60] QDs hybrid Trinitrotoluene 520/650 One reference and one response signals FRET Yellow‐ green to red – – [61] gQDs@SiO 2 @ rQDs Trinitrotoluene 508/625 One reference and one response signals Electron transfer Red to green 0–5 – [62] CdTe@SiO 2 @ F‐PDA/MIPs 4‐nitrophenol 529/664 One reference and one response signals Electron transfer Green to red 0–0.55 – [63] Eu‐MoS 2 QDs 3'‐diphosphate‐ 5‐diphophate 410/616 One reference and one response signals AE Bluish violet to red 0–250 23.8 [64] CDs/CuNCs Dopamine 440/640 One reference and one response signals Electron transfer Blue to red 0–100 – [65] DNA‐CuNC/CDs Arginine/ acetaminophen 440/670 One reference and one response signals – Blue to red/red to blue 0–70 – [66] CDs/CdTe QDs Spermine 470/690 One reference and one response signals Aggregation Shallow pink to blue 0–1 [67] CDs Fungicide cymoxanil 435/520 Two reversed signals IFE and aggregation Green to blue 0–0.5 [68] rQDs@SiO 2 @CDs Thiram 453/644 One reference and one response signals FRET Pink to blue 0–10 0.059 [69] CD‐QD@SiO 2 Ascorbic acid 450/630 One reference and one response signals Electron transfer Red to blue 0–150 10 [70] CDs H 2 O 467/525 Two reversed signals Excited‐state intramolecular proton transfer Blue to green 0–100/ % – [71] QDs@SiO 2 ‐CDs Glucose 445/630 One reference and one response signals Electron transfer Blue to deep red 0–500 – [72] rQDs@SiO 2 @ gQDs Cysteine/ homocysteine 545/645 One reference and one response signals Analytes‐ induced displacement of the quencher Red to green 0–500/ 0–800 – [73] CDs‐QDs Folic acid 468/587 Two reversed signals Hydrogen bonds Pink to blue 0–900 – [74] Peptide‐SA‐QD Conjugates Benzothiostrobin 525/623 Two reversed signals Competitive and noncompetitive immunoassays Green to red 0–0.25 0.00042 [75a] [a] AE: antenna effect, IFE: inner filtration effect, AETE: absorbance‐energy transfer‐emission. Wiley‐VCH GmbH Yang et†al. constructed a novel ratiometric FL test strip for visual assay of TC. [45] Bai et†al. established a novel PONT paper by using Eu 3+ /N, B−Ti 3 C 2 MQDs system. Upon addition of TC in the test paper, the blue FL of N,B−Ti 3 C 2 MQDs (nitrogen and boron co‐doped Ti 3 C 2 MXene QDs) was quenched and red FL of Eu 3+ was enhanced gradually, the color hue and R/B change could be recognized by naked eye and smartphone respectively. [46] Jia et†al. prepared a test strip‐based sensor for identifying TC through a chromaticity analysis application. [47] With a customized smartphone‐based portable measurement chamber and MoS 2 NPs@Gmp/Eu‐Cit‐based test paper, Sun et†al. put forward to a promising tool for TC determination. [48] Wu et†al. prepared a test paper for sensing TC by fixing ZnO/Eu on the filter paper, not only realizing visual analysis, but also achieving on‐site and real‐time quantitative analysis assisted with a mobile phone app. [49] Zhang et†al. constructed a smartphone‐assisted paper‐based PONT platform through utilizing a color scanning app. [50] The FL color of the strip varied from blue to red with increasing concentration of TC, which could be recognized by human vision or the smartphone. Fan et†al. preliminarily explored a portable visual paper sensor with La‐CDs as probes for sensing TC or OTC. [51] Chen et†al. applied a test paper for the quantitative assay of OTC with a LOD of 0.5†μM based on a digital camera installed with a color detector app. [52] Liu et†al. established a convenient FL test strip which can be identified by naked eyes or smartphone; the FL color of the strip altered from blue to green as the concentration of doxycycline increased. [53] Gui et†al. used CDs/SiDs‐BPMA‐Cu 2+ as an effective ratiometric FL probe to detect ciprofloxacin (CIP), the FL color changed between red and blue. Based on CIP‐induced FL signals responses corresponding color, a filter paper‐based test strip by using the probe was applied for naked‐eye visual detection of CIP. [54] The features of this test paper and other test papers for antibiotics were described in Table 3 . Table 3 Visual hue recognition for PONT of biomarkers by using semiconductor/carbon QD‐based test paper/strip. Probe Analyte λ em [nm] Type of FL signals Mechanism Color effect Concentration range of visual detection LOD or discernable scale Ref gQDs and Cy3 Oligonucleotide 525/570 One reference and one response signals FRET Green to yellow 0–45†pM 450†fM [77] RF‐QDs‐DA Tyrosinase 542/657 One reference and one response signals Electron transfer Yellow to red 0–5†μg/mL – [78] N‐GQDs and DA‐rQDs Tyrosinase 644/440 One reference and one response signals Electron transfer Red to blue 0–0.64†U/mL – [79] QDs‐Rox Telomerase 524/605 One reference and one response signals Quenching caused by oxidation Red to yellow‐green 10–1000†cells – [80] C‐MIPs@FITC Ovalbumin 454/520 One reference and one response signals Hydrogen bond interaction Blue to green 0–2†μM 15.4†nM [81] PADs, gQD‐sDNA1 and rQD‐sDNA2 miRNA‐21 and circRNA‐ HIAT1 453/512 or 610 Two reversed signals Hybridization and FRET Blue to green/Blue to red 0–2†nM/ 0–2†nM 3.5/2.7†fM [82] 450‐NBs and 655‐QDs H‐IgG 450/655 One reference and one response signals Immunoassays Blue to red 0–1000†ng/mL – [83] SAQ‐mAb1 and mAb2‐gQD heart‐type fatty acid binding protein 524/626 Two reversed signals IFE and immunoassays Green to orange red 0–50†ng/mL 1†ng/mL [84] Wiley‐VCH GmbH At present, due to the great harm of anthrax, 2,6‐Dipicolinic acid (DPA), a biomarker for Bacillus anthracis and B. anthras has been of interest to analytical chemists. For instance, Rong et†al. integrated Eu‐CDs with filter paper, and prepared a test paper for naked‐eye recognition of DPA under a 254†nm UV lamp. [55] Rong also produced a paper‐based test strip by using functionalized manganese‐doped CDs for visual detection of DPA with a LOD of 1†μM. [56] Subsequently, Rong et†al. prepared a convenient boron carbon oxynitride QD test paper for visual determination of DPA through using commercial fiber filer paper as substrate. [57] Liu et.†al. designed a portable and sensitive CDs‐Tb‐based test strip for naked‐eye assay of DPA with a ultra‐low LOD of 0.5†nM. [58] Yang et†al. achieved visual PONT of DPA based on paper‐based analytical device by printing the probe solution into polyvinylidene fluoride (PVDF) film. [59] In order to obtain an economic, sensitive and naked‐eye FL test paper for determining DPA, our group designed a Eu 3+ functionalized carbon dots (CDs‐Eu) as a novel ratiometric FL probe, a test paper was further prepared for visual and quantitative PONT of DPA by utilizing a phone equipped with an app named "Color Analyzer". The detection range and LOD of the papered‐based sensors are 1–30†μM and 67†nM (Figure 4a ). [60] These reported test papers mentioned above all used lanthanides ions as response signals and QDs as reference signals to construct single‐signal‐response ratiometric FL system for DPA. The evolutionary sensitivity of color hue is intrinsically limited, thus affect the sensitivity of detection by naked eye or smartphone. Figure 4 (a) The application of the visible identification of DPA by paper‐based sensor and mobile phone. Reproduced with permission from Ref.†[60] Copyright 2020, Elsevier B.V. (b) Smartphone sensing platform based on 3D printing for thiram analysis and corresponding detection mechanism. Reproduced with permission from Ref.†[69] Copyright 2020, American Chemical Society. Among the above‐mentioned fluorescent test papers for antibiotics and DPA, the Eu 3+ or Tb 3+ modified/doped semiconductor/carbon QDs presented higher sensitivity than other probes owing to following reason. Some of antibiotics and DPA can acts as the antenna group to absorb the excitation light based on the antenna effect after the combination of the target molecule of Eu 3+ or Tb 3+ . It was noticed that most of these test papers with signals showed color change from blue to red. More different color‐transition modes between two different signals or three signals need to be explored. In addition to antibiotic and DPA, paper‐based hue recognition for PONT of other various small molecules have also been extensively explored. For instance, a new technique of PONT for trinitrotoluene (TNT) is need for homeland security needs against terrorism. Zhang et†al. designed a dual‐emission QDs hybrid and QDs hybrid‐based test paper for sensing TNT. The hybrid was prepared by embedding the red‐emitting QDs in silica nanoparticles and covalently linking the green‐emitting QDs to the silica surface. In the presence of TNT, the red QDs remained constant, the green QDs functionalized with polyamine can selectively bind TNT via the formation of the Meisenheimer complex, resulting in the green FL quenching due to FRET. The different FL intensity ratios displayed different color between yellow‐green and red, and versus different amounts of TNT. In addition, Qian et†al. prepared a gQDs@SiO 2 @rQDs nanohybrid and nanohybrid‐based test paper for visual sensing of trinitrotoluene. [62] The internal rQDs were as stable references, external bQDs can react with TNT to form the Meisenheimer complex, thus leading to the FL quenching of rQDs owing to a fact that electrons of rQDs transferred to TNT molecule. Compared with Zhang's nanohybrid, this nanohybrid was more sensitive for TNT. As a result of "coffee ring effect", the performance of filter papers‐based common FL test strips is restricted in visual detection. To overcome this issue, Xu et†al. prepared a ratiometric FL test strip based on the PVDF hydrophobic membrane and probe for naked‐eye assay of 4‐nitrophenol. [63] The hydrophobic PVDF membrane with colorless FL background efficiently overpowered this "coffee ring effect" and background interference. A comparison experiment of visual detection demonstrated that light color variations of the PVDF fluorescent test strip were recognizable and more evident than that with a conventional filter paper. Jiang and his researchers developed a visual quantitative platform for detecting pesticide composed of a test paper strip, a cellphone and dark cavity with a UV lamp (Figure 4b ). [69] The blue FL could be quenched by gold nanoparticles and then recovered with pesticide. And red FL of rQDs was just as a reference signal. R/B values of captured pictures could be used for pesticide quantification with a sensitive LOD of 5†nM. The dark cavity and optical filter of the designed platform was used to eliminate the interference of surroundings. Rong et†al. fabricated a facile and portable Eu‐MoS 2 QDs test paper for naked‐eye assay of 3'‐diphosphate‐5‐diphophate. [64] He et†al. constructed CDs/CuNCs‐based test paper for visual detection of dopamine on filter paper. Each FL color of test paper was assigned to the corresponding concentration of dopamine in the aqueous suspension of sample‐nanohybrid mixtures. [65] Bu et†al. synthesized DNA‐CuNC/CD complex as a dual‐emission ratiometric probe to determine both arginine and acetaminophen, by placing this probe in cuvettes or dropping it onto filter paper strips. [66] Fu et†al. designed a ratiometric fluorescent paper as an onsite, fast, economical approach for visual sensing of spermine, as well as freshness of pork respectively. The test paper used CdTe QDs and CNDs as a response labels and internal reference labels [67] Zhao et†al. designed a ratiometric FL sensor based on CD‐QD@SiO 2 for ascorbic acid detection with satisfactory results in fruit juice. [70] Yin et†al. synthesized blue FL CNDs as an effective ratiometric sensor for sensing water in organic solvents. And cellphone‐based quantitative detection was also achieved by CNDs‐loaded test papers through a color processing application. [71] Huang et†al. demonstrated test papers printed by a QDs@SiO 2 ‐CDs ink for glucose sensing, thus enabled a potential PONT application. [72] Blood glucose was determined by this test paper and a standard glucometer, the obtained result was almost same. He et†al. prepared a paper‐based sensor based on CDs‐QDs to demonstrate a suitable and straightforward approach to visually detect folic acid. [74] In order to obtain a stable, bright and tunable ratiometric probe, our group assembled two different hydrophobic QDs into a silica frame based on thiol‐metal coordination, thereby formed a dual‐signal hierarchical luminescent silica composite. [75b] Compared with other nanohybrids, the ratio ( I 627 / I 528 ) and color hue of the generated nanohybrid can be accurately formulated by varying the amounts of these two QDs. Thanks to the analyte‐induced IFE between gold nanoparticles and nanohybrid, a ratiometric system with two reversed signals and a logic gate strategy were successfully constructed for the visual determination of melamine with a color hue evolution from green to orange‐red. Lately, a new unconstrained ratiometric signal approach was successfully applied to immunochromatographic strips for visual determination of benzothiostrobin by Chen and co‐workers. [75a] This innovation possessed important reference value for hue recognition of small molecules by lateral flow immunoassay (LFIA). The study can be widely utilized to FL or colorimetry reading by fluorescent or colorimetric labels. They amalgamated the negative‐readout. competitive and positive‐readout noncompetitive immunoassays into one test through utilizing various color markers, including labeled and anti‐immunocomplex peptides. As the content of benzothiostrobin increased, the green FL signal or red colorimetric signal on T line decreased, the red FL signal or blue colorimetric signal increased simultaneously. Moreover, a color reference card was constructed for visual judgment based on such distinct color hue changes from green to red (fluorescent mode) or red to blue (colorimetric mode). After adaptation with a portable smartphone, the quantitative detection was implemented with high sensitivity and good accuracy. The features of the test papers were described in Table 4 . Table 4 Visual hue recognition for PONT of pH by using semiconductor/carbon QD‐based test paper. Probe Range λ em [nm] Type of FL signals Mechanism Color effect Resolution Ref gQD@SiO 2 / GO 5.5–7.5 520/590 One reference and one response signals Conformational changes Green to red 0.1†pH [85] AC‐CDs 1–14 440/510/620 Three response signals The pyridine on the surface of ACis was easy to be protonated and deprotonated. Bright green to grayish and blue to purple to yellow – [86] CDs 1 1–3 514/600 One reference and one response signals Dynamic quenching process Pink to green 0.2‐pH [87] Cu‐CDs 3–8.7 436/520 One reference and one response signals Surface passivation effect Green to blue – [88] Wiley‐VCH GmbH The development of ratiometric fluorescent probes for gaseous molecules is of great challenge, Wang and his group have developed QDs‐based ratiometric fluorescent probes for gaseous molecules. They designed a fluorescence nanohybrid probe with one response signal and one reference signal at 452 and 657†nm separately for sensing gaseous hydrogen sulfide. [76a] The probe was prepared by encapsulating red 3‐mercaptopropionic acid modified CdTe QDs in silica shells and then modifying the silica surface with a blue organic molecules azidocoumarin‐4‐acetic acid. They also designed a nanohybrid with one response signal and one reference signal at 665 and 460†nm separately for visual determination of nitrogen dioxide. The nanohybrid was prepared by conjugating through coupling reaction between carboxyl groups and amino groups on the surface of rede‐missive QDs and blue fluorescent CNDs. [76b] In general, the combination of sensor with paper substance to prepare test paper is more conducive for PONT of gaseous molecules. More efforts need to be made for designing QDs‐based ratiometric FL test paper for PONT of gaseous molecules. 3.4 Biomarkers Paper‐based bio‐detection sensors based on ratiometric FL technology and semiconductor/carbon QDs have also been reported. For instance, under a 365†nm UV lamp, Noor and Krull implemented the RGB color space of a digicam for quantifiable ratiometric conversion of nuclei acid hybridization into a paper substrate, which used fixed QDs as donors and Cy3 as an acceptor in FRET. [77] Yan et†al. designed a test paper by using a ratiometric probe contained of red and green QDs for fluorescent visual detection of tyrosinase with a color evolution from yellow‐green to red. [78] Based on a same detection mechanism of electron transfer, Qu et†al. also fabricated a test strip by pre‐fixing red‐emitting dopamine‐modulated CdTe QDs and blue‐emitting graphene QDs on a filter paper for visual detection of tyrosinase activity with consecutive color change between red and blue. [79] Ma et†al. prepared a simple paper‐based platform for analyzing telomerase activity to diagnose bladder cancer simply and sensitively. [80] The FL color varied from red to yellow‐green with the increasing content of telomerase reaction products, which realized naked‐eye semiquantitative detection. Wang et†al. prepared a filter paper‐based test strip by using C–MIPs@FITC for sensitive naked‐eye assay of ovalbumin. [81] Liu et†al. demonstrated a paper‐based platform with two reversed signals for visual detections of gastric cancer‐related microRNA‐21 (miRNA‐21) and circular RNA from hippocampus abundant transcript 1 gene (circRNA‐HIAT1) (Figure 5a ) [82] Upon the addition of analyte, long DNA strands were generated by the trigger of rolling circle amplifications for the assemblies of green‐/red‐emissive labels (Figure 5b ). Therefore, distinct color variation of the PADs was realized (Figure 5c ). Figure 5 (a) Cellulose paper modified with citric acid and on‐site synthesis of CDs. (b) miRNA‐21 and circRNA‐HIAT1 activated RCA and fluorescent marking of PADs. (c) Imaging of the PADs arrays for FL color analysis of miRNA‐21 and circRNA‐HIAT1. Reproduced with permission from Ref.†[82] Copyright 2021, Elsevier B.V. (d) Illustration of visual and quantitative detection of H‐FABP based on RFLFIA and traditional FLFIA. Reproduced with permission from Ref.†[84] Copyright 2021, Wiley‐VCH For the purpose of enhancing visual assay capacity of conventional FL immunochromatographic assay, our group demonstrated a ratiometric fluorescent lateral flow immunoassay (RFLFIA) strip for detecting heart‐type fatty acid‐binding protein (H‐FABP) visually. [84] As shown in Figure 5d , the RFLFIA strip with two reversed signals was designed with an appropriate reporter and capture probes consisting of two kinds of QDs conjugated with two different types of H‐FABP antibodies. The detection principle of RFLFIA referred to a target‐induced IFE between mentioned reporter and capture probes, finally generated a transformation of FL color hue, which was used for naked‐eyed semiquantitative analysis and accurate quantitative analysis via combining with a cellphone installed with a "Color Picker" app. A detection platform including dark cavity and optical filter was also designed to eliminate the interference of surroundings. Other detection mechanisms such as FRET or electron transfer and color modulation mode between blue and red, or cyan and red will be effective approaches to enhance visual assay capacity of FL immunochromatographic assay. In addition, up to now, ratiometric FL technology with three different signals (blue, green, red) was not applied to FL immunochromatographic assay. It will be an interesting and challenging task. The features of mentioned test papers for biomarkers were described in Table 3. 3.5 pH Recently, ratiometric FL pH test papers based on QDs have been investigated for visual detection owing to the excellent FL property, easy production, economical, and convenient merit. For instance, Liu et†al. reported a supramolecular assembly based on aptamer, QD and graphene oxide, and it can be used to fabricate test strips for visual PONT of pH, which displayed fluorescent green‐to‐red color hue changes. [85] With an inexpensive UV lamp, the ratiometric sensory systems enable the detection of analytes with the naked eye that is as easy as colorimetric pH test paper, but with greater sensitivity. In addition, the FL of the nanosensor was tolerant of the presence of various urine components, which presented its practicability for sensing pH in urine samples. With a purpose of developing multi‐color emission CNDs, which can avoid outside fluorescent interference, Zhang et†al. synthesized tricolor emissive CNDs with satisfactory pH‐sensitive feature, which were used to fabricate pH strips by using this CND as probe and filter paper as substrate. The papers displayed significant color evolution from bright green to grayish and blue to purple to yellow with increasing pH value. [86] Although the range of color transitions was very wide, the sensitivity and reliability of the test paper were not studied. Zhao et†al. demonstrated a novel CD‐based test paper with high sensitivity for pH. And the color hue changed from pink to green with the increasing of pH from 1 to 3. [87] The high stability of CNDs endow the test paper with reliability for sensing pH. Yang et†al. synthesized dual‐emission Cu‐doped carbon dots (Cu‐CDs) by a one‐pot hydrothermal method. [88] The Cu‐CDs coated fluorescent paper was also prepared, which can visually monitor pH from 4 to 7.9 in urine with color hue transition from green to blue. The features of mentioned test papers for pH were described in Table 4. 3.1 Cations The higher concentration of metal cations like Hg 2+ and As 3+ induces various issues associated with healthiness and environment around the world. [15] Some metal ions including Pb 2+ , Hg 2+ , Cd 2+ , As 3+ and Cu 2+ can trigger different disease including osteoporosis, Alzheimer's disease, Parkinson's disease, Minamata disease, endocrine system disturbance and others. [16] The metal ions like As 3+ , Cr 3+ , Pb 2+ , and Cd 2+ give rise to severe bodily injury in spite of their trace level. However, these metal cations had been released from mining, industrial discharges, household effluent, and other garbage to pollute surroundings. Recently, the monitoring of certain metal cations in sewage and solid samples has been well implemented by the semiconductor/carbon QD‐based ratiometric FL test paper in research lab and industrial factories. Zhang and Jiang accomplished many work about color recognition‐based assay and have contributed to the development of visual test paper in color modulation strategies, detection mechanisms, and preparation methods. [17a] Zhang et†al. reported a sensitive fluorescent test paper for detecting As 3+ ions. [17b] As 3+ ions reacted with GSH/DTT‐QDs to form As−S bonds, thus induced the aggregation and FL quenching of this red QDs; meanwhile the cyan FL of CDs remained unchanged. It should be emphasized that the range of color variation between cyan and red was wider than green and red, green and blue, blue and red. So, a broad scope of sequential color hue variation for the hue recognition of As 3+ ions was achieved. These test papers were applied for the detection of As 3+ ions in water samples without pretreatments, the obtained satisfactory visual effect demonstrated its practicability. Zhang and colleagues also developed a ratiometric FL test paper using red‐emission CdTe QDs and blue‐emission CDs. [18] The test paper was applied for ratiometric detection of Cu 2+ ions; the FL color hue of the printed pattern "Copper 2+ " gradually changed from pink to blue through cyan. Although these mentioned test papers showed favorable dose‐sensitive color evolution, inferior compatibility between QDs and CDs made the preparation approach tedious. CdTe QDs possessed environmental toxicity. These issues limited the use of test papers. In order to solve these issues, Zhang put forward a ratiometric FL strategy on test paper using blue CD (b‐CD) and red CD (r‐CD) probes. (Figure 3A ). [19] The sensing mechanism of the test papers for Cu 2+ ions were described as follows: The surfaces of b‐CDs and r‐CDs had −COOH and p‐PDA ligands respectively, Cu 2+ ions will interact with b‐CDs and r‐CDs based on the surface complexing reaction. A new absorption band corresponding to the coordination of Cu 2+ with r‐CDs displayed a large spectrum overlapping with the emission of b‐CDs. Thanks to the FRET effect between b‐CDs and the Cu 2+ ‐p‐PDA complex at the surface of r‐CDs, the blue FL signal was quenched; meanwhile the red FL signal remained unchanged. The ability of this test paper to monitor Cu 2+ ions in water by the direct observation of FL colors under an excitation of 365†nm was tested; the test paper presented a sequential wide change from blue to orange‐red in the presence of increasing amounts of Cu 2+ , and had a visual LOD of 25†nM. In these studies, Zhang used an inkjet‐printing approach to prepare fluorescent test paper owing to following advantages. The uploaded amounts of probes are controllable, and the uniformity of obtained test papers can be guaranteed. Moreover, the patterns of printed probes on substrates can also be designed by computer. Figure 3 (a) Mechanism and process of ratiometric fluorescent test paper for Cu 2+ assay by a content sensitive color hue variation. Reproduced with permission from Ref.†[19] Copyright 2017, American Chemical Society. (b) Visual platform for ultrasensitive monitoring of endogenous Cu 2+ in human urine. Reproduced with permission from Ref.†[24] Copyright 2017, Elsevier B.V. (c) Major modules of the "Concentration Detection" app and (d) Smartphone based platform device for uranyl ion detection. Reproduced with permission from Ref.†[38] Copyright 2018, American Chemical Society. (e) Schematic illustration of the visual ratiometric detection system for CN − sensing and (f) hue response of prepared test paper versus CN − concentration. Reproduced with permission from Ref.†[43] Copyright 2019, Elsevier B.V. Other researchers have likewise established paper‐based sensors for hue recognition of Cu 2+ ions using QDs‐based probes. Wu and colleagues prepared a paper‐based sensor through drenching cellulose acetate membrane in probe solution. It was used for on‐site visual semi‐quantitative assay of Cu 2+ ions in red wine examples by hue recognition. [21] Wu also prepared a MOF/CdTe QDs‐based test paper for visual determination of Cu 2+ and Hg 2+ ions. [22a] Although the sensitivity and selectivity of these two test papers were not so good as other studies, on‐site and rapid determination of targets in red wine without any pretreatment procedure was realized successfully. Wang presented a simple approach to prepare fluorescent test paper with appropriate proportion of two CNDs. The strips were utilized for naked‐eye detection of Cu 2+ ions with a color change from orange‐red to blue. [23] The ratiometric FL test paper for cations has been widely created using different sensing probes, but it still remains a challenging task to prepare a test paper with multicolor variation with target dosages for definite determination. Du and Zhang reported a profuse color‐evolution‐based ratiometric FL test paper with red, green and blue three signals for visual monitoring of Cu 2+ ions in human urine. [24] As shown in Figure 3b , the tricolor probe system was obtained by mixing bCDs, gQDs and rQDs through volume ratio of 3 : 9 : 4. And the probe system‐based test papers were fabricated by inkjet‐printing approach. In the presence of Cu 2+ ions, gQDs and rQDs were simultaneously quenched whereas the FL of bCDs remains steady. Due to the change of ratio of FL intensities at three emission wavelengths, an abundant and wide color transformation (shallow pink‐light salmon‐dark orange‐olive drab‐dark olive green‐slate blue‐royal blue‐dark blue) was observed. Just by a pathway of dilution, the concentration of human urine samples can be distinguished by recognition of color hue of the test paper under a 365†nm UV lamp. The distinguished dosage scale is 6†nM in human urine, which cannot be achieved by other reported QDs‐based test papers for Cu 2+ ions. This study demonstrated that ratio FL test paper with three different signals not only had high sensitivity, but also possessed more profuse and wider color variations compared with two signals. Wang and co‐workers developed ratiometric FL nanohybrids by covalently linking prepared green QDs or Au NCs to the surface of silica nanoparticles embedded with red QDs.[ 22b , 37b ] The nanohybrid was used for on‐site visual determination of Cu 2+ and Pb 2+ ions respectively. And a probe‐based PVA‐film sensor was also constructed for sensing Pb 2+ ions. [37b] Wang and co‐workers also designed a dual‐emissive nanohybrid by hybridizing CNDs and gold nanoclusters. [26] The nanohybrid was applied for preparing test paper for the visualization of Hg 2+ ions by the distinct FL color change. The uranyl ion is regarded as an important index for nuclear industrial wastewater. It is challenging to sensitively and rapidly detect UO 2 2+ ions. For the purpose of achieving visual, rapid, on‐site, and sensitive assay of UO 2 2+ ions, Wang and colleagues designed a cellphone‐based portable platform including a 3D‐printed accessory, a smartphone installed with an app called "Concentration Detection" and a test strip (Figure 3d ). [38] Upon the addition of UO 2 2+ ions into test strip, the response signal of CdTe QDs was quenched by photo‐induced electron transfer, meanwhile, the reference signal of CNDs remained stable, thus generated significant variations in FL intensity ratio ( I 525 / I 640 ) and color of test strip. Compared with other studies, a significant advantage of this study is that after the detection process was accomplished, the colors of reference and sample were revealed synchronously in a smartphone on the result page, and the concentration of UO 2 2+ ions was computed and displayed clearly (Figure 3c and Figure†S1 in Supporting Information). Thanks to the self‐calibration capacity of ratiometric technology and designed platform device, interference such as ambient light was eliminated to a large extent; the platform for uranyl detection had been successfully applied in various real water samples. It should be noted that the interference from Cu 2+ and Ag + ions should be eliminated by the addition of sodium sulfide. Other researchers also made a lot of contributions in designing semiconductor or/and carbon QD‐based ratiometric FL test paper. For instance, Jiang and his researchers proposed a fluorescent test paper for visual detection of Hg 2+ ions by a printing process, which used CND as ink and filter paper as carrier. [29] Kim et†al. designed rhodamine‐appended CNDs‐based paper sensor for analyzing Al 3+ ions, and distinct change of color hue from blue to greenish‐yellow of the sensor can been recognized by the naked eye. [30] Wu et†al. demonstrated the employment of test paper for Fe 3+ ions sensing. [33] You et†al. illustrated a paper‐based ratiometric procedure for detection of Ag + ions, which used CdTe QDs and CNDs as probes. [35] Here, the mechanism is based on ion‐exchanging between red CdTe QDs and Ag + . On the basis of dual‐signal QD‐poly(dA)‐TSPP probes and Zn 2+ ‐chelation‐induced spectral modulation, Liu et†al. prepared a test strip with two reversed signals for on‐site naked‐eye detection of Zn 2+ ions. [36] Dong and group members constructed a fluorescent paper by doping dual‐emitting CNDs onto the cellulosic filter paper. The hue of FL color changed from pink to cyan with increasing concentration of Pb 2+ ions. [37a] On the whole, the detection mechanism of these semiconductor/carbon QD‐based test paper for cations based on FRET or electron transfer were more sensitive than other detection mechanisms such as aggregation. However, the design scheme was complicated, the distance between donor and acceptor should be regulatory, and the relative orientation of their transition dipole moments will influence the performance of detection. In addition, it was noticed that ratiometric FL test paper with two reversed signals or three signals was reported rarely: the construction of paper‐based ratiometric FL sensors with two reversed signals or three signals are demanded for improving the sensitivity and broadening color variations. 3.2 Anions The undue utilization of anions in various manufacturing industry and approaches and widespread discharge of anions into physical environment taints the natural land as well as water. This results in hazardous effects to humans and animals. Therefore, rapid, simple, on‐site visual sensors are highly demanded for sensing these anions. At present, various anions have been detected by QDs‐based FL sensors using hue recognition strategy. Zhang and Jiang designed a ratiometric fluorescent strip for sensitive, selective, naked‐eye detection of F − anions. [40] The strips used a F − ‐response organic probe and F − ‐inert CdTe QDs as signals. In the presence of different concentrations of F − , the test paper presented different FL colors with a minimum visible amount of 6†μM by naked eye. Zhang and co‐worker presented a N,S‐CD stained paper‐based test paper, which had one reference signal and one response signal for ClO − ions. [41] As the increase of concentration of ClO − anions, the FL color gradually altered from yellow to blue, which proved its potential of semi‐quantitative analysis. Zhang et†al. prepared fluorescent test strips through fixing mixed GCD/RCD/Eu 3+ buffer solutions on filter paper, of which GCD and RCD were as response and reference labels respectively. Upon the addition of inorganic phosphates, the FL color steadily varied from red to green with an observed dosage of 5†μM. [42] A standard reference card was also established for visual identification of different concentrations of inorganic phosphates on fluorescent test paper. Under a 365‐nm UV light, the trend of color changes in detection of spiked samples is similar to the card, which verified its capacity of semi‐quantitative visual detection of inorganic phosphates. CN − anions are recognized as hazardous substance in environmental and biological processes. Although many fluorescent probes had been designed for detecting CN − anions, it was a challenge to construct a CN − fluorescent assay which not only had the advantages of high sensitivity and selectivity of organic probes, but also held the good water solubility and stability of nanoprobes. In order to accomplish this challenge, our group designed a specific dual‐emission nano‐system for ultrasensitive detection of CN − ions. [43] The system used blue fluorescent CNDs as an inert reference and red fluorescent N‐acetyl‐L‐cysteine‐capped CdTe QD as a reporter. As shown in Figure 3e , the FL of N‐Acetyl‐L‐cysteine (NALC)‐capped CdTe QDs was first quenched by addition of Cu 2+ , then the introduction of carbon dots constituted a ratiometric FL detection system. Upon addition of CN − , CN − coordinated with Cu 2+ to form [Cu(CN) n ] (n−1)− complex, which caused the Cu 2+ to detach from the QDs, resulting in FL recovery. Meanwhile the CN − undergoes a nucleophilic addition reaction with the carbonyl group in the NALC, which passivated trap states and enhanced the FL of the QDs obviously. The blue emission of CDs was as a stable internal reference. Because of Cu 2+ ‐promoted complexation and addition reaction as recognition units, the presented method showed excellent selectivity and sensitivity for CN − ions. Furthermore, a fluorescent test paper fabricated with the detection system was applied for sensing CN − ions in water and cassava samples with pretreatment under 365†nm UV light. For quantitative detection, a "Color Analyzer" app installed in a phone was used to read the picture of the test strip and then transform it into hue values. Here the hue values was employed to describe the standard calibration versus various contents of CN − ions (Figure 3f ) with a LOD of 0.284†μM. On the whole, the semiconductor/carbon QDs‐based ratiometric FL test papers for visual sensing of anions are rarely reported. New detection mechanisms, patterns and materials are needed for broadening the application scope of ratiometric FL test papers for anions. And the challenge about specificity of test paper toward specific ions need to be overcome. 3.3 Small molecules Unreasonable misuse of antibiotics may cause durable and frequent ingestion of antibiotics into anatomy and injure the human body. Therefore, the remnants of antibiotics in food and the environment needs to be monitored for assuring food security and bodily health. Recently, various ratiometric FL test papers based on lanthanides‐modificatory QDs or CNDs with two reversed signals have been developed for PONT of tetracycline (TC) and oxytetracycline (OTC). For instance, Hu et†al. prepared a ratiometric fluorescent test paper for sensitive, precise and visual detection of TC, which used BCDs‐Eu/CMP‐cit as the probe. [44] The features of this test paper were described in Table 2 . Table 2 Visual hue recognition for PONT of small molecules by using semiconductor/carbon QD‐based test paper/strip. Probe Analyte λ em [nm] Type of FL signals Mechanism [a] Color effect Concentration range of visual detection [μM] LOD or discernable scale [μM] Ref BCDsEu/ CMP‐cit Tetracycline 465/620 Two reversed signals AE Bright blue/ek; to red 0–1.5 0.05 [44] BNQD‐Eu 3+ Tetracyclines 414/616 Two reversed signals IFE and PET as well as the AE Blue to red 0–20 – [45] Eu3 + /N, B‐Ti 3 C 2 MQDs Tetracycline 448/616 Two reversed signals IFE and AE Blue to red 0–20 – [46] Atta‐CDs‐Eu Tetracycline 445/617 Two reversed signals IFE and AE Blue to red 0–2 – [47] MoS 2 NPs@Gmp/ Eu‐Cit Tetracycline 430/617 Two reversed signals IFE and PET Blue to red 0–80 0.127 [48] ZnO/Eu Tetracycline 530/616 Two reversed signals AE Yellow to red 0–30 0.5 [49] SiQDs‐Cit‐Eu Tetracycline 455/617 Two reversed signals AE Blue to red 0–30 0.25 [50] La‐CDs Tetracyclines 450/515 Two reversed signals Static quenching or/and dynamic quenching Blue to green 0–1039.5 – [51] CD@AMP/ Eu NCPs Oxytetracycline 430/615 Two reversed signals IFE Blue to red 0–100 0.5 [52] BCNO QDs‐Ca 2+ Doxycycline 425/520 Two reversed signals IFE Blue to green 0–1039.5 1 [53] CDs/SiDs‐BPMA‐ Cu 2+ Ciprofloxacin 425/660 One reference and one response signals PET Slight‐blue to deep‐red 0–100 – [54] Eu‐CDs 2,6‐dipicolinic acid 475/615 One reference and one response signals AETE Blue to red 0–200 1 [55] FMn‐CDs 2,6‐dipicolinic acid 461/616 One reference and one response signals AETE Blue to red 0–200 1 [56] BCNO QDs 2,6‐dipicolinic acid 475/616 One reference and one response signals AETE Blue to red 0–700 1 [57] CDs‐Tb 2,6‐dipicolinic acid 459/495 One reference and one response signals Energy transfer Yellow to dark 0–5 0.0005 [58] hPEI‐CD‐EDTA‐ Eu 3+ 2,6‐dipicolinic acid 440/616 One reference and one response signals FRET Blue to purple 0–500 0.5 [59] CDs‐Eu 2,6‐dipicolinic acid 530/615 One reference and one response signals AE Green to red 0–50 0.067 [60] QDs hybrid Trinitrotoluene 520/650 One reference and one response signals FRET Yellow‐ green to red – – [61] gQDs@SiO 2 @ rQDs Trinitrotoluene 508/625 One reference and one response signals Electron transfer Red to green 0–5 – [62] CdTe@SiO 2 @ F‐PDA/MIPs 4‐nitrophenol 529/664 One reference and one response signals Electron transfer Green to red 0–0.55 – [63] Eu‐MoS 2 QDs 3'‐diphosphate‐ 5‐diphophate 410/616 One reference and one response signals AE Bluish violet to red 0–250 23.8 [64] CDs/CuNCs Dopamine 440/640 One reference and one response signals Electron transfer Blue to red 0–100 – [65] DNA‐CuNC/CDs Arginine/ acetaminophen 440/670 One reference and one response signals – Blue to red/red to blue 0–70 – [66] CDs/CdTe QDs Spermine 470/690 One reference and one response signals Aggregation Shallow pink to blue 0–1 [67] CDs Fungicide cymoxanil 435/520 Two reversed signals IFE and aggregation Green to blue 0–0.5 [68] rQDs@SiO 2 @CDs Thiram 453/644 One reference and one response signals FRET Pink to blue 0–10 0.059 [69] CD‐QD@SiO 2 Ascorbic acid 450/630 One reference and one response signals Electron transfer Red to blue 0–150 10 [70] CDs H 2 O 467/525 Two reversed signals Excited‐state intramolecular proton transfer Blue to green 0–100/ % – [71] QDs@SiO 2 ‐CDs Glucose 445/630 One reference and one response signals Electron transfer Blue to deep red 0–500 – [72] rQDs@SiO 2 @ gQDs Cysteine/ homocysteine 545/645 One reference and one response signals Analytes‐ induced displacement of the quencher Red to green 0–500/ 0–800 – [73] CDs‐QDs Folic acid 468/587 Two reversed signals Hydrogen bonds Pink to blue 0–900 – [74] Peptide‐SA‐QD Conjugates Benzothiostrobin 525/623 Two reversed signals Competitive and noncompetitive immunoassays Green to red 0–0.25 0.00042 [75a] [a] AE: antenna effect, IFE: inner filtration effect, AETE: absorbance‐energy transfer‐emission. Wiley‐VCH GmbH Yang et†al. constructed a novel ratiometric FL test strip for visual assay of TC. [45] Bai et†al. established a novel PONT paper by using Eu 3+ /N, B−Ti 3 C 2 MQDs system. Upon addition of TC in the test paper, the blue FL of N,B−Ti 3 C 2 MQDs (nitrogen and boron co‐doped Ti 3 C 2 MXene QDs) was quenched and red FL of Eu 3+ was enhanced gradually, the color hue and R/B change could be recognized by naked eye and smartphone respectively. [46] Jia et†al. prepared a test strip‐based sensor for identifying TC through a chromaticity analysis application. [47] With a customized smartphone‐based portable measurement chamber and MoS 2 NPs@Gmp/Eu‐Cit‐based test paper, Sun et†al. put forward to a promising tool for TC determination. [48] Wu et†al. prepared a test paper for sensing TC by fixing ZnO/Eu on the filter paper, not only realizing visual analysis, but also achieving on‐site and real‐time quantitative analysis assisted with a mobile phone app. [49] Zhang et†al. constructed a smartphone‐assisted paper‐based PONT platform through utilizing a color scanning app. [50] The FL color of the strip varied from blue to red with increasing concentration of TC, which could be recognized by human vision or the smartphone. Fan et†al. preliminarily explored a portable visual paper sensor with La‐CDs as probes for sensing TC or OTC. [51] Chen et†al. applied a test paper for the quantitative assay of OTC with a LOD of 0.5†μM based on a digital camera installed with a color detector app. [52] Liu et†al. established a convenient FL test strip which can be identified by naked eyes or smartphone; the FL color of the strip altered from blue to green as the concentration of doxycycline increased. [53] Gui et†al. used CDs/SiDs‐BPMA‐Cu 2+ as an effective ratiometric FL probe to detect ciprofloxacin (CIP), the FL color changed between red and blue. Based on CIP‐induced FL signals responses corresponding color, a filter paper‐based test strip by using the probe was applied for naked‐eye visual detection of CIP. [54] The features of this test paper and other test papers for antibiotics were described in Table 3 . Table 3 Visual hue recognition for PONT of biomarkers by using semiconductor/carbon QD‐based test paper/strip. Probe Analyte λ em [nm] Type of FL signals Mechanism Color effect Concentration range of visual detection LOD or discernable scale Ref gQDs and Cy3 Oligonucleotide 525/570 One reference and one response signals FRET Green to yellow 0–45†pM 450†fM [77] RF‐QDs‐DA Tyrosinase 542/657 One reference and one response signals Electron transfer Yellow to red 0–5†μg/mL – [78] N‐GQDs and DA‐rQDs Tyrosinase 644/440 One reference and one response signals Electron transfer Red to blue 0–0.64†U/mL – [79] QDs‐Rox Telomerase 524/605 One reference and one response signals Quenching caused by oxidation Red to yellow‐green 10–1000†cells – [80] C‐MIPs@FITC Ovalbumin 454/520 One reference and one response signals Hydrogen bond interaction Blue to green 0–2†μM 15.4†nM [81] PADs, gQD‐sDNA1 and rQD‐sDNA2 miRNA‐21 and circRNA‐ HIAT1 453/512 or 610 Two reversed signals Hybridization and FRET Blue to green/Blue to red 0–2†nM/ 0–2†nM 3.5/2.7†fM [82] 450‐NBs and 655‐QDs H‐IgG 450/655 One reference and one response signals Immunoassays Blue to red 0–1000†ng/mL – [83] SAQ‐mAb1 and mAb2‐gQD heart‐type fatty acid binding protein 524/626 Two reversed signals IFE and immunoassays Green to orange red 0–50†ng/mL 1†ng/mL [84] Wiley‐VCH GmbH At present, due to the great harm of anthrax, 2,6‐Dipicolinic acid (DPA), a biomarker for Bacillus anthracis and B. anthras has been of interest to analytical chemists. For instance, Rong et†al. integrated Eu‐CDs with filter paper, and prepared a test paper for naked‐eye recognition of DPA under a 254†nm UV lamp. [55] Rong also produced a paper‐based test strip by using functionalized manganese‐doped CDs for visual detection of DPA with a LOD of 1†μM. [56] Subsequently, Rong et†al. prepared a convenient boron carbon oxynitride QD test paper for visual determination of DPA through using commercial fiber filer paper as substrate. [57] Liu et.†al. designed a portable and sensitive CDs‐Tb‐based test strip for naked‐eye assay of DPA with a ultra‐low LOD of 0.5†nM. [58] Yang et†al. achieved visual PONT of DPA based on paper‐based analytical device by printing the probe solution into polyvinylidene fluoride (PVDF) film. [59] In order to obtain an economic, sensitive and naked‐eye FL test paper for determining DPA, our group designed a Eu 3+ functionalized carbon dots (CDs‐Eu) as a novel ratiometric FL probe, a test paper was further prepared for visual and quantitative PONT of DPA by utilizing a phone equipped with an app named "Color Analyzer". The detection range and LOD of the papered‐based sensors are 1–30†μM and 67†nM (Figure 4a ). [60] These reported test papers mentioned above all used lanthanides ions as response signals and QDs as reference signals to construct single‐signal‐response ratiometric FL system for DPA. The evolutionary sensitivity of color hue is intrinsically limited, thus affect the sensitivity of detection by naked eye or smartphone. Figure 4 (a) The application of the visible identification of DPA by paper‐based sensor and mobile phone. Reproduced with permission from Ref.†[60] Copyright 2020, Elsevier B.V. (b) Smartphone sensing platform based on 3D printing for thiram analysis and corresponding detection mechanism. Reproduced with permission from Ref.†[69] Copyright 2020, American Chemical Society. Among the above‐mentioned fluorescent test papers for antibiotics and DPA, the Eu 3+ or Tb 3+ modified/doped semiconductor/carbon QDs presented higher sensitivity than other probes owing to following reason. Some of antibiotics and DPA can acts as the antenna group to absorb the excitation light based on the antenna effect after the combination of the target molecule of Eu 3+ or Tb 3+ . It was noticed that most of these test papers with signals showed color change from blue to red. More different color‐transition modes between two different signals or three signals need to be explored. In addition to antibiotic and DPA, paper‐based hue recognition for PONT of other various small molecules have also been extensively explored. For instance, a new technique of PONT for trinitrotoluene (TNT) is need for homeland security needs against terrorism. Zhang et†al. designed a dual‐emission QDs hybrid and QDs hybrid‐based test paper for sensing TNT. The hybrid was prepared by embedding the red‐emitting QDs in silica nanoparticles and covalently linking the green‐emitting QDs to the silica surface. In the presence of TNT, the red QDs remained constant, the green QDs functionalized with polyamine can selectively bind TNT via the formation of the Meisenheimer complex, resulting in the green FL quenching due to FRET. The different FL intensity ratios displayed different color between yellow‐green and red, and versus different amounts of TNT. In addition, Qian et†al. prepared a gQDs@SiO 2 @rQDs nanohybrid and nanohybrid‐based test paper for visual sensing of trinitrotoluene. [62] The internal rQDs were as stable references, external bQDs can react with TNT to form the Meisenheimer complex, thus leading to the FL quenching of rQDs owing to a fact that electrons of rQDs transferred to TNT molecule. Compared with Zhang's nanohybrid, this nanohybrid was more sensitive for TNT. As a result of "coffee ring effect", the performance of filter papers‐based common FL test strips is restricted in visual detection. To overcome this issue, Xu et†al. prepared a ratiometric FL test strip based on the PVDF hydrophobic membrane and probe for naked‐eye assay of 4‐nitrophenol. [63] The hydrophobic PVDF membrane with colorless FL background efficiently overpowered this "coffee ring effect" and background interference. A comparison experiment of visual detection demonstrated that light color variations of the PVDF fluorescent test strip were recognizable and more evident than that with a conventional filter paper. Jiang and his researchers developed a visual quantitative platform for detecting pesticide composed of a test paper strip, a cellphone and dark cavity with a UV lamp (Figure 4b ). [69] The blue FL could be quenched by gold nanoparticles and then recovered with pesticide. And red FL of rQDs was just as a reference signal. R/B values of captured pictures could be used for pesticide quantification with a sensitive LOD of 5†nM. The dark cavity and optical filter of the designed platform was used to eliminate the interference of surroundings. Rong et†al. fabricated a facile and portable Eu‐MoS 2 QDs test paper for naked‐eye assay of 3'‐diphosphate‐5‐diphophate. [64] He et†al. constructed CDs/CuNCs‐based test paper for visual detection of dopamine on filter paper. Each FL color of test paper was assigned to the corresponding concentration of dopamine in the aqueous suspension of sample‐nanohybrid mixtures. [65] Bu et†al. synthesized DNA‐CuNC/CD complex as a dual‐emission ratiometric probe to determine both arginine and acetaminophen, by placing this probe in cuvettes or dropping it onto filter paper strips. [66] Fu et†al. designed a ratiometric fluorescent paper as an onsite, fast, economical approach for visual sensing of spermine, as well as freshness of pork respectively. The test paper used CdTe QDs and CNDs as a response labels and internal reference labels [67] Zhao et†al. designed a ratiometric FL sensor based on CD‐QD@SiO 2 for ascorbic acid detection with satisfactory results in fruit juice. [70] Yin et†al. synthesized blue FL CNDs as an effective ratiometric sensor for sensing water in organic solvents. And cellphone‐based quantitative detection was also achieved by CNDs‐loaded test papers through a color processing application. [71] Huang et†al. demonstrated test papers printed by a QDs@SiO 2 ‐CDs ink for glucose sensing, thus enabled a potential PONT application. [72] Blood glucose was determined by this test paper and a standard glucometer, the obtained result was almost same. He et†al. prepared a paper‐based sensor based on CDs‐QDs to demonstrate a suitable and straightforward approach to visually detect folic acid. [74] In order to obtain a stable, bright and tunable ratiometric probe, our group assembled two different hydrophobic QDs into a silica frame based on thiol‐metal coordination, thereby formed a dual‐signal hierarchical luminescent silica composite. [75b] Compared with other nanohybrids, the ratio ( I 627 / I 528 ) and color hue of the generated nanohybrid can be accurately formulated by varying the amounts of these two QDs. Thanks to the analyte‐induced IFE between gold nanoparticles and nanohybrid, a ratiometric system with two reversed signals and a logic gate strategy were successfully constructed for the visual determination of melamine with a color hue evolution from green to orange‐red. Lately, a new unconstrained ratiometric signal approach was successfully applied to immunochromatographic strips for visual determination of benzothiostrobin by Chen and co‐workers. [75a] This innovation possessed important reference value for hue recognition of small molecules by lateral flow immunoassay (LFIA). The study can be widely utilized to FL or colorimetry reading by fluorescent or colorimetric labels. They amalgamated the negative‐readout. competitive and positive‐readout noncompetitive immunoassays into one test through utilizing various color markers, including labeled and anti‐immunocomplex peptides. As the content of benzothiostrobin increased, the green FL signal or red colorimetric signal on T line decreased, the red FL signal or blue colorimetric signal increased simultaneously. Moreover, a color reference card was constructed for visual judgment based on such distinct color hue changes from green to red (fluorescent mode) or red to blue (colorimetric mode). After adaptation with a portable smartphone, the quantitative detection was implemented with high sensitivity and good accuracy. The features of the test papers were described in Table 4 . Table 4 Visual hue recognition for PONT of pH by using semiconductor/carbon QD‐based test paper. Probe Range λ em [nm] Type of FL signals Mechanism Color effect Resolution Ref gQD@SiO 2 / GO 5.5–7.5 520/590 One reference and one response signals Conformational changes Green to red 0.1†pH [85] AC‐CDs 1–14 440/510/620 Three response signals The pyridine on the surface of ACis was easy to be protonated and deprotonated. Bright green to grayish and blue to purple to yellow – [86] CDs 1 1–3 514/600 One reference and one response signals Dynamic quenching process Pink to green 0.2‐pH [87] Cu‐CDs 3–8.7 436/520 One reference and one response signals Surface passivation effect Green to blue – [88] Wiley‐VCH GmbH The development of ratiometric fluorescent probes for gaseous molecules is of great challenge, Wang and his group have developed QDs‐based ratiometric fluorescent probes for gaseous molecules. They designed a fluorescence nanohybrid probe with one response signal and one reference signal at 452 and 657†nm separately for sensing gaseous hydrogen sulfide. [76a] The probe was prepared by encapsulating red 3‐mercaptopropionic acid modified CdTe QDs in silica shells and then modifying the silica surface with a blue organic molecules azidocoumarin‐4‐acetic acid. They also designed a nanohybrid with one response signal and one reference signal at 665 and 460†nm separately for visual determination of nitrogen dioxide. The nanohybrid was prepared by conjugating through coupling reaction between carboxyl groups and amino groups on the surface of rede‐missive QDs and blue fluorescent CNDs. [76b] In general, the combination of sensor with paper substance to prepare test paper is more conducive for PONT of gaseous molecules. More efforts need to be made for designing QDs‐based ratiometric FL test paper for PONT of gaseous molecules. 3.4 Biomarkers Paper‐based bio‐detection sensors based on ratiometric FL technology and semiconductor/carbon QDs have also been reported. For instance, under a 365†nm UV lamp, Noor and Krull implemented the RGB color space of a digicam for quantifiable ratiometric conversion of nuclei acid hybridization into a paper substrate, which used fixed QDs as donors and Cy3 as an acceptor in FRET. [77] Yan et†al. designed a test paper by using a ratiometric probe contained of red and green QDs for fluorescent visual detection of tyrosinase with a color evolution from yellow‐green to red. [78] Based on a same detection mechanism of electron transfer, Qu et†al. also fabricated a test strip by pre‐fixing red‐emitting dopamine‐modulated CdTe QDs and blue‐emitting graphene QDs on a filter paper for visual detection of tyrosinase activity with consecutive color change between red and blue. [79] Ma et†al. prepared a simple paper‐based platform for analyzing telomerase activity to diagnose bladder cancer simply and sensitively. [80] The FL color varied from red to yellow‐green with the increasing content of telomerase reaction products, which realized naked‐eye semiquantitative detection. Wang et†al. prepared a filter paper‐based test strip by using C–MIPs@FITC for sensitive naked‐eye assay of ovalbumin. [81] Liu et†al. demonstrated a paper‐based platform with two reversed signals for visual detections of gastric cancer‐related microRNA‐21 (miRNA‐21) and circular RNA from hippocampus abundant transcript 1 gene (circRNA‐HIAT1) (Figure 5a ) [82] Upon the addition of analyte, long DNA strands were generated by the trigger of rolling circle amplifications for the assemblies of green‐/red‐emissive labels (Figure 5b ). Therefore, distinct color variation of the PADs was realized (Figure 5c ). Figure 5 (a) Cellulose paper modified with citric acid and on‐site synthesis of CDs. (b) miRNA‐21 and circRNA‐HIAT1 activated RCA and fluorescent marking of PADs. (c) Imaging of the PADs arrays for FL color analysis of miRNA‐21 and circRNA‐HIAT1. Reproduced with permission from Ref.†[82] Copyright 2021, Elsevier B.V. (d) Illustration of visual and quantitative detection of H‐FABP based on RFLFIA and traditional FLFIA. Reproduced with permission from Ref.†[84] Copyright 2021, Wiley‐VCH For the purpose of enhancing visual assay capacity of conventional FL immunochromatographic assay, our group demonstrated a ratiometric fluorescent lateral flow immunoassay (RFLFIA) strip for detecting heart‐type fatty acid‐binding protein (H‐FABP) visually. [84] As shown in Figure 5d , the RFLFIA strip with two reversed signals was designed with an appropriate reporter and capture probes consisting of two kinds of QDs conjugated with two different types of H‐FABP antibodies. The detection principle of RFLFIA referred to a target‐induced IFE between mentioned reporter and capture probes, finally generated a transformation of FL color hue, which was used for naked‐eyed semiquantitative analysis and accurate quantitative analysis via combining with a cellphone installed with a "Color Picker" app. A detection platform including dark cavity and optical filter was also designed to eliminate the interference of surroundings. Other detection mechanisms such as FRET or electron transfer and color modulation mode between blue and red, or cyan and red will be effective approaches to enhance visual assay capacity of FL immunochromatographic assay. In addition, up to now, ratiometric FL technology with three different signals (blue, green, red) was not applied to FL immunochromatographic assay. It will be an interesting and challenging task. The features of mentioned test papers for biomarkers were described in Table 3. 3.5 pH Recently, ratiometric FL pH test papers based on QDs have been investigated for visual detection owing to the excellent FL property, easy production, economical, and convenient merit. For instance, Liu et†al. reported a supramolecular assembly based on aptamer, QD and graphene oxide, and it can be used to fabricate test strips for visual PONT of pH, which displayed fluorescent green‐to‐red color hue changes. [85] With an inexpensive UV lamp, the ratiometric sensory systems enable the detection of analytes with the naked eye that is as easy as colorimetric pH test paper, but with greater sensitivity. In addition, the FL of the nanosensor was tolerant of the presence of various urine components, which presented its practicability for sensing pH in urine samples. With a purpose of developing multi‐color emission CNDs, which can avoid outside fluorescent interference, Zhang et†al. synthesized tricolor emissive CNDs with satisfactory pH‐sensitive feature, which were used to fabricate pH strips by using this CND as probe and filter paper as substrate. The papers displayed significant color evolution from bright green to grayish and blue to purple to yellow with increasing pH value. [86] Although the range of color transitions was very wide, the sensitivity and reliability of the test paper were not studied. Zhao et†al. demonstrated a novel CD‐based test paper with high sensitivity for pH. And the color hue changed from pink to green with the increasing of pH from 1 to 3. [87] The high stability of CNDs endow the test paper with reliability for sensing pH. Yang et†al. synthesized dual‐emission Cu‐doped carbon dots (Cu‐CDs) by a one‐pot hydrothermal method. [88] The Cu‐CDs coated fluorescent paper was also prepared, which can visually monitor pH from 4 to 7.9 in urine with color hue transition from green to blue. The features of mentioned test papers for pH were described in Table 4. 4 Conclusions and Perspectives Various semiconductor/carbon QDs have been utilized as core sensing units to combine with paper‐based substrates for fabricating ratiometric FL platforms/sensors for PONT using hue recognition. On the potential toxicity of semiconductor QDs, which is a general concern for various applications, one can see that in recent years researchers are increasingly inclined to use non‐toxic semiconductor QDs or to reduce the toxicity of the semiconductor QDs‐based probes through post‐modification. Although CDs are non‐toxic, the photochemical properties of CDs are not as good as traditional semiconductor QDs in photoluminescence quantum yield and FL emission band width. Therefore, seeking the preparation of semiconductor/carbon QDs with improved properties is essential. In addition, semiconductor/carbon QDs‐based test strips are meant to be used by non‐specialists or customers, who dislike carrying around specific additional containers for collecting hazardous waste (which in turn would make the assay much more cumbersome). Moreover, once in the hands of non‐experts, test strips are easily thrown away; the strips contain QDs will pollute the environment which clearly contradicts the UN′s sustainability goals. Thus, reusable or/and biodegradable semiconductor/carbon QDs‐based test strips are in high demand for solving these problems. The construction of probes on different paper‐based substrates are done mainly through straightforward deposition or inkjet printing to make efficient and sensitive fluorescent paper‐based sensors. Generally, the paper‐based substrates are unprocessed filter paper and A4 paper; it will be another effective approach to improve the effect of hue recognition‐based assay by changing the type of substrate or modifying the surface of the substrate. Moreover, the FL color of these sensors are further used for detecting analyte qualitatively and semi‐quantitatively by the naked eye, or quantitatively by recording and converting obtained images using digital analysis devices. Smartphones display wonderful ability in recording and transforming FL signals by using appropriate procedures and color space. In addition, it has great potential allowing for the transfer of testing result to a monitoring center, or a doctor, through a wireless 4G or 5G network. In this review, we sum up and discuss different applications of cations, anions, small molecules, biomarkers and pH from biological, clinical, food, and environmental samples, aiming at providing a fine notion for researchers to select the investigation direction or initiate novel research in hue recognition‐related work. Conflict of interest The authors declare no conflict of interest. 5 Biographical Information Jing Wang was born in Zhejiang Province, China, in 1986. He received his B.S. and PhD degree in 2008 and 2013, respectively in Huazhong Agricultural University. Presently he is working as an associate professor at Zhejiang University of Technology. His scientific interests focus on functionalized photoelectric nanomaterials for point‐of‐care testing applications . Biographical Information Daquan Li received his B.S. degree in materials science and engineering from HuangShan University in 2016. Then he obtained his PhD degree in chemical engineering and technology from Zhejiang University of Technology in 2021. He is currently working as a postdoctoral fellow at Zhejiang University of Technology. His main interest focuses on the synthesis of Quantum dots‐based fluorescent sensors for various point‐of‐need testing applications . Supporting information As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Supporting Information Click here for additional data file. Data Availability Statement Data sharing is not applicable to this article as no new data were created or analyzed in this study.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8367587/

The Role of Exercise in Reducing Hyperlipidemia-Induced Neuronal Damage in Apolipoprotein E-Deficient Mice

Hyperlipidemia causes nervous system-related diseases. Exercise training has developed into an established evidence-based treatment strategy that is beneficial for neuronal injury. This study investigated the effect of exercise on hyperlipidemia-induced neuronal injury in apolipoprotein E-deficient (ApoE −/− ) mice. Male ApoE −/− mice (age: 8 weeks) were randomly divided into four groups as follows: mice fed a normal diet (ND), normal diet+swimming training (ND+S), high-fat diet (HD), and high-fat diet+swimming (HD+S). Exercise training consisted of swimming for 40 min/day, 5 days/week for 12 weeks. After 12 weeks, we measured serum levels of total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-c). We also evaluated glial fibrillary acidic protein (GFAP) expression levels using immunohistochemistry, real-time PCR, and immunoblotting. In addition, NLR family pyrin domain-containing 3 (NLRP3), interleukin- (IL-) 18, caspase-1, Bax, Bcl-2, and phosphorylated extracellular signal-regulated kinase (p-ERK) expression levels were measured using immunoblotting. Serum levels of TG, TC, and LDL-c were lower in ApoE −/− HD+S mice than in ApoE −/− HD mice. Immunohistochemistry, real-time PCR, and immunoblotting showed increased levels of GFAP in the ApoE −/− HD group. Immunoblotting revealed increased levels of NLRP3, IL-18, caspase-1, Bax, Bcl-2, and p-ERK in the ApoE −/− HD group; however, they were significantly suppressed in the ApoE −/− HD+S group. Therefore, exercise has protective effects against neuronal injury caused by hyperlipidemia. 1. Introduction Hyperlipidemia is a lipid metabolism disorder that causes elevated serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c), and triglyceride (TG) levels and/or decreased high-density lipoprotein cholesterol levels. Many studies have revealed that hyperlipidemia is a major risk factor for atherosclerosis, cardiocerebrovascular disease, chronic kidney damage, and fatty liver disease [ 1 , 2 ]. There is considerable evidence that hyperlipidemia also leads to neuroinflammation, followed by neuronal damage [ 3 , 4 ]. Numerous studies have reported that hyperlipidemia may cause nervous system-related diseases [ 5 , 6 ]. For example, hyperlipidemia has been reported to be related to neurodegenerative diseases such as Alzheimer's and Niemann-Pick disease [ 7 , 8 ]. Paul et al. also reported that hypercholesterolemia increased midbrain dopaminergic neurodegeneration in a mouse model of Parkinson's disease [ 9 ]. Hyperlipidemia is an independent risk factor for dementia [ 10 ], and high-fat diet- (HD-) induced obesity is associated with an increased risk of type 2 diabetes and impaired neural functions [ 11 ]. A review article concluded that aerobic exercise and the combination of aerobic and resistance training have effects on the serum levels of cholesterol and lipids [ 12 ]. In addition, exercise reduced neuronal injury in several studies. For example, treadmill training significantly increased the formation of lumbar spinal synapses [ 13 ]. Similarly, step training after transection of newborn rat spinal cords can increase motor neuron synapse activation [ 14 ]. Apolipoprotein E-deficient (ApoE −/− ) mice, a well-established animal model of hyperlipidemia, have been used extensively to study the effects of atherosclerosis and renal injury [ 15 – 17 ]. Therefore, we established a hyperlipidemia-induced neuron injury model in ApoE −/− mice by administering a HD and subjecting the mice to exercise in the form of swimming for 40 min/day, 5 days/week for 12 weeks. This study determined the effect of exercise on hyperlipidemia-induced neuronal injury in ApoE −/− mice and the specific mechanisms involved. 2. Materials and Methods 2.1. Animals Eight-week-old male ApoE −/− mice were obtained from Beijing Vital River Laboratories Animal Technology Co., Ltd. (Beijing, China). All mice were given ad libitum access to food and water and were housed in a room having 40-60% humidity, at 24-26°C with a 12 h light/dark cycle. ApoE −/− mice were divided randomly into four groups of 7 mice each: normal diet (ND), normal diet+exercise training (ND+S), high-fat diet (HD), and high-fat diet+swimming exercise training (HD+S). The HD mouse food comprised 1.25% ( w / w ) cholesterol, 22.5% ( w / w ) protein, 20.0% ( w / w ) cocoa fat, and 45.0% carbohydrate (Jiangsu Medicience, Jiangsu, China). One week prior to administration of the test diets, exercise training was initiated in an experimental swimming pool (temperature, 30°C; water depth, 44 cm; and radius, 120 cm). The progressive exercise program initially involved swimming for 5–10 min and was gradually extended to 30 min/day. When the test diets were implemented, the mice were subjected to formal swimming exercise for 40 min/day, 5 days/week for 12 weeks. All animal experiments were approved by Gongyi People's Hospital. 2.2. Biochemical Measurements Blood samples were taken from the abdominal aorta of rats, and serum was stored at -80°C. TC, TG, and LDL-c levels were measured using enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocols. The TC, TG, and LDL-c concentrations were calculated based on measurements of optical density at the respective wavelengths for each compound according to the manufacturer's protocol. 2.3. Immunohistochemistry The brains of all mice were perfusion-fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) following a heparinized saline flush. The brains were dehydrated and embedded in paraffin. Serial 7 μ m coronal sections were cut using a microtome. Paraffin sections of the hippocampus were used for the immunohistochemical analysis, which was performed using the Histone Simple stain kit (Nichirei, Tokyo, Japan) according to the manufacturer's instructions. Paraffin-embedded sections were deparaffinized with xylene and then rehydrated in a descending series of ethanol washes. The sections were treated for 15 min with 3% H 2 O 2 in methanol to inactivate endogenous peroxidases and then incubated at 4°C overnight with a primary antibody against glial fibrillary acidic protein (GFAP; rabbit anti-GFAP, 1 : 500; Z0334, Dako, Carpinteria, CA, USA). All sections were examined microscopically using a BX40 upright light microscope (Olympus, Tokyo, Japan). 2.4. Western Blotting Mice in each group were euthanized by intraperitoneal injection of an overdose of sodium pentobarbital. The hippocampus and cortex were isolated from each brain ( n = 5 in each group). Briefly, the hippocampus was homogenized (1 : 5, w : v ) in ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate, protease inhibitor cocktail, and phosphatase inhibitor cocktail (1 : 100 each; Nacalai Tesque, Kyoto, Japan). The resulting homogenates were centrifuged at 12,000 × g for 30 min at 4°C, the supernatants were collected, and total protein levels were determined using a bicinchoninic acid assay kit (Pierce, Rockford, IL, USA). Proteins (15 μ g) were separated on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes in a wet transfer device (30 V, 1 h). Membranes were preincubated in 5% bovine serum albumin for 2 h and then incubated with the following primary antibodies overnight at 4°C: rabbit anti-GFAP, anti-NLRP3, anti-caspase-1, anti-interleukin- (IL-) 18, anti-Bax, anti-Bcl2, and anti-p-ERK (all at 1 : 1000 dilution and from ProteinTech Group, Rosemont, IL, USA). After incubation with horseradish peroxidase-conjugated anti-rabbit secondary antibodies, as appropriate, for 1 h (1 : 5000; KPL, Gaithersburg, MD, USA), the membranes were reacted with an enhanced chemiluminescence reagent (New England Lab, Woburn, MA, USA). Finally, specific protein bands were visualized by using the ImageQuant LAS 4000 imaging system (GE Healthcare Life Sciences, Issaquah, WA, USA). Intensities of the protein bands were quantified using ImageJ software (NIH; https://imagej.nih.gov/ij/ ). 2.5. RNA Isolation and Real-Time RT-PCR Total DNA was isolated from the cerebral cortex and hippocampus tissues. Then, according to the manufacturer's protocol, TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kits (TransGen, Beijing, China) were used to prepare complementary DNA. The GFAP gene expressions were analyzed in terms of quantity by running RT-PCR by the use of fluorescent SYBR Green technology. Relative expression of the GFAP gene was normalized to β -actin. The primer was as follows: β -actin: forward primer: 5-CGATGCCCTGAGGGTCTTT-3′ and reverse primer: 5′-TGGATGCCACAGGATTCCAT-3′, and GFAP: forward primer: 5′-TTGCTGGAGGGCGAAGAAA-3′ and reverse primer: 5′-AGGGAGAGCTGGCAGG-3′. 2.6. Statistical Analyses Data are presented as the mean ± standard error of mean and were analyzed using SPSS software version 23.0 (IBM, Chicago, IL, USA). Intergroup differences were determined by an analysis of variance and Tukey's post hoc test. P < 0.05 was regarded as significant. 2.1. Animals Eight-week-old male ApoE −/− mice were obtained from Beijing Vital River Laboratories Animal Technology Co., Ltd. (Beijing, China). All mice were given ad libitum access to food and water and were housed in a room having 40-60% humidity, at 24-26°C with a 12 h light/dark cycle. ApoE −/− mice were divided randomly into four groups of 7 mice each: normal diet (ND), normal diet+exercise training (ND+S), high-fat diet (HD), and high-fat diet+swimming exercise training (HD+S). The HD mouse food comprised 1.25% ( w / w ) cholesterol, 22.5% ( w / w ) protein, 20.0% ( w / w ) cocoa fat, and 45.0% carbohydrate (Jiangsu Medicience, Jiangsu, China). One week prior to administration of the test diets, exercise training was initiated in an experimental swimming pool (temperature, 30°C; water depth, 44 cm; and radius, 120 cm). The progressive exercise program initially involved swimming for 5–10 min and was gradually extended to 30 min/day. When the test diets were implemented, the mice were subjected to formal swimming exercise for 40 min/day, 5 days/week for 12 weeks. All animal experiments were approved by Gongyi People's Hospital. 2.2. Biochemical Measurements Blood samples were taken from the abdominal aorta of rats, and serum was stored at -80°C. TC, TG, and LDL-c levels were measured using enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocols. The TC, TG, and LDL-c concentrations were calculated based on measurements of optical density at the respective wavelengths for each compound according to the manufacturer's protocol. 2.3. Immunohistochemistry The brains of all mice were perfusion-fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) following a heparinized saline flush. The brains were dehydrated and embedded in paraffin. Serial 7 μ m coronal sections were cut using a microtome. Paraffin sections of the hippocampus were used for the immunohistochemical analysis, which was performed using the Histone Simple stain kit (Nichirei, Tokyo, Japan) according to the manufacturer's instructions. Paraffin-embedded sections were deparaffinized with xylene and then rehydrated in a descending series of ethanol washes. The sections were treated for 15 min with 3% H 2 O 2 in methanol to inactivate endogenous peroxidases and then incubated at 4°C overnight with a primary antibody against glial fibrillary acidic protein (GFAP; rabbit anti-GFAP, 1 : 500; Z0334, Dako, Carpinteria, CA, USA). All sections were examined microscopically using a BX40 upright light microscope (Olympus, Tokyo, Japan). 2.4. Western Blotting Mice in each group were euthanized by intraperitoneal injection of an overdose of sodium pentobarbital. The hippocampus and cortex were isolated from each brain ( n = 5 in each group). Briefly, the hippocampus was homogenized (1 : 5, w : v ) in ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate, protease inhibitor cocktail, and phosphatase inhibitor cocktail (1 : 100 each; Nacalai Tesque, Kyoto, Japan). The resulting homogenates were centrifuged at 12,000 × g for 30 min at 4°C, the supernatants were collected, and total protein levels were determined using a bicinchoninic acid assay kit (Pierce, Rockford, IL, USA). Proteins (15 μ g) were separated on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes in a wet transfer device (30 V, 1 h). Membranes were preincubated in 5% bovine serum albumin for 2 h and then incubated with the following primary antibodies overnight at 4°C: rabbit anti-GFAP, anti-NLRP3, anti-caspase-1, anti-interleukin- (IL-) 18, anti-Bax, anti-Bcl2, and anti-p-ERK (all at 1 : 1000 dilution and from ProteinTech Group, Rosemont, IL, USA). After incubation with horseradish peroxidase-conjugated anti-rabbit secondary antibodies, as appropriate, for 1 h (1 : 5000; KPL, Gaithersburg, MD, USA), the membranes were reacted with an enhanced chemiluminescence reagent (New England Lab, Woburn, MA, USA). Finally, specific protein bands were visualized by using the ImageQuant LAS 4000 imaging system (GE Healthcare Life Sciences, Issaquah, WA, USA). Intensities of the protein bands were quantified using ImageJ software (NIH; https://imagej.nih.gov/ij/ ). 2.5. RNA Isolation and Real-Time RT-PCR Total DNA was isolated from the cerebral cortex and hippocampus tissues. Then, according to the manufacturer's protocol, TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kits (TransGen, Beijing, China) were used to prepare complementary DNA. The GFAP gene expressions were analyzed in terms of quantity by running RT-PCR by the use of fluorescent SYBR Green technology. Relative expression of the GFAP gene was normalized to β -actin. The primer was as follows: β -actin: forward primer: 5-CGATGCCCTGAGGGTCTTT-3′ and reverse primer: 5′-TGGATGCCACAGGATTCCAT-3′, and GFAP: forward primer: 5′-TTGCTGGAGGGCGAAGAAA-3′ and reverse primer: 5′-AGGGAGAGCTGGCAGG-3′. 2.6. Statistical Analyses Data are presented as the mean ± standard error of mean and were analyzed using SPSS software version 23.0 (IBM, Chicago, IL, USA). Intergroup differences were determined by an analysis of variance and Tukey's post hoc test. P < 0.05 was regarded as significant. 3. Results 3.1. Metabolic Characterization The metabolic characteristics of the animals are shown in Figure 1 . Body weights did not differ significantly among the four groups. The levels of LDL-c, TC, and TGs were significantly increased ( P < 0.05) in the ApoE −/− HD groups compared with the ND and ND+S groups. In addition, the levels of LDL-c and TC were decreased in the HD+S group compared to the HD group. However, TG levels did not show this decrease. These results suggest that exercise was effective in reducing TC and LDL levels in HD mice. 3.2. Exercise Inhibits the Increased GFAP Expression Caused by Hyperlipidemia in the Cerebral Cortical Layer and Hippocampal Area To investigate the pathological changes in hyperlipidemia-induced neuroinflammation, we performed GFAP immunohistochemical staining ( Figure 2 ) of different brain tissues (cerebral cortex, hippocampal CA1 area, hippocampal CA3 area, and dentate gyrus). The results showed that GFAP-positive cells were increased in the ApoE −/− HD group compared with the ND and ND+S groups. Interestingly, swimming exercise (HD+S group) decreased the number of GFAP-positive cells. In addition, in the ND group, GFAP-positive glial cells had small nuclei and short protrusions, while in the HD group, glial cells were activated, the nuclei were enlarged and rounded, and the protrusions were elongated. After exercise, glial cell activity decreased, the nuclei became smaller, and the protrusions shrank compared to the HD group. Furthermore, real-time PCR and western blot analysis of GFAP showed that the expression of GFAP was inhibited by exercise ( Figure 3 ). 3.3. Exercise Inhibits Hyperlipidemia-Induced Neuroinflammation To evaluate the involvement of proinflammatory cytokines and cell death factors in neuronal tissues from each of the four groups, NLRP3, IL-18, and caspase-1 protein expression were measured using western blotting ( Figure 4 ). All three proteins were increased in the HD group compared to the ND group. However, these increases were attenuated in the HD+S group. 3.4. Exercise Inhibits Apoptosis Induced by Hyperlipidemia To evaluate apoptosis in neuronal tissues of the four experimental groups, Bax and Bcl-2 protein expression was measured using western blotting ( Figure 5 ). The Bax protein level was higher in the HD group than in the ND group. This increase was attenuated in the HD+S group. Interestingly, the expression of Bcl-2 showed the opposite trend. Compared to the ND group, Bcl-2 expression was decreased, and this decrease was attenuated by exercise in the HD+S group. 3.5. p-ERK Signaling Pathway Western blotting analysis of p-ERK was used to investigate neuronal damage caused by hyperlipidemia ( Figure 6 ). p-ERK protein expression in neuronal tissues was lower in ApoE −/− HD+S mice than in ApoE −/− HD mice. 3.1. Metabolic Characterization The metabolic characteristics of the animals are shown in Figure 1 . Body weights did not differ significantly among the four groups. The levels of LDL-c, TC, and TGs were significantly increased ( P < 0.05) in the ApoE −/− HD groups compared with the ND and ND+S groups. In addition, the levels of LDL-c and TC were decreased in the HD+S group compared to the HD group. However, TG levels did not show this decrease. These results suggest that exercise was effective in reducing TC and LDL levels in HD mice. 3.2. Exercise Inhibits the Increased GFAP Expression Caused by Hyperlipidemia in the Cerebral Cortical Layer and Hippocampal Area To investigate the pathological changes in hyperlipidemia-induced neuroinflammation, we performed GFAP immunohistochemical staining ( Figure 2 ) of different brain tissues (cerebral cortex, hippocampal CA1 area, hippocampal CA3 area, and dentate gyrus). The results showed that GFAP-positive cells were increased in the ApoE −/− HD group compared with the ND and ND+S groups. Interestingly, swimming exercise (HD+S group) decreased the number of GFAP-positive cells. In addition, in the ND group, GFAP-positive glial cells had small nuclei and short protrusions, while in the HD group, glial cells were activated, the nuclei were enlarged and rounded, and the protrusions were elongated. After exercise, glial cell activity decreased, the nuclei became smaller, and the protrusions shrank compared to the HD group. Furthermore, real-time PCR and western blot analysis of GFAP showed that the expression of GFAP was inhibited by exercise ( Figure 3 ). 3.3. Exercise Inhibits Hyperlipidemia-Induced Neuroinflammation To evaluate the involvement of proinflammatory cytokines and cell death factors in neuronal tissues from each of the four groups, NLRP3, IL-18, and caspase-1 protein expression were measured using western blotting ( Figure 4 ). All three proteins were increased in the HD group compared to the ND group. However, these increases were attenuated in the HD+S group. 3.4. Exercise Inhibits Apoptosis Induced by Hyperlipidemia To evaluate apoptosis in neuronal tissues of the four experimental groups, Bax and Bcl-2 protein expression was measured using western blotting ( Figure 5 ). The Bax protein level was higher in the HD group than in the ND group. This increase was attenuated in the HD+S group. Interestingly, the expression of Bcl-2 showed the opposite trend. Compared to the ND group, Bcl-2 expression was decreased, and this decrease was attenuated by exercise in the HD+S group. 3.5. p-ERK Signaling Pathway Western blotting analysis of p-ERK was used to investigate neuronal damage caused by hyperlipidemia ( Figure 6 ). p-ERK protein expression in neuronal tissues was lower in ApoE −/− HD+S mice than in ApoE −/− HD mice. 4. Discussion In this study, we explored the protective effects of exercise using a model of hyperlipidemia-induced neuronal injury in ApoE −/− mice. The results demonstrated that exercise had a protective effect on this injury by affecting proinflammatory cytokine levels, apoptosis, GFAP expression, and the p-ERK pathway. No significant variation was observed in body weights among the four groups of mice. However, compared with ApoE −/− ND group mice, higher LDL-c and TC levels were observed in ApoE −/− HD group mice. This result indicated that the hyperlipidemia mouse model was established successfully by a high-fat diet. A review article noted that exercise has an effect on the serum levels of cholesterol and lipids [ 18 ]. Interestingly, LDL-c and TC levels were significantly lower in the ApoE −/− HD+S group than in the ApoE −/− HD group, suggesting that exercise had a protective effect against lipid deposition caused by a HD. However, the effect of exercise on hyperlipidemia-induced neuronal injury remained unclear. Immunohistochemistry, real-time PCR, and immunoblotting were performed to evaluate GFAP levels for understanding the mechanism of neuronal damage caused by hyperlipidemia. Astrocytes play a significant role in maintaining the physiological functions of the blood-brain barrier and in regulating the metabolism of lipids in the brain [ 19 ]. GFAP, an intermediate filament protein that is primarily expressed in astrocytes, is a key marker of mature astrocytes [ 20 ]. Many studies have shown that overexpression of GFAP is associated with neuronal damage. Stolmeier et al. reported that increases in GFAP and Ibal antagonized hippocampal function [ 21 ]. In the current study, the expression of GFAP was increased in the HD group. This showed that hyperlipidemia activated glial cells and caused neuronal damage. Importantly, this effect was suppressed by exercise. Hyperlipidemia-induced inflammation plays a crucial role in the development of cardiac damage, ischemic stroke, and brain injury [ 22 ]. Neuroinflammation has been identified as a causative factor of multiple neurological diseases [ 23 , 24 ]. The nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome is abundantly expressed in the central nervous system and is the most studied inflammasome in this system [ 25 , 26 ]. Activating the NLRP3 inflammasome leads to the activation of caspase-1. Subsequently, activated caspase-1 causes the production of IL-1 β and IL-18, as well as proinflammatory cytokines, and mediates rapid cell death. IL-1 β and IL-18 drive inflammatory responses through a variety of downstream signaling pathways that result in neuronal injury [ 12 , 27 , 28 ]. Results of the current study showed that NLRP3, caspase-1, and IL-18 levels were increased in the HD group. In this study, swimming, as a treatment, reduced the expression of NLRP3, caspase-1, and IL-18, thereby indicating that exercise can act as a protective agent against hyperlipidemia-induced neuronal injury. Apoptosis plays an important role in various neuronal injuries [ 29 – 31 ]. Kim et al. found that berberine treatment inhibited brain inflammation in poloxamer 407-treated hyperlipidemic rats by inhibiting apoptosis [ 32 ]. The key regulators of apoptosis are members of the Bcl-2 family of proteins; this protein family contains a variety of proapoptotic (such as Bax and Bak) and antiapoptotic (such as Bcl-2, Bcl-xL, and Bcl-w) proteins [ 33 , 34 ]. In the current study, compared to the ND group, Bax protein levels were increased and Bcl-2 protein levels decreased in the HD group. It is worth noting that exercise inhibited the expression of Bax and enhanced the expression of Bcl-2, suggesting that apoptosis was inhibited. The mitogen-activated protein kinase/ERK pathway participates in every stage of cell growth and development, including cell proliferation, differentiation, migration, senescence, and apoptosis [ 35 ]. In white matter-lesioned rats, activation of the mitogen-activated protein kinase/ERK pathway promotes neuronal apoptosis, thereby worsening the condition [ 36 ]. In the current study, we found that p-ERK expression was significantly increased in the HD group. This showed that hyperlipidemia caused neuronal injury by activating the p-ERK signaling pathway, and exercise can inhibit this effect. 5. Conclusions In conclusion, the results of the present study showed that exercise treatment had a protective effect on hyperlipidemia-induced neuronal injury in ApoE −/− mice. Exercise reduced the increases in serum LDL-c and TC and protected against neuronal damage by inhibiting GFAP expression, inflammation, apoptosis, and activation of the p-ERK signaling pathway. The findings of this study could be beneficial in developing novel strategies for the prevention and treatment of neuronal injury. Data Availability All datasets are available from the corresponding author upon reasonable request. Conflicts of Interest The authors declare that there is no conflict of interest regarding the publication of this paper.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7123672/

Clinical Management of Patients Infected with Highly Pathogenic Microorganisms

The clinical management of high consequence infectious diseases (HCID) poses an immense challenge, seen largely varying standards in terms of infection prevention control (IPC) as well as in quality of clinical care. This chapter gives an overview of possible treatment as well as IPC options. Lessons learned within the German Permanent Working Group of Competence and Treatment Centres for highly infectious, life-threatening diseases (STAKOB) are taken into account. Introduction Providing clinical care for patients suffering from high-consequence infectious disease (HCID) is challenging. Medical therapy for patients suffering from HCID may frequently be sophisticated and complex but lacking in evidence base and in extensive experience. Despite maximal intensive care treatment that is administered, including the most advanced resources, mortality among such patients is mostly very high. On the other hand, the natural impulse as a medical practitioner to provide immediate and efficient medical care to the patient may be compromised by the need for health care workers' (HCW) protection with personal protective equipment (PPE). Biological weapons agents are frequently rare, highly virulent microorganisms causative for HCID (Table 9.1 ). Table 9.1 Causative agents of HCID potentially requiring high level containment Agent Disease(s) US Biosafety Level (BSL) requirement a Middle East respiratory syndrome coronavirus (MERS-CoV) Middle East respiratory syndrome (MERS) 3 Severe acute respiratory syndrome coronavirus (SARS-CoV) Severe acute respiratory syndrome (SARS) 3 Yersinia pestis Pneumonic plague Bubonic plague Plague meningitis Plague sepsis Other forms of plague 3 Variola virus (VARV) Smallpox 4 Monkeypox virus (MPXV) Monkeypox 4 Crimean-Congo hemorrhagic fever virus (CCHFV) Crimean-Congo hemorrhagic fever (CCHF) 4 Ebola Virus (EBOV) Sudan Virus (SUDV) Taï Forest Virus (TAFV) Bundibugyo Virus (BDBV) Ebola virus disease (EVD) 4 Marburg Virus (MARV) Ravn Virus (RAVV) Marburg virus disease (MVD) 4 Lassa virus (LASV) Lassa fever 4 Other viruses for which US BSL requirement is 4 and which are causative of viral hemorrhagic fevers (e.g., Junín Virus, Machupo Virus) a Laboratory biosafety levels according to: https://www.cdc.gov/biosafety/publications/bmbl5/bmbl5_sect_iv.pdf This chapter focuses primarily on individual patient care with a high level of both patient care and infection prevention and control. Mass casualty management strategies for HCID are discussed as well. Initial Approach Towards an HCID Patient Potentially Requiring High Containment Isolation The initial approach towards an HCID patient differs. Scenarios might be: A patient turns up at a medical facility on her/his own initiative as a suspect case This might include the so-called "worried well" who did not encounter a significant source of infection. Particularly in the case of a mass casualty scenario, the "worried well" may slow down clinical management of genuinely affected patients [ 1 ]. Furthermore, a significant role is played by patient and staff flow as well as structural capacities of the hospital. (b) A patient is brought to a medical facility by ambulance as a suspected case This scenario is probably managed most easily. Patient and staff flow, area management, and protective measures (such as donning PPE) are regulated quite restrictively in Standard Operating Procedures (SOPs) of high-level isolation units as well as of other tertiary care hospitals. (c) A patient is admitted to hospital for a differential diagnosis (e.g., malaria, influenza). An HCID is diagnosed later on in the hospital or after discharge Secondary transmissions of HCID in Africa and the Western World frequently follow this scenario. For example, In May 2015, a patient travelled from Liberia via Morocco to the USA, where he was admitted to hospital due to a sore throat, fever, and tiredness. He reportedly did not indicate travel to Western Africa and was discharged home the same day. Three days later, he was re-admitted due to clinical deterioration and tested positive for Lassa virus (LASV) infection. He succumbed to Lassa fever [ 2 ]. A patient who was initially diagnosed with malaria was medically evacuated from Togo to Germany in March 2016. After his death, his body was retrospectively tested and found to be infected with LASV. The mortician who handled the corpse contracted the virus, developed Lassa fever, was administered experimental treatment with ribavirin and favipiravir and survived [ 3 , 4 ]. In August 2016, a man presented to a university hospital in Madrid, Spain, with a history of high fever, abdominal pain, malaise, nausea, and diarrhea. His clinical condition deteriorated, he developed multi-organ failure, and died in spite of intensive care treatment. All tests for routine infections were negative. Retrospectively, he was diagnosed with Crimean-Congo hemorrhagic fever (CCHF). A nurse who had assisted with the endotracheal intubation of the index patient contracted CCHFV, was treated with ribavirin, and survived [ 5 ]. Patients might present to a health care facility of any level. It is probable that the initial physician confronted with an HCID patient sees an HCID clinical picture for the first time in her or his life. Hence it is crucial to have immediate technical support by medical professionals experienced in infectious diseases as well as reference hospitals close by. Numbers and bed capacities of high-level isolation units (HLIU) differ largely between countries [ 6 ]. During the 2013–2016 Ebola virus disease (EVD) outbreak in Western Africa, medical evacuation of non-EU/non-US citizens was an issue due to the scarcity of suitable isolation facilities in their home countries [ 7 ]. Infection prevention and control measures are crucial. Please refer to Chap. 10.1007/978-3-030-03053-7_8 for further instructions on universal precautions. The particular approach differs according to the suspected pathogen. Causative pathogens of viral hemorrhagic fevers (VHFs) such as Ebola virus (EBOV) and LASV have a relatively low stability, and fluid transmission plays the key role [ 8 ]; hence national and international guidelines on EVD recommend a safety distance of 1.5–2 m to avoid transmission via macro droplets in case PPE is not worn—such as in patient screening [ 9 , 10 ]. If physical contact to the patient and/or her/his bodily fluids has to occur, wearing PPE is of utmost importance. For PPE recommendations please refer to the Sect. 9. 7 . Health care facilities of any level should include the initial approach towards suspected and confirmed HCID cases in their emergency preparedness plans and SOPs. These procedures might differ largely between hospitals, depending on factors such as human resources and the architectural setup of the medical facilities. Area Management The location where an HCID patient receives medical care is potentially highly contaminated. PPE should be worn at any time. Areas in which there is a low risk of infection and which serve as a supply base (e.g., storage room, staff room) versus sites that are high-risk zones (places where patient care takes place) should be defined very clearly. The 2013–2016 EVD outbreak has shown that poor area management can be a significant source of infection [ 11 ]. There are different concepts of area management. For example, In EVD treatment centers in field settings, usually a "low-risk zone" and "high-risk zone" are defined. A differentiation between a red zone (contaminated patient area), a yellow zone (inner chamber), and a green zone (outer chamber/non-contaminated area) is made in the context of occupational health dealing with HCID in Germany [ 12 ]. The inner chamber ("yellow zone") allows space for PPE decontamination and doffing (Fig. 9.1 ). Fig. 9.1 Example of Area Management: Medical evacuation aircraft "Robert Koch". The patient is treated in the red zone by staff wearing personal protective equipment (PPE). The yellow zone is designated for PPE decontamination and doffing. Staff can don PPE and store supplies in the green zone Supportive Treatment Supportive treatment should be based on clear plans and guidelines, such as those for intensive care and sepsis management ( http://www.survivingsepsis.org ). Furthermore, national and international (WHO) specific guidelines and therapeutic recommendations for the particular disease should be consulted. Intensive care largely depends on available resources in the limited nursing setting. There are indications that intensive care may improve clinical outcomes: Out of 20 patients who were medically evacuated during the 2013–2016 EVD outbreak, 16 survived, yielding a case–fatality ratio (CFR) of 20% [ 13 ]. On the other hand, the overall case–fatality ratio for all confirmed cases in Guinea, Sierra Leone, and Liberia with recorded clinical outcomes was 63% [ 14 ]. Even more, case fatality ratios for Marburg virus disease (MVD) fluctuate between ~22% for an outbreak which occurred in West Germany in 1967 (supportive treatment and frequent blood samplings for clinical surveillance were performed at that time) and ~88% for an outbreak in Uíge/Angola in 2005 [ 15 , 16 ]. One means of intensive care not listed in Table 9.2 is extracorporeal membrane oxygenation, the use of which has been documented for one case of Middle East respiratory syndrome (MERS) treated in France [ 19 ]. Severe acute respiratory syndrome (SARS), MERS, and pneumonic plague mainly affect the respiratory system. A long-term follow-up study indicates that patients affected by adult respiratory distress syndrome (ARDS) can benefit from extracorporeal membrane oxygenation [ 20 ]. Equipment and qualified staff are available in designated extracorporeal membrane oxygenation centers. A directory of these centers is available at https://www.elso.org/ . Table 9.2 Supportive care measures reportedly delivered at EVD treatment facilities during the 2013–2016 Western African outbreak [ 13 , 17 , 18 ] Facility Supportive care measures available Goderich Treatment Centre (Sierra Leone) Constant bedside nursing Continuous blood pressure/heart rate/respiratory rate monitoring Pulse oximetry Arterial and venous cannulation Nasogastric tube feeding Invasive ventilation Continuous renal replacement therapy Diagnostic biochemistry and hematology Ultrasonography Plain radiography Mathaska Ebola Treatment Unit (Sierra Leone) Nasogastric tube feeding Bedside ultrasound Intraosseous cannulation for intravenous fluid resuscitation Frankfurt High-Level Isolation Unit (Germany) Constant bedside nursing Continuous blood pressure/heart rate/respiratory rate monitoring Pulse oximetry Peripheral arterial and venous cannulation Central venous cannulation/placement of Sheldon Catheter Invasive ventilation Renal replacement therapy Diagnostic biochemistry and hematology Ultrasonography Plain radiography Specific Treatment Specific treatment for HCID is often still experimental and/or only administered as compassionate use. This lack of progress is partly due to a lack of patients and costly large-scale clinical trials, hence precluding regular drug approval by national drug authorities. For example, the use of ribavirin as a treatment of Lassa fever is mainly based on a small clinical study without a control group performed in the 1980s [ 21 ]. However, none of the HCID dealt with in this chapter are listed as neglected tropical diseases, according to WHO [ 22 ]. Examples of specific treatments for HCID are indicated in Table 9.3 . Table 9.3 Examples of specific treatments, post-exposure prophylaxis and vaccines for HCID (all experimental) Disease(s) Treatment Post-exposure prophylaxis Vaccines International specific treatment guidelines Pneumonic plague Quinolones Aminoglycosides Tetracyclines Cotrimoxazole [ 23 , 24 ] Quinolones, cotrimoxazole [ 23 , 24 ] Sub-unit vaccine based on the F1- and V-antigens [ 25 ] WHO guidelines available [ 23 ] Smallpox Brincidofovir [ 26 ] Tecovirimat (ST-246) [ 27 ] Smallpox vaccine [ 28 ] Smallpox vaccine (WHO Emergency stockpile 'SVES' available) [ 29 ] Crimean–Congo hemorrhagic fever Ribavirin [ 30 ] Favipiravir [ 31 ] WHO guidelines in development [ 32 ] Ebola virus disease a ZMAPP [ 33 ] Favipiravir [ 34 ] VSV-ZEBOV vaccine [ 35 ] VSV-ZEBOV ChAd3-vaccine [ 36 ] WHO guidelines available [ 37 ] Lassa fever Ribavirin [ 21 ] Favipiravir [ 38 ] Ribavirin [ 39 ] For more details, please consult chapters dealing with the particular disease as well as national and international guidelines a In the context of the 2013–2016 Western African EVD outbreak, research activities were mainly focused on Ebola Virus (EBOV) and did not target other ebolaviruses (SUDV, TADF, BDBV) or marburgviruses (MARV, RAVV). For future epidemics caused by filoviruses other than EBOV, there are thus still significant research gaps concerning specific treatment and vaccination [ 40 ] National and international guidelines which mention specific treatments against HCID are scarce. Some of the above-mentioned specific treatments are more frequently used for treating more 'common' diseases, making available more safety data and facilitating easier drug procurement, for example: Antibiotics such as quinolones/tetracyclines/cotrimoxazole against bacterial infections Oral ribavirin against hepatitis C and Favipiravir against influenza. On the other hand, drug procurement and stockpiling of rarely used drugs or those commercially undeveloped (so-called 'orphan drugs') are a challenge. The fact that synergies are not implemented but country and state authorities tend to do 'their own thing' can be detrimental for efficient stockpiling strategies [ 41 ]. Drug procurement strategies should take into account established contacts to the drug manufacturer and to the public health/drug/customs authorities and, if applicable, should also consider benefitting from the compassionate use program. For examples of such programs, please refer to [ 42 – 44 ]. Dead Body Management Handling dead bodies is a high-risk task. Staff managing dead bodies (e.g., morticians) are rarely trained at the same level as medical practitioners, although they may be exposed to the same risks. As for EVD, the highest viral loads can be found in dead bodies [ 45 ]. Having well-trained staff and PPE available is crucial. All procedures that involve any contact with the dead body and/or body fluids should be performed while wearing PPE. All avoidable procedures (e.g., embalming) should be avoided. The body should be placed in a body bag. Cultural sensitivities should be respected when handling a dead body as the evacuation of a dead body can be traumatizing for the population. For example, during the Western African EVD outbreak, burial teams were attacked [ 46 ]. Exact procedures for decontamination of the dead body, body bag, and surroundings as well as exact burial procedures depend on the causative pathogen. For example, Yersinia pestis , though sensitive in aerosols, can survive in carcasses for up to 2 months at 35 °C [ 47 ]. ICRC guidelines for dead body management in disaster contexts were updated recently [ 48 ]. A specific WHO dead body management/burial guideline for EVD is available [ 49 ]. Personal Protective Equipment (PPE) For further instructions on universal precautions, please refer to the Chap. 10.1007/978-3-030-03053-7_8 in this book. PPE should be safe, user-friendly, and appropriate. Choosing the 'appropriate' PPE is complex because of to the following elements: National and international PPE recommendations vary. For example, during the Western African EVD outbreak, differences between PPE standards of WHO, CDC, MSF, and other actors were the subject of debate [ 50 ]. The resources in terms of materials and qualified staff are different from place to place. National work security regulations differ (e.g., restricting duration of shifts). In the event of a possible bioweapons attack, the causative agent might be unknown (unknown pathogen or, even more complex, generally unknown chemical/biological/radiological/nuclear CBRN-spectrum agent) or multi resistant (example: multi-resistant Y. pestis ). Particle sizes for different pathogens differ significantly, compromising the choice of the appropriate respirator. For example, if a chemical agent cannot be excluded in a scenario of a generally unknown CBRN-spectrum agent, a self-contained breathing apparatus should be considered. Types of PPE are PPE with gown/overall , FFP2/3 (or N95/N98) respirator, double gloving, goggles/face shield and other facultative items (e.g., apron, rubber boots): This PPE is inexpensive, widely available, and WHO standards have been set for clinical care for patients infected with filoviruses such as EBOV: http://www.who.int/csr/resources/publications/ebola/ppe-guideline/en/ . The challenge is that this PPE is composed of singular items, each of which was usually tested according to norms (in the European Union: EN standards). In practice, due to the complexity of donning, doffing, and decontaminating PPE, errors which compromised biosafety have been documented [ 51 ]. Furthermore, particular agents for decontamination (e.g., peracetic acid) cannot be used due to lack of filtering of chemical agents by FFP2/3 (or N95) respirators. Finally, because of the lack of ventilation, exhaustion due to heat is frequent and the wearing time thus limited [ 52 ]. Powered Air Purifying Respirator (PAPR) is frequently used in European HLIU and, as for the EU, defined by EN standard 12,941 [ 53 ]. Depending on the exact type, they are mostly easy to don and doff. As ventilated equipment, PAPR is comfortable to wear, allowing longer working times [ 54 ]. Decontamination can be performed by, for instance, taking a shower with a decontaminating agent, provided that this agent is compatible with the filter chosen. However, PAPR is quite expensive and hence may not be available in resource-poor settings. Self-Contained Breathing Apparatus is frequently used by fire brigades in Western countries (e.g., for intervening in smoky locations or those contaminated by chemical agents). This apparatus is heavy, complex to use, allows only very limited time of operation while wearing it, and is, according to national work security regulations, only to be used by staff who underwent a medical examination for aptitude. The choice of a self-contained breathing apparatus should hence be reserved to settings in which the presence of an agent leaking through the filters of other PPE (e.g. PAPR) cannot be excluded [ 54 ]. The choice of PPE for different procedures in environmental and health care settings should be circumscribed as clearly as possible before an HCID-related event takes place. Case example 1 : Choice of PPE in HLIU in Germany The choice of PPE in Germany is mainly prescribed by regulations (e.g., Ordinance on biological substances or technical regulations for biological substances—TRBA). Annex 1 of TRBA No. 250 describes the infection prevention and control measures that have to be undertaken in an HLIU. There, PAPR according to the norm EN 12941 is set forth as mandatory. Each one of the seven German HLIU has its individual PAPR. Integrated systems as well as a combination of an overall and a ventilated hood are used. Case example 2 : Choice of PPE in mass casualty management in Israel The type of PPE to be used in Israel by medical first responders and hospital teams in each type of emergency scenario is determined by the Israeli Ministry of Health. The decision is made based on the lethality of each agent, availability or lack of a specific effective treatment, extent of morbidity, and level of infectivity. In case of agents that are not communicable from person to person (e.g., Bacillus anthracis ), standard droplet protection is required consisting of surgical masks, protective glasses/facial protection, and a gown while performing actions that involve aerosol exposure (e.g. suction of secretions). During pandemics, aerosol protection is required especially if the lethality of the disease is high relative to seasonal influenza. This protection includes surgical masks (to be replaced at least every 4 h); gowns over clothes; gloves to prevent contact with mucous membranes, blood secretions, and fluids; and protective goggles only when performing activities that pose a risk of spraying onto mucous membranes. In cases when aerosolization is likely, N95 masks are required (e.g., cases of airborne infection). Only very severely ill patients are treated by staff donning the strictest PPE against aerosol agents, including negative-pressure protective systems and ventilated hoods. Decontamination and Disinfection This chapter deals with decontamination and disinfection in the context of clinical management. For general advice on decontaminating capabilities and facilities, please refer to Chap. 10.1007/978-3-030-03053-7_6 in this book. The decontaminating agent should be chosen depending on the pathogen and site (PPE, surface, medical equipment, skin/mucous membranes or eyes, environmental decontamination). International and national recommendations concerning decontaminating agents should be taken into account, considering the pathogen (bacteria, viruses, or fungi). The 'worst-case pathogen' is Bacillus anthracis due to its extreme stability in most environments [ 55 ]. Concerning PPE decontamination , detailed documentation is available concerning its application and decontamination at a CBRN site ('hot zone') [ 56 ]. On the other hand, the decision of how to decontaminate PPE after having performed clinical management of HCID patients is complex: Concerning EVD, CDC currently recommends disinfection of gloved hands as well as of visibly soiled parts of the PPE. PAPR should be cleaned and disinfected in accordance with the manufacturer's recommendations [ 57 ]. As for high-level isolation units in Germany, PAPR is systematically decontaminated after use. For example, when an EVD case was cared for in Hamburg/Germany in 2014, the PPE was decontaminated after use by two shower cycles with 2% perchloric acid for 2 min, a residence time of 7 min, and rinsing off of acid residues by showering with water [ 17 ]. Surface decontamination can be complex if the surface is porous and/or if medical equipment is involved. Krauter et al. [ 58 ] discuss general decontamination strategies following a bio-contamination event. The decontamination of medical devices and the needed measures are, in detail, discussed by WHO [ 59 ] and RKI [ 60 ]; differentiating between: non-critical equipment which neither invades the skin nor comes in contact with mucous membranes (e.g., ECG electrodes) semi-critical equipment which comes in contact with mucous membranes (e.g., speculum, gastroscope), and critical equipment which is invasive (e.g., retractors, trocars). The relevant guidelines should be consulted to decontaminate medical devices adequately. Decontamination of skin, mucous membranes, or eyes applies mainly in the context of immediate contamination with a bioweapons, such as B. anthracis spores. Please refer to Chap. 10.1007/978-3-030-03053-7_6 in this book as well as Krauter et al. [ 58 ]. Disinfection of skin, mucous membranes, or eyes is subject to universal precautions (see Chap. 10.1007/978-3-030-03053-7_8 in this book). A particular challenge can be the disinfection of mucous membranes and eyes due to their vulnerability. For disinfection of mucous membranes, the Framework Ebola Virus Disease document in Germany recommends octenidine dihydrochloride/phenoxyethanol or chlorhexidine-containing drugs or povidone–iodine complexes (7.5%) (e.g. Octenisept®, Skinsept mucosa®, Braunol®). For application on the eyes, 5% povidone–iodine complex can be suitable [ 61 ]. A particular scenario might be an accidental exposure to bodily fluids, for instance by a splash of infective bodily fluids on unprotected skin or into the eyes, or by a needle stick injury. Medical facilities should have clear SOPs for such incidents. For environmental decontamination, please refer to Chap. 10.1007/978-3-030-03053-7_6 in this book. This topic is furthermore discussed in detail by Franco and Bouri [ 55 ]. Practical Case Examples: Lassa Fever and Ebola Virus Disease in Germany Ebola Virus Disease in Germany in 2014 Health care worker (HCW) infections were an important and very sad effect of the Western African EVD outbreak. As of 31 March 2015, 815 HCW had contracted EVD. Out of 635 health care workers for whom the outcome was available, 418 (65.9%) died [ 62 ]. In total, three medically evacuated HCW were treated in HLIU in Germany in 2014, two of whom survived. The patients were administered experimental treatments and likely benefited from intensive care. Infection prevention and control was thoroughly maintained. Staff caring for the patients wore PAPR as PPE. Neither secondary infections nor imported cases occurred in Germany [ 17 , 18 ]. In spite of this fortunate epidemiologic 'outcome', there was an emotional debate in Germany driven by fear [ 63 ]. Public health authorities made a tremendous effort to enhance emergency preparedness towards the disease, and an elaborate document 'Framework Ebola Virus Disease' was released led by Robert Koch Institute, Germany RKI [ 64 ]. A sophisticated medical evacuation airplane with resources for performing comprehensive intensive care measures aboard was designed, launched in November 2014 and, because of the end of the humanitarian crisis related to EVD, dismantled in [ 65 , 66 ]. 2. Lassa Fever in Germany 2015 In February 2016, a 46-year-old HCW who worked in Togo fell sick with developing fever, malaise, and a sore throat. He was admitted to a local hospital where treatment for suspected malaria was started immediately. Because of clinical deterioration he was medically evacuated from Togo to Germany nearly 2 weeks later. He was admitted to the University Hospital of Cologne, Germany. After arriving at this hospital, the clinical condition of the patient deteriorated dramatically, resulting in the patient's death shortly after hospital admission. The post-mortem examination did not reveal an underlying infectious disease. Simultaneously, after histologic examination of liver specimens of the index case a viral hemorrhagic fever was suspected. Blood and liver samples were sent to the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany, for further analysis. The institute confirmed LASV infection by PCR. The body was hence cremated [ 67 ]. A mortician who handled the primary case's corpse, and who was reported to have worn gloves and did not recall being exposed to body fluids, contracted the disease. He had overlapping clinical signs of an upper respiratory tract infection at the time of contact. Eight days after exposure, a first PCR for LASV was negative. A subsequent PCR was positive and the patient was admitted to the isolation unit in Frankfurt. He received experimental treatment with ribavirin and favipiravir, clinically improved with resolving fever from the 7th day of disease onwards, and survived [ 4 , 68 ]. This was the first recorded transmission of LASV infection outside Africa. The case underlines the need for rapid and robust diagnostics of HCIDs and for maintaining proper infection prevention and control measures by using PPE whenever HCIDs cannot be ruled out. The incident also demonstrated that larger outbreaks can be prevented successfully when the Health Protection Authorities (HPA) react quickly and efficiently and install effective infection control measures. Difficulties were related to the unreliableness of contact persons who did not adhere to communicated control measures, thereby provoking an increase in the number of contacts. Also, one explicit lesson learned was that information between different authorities involved should be shared rapidly, facilitated by a central coordination [ 69 ]. Role of the Public Health Sector The interface between HCID expertise in clinical management and public health sectors should be well established. For example, for referring a patient, a health care facility might need an isolation transport ambulance—and hence the assistance of the public health service for coordination. For contact-tracing activities, the public health service requires information that is gathered by the health care facilities from affected people. HPA are responsible for responding to any problem concerning the infected patient outside of health care facilities, as well as for limiting secondary transmissions. Despite an increasing knowledge about HCID, major challenges still remain for the physicians and HPA who treat these patients. In Germany, the Permanent Working Group of Competence and Treatment Centres for highly infectious, life-threatening diseases (STAKOB) was established in 2014. It regroups HCID experts from all seven HLIU—mainly infectious disease specialists—and from seven corresponding HPA [ 70 ]. The STAKOB members HLIU and HPA are located dispersedly on the state level to assure proximity. Additionally, specialists from the Federal Institute for Vaccines and Biomedicines, the medical services from the Federal Foreign Office, the medical service of the German Federal Armed Forces, and others participate in the activities. The STAKOB office is affiliated to the Robert Koch Institute (RKI). Regular meetings are held twice a year, and conference calls are made supplementarily and ad hoc in the event of an occurring crisis. In addition to professional exchange and emergency preparedness activities, STAKOB was involved in the provision of medical care to the three EVD cases evacuated to Germany in 2014 and in the response to Lassa Fever that occurred in Germany in 2015 (see case descriptions above). Particularities in HCID Mass Casualty Management: The Israeli Example Surge Capacity An HCID outbreak might rapidly develop andrequire management of mass casualties that may present to the hospitals. The influx of patients to the emergency departments (EDs) necessitates preparedness to expand surge capacities of the varied hospital facilities including the ED, intensive care units, inpatient departments, and laboratory capacities [ 71 , 72 ]. Nevertheless, compared to natural disasters or other types of emergency situations, an HCID outbreak presents different challenges, as the patients do not necessarily present to the emergency departments simultaneously and for a short period of time but rather do so over a prolonged time span [ 65 ]. The patients may present a high risk for transmitting the infection and thus pose a risk for spreading the disease to the personnel or other patients that are hospitalized. Therefore, those managing the event need to take into account unique elements of surge capacity such as designated infrastructure and equipment (i.e., isolation facilities), communication measures, and means for infection control [ 65 , 73 ]. It is recommended that establishment of a designated "biological ED" be considered, separate from the routine EDs, to facilitate safety measures and prevent potential infection of staff and other patients that present to the hospital concurrently. Regardless of the decision to operate a designated "biological ED" or admit patients in the routine ED, expansion of surge capacity of place, resources, infrastructure, and medical personnel should be instated to enable management of the numerous patients that are expected [ 72 , 74 ]. The overload of inpatients may necessitate application of modifications in the hospitalization policy, compared to routine management, to optimize resource management [ 75 , 76 ]. For example, the proportion of patients who require ventilation may rise to a level of 3–4 times the average number during routine periods, rendering the Intensive Care Units (ICUs) incapable of treating all the patients. Expansion of ventilation capacities is needed, based on designation of sites that consist of needed infrastructure for ventilation, such as in ambulatory surgical departments or step-down units. Elective and ambulatory activities may be reduced and, in extreme situations, may be ceased altogether. General hospitalization guidelines may be modified, such as restricting hospitalization periods after childbirth or surgeries, and may adopt altered standards of care [ 76 ]. The resources that will be made available resulting from the modified operations can then be redirected to the HCID mass casualty management. To overcome the shortage of hospital beds, additional facilities within the hospital may need to be involved as admitting or hospitalization sites. Hospital temporary wards may thus be installed in designated sites such as dining rooms, ambulatory clinics, and libraries or other areas that are sufficiently spacious to be converted for this purpose. Regardless, the scope of inpatients may exceed the overall capacities of the hospitals, necessitating operation of auxiliary facilities such as geriatric hospitals, nursing homes, community clinics, or even hotels, which may be temporarily used as extension hospital sites [ 77 , 78 ]. Triage Triage of patients is a crucial component of managing an HCID mass casualty event to prevent overload and facilitate an effective management of resources [ 79 ]. The main objective of triage processes is to facilitate saving as many salvageable patients as possible by identifying those that will survive as a result of the administered medical attention who would otherwise not live [ 79 , 80 ]. Triage during HCID events focuses on sorting those severely and critically ill patients who require immediate medical intervention from other casualties whose treatment may be delayed or administered in alternative facilities [ 81 ]. Another objective of triage in HCID situations is to use the available resources (i.e., manpower, equipment, or infrastructure) optimally to mitigate chaos and restore order, so as to resume routine operations and levels of medical care in the shortest time possible [ 82 ]. The triage process may represent a significant challenge to health care personnel as it is conducted under potentially stressful circumstances when many patients present to the hospital simultaneously. As the staff are required to work under biosafety precautions and regulations, deviations from the routine mode of operation pose a complex challenge. Modifications to the routine mode of patient admittance are instated, and the staff operate while wearing personal protective gear that may be perceived as uncomfortable or daunting [ 83 , 84 ]. A triage site must be defined to direct the patients to the appropriate ED. A senior internist or pediatrician (according to the designation of the site) should be appointed as Triage Medical Officer, responsible for managing the triage operations and directing each patient to the appropriate treatment site. Maintaining calm, adhering to principles of emergency response, and leading the initial sorting of the patients is a vital component of an effective emergency management of an HCID event [ 85 ]. Patients who are referred to the hospital by official agencies, such as the emergency medical services (EMS) or their primary physicians, should be directed immediately to the "biological ED"; patients who present to the hospital independently should be referred to the Biological Site according to the decision of the Triage Medical Officer. Initial treatment in the ED is targeted to provide urgent (life-saving) treatment and to decide whether the patient requires hospitalization or may be discharged to be cared for in the community [ 83 ]. The triage must be administered strictly; patients may be referred from the ED to hospitalization only following the decision of a senior physician. Therefore, hospitals need to ensure the ongoing presence of senior staff in the ED around the clock as long as the situation persists [ 71 ]. Deployment of a Hospital Incident Command Center As the significant influx of patients may overwhelm the hospital's capacities and to achieve optimal management of resources, an Incident Command Center needs to be established in an HCID epidemic [ 72 ]. The Incident Command Center is responsible for providing support to the medical teams to enable them to administer optimal medical care, despite the shortage in resources [ 71 ]. The main tasks of this Center are to update and direct the hospital's staff concerning the mode of operation, both clinically and organizationally; maintain ongoing contact with internal and external bodies; manage appropriate allocation of resources including manpower and equipment; monitor admission of patients; manage allocation of protective measures for the personnel and inpatients; and coordinate reinforcements of imaging and laboratory capacities. Isolation Facilities in the Mass Casualty Management Context Technical requirements to ensure capacity to treat patients with HCID have been defined and include components such as negative-pressure airborne infection isolation facilities and aerosol-tight doors and windows [ 86 , 87 ]. Although many countries have installed such measures that allow them safely to admit and treat patients infected with biological agents, only few of them have facilities that allow for a massive influx of such patients. It is therefore expected that the surge of patients presenting to the hospitals will significantly surpass the available isolation capacities [ 6 , 88 ]. Considering the high cost of installing isolation facilities that comply with the strict requirements of suitability to manage HCID events, it stands to reason that the current infrastructure will not change dramatically. Accordingly, a modified mode of operation is needed to facilitate effectively the management of a mass casualty HCID event while maintaining the safety of the staff and other inpatients of the hospital, such as designating areas in the hospital that will service exclusively HCID patients. Miller [ 87 ] have reported that it is possible to design a temporarily designated negative-pressure isolation ward and operate it in a separate pre-determined location within the hospital. In their study, though, this site was activated over a period of only 24 h, and they have expressed some doubts as to whether such a facility may also be functional during a longer span of time. Reinforcement of Resources Staff Reinforcement of hospital personnel should be recruited from two main sources: internal and external. Internal reinforcement should be based on a freeze of vacations, training and education programs, and participation in congresses or other activities external to the medical facility; modification/expansion of working shifts (following agreement with the staff and/or unions); and re-direction of personnel who were formerly assigned to elective or ambulatory services to the newly established wards [ 89 ]. External reinforcement may include medical or nursing students as well as recruitment of retired personnel who are still proficient in their basic profession. The hospital should identify areas/activities that may be performed by volunteers from the community and identify such potential volunteers who are available in the vicinity of the hospital. Those managing personnel must consider not only the number of staff needed at any point in time, over a prolonged period of days or weeks, but must also plan to cope with the fears and anxiety that often accompany outbreaks of HCID [ 65 ]. The severity of the disease, lack of effective treatments, and the potentially high transmissibility may create uncertainty among the staff concerning their safety and even distrust of the system at large and specifically the hospital administration [ 90 ]. Equipment As the routine medical equipment and devices may be insufficient in mass casualty events, each hospital needs to plan ways of reinforcing vital means, including ventilation machines and PPE [ 74 ]. As much as possible, electronic ventilators should be used, but because of the extreme numbers of patients which may require ventilation, manual ventilators may need to be used. According to the type of pathogen, appropriate filters will need to be installed in the ventilators. Plans for procurement of relevant medications should also be developed according to the estimated number of patients expected to present to the hospital. Nonetheless, under austere conditions, some procedures may not be provided considering the scarcity of resources; for example, it may be decided that extracorporeal membrane oxygenation will not be provided during an HCID mass casualty event, despite its effective use during the 2009 H1N1 influenza pandemic [ 71 ]. Decision making concerning types and amounts of equipment that should be purchased to maintain the capacity to manage an HCID needs to consider the risk, surge capacity, and costs of stockpiling and managing the equipment [ 74 ]. A significant challenge is balancing between the need to maintain continuous preparedness while minimizing waste and the need to destroy (and consequently substitute) drugs or other materials that have expired [ 71 , 74 ]. Training Training and education are crucial for ensuring the ability of medical professionals to manage HCID mass casualty events [ 91 ]. The knowledge required of hospital personnel during an HCID epidemic encompasses both familiarity with the approach to a single/individual patient and his/her contacts and the overall organizational preparedness for mass casualties, increase of encounters of medical/nursing staff with patients, and the need to provide medical care in ambulatory and modified hospital conditions [ 92 , 93 ]. Medical as well as organizational aptitude is required [ 77 , 93 ]. Three main levels have been recognized as significant for inclusion in training programs for HCID events: (1) information concerning the varied biological agents, their consequences, the courses of the disease, and means to prevent and/or mitigate its infectivity; (2) ways to develop case definitions, criteria for hospitalization, medical protocols for in-house and ambulatory care, and provision of care for patients' contacts (including staff and family members); (3) policy and decision-making processes [ 91 , 94 ]. All hospital staff should be trained in the basic level of required knowledge, medical personnel need also to be trained in the second level, while managers of the EDs and the various hospital departments need to be trained in all three levels of knowledge and competencies. HCIDs present a significant challenge to HCW because of potentially extreme working conditions, close encounters with infectious patients which may involve a substantial exposure to a lethal disease, preventive care that may be only partly effective, and high psychological pressure resulting from a dual commitment to the family and the workplace. Hospitals' training programs should accordingly focus not only on knowledge and competencies of the staff but also relate to the personnel's expected perceptions and attitudes [ 95 , 96 ]. Crisis Communication Risk communication is a vital component of managing a mass casualty HCID event, targeted to reduce uncertainty, confusion, and fear [ 87 , 97 ]. Reporting of hospital staff to work during HCID may be challenging, especially when they perceive a threat to their own or their families' well-being. Based on lessons identified during former HCID events, such as SARS, influenza, or EVD outbreaks, elements which impact on staff's willingness to report to work include perceived cohesiveness and sense of belonging to their team/place of work, and a feeling of a "mission" and commitment to patients and co-workers. Hospital administrators should communicate to the staff the criticality of their tasks, the perception of their contribution as vital for saving lives and for the continuous functioning of the medical facility, and its significant impact on medical consequences [ 98 ]. Risk communication should also include reference to actions that were taken to preserve their health and safety, such as preventive measures (physical protection, isolation facilities, infection control means, or preventive medications). Communicating messages during an HCID event must be based on relaying accurate and trustworthy information, conveyed calmly to avoid the creation of confusion and panic, and respectfully regarding the diversity prevailing among the population, including persons with special needs [ 65 , 99 ]. Both the medical teams and the public need to be updated continually concerning the HCID event, its development, and potential consequences, as a dynamic process, transparently and credibly [ 98 , 100 ]. The risk communication messages should be developed and published differentially according to the target population: the patients, medical personnel, family members of the staff or the patients, managers of interface agencies that operate in the vicinity of the hospital, decision and policy-makers, or the public at large [ 101 ]. Proactive risk communication should be adopted to achieve wide coverage. Clear, easily understandable information should be distributed concerning questions such as who should seek medical care, who needs to be vaccinated, who should approach the hospital or alternative facilities, or what means should be used for evacuation to hospitals (e.g., ambulances or private cars) [ 102 ]. Use of a tele-information center is recommended to provide the concerned public with answers to their worries. Leaflets delineating answers to most frequently asked questions should be prepared, translated into languages of the major fractions of the local population. This information should be uploaded to the hospitals' websites to be readily available to all interested parties. Proficient spokespersons should be available to relay information and updated data to local media and, through them, to the population. The spokespersons and the administrators of the hospitals should collaborate closely with other entities and authorities that operate in the community to coordinate the messaging and ensure a synchronized risk communication policy [ 87 , 102 ]. Reporting (Internal and External) Effective management of an HCID event is dependent on early identification of the first patients before the outbreak reaches uncontainable magnitudes; this can only be achieved by sharing information concerning patients or suspected contacts [ 103 , 104 ]. Once the outbreak has created mass casualties, the need for early detection and identification evolves into a vital need for sharing information concerning the patients, their differential diagnoses, and efficiency of treatments that were administered, in an attempt to contain the event and/or mitigate its effects [ 103 , 105 ]. Patient charts should also be maintained during an HCID, similar to routine procedures. Nonetheless, additional reporting is needed to manage the situation including Epidemiological Investigation Forms that delineate the patients' details, areas where they stayed during the event, tentative diagnosis, and treatment that was administered. The hospital needs to record and report the number of patients admitted to the EDs, the number of suspected patients infected with biological agents, the number of patients hospitalized in ICUs (adult and pediatric), and the number of deaths from HCID. Aggregation and analysis of unbiased data will facilitate their interpretation and subsequently their utilization for decision making concerning ways to manage the situation [ 105 ]. The analyzed data should be further disseminated and shared transparently to promote scientific collaboration and effective systemic management [ 103 ]. Risk Assessment and Risk Reduction Outbreaks of HCID which cause numerous casualties constitute a continuous global risk; as the consequences of such events may be devastating to humans and infrastructures, risk assessment and risk reduction must be intrinsic components of a holistic risk management program [ 106 , 107 ]. Managing mass casualty HCID events necessitates a robust organizational and preparedness program, including vigorous risk assessment and decision-making processes [ 108 ]. Risk assessment is vital for both planning and response phases to facilitate decision making and implementation of effective interventions [ 98 , 109 ]. As the health caregivers may be at risk of being infected, either primarily by the HCID agent or from secondary transmission of the pathogen by the patients they treat, each hospital must have the capacity to identify the risk, assess its potential damage, and implement measures to reduce the risk and ensure the safety of its manpower [ 99 ]. Risk reduction in HCID events is dependent on hospital personnel's strict adherence to infection control procedures [ 65 ]. If such measures are not implemented as needed, the hospitals themselves may contribute to spreading the pathogen rather than containing or controlling its evolvement. Previous studies have shown that during the SARS outbreak in both Canada and Taiwan, over 75% of the cases could be traced back to virus transmission within hospitals [ 110 ]. Ethical Dilemmas The goal of caring for patients is founded on the ethical objective to provide the optimal protection of lives, health, and welfare of the population in the most effective and successful manner. Ethical management considers presence of factors that may not be known or accurately estimated prior to the event [ 111 ], such as the biological features of the agent (lethality, epidemiology, infectivity, and sensitivity to antiviral treatments); characteristics of the population (density, scope of risk, and age distribution); and behavioral patterns of the population. Major ethical aspects which need to be considered include allocation of medical resources under austerity, limitation of civil rights, enforcing professional ethics of medical teams during an HCID event, competing commitments of health care providers to their place of work versus responsibility to their families and themselves, and integrating ethical as well as religious aspects in decision-making processes [ 111 , 112 ]. Bio-ethical professionals should be involved in the decision-making processes. Surge Capacity An HCID outbreak might rapidly develop andrequire management of mass casualties that may present to the hospitals. The influx of patients to the emergency departments (EDs) necessitates preparedness to expand surge capacities of the varied hospital facilities including the ED, intensive care units, inpatient departments, and laboratory capacities [ 71 , 72 ]. Nevertheless, compared to natural disasters or other types of emergency situations, an HCID outbreak presents different challenges, as the patients do not necessarily present to the emergency departments simultaneously and for a short period of time but rather do so over a prolonged time span [ 65 ]. The patients may present a high risk for transmitting the infection and thus pose a risk for spreading the disease to the personnel or other patients that are hospitalized. Therefore, those managing the event need to take into account unique elements of surge capacity such as designated infrastructure and equipment (i.e., isolation facilities), communication measures, and means for infection control [ 65 , 73 ]. It is recommended that establishment of a designated "biological ED" be considered, separate from the routine EDs, to facilitate safety measures and prevent potential infection of staff and other patients that present to the hospital concurrently. Regardless of the decision to operate a designated "biological ED" or admit patients in the routine ED, expansion of surge capacity of place, resources, infrastructure, and medical personnel should be instated to enable management of the numerous patients that are expected [ 72 , 74 ]. The overload of inpatients may necessitate application of modifications in the hospitalization policy, compared to routine management, to optimize resource management [ 75 , 76 ]. For example, the proportion of patients who require ventilation may rise to a level of 3–4 times the average number during routine periods, rendering the Intensive Care Units (ICUs) incapable of treating all the patients. Expansion of ventilation capacities is needed, based on designation of sites that consist of needed infrastructure for ventilation, such as in ambulatory surgical departments or step-down units. Elective and ambulatory activities may be reduced and, in extreme situations, may be ceased altogether. General hospitalization guidelines may be modified, such as restricting hospitalization periods after childbirth or surgeries, and may adopt altered standards of care [ 76 ]. The resources that will be made available resulting from the modified operations can then be redirected to the HCID mass casualty management. To overcome the shortage of hospital beds, additional facilities within the hospital may need to be involved as admitting or hospitalization sites. Hospital temporary wards may thus be installed in designated sites such as dining rooms, ambulatory clinics, and libraries or other areas that are sufficiently spacious to be converted for this purpose. Regardless, the scope of inpatients may exceed the overall capacities of the hospitals, necessitating operation of auxiliary facilities such as geriatric hospitals, nursing homes, community clinics, or even hotels, which may be temporarily used as extension hospital sites [ 77 , 78 ]. Triage Triage of patients is a crucial component of managing an HCID mass casualty event to prevent overload and facilitate an effective management of resources [ 79 ]. The main objective of triage processes is to facilitate saving as many salvageable patients as possible by identifying those that will survive as a result of the administered medical attention who would otherwise not live [ 79 , 80 ]. Triage during HCID events focuses on sorting those severely and critically ill patients who require immediate medical intervention from other casualties whose treatment may be delayed or administered in alternative facilities [ 81 ]. Another objective of triage in HCID situations is to use the available resources (i.e., manpower, equipment, or infrastructure) optimally to mitigate chaos and restore order, so as to resume routine operations and levels of medical care in the shortest time possible [ 82 ]. The triage process may represent a significant challenge to health care personnel as it is conducted under potentially stressful circumstances when many patients present to the hospital simultaneously. As the staff are required to work under biosafety precautions and regulations, deviations from the routine mode of operation pose a complex challenge. Modifications to the routine mode of patient admittance are instated, and the staff operate while wearing personal protective gear that may be perceived as uncomfortable or daunting [ 83 , 84 ]. A triage site must be defined to direct the patients to the appropriate ED. A senior internist or pediatrician (according to the designation of the site) should be appointed as Triage Medical Officer, responsible for managing the triage operations and directing each patient to the appropriate treatment site. Maintaining calm, adhering to principles of emergency response, and leading the initial sorting of the patients is a vital component of an effective emergency management of an HCID event [ 85 ]. Patients who are referred to the hospital by official agencies, such as the emergency medical services (EMS) or their primary physicians, should be directed immediately to the "biological ED"; patients who present to the hospital independently should be referred to the Biological Site according to the decision of the Triage Medical Officer. Initial treatment in the ED is targeted to provide urgent (life-saving) treatment and to decide whether the patient requires hospitalization or may be discharged to be cared for in the community [ 83 ]. The triage must be administered strictly; patients may be referred from the ED to hospitalization only following the decision of a senior physician. Therefore, hospitals need to ensure the ongoing presence of senior staff in the ED around the clock as long as the situation persists [ 71 ]. Deployment of a Hospital Incident Command Center As the significant influx of patients may overwhelm the hospital's capacities and to achieve optimal management of resources, an Incident Command Center needs to be established in an HCID epidemic [ 72 ]. The Incident Command Center is responsible for providing support to the medical teams to enable them to administer optimal medical care, despite the shortage in resources [ 71 ]. The main tasks of this Center are to update and direct the hospital's staff concerning the mode of operation, both clinically and organizationally; maintain ongoing contact with internal and external bodies; manage appropriate allocation of resources including manpower and equipment; monitor admission of patients; manage allocation of protective measures for the personnel and inpatients; and coordinate reinforcements of imaging and laboratory capacities. Isolation Facilities in the Mass Casualty Management Context Technical requirements to ensure capacity to treat patients with HCID have been defined and include components such as negative-pressure airborne infection isolation facilities and aerosol-tight doors and windows [ 86 , 87 ]. Although many countries have installed such measures that allow them safely to admit and treat patients infected with biological agents, only few of them have facilities that allow for a massive influx of such patients. It is therefore expected that the surge of patients presenting to the hospitals will significantly surpass the available isolation capacities [ 6 , 88 ]. Considering the high cost of installing isolation facilities that comply with the strict requirements of suitability to manage HCID events, it stands to reason that the current infrastructure will not change dramatically. Accordingly, a modified mode of operation is needed to facilitate effectively the management of a mass casualty HCID event while maintaining the safety of the staff and other inpatients of the hospital, such as designating areas in the hospital that will service exclusively HCID patients. Miller [ 87 ] have reported that it is possible to design a temporarily designated negative-pressure isolation ward and operate it in a separate pre-determined location within the hospital. In their study, though, this site was activated over a period of only 24 h, and they have expressed some doubts as to whether such a facility may also be functional during a longer span of time. Reinforcement of Resources Staff Reinforcement of hospital personnel should be recruited from two main sources: internal and external. Internal reinforcement should be based on a freeze of vacations, training and education programs, and participation in congresses or other activities external to the medical facility; modification/expansion of working shifts (following agreement with the staff and/or unions); and re-direction of personnel who were formerly assigned to elective or ambulatory services to the newly established wards [ 89 ]. External reinforcement may include medical or nursing students as well as recruitment of retired personnel who are still proficient in their basic profession. The hospital should identify areas/activities that may be performed by volunteers from the community and identify such potential volunteers who are available in the vicinity of the hospital. Those managing personnel must consider not only the number of staff needed at any point in time, over a prolonged period of days or weeks, but must also plan to cope with the fears and anxiety that often accompany outbreaks of HCID [ 65 ]. The severity of the disease, lack of effective treatments, and the potentially high transmissibility may create uncertainty among the staff concerning their safety and even distrust of the system at large and specifically the hospital administration [ 90 ]. Equipment As the routine medical equipment and devices may be insufficient in mass casualty events, each hospital needs to plan ways of reinforcing vital means, including ventilation machines and PPE [ 74 ]. As much as possible, electronic ventilators should be used, but because of the extreme numbers of patients which may require ventilation, manual ventilators may need to be used. According to the type of pathogen, appropriate filters will need to be installed in the ventilators. Plans for procurement of relevant medications should also be developed according to the estimated number of patients expected to present to the hospital. Nonetheless, under austere conditions, some procedures may not be provided considering the scarcity of resources; for example, it may be decided that extracorporeal membrane oxygenation will not be provided during an HCID mass casualty event, despite its effective use during the 2009 H1N1 influenza pandemic [ 71 ]. Decision making concerning types and amounts of equipment that should be purchased to maintain the capacity to manage an HCID needs to consider the risk, surge capacity, and costs of stockpiling and managing the equipment [ 74 ]. A significant challenge is balancing between the need to maintain continuous preparedness while minimizing waste and the need to destroy (and consequently substitute) drugs or other materials that have expired [ 71 , 74 ]. Training Training and education are crucial for ensuring the ability of medical professionals to manage HCID mass casualty events [ 91 ]. The knowledge required of hospital personnel during an HCID epidemic encompasses both familiarity with the approach to a single/individual patient and his/her contacts and the overall organizational preparedness for mass casualties, increase of encounters of medical/nursing staff with patients, and the need to provide medical care in ambulatory and modified hospital conditions [ 92 , 93 ]. Medical as well as organizational aptitude is required [ 77 , 93 ]. Three main levels have been recognized as significant for inclusion in training programs for HCID events: (1) information concerning the varied biological agents, their consequences, the courses of the disease, and means to prevent and/or mitigate its infectivity; (2) ways to develop case definitions, criteria for hospitalization, medical protocols for in-house and ambulatory care, and provision of care for patients' contacts (including staff and family members); (3) policy and decision-making processes [ 91 , 94 ]. All hospital staff should be trained in the basic level of required knowledge, medical personnel need also to be trained in the second level, while managers of the EDs and the various hospital departments need to be trained in all three levels of knowledge and competencies. HCIDs present a significant challenge to HCW because of potentially extreme working conditions, close encounters with infectious patients which may involve a substantial exposure to a lethal disease, preventive care that may be only partly effective, and high psychological pressure resulting from a dual commitment to the family and the workplace. Hospitals' training programs should accordingly focus not only on knowledge and competencies of the staff but also relate to the personnel's expected perceptions and attitudes [ 95 , 96 ]. Staff Reinforcement of hospital personnel should be recruited from two main sources: internal and external. Internal reinforcement should be based on a freeze of vacations, training and education programs, and participation in congresses or other activities external to the medical facility; modification/expansion of working shifts (following agreement with the staff and/or unions); and re-direction of personnel who were formerly assigned to elective or ambulatory services to the newly established wards [ 89 ]. External reinforcement may include medical or nursing students as well as recruitment of retired personnel who are still proficient in their basic profession. The hospital should identify areas/activities that may be performed by volunteers from the community and identify such potential volunteers who are available in the vicinity of the hospital. Those managing personnel must consider not only the number of staff needed at any point in time, over a prolonged period of days or weeks, but must also plan to cope with the fears and anxiety that often accompany outbreaks of HCID [ 65 ]. The severity of the disease, lack of effective treatments, and the potentially high transmissibility may create uncertainty among the staff concerning their safety and even distrust of the system at large and specifically the hospital administration [ 90 ]. Equipment As the routine medical equipment and devices may be insufficient in mass casualty events, each hospital needs to plan ways of reinforcing vital means, including ventilation machines and PPE [ 74 ]. As much as possible, electronic ventilators should be used, but because of the extreme numbers of patients which may require ventilation, manual ventilators may need to be used. According to the type of pathogen, appropriate filters will need to be installed in the ventilators. Plans for procurement of relevant medications should also be developed according to the estimated number of patients expected to present to the hospital. Nonetheless, under austere conditions, some procedures may not be provided considering the scarcity of resources; for example, it may be decided that extracorporeal membrane oxygenation will not be provided during an HCID mass casualty event, despite its effective use during the 2009 H1N1 influenza pandemic [ 71 ]. Decision making concerning types and amounts of equipment that should be purchased to maintain the capacity to manage an HCID needs to consider the risk, surge capacity, and costs of stockpiling and managing the equipment [ 74 ]. A significant challenge is balancing between the need to maintain continuous preparedness while minimizing waste and the need to destroy (and consequently substitute) drugs or other materials that have expired [ 71 , 74 ]. Training Training and education are crucial for ensuring the ability of medical professionals to manage HCID mass casualty events [ 91 ]. The knowledge required of hospital personnel during an HCID epidemic encompasses both familiarity with the approach to a single/individual patient and his/her contacts and the overall organizational preparedness for mass casualties, increase of encounters of medical/nursing staff with patients, and the need to provide medical care in ambulatory and modified hospital conditions [ 92 , 93 ]. Medical as well as organizational aptitude is required [ 77 , 93 ]. Three main levels have been recognized as significant for inclusion in training programs for HCID events: (1) information concerning the varied biological agents, their consequences, the courses of the disease, and means to prevent and/or mitigate its infectivity; (2) ways to develop case definitions, criteria for hospitalization, medical protocols for in-house and ambulatory care, and provision of care for patients' contacts (including staff and family members); (3) policy and decision-making processes [ 91 , 94 ]. All hospital staff should be trained in the basic level of required knowledge, medical personnel need also to be trained in the second level, while managers of the EDs and the various hospital departments need to be trained in all three levels of knowledge and competencies. HCIDs present a significant challenge to HCW because of potentially extreme working conditions, close encounters with infectious patients which may involve a substantial exposure to a lethal disease, preventive care that may be only partly effective, and high psychological pressure resulting from a dual commitment to the family and the workplace. Hospitals' training programs should accordingly focus not only on knowledge and competencies of the staff but also relate to the personnel's expected perceptions and attitudes [ 95 , 96 ]. Crisis Communication Risk communication is a vital component of managing a mass casualty HCID event, targeted to reduce uncertainty, confusion, and fear [ 87 , 97 ]. Reporting of hospital staff to work during HCID may be challenging, especially when they perceive a threat to their own or their families' well-being. Based on lessons identified during former HCID events, such as SARS, influenza, or EVD outbreaks, elements which impact on staff's willingness to report to work include perceived cohesiveness and sense of belonging to their team/place of work, and a feeling of a "mission" and commitment to patients and co-workers. Hospital administrators should communicate to the staff the criticality of their tasks, the perception of their contribution as vital for saving lives and for the continuous functioning of the medical facility, and its significant impact on medical consequences [ 98 ]. Risk communication should also include reference to actions that were taken to preserve their health and safety, such as preventive measures (physical protection, isolation facilities, infection control means, or preventive medications). Communicating messages during an HCID event must be based on relaying accurate and trustworthy information, conveyed calmly to avoid the creation of confusion and panic, and respectfully regarding the diversity prevailing among the population, including persons with special needs [ 65 , 99 ]. Both the medical teams and the public need to be updated continually concerning the HCID event, its development, and potential consequences, as a dynamic process, transparently and credibly [ 98 , 100 ]. The risk communication messages should be developed and published differentially according to the target population: the patients, medical personnel, family members of the staff or the patients, managers of interface agencies that operate in the vicinity of the hospital, decision and policy-makers, or the public at large [ 101 ]. Proactive risk communication should be adopted to achieve wide coverage. Clear, easily understandable information should be distributed concerning questions such as who should seek medical care, who needs to be vaccinated, who should approach the hospital or alternative facilities, or what means should be used for evacuation to hospitals (e.g., ambulances or private cars) [ 102 ]. Use of a tele-information center is recommended to provide the concerned public with answers to their worries. Leaflets delineating answers to most frequently asked questions should be prepared, translated into languages of the major fractions of the local population. This information should be uploaded to the hospitals' websites to be readily available to all interested parties. Proficient spokespersons should be available to relay information and updated data to local media and, through them, to the population. The spokespersons and the administrators of the hospitals should collaborate closely with other entities and authorities that operate in the community to coordinate the messaging and ensure a synchronized risk communication policy [ 87 , 102 ]. Reporting (Internal and External) Effective management of an HCID event is dependent on early identification of the first patients before the outbreak reaches uncontainable magnitudes; this can only be achieved by sharing information concerning patients or suspected contacts [ 103 , 104 ]. Once the outbreak has created mass casualties, the need for early detection and identification evolves into a vital need for sharing information concerning the patients, their differential diagnoses, and efficiency of treatments that were administered, in an attempt to contain the event and/or mitigate its effects [ 103 , 105 ]. Patient charts should also be maintained during an HCID, similar to routine procedures. Nonetheless, additional reporting is needed to manage the situation including Epidemiological Investigation Forms that delineate the patients' details, areas where they stayed during the event, tentative diagnosis, and treatment that was administered. The hospital needs to record and report the number of patients admitted to the EDs, the number of suspected patients infected with biological agents, the number of patients hospitalized in ICUs (adult and pediatric), and the number of deaths from HCID. Aggregation and analysis of unbiased data will facilitate their interpretation and subsequently their utilization for decision making concerning ways to manage the situation [ 105 ]. The analyzed data should be further disseminated and shared transparently to promote scientific collaboration and effective systemic management [ 103 ]. Risk Assessment and Risk Reduction Outbreaks of HCID which cause numerous casualties constitute a continuous global risk; as the consequences of such events may be devastating to humans and infrastructures, risk assessment and risk reduction must be intrinsic components of a holistic risk management program [ 106 , 107 ]. Managing mass casualty HCID events necessitates a robust organizational and preparedness program, including vigorous risk assessment and decision-making processes [ 108 ]. Risk assessment is vital for both planning and response phases to facilitate decision making and implementation of effective interventions [ 98 , 109 ]. As the health caregivers may be at risk of being infected, either primarily by the HCID agent or from secondary transmission of the pathogen by the patients they treat, each hospital must have the capacity to identify the risk, assess its potential damage, and implement measures to reduce the risk and ensure the safety of its manpower [ 99 ]. Risk reduction in HCID events is dependent on hospital personnel's strict adherence to infection control procedures [ 65 ]. If such measures are not implemented as needed, the hospitals themselves may contribute to spreading the pathogen rather than containing or controlling its evolvement. Previous studies have shown that during the SARS outbreak in both Canada and Taiwan, over 75% of the cases could be traced back to virus transmission within hospitals [ 110 ]. Ethical Dilemmas The goal of caring for patients is founded on the ethical objective to provide the optimal protection of lives, health, and welfare of the population in the most effective and successful manner. Ethical management considers presence of factors that may not be known or accurately estimated prior to the event [ 111 ], such as the biological features of the agent (lethality, epidemiology, infectivity, and sensitivity to antiviral treatments); characteristics of the population (density, scope of risk, and age distribution); and behavioral patterns of the population. Major ethical aspects which need to be considered include allocation of medical resources under austerity, limitation of civil rights, enforcing professional ethics of medical teams during an HCID event, competing commitments of health care providers to their place of work versus responsibility to their families and themselves, and integrating ethical as well as religious aspects in decision-making processes [ 111 , 112 ]. Bio-ethical professionals should be involved in the decision-making processes.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9845045/

Identification and Analysis of Potential Immune-Related Biomarkers in Endometriosis

Background Endometriosis is an inflammatory gynecological disease leading to deep pelvic pain, dyspareunia, and infertility. The pathophysiology of endometriosis is complex and depends on a variety of biological processes and pathways. Therefore, there is an urgent need to identify reliable biomarkers for early detection and accurate diagnosis to predict clinical outcomes and aid in the early intervention of endometriosis. We screened transcription factor- (TF-) immune-related gene (IRG) regulatory networks as potential biomarkers to reveal new molecular subgroups for the early diagnosis of endometriosis. Methods To explore potential therapeutic targets for endometriosis, the Gene Expression Omnibus (GEO), Immunology Database and Analysis Portal (ImmPort), and TF databases were used to obtain data related to the recognition of differentially expressed genes (DEGs), differentially expressed IRGs (DEIRGs), and differentially expressed TFs (DETFs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the DETFs and DEIRGs. Then, DETFs and DEIRGs were further validated in the external datasets of GSE51981 and GSE1230103. Then, we used quantitative real-time polymerase chain reaction (qRT-PCR) to verify the hub genes. Simultaneously, the Pearson correlation analysis and protein-protein interaction (PPI) analyses were used to indicate the potential mechanisms of TF-IRGs at the molecular level and obtain hub IRGs. Finally, the receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic value of the hub IRGs. Results We screened a total of 94 DETFs and 121 DEIRGs in endometriosis. Most downregulated DETFs showed decreased expression in the endometria of moderate/severe endometriosis patients. The top-ranked upregulated DEIRGs were upregulated in the endometra of infertile women. Functional analysis showed that DETFs and DEIRGs may be involved in the biological behaviors and pathways of endometriosis. The TF-IRG PPI network was successfully constructed. Compared with the control group, high C3, VCAM1, ITGB2, and C3AR1 expression had statistical significance in endometriosis among the hub DEIRGs. They also showed higher sensitivity and specificity by ROC analysis for the diagnosis of endometriosis. Finally, compared with controls, C3 and VCAM1 were highly expressed in endometriosis tissue samples. In addition, they also showed high specificity and sensitivity for diagnosing endometriosis. Conclusion Overall, we discovered the TF-IRG regulatory network and analyzed 4 hub IRGs that were closely related to endometriosis, which contributes to the diagnosis of endometriosis. Additionally, we verified that DETFs or DEIRGs were associated with the clinicopathological features of endometriosis, and external datasets also confirmed the hub IRGs. Finally, C3 and VCAM1 were highly expressed in endometriosis tissue samples compared with controls and may be potential biomarkers of endometriosis, which are helpful for the early diagnosis of endometriosis. Background Endometriosis is an inflammatory gynecological disease leading to deep pelvic pain, dyspareunia, and infertility. The pathophysiology of endometriosis is complex and depends on a variety of biological processes and pathways. Therefore, there is an urgent need to identify reliable biomarkers for early detection and accurate diagnosis to predict clinical outcomes and aid in the early intervention of endometriosis. We screened transcription factor- (TF-) immune-related gene (IRG) regulatory networks as potential biomarkers to reveal new molecular subgroups for the early diagnosis of endometriosis. Methods To explore potential therapeutic targets for endometriosis, the Gene Expression Omnibus (GEO), Immunology Database and Analysis Portal (ImmPort), and TF databases were used to obtain data related to the recognition of differentially expressed genes (DEGs), differentially expressed IRGs (DEIRGs), and differentially expressed TFs (DETFs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the DETFs and DEIRGs. Then, DETFs and DEIRGs were further validated in the external datasets of GSE51981 and GSE1230103. Then, we used quantitative real-time polymerase chain reaction (qRT-PCR) to verify the hub genes. Simultaneously, the Pearson correlation analysis and protein-protein interaction (PPI) analyses were used to indicate the potential mechanisms of TF-IRGs at the molecular level and obtain hub IRGs. Finally, the receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic value of the hub IRGs. Results We screened a total of 94 DETFs and 121 DEIRGs in endometriosis. Most downregulated DETFs showed decreased expression in the endometria of moderate/severe endometriosis patients. The top-ranked upregulated DEIRGs were upregulated in the endometra of infertile women. Functional analysis showed that DETFs and DEIRGs may be involved in the biological behaviors and pathways of endometriosis. The TF-IRG PPI network was successfully constructed. Compared with the control group, high C3, VCAM1, ITGB2, and C3AR1 expression had statistical significance in endometriosis among the hub DEIRGs. They also showed higher sensitivity and specificity by ROC analysis for the diagnosis of endometriosis. Finally, compared with controls, C3 and VCAM1 were highly expressed in endometriosis tissue samples. In addition, they also showed high specificity and sensitivity for diagnosing endometriosis. Conclusion Overall, we discovered the TF-IRG regulatory network and analyzed 4 hub IRGs that were closely related to endometriosis, which contributes to the diagnosis of endometriosis. Additionally, we verified that DETFs or DEIRGs were associated with the clinicopathological features of endometriosis, and external datasets also confirmed the hub IRGs. Finally, C3 and VCAM1 were highly expressed in endometriosis tissue samples compared with controls and may be potential biomarkers of endometriosis, which are helpful for the early diagnosis of endometriosis. 1. Introduction Endometriosis is an inflammatory gynecological disease characterized by the presence of endometrial tissues outside the uterus [ 1 ]. It affects approximately 10% of females in their reproductive years leading to a heavy financial burden on patients [ 2 ]. The typical clinical symptoms are chronic pelvic pain, dysmenorrhea, dyspareunia, and infertility, causing a decrease in patients' quality of life [ 3 ]. In addition, endometriosis surgery is the second most common surgery in premenopausal women. The occurrence and development of endometriosis are usually slow and are related to the local inflammatory response, proliferation, invasion, and angiogenesis of endometrial cells [ 4 ]. There are many theories about the etiology of endometriosis, but no exact theory can explain the pathogenesis of endometriosis [ 5 ]. Among the theories, the most prevalent is Sampson's theory of retrograde menstruation proposed in 1921. However, there are many arguments against this theory [ 6 ]. Because 90% of women have menstrual reflux, only 10% of women suffer from endometriosis. Although our understanding of endometriosis is growing, the exact molecular mechanisms underlying this tumor-like disease are still far from being understood. The pathophysiology of the occurrence and development of endometriosis is complex and depends on a variety of biological processes and pathways [ 3 ]. Therefore, there is an urgent need to determine reliable diagnostic biomarkers to predict early diagnosis and clinical severity. The immune system plays a major role in survival in the pelvic microenvironment, including causing immune tolerance, depressing immunosurveillance, and escaping phagocytosis by immune cells [ 7 ]. Previous studies have indicated that immune-related genes (IRGs) play an important role in the complex regulatory network of tumors [ 8 ], and they have been explored to indicate the development of tumor immunity and the pathophysiological mechanisms of tumors [ 9 ], such as ovarian cancer. Emerging evidence has shown that women with endometriosis not only have a changed immune status of the endometrium but also have an altered peripheral immune system [ 10 ]. Consistent with the changes in the peritoneal environment of endometriosis, a large number of immune cells, inflammatory factors, and relevant cytokines have also been recruited to contribute to the abnormal immune environment in endometriosis [ 11 , 12 ]. Nevertheless, the relationship between IRGs and the diagnosis of endometriosis patients is not clear, and further research is needed. This study is aimed at exploring the potential correlation between the onset of endometriosis and IRGs, which are potential molecular biomarkers to reveal new molecular subgroups for the early diagnosis of endometriosis. Some transcription factors (TFs) are closely related to IRGs and can also regulate the function of IRGs in some diseases. Aberrant TF-IRGs could influence the various processes of tumor development. Additionally, the differential expression of TFs and their downstream target genes has been found to be related to the progression of endometriosis. Previous studies have shown that IRGs act as important regulators in diverse pathological processes. Therefore, studying the role of IRGs and their related molecular mechanisms in endometriosis is crucial, which is beneficial for exploring the pathogenesis of endometriosis and detecting more effective potential diagnostic markers. 2. Materials and Methods 2.1. Preparation and Processing of TF and IRG Data in Endometriosis We searched two online TF datasets and downloaded 1665 TFs from the Human Transcription Factor Database (Human TFDB) [ 13 ] and 1639 TFs from the Human Transcription Factors Database [ 14 ]. The 1508 TFs obtained by the intersection of these two TF databases were used in our research on endometriosis. We constructed a diagnostic signature from the IRGs downloaded from the Immunology Database and Analysis Portal (ImmPort) database ( http://www.immport.org ) [ 15 ]. We used the Gene Expression Omnibus (GEO) database ( https://www.ncbi.nlm.nih.gov/geo/ ) to analyze gene expression datasets. A total of 1871 series of endometriosis were retrieved from the database. We selected five GEO datasets (GSE7305, GSE7307, GSE51981, GSE1230103, and GSE23339) after filtering. We matched the gene symbols of the data with the corresponding GEO platforms (GPL). In total, 10 endometriosis cases and 10 control samples were obtained from GSE7305, while 23 endometriosis patients' specimens and 18 control specimens were acquired from GSE7307. Both two expression microarrays were based on the GPL570 ((HG-U133_Plus_2) Affymetrix Human Genome U133 Plus 2.0 Array) platform. Moreover, GSE51981 and GSE120103, based on the GPL570 ((HG-U133_Plus_2) Affymetrix Human Genome U133 Plus 2.0 Array) and GPL6480 (Agilent-014850 Whole Huma Genome Microarray 4x44K G4112F) platform, respectively, were chosen for further validation. All of the data are freely available online. 2.2. Identification of DETFs and DEIRGs in Endometriosis We selected two GEO gene expression datasets (GSE7305 and GSE7307) and divided the above data into the endometriosis group and the control group. First, the differentially expressed genes (DEGs) between the endometriosis and control samples were identified using the GEO2R online analysis tool ( https://www.ncbi.nlm.nih.gov/geo/geo2r/ ), and the genes met the cutoff criteria based on the criteria of P 0.9. Subsequently, the visualized PPI network was constructed by Cytoscape software (version 3.7.1). The Hmisc R package (version 4.4.2) ( https://hbiostat.org/R/doc/sintro.pdf ) was utilized to test the correlations between DETFs and DEIRGs with the cutoff criteria set as correlation coefficient > 0.5 and P 0.9. Subsequently, the visualized PPI network was constructed by Cytoscape software (version 3.7.1). The Hmisc R package (version 4.4.2) ( https://hbiostat.org/R/doc/sintro.pdf ) was utilized to test the correlations between DETFs and DEIRGs with the cutoff criteria set as correlation coefficient > 0.5 and P < 0.001. The Molecular Complex Detection (MCODE) plugin of Cytoscape software was utilized to recognize the most prominent clustering modules. Functional enrichment analysis of the genes in individual modules was achieved by DAVID, an online tool ( https://david.ncifcrf.gov/ ), with a significance threshold of P < 0.05. Nodes with higher connectivity tend to be more important for maintaining the stability of the entire network. Therefore, cytoHubba, a plugin in Cytoscape, was used to screen out hub genes. 2.5. Collection of Human Tissues Ectopic endometrium tissues were collected from chocolate cyst in endometriosis to identify hub genes expression ( n = 12). Endometrium tissue from patients undergoing surgery for uterine fibroids served as a control group ( n = 12). All patients in our study with or without endometriosis had no menstrual disorders. Patients who had received hormone therapy or other serious diseases were not included in this study. All tissue samples obtained were approved by the Ethics Commission of Harbin Medical University (202106). 2.6. Reverse Transcription and Quantitative Real-Time Polymerase Chain Reaction 12 endometriosis samples and 12 controls were frozen in liquid nitrogen, and total RNA was extracted using the TRIzol ® reagent (15596026, America). The relative expression of VCAM1, ITGB2, C3AR1, and C3 mRNA was normalized to GAPDH, and calculated using the 2 −ΔΔCt method (ΔCt = Ct targetgene − Ct internalcontrol ). The total RNA was used only if the A260/280 ratio of the absorbances was between 1.8 and 2.2 when measured by spectrophotometry. Reverse transcription was performed at 42°C (15 min) followed by 95°C (3 min), then in a 10 ml SYBR reaction system using the Talent qPCR PreMix (FP209-02, China) with 1 cycle of 95°C for 3 minutes, and 40 cycles of 95°C for 5 seconds and 60°C for 15 seconds. We identified the target mRNA sequences with ideal melting curves and sizes. Sequences of the primers are shown in Table 1 . 2.7. Statistical Analysis The method used to compare DETF expression in different severity groups of endometriosis was unpaired Student's t test. At the same time, the comparison method between the expression of DEIRGs in infertile and fertile endometriosis was unpaired Student's t test. The pROC R package (version 1.18.0) [ 18 ] was used to evaluate the sensitivity and specificity of DETFs and DEIRGs in the diagnosis of endometriosis. P < 0.05 was considered statistically significant in our study. 3. Results 3.1. Identification of DETFs and DEIRGs in Endometriosis We obtained 1508 intersecting TFs from Human TFDB and the Human Transcription Factors database ( Figure 1(a) ). Subsequently, we chose gene expression datasets from the GEO datasets. GSE7305 and GSE7307 were selected to identify DEGs because both included the endometrial samples with or without endometriosis. Based on the criteria of P < 0.05 and |log2FC| ≥1 [ 13 ], a total of 1141 DEGs from GSE7305 and GSE7307 were acquired by the GEO2R analysis tool, including 525 upregulated genes and 616 downregulated genes (Figures 1(b) and 1(c) ). The DEGs were visualized by volcano plots in GSE7305 and GSE7307 (Figures 2(a) and 2(b) ). Then, the results were intersected with 1508 TFs, identifying the 94 DETFs (35 upregulated DETFs and 59 downregulated DETFs) (Figures 2(c) and 2(d) ). Similarly, 1793 unique IRGs were downloaded from the ImmPort database, and 111 DEIRGs (80 upregulated genes and 31 downregulated genes) were obtained from the intersection of the IRGs and the DEGs (Figures 2(e) and 2(f) ). The DETFs and DEIRGs were visualized by a heatmap in GSE7305 and GSE7307, and there was a clear division between the endometriosis and control groups (Figures 3 – 6 ). 3.2. Validation of the DETFs in Different Severity Groups of Endometriosis To verify the credibility and applicability of the DETFs, we selected the external dataset GSE51981, which contained endometriosis samples with different severities. We obtained the 40 most highly expressed DETFs (14 upregulated DETFs and 26 downregulated DETFs) in 10 randomly selected samples from the minimal/mild group and moderate/severe group in GSE7305 and GSE7307. As shown in Figure 7(a) , we found that a series of upregulated DETFs were still highly expressed in the moderate/severe group, and these upregulated DETFs could predict the severity of endometriosis by receiver operating characteristic (ROC) curve analysis ( P < 0.05) ( Figure 8(a) ). Downregulated DETFs had a more pronounced advantage in predicting endometriosis severity than upregulated DETFs, and most of the downregulated DETFs remained expressed at low levels in the moderate/severe group ( P < 0.05) ( Figure 7(b) ). In addition, the ROC curve provided powerful evidence to support this view, with area under the curve (AUC) values all over 0.7( Figure 8(b) ). 3.3. The Expression of DEIRGs in Women with Endometriosis with or without Infertility The GSE120103 dataset was chosen for subsequent validation because it included infertile and fertile females with endometriosis, and we obtained the top 40 DEIRGs expressed in it. Interestingly, most of the upregulated DEIRGs were increased in infertile females with endometriosis, while some downregulated DEIRGs were expressed at low levels in the endometria of infertile women ( P < 0.05) (Figures 9(a) and 9(b) ). For this result, we conducted ROC analysis to evaluate the values of DEIRGs in the diagnosis of endometriosis, and the AUC also verified the sensitivity and specificity of DEIRGs with P < 0.05 (Figures 10(a) and 10(b) ). 3.4. Functional Enrichment Analysis of DETFs and DEIRGs To indicate the biological properties of DETFs and DEIRGs, functional analysis was performed, including GO functional and KEGG encrichment analyses. The enriched GO terms were divided into BP, CC, and MF ontologies. The GO functional enrichment results of the DETFs were mainly enriched in the BP ontology. BP analysis showed that the DETFs were significantly enriched in reproductive structure or system development, cell fate commitment, and anterior/posterior pattern specification. For the cell component, the DETFs were enriched in the transcription regulator complex and nuclear speck RNA polymerase II transcription regulator complex. The MF ontology of DETFs was mainly related to ligand-activated transcription factor activity, RNA polymerase II-specific DNA-binding transcription factor binding, and nuclear receptor binding or activity ( Figure 11(a) ). For the KEGG analysis, the DETFs were mainly enriched in the signaling pathways associated with transcriptional misregulation in cancer and the Notch signaling pathway ( Figure 11(b) ). Likewise, the DEIRGs were also enriched in the regulation chemotaxis, lipase or phospholipase activity, cytoplasmic vesicle lumen, external side of plasma membrane, cytokine activity, and nuclear receptor activity ( Figure 11(c) ). The KEGG pathways of the DEIRGs were mainly enriched in viral protein interactions with cytokines and cytokine receptors, the PI3K-Akt signaling pathway, and the MAPK signaling pathway ( Figure 11(d) ). 3.5. PPI Network Construction and Pearson's Correlation Analysis Protein interactions between the DETFs and DEIRGs were constructed using the STRING online database, and the PPI network was constructed using Cytoscape. Five subnetworks were recognized. Therefore, we obtained TF-IRG regulatory networks containing 44 nodes and 73 edges to accurately illustrate the regulatory relationships between the DETFs and DEIRGs ( Figure 12(a) ). Pearson's correlation analysis was used to analyze the TF-IRG regulatory network, and most nodes were correlated with other nodes at the expression level with P < 0.001 ( Figure 12(b) ). The MCODE plugin of Cytoscape was used to complement the module analysis, with the corresponding modules shown in Figure 13 . Furthermore, the most significantly enriched functional modules were those related to complement and coagulation cascades, Staphylococcus aureus infection, proteoglycans in cancer, focal adhesion, and the Rap1 signaling pathway ( Table 2 ). Subsequently, we used the cytoHubba plugin of Cytoscape to identify hub genes according to the three most important topological features in network analysis, including degree, betweenness, and closeness. We then ranked the top ten nodes for each set of the three different topological measurements ( Table 3 ). As a result, we obtained five nodes (CXCL2, C3, VCAM1, ITGB2, and C3AR1) in all three of the lists ( Figure 12(c) ). These five DEIRGs can therefore be considered hub genes in the regulatory network. 3.6. Identification and Validation of Hub IRGs As shown in GSE7305 and GSE7307, the expression of each hub IRG was significantly higher in the endometriosis group than in the control group in the box plot ( P < 0.05) (Figures 14(a) and 14(b) ). The GSE23339 dataset (GPL6102 Illumina human-6 v2.0 expression beadchip) and the publicly accessible ENDOMET Turku Endometriosis Database were also used to verify the DEIRGs (Figures 14(c) and 14(d) ). However, in the additional database validation of GSE23339, CXCL2 was not statistically significant. Thus, four hub genes were obtained (C3, VCAM1, ITGB2, and C3AR1). In view of the above results, we verified the above four target genes by qRT-PCR, and the highly expressed C3 and VCAM1 were statistically significant in endometriosis, and the AUCs were 0.96 and 0.76 ( Figure 14(e) ). However, ITGB2 and C3AR1 were not statistically significant (Figure S1 ). The AUCs calculated from ROC analysis in GSE7305 ( Figure 15(a) ), GSE7307 ( Figure 15(b) ), and GSE23339 ( Figure 15(c) ) were used to evaluate the diagnostic value of endometriosis. The AUC values of all four hub DEIRGs were over 80%, which meant that the hub DEIRGs played a critical role as novel biomarkers for endometriosis. 3.1. Identification of DETFs and DEIRGs in Endometriosis We obtained 1508 intersecting TFs from Human TFDB and the Human Transcription Factors database ( Figure 1(a) ). Subsequently, we chose gene expression datasets from the GEO datasets. GSE7305 and GSE7307 were selected to identify DEGs because both included the endometrial samples with or without endometriosis. Based on the criteria of P < 0.05 and |log2FC| ≥1 [ 13 ], a total of 1141 DEGs from GSE7305 and GSE7307 were acquired by the GEO2R analysis tool, including 525 upregulated genes and 616 downregulated genes (Figures 1(b) and 1(c) ). The DEGs were visualized by volcano plots in GSE7305 and GSE7307 (Figures 2(a) and 2(b) ). Then, the results were intersected with 1508 TFs, identifying the 94 DETFs (35 upregulated DETFs and 59 downregulated DETFs) (Figures 2(c) and 2(d) ). Similarly, 1793 unique IRGs were downloaded from the ImmPort database, and 111 DEIRGs (80 upregulated genes and 31 downregulated genes) were obtained from the intersection of the IRGs and the DEGs (Figures 2(e) and 2(f) ). The DETFs and DEIRGs were visualized by a heatmap in GSE7305 and GSE7307, and there was a clear division between the endometriosis and control groups (Figures 3 – 6 ). 3.2. Validation of the DETFs in Different Severity Groups of Endometriosis To verify the credibility and applicability of the DETFs, we selected the external dataset GSE51981, which contained endometriosis samples with different severities. We obtained the 40 most highly expressed DETFs (14 upregulated DETFs and 26 downregulated DETFs) in 10 randomly selected samples from the minimal/mild group and moderate/severe group in GSE7305 and GSE7307. As shown in Figure 7(a) , we found that a series of upregulated DETFs were still highly expressed in the moderate/severe group, and these upregulated DETFs could predict the severity of endometriosis by receiver operating characteristic (ROC) curve analysis ( P < 0.05) ( Figure 8(a) ). Downregulated DETFs had a more pronounced advantage in predicting endometriosis severity than upregulated DETFs, and most of the downregulated DETFs remained expressed at low levels in the moderate/severe group ( P < 0.05) ( Figure 7(b) ). In addition, the ROC curve provided powerful evidence to support this view, with area under the curve (AUC) values all over 0.7( Figure 8(b) ). 3.3. The Expression of DEIRGs in Women with Endometriosis with or without Infertility The GSE120103 dataset was chosen for subsequent validation because it included infertile and fertile females with endometriosis, and we obtained the top 40 DEIRGs expressed in it. Interestingly, most of the upregulated DEIRGs were increased in infertile females with endometriosis, while some downregulated DEIRGs were expressed at low levels in the endometria of infertile women ( P < 0.05) (Figures 9(a) and 9(b) ). For this result, we conducted ROC analysis to evaluate the values of DEIRGs in the diagnosis of endometriosis, and the AUC also verified the sensitivity and specificity of DEIRGs with P < 0.05 (Figures 10(a) and 10(b) ). 3.4. Functional Enrichment Analysis of DETFs and DEIRGs To indicate the biological properties of DETFs and DEIRGs, functional analysis was performed, including GO functional and KEGG encrichment analyses. The enriched GO terms were divided into BP, CC, and MF ontologies. The GO functional enrichment results of the DETFs were mainly enriched in the BP ontology. BP analysis showed that the DETFs were significantly enriched in reproductive structure or system development, cell fate commitment, and anterior/posterior pattern specification. For the cell component, the DETFs were enriched in the transcription regulator complex and nuclear speck RNA polymerase II transcription regulator complex. The MF ontology of DETFs was mainly related to ligand-activated transcription factor activity, RNA polymerase II-specific DNA-binding transcription factor binding, and nuclear receptor binding or activity ( Figure 11(a) ). For the KEGG analysis, the DETFs were mainly enriched in the signaling pathways associated with transcriptional misregulation in cancer and the Notch signaling pathway ( Figure 11(b) ). Likewise, the DEIRGs were also enriched in the regulation chemotaxis, lipase or phospholipase activity, cytoplasmic vesicle lumen, external side of plasma membrane, cytokine activity, and nuclear receptor activity ( Figure 11(c) ). The KEGG pathways of the DEIRGs were mainly enriched in viral protein interactions with cytokines and cytokine receptors, the PI3K-Akt signaling pathway, and the MAPK signaling pathway ( Figure 11(d) ). 3.5. PPI Network Construction and Pearson's Correlation Analysis Protein interactions between the DETFs and DEIRGs were constructed using the STRING online database, and the PPI network was constructed using Cytoscape. Five subnetworks were recognized. Therefore, we obtained TF-IRG regulatory networks containing 44 nodes and 73 edges to accurately illustrate the regulatory relationships between the DETFs and DEIRGs ( Figure 12(a) ). Pearson's correlation analysis was used to analyze the TF-IRG regulatory network, and most nodes were correlated with other nodes at the expression level with P < 0.001 ( Figure 12(b) ). The MCODE plugin of Cytoscape was used to complement the module analysis, with the corresponding modules shown in Figure 13 . Furthermore, the most significantly enriched functional modules were those related to complement and coagulation cascades, Staphylococcus aureus infection, proteoglycans in cancer, focal adhesion, and the Rap1 signaling pathway ( Table 2 ). Subsequently, we used the cytoHubba plugin of Cytoscape to identify hub genes according to the three most important topological features in network analysis, including degree, betweenness, and closeness. We then ranked the top ten nodes for each set of the three different topological measurements ( Table 3 ). As a result, we obtained five nodes (CXCL2, C3, VCAM1, ITGB2, and C3AR1) in all three of the lists ( Figure 12(c) ). These five DEIRGs can therefore be considered hub genes in the regulatory network. 3.6. Identification and Validation of Hub IRGs As shown in GSE7305 and GSE7307, the expression of each hub IRG was significantly higher in the endometriosis group than in the control group in the box plot ( P < 0.05) (Figures 14(a) and 14(b) ). The GSE23339 dataset (GPL6102 Illumina human-6 v2.0 expression beadchip) and the publicly accessible ENDOMET Turku Endometriosis Database were also used to verify the DEIRGs (Figures 14(c) and 14(d) ). However, in the additional database validation of GSE23339, CXCL2 was not statistically significant. Thus, four hub genes were obtained (C3, VCAM1, ITGB2, and C3AR1). In view of the above results, we verified the above four target genes by qRT-PCR, and the highly expressed C3 and VCAM1 were statistically significant in endometriosis, and the AUCs were 0.96 and 0.76 ( Figure 14(e) ). However, ITGB2 and C3AR1 were not statistically significant (Figure S1 ). The AUCs calculated from ROC analysis in GSE7305 ( Figure 15(a) ), GSE7307 ( Figure 15(b) ), and GSE23339 ( Figure 15(c) ) were used to evaluate the diagnostic value of endometriosis. The AUC values of all four hub DEIRGs were over 80%, which meant that the hub DEIRGs played a critical role as novel biomarkers for endometriosis. 4. Discussion Endometriosis is a common benign gynecological disorder characterized by immunity, inflammation, and hormone dependence. Previous studies on endometriosis have mainly focused on TFs [ 19 ] or IRGs [ 20 ], and there have been few studies on TF-related IRGs in endometriosis. TF-related IRGs not only function in immunity regulation but can also be used as prognostic biomarkers and play a key role in the development of cancer [ 21 ]. The exact molecular mechanisms of endometriosis are still unclear, and the current treatments are limited. Therefore, the discovery of new therapeutic targets and potential diagnostic biomarkers remains a research focus. With the rapid development of the high-throughput methods and data analysis of various databases, Bohler et al. have focused on bioinformatics analysis, which can also serve as the basis for molecular biology experiments of endometriosis [ 22 ]. This study mainly analyzed DETFs and DEIRGs in endometriosis by bioinformatics methods and analyzed the expression of DETFs and DEIRGs in the endometria of women with different disease severities and infertility statuses. In addition, the enrichment analysis and networks were performed and constructed on DETFs and DEIRGs to discover valuable TFs, IRGs, and related endometriosis pathways. Among patients with different severities of endometriosis, upregulated DETFs were more highly expressed in patients with moderate/severe endometriosis. However, downregulated DETFs presented the opposite trend. We believe that this phenomenon may be related to the degree of macrophage infiltration of different severities of endometriosis. Compared with the r-AFS stage I-II of endometriosis, the proportion of M2 macrophages in stage III-IV endometriosis was higher, suggesting that the degree of M2 macrophage infiltration was related to the severity of endometriosis [ 23 ]. M2 macrophages have anti-inflammatory properties, promote wound healing, promote fibrosis, and enable the immune escape of ectopic endometrium [ 24 ]. In addition, M2 macrophages produce matrix metalloproteinases (MMPs), such as MMP 9, which promote the ectopic growth and progression of endometrial cells by degrading the extracellular matrix and enhancing intercellular adhesion [ 25 , 26 ]. At the same time, the results of DETF enrichment analysis also suggested that they may affect the activation of macrophages in endometriosis, such as the Notch pathway, which was consistent with the findings of previous research [ 27 ]. In fertile or infertile women with endometriosis, most of the upregulated DEIRGs were also highly expressed in infertile women, whereas only 4 downregulated DEIRGs were significantly expressed at a low level in the infertile group. Immunity, inflammation, and DEGs have important implications in infertile patients. These factors may affect the expression of IRGs, and that they and IRGs coregulate to influence susceptibility in patients with endometriosis-related infertility. A previous study found that BDNF (Met) single-nucleotide polymorphism, an IRG, was associated with endometriosis-related infertility women, suggesting that low levels of BDNF may be responsible for poor in vitro fertilization (IVF) outcomes in infertile patients with the BDNF (Met/Met) genotype [ 28 ]. Yin et al. studied another IRG, PTX3, which is also associated with endometriosis-related infertility [ 29 ]. In our study, IGF1 had lower expression levels in infertile women with endometriosis, and it had the ability to attenuate oocyte and embryo development resulting in endometriotic infertility, as reported in the study of Ding et al. [ 30 ]. At the same time, ESR1 showed low expression, and related studies suggested that ESR1 can affect the possibility of pregnancy in infertile patients with endometriosis [ 31 ]. In this study, the GO and KEGG enrichment analyses showed that the IRGs were mainly related to immune-related functions and pathways, such as the external side of the plasma membrane, cytokine activity and nuclear receptor activity, the PI3K-Akt signaling pathway, and the MAPK signaling pathway. In general, the external side of the plasma membrane plays an important role in endometriosis immunity. Antigens derived from the plasma membrane might directly assay reactive autoantibodies to indicate the immunoreactivity of endometriosis severity [ 32 ]. Cytokine activity, such as proinflammatory cytokines (IL-1 β , IL-6) and tumor growth factor-beta (TNF- β ), plays an important role in evading immune surveillance and predicting the disease severity of endometriosis [ 33 , 34 ]. In addition, upregulation of MAPK subfamilies promoted the occurrence of endometriosis by influencing the function of various cytokines, including IL-6 and IL-8 [ 35 ]. The PI3K-Akt and MAPK pathways are interconnected with each other [ 36 , 37 ]. The activation of the PI3K-Akt signaling pathway and the ERK-related intracellular MAPK signaling pathway was correlated with endometriosis [ 36 ], and both were shown to be involved in the immunity [ 38 ]. In addition, our research further identified the involvement of IRGs in the regulation of the PI3K-Akt signaling pathway and MAPK signaling pathway in endometriosis. A previous study showed that upregulation of the adaptor protein SHC1 had the ability to activate the PI3K-Akt and/or MAPK pathways in endometriosis samples [ 39 ]. The activation of the PI3K-Akt and MAPK pathways was associated with the immune-related pathway, nuclear factor- κ B (NF- κ B) signaling pathway in endometriosis cells [ 40 ]. Therefore, the study of IRGs in endometriosis is essential. These findings shed light on the screening of new potential biomarkers and the early diagnosis of endometriosis. A previous study reported a TF-targeted gene network indicating the onset of endometriosis [ 41 ]. To further investigate the possible underlying molecular regulatory mechanisms, a TF-IRG network was constructed to study the mechanism of endometriosis. A total of 39 IRGs (RND3, PLK2, AURKA, RCAN1, EZH2, etc.) were selected to analyze the TF-IRG PPI network. Five DETFs (IRF6, EGR1, FOSB, JUNB, and MECOM) were connected with several IRGs. The genes in the PPI network were closely linked and cross regulated with each other. For instance, in the multifunctional network, IRF6/BST2 was involved in the regulation of immunity. Currently, there are many related studies on IRF6 and BST2 in immunity. Aberrant DNA methylation of IRF6 and BST2 in CD4+ T cells induced autoimmune responses [ 42 ]. Meanwhile, Figure 13 shows the predicted binding sites of IRF6 and BST2, which suggests that IRF6 and BST2 may function through mutual regulation. In a human papillomavirus type 16 (HPV16) study of host immunity, inhibition of IRF6 was responsible for immune escape from HPV16 blocking IL-1 β secretion [ 43 ]. In this study, we found that BST2/CD317 in combination with TLR agonists specifically presented Ag by plasmacytoid dendritic cells in vivo, which contributed to the strong cellular and humoral immune responses [ 44 ]. However, the detailed mechanism of the PPI network should be elucidated in the future. Our findings provide an informatics basis for future research in this direction. In this research, we mainly aimed to construct an IRG-related diagnostic model, which was established based on DEGs. ROC analysis revealed that four IRGs can be used as potential biomarkers of endometriosis, which also demonstrated the feasibility in terms of the AUC, a signal for endometriosis occurrence. Recently, C3 was considered a candidate diagnostic biomarker of endometrosis, and its expression was correlated with the engraftment of the endometriotic cysts [ 45 ]. The overexpression of VCAM-1 on the peritoneum of endometriosis had been revealed by Schutt et al. [ 46 ]. The increased expression of ITGB2 had been previously reported in endometriosis tissues compared with normal tissues [ 47 ], and high C3AR1 expression might be used as a diagnostic factor for the endometriosis-associated malignant phenotype [ 5 ]. In this research, two hub DEIRGs (C3 and VCAM1) with diagnostic value were obtained. However, this research had some limitations. First, the applicability of the diagnostic model needs to be validated in a larger sample population in future studies. Second, we will continue to complete the molecular mechanism study on the role of IRGs in endometriosis. 5. Conclusion The TF-IRG network could be used to present novel prospective molecular mechanisms underlying the development of tumors [ 45 ]. However, studies of the regulatory mechanisms underlying TFs and IRGs in endometriosis are still in progress. In our study, IRGs were used to construct a diagnostic model to predict the onset of endometriosis patients by bioinformatics analysis. ROC analysis confirmed that the diagnostic value of hub genes (C3 and VCAM1) was clinically feasible. Additionally, the TF-IRG regulatory network broadened the horizon for research concerning the pathogenesis of endometriosis. Data Availability All of the data we used in this study were publicly available as described in the methods section and can be found in online Github page: https://github.com/zgm19661026/zgm19661026.git . Ethical Approval This research has been conducted using publicly available datasets, and no ethical approval was required. All tissue samples obtained were approved by the Ethics Commission of Harbin Medical University (202106). Conflicts of Interest The authors declare that they have no competing interests. Authors' Contributions GMZ designed all the study. YNH and JXL performed the data processing and experimental analysis. YJQ, LYS, XBZ, and HW drafted the manuscript. All authors reviewed and approved the final version of the manuscript. Yanan He and Jixin Li contributed equally to this work. Supplementary Materials Supplementary Materials Figure S1: the differences in the mRNA expression levels of ITGB2 and C3AR1 by qRT-PCR between the endometriosis tissues and the controls. (A) ITGB2. (B) C3AR1. Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041481/

Considering Protonation as a Post-translational Modification Regulating Protein Structure and Function

Post-translational modification of proteins is an evolutionarily conserved mechanism for regulating activity, binding affinities and stability. Compared with established post-translational modifications such as phosphorylation or uniquitination, post-translational modification by protons within physiological pH ranges is a less recognized mechanism for regulating protein function. By changing the charge of amino acid side chains, post-translational modification by protons can drive dynamical changes in protein conformation and function. Addition and removal of a proton is rapid and reversible and in contrast to most other post-translational modifications does not require an enzyme. Signaling specificity is achieved by only a minority of sites in proteins titrating within the physiological pH range. Here, we examine the structural mechanisms and functional consequences of proton post-translational modification of pH-sensing proteins regulating different cellular processes.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3253120/

A Novel Strategy for Development of Recombinant Antitoxin Therapeutics Tested in a Mouse Botulism Model

Antitoxins are needed that can be produced economically with improved safety and shelf life compared to conventional antisera-based therapeutics. Here we report a practical strategy for development of simple antitoxin therapeutics with substantial advantages over currently available treatments. The therapeutic strategy employs a single recombinant 'targeting agent' that binds a toxin at two unique sites and a 'clearing Ab' that binds two epitopes present on each targeting agent. Co-administration of the targeting agent and the clearing Ab results in decoration of the toxin with up to four Abs to promote accelerated clearance. The therapeutic strategy was applied to two Botulinum neurotoxin (BoNT) serotypes and protected mice from lethality in two different intoxication models with an efficacy equivalent to conventional antitoxin serum. Targeting agents were a single recombinant protein consisting of a heterodimer of two camelid anti-BoNT heavy-chain-only Ab V H (VHH) binding domains and two E-tag epitopes. The clearing mAb was an anti-E-tag mAb. By comparing the in vivo efficacy of treatments that employed neutralizing vs. non-neutralizing agents or the presence vs. absence of clearing Ab permitted unprecedented insight into the roles of toxin neutralization and clearance in antitoxin efficacy. Surprisingly, when a post-intoxication treatment model was used, a toxin-neutralizing heterodimer agent fully protected mice from intoxication even in the absence of clearing Ab. Thus a single, easy-to-produce recombinant protein was as efficacious as polyclonal antiserum in a clinically-relevant mouse model of botulism. This strategy should have widespread application in antitoxin development and other therapies in which neutralization and/or accelerated clearance of a serum biomolecule can offer therapeutic benefit. Introduction The presence of toxins in circulation is the cause of a wide variety of human and animal illnesses. Antitoxins are therapeutic agents that reduce further development of symptoms in patients that have been exposed to a toxin. Typically, antitoxins are the antisera obtained from large animals that were immunized with inactivated toxin [1] , [2] . More recently, some antitoxin therapies have been developed using one or more antitoxin mAbs [3] , [4] , [5] , [6] . Antisera and mAbs can be difficult to produce economically at scale, usually require long development times and often have problematic quality control, shelf-life and safety issues. New therapeutic strategies to develop and prepare antitoxins are needed. Antitoxins function through two key mechanisms; neutralization of toxin function and clearance of toxin from the body. Toxin neutralization can occur through processes such as inhibition of enzymatic activity and prevention of binding to cellular receptors. Antibody mediated clearance from serum is thought to occur subsequent to the binding of multiple antibodies to the target antigen [7] , [8] , [9] , [10] . Multimeric antibody decoration of the target is considered necessary to permit binding to low affinity Fc receptors [8] , [10] . An ideal antitoxin therapeutic will both promote toxin neutralization to immediately block further toxin activity and accelerate toxin clearance to eliminate future pathology if neutralization becomes reversed. Clostridium botulinum neurotoxin (BoNT) is a National Institute of Allergy and Infectious Diseases (NIAID) Category A priority pathogen which can cause botulism, a potentially lethal flaccid paralysis. Currently, the only treatments for botulism are antitoxins. Polyclonal antitoxin sera are available to treat infants (BabyBIG [11] ) or adults (HBAT [12] ) that become exposed to BoNT and these can prevent further development of paralysis. Once serious paralysis has occurred, though, palliative care is the only available option [13] . Some laboratories are working to develop monoclonal antibodies (mAbs) as possible antitoxin alternatives to polyclonal antisera [3] , [14] , [15] , [16] , [17] . Nowakowski et al [3] found that effective protection of mice against high dose challenge of BoNT serotype A (BoNT/A) required co-administration of three antitoxin mAbs, presumably to promote clearance. We previously demonstrated that administration of a pool of three or more small binding agents, each produced with a common epitopic tag, dramatically reduced serum levels of a toxin when co-administered with an anti-tag mAb [18] . The tagged binding agents directed the binding of anti-tag mAb to multiple sites on the toxin, thus indirectly decorating the toxin with Ab Fc domains and leading to its clearance through the liver. The use of small binding agents to direct the decoration of toxin with Ab permits new strategies for the development of agents with improved commercial properties. One binding agent scaffold with excellent properties is the camelid heavy-chain-only Ab V H (VHH) domain. VHHs are small (∼12 kD), easy to produce, and generally more stable than conventional antibody fragments [19] , [20] . They are often found to have unusual epitope specificities, particularly an improved ability to bind active site pockets to produce enzyme inhibition [21] . Because of the many favourable properties of VHHs, they have become widely used in research and show clear commercial potential [22] , [23] . Here we show that a single recombinant heterodimeric binding agent consisting of two high-affinity BoNT binding VHH agents and two epitopic tags, co-administered with an anti-tag mAb, protected mice from lethality with an efficacy equivalent to conventional BoNT antitoxin serum in two different in vivo assays. Studies comparing neutralizing or non-neutralizing binding agents administered with or without clearing Ab provide a unique method for evaluating the relative contributions of toxin neutralization and toxin clearance to antitoxin efficacy. We show that toxin neutralization and toxin clearance both contribute significantly to antitoxin efficacy in mice. Using the heterodimer antitoxin strategy, toxin neutralization or toxin clearance alone proved to be sufficient to protect mice from BoNT intoxication in a therapeutically relevant, post-intoxication assay. Results Identification and characterization of anti-BoNT VHHs Serum clearance of the protein, Botulinum neurotoxin serotype A (BoNT/A), can be dramatically accelerated by administering a pool of different epitopically-tagged single-chain Ig variable fragment (scFv) domain binding agents together with an anti-tag mAb [18] . To determine whether similar results could be obtained using a more commercially and clinically acceptable binding agent, a panel of camelid heavy-chain-only Vh (VHH) binding agents were obtained having high affinity for BoNT/A holotoxin. In addition, VHHs were obtained that bind to BoNT serotype B (BoNT/B) holotoxin to permit efficacy testing on a second pathogenic serum protein. Competition ELISAs were used to identify the VHHs with the highest apparent affinity for unique epitopes on BoNT/A and BoNT/B leading to the selection of seven BoNT/A VHHs ( Figure S1A ) and four BoNT/B VHHs ( Figure S1B ). Each VHH was purified from E. coli as a thioredoxin fusion protein containing a single carboxyl-terminal epitopic tag (E-tag) (SDS-PAGE shown in Figure S2A ). The seven unique BoNT/A binding VHHs were further characterized for their target affinity by surface plasmon resonance (SPR) and their ability to prevent intoxication of primary neurons in culture ( Table 1 ; Figure 1 ). All VHHs displayed good affinity for their toxin targets with Kd99.99%) of BoNT/A when co-administered to a mouse. Recombinant antitoxin efficacy in a clinically relevant post-intoxication assay Assays in which varying doses of toxins are co-administered with antitoxin agents permit sensitive quantification of antitoxin efficacy but do not accurately reflect the typical clinical situation. To test antitoxin agents in a more clinically relevant assay, mice were administered with 10 LD 50 of BoNT/A by intraperitoneal administration and, at various times later, administered intravenously with test agents. As a positive control, we used a potent sheep anti-BoNT/A serum at a dose previously demonstrated to protect 100% of mice from lethality when co-administered with 10,000 LD 50 of BoNT/A (not shown). Two different anti-BoNT/A VHH heterodimers were tested; the non-neutralizing ciA-F12/D12(2E) heterodimer ( Figure 5A ) and the neutralizing ciA-H7/B5(2E) heterodimer ( Figure 5B ). Each heterodimer contained two copies of E-tag and was tested both with and without the anti-E-tag clearing Ab. The non-neutralizing heterodimer had little or no antitoxin efficacy in the absence of clearing Ab, yet when in the presence of this agent it displayed an efficacy nearly equivalent to the positive control sheep antiserum. These results show that toxin clearance alone is sufficient to protect mice from a low dose BoNT challenge (10 LD 50 ), even when the agents are administered several hours post-intoxication. 10.1371/journal.pone.0029941.g005 Figure 5 BoNT/A intoxication is prevented by administering a single anti-BoNT/A VHH heterodimer in a clinically-relevant post-intoxication mouse model. A 10 LD 50 dose of toxin was administered by intraperitoneal injection either 1.5 or 3 hours prior to intravenous administration of antitoxin agents as indicated. Symptoms of BoNT/A intoxication and lethality were monitored following post-intoxication administration of double-tagged heterodimers of neutralizing or non-neutralizing anti-BoNT/A VHHs+/−clearing Ab. The time to death is plotted as % survival as a function of time following administration of toxin. Time to death for mice given no agents or the positive control sheep antitoxin are also shown. ( A ) The double-tagged heterodimer consisted of two non-neutralizing VHHs, ciA-F12 and ciA-D12 (F12/D12(2E)) and was administered with or without anti-E-tag clearing mAb (αE) as indicated. ( B ) The double-tagged heterodimer consisted of two BoNT/A neutralizing VHHs, ciA-H7 and ciA-B5 (H7/B5(2E)) and was administered with or without anti-E-tag clearing mAb (αE) as indicated. Surprisingly, the neutralizing heterodimer was highly effective as an antitoxin in this assay whether or not clearing Ab was included ( Figure 5B ). The double-tagged toxin neutralizing heterodimer possessed an antitoxin efficacy equivalent to polyclonal antitoxin even when administered in the absence of anti-tag clearing Ab. These data strongly suggest that BoNT neutralization is sufficient for full antitoxin efficacy when tested in a clinically relevant post-intoxication assay with low dose toxin challenge. The results show that a single protein composed simply of two toxin-neutralizing VHHs has the potential to be as effective as antitoxin sera in clinical situations. Antitoxin efficacy of a double-tagged heterodimer targeting BoNT/B The use of double-tagged heterodimer antitoxins was extended to a different toxin target by using VHHs recognizing unique epitopes on BoNT/B holotoxin ( Figure S1B ). Two of the VHHs, ciB-A11 and B5, were the most potent in vivo in monomer pool studies (not shown) and were selected for expression as a double-tagged heterodimer (ciB-A11/B5(2E)) (sequence in Figure S1E ). This agent fully protected mice against 1000 LD 50 of BoNT/B in the presence of clearing Ab ( Figure 6A ). In the clinically relevant post-intoxication assay, ciB-A11/B5(2E) was only partially effective in the absence of clearing Ab indicating that the heterodimer is not potent at toxin neutralization. Furthermore, the affinity of this heterodimer for BoNT/B (Kd∼5 nM, Table 1 ) was weaker than the affinity of either of the two component monomers (Kd∼1 nM each) suggesting that the recombinant heterodimer was not fully functional. Despite this, when the heterodimer was administered with clearing Ab, the treatment was at least as effective as sheep anti-BoNT/B polyclonal antiserum in preventing BoNT/B lethality ( Figure 6B ). These results demonstrate the efficacy of the heterodimer VHH antitoxin strategy for a second BoNT serotype and suggest the strategy will be effective for treating exposure to the other BoNT serotypes as well as to other pathogenic biomolecules that may occur in patient serum. 10.1371/journal.pone.0029941.g006 Figure 6 BoNT/B intoxication is prevented by heterodimer antitoxin agents in two models of BoNT/B intoxication in mice. Protection of mice from BoNT/B lethality by administration of a double-tagged heterodimer of anti-BoNT/B VHHs ciB-A11 and ciB-B5 (A11/B5(2E))+/−clearing Ab. Time to death is plotted as % survival as a function of time. An asterisk indicates that mice did not display any symptoms of intoxication. The results shown are combined from two replicate studies. ( A ) Groups of mice were co-administered the indicated LD 50 dose of BoNT/B together with no agents or the A11/B5(2E) heterodimer VHH with or without anti-E-tag clearing Ab (αE). ( B ) Groups of mice were administered a 10 LD 50 dose of BoNT/B and three hours later administered no agents, sheep anti-BoNT/B antiserum or the A11/B5(2E) heterodimer VHH with or without anti-E-tag clearing Ab (αE). Identification and characterization of anti-BoNT VHHs Serum clearance of the protein, Botulinum neurotoxin serotype A (BoNT/A), can be dramatically accelerated by administering a pool of different epitopically-tagged single-chain Ig variable fragment (scFv) domain binding agents together with an anti-tag mAb [18] . To determine whether similar results could be obtained using a more commercially and clinically acceptable binding agent, a panel of camelid heavy-chain-only Vh (VHH) binding agents were obtained having high affinity for BoNT/A holotoxin. In addition, VHHs were obtained that bind to BoNT serotype B (BoNT/B) holotoxin to permit efficacy testing on a second pathogenic serum protein. Competition ELISAs were used to identify the VHHs with the highest apparent affinity for unique epitopes on BoNT/A and BoNT/B leading to the selection of seven BoNT/A VHHs ( Figure S1A ) and four BoNT/B VHHs ( Figure S1B ). Each VHH was purified from E. coli as a thioredoxin fusion protein containing a single carboxyl-terminal epitopic tag (E-tag) (SDS-PAGE shown in Figure S2A ). The seven unique BoNT/A binding VHHs were further characterized for their target affinity by surface plasmon resonance (SPR) and their ability to prevent intoxication of primary neurons in culture ( Table 1 ; Figure 1 ). All VHHs displayed good affinity for their toxin targets with Kd99.99%) of BoNT/A when co-administered to a mouse. Recombinant antitoxin efficacy in a clinically relevant post-intoxication assay Assays in which varying doses of toxins are co-administered with antitoxin agents permit sensitive quantification of antitoxin efficacy but do not accurately reflect the typical clinical situation. To test antitoxin agents in a more clinically relevant assay, mice were administered with 10 LD 50 of BoNT/A by intraperitoneal administration and, at various times later, administered intravenously with test agents. As a positive control, we used a potent sheep anti-BoNT/A serum at a dose previously demonstrated to protect 100% of mice from lethality when co-administered with 10,000 LD 50 of BoNT/A (not shown). Two different anti-BoNT/A VHH heterodimers were tested; the non-neutralizing ciA-F12/D12(2E) heterodimer ( Figure 5A ) and the neutralizing ciA-H7/B5(2E) heterodimer ( Figure 5B ). Each heterodimer contained two copies of E-tag and was tested both with and without the anti-E-tag clearing Ab. The non-neutralizing heterodimer had little or no antitoxin efficacy in the absence of clearing Ab, yet when in the presence of this agent it displayed an efficacy nearly equivalent to the positive control sheep antiserum. These results show that toxin clearance alone is sufficient to protect mice from a low dose BoNT challenge (10 LD 50 ), even when the agents are administered several hours post-intoxication. 10.1371/journal.pone.0029941.g005 Figure 5 BoNT/A intoxication is prevented by administering a single anti-BoNT/A VHH heterodimer in a clinically-relevant post-intoxication mouse model. A 10 LD 50 dose of toxin was administered by intraperitoneal injection either 1.5 or 3 hours prior to intravenous administration of antitoxin agents as indicated. Symptoms of BoNT/A intoxication and lethality were monitored following post-intoxication administration of double-tagged heterodimers of neutralizing or non-neutralizing anti-BoNT/A VHHs+/−clearing Ab. The time to death is plotted as % survival as a function of time following administration of toxin. Time to death for mice given no agents or the positive control sheep antitoxin are also shown. ( A ) The double-tagged heterodimer consisted of two non-neutralizing VHHs, ciA-F12 and ciA-D12 (F12/D12(2E)) and was administered with or without anti-E-tag clearing mAb (αE) as indicated. ( B ) The double-tagged heterodimer consisted of two BoNT/A neutralizing VHHs, ciA-H7 and ciA-B5 (H7/B5(2E)) and was administered with or without anti-E-tag clearing mAb (αE) as indicated. Surprisingly, the neutralizing heterodimer was highly effective as an antitoxin in this assay whether or not clearing Ab was included ( Figure 5B ). The double-tagged toxin neutralizing heterodimer possessed an antitoxin efficacy equivalent to polyclonal antitoxin even when administered in the absence of anti-tag clearing Ab. These data strongly suggest that BoNT neutralization is sufficient for full antitoxin efficacy when tested in a clinically relevant post-intoxication assay with low dose toxin challenge. The results show that a single protein composed simply of two toxin-neutralizing VHHs has the potential to be as effective as antitoxin sera in clinical situations. Antitoxin efficacy of a double-tagged heterodimer targeting BoNT/B The use of double-tagged heterodimer antitoxins was extended to a different toxin target by using VHHs recognizing unique epitopes on BoNT/B holotoxin ( Figure S1B ). Two of the VHHs, ciB-A11 and B5, were the most potent in vivo in monomer pool studies (not shown) and were selected for expression as a double-tagged heterodimer (ciB-A11/B5(2E)) (sequence in Figure S1E ). This agent fully protected mice against 1000 LD 50 of BoNT/B in the presence of clearing Ab ( Figure 6A ). In the clinically relevant post-intoxication assay, ciB-A11/B5(2E) was only partially effective in the absence of clearing Ab indicating that the heterodimer is not potent at toxin neutralization. Furthermore, the affinity of this heterodimer for BoNT/B (Kd∼5 nM, Table 1 ) was weaker than the affinity of either of the two component monomers (Kd∼1 nM each) suggesting that the recombinant heterodimer was not fully functional. Despite this, when the heterodimer was administered with clearing Ab, the treatment was at least as effective as sheep anti-BoNT/B polyclonal antiserum in preventing BoNT/B lethality ( Figure 6B ). These results demonstrate the efficacy of the heterodimer VHH antitoxin strategy for a second BoNT serotype and suggest the strategy will be effective for treating exposure to the other BoNT serotypes as well as to other pathogenic biomolecules that may occur in patient serum. 10.1371/journal.pone.0029941.g006 Figure 6 BoNT/B intoxication is prevented by heterodimer antitoxin agents in two models of BoNT/B intoxication in mice. Protection of mice from BoNT/B lethality by administration of a double-tagged heterodimer of anti-BoNT/B VHHs ciB-A11 and ciB-B5 (A11/B5(2E))+/−clearing Ab. Time to death is plotted as % survival as a function of time. An asterisk indicates that mice did not display any symptoms of intoxication. The results shown are combined from two replicate studies. ( A ) Groups of mice were co-administered the indicated LD 50 dose of BoNT/B together with no agents or the A11/B5(2E) heterodimer VHH with or without anti-E-tag clearing Ab (αE). ( B ) Groups of mice were administered a 10 LD 50 dose of BoNT/B and three hours later administered no agents, sheep anti-BoNT/B antiserum or the A11/B5(2E) heterodimer VHH with or without anti-E-tag clearing Ab (αE). Discussion This manuscript reports a new approach to the development of antitoxins that employs a single recombinant protein (double-tagged VHH heterodimer) to promote toxin decoration with multiple copies of a single monoclonal antibody (anti-tag mAb) leading to its neutralization and clearance from the body. The approach should have general applicability for clinical situations in which neutralization and/or clearance of a circulating pathogenic biomolecule will result in therapeutic benefit. Earlier studies had shown that a pool of scFv domain binding agents with specificity for BoNT/A, each containing a common epitopic tag, could direct the decoration of the toxin with multiple anti-tag Abs leading to its clearance via the liver with an efficacy in mouse assays equivalent to conventional polyclonal antitoxin sera [18] . The scFvs served as the toxin targeting agents and the anti-tag mAb served as the clearing agent. Here we show that camelid VHH binding domains, with multiple commercial advantages over scFvs [22] , [23] , also serve effectively as the toxin targeting agents. One important advantage of VHHs is the ability to express these agents as heterodimers in which each VHH remains fully functional. This makes possible the fusion of two VHHs that bind to different epitopes on the same toxin target. Incorporation of two epitope tags on the heterodimer permits decoration of the toxin with two clearing Abs at each epitope, a total of four mAbs on the toxin to promote efficient toxin clearance. Since each double-tagged heterodimeric binding agent binds to only two mAbs, the agent itself should not be effectively cleared by low affinity Fc receptors unless bound to toxin. The heterodimers also proved to have much greater apparent affinity for toxin than the equivalent pool of two monomers and this likely contributed to the substantial gains in antitoxin efficacy achieved with the heterodimer antitoxins in mouse models of BoNT intoxication. The ability of antitoxin antibodies to protect animals from the symptoms of toxin exposure can be influenced by several factors. The dose of antitoxin agent and the timing of antitoxin administration relative to exposure to toxin are obvious factors that influence efficacy. In addition, the affinity of the antibodies for the toxin will influence the ability of the antibody to bind (k on ) and remain bound (k off ) to the toxin and exert its effect. The further ability of the antibody to inhibit the enzymatic activity of the toxin and/or prevent its entry into target cells (i.e. neutralization) also must be expected to play a key role. Finally, the ability of the antibodies to promote the clearance of the toxin from the serum will permit the antitoxin to reduce the pool of toxin in circulation. Little is known about the relative importance neutralization vs clearance mechanisms to antitoxin efficacy. In this study, insight into antitoxin mechanism was possible since experiments were performed that separately tested the roles of toxin neutralization and clearance in determining antitoxin efficacy. The role of neutralization could be assessed by comparing the efficacy of VHHs that are non-neutralizing with toxin neutralizing agents. The role of clearance could be analyzed by comparing antitoxin efficacy of identical non-neutralizing VHH agents in the presence or absence of the anti-tag clearing antibody. The results clearly demonstrate that agents possessing potent BoNT neutralizing activity can be very effective in mouse intoxication models in the absence of clearance. Similarly, agents that have little or no antitoxin efficacy in the absence of clearing Ab can become highly effective simply by co-administration of clearing Ab to promote toxin clearance. Furthermore, our results indicate that combining neutralization and clearance is essential for maximal antitoxin efficacy when the toxin doses are very high. Interestingly, when low BoNT doses were employed, such as the 10 LD 50 dose used in the clinically-relevant post-intoxication model, toxin clearance or neutralization alone were each sufficient and about equally effective. Presumably this occurs because clearance or neutralization alone are each capable of sufficiently reducing the level of active BoNT when the antitoxin is administered during the 'window of opportunity' [25] that exists between exposure and the onset of irreversible symptoms. At much higher BoNT doses, both toxin clearance and neutralization appear necessary to permit survival. The toxins employed in this study were Botulinum neurotoxins (BoNTs) and additional studies will be necessary to assess the extent to which the heterodimer binding agent antitoxin strategy described here will prove efficacious for other toxins. BoNTs are extremely potent with exquisite specificity for neurons and normally remain in circulation until they enter a neuron or are naturally cleared. Because of the high potency of BoNT, measurable toxicity occurs with extremely small amounts of circulating toxin. With less potent toxins, intoxication requires higher doses of toxin and thus higher concentrations of Ab are required for antitoxin efficacy. For toxins with less target cell specificity, such as those from Clostridium difficile and Escherichia coli or ricin, the toxins are likely to spend shorter time in circulation and, in these cases, toxin neutralization may be more important to efficacy than toxin clearance. Where the toxins are active on cells with low affinity Fc receptors involved in toxin clearance, antitoxins that promote clearance may not be of benefit. For these reasons, it is difficult to predict whether the most effective heterodimer antitoxin strategy should promote toxin clearance or focus entirely on toxin neutralization. One concern with the use of heterodimer binding agents is the possibility that the binding agents will be immunogenic and elicit an immune response that reduces or eliminates the therapeutic efficacy. VHHs are not considered to be strongly immunogenic and the immunogenicity can by reduced further by introducing targeted mutations [26] . Alternative non-Ab binding agents such as DARPins, Anticalins or AdNectins [27] should be able to replace VHHs if sufficient target affinity can be achieved and these are specifically designed to be poor immunogens. Some immunogenicity may be tolerable and may even improve efficacy by promoting target clearance. Studies reported here have demonstrated that a single heterodimer protein composed of two distinct toxin neutralizing VHHs has efficacy equivalent to polyclonal antitoxin serum in a clinically-relevant post-intoxication BoNT lethality assay. The ability to prevent intoxication with a single polypeptide substantially simplifies the commercial production of the antitoxin and makes possible genetic delivery approaches such as with DNA or viral vectors. Improved therapeutic efficacy is possible by promoting the clearance of the pathogenic biomolecule target and this can be achieved by producing the heterodimer with two copies of an epitopic tag and co-administering the agent with an anti-tag clearing mAb. This results in the decoration of the target with up to four mAbs which leads to clearance presumably by a low affinity FcR-dependent pathway. The clearing mAb itself could be made unnecessary by producing the heterodimer fused to a peptide or VHH that binds to low-affinity FcR. Using these varied strategies, it should be possible to develop new and versatile therapeutic approaches that permit the neutralization and/or clearance of one or more targeted pathogenic biomolecules from the circulation of patients. Materials and Methods Ethics statement All studies were carried out in strict accordance with the recommendations delineated in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The procedures used were approved by the Tufts University Institutional Animal Care and Use Committee (IACUC) and were performed under Protocols #G2010-60 and G874-07. Toxins and reagents Botulinum neurotoxin serotype A1 (BoNT/A) and serotype B (BoNT/B) were obtained from Metabiologics Inc. Each batch of toxin was assayed to establish LD 50 dose. BoNT complex was used for animal studies and BoNT holotoxin was used for the cell-based studies. Purified recombinant BoNT serotype A1 and B holotoxins containing mutations rendering them catalytically inactive (ciBoNTA, ciBoNTB) were produced as previously described [28] . Sheep anti-BoNT/A1 antiserum was produced by immunization of sheep with BoNT/A1 toxoid followed by BoNT/A1 holotoxin. Less than 1 µl of this sheep antitoxin serum protects mice from lethality when co-administered with 10,000 LD 50 of BoNT/A1. Reagents for Western blotting were purchased from KPL. Antibodies used were rabbit anti-SNAP25 antibody (Sigma); goat anti-rabbit HRP antiserum (Sigma); anti-E-tag mAb (Phadia); HRP anti-E-tag mAb (GE Healthcare). All studies with holotoxin were performed within a Select Agent laboratory registered with the CDC. Alpaca immunization and VHH-display library preparation Two alpacas were immunized with ciBoNTA and two with ciBoNTB. The immunization regimen employed 100 µg of protein in the primary immunization and 50 µg in three subsequent boosting immunizations at about 3 weekly intervals in alum/CpG adjuvant. Five days following the final boost, blood was obtained for lymphocyte preparation and VHH-display phage libraries were prepared from the immunized alpacas as previously described [29] , [30] . More than 10 6 independent clones were prepared from B cells of alpacas successfully immunized with each of the BoNT immunogens. Anti-BoNT VHH identification and preparation The VHH-display phage libraries were panned for binding to ciBoNTA or ciBoNTB targets that were coated onto a well of a 12 well plate. Coating was performed by overnight incubation with one ml of a 5 µg/ml target solution in PBS at 4°C followed by washing with PBS and 2 hrs incubation at 37°C with blocking agent (4% non-fat dried milk powder in PBS). Panning, phage recovery and clone fingerprinting were performed as previously described [29] , [30] . A total of 192 and 142 VHH clones were identified as strong positives for binding to BoNT/A and BoNT/B respectively based on phage ELISA signals. Of the strong positives, 62 unique DNA fingerprints were identified among the VHHs selected for binding to BoNT/A and 32 for VHHs selected for binding to BoNT/B. DNA sequences of the VHH coding regions was obtained for each phage clone and compared for homologies. Based on this analysis, 12 of the anti-BoNT/A VHHs and 11 anti-BoNT/B were identified as unlikely to have common B cell clonal origins and selected for protein expression. Expression and purification of VHHs in E. coli as recombinant thioredoxin (Trx) fusion proteins containing hexahistidine was performed as previously described [30] . For heterodimers, DNA encoding two different VHHs were joined in frame downstream of Trx and separated by DNA encoding a 15 amino acid flexible spacer ((GGGGS) 3 ). All VHHs were expressed with a carboxyl terminal E-tag epitope. Some expression constructions were engineered to contain a second copy of the E-tag by introducing the coding DNA in frame between the Trx and VHH domains. An example of a Trx fusion to a VHH heterodimer with two E-tags is shown in Figure S1C . A third E-tag was introduced in frame within the DNA encoding the flexible spacer of the heterodimer containing ciA-D12 and ciA-F12 to create a triple tagged heterodimer (D12/F12(3E)). VHH target binding competition analysis Phage displaying individual VHHs were prepared and titered by phage dilution ELISA [29] for recognition of ciBoNTA or ciBoNTB using HRP/anti-M13 Ab for detection. A dilution was selected for each phage preparation that produced a signal near the top of the linear range of the ELISA signal. The selected phage dilution (100 µl) for each VHH-displayed phage preparation were added to a 96 well plate that has been coated with ciBoNTA or ciBoNTB and then pre-incubated for 30 minutes with 100 µl of a 10 µg/ml solution containing a purified Trx/VHH fusion protein test agent or control in PBS. After an hour, the wells were washed and phage binding was detected as above. Test VHHs that reduced target binding of phage-displayed VHHs by less than two fold vs controls were considered to recognize distinct epitopes. Positive controls were performed in which the Trx/VHH competitor contained the same VHH as displayed on phage and typically reduced the ELISA signal >95%. Characterization of VHH binding properties VHHs were tested for binding to native or atoxic mutant BoNT holotoxins by standard ELISA using plates coated with 100 µl of 1 µg/ml protein. VHHs were also tested for recognition of BoNT subunits by a dilution series ELISA (10 µg/ml, 1∶5 dilutions) using plates coated by overnight incubation at 4°C with 5 µg/ml purified recombinant BoNT light chain [30] or 1 µg/ml BoNT heavy chain. VHH binding was detected with HRP-anti-E-tag mAb (GE Healthcare). VHHs were also characterized for recognition of subunits by Western blotting on BoNT holotoxin following standard SDS-PAGE (4–20% gel) with samples boiled in SDS sample buffer under reducing conditions (5% βME). VHHs were incubated with filters at 10 µg/ml and bound VHH detected with HRP-anti-E-tag mAb (GE Healthcare) by standard procedures. Kinetic analysis by surface plasmon resonance Studies to assess the kinetic parameters of the VHHs were performed using a ProteOn XPR36 Protein Interaction Array System (Bio-Rad, Hercules, CA) after immobilization of ciBoNTA or ciBoNTB by amine coupling chemistry using the manufacturer recommended protocol. Briefly, after activation of a GLH (high protein immobilization capacity) chip surface with a mixture of 0.4 M ethyl(dimethylaminopropyl) carbodiimide (EDC) and 0.1 M N-hydroxysulfosuccinimide (sulfo-NHS) injected for 300 s at 30 µL/min, ciBoNTA or ciBoNTB was immobilized by passing a 60 µg/mL solution of the protein at pH 5 over the surface for 180 s at 25 µL/min. The surface was deactivated with a 30 µL/min injection of 1 M ethanolamine for 300 s. A concentration series for each VHH (between 2.5 nM and 1000 nM, optimized for each antibody fragment) was passed over the surface at 100 µL/min for 60 s, then dissociation was recorded for 600 s or 1200 s. The surface was then regenerated with a 36 s injection of 10 mM glycine, pH 2.0 at 50 µL/min. Running buffer for these studies was 10 mM Hepes, pH 7.4, 150 mM NaCl, 0.005% Tween-20. Data was evaluated with ProteOn Manager software (version 2.1.2) using the Langmuir interaction model. BoNT neutralization assay using primary neurons Neuronal granule cells from the pooled cerebella of either 7–8 day old Sprague-Dawley rats or 5–7 day old CD-1 mice were harvested as described by Skaper et al [31] and cultured in 24 well plates as described by Eubanks et al [32] . After at least a week of culture the well volumes were adjusted to 0.5 ml containing various VHH dilutions or buffer controls followed immediately by addition of BoNT/A in 0.5 ml to a final 10 pM. After overnight at 37°C, cells were harvested and the extent of SNAP25 cleavage assessed by Western blot as previously described [32] . Standard mouse toxin lethality assay Female CD1 mice 15–17 g each (Charles River Labs) were received 5 days prior to use. One day prior to initiation of study, mice were weighed and placed into groups in an effort to minimize inter-group weight variation. Appropriate dilutions of the test agents were prepared in PBS. BoNT holotoxins were separately prepared in PBS+0.2% gelatin (Sigma) at the desired doses. 600 µl of test agent and 600 µl of the toxin were combined and incubated at room temperature for 30 minutes. 200 µl of the mixture was administered by intravenous injection at time 0 to mice in groups of five. Mice were monitored at least four times per day and scored for overall disposition, severity of abdominal breathing, presence of open-mouth breathing, activity level, presence of lethargy, and mortality. Moribund mice were euthanized. Time to onset of symptoms and time to death were established for each mouse [33] . Mouse toxin lethality assay with agents administered post-intoxication This assay is a modification of an assay developed by Cheng et al [25] . Groups of mice were prepared as above. Mice were administered 10 LD 50 of BoNT/A by intraperitoneal injection. At indicated times post-intoxication, mice were administered 200 ul of test agent in PBS by intravenous injection. Mice were monitored for symptoms of intoxication as above. Ethics statement All studies were carried out in strict accordance with the recommendations delineated in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The procedures used were approved by the Tufts University Institutional Animal Care and Use Committee (IACUC) and were performed under Protocols #G2010-60 and G874-07. Toxins and reagents Botulinum neurotoxin serotype A1 (BoNT/A) and serotype B (BoNT/B) were obtained from Metabiologics Inc. Each batch of toxin was assayed to establish LD 50 dose. BoNT complex was used for animal studies and BoNT holotoxin was used for the cell-based studies. Purified recombinant BoNT serotype A1 and B holotoxins containing mutations rendering them catalytically inactive (ciBoNTA, ciBoNTB) were produced as previously described [28] . Sheep anti-BoNT/A1 antiserum was produced by immunization of sheep with BoNT/A1 toxoid followed by BoNT/A1 holotoxin. Less than 1 µl of this sheep antitoxin serum protects mice from lethality when co-administered with 10,000 LD 50 of BoNT/A1. Reagents for Western blotting were purchased from KPL. Antibodies used were rabbit anti-SNAP25 antibody (Sigma); goat anti-rabbit HRP antiserum (Sigma); anti-E-tag mAb (Phadia); HRP anti-E-tag mAb (GE Healthcare). All studies with holotoxin were performed within a Select Agent laboratory registered with the CDC. Alpaca immunization and VHH-display library preparation Two alpacas were immunized with ciBoNTA and two with ciBoNTB. The immunization regimen employed 100 µg of protein in the primary immunization and 50 µg in three subsequent boosting immunizations at about 3 weekly intervals in alum/CpG adjuvant. Five days following the final boost, blood was obtained for lymphocyte preparation and VHH-display phage libraries were prepared from the immunized alpacas as previously described [29] , [30] . More than 10 6 independent clones were prepared from B cells of alpacas successfully immunized with each of the BoNT immunogens. Anti-BoNT VHH identification and preparation The VHH-display phage libraries were panned for binding to ciBoNTA or ciBoNTB targets that were coated onto a well of a 12 well plate. Coating was performed by overnight incubation with one ml of a 5 µg/ml target solution in PBS at 4°C followed by washing with PBS and 2 hrs incubation at 37°C with blocking agent (4% non-fat dried milk powder in PBS). Panning, phage recovery and clone fingerprinting were performed as previously described [29] , [30] . A total of 192 and 142 VHH clones were identified as strong positives for binding to BoNT/A and BoNT/B respectively based on phage ELISA signals. Of the strong positives, 62 unique DNA fingerprints were identified among the VHHs selected for binding to BoNT/A and 32 for VHHs selected for binding to BoNT/B. DNA sequences of the VHH coding regions was obtained for each phage clone and compared for homologies. Based on this analysis, 12 of the anti-BoNT/A VHHs and 11 anti-BoNT/B were identified as unlikely to have common B cell clonal origins and selected for protein expression. Expression and purification of VHHs in E. coli as recombinant thioredoxin (Trx) fusion proteins containing hexahistidine was performed as previously described [30] . For heterodimers, DNA encoding two different VHHs were joined in frame downstream of Trx and separated by DNA encoding a 15 amino acid flexible spacer ((GGGGS) 3 ). All VHHs were expressed with a carboxyl terminal E-tag epitope. Some expression constructions were engineered to contain a second copy of the E-tag by introducing the coding DNA in frame between the Trx and VHH domains. An example of a Trx fusion to a VHH heterodimer with two E-tags is shown in Figure S1C . A third E-tag was introduced in frame within the DNA encoding the flexible spacer of the heterodimer containing ciA-D12 and ciA-F12 to create a triple tagged heterodimer (D12/F12(3E)). VHH target binding competition analysis Phage displaying individual VHHs were prepared and titered by phage dilution ELISA [29] for recognition of ciBoNTA or ciBoNTB using HRP/anti-M13 Ab for detection. A dilution was selected for each phage preparation that produced a signal near the top of the linear range of the ELISA signal. The selected phage dilution (100 µl) for each VHH-displayed phage preparation were added to a 96 well plate that has been coated with ciBoNTA or ciBoNTB and then pre-incubated for 30 minutes with 100 µl of a 10 µg/ml solution containing a purified Trx/VHH fusion protein test agent or control in PBS. After an hour, the wells were washed and phage binding was detected as above. Test VHHs that reduced target binding of phage-displayed VHHs by less than two fold vs controls were considered to recognize distinct epitopes. Positive controls were performed in which the Trx/VHH competitor contained the same VHH as displayed on phage and typically reduced the ELISA signal >95%. Characterization of VHH binding properties VHHs were tested for binding to native or atoxic mutant BoNT holotoxins by standard ELISA using plates coated with 100 µl of 1 µg/ml protein. VHHs were also tested for recognition of BoNT subunits by a dilution series ELISA (10 µg/ml, 1∶5 dilutions) using plates coated by overnight incubation at 4°C with 5 µg/ml purified recombinant BoNT light chain [30] or 1 µg/ml BoNT heavy chain. VHH binding was detected with HRP-anti-E-tag mAb (GE Healthcare). VHHs were also characterized for recognition of subunits by Western blotting on BoNT holotoxin following standard SDS-PAGE (4–20% gel) with samples boiled in SDS sample buffer under reducing conditions (5% βME). VHHs were incubated with filters at 10 µg/ml and bound VHH detected with HRP-anti-E-tag mAb (GE Healthcare) by standard procedures. Kinetic analysis by surface plasmon resonance Studies to assess the kinetic parameters of the VHHs were performed using a ProteOn XPR36 Protein Interaction Array System (Bio-Rad, Hercules, CA) after immobilization of ciBoNTA or ciBoNTB by amine coupling chemistry using the manufacturer recommended protocol. Briefly, after activation of a GLH (high protein immobilization capacity) chip surface with a mixture of 0.4 M ethyl(dimethylaminopropyl) carbodiimide (EDC) and 0.1 M N-hydroxysulfosuccinimide (sulfo-NHS) injected for 300 s at 30 µL/min, ciBoNTA or ciBoNTB was immobilized by passing a 60 µg/mL solution of the protein at pH 5 over the surface for 180 s at 25 µL/min. The surface was deactivated with a 30 µL/min injection of 1 M ethanolamine for 300 s. A concentration series for each VHH (between 2.5 nM and 1000 nM, optimized for each antibody fragment) was passed over the surface at 100 µL/min for 60 s, then dissociation was recorded for 600 s or 1200 s. The surface was then regenerated with a 36 s injection of 10 mM glycine, pH 2.0 at 50 µL/min. Running buffer for these studies was 10 mM Hepes, pH 7.4, 150 mM NaCl, 0.005% Tween-20. Data was evaluated with ProteOn Manager software (version 2.1.2) using the Langmuir interaction model. BoNT neutralization assay using primary neurons Neuronal granule cells from the pooled cerebella of either 7–8 day old Sprague-Dawley rats or 5–7 day old CD-1 mice were harvested as described by Skaper et al [31] and cultured in 24 well plates as described by Eubanks et al [32] . After at least a week of culture the well volumes were adjusted to 0.5 ml containing various VHH dilutions or buffer controls followed immediately by addition of BoNT/A in 0.5 ml to a final 10 pM. After overnight at 37°C, cells were harvested and the extent of SNAP25 cleavage assessed by Western blot as previously described [32] . Standard mouse toxin lethality assay Female CD1 mice 15–17 g each (Charles River Labs) were received 5 days prior to use. One day prior to initiation of study, mice were weighed and placed into groups in an effort to minimize inter-group weight variation. Appropriate dilutions of the test agents were prepared in PBS. BoNT holotoxins were separately prepared in PBS+0.2% gelatin (Sigma) at the desired doses. 600 µl of test agent and 600 µl of the toxin were combined and incubated at room temperature for 30 minutes. 200 µl of the mixture was administered by intravenous injection at time 0 to mice in groups of five. Mice were monitored at least four times per day and scored for overall disposition, severity of abdominal breathing, presence of open-mouth breathing, activity level, presence of lethargy, and mortality. Moribund mice were euthanized. Time to onset of symptoms and time to death were established for each mouse [33] . Mouse toxin lethality assay with agents administered post-intoxication This assay is a modification of an assay developed by Cheng et al [25] . Groups of mice were prepared as above. Mice were administered 10 LD 50 of BoNT/A by intraperitoneal injection. At indicated times post-intoxication, mice were administered 200 ul of test agent in PBS by intravenous injection. Mice were monitored for symptoms of intoxication as above. Supporting Information Figure S1 Protein sequences of anti-BoNT/A and anti-BoNT/B VHH monomers and heterodimers. ( A ) Protein sequences of VHHs recognizing unique epitopes on BoNT/A (ciA) are shown aligned for homology. Regions represented by dashes are gaps. ( B ) Protein sequences of VHHs recognizing unique epitopes on BoNT/B (ciB) are shown aligned for homology. Regions represented by dashes are gaps. ( C ) Protein sequences of three VHHs recognizing the same BoNT/A epitope as ciA-H7 are shown aligned for homology. VHHs in A and B also contain Q(L/V)QLVE at the amino end that is encoded by the PCR primer used to generate the VHH-display library [34] . The eight amino acids shown at the carboxyl end are encoded by either the short hinge or long hinge PCR primer that were used to generate the library [34] . ( D ) Schematic diagram of the domain structure of a double-tagged VHH heterodimer protein. Proteins were expressed in pET32b with an amino-terminal E. coli thioredoxin. Domain abbreviations used were: Trx, E. coli thioredoxin; 6H, hexahistidine domain including enterokinase cleavage site ( DDDDK ); E, E-tag peptide; VHH-1, first VHH; fs, flexible spacer domain (( GGGGS ) 5 ); VHH-2 second VHH. Relative domain sizes in the diagram are approximate. ( E ) Protein sequences of the entire translation product of three recombinant VHH heterodimers containing two copies of E-tag. The E-tag sequences ( GAPVPYPDPLEPR ) are underlined. The amino acid sequences preceding the first E-tag in each protein contains the thioredoxin fusion partner and hexahistidine encoded by the pET32b expression vector. The VHH sequences are flanked by the two E-tag peptides and separated by the unstructured spacer (( GGGGS ) 3 ). (TIF) Click here for additional data file. Figure S2 SDS-PAGE analysis of purified VHH monomers and heterodimers. Following gel electrophoresis of 1 µg of the indicated purified proteins, gels were stained for protein. ( A ) VHH monomers recognizing BoNT/A (ciA-). ( B ) VHH heterodimers recognizing BoNT/A (ciA-) or BoNT/B (ciB-) in which the two indicated VHHs are expressed with the first VHH at the amino end and the second VHH at the carboxyl end. An E indicates the presence and position of peptide E-tags relative to the VHHs. The migration positions of molecular weight markers are shown with arrows. (TIF) Click here for additional data file. Figure S3 Time to death plots following co-injection BoNT/A and pools of four or six anti-BoNT/A VHHs+clearing Ab (αE). The contents of the pool of ciA-VHHs or control (no agents) that was administered to the mice is indicated by arrows. An asterisk indicates that mice did not display any symptoms of intoxication. (TIF) Click here for additional data file. Figure S4 Antitoxin efficacy of non-neutralizing anti-BoNT/A VHH heterodimer ciA-F12/D12 containing one, two or three E-tag epitopes when co-administered with anti-E-tag clearing Ab and BoNT/A. The % survival is plotted as a function of time for groups of five mice. Groups of mice were administered 20 pmoles of the heterodimer of VHHs ciA-F12 and ciA-D12 (F12/D12) containing one (1E), two (2E) or three (3E) copies of the E-tag epitope as indicated by arrows. Another group of mice received a pool of the two monomer VHHs (20 pm each), ciA-F12 and ciA-D12. The toxin dose is indicated in LD 50 . All mice received 60 pm of anti-E-tag clearing Ab (αE). (TIF) Click here for additional data file. Figure S5 Antitoxin efficacy of non-neutralizing anti-BoNT/A VHH heterodimer ciA-F12/D12 containing two copies of E-tag and co-administered with BoNT/A and varying doses of anti-E-tag clearing Ab. The % survival is plotted as a function of time for groups of five mice. Groups of mice were co-administered BoNT/A, 20 pmoles of the non-neutralizing VHH heterodimer ciA-F12/D12 containing two copies of E-tag (F12/D12(2E)) or no agents and anti-E-tag mAb as the indicated dose. The toxin dose is indicated in LD 50 . (TIF) Click here for additional data file. Figure S6 Titration of the BoNT/A antitoxin efficacy of the neutralizing anti-BoNT/A VHH heterodimer co-administered with clearing Ab. The % survival is plotted as a function of time for groups of five mice. Groups of mice were administered 1000 LD 50 of BoNT/A (∼0.3 pmoles) and either no agents or 40, 13, 4.4 or 1.5 pmoles of the double-tagged BoNT/A neutralizing VHH heterodimer, ciA-H7/B5(2E). (TIF) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127534/

Promises and pitfalls for recombinant oligoclonal antibodies-based therapeutics in cancer and infectious disease

Graphical abstract Introduction Combinations of monoclonal antibodies provide promising opportunities to increase patient benefit beyond that already observed with individual mAbs across numerous disease areas including oncology, infectious diseases, and autoimmunity [ 1 , 2 , 3 , 4 , 5 ]. Such combinations generally retain the advantages of individual mAbs, including well-characterized cell-intrinsic and cell-extrinsic mechanism(s) of action [ 6 , 7• ], established manufacturing processes, and acceptable safety profiles [ 8 , 9 ]. As the number of approved antibodies increases — seven new mAbs were approved in 2015, nearly 50 mAbs are in late stage clinical development ( i.e . Phase 3), and approximately 60–80 new mAbs enter Phase 1 every year [ 10 ] — it is likely that the number of clinical studies of antibody combinations will similarly increase. Oligoclonal antibodies are a subset of the broader field of antibody combinations. Unlike traditional combinations of mAbs, in which approval is sought for the combined use of two or more separate drug products, oligoclonal antibodies are developed as a single drug product that is defined by the mixture of mAbs at a specified formulation ratio. The biological rationale for and the clinical application of combinations of anti-cancer mAbs is well-established with numerous studies across solid and hematological cancers [ 3 , 4 , 11 , 12 ]. To date, however, only two pairs of antibodies have received regulatory approval for use in combination — the HER2-targeted mAbs trastuzumab and pertuzumab in breast cancer [ 13 , 14 ] and the immunomodulatory mAbs ipilimumab and nivolumab in melanoma [ 15 ]. These two successes stand apart from the multitude of studies that have assessed the clinical benefit for the combined use of monoclonal antibodies. Those studies notably include the combined use of approved mAbs against EGFR (cetuximab, panitumumab), HER2 (trastuzumab), and VEGF (bevacizumab) across cancer indications [ 16 , 17 , 18 , 19 ] and combined use of these agents with investigational mAbs against targets such as IGF1R or HGF/cMet [ 20 ]. Clinical data with combinations of immunomodulatory mAbs are still immature but such studies remain an ongoing and exciting area for development of antibody combinations [ 4 , 21•• , 22 ]. While combined use of separate drug products is common in oncology, only two oligoclonal mixtures of mAbs are in clinical development — MM-151 (three anti-EGFR IgG1) [ 23 •• ] and Sym004 (two anti-EGFR IgG1) [ 24 , 25 ]. The rationale and potential application of oligoclonal mAbs could be paralleled within the context of infectious diseases. Anti-infective antibodies are an alternative therapeutic modality to antimicrobials such as conventional antibiotics and peptides [ 26 , 27 ]; however, efforts to develop anti-infective antibodies have lagged behind those for cancer [ 28 , 29 ]. Only two monoclonal antibodies are approved in this disease area — palivizumab for the prevention of RSV infections in premature and at risk newborns and raxibacumab for the treatment of inhalational anthrax. In addition to the ongoing development of new mAbs, including radiolabeled mAbs [ 30 ], there are numerous ongoing preclinical and clinical studies that are assessing the utility of traditional and oligoclonal combinations of anti-infective antibodies [ 28 , 29 ]. Discussion Oligoclonal antibody combinations in infectious diseases: the status quo ? Development of effective therapeutics against bacterial and viral targets must contend with a variety of primary and adaptive mechanisms that limit activity and/or duration of response. Oligoclonal antibodies have been recognized as a potential solution to these challenges and a significant number of candidates are undergoing development for infectious diseases ( Table 1 ). Table 1 Oligoclonal antibodies in development for infectious diseases. Name Format Target Dev. Status Company Ref. CL184 Oligoclonal (2) Rabies G protein Phase 2 c Crucell [ 40 , 41 ] 2G12+4E10+2F5 Oligoclonal (3) a HIV-1 Env Phase 2 c ETH/USZ/Polymun [ 110 ] XTL-001 Oligoclonal (2) HBV HBsAg Phase 2 c Cubist [ 42 ] CT-P27 Oligoclonal (2) Group 1 and 2 Influenza A HAs Phase 2 Celltrion cαStx1/cαStx2 (Shigamab) Oligoclonal (2) Shiga toxins 1 and 2 Phase 2 Bellus Health/Thallion Pharmaceuticals [ 34 ] Zmapp Oligoclonal (3) Ebola virus GP protein Phase 1/2 Mapp BioPharmaceutical and NIH [ 48 , 49• ] XOMA 3AB Oligoclonal (3) Botulinum toxin A Phase 1 Xoma and UCSF [ 44 , 45 , 46•• ] ASN100 Oligoclonal (2) S. aureus AT and leukotoxins Phase 1 Arsanis [ 33 ] Sym003 Oligoclonal (6) RSV Preclinical Symphogen Sym002 Oligoclonal (26) Vaccinia virus Preclinical Symphogen [ 52 , 53 ] SYN-005 Oligoclonal (2) Pertussis toxin Preclinical Univ. of Texas and Synthetic Biologics [ 47 ] REGN3051 + REGN3048 Oligoclonal (2) b MERS-CoV S protein Preclinical Regeneron [ 43 ] MEDI0195 Oligoclonal (2) C. difficile toxins A and B Preclinical MedImmune and Progenics [ 32 ] RVC20/RVC58 Oligoclonal (2) b Rabies G protein Preclinical Humabs BioMed in press a Combined at time of administration (serial infusions). b Based on available reports, this combination is expected to be developed as an oligoclonal antibody. c Based on available reports, development has been discontinued. Complex pathogens, such as bacteria, harbor redundant cell surface and/or secreted virulence factors and have evolved resistance to available drugs, as demonstrated by the increasing prevalence of multi-drug resistant Gram-positive and Gram-negative bacteria in nosocomial infections. These pathogens can be targeted, in terms of strain coverage and involvement of multiple mechanisms of action, more effectively by the combination of multiple antibodies. This is exemplified by the development of two oligoclonal mixtures of two mAbs targeting Clostridium difficile exotoxins A and B [ 31•• , 32 ]. Another example is the oligoclonal mixture of two mAbs to fight pneumonia and bloodstream infections caused by Staphylococcus aureus — the Asn-1 mAb which broadly reacts with alpha-toxin and the F-components of three leukotoxins (LukSF, LukED and HlgB) and a second mAb targeting leukotoxin LukAB [ 33 ]. Yet another example is the oligoclonal mixture of two antibodies against Shiga toxin 1 and 2, which are produced by Escherichia coli and cause hemorrhagic colitis and hemolytic-uremic syndrome [ 34 ]. The need for a combination of multiple antibodies also applies to serotype-dependent bacterial targets, such as the O-antigen of lipopolysaccharide in Gram-negative bacteria. In this context, targeting only one of the several serotypes would provide an overall insufficient coverage ( e.g . the O11-specific IgM antibody panobacumab against P. aeruginosa [ 35 , 36 ]). Oligoclonal mixtures have also been assessed against viral pathogens in order to neutralize highly variable and continuously drifting targets. For example, a mixture of two anti-infective antibodies called CT-P27 was necessary to target the hemagglutinins of both group 1 and group 2 subtypes of Influenza A and presents an alternative to rare antibodies capable of broadly neutralizing all subtypes [ 37 , 38 ]. In general, antibodies broadly reacting with highly variable viruses, such as influenza virus or HIV-1, are also characterized by the recognition of sites of vulnerability, reducing the risk of pathogen escape [ 39 ]. In the case of less variable pathogens, the need for antibody combinations is driven by the risk of rare natural escape variants or the risk of selecting in vivo escape mutants. The use of two antibodies against non-overlapping sites was considered a possible solution to this problem and has been demonstrated for rabies virus [ 40 , 41 ], HBV [ 42 ], and MERS-CoV [ 43 ]. There is no general scientific rule governing the rational selection of the appropriate number of antibodies in an oligoclonal mixture. There are examples where the combinations of multiple antibodies, typically three, showed a significant synergistic effect in vivo , such as in the case of antibodies against botulinum neurotoxin [ 44 , 45 , 46•• ] and pertussis toxin [ 47 ]. Another example is the case of the three antibodies composing the Zmapp preparation which has been utilized on a compassionate basis, and recently in a randomized trial (that however failed to show a statistically significant effect [ 48 ]), to treat Ebola virus infections from the recent outbreak in West Africa [ 49 • ]. The suggested mechanisms responsible for the observed synergistic effects were the increase in functional antibody binding affinity, the combination of direct neutralization and antibody effector functions, and Fc-dependent hepatic clearance of immune-complexes similar to what was recently observed for IgE and GM-CSF [ 50 , 51 ]. The generation of high-order oligoclonal mixtures (also named 'polyclonal', due to the high number of antibodies in the mixture) has been pioneered by a limited number of companies, including Symphogen, as demonstrated by their mixtures of 26 mAbs to target vaccinia virus (Sym002) and six mAbs against RSV (Sym003) [ 52 , 53 ]. The rationale for such high-order mixtures, both of which have now ceased clinical development, was stated to be the recapitulation of the diversity and specificity of the human antibody immune response. Oligoclonal antibody combinations in oncology: overcoming tumor heterogeneity and plasticity The diversity of monoclonal antibody-based therapeutics for the treatment of cancer encompasses a wide class of agents including anti-tumor mAbs ( e.g. trastuzumab, pertuzumab, cetuximab, rituximab), inhibitors of angiogenesis ( e.g . bevacizumab), immunotherapies ( e.g . ipilimumab, nivolumab), antibody-drug conjugates ( e.g . ado-trastuzumab emtansine, brentuximab vedotin), and a host of novel formats [ 6 , 7• , 21•• , 54 , 55 ]. These mAbs function through a variety of overlapping mechanisms that notably include the perturbation of cell signaling responses, engagement of immune-effector activities ( e.g . antibody-dependent cellular cytotoxicity), and modulation of the host immune response [ 7 • ]. The application of combinations of these agents is the focus of intense preclinical and clinical development [ 4 , 11 , 12 , 21•• , 56 ], as highlighted by the necessity for programmatic keyword searches across public databases to generate a more complete accounting of such combinations [ 11 , 12 ]. The development of antibody combinations is contemporaneous with, if not intimately dependent upon, advances in the molecular profiling technologies that have led to a revolution in the understanding of the genomic and proteomic landscapes across cancer and the host immune system ( e.g . next-generation sequencing, multiplex immunohistochemistry) [ 57 , 58 , 59 , 60 , 61 ]. These efforts are a linchpin in precision medicine strategies and have revealed a universe of driver genes and redundancies within and across cell signaling pathways that inform the selection of appropriate therapeutic combinations and the identification of predictive biomarkers. More recently, longitudinal analysis of patient samples across the course of treatment(s) has revealed the challenges and opportunities in developing therapeutic strategies that overcome or exploit tumor heterogeneity, evolution, and plasticity [ 62 , 63 , 64 , 65 ]. In this regard, oncology shares a core biological context with infectious diseases [ 66 ] and thus the necessity to both recapitulate this context in preclinical testing [ 67 , 68 , 69 , 70 ] and monitor tumor evolution during the course of treatment ( e.g . via liquid biopsy analysis of cell-free DNA) [ 71 , 72 , 73 , 74 , 75 ]. The use of oligoclonal antibodies in oncology is still emerging and there are relatively few candidates in development ( Table 2 ). Receptor plasticity and functional redundancy are two exemplary phenomena addressed by current oligoclonal antibodies and these are also analogous to the biology of infectious diseases. These aspects are perhaps no better studied than for the ErbB receptors (EGFR, HER2/ErbB2, HER3/ErbB3) and the downstream MAPK and AKT effector pathways. An impressive number of preclinical and clinical studies have evaluated combinations of targeted therapies, including both antibodies and small-molecule inhibitors ( e.g . tyrosine kinase inhibitors), within this core cancer pathway [ 76 , 77 , 78 , 79 ]. Table 2 Oligoclonal antibodies in development for oncology. Table 2 Name Format Target Dev. Status Company Ref. MM-151 Oligoclonal (3) EGFR Phase 1/2 Merrimack Pharmaceuticals. [ 23 •• ] MM-121 + MM-151 Use in combination of mAb and oligoclonal (3) ErbB3 and EGFR Phase 1 Merrimack Pharmaceuticals [ 115 ] Sym004 Oligoclonal (2) EGFR Phase 2 Symphogen [ 24 , 25 ] Sym013 (Pan-HER) Oligoclonal (6) EGFR, ErbB2, ErbB3 Preclinical Symphogen [ 88 •• ] First, monospecific oligoclonal mixtures have been demonstrated to maintain activity in the presence of somatic mutations in the extracellular domains of EGFR and HER2 receptors that function as markers of primary insensitivity or acquired resistance to single mAbs (cetuximab, panitumumab, trastuzumab) [ 80 , 81 , 82 ]. This is an additional mechanism of action for monospecific oligoclonal mixtures beyond those already described — robust inhibition of cell signaling, enhanced down-regulation of receptor, and increased activation of complement-dependent cytotoxicity [ 23•• , 56 , 77 , 78 , 83 , 84 ]. Notably, the MM-151 oligoclonal mixture of three anti-EGFR IgG1 antibodies has been demonstrated to maintain activity in cell lines and patient-derived models harboring EGFR extracellular domain mutations that inhibit binding of single antibodies. Longitudinal analysis of cell-free DNA ('liquid biopsies') of a sub-cohort of patients treated with MM-151 revealed changes in the allelic frequencies of EGFR mutations during the course of treatment [ 85 •• ]. We hypothesize that mixtures of multiple mAbs with single-agent activities, as is the case for MM-151, are perhaps required to generate sufficient therapeutic redundancy to overcome receptor plasticity that inhibits the binding of one or more of the mAbs. Second, combinations of antibodies with specificities for different ErbB receptors have been demonstrated in preclinical models to overcome both primary and adaptive functional redundancy via signaling elsewhere in the ErbB/MAPK/AKT network [ 76 , 86 , 87 ]. To date, however, combinations of approved therapeutics against these targets, such as trastuzumab (HER2) and cetuximab (EGFR), have not shown sufficient clinical activity to seek approval for the combination [ 76 ]. The Sym013 oligoclonal mixture of six antibodies against EGFR, HER2, and HER3 (two mAbs per receptor) represents a novel approach to overcoming functional redundancy across multiple targets [ 88 •• ]. It remains to be seen how any future clinical development with Sym013 (or more generally, any multi-specific oligoclonal antibodies) will incorporate a diagnostic strategy to identify patients whose tumors are truly dependent upon the target antigens ( e.g . receptors) or are likely to adapt to treatment against one antigen via the remaining antigen(s). Polyclonal and bispecific antibodies as alternatives to monoclonal antibody combinations The humoral immune response generates polyclonal antibody responses targeting multiple epitopes and mediating a broad variety of effector functions. The use of passive serotherapy was pioneered by Emil v. Behring and Kitasato Shibasaburō in the early 1890s when they showed that hyperimmune sera of animal origin could protect against diphtheria and tetanus [ 89 ]. This approach, in some cases replaced by the use of hyperimmune immunoglobulin preparations, is still used to treat several infectious diseases [ 90 , 91 , 92 , 93 , 94• ]. While no polyclonal antibody therapies have entered clinical development in oncology, some technological hurdles have been addressed to enable these products, such as development of methodologies to generate libraries of polyclonal antibodies [ 95 ]. The use of blood-derived polyclonal antibodies faces the obstacles of limited availability, the risk of blood-borne disease transmission, batch-to-batch variability and, more importantly, low specific activity because only a very small fraction of the antibodies are specific for the antigen of interest (thus requiring large doses for efficacy) [ 93 ]. One of the advantages of recombinant oligoclonal antibody mixtures over the 'natural' polyclonal response is that in this case each of the antibodies of the cocktail can be selected, engineered and tuned for high affinity, neutralizing activity and optimized effector functions. This design avoids the sinking effect of decoy epitopes and thus allows the development of antibody-based products able to exceed the potency of 'natural' polyclonal antibodies [ 96 , 97 ]. Another advantage for oligoclonal antibodies is the ability to rationally define an optimal formulation ratio on the basis of systematic preclinical studies ( e.g. using pairwise titrations of purified component antibodies) without the significant inconvenience of generating and screening a multitude of polyclonal expression variants. The combination of multiple specificities into a single molecule represents an alternative therapeutic strategy to oligoclonal antibodies and a wide number of multi-specific (often bispecific) antibody formats have progressed into clinical development [ 98 , 99 ]. Two bispecific antibodies are approved for use in oncology — catumaxomab (in EU) and blinatumomab (in US) — and both utilize a bispecific format to retarget T cells (anti-CD3) to tumor cells via engagement of HER2 or EpCAM, respectively. To date, no multi-specific products are approved for the treatment of infectious diseases. One promising candidate in early clinical development is MEDI3902, which targets two Pseudomonas aeruginosa cell-surface factors, Psl and PcrV, and has showed enhanced activity in comparison to the combination of the parental antibodies [ 100 ]. There is no evidence to suggest that multi-specific antibodies harbor a general advantage over combinations of monospecific antibodies. To the contrary, it is understood that the selection of the appropriate therapeutic format is dependent upon factors such as target engagement ( e.g . is targeting required? ) and biological context ( e.g . are antigens co-expressed on the same cell ? If antigens are expressed on distinct cells, are these cells in close proximity? ) that must be considered early in preclinical development [ 54 , 101 ]. There are examples where antibody mixtures showed higher efficacy as compared to the corresponding bispecific antibodies, such as in the case of a combination of antibodies targeting a cell-surface molecule and a secreted toxin (DC, unpublished results). In this scenario, one of the two targets might exhibit a sinking effect over the second antibody. The regulatory landscape around the development of oligoclonal antibodies Polyclonal antibodies are regulated by the Center for Biologics Evaluation and Research (CBER). These regulations require characterization of the bulk activity of the product, but not the individual antibodies. A similar consideration applies to the regulation of multivalent vaccines, which does not require assessment of individual vaccine antigen components [ 102 ]. However, recombinant biologics are regulated by the Center for Drug Evaluation and Research (CDER) and different rules apply. In general, oligoclonal recombinant antibodies are regulated according to the combination drug rules of FDA and EMA guidelines [ 103 , 104 , 105 ]. This rule indicates that the components of a combination product have to be assessed individually as well as in combination and, generally, this may require assessment of safety and pharmacokinetics, and potentially efficacy, in multi-arm clinical studies [ 104 , 106 ]. The assessment of individual antibody components in late stage multi-arm trials poses obvious clinical and financial obstacles, especially if all combinatorial 'sub-mixtures' are included. Importantly for development of such therapeutics, the current guidance does specify that this requirement may be waived on ethical grounds if there are sufficient preclinical or clinical data indicating that the monotherapies would likely be ineffective or in cases where primary and/or acquired resistance are a significant concern. Indeed, the FDA on several occasions has allowed oligoclonal antibodies to be clinically tested as single products in Phase 1 and Phase 2 studies (such as for rabies [ 41 ], botulinum [ 44 ], the rhesus D antigen [ 107 ] and for the MM-151 and Sym004 anti-EGFR therapeutics in cancer). As no oligoclonal antibodies have, to date, filed for regulatory approval, we recommend that sponsors engage regulators early and often during development of these products and, as warranted, share their experiences with the community. Manufacturing of oligoclonal antibodies The production of recombinant oligoclonal antibodies is largely an extension of well-established practices [ 8 , 9 ] utilized for individual mAbs [ 108 ]. To date, the majority of disclosed oligoclonal antibody products utilize a parallel GMP manufacturing approach in which mAbs are expressed and purified individually and then subsequently formulated in a single vial [ 56 ]. Rarely, the mAbs are combined at the point of administration, as illustrated by the early clinical evaluation of three HIV-1 broadly neutralizing antibodies administered by serial infusions [ 109 , 110 ]. The so-called 'single pot' strategy represents an alternative approach for the production of oligoclonal mixtures. As the component antibodies are inherently combined downstream, the single pot production strategy utilizes a mixture of antibody-producing cell lines in a single bioreactor. Several single pot technology platforms have been developed to overcome the challenges of reproducible cell growth and constant antibody ratios. Merus has developed a platform that utilizes PER.C6 cells stably expressing one common light chain and three heavy chains that is limited to the production of oligoclonal antibodies that do not include antigen specificity via the light chains [ 111 ]. A second approach was developed by Symphogen and is based on the site specific integration of the antibody expression cassette in order to favor consistent growth and expression ( e.g . expression of 25 antibodies in the Sym001 product, called rozrolimupab, tested in Phase 2 in 2012 for the treatment of Primary Immune Thrombocytopenia Purpura [ 107 , 112 ]). A variant to this second approach, also developed at Symphogen, is based on the random integration and selection of high expressing stable clones to be used in the single pot production, as shown in the case of the Sym002 (RSV) and Sym003 (vaccinia virus) oligoclonal antibody products [ 112 ]. Practical considerations for the development of oligoclonal antibodies The development of oligoclonal antibodies involves several unique challenges that must be considered at key decision points starting from early therapeutic design and preclinical testing and continuing through clinical studies ( Figure 1 ). The considerations described below represent some, but certainly not all, of the challenges. • Pharmacokinetics of the component antibodies. An oligoclonal antibody product consists of a formulation of component mAbs at a ratio (or within a range of ratios) that is defined at the time of the initial regulatory filing ( e.g . IND). However, it is problematic to assume that this formulation ratio will be maintained in the patient, due to the potential for differential pharmacokinetics or ADME ( i.e . absorption, distribution, metabolism and excretion) for each antibody. The characterization and mitigation of this issue must be an essential factor in the development of all oligoclonal antibodies. In preclinical research, the performance of the mixture should be assessed over a physiologically relevant range of ratios to ensure that activity is maintained. In clinical studies, the selection of the dose and the dosing interval should consider the pharmacokinetics of the individual component antibodies (using antibody-specific assays) to ensure that active ratios are maintained over time and across patients. This issue is exemplified by the use of oligoclonal antibodies against multiple antigens that are described to mediate different degrees of target-mediated drug disposition (TMDD). For example, it can be reasonably expected that the equimolar ratio of the six antibodies in the Sym013 combination will vary over time due to significant (via EGFR) or negligible TMDD (via HER2/ErbB2 and HER3/ErbB3). These risks may apply also to anti-infective antibodies where the multiple antigens targeted could be displayed at different and variable levels, depending on multiple factors that influence pathogen growth and clearance. • Overlapping toxicity profiles. Similar to combination drug treatment, oligoclonal antibodies harbor the potential for overlapping toxicity profiles that may be difficult to interpret and cannot be mitigated in the clinic by the adjustment of the defined formulation ratio (as is regularly done for combined use of separate mAb drug products). This is perhaps most pressing for anti-cancer antibodies that must contend with self-recognition and less pressing for anti-infective antibodies that have the inherent advantage of specificity for pathogenic antigens. Indeed, anti-infective oligoclonal antibodies are commonly tested for safety in Phase 1 studies in healthy patients. While the two instances of approved combined use of separate mAbs in oncology utilize the monotherapy dose for each mAb ( e.g . trastuzumab + pertuzumab; nivolumab + ipilimumab), rigorous preclinical and early clinical toxicology studies should be performed with oligoclonal antibodies to identify issues that would motivate reformulation (likely necessitating a new IND filing) or discontinuation. • Overlapping or elusive mechanisms of action. Oligoclonal antibodies have demonstrated several mechanisms of action that are unique to the mixture such as engagement of immune responses, enhanced receptor down-regulation, increased clearance of immune complexes, or tolerance for escape variants and other acquired resistance mechanisms. Characterization of these emergent properties , both individually or in combination, is not trivial and is complicated by the need for comprehensive model systems. For example, standard human xenograft tumor models will mask enhanced immune activities (if implanted in an immune-deficient mouse) or other tumor microenvironment effects (if one or more antibodies do not cross-react to the mouse antigen). The oligoclonal characterization strategy should be considered early in development as it will likely inform decisions in the therapeutic design and selection of the formulation ratio ( e.g. should species cross-reactivity be considered in lead selection? ). • Translation of preclinical findings to clinical development. The inherent risk in the development of oligoclonal antibodies is the necessity to have a complete and comprehensive preclinical data package to support the combination of antibodies and the formulation ratio. Beyond commercial and manufacturing considerations, this is the key differentiator from combinations of separate antibody products. Those developing oligoclonal antibodies would be wise to prioritize both early preclinical efforts and ongoing translational research. The late-stage failure of the combination of two antibodies against Clostridium difficile toxins A and B (actoxumab + bezlotoxumab), while not technically an oligoclonal antibody, is a cautionary tale for all oligoclonal antibodies. This experience highlights the need to clearly demonstrate the necessity of the oligoclonal antibody modality in rigorous preclinical studies with relevant models. Initial animal studies had demonstrated enhanced activity for the combination [ 113 ] and motivated a Phase 2 trial that assessed the activity of the combination, but not of the individual antibodies [ 31 •• ]. However, later results from two comparative Phase 3 clinical trials (MODIFY I and II) revealed no benefit of the combination over the toxin B-specific mAb alone (bezlotoxumab). Indeed, subsequent animal studies using isogenic toxin A and B mutants of a virulent C. difficile strain demonstrated that only toxin B is essential for virulence [ 114 ]. Figure 1 Practical considerations for the development of oligoclonal antibodies. (a) The underlying biology or disease context should inform the therapeutic design and generation of proof-of-concept molecules. A key consideration will be the selection of mono-specific or multi-specific antibodies. (b) Comprehensive preclinical assessment should be performed in relevant model systems to characterize the performance of the individual mAbs and the mixture (and any emergent properties of the mixture). Findings will likely, and iteratively, inform further therapeutic design. (c) The selection of the formulation ratio (the ratio of antibodies within the mixture) is perhaps the most critical decision point for the development of oligoclonal antibodies and is strongly coupled to preclinical testing, manufacturing considerations, and later clinical development. (d) There are numerous manufacturing strategies for oligoclonal antibodies, notably parallel GMP and single pot, and these will be informed by the formulation ratio and commercial considerations. The assessment of the pharmacokinetics and safety in preclinical (e) and clinical (f) studies should consider the individual mAbs as well as the mixture. Unexpected findings, such as more rapid clearance or a limited therapeutic window for an individual antibody, may necessitate a costly and time consuming reconsideration of the formulation ratio. (g) The clinical development strategy should include a strong consideration of the regulatory landscape to ensure that sufficient data will be generated to support the mixture ( e.g . improved activity of the mixture versus the individual antibodies) and/or an application for a waiver to the combination drug rule. Oligoclonal antibody combinations in infectious diseases: the status quo ? Development of effective therapeutics against bacterial and viral targets must contend with a variety of primary and adaptive mechanisms that limit activity and/or duration of response. Oligoclonal antibodies have been recognized as a potential solution to these challenges and a significant number of candidates are undergoing development for infectious diseases ( Table 1 ). Table 1 Oligoclonal antibodies in development for infectious diseases. Name Format Target Dev. Status Company Ref. CL184 Oligoclonal (2) Rabies G protein Phase 2 c Crucell [ 40 , 41 ] 2G12+4E10+2F5 Oligoclonal (3) a HIV-1 Env Phase 2 c ETH/USZ/Polymun [ 110 ] XTL-001 Oligoclonal (2) HBV HBsAg Phase 2 c Cubist [ 42 ] CT-P27 Oligoclonal (2) Group 1 and 2 Influenza A HAs Phase 2 Celltrion cαStx1/cαStx2 (Shigamab) Oligoclonal (2) Shiga toxins 1 and 2 Phase 2 Bellus Health/Thallion Pharmaceuticals [ 34 ] Zmapp Oligoclonal (3) Ebola virus GP protein Phase 1/2 Mapp BioPharmaceutical and NIH [ 48 , 49• ] XOMA 3AB Oligoclonal (3) Botulinum toxin A Phase 1 Xoma and UCSF [ 44 , 45 , 46•• ] ASN100 Oligoclonal (2) S. aureus AT and leukotoxins Phase 1 Arsanis [ 33 ] Sym003 Oligoclonal (6) RSV Preclinical Symphogen Sym002 Oligoclonal (26) Vaccinia virus Preclinical Symphogen [ 52 , 53 ] SYN-005 Oligoclonal (2) Pertussis toxin Preclinical Univ. of Texas and Synthetic Biologics [ 47 ] REGN3051 + REGN3048 Oligoclonal (2) b MERS-CoV S protein Preclinical Regeneron [ 43 ] MEDI0195 Oligoclonal (2) C. difficile toxins A and B Preclinical MedImmune and Progenics [ 32 ] RVC20/RVC58 Oligoclonal (2) b Rabies G protein Preclinical Humabs BioMed in press a Combined at time of administration (serial infusions). b Based on available reports, this combination is expected to be developed as an oligoclonal antibody. c Based on available reports, development has been discontinued. Complex pathogens, such as bacteria, harbor redundant cell surface and/or secreted virulence factors and have evolved resistance to available drugs, as demonstrated by the increasing prevalence of multi-drug resistant Gram-positive and Gram-negative bacteria in nosocomial infections. These pathogens can be targeted, in terms of strain coverage and involvement of multiple mechanisms of action, more effectively by the combination of multiple antibodies. This is exemplified by the development of two oligoclonal mixtures of two mAbs targeting Clostridium difficile exotoxins A and B [ 31•• , 32 ]. Another example is the oligoclonal mixture of two mAbs to fight pneumonia and bloodstream infections caused by Staphylococcus aureus — the Asn-1 mAb which broadly reacts with alpha-toxin and the F-components of three leukotoxins (LukSF, LukED and HlgB) and a second mAb targeting leukotoxin LukAB [ 33 ]. Yet another example is the oligoclonal mixture of two antibodies against Shiga toxin 1 and 2, which are produced by Escherichia coli and cause hemorrhagic colitis and hemolytic-uremic syndrome [ 34 ]. The need for a combination of multiple antibodies also applies to serotype-dependent bacterial targets, such as the O-antigen of lipopolysaccharide in Gram-negative bacteria. In this context, targeting only one of the several serotypes would provide an overall insufficient coverage ( e.g . the O11-specific IgM antibody panobacumab against P. aeruginosa [ 35 , 36 ]). Oligoclonal mixtures have also been assessed against viral pathogens in order to neutralize highly variable and continuously drifting targets. For example, a mixture of two anti-infective antibodies called CT-P27 was necessary to target the hemagglutinins of both group 1 and group 2 subtypes of Influenza A and presents an alternative to rare antibodies capable of broadly neutralizing all subtypes [ 37 , 38 ]. In general, antibodies broadly reacting with highly variable viruses, such as influenza virus or HIV-1, are also characterized by the recognition of sites of vulnerability, reducing the risk of pathogen escape [ 39 ]. In the case of less variable pathogens, the need for antibody combinations is driven by the risk of rare natural escape variants or the risk of selecting in vivo escape mutants. The use of two antibodies against non-overlapping sites was considered a possible solution to this problem and has been demonstrated for rabies virus [ 40 , 41 ], HBV [ 42 ], and MERS-CoV [ 43 ]. There is no general scientific rule governing the rational selection of the appropriate number of antibodies in an oligoclonal mixture. There are examples where the combinations of multiple antibodies, typically three, showed a significant synergistic effect in vivo , such as in the case of antibodies against botulinum neurotoxin [ 44 , 45 , 46•• ] and pertussis toxin [ 47 ]. Another example is the case of the three antibodies composing the Zmapp preparation which has been utilized on a compassionate basis, and recently in a randomized trial (that however failed to show a statistically significant effect [ 48 ]), to treat Ebola virus infections from the recent outbreak in West Africa [ 49 • ]. The suggested mechanisms responsible for the observed synergistic effects were the increase in functional antibody binding affinity, the combination of direct neutralization and antibody effector functions, and Fc-dependent hepatic clearance of immune-complexes similar to what was recently observed for IgE and GM-CSF [ 50 , 51 ]. The generation of high-order oligoclonal mixtures (also named 'polyclonal', due to the high number of antibodies in the mixture) has been pioneered by a limited number of companies, including Symphogen, as demonstrated by their mixtures of 26 mAbs to target vaccinia virus (Sym002) and six mAbs against RSV (Sym003) [ 52 , 53 ]. The rationale for such high-order mixtures, both of which have now ceased clinical development, was stated to be the recapitulation of the diversity and specificity of the human antibody immune response. Oligoclonal antibody combinations in oncology: overcoming tumor heterogeneity and plasticity The diversity of monoclonal antibody-based therapeutics for the treatment of cancer encompasses a wide class of agents including anti-tumor mAbs ( e.g. trastuzumab, pertuzumab, cetuximab, rituximab), inhibitors of angiogenesis ( e.g . bevacizumab), immunotherapies ( e.g . ipilimumab, nivolumab), antibody-drug conjugates ( e.g . ado-trastuzumab emtansine, brentuximab vedotin), and a host of novel formats [ 6 , 7• , 21•• , 54 , 55 ]. These mAbs function through a variety of overlapping mechanisms that notably include the perturbation of cell signaling responses, engagement of immune-effector activities ( e.g . antibody-dependent cellular cytotoxicity), and modulation of the host immune response [ 7 • ]. The application of combinations of these agents is the focus of intense preclinical and clinical development [ 4 , 11 , 12 , 21•• , 56 ], as highlighted by the necessity for programmatic keyword searches across public databases to generate a more complete accounting of such combinations [ 11 , 12 ]. The development of antibody combinations is contemporaneous with, if not intimately dependent upon, advances in the molecular profiling technologies that have led to a revolution in the understanding of the genomic and proteomic landscapes across cancer and the host immune system ( e.g . next-generation sequencing, multiplex immunohistochemistry) [ 57 , 58 , 59 , 60 , 61 ]. These efforts are a linchpin in precision medicine strategies and have revealed a universe of driver genes and redundancies within and across cell signaling pathways that inform the selection of appropriate therapeutic combinations and the identification of predictive biomarkers. More recently, longitudinal analysis of patient samples across the course of treatment(s) has revealed the challenges and opportunities in developing therapeutic strategies that overcome or exploit tumor heterogeneity, evolution, and plasticity [ 62 , 63 , 64 , 65 ]. In this regard, oncology shares a core biological context with infectious diseases [ 66 ] and thus the necessity to both recapitulate this context in preclinical testing [ 67 , 68 , 69 , 70 ] and monitor tumor evolution during the course of treatment ( e.g . via liquid biopsy analysis of cell-free DNA) [ 71 , 72 , 73 , 74 , 75 ]. The use of oligoclonal antibodies in oncology is still emerging and there are relatively few candidates in development ( Table 2 ). Receptor plasticity and functional redundancy are two exemplary phenomena addressed by current oligoclonal antibodies and these are also analogous to the biology of infectious diseases. These aspects are perhaps no better studied than for the ErbB receptors (EGFR, HER2/ErbB2, HER3/ErbB3) and the downstream MAPK and AKT effector pathways. An impressive number of preclinical and clinical studies have evaluated combinations of targeted therapies, including both antibodies and small-molecule inhibitors ( e.g . tyrosine kinase inhibitors), within this core cancer pathway [ 76 , 77 , 78 , 79 ]. Table 2 Oligoclonal antibodies in development for oncology. Table 2 Name Format Target Dev. Status Company Ref. MM-151 Oligoclonal (3) EGFR Phase 1/2 Merrimack Pharmaceuticals. [ 23 •• ] MM-121 + MM-151 Use in combination of mAb and oligoclonal (3) ErbB3 and EGFR Phase 1 Merrimack Pharmaceuticals [ 115 ] Sym004 Oligoclonal (2) EGFR Phase 2 Symphogen [ 24 , 25 ] Sym013 (Pan-HER) Oligoclonal (6) EGFR, ErbB2, ErbB3 Preclinical Symphogen [ 88 •• ] First, monospecific oligoclonal mixtures have been demonstrated to maintain activity in the presence of somatic mutations in the extracellular domains of EGFR and HER2 receptors that function as markers of primary insensitivity or acquired resistance to single mAbs (cetuximab, panitumumab, trastuzumab) [ 80 , 81 , 82 ]. This is an additional mechanism of action for monospecific oligoclonal mixtures beyond those already described — robust inhibition of cell signaling, enhanced down-regulation of receptor, and increased activation of complement-dependent cytotoxicity [ 23•• , 56 , 77 , 78 , 83 , 84 ]. Notably, the MM-151 oligoclonal mixture of three anti-EGFR IgG1 antibodies has been demonstrated to maintain activity in cell lines and patient-derived models harboring EGFR extracellular domain mutations that inhibit binding of single antibodies. Longitudinal analysis of cell-free DNA ('liquid biopsies') of a sub-cohort of patients treated with MM-151 revealed changes in the allelic frequencies of EGFR mutations during the course of treatment [ 85 •• ]. We hypothesize that mixtures of multiple mAbs with single-agent activities, as is the case for MM-151, are perhaps required to generate sufficient therapeutic redundancy to overcome receptor plasticity that inhibits the binding of one or more of the mAbs. Second, combinations of antibodies with specificities for different ErbB receptors have been demonstrated in preclinical models to overcome both primary and adaptive functional redundancy via signaling elsewhere in the ErbB/MAPK/AKT network [ 76 , 86 , 87 ]. To date, however, combinations of approved therapeutics against these targets, such as trastuzumab (HER2) and cetuximab (EGFR), have not shown sufficient clinical activity to seek approval for the combination [ 76 ]. The Sym013 oligoclonal mixture of six antibodies against EGFR, HER2, and HER3 (two mAbs per receptor) represents a novel approach to overcoming functional redundancy across multiple targets [ 88 •• ]. It remains to be seen how any future clinical development with Sym013 (or more generally, any multi-specific oligoclonal antibodies) will incorporate a diagnostic strategy to identify patients whose tumors are truly dependent upon the target antigens ( e.g . receptors) or are likely to adapt to treatment against one antigen via the remaining antigen(s). Polyclonal and bispecific antibodies as alternatives to monoclonal antibody combinations The humoral immune response generates polyclonal antibody responses targeting multiple epitopes and mediating a broad variety of effector functions. The use of passive serotherapy was pioneered by Emil v. Behring and Kitasato Shibasaburō in the early 1890s when they showed that hyperimmune sera of animal origin could protect against diphtheria and tetanus [ 89 ]. This approach, in some cases replaced by the use of hyperimmune immunoglobulin preparations, is still used to treat several infectious diseases [ 90 , 91 , 92 , 93 , 94• ]. While no polyclonal antibody therapies have entered clinical development in oncology, some technological hurdles have been addressed to enable these products, such as development of methodologies to generate libraries of polyclonal antibodies [ 95 ]. The use of blood-derived polyclonal antibodies faces the obstacles of limited availability, the risk of blood-borne disease transmission, batch-to-batch variability and, more importantly, low specific activity because only a very small fraction of the antibodies are specific for the antigen of interest (thus requiring large doses for efficacy) [ 93 ]. One of the advantages of recombinant oligoclonal antibody mixtures over the 'natural' polyclonal response is that in this case each of the antibodies of the cocktail can be selected, engineered and tuned for high affinity, neutralizing activity and optimized effector functions. This design avoids the sinking effect of decoy epitopes and thus allows the development of antibody-based products able to exceed the potency of 'natural' polyclonal antibodies [ 96 , 97 ]. Another advantage for oligoclonal antibodies is the ability to rationally define an optimal formulation ratio on the basis of systematic preclinical studies ( e.g. using pairwise titrations of purified component antibodies) without the significant inconvenience of generating and screening a multitude of polyclonal expression variants. The combination of multiple specificities into a single molecule represents an alternative therapeutic strategy to oligoclonal antibodies and a wide number of multi-specific (often bispecific) antibody formats have progressed into clinical development [ 98 , 99 ]. Two bispecific antibodies are approved for use in oncology — catumaxomab (in EU) and blinatumomab (in US) — and both utilize a bispecific format to retarget T cells (anti-CD3) to tumor cells via engagement of HER2 or EpCAM, respectively. To date, no multi-specific products are approved for the treatment of infectious diseases. One promising candidate in early clinical development is MEDI3902, which targets two Pseudomonas aeruginosa cell-surface factors, Psl and PcrV, and has showed enhanced activity in comparison to the combination of the parental antibodies [ 100 ]. There is no evidence to suggest that multi-specific antibodies harbor a general advantage over combinations of monospecific antibodies. To the contrary, it is understood that the selection of the appropriate therapeutic format is dependent upon factors such as target engagement ( e.g . is targeting required? ) and biological context ( e.g . are antigens co-expressed on the same cell ? If antigens are expressed on distinct cells, are these cells in close proximity? ) that must be considered early in preclinical development [ 54 , 101 ]. There are examples where antibody mixtures showed higher efficacy as compared to the corresponding bispecific antibodies, such as in the case of a combination of antibodies targeting a cell-surface molecule and a secreted toxin (DC, unpublished results). In this scenario, one of the two targets might exhibit a sinking effect over the second antibody. The regulatory landscape around the development of oligoclonal antibodies Polyclonal antibodies are regulated by the Center for Biologics Evaluation and Research (CBER). These regulations require characterization of the bulk activity of the product, but not the individual antibodies. A similar consideration applies to the regulation of multivalent vaccines, which does not require assessment of individual vaccine antigen components [ 102 ]. However, recombinant biologics are regulated by the Center for Drug Evaluation and Research (CDER) and different rules apply. In general, oligoclonal recombinant antibodies are regulated according to the combination drug rules of FDA and EMA guidelines [ 103 , 104 , 105 ]. This rule indicates that the components of a combination product have to be assessed individually as well as in combination and, generally, this may require assessment of safety and pharmacokinetics, and potentially efficacy, in multi-arm clinical studies [ 104 , 106 ]. The assessment of individual antibody components in late stage multi-arm trials poses obvious clinical and financial obstacles, especially if all combinatorial 'sub-mixtures' are included. Importantly for development of such therapeutics, the current guidance does specify that this requirement may be waived on ethical grounds if there are sufficient preclinical or clinical data indicating that the monotherapies would likely be ineffective or in cases where primary and/or acquired resistance are a significant concern. Indeed, the FDA on several occasions has allowed oligoclonal antibodies to be clinically tested as single products in Phase 1 and Phase 2 studies (such as for rabies [ 41 ], botulinum [ 44 ], the rhesus D antigen [ 107 ] and for the MM-151 and Sym004 anti-EGFR therapeutics in cancer). As no oligoclonal antibodies have, to date, filed for regulatory approval, we recommend that sponsors engage regulators early and often during development of these products and, as warranted, share their experiences with the community. Manufacturing of oligoclonal antibodies The production of recombinant oligoclonal antibodies is largely an extension of well-established practices [ 8 , 9 ] utilized for individual mAbs [ 108 ]. To date, the majority of disclosed oligoclonal antibody products utilize a parallel GMP manufacturing approach in which mAbs are expressed and purified individually and then subsequently formulated in a single vial [ 56 ]. Rarely, the mAbs are combined at the point of administration, as illustrated by the early clinical evaluation of three HIV-1 broadly neutralizing antibodies administered by serial infusions [ 109 , 110 ]. The so-called 'single pot' strategy represents an alternative approach for the production of oligoclonal mixtures. As the component antibodies are inherently combined downstream, the single pot production strategy utilizes a mixture of antibody-producing cell lines in a single bioreactor. Several single pot technology platforms have been developed to overcome the challenges of reproducible cell growth and constant antibody ratios. Merus has developed a platform that utilizes PER.C6 cells stably expressing one common light chain and three heavy chains that is limited to the production of oligoclonal antibodies that do not include antigen specificity via the light chains [ 111 ]. A second approach was developed by Symphogen and is based on the site specific integration of the antibody expression cassette in order to favor consistent growth and expression ( e.g . expression of 25 antibodies in the Sym001 product, called rozrolimupab, tested in Phase 2 in 2012 for the treatment of Primary Immune Thrombocytopenia Purpura [ 107 , 112 ]). A variant to this second approach, also developed at Symphogen, is based on the random integration and selection of high expressing stable clones to be used in the single pot production, as shown in the case of the Sym002 (RSV) and Sym003 (vaccinia virus) oligoclonal antibody products [ 112 ]. Practical considerations for the development of oligoclonal antibodies The development of oligoclonal antibodies involves several unique challenges that must be considered at key decision points starting from early therapeutic design and preclinical testing and continuing through clinical studies ( Figure 1 ). The considerations described below represent some, but certainly not all, of the challenges. • Pharmacokinetics of the component antibodies. An oligoclonal antibody product consists of a formulation of component mAbs at a ratio (or within a range of ratios) that is defined at the time of the initial regulatory filing ( e.g . IND). However, it is problematic to assume that this formulation ratio will be maintained in the patient, due to the potential for differential pharmacokinetics or ADME ( i.e . absorption, distribution, metabolism and excretion) for each antibody. The characterization and mitigation of this issue must be an essential factor in the development of all oligoclonal antibodies. In preclinical research, the performance of the mixture should be assessed over a physiologically relevant range of ratios to ensure that activity is maintained. In clinical studies, the selection of the dose and the dosing interval should consider the pharmacokinetics of the individual component antibodies (using antibody-specific assays) to ensure that active ratios are maintained over time and across patients. This issue is exemplified by the use of oligoclonal antibodies against multiple antigens that are described to mediate different degrees of target-mediated drug disposition (TMDD). For example, it can be reasonably expected that the equimolar ratio of the six antibodies in the Sym013 combination will vary over time due to significant (via EGFR) or negligible TMDD (via HER2/ErbB2 and HER3/ErbB3). These risks may apply also to anti-infective antibodies where the multiple antigens targeted could be displayed at different and variable levels, depending on multiple factors that influence pathogen growth and clearance. • Overlapping toxicity profiles. Similar to combination drug treatment, oligoclonal antibodies harbor the potential for overlapping toxicity profiles that may be difficult to interpret and cannot be mitigated in the clinic by the adjustment of the defined formulation ratio (as is regularly done for combined use of separate mAb drug products). This is perhaps most pressing for anti-cancer antibodies that must contend with self-recognition and less pressing for anti-infective antibodies that have the inherent advantage of specificity for pathogenic antigens. Indeed, anti-infective oligoclonal antibodies are commonly tested for safety in Phase 1 studies in healthy patients. While the two instances of approved combined use of separate mAbs in oncology utilize the monotherapy dose for each mAb ( e.g . trastuzumab + pertuzumab; nivolumab + ipilimumab), rigorous preclinical and early clinical toxicology studies should be performed with oligoclonal antibodies to identify issues that would motivate reformulation (likely necessitating a new IND filing) or discontinuation. • Overlapping or elusive mechanisms of action. Oligoclonal antibodies have demonstrated several mechanisms of action that are unique to the mixture such as engagement of immune responses, enhanced receptor down-regulation, increased clearance of immune complexes, or tolerance for escape variants and other acquired resistance mechanisms. Characterization of these emergent properties , both individually or in combination, is not trivial and is complicated by the need for comprehensive model systems. For example, standard human xenograft tumor models will mask enhanced immune activities (if implanted in an immune-deficient mouse) or other tumor microenvironment effects (if one or more antibodies do not cross-react to the mouse antigen). The oligoclonal characterization strategy should be considered early in development as it will likely inform decisions in the therapeutic design and selection of the formulation ratio ( e.g. should species cross-reactivity be considered in lead selection? ). • Translation of preclinical findings to clinical development. The inherent risk in the development of oligoclonal antibodies is the necessity to have a complete and comprehensive preclinical data package to support the combination of antibodies and the formulation ratio. Beyond commercial and manufacturing considerations, this is the key differentiator from combinations of separate antibody products. Those developing oligoclonal antibodies would be wise to prioritize both early preclinical efforts and ongoing translational research. The late-stage failure of the combination of two antibodies against Clostridium difficile toxins A and B (actoxumab + bezlotoxumab), while not technically an oligoclonal antibody, is a cautionary tale for all oligoclonal antibodies. This experience highlights the need to clearly demonstrate the necessity of the oligoclonal antibody modality in rigorous preclinical studies with relevant models. Initial animal studies had demonstrated enhanced activity for the combination [ 113 ] and motivated a Phase 2 trial that assessed the activity of the combination, but not of the individual antibodies [ 31 •• ]. However, later results from two comparative Phase 3 clinical trials (MODIFY I and II) revealed no benefit of the combination over the toxin B-specific mAb alone (bezlotoxumab). Indeed, subsequent animal studies using isogenic toxin A and B mutants of a virulent C. difficile strain demonstrated that only toxin B is essential for virulence [ 114 ]. Figure 1 Practical considerations for the development of oligoclonal antibodies. (a) The underlying biology or disease context should inform the therapeutic design and generation of proof-of-concept molecules. A key consideration will be the selection of mono-specific or multi-specific antibodies. (b) Comprehensive preclinical assessment should be performed in relevant model systems to characterize the performance of the individual mAbs and the mixture (and any emergent properties of the mixture). Findings will likely, and iteratively, inform further therapeutic design. (c) The selection of the formulation ratio (the ratio of antibodies within the mixture) is perhaps the most critical decision point for the development of oligoclonal antibodies and is strongly coupled to preclinical testing, manufacturing considerations, and later clinical development. (d) There are numerous manufacturing strategies for oligoclonal antibodies, notably parallel GMP and single pot, and these will be informed by the formulation ratio and commercial considerations. The assessment of the pharmacokinetics and safety in preclinical (e) and clinical (f) studies should consider the individual mAbs as well as the mixture. Unexpected findings, such as more rapid clearance or a limited therapeutic window for an individual antibody, may necessitate a costly and time consuming reconsideration of the formulation ratio. (g) The clinical development strategy should include a strong consideration of the regulatory landscape to ensure that sufficient data will be generated to support the mixture ( e.g . improved activity of the mixture versus the individual antibodies) and/or an application for a waiver to the combination drug rule. Conclusions The conceptual utility of oligoclonal antibodies for the treatment of infectious diseases or cancer is supported by the growing list of investigational agents undergoing clinical development in both of these disease areas. Nevertheless, such therapeutics have yet to establish clinical utility and no such products have received regulatory approval, to date. Increased scientific and development focus on oligoclonal antibodies, however, suggest that the field is approaching a watershed moment. In this review, we highlight biological contexts and practical considerations that existing oligoclonal antibodies have realized throughout their development programs. In particular, we note the importance of a comprehensive preclinical data package to support both the oligoclonal format and the formulation ratio as a key differentiator from traditional antibody combinations that can be readily adjusted based upon comparative clinical studies. Comprehensive preclinical and clinical strategies to address these considerations will likely have a positive effect on the development of oligoclonal antibodies across disease areas. References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: • of special interest •• of outstanding interest

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149597/

Manufacturing Vaccines for an Emerging Viral Infection–Specific Issues Associated with the Development of a Prototype SARS Vaccine

Introduction In late 2002, several hundred cases of a severe atypical pneumonia were reported in the Guangdong Province of the People's Republic of China. By the first quarter of 2003, similar cases were reported in Hong Kong and sporadically throughout South-East Asia and Canada. This severe disease, with a mortality of 5–10%, spread rapidly around the world and in April 2003, 25 countries on 5 different continents had reported cases [World Health Organization (WHO), accumulative SARS cases]. As a result, the WHO issued on March 2003 a global alert for the illness that would be known as "Severe Acute Respiratory Syndrome" (SARS) (WHO SARS alert). Within the same time frame, secondary cases of SARS were being identified in health care workers and family members who had close contact with patients suffering from this severe respiratory illness. By the second week of June, using the WHO case definition (WHO case description), approximately 8000 SARS cases and 774 SARS-related deaths had been reported to the WHO. While the first wave of the SARS epidemic seemed to have reached its conclusion, it was completely unclear how the spread of the virus would evolve. There was no clear understanding of the animal reservoir and the impact of virus mutation and unapparent infections. Different situations could be envisaged; one scenario was that virulent SARS-coronavirus (SARS-CoV) would persist and become endemic. Another possibility was that other epidemic waves would occur or, finally, that the virus would disappear. Taking into account all the uncertainties and anticipating the worst-case scenario, many laboratories and vaccine manufacturers started working on a vaccine approach against SARS infection, largely based on what was known from animal CoVs. In this chapter, we will discuss the necessity for international cooperation and the importance of discretionary funding for rapidly developing a prototype vaccine candidate. We will review the decision-making process, the strategic choices made in terms of vaccine candidate, adjuvants, working conditions, and the safety precautions implemented at the beginning and throughout the entire production process of the SARS vaccine. In addition, we will discuss the unique challenges associated with moving a vaccine such as SARS through the regulatory process. Role of International Collaboration and U.S. National Institute of Allergy and Infectious Diseases (NIAID)—Lessons Learned With the uncertainty about the possible extent of the spread of the SARS outbreak in early 2003, there was substantial international collaboration first in the isolation and identification of the CoV, sharing of sequence data, and eventually the sharing of seed virus that would be suitable for vaccine development. In the case of the sanofi pasteur vaccine, the prototype virus was supplied by the Centers for Disease Control and Prevention (CDC) (Utah Virus P2/Vero cell P143). Furthermore, in this spirit of collaboration, sanofi pasteur provided stocks of the Vero cells for the virus isolation to better ensure that any vaccine made from these cells would be acceptable from a regulatory perspective. These were the same Vero cells as are routinely used in the commercial production of inactivated polio vaccine. In addition, there was rapid sharing of immunological reagents to ensure that the plaque-purified prototype seed virus accurately reflected the circulating epidemic SARS-CoV. One particular difficulty with rapidly emerging infections such as SARS, of course, is that they may occur anytime of the year and are unlikely to be aligned with the annual process for assigning financial and human resources and project prioritization. In order to circumvent these problems, once it became clear that the SARS-CoV could grow in Vero cells, the National Institute for Allergy and Infectious Diseases (NIAID) in the United States focused their request on several companies that had experience in large-scale Vero cell culture and had the experience to work with viruses at biosafety level 3 (BSL3) containment. Our first contact with NIAID on the possibility of developing an inactivated vaccine occurred in the first quarter of 2003. Sanofi pasteur was one of only two companies that satisfied the NIAID's eligibility criteria. The solicitation and funding mechanism that NIAID employed was a directed Request for Proposals (RFP). This is sometimes called a Letter Contract by virtue of the fact that NIAID initiated contact with the companies first by letter outlining the project objectives, infrastructure requirements, and deliverables. The advantage of this mechanism, for diseases such as SARS, is that the absolute eligibility criteria, vaccine characteristics, and project objectives are clearly defined; the proposal review cycle is very short; and the necessary financial resources are immediately available. In this particular case, the RFP was received by us on May 23, and we submitted our project plan and budget by June 16. Preliminary work began in July and August, and by the third week of September 2003, we had a fully executed contract. By October 2004, our inactivated prototype SARS vaccine was filled and available for clinical assessment. Another positive aspect of this mechanism is that involvement of such a large research organization provides the potential to access the expertise of many different investigators, as technical problems may arise. Unlike traditional research grants, these types of contracts have very short duration and, consistent with the sense of urgency, progress against objectives is monitored on a weekly basis, perhaps, contributing to the fact that our project finished almost a month ahead of schedule. Importantly, this type of mechanism does not compete with academic researchers for funding but allows these researchers to develop more basic science proposals of longer duration that can be reviewed and funded by other established mechanisms. Perhaps, among others, there are three important lessons about responding to emerging threats that can be learned from the SARS experience. First, information must be shared quickly and transparently. The somewhat surprising observation that the SARS-CoV grew well in Vero cells was important in prioritizing the development of an inactivated, Vero cell–derived virus. Second, there should be an ongoing dialogue between the national research organizations and their potential industrial partners to understand what capacity and experience could be urgently brought to bear in a crisis situation. Although there are potential issues with competitive intelligence, it remains important that information about industrial capability should be shared. Third, funding organizations should have in place a mechanism for rapid proposal solicitation, review and monitoring, and also adequate discretionary funding that could support new vaccine projects in an urgent manner. Prior to the Start of the Laboratory Work As early as April 25, 2003, the WHO and the CDC in the United States (WHO guidelines, CDC) recommended laboratories and vaccine manufacturers to handle SARS-CoV specimens using Standard BSL3 work practices. It is not exceptional for vaccine manufacturers to work in BSL3 facilities for the production of certain vaccines, e.g., rabies, Japanese encephalitits, or polio viruses. Some of these commercial vaccines have been made for decades. For these vaccines, robust production processes and standard operating procedures have been put in place. The experience gained on the decontamination and inactivation of viral vaccines during the production process and the quality control (QC) are all important. Moreover, the personnel working at the different stages of such a vaccine production are typically vaccinated, and revaccination procedures are in place in case of major accidents (i.e., boost after potential rabies contamination). In case of an emerging virus such as SARS, the situation is completely different. Although the BSL3 experience will be fully exploited, all processes and procedures need to be discussed, evaluated, validated, and implemented. For each emerging virus, this exercise needs to be repeated and specific conditions must to be adopted according to the unique characteristics of the new virus. Consistent with the WHO and CDC recommendations, at sanofi pasteur live SARS-CoV was handled in a BSL3 facility. Since no treatment or vaccine was available against SARS, we decided to work in "BSL3 plus" laboratory conditions. Essentially our basic level of containment was BSL3 incorporating several BSL4 working practices: clothing change before entering the facilities, shower on exit, and all material decontaminated before exit from the facilities. Also, all steps, where the product was handled in open phase, were performed within Class III Biological Safety Cabinets (CSB). To prevent accidental contamination, the laboratory workers wore a positive pressure personal mask. These precautions were considered necessary taking into consideration the large amount of live virus handled (20–50 l) and finally turned out to be valid as the risk for contamination existed as was shown in the laboratory accidents in Singapore, Taiwan, and Beijing in China ( Senior, 2003 ; Normile, 2004 ; Orellana, 2004 ). Furthermore, as described below, the decontamination experiments demonstrated that the SARS-CoV is an extremely resistant virus. For the medical follow-up of the BSL3-trained personal involved in the SARS vaccine development, it was of critical importance to be able to distinguish the symptoms of respiratory distress caused by SARS or other respiratory agents. It was therefore decided that the BSL3 laboratory workers should be selected in accordance with their immune status and would be immunized with Streptococcus pneumoniae and influenza vaccines, if appropriate. In case a worker would present symptoms, a procedure had been put in place to isolate the worker using a high-efficiency particulate air (HEPA) filter mask before being transported to a nearby hospital where a special, dedicated negative pressure hospital room had been prepared. Starting the Laboratory Work: First Steps Definition of the Vaccine Profile The very first decision concerned the choice of vaccine immunization strategy. In the intense early days of the epidemic a variety of approaches were considered. These included inactivated vaccines, subunit products, DNA (either alone or in combination as part of a prime-boost strategy), vectored vaccines, and live attenuated candidates. Similarly, alternative routes of vaccine administration (inoculation, aerosol, etc.) and formulation (adjuvants, etc.) were considered. In fact, several live attenuated and killed vaccines for veterinary CoVs are already marketed: e.g., a live attenuated and killed vaccine against the chicken infectious bronchitis virus ( Ladman et al., 2002 ), a live modified virus against canine CoV ( Pratelli et al., 2004 ). Ultimately, however, the crucial factor in the decision-making process was likely development timelines. At the time of this decision, the epidemic was prevalent and there was a sense of urgency that a prototype vaccine had to be developed as soon as possible. Embedded in this decision-making process was the realization that we were seeking a vaccine candidate that largely used existing, conventional technology and that could be made in significant quantities, if necessary. In the context of the likelihood for rapid development and the possibility for large-scale production, the decision was taken to produce a monovalent, whole, inactivated, aluminum-adjuvanted SARS vaccine for intramuscular injection. From our perspective, a whole, inactivated vaccine was a logical first choice taking into consideration our extensive experience with inactivated vaccines such as inactivated poliovirus, rabies, and hepatitis A vaccines. We were also encouraged by the fact that SARS-CoV grew very well in Vero cells. Sanofi pasteur has a long industrial experience with these particular cells and developed cell banks that are validated and registered around the world. Even for such a direct experience-based approach, we realized that it would take 12 months to make a good manufacturing practice (GMP) clinical lot for Phase 1 and 2 clinical studies. Killed vaccines need, in many cases, adjuvantation. In our case, the adjuvant of choice was aluminum hydroxide. Again, in the situation where a new virus is emerging, there is no time to evaluate new adjuvants. Our preference was to use aluminum hydroxide since it is well known, used in many vaccines, and well accepted by regulatory authorities. SARS Inactivation and Testing Reagents When the laboratory work on the SARS-CoV vaccine development started, no data were available on the inactivation characteristics of the virus. One of the priorities was to identify the conditions to fully inactivate the virus for vaccine development, but also for decontamination of equipment, facilities, and waste decontamination. The results, as described in more detail below, were unexpected. The SARS-CoV is an extremely resistant virus and several of the routine decontamination working practices cannot be applied for this virus. These results demonstrate how important it is to immediately perform decontamination testing and adapt decontamination practices and strategies for each specific (emerging) virus. At the time the development of a vaccine against an emerging virus is initiated, it is very unlikely that routine tests and reagents are available. This was indeed the case for SARS. Therefore, our first experiments were dedicated to develop routine tests that are required for vaccine development and the analysis of the host response after immunization. These include neutralization tests, enzyme-linked immunosorbent assays (ELISA), polymerase chain reactions (PCRs), and others. Well-validated reagents are needed as reference standards for essential laboratory tests as polyclonal and monoclonal antibodies, recombinant proteins, and PCR primer pairs. One of the most sensitive issues was how to select an appropriate animal model to evaluate the candidate vaccines. For example, we observed that NMRI mice gave a very heterogeneous response whereas Balb-C or C57BL/6J mice responded uniformly to immunization with the vaccine candidate. Also, guinea pigs appeared to be a good model for the evaluation of immune responses. Beyond immunogenicity, it is important to work with an animal model that is appropriate for challenge studies, as an assessment of vaccine efficacy. This is especially important for emerging infections since efficacy studies in humans may not be possible. In case of the SARS, the Macaca fascicularis was identified very early on ( Fouchier et al., 2003 ) as a likely predictive, challenge model. All of the work was done in constant communication with the regulatory authorities. Our experience with SARS reinforces the idea that manufacturers should be encouraged to open and maintain an active dialogue with regulatory officials, very early in the development process. Results of the Decontamination Experiments Viruses can be inactivated by several methods, based on either physical or chemical mechanisms. We investigated five decontamination methods that are currently used for equipment, facilities, and waste decontamination: heat, alkaline treatment, sodium hypochlorite treatment, gaseous formol fumigation, and drying. It should be stressed that the data presented here have been obtained under our specific experimental conditions. Indeed, the virus sensitivity to inactivation depends on the virus environment and concentration. Thus, the methods presented here were validated with our specific suspensions and experimental conditions, and appropriate precautions should be taken when manipulating SARS-CoV under other laboratory conditions. Inactivation experiments using different pH values gave rise to unexpected results. When the pH was adjusted to pH13 or pH13.5, a strong decrease in the viral infectivity titer can be observed. However, the virus is not totally inactivated and even after 6 h of alkaline treatment, infectious particles can still be observed. Total inactivation is observed only after 24 h of treatment. These data are surprising as enveloped viruses are usually sensitive to such drastic alkaline conditions. The results from the experiments performed to evaluate the viral loss of the SARS-CoV due to drying on glass surface were also surprising: 35–42 days were necessary to inactivate the virus to the detection limit of the technique. Other viruses, such as polio or rabies, can be inactivated by drying durations of approximately 72 and 144 h, respectively. In our experience, the SARS-CoV is the most resistant virus ever described in an industrial setting. The gaseous formaldehyde fumigation is a viral decontamination technique widely used throughout the world. We found that formol fumigation is totally inefficient on dried virus. However, virus in solution is efficiently inactivated. The two decontamination techniques tested here, drying and formaldehyde fumigation, reinforce the necessity to decontaminate the working areas in the laboratory, as well as the equipment, very frequently in order to avoid any drying of viral suspension onto glass or any other surface. Finally, sodium hypochlorite (2°Cl) and heat treatment were evaluated. The effect of sodium hypochlorite on dried virus is very rapid and efficient, as no infectious viral particles were recovered after washing the surface with sodium hypochlorite. Thermal decontamination was shown to be efficient at both 58 and 68°C. To achieve total inactivation of the SARS-CoV, 1 h heating at 68°C and 2 h heating at 58°C are necessary. Considering the decontamination data, the following strategy was put in place. All solid waste, as well as the equipment that could resist such a drastic treatment, was autoclaved. For liquid waste, the solutions were subjected to alkaline pH treatment for at least 2 h and then transferred into a tank for thermal decontamination at 105°C for 30 min. The laboratory facilities, and the equipment that could not be autoclaved, were decontaminated with 2°Cl sodium hypochlorite solutions, before being fumigated with gaseous formol. The data on decontamination of the SARS-CoV presented here show that existing decontamination strategies cannot be directly extrapolated to emerging viruses and that these inactivation conditions should be determined empirically for each virus. Definition of the Vaccine Profile The very first decision concerned the choice of vaccine immunization strategy. In the intense early days of the epidemic a variety of approaches were considered. These included inactivated vaccines, subunit products, DNA (either alone or in combination as part of a prime-boost strategy), vectored vaccines, and live attenuated candidates. Similarly, alternative routes of vaccine administration (inoculation, aerosol, etc.) and formulation (adjuvants, etc.) were considered. In fact, several live attenuated and killed vaccines for veterinary CoVs are already marketed: e.g., a live attenuated and killed vaccine against the chicken infectious bronchitis virus ( Ladman et al., 2002 ), a live modified virus against canine CoV ( Pratelli et al., 2004 ). Ultimately, however, the crucial factor in the decision-making process was likely development timelines. At the time of this decision, the epidemic was prevalent and there was a sense of urgency that a prototype vaccine had to be developed as soon as possible. Embedded in this decision-making process was the realization that we were seeking a vaccine candidate that largely used existing, conventional technology and that could be made in significant quantities, if necessary. In the context of the likelihood for rapid development and the possibility for large-scale production, the decision was taken to produce a monovalent, whole, inactivated, aluminum-adjuvanted SARS vaccine for intramuscular injection. From our perspective, a whole, inactivated vaccine was a logical first choice taking into consideration our extensive experience with inactivated vaccines such as inactivated poliovirus, rabies, and hepatitis A vaccines. We were also encouraged by the fact that SARS-CoV grew very well in Vero cells. Sanofi pasteur has a long industrial experience with these particular cells and developed cell banks that are validated and registered around the world. Even for such a direct experience-based approach, we realized that it would take 12 months to make a good manufacturing practice (GMP) clinical lot for Phase 1 and 2 clinical studies. Killed vaccines need, in many cases, adjuvantation. In our case, the adjuvant of choice was aluminum hydroxide. Again, in the situation where a new virus is emerging, there is no time to evaluate new adjuvants. Our preference was to use aluminum hydroxide since it is well known, used in many vaccines, and well accepted by regulatory authorities. SARS Inactivation and Testing Reagents When the laboratory work on the SARS-CoV vaccine development started, no data were available on the inactivation characteristics of the virus. One of the priorities was to identify the conditions to fully inactivate the virus for vaccine development, but also for decontamination of equipment, facilities, and waste decontamination. The results, as described in more detail below, were unexpected. The SARS-CoV is an extremely resistant virus and several of the routine decontamination working practices cannot be applied for this virus. These results demonstrate how important it is to immediately perform decontamination testing and adapt decontamination practices and strategies for each specific (emerging) virus. At the time the development of a vaccine against an emerging virus is initiated, it is very unlikely that routine tests and reagents are available. This was indeed the case for SARS. Therefore, our first experiments were dedicated to develop routine tests that are required for vaccine development and the analysis of the host response after immunization. These include neutralization tests, enzyme-linked immunosorbent assays (ELISA), polymerase chain reactions (PCRs), and others. Well-validated reagents are needed as reference standards for essential laboratory tests as polyclonal and monoclonal antibodies, recombinant proteins, and PCR primer pairs. One of the most sensitive issues was how to select an appropriate animal model to evaluate the candidate vaccines. For example, we observed that NMRI mice gave a very heterogeneous response whereas Balb-C or C57BL/6J mice responded uniformly to immunization with the vaccine candidate. Also, guinea pigs appeared to be a good model for the evaluation of immune responses. Beyond immunogenicity, it is important to work with an animal model that is appropriate for challenge studies, as an assessment of vaccine efficacy. This is especially important for emerging infections since efficacy studies in humans may not be possible. In case of the SARS, the Macaca fascicularis was identified very early on ( Fouchier et al., 2003 ) as a likely predictive, challenge model. All of the work was done in constant communication with the regulatory authorities. Our experience with SARS reinforces the idea that manufacturers should be encouraged to open and maintain an active dialogue with regulatory officials, very early in the development process. Results of the Decontamination Experiments Viruses can be inactivated by several methods, based on either physical or chemical mechanisms. We investigated five decontamination methods that are currently used for equipment, facilities, and waste decontamination: heat, alkaline treatment, sodium hypochlorite treatment, gaseous formol fumigation, and drying. It should be stressed that the data presented here have been obtained under our specific experimental conditions. Indeed, the virus sensitivity to inactivation depends on the virus environment and concentration. Thus, the methods presented here were validated with our specific suspensions and experimental conditions, and appropriate precautions should be taken when manipulating SARS-CoV under other laboratory conditions. Inactivation experiments using different pH values gave rise to unexpected results. When the pH was adjusted to pH13 or pH13.5, a strong decrease in the viral infectivity titer can be observed. However, the virus is not totally inactivated and even after 6 h of alkaline treatment, infectious particles can still be observed. Total inactivation is observed only after 24 h of treatment. These data are surprising as enveloped viruses are usually sensitive to such drastic alkaline conditions. The results from the experiments performed to evaluate the viral loss of the SARS-CoV due to drying on glass surface were also surprising: 35–42 days were necessary to inactivate the virus to the detection limit of the technique. Other viruses, such as polio or rabies, can be inactivated by drying durations of approximately 72 and 144 h, respectively. In our experience, the SARS-CoV is the most resistant virus ever described in an industrial setting. The gaseous formaldehyde fumigation is a viral decontamination technique widely used throughout the world. We found that formol fumigation is totally inefficient on dried virus. However, virus in solution is efficiently inactivated. The two decontamination techniques tested here, drying and formaldehyde fumigation, reinforce the necessity to decontaminate the working areas in the laboratory, as well as the equipment, very frequently in order to avoid any drying of viral suspension onto glass or any other surface. Finally, sodium hypochlorite (2°Cl) and heat treatment were evaluated. The effect of sodium hypochlorite on dried virus is very rapid and efficient, as no infectious viral particles were recovered after washing the surface with sodium hypochlorite. Thermal decontamination was shown to be efficient at both 58 and 68°C. To achieve total inactivation of the SARS-CoV, 1 h heating at 68°C and 2 h heating at 58°C are necessary. Considering the decontamination data, the following strategy was put in place. All solid waste, as well as the equipment that could resist such a drastic treatment, was autoclaved. For liquid waste, the solutions were subjected to alkaline pH treatment for at least 2 h and then transferred into a tank for thermal decontamination at 105°C for 30 min. The laboratory facilities, and the equipment that could not be autoclaved, were decontaminated with 2°Cl sodium hypochlorite solutions, before being fumigated with gaseous formol. The data on decontamination of the SARS-CoV presented here show that existing decontamination strategies cannot be directly extrapolated to emerging viruses and that these inactivation conditions should be determined empirically for each virus. The Production Process Viral Source and Raw Material The SARS-CoV seed virus isolate was provided on August 25, 2003, by the CDC. This isolate (the so-called UTAH strain) was made from the sputum from an acutely ill U.S. traveler who had apparently been exposed in Hong Kong. This isolate was fully sequenced by the CDC and shown to be virtually identical to the Urbani strain of SARS-CoV. To obtain an original seed virus, in full accordance with Food and Drug Administration (FDA) requirements, sanofi pasteur provided certified Vero cells to the CDC, who performed the isolation of the virus and made two passages before sending the virus to sanofi pasteur. Upon receipt, the virus underwent two additional passages, and was plaque purified. This plaque purification step is of importance to limit the risk of adventitious agents during the subsequent expansion of the virus. In contrast, there is also a risk that in selecting a plaque, it may differ significantly from the uncloned vaccine. To evaluate the latter, it was decided to compare cloned vs. uncloned virus in terms of virus sequence and immunogenicity in guinea pigs. It was demonstrated that the two candidate vaccines were totally similar. Following this verification, the selected clone (VVNFL11) was passaged eight additional times for adaptation. From our experience with viruses to be used to prepare an inactivated viral vaccine at industrial scale, there is a need to reach a titer of virus >7.0 log/ml. Indeed, with the SARS-CoV, we obtained a consistent titer of around 7.3 log 10 TCID 50 /ml. In Vero cells, distinct cytopathic effect (CPE) is always observed at day 2 or 3 post-infection. These first experiments encouraged us that the prototype vaccine could be produced in Vero cells using a single harvest totally compatible with our experience with inactivated poliovirus. Raw materials used to develop the candidate vaccine (serum and trypsin) were selected in accordance with current regulation. Calf serum was imported from Australia, and gamma irradiated prior to use. The trypsin was from porcine origin and also gamma irradiated. Extensive evaluation for adventitious agents was performed on the raw material, which included the search for cytopathogenic agents, hemadsorbing agents, and specific viral contaminants as bluetongue, reovirus, rabies, parainfluenza type 3, specific bovine viruses [adenovirus, parvovirus, respiratory syncytial virus (RSV)], bovine viral diarrhea, rhinotracheitis virus), and porcine viruses (parvovirus, adenovirus; transmissible gastroenteritis virus; hepatitis E virus, rabies virus, and porcine pestis virus). Manufacturing of the Viral Seed Lots The viral seed lots were produced in Vero cells. QC testing is a major step in the qualification of such a seed. Of all the different tests performed (identification of the Vero cells, sterility, mycoplasma, titer, and contaminating viruses), the research of contaminating virus(es), also called adventitious agents, represents the most crucial step. To detect adventitious agents, sensitive cell culture monolayers of CV-1 cells, human diploid MRC-5 cells, and chick embryo fibroblasts (CEFs) were inoculated with the crude viral suspension and are observed for induced CPE and/or hemadsorption. CV-1 cells are used in these tests, as they are of the same species and the same origin as the cells used for vaccine manufacturing. MRC-5 cells are human diploid cells and can potentially reveal other viruses able to infect human cells. And finally, taking into consideration the origin of the specimen (pulmonary syndrome) we added CEF, which are known to be sensitive to the infection of several respiratory viruses such as influenza virus and RSV. At the same time, adventitious agent testing was performed on control cells (search for hemagglutining or hemadsorbant viruses) and on the supernatant of the control cells (search for adventitious agents) by inoculation of the three cell lines: Vero, MRC-5, and CEF. Adventitious agent testing was also done in vivo by inoculation in suckling mice, mice, and guinea pigs, as well as the allantoïc cavity and yolk sac of embryonated chicken eggs. Complementary to the conventional methods to qualify viral seed, as described above, extensive characterization was performed using PCR testing. The PCR tests are listed in Table 11.1 . Table 11.1 List of viruses for which the crude SARS-CoV viral suspension was monitored by PCR Detection of viruses by PCR Adenovirus Bovine and porcine circovirus Bovine herpesvirus—I, IV Bovine polyoma virus Cytomegalovirus Epstein-Barr virus Hepatitis A virus Hepatitis B virus Hepatitis C virus Human herpes virus (HHV)-6 and HHV-7 HHV-8 Human immunodeficiency virus (HIV)-1 and HIV-2 Human respiratory syncytial virus Herpes simplex virus Human T-cell leukemia virus type-1 (HTLV-1) Human T-cell leukemia virus type-2 (HTLV-2) Human papilloma virus Human parainfluenza virus—1, 2, 3 Human polyoma virus Metapneumovirus Parvovirus B19 Simian spumavirus Simian immunodeficiency virus Simian retrovirus—1, 2, 3/SMRV Simian T-cell lymphotropic virus Simian virus 40 All tests were performed in accordance with international requirements, showing that no adventitious agents were detected. Results of the Inactivation Experiments The difficulty with inactivating viruses remains the balance between fully validated inactivation and preservation of immunogenicity or epitopes associated with protection. It is well known that reagents used to inactivate viruses [betapropiolactone (BPL), formaldehyde] can change the outer membrane antigens with the risk of a reduced immunogenicity of the vaccine. For the inactivation of SARS-CoV, we chose to test BPL. Assays were performed to determine the best BPL concentration for inactivation while maintaining a good immune response in mice. Virus inactivation was performed using three different BPL concentrations: 1/2000, 1/3000, and 1/4000 (v/v). In our experience, a 1/4000 dilution of BPL was the optimal concentration for the inactivation of SARS-CoV based on a balance between total inactivation and maintenance of antigenic properties. The 1/4000 BPL dilution is similar to what is used for rabies vaccine inactivation. The usual way to measure viral inactivation is by kinetic studies, i.e., reduction in virus infectivity. This technique has a detection limit of 1.5 log 10 TCID 50 /ml. The kinetics of inactivation was performed at 0, 5, 30 min, and 1, 2, 3, 4, 6, 8, and 24 h, and it was shown that inactivation below the limit of detection was obtained after 6 h. In order to increase the detection sensitivity, an amplification test was also performed. For this amplification, Vero cells are incubated with a portion of the viral solution following inactivation for different periods of time. After 7 days of incubation, the cells are trypsinized and cultivated for additional 14 days. At the different stages of amplification, the cells were microscopically observed for CPE and at the end of the incubation (day 21) an immunocolorimetric assay test was performed. Our data demonstrated that the virus is fully inactivated by 12 h, not 6 h of BPL treatment, demonstrating that the amplification test is much more sensitive. To complete the validation of the amplification test, the minimum limit of detectable infectious viral particles was determined. This was done by spiking the inactivated vaccine with different concentrations of live virus and incubating with Vero cells. Using this approach, it was possible to establish the minimum virus detection as 1 pfu. Based on these data, it was concluded that inactivation with 1/4000 BPL dilution for 12 h fully inactivates the SARS-CoV batches. To ensure a very large safety margin, we adopted an inactivation period of 24 h for the SARS-CoV vaccine production process. Viral Source and Raw Material The SARS-CoV seed virus isolate was provided on August 25, 2003, by the CDC. This isolate (the so-called UTAH strain) was made from the sputum from an acutely ill U.S. traveler who had apparently been exposed in Hong Kong. This isolate was fully sequenced by the CDC and shown to be virtually identical to the Urbani strain of SARS-CoV. To obtain an original seed virus, in full accordance with Food and Drug Administration (FDA) requirements, sanofi pasteur provided certified Vero cells to the CDC, who performed the isolation of the virus and made two passages before sending the virus to sanofi pasteur. Upon receipt, the virus underwent two additional passages, and was plaque purified. This plaque purification step is of importance to limit the risk of adventitious agents during the subsequent expansion of the virus. In contrast, there is also a risk that in selecting a plaque, it may differ significantly from the uncloned vaccine. To evaluate the latter, it was decided to compare cloned vs. uncloned virus in terms of virus sequence and immunogenicity in guinea pigs. It was demonstrated that the two candidate vaccines were totally similar. Following this verification, the selected clone (VVNFL11) was passaged eight additional times for adaptation. From our experience with viruses to be used to prepare an inactivated viral vaccine at industrial scale, there is a need to reach a titer of virus >7.0 log/ml. Indeed, with the SARS-CoV, we obtained a consistent titer of around 7.3 log 10 TCID 50 /ml. In Vero cells, distinct cytopathic effect (CPE) is always observed at day 2 or 3 post-infection. These first experiments encouraged us that the prototype vaccine could be produced in Vero cells using a single harvest totally compatible with our experience with inactivated poliovirus. Raw materials used to develop the candidate vaccine (serum and trypsin) were selected in accordance with current regulation. Calf serum was imported from Australia, and gamma irradiated prior to use. The trypsin was from porcine origin and also gamma irradiated. Extensive evaluation for adventitious agents was performed on the raw material, which included the search for cytopathogenic agents, hemadsorbing agents, and specific viral contaminants as bluetongue, reovirus, rabies, parainfluenza type 3, specific bovine viruses [adenovirus, parvovirus, respiratory syncytial virus (RSV)], bovine viral diarrhea, rhinotracheitis virus), and porcine viruses (parvovirus, adenovirus; transmissible gastroenteritis virus; hepatitis E virus, rabies virus, and porcine pestis virus). Manufacturing of the Viral Seed Lots The viral seed lots were produced in Vero cells. QC testing is a major step in the qualification of such a seed. Of all the different tests performed (identification of the Vero cells, sterility, mycoplasma, titer, and contaminating viruses), the research of contaminating virus(es), also called adventitious agents, represents the most crucial step. To detect adventitious agents, sensitive cell culture monolayers of CV-1 cells, human diploid MRC-5 cells, and chick embryo fibroblasts (CEFs) were inoculated with the crude viral suspension and are observed for induced CPE and/or hemadsorption. CV-1 cells are used in these tests, as they are of the same species and the same origin as the cells used for vaccine manufacturing. MRC-5 cells are human diploid cells and can potentially reveal other viruses able to infect human cells. And finally, taking into consideration the origin of the specimen (pulmonary syndrome) we added CEF, which are known to be sensitive to the infection of several respiratory viruses such as influenza virus and RSV. At the same time, adventitious agent testing was performed on control cells (search for hemagglutining or hemadsorbant viruses) and on the supernatant of the control cells (search for adventitious agents) by inoculation of the three cell lines: Vero, MRC-5, and CEF. Adventitious agent testing was also done in vivo by inoculation in suckling mice, mice, and guinea pigs, as well as the allantoïc cavity and yolk sac of embryonated chicken eggs. Complementary to the conventional methods to qualify viral seed, as described above, extensive characterization was performed using PCR testing. The PCR tests are listed in Table 11.1 . Table 11.1 List of viruses for which the crude SARS-CoV viral suspension was monitored by PCR Detection of viruses by PCR Adenovirus Bovine and porcine circovirus Bovine herpesvirus—I, IV Bovine polyoma virus Cytomegalovirus Epstein-Barr virus Hepatitis A virus Hepatitis B virus Hepatitis C virus Human herpes virus (HHV)-6 and HHV-7 HHV-8 Human immunodeficiency virus (HIV)-1 and HIV-2 Human respiratory syncytial virus Herpes simplex virus Human T-cell leukemia virus type-1 (HTLV-1) Human T-cell leukemia virus type-2 (HTLV-2) Human papilloma virus Human parainfluenza virus—1, 2, 3 Human polyoma virus Metapneumovirus Parvovirus B19 Simian spumavirus Simian immunodeficiency virus Simian retrovirus—1, 2, 3/SMRV Simian T-cell lymphotropic virus Simian virus 40 All tests were performed in accordance with international requirements, showing that no adventitious agents were detected. Results of the Inactivation Experiments The difficulty with inactivating viruses remains the balance between fully validated inactivation and preservation of immunogenicity or epitopes associated with protection. It is well known that reagents used to inactivate viruses [betapropiolactone (BPL), formaldehyde] can change the outer membrane antigens with the risk of a reduced immunogenicity of the vaccine. For the inactivation of SARS-CoV, we chose to test BPL. Assays were performed to determine the best BPL concentration for inactivation while maintaining a good immune response in mice. Virus inactivation was performed using three different BPL concentrations: 1/2000, 1/3000, and 1/4000 (v/v). In our experience, a 1/4000 dilution of BPL was the optimal concentration for the inactivation of SARS-CoV based on a balance between total inactivation and maintenance of antigenic properties. The 1/4000 BPL dilution is similar to what is used for rabies vaccine inactivation. The usual way to measure viral inactivation is by kinetic studies, i.e., reduction in virus infectivity. This technique has a detection limit of 1.5 log 10 TCID 50 /ml. The kinetics of inactivation was performed at 0, 5, 30 min, and 1, 2, 3, 4, 6, 8, and 24 h, and it was shown that inactivation below the limit of detection was obtained after 6 h. In order to increase the detection sensitivity, an amplification test was also performed. For this amplification, Vero cells are incubated with a portion of the viral solution following inactivation for different periods of time. After 7 days of incubation, the cells are trypsinized and cultivated for additional 14 days. At the different stages of amplification, the cells were microscopically observed for CPE and at the end of the incubation (day 21) an immunocolorimetric assay test was performed. Our data demonstrated that the virus is fully inactivated by 12 h, not 6 h of BPL treatment, demonstrating that the amplification test is much more sensitive. To complete the validation of the amplification test, the minimum limit of detectable infectious viral particles was determined. This was done by spiking the inactivated vaccine with different concentrations of live virus and incubating with Vero cells. Using this approach, it was possible to establish the minimum virus detection as 1 pfu. Based on these data, it was concluded that inactivation with 1/4000 BPL dilution for 12 h fully inactivates the SARS-CoV batches. To ensure a very large safety margin, we adopted an inactivation period of 24 h for the SARS-CoV vaccine production process. Animal Models Since a direct efficacy trial in humans will be impossible, because of a lack of naturally circulating SARS-CoV, the licensure of a SARS-CoV vaccine will depend on surrogate markers. Recently (as described below) the FDA adopted the Animal Efficacy Rule that envisions that under such circumstances, demonstration of efficacy can be performed in two animal models. For SARS-CoV vaccine development, monkeys and ferrets can be used to evaluate candidate vaccine. Both animal models show pathology in the lungs upon autopsy. The immunogenicity of the SARS vaccine was evaluated in nonhuman primates, M. fascicularis, and ferrets. Both animal models are susceptible to infection, do show some signs of disease (lethargy), and show signs of pulmonary lesions upon histological examination ( Fouchier et al., 2003 ; Martina et al., 2003 ; ter Meulen et al., 2004 ; Rowe et al., 2004 ; McAuliffe et al., 2004 ). Different doses of the SARS vaccine (6 or 7 log 10 TCID 50 /ml) were injected in the presence or absence of aluminum hydroxide. Two intramuscular injections were performed at a one month interval. Regarding the humoral response, sustained levels of ELISA and serum-neutralizing virus-specific antibodies were elicited in vaccinated monkeys and ferrets. A significant dose–effect relationship could be demonstrated. Moreover, a strong adjuvant effect of aluminum hydroxide was evidenced for each vaccine dose and proved in most cases to be highly significant. In order to evaluate the efficacy of the SARS vaccine, immunized monkeys and ferrets were challenged intratracheally with a heterologous Hong Kong SARS-CoV strain (Coronovative, Rotterdam, The Netherlands). Monkeys immunized with 6 or 7 log 10 of inactivated virus were protected as measured by RT-PCR and viral titration on lung samples five days post-challenge. The ferrets were protected at the lower immunization dose of 5 or 6 log 10 . Based on our experience to date, the inactivated, adjuvanted SARS-CoV prototype vaccine seems to be a good candidate for further evaluation in Phase 1 studies. Regulatory Approval Process—some Unique Aspects of SARS and other Emerging Diseases As with other vaccines, vaccines for SARS and other emerging threats need to follow a structured pattern of regulatory development. The initial stages would be very similar to those followed for vaccines under development for conventional infectious diseases. In the United States, the earliest stages would include the development of sufficient preclinical information about the vaccine to allow the preparation of an investigational new drug (IND) application for submission to the FDA (see Chapter 13). The IND may have information unique to the vaccine candidate but should include information about the rationale for the vaccine design, the source of the virus and other components, the manufacture of the active vaccine component, formulation, preliminary characterization of the vaccine including purity and potential contaminants, immunological testing, and animal testing, including toxicology. Even at this early stage, the vaccine should be made under GMP conditions and other laboratory work conducted under good laboratory practice (GLP) conditions, as appropriate. The IND application should also include important information about the Phase 1 clinical design, focusing on how the safety will be monitored and a discussion of any potential adverse reactions based on the experience with vaccines that have similar components or methods of preparation. There should be an opportunity to outline the vaccine concept and Phase 1 clinical study at a pre-IND meeting that often provides the opportunity to receive the input and concerns of the regulatory agency. Following approval of the IND, vaccines such as SARS can progress to a conventionally designed Phase 1 study. Typically, this Phase 1 clinical study is descriptive and would include a small number of healthy young adults with the emphasis on monitoring the safety of the vaccine (local and systemic reactions). Often, the first immunologic assessment is part of this study. Following successful completion of the Phase 1, as with other vaccines, a SARS vaccine candidate could move forward to Phase 2. During this phase, in addition to safety monitoring, dose-ranging studies are conducted in much larger groups of individuals and the vaccine should meet predefined primary and secondary endpoints. If the Phase 2 is successful, following a pre-Phase 3 meeting, clinical studies are conducted in a greater number of subjects during which less frequent reactions can be detected and the efficacy or effectiveness of the vaccine determined. As part of this Phase 3 evaluation, the consistency of sequential lots of the vaccine are typically compared in order to ensure that the vaccine can be reproducibly manufactured. Obviously, as the vaccine progresses clinically from Phase 1 to Phase 3, the size of the lots of vaccine often increases and the manufacturing and in-process and release testing specifications become increasingly well defined, so as to guarantee that the vaccine can consistently be made at a commercially useful scale. If all three phases of clinical development are successful, the manufacturer may then submit a Biologics License Application (BLA), which is a very extensive compilation of all of the information relating to the development and manufacture of the vaccine. As suggested in the Animal Models section above, the unique challenge for SARS and other emerging threats, whether anthrax or Ebola viruses, is that it may not be possible to conduct Phase 3 clinical studies to determine the effectiveness of the vaccine. For these agents, it would be too dangerous to conduct challenge studies in humans and the prevalence of the disease is either nonexistent, sporadic, or too small to allow the development of a reasonable clinical protocol. In anticipation of this problem, largely in the face of potential bioterrorist agents, in 2002, the FDA adapted the so-called Animal Rule [see Federal Register, May 31, 2002 (Volume 67, Number 105)]. Under this guidance, new drugs or biological products that are intended to prevent serious or life-threatening conditions may be approved on evidence of effectiveness derived from appropriate studies in animals and any additional supporting data, if controlled clinical studies cannot be conducted in human volunteers and field trials are not possible. In order to satisfy this alternative mechanism, however, several criteria must be met. First, there is a reasonably well-understood pathophysiological mechanism that can be ameliorated or prevented by the product. Second, the effect is demonstrated in more than one animal species, unless it is demonstrated in a single species that represents a sufficiently well-characterized animal model. Third, the animal study endpoint is clearly related to the desired benefit in humans. And finally, the data are sufficiently well understood to allow selection of an effective dose in humans. It is therefore reasonable to expect that the effectiveness of the product in animal model(s) is a reliable indicator of its effectiveness in humans. Obviously, it is too early to know whether the SARS vaccine candidate as described in this chapter will move forward and be able to meet all of the criteria of the Animal Rule. In particular, SARS vaccine development is hindered by relatively little information about human CoVs in general. Until the rapid emergence of SARS, most of the basic research was focused on animal CoVs and our inactivated SARS vaccine candidate described in this chapter is exclusively based on experience with vaccines to animal CoVs. Certainly, it is too early to conclude whether the ferret and/or M. fascicularis is/are the most appropriate model(s) for human SARS infections. As a result, except for clinical cases documented during the outbreak, there is relatively little information about SARS pathogenesis and correlates of immunity. Another difficult aspect is that a feline infectious peritonitis (FIP) vaccine was actually harmful to the health of the immunized cats upon challenge with wild-type FIP virus ( Weiss and Scott, 1981 ). Before moving forward with approval, therefore, it will be very important to determine whether these adverse outcomes can be prompted or mimicked by any of the SARS vaccine candidates. Conclusions What Were the Parameters for the Rapid Development of the SARS Vaccine? The production of a GMP clinical lot of a mono­valent, whole, inactivated, aluminum hydroxide-adjuvanted SARS-CoV vaccine took 12 months. In terms of vaccine development, this is extremely rapid. Several factors contributed to these short timelines. The grants made available by the NIAID for the development of a SARS vaccine completely changed the classical environment, allowing vaccine industries to start almost immediately the development of a new vaccine. Indeed, the development of a new vaccine can only be done to the detriment of other vaccine developments, mobilizing teams and facilities. From a technical point of view, the choice of a classical vaccine development strategy using conventional procedures, such as Vero cell culture for viral propagation and BPL inactivation, was a decisive factor to success. Importantly, we were able to quickly recruit a volunteer workforce that was both familiar with the technology and trained to work in a BSL3-plus environment. A close collaboration with the reference laboratory, the CDC's Influenza Branch, where the SARS-CoV was isolated, was essential. We provided certified Vero cells to the CDC, which allowed us, upon receipt of the purified strain from the CDC, to re-isolate the SARS-CoV under conditions making the prompt start of a vaccine development possible. When initiating vaccine development against a new emerging infectious agent, the problem of availability of reagents and routine tests to perform biological and molecular studies must be addressed. It is obvious, that at the beginning of such development, there are no such reagents or commercial kits available. As a consequence, the first step in the SARS-CoV project was to prepare the different reagents (antisera and monoclonal antibodies) and the appropriate tests (viral titration, PCR, ELISA, immunofluorescence assay, etc.). Finally, a series of preliminary experiments on monkeys (three months after the start of the project) had given guidance whether it was appropriate to use an inactivated vaccine, as well as to the choice of the adjuvant. Constant communication with regulatory authorities has allowed the validation of this strategy from the beginning of the project. This communication was also very important for the qualification of the viral seed lots. The qualification of the Vero cells was not an issue as several vaccines are already produced in Vero cells, but this was obviously not the case for the viral seeds. Two major obstacles had to be overcome: (1) the realization that the animal testing had to be done in BSL3 facilities by BSL3-trained personnel, and (2) the search for adventitious agents using general classical tests (search for adventitious viruses on cells and in animals) and specific tests (PCR). For the latter, there was no list available and the final testing to be performed was under the responsibility of health authorities. This resulted in a rather exhaustive list of PCR testing. Can We Shorten the Timelines Even More When Facing an Emerging Pathogen? It is likely that epidemics will emerge in the future from unrecognized sources and some of these will be highly pathogenic for humans. These pathogens will be categorized as BSL3 or BSL4 pathogens needing high security level laboratories as well as specialized personnel. How to manipulate these pathogens that are highly pathogenic, in large quantities? To face the emergence of new pathogens, dedicated structures are needed with the right equipment and trained personnel. From an industrial perspective, this seems not compatible with the need and use of trained personnel and facilities that do have a constant activity to assure the production of existing vaccines and the development of new vaccines. Such emergency structures could be set up and maintained by national reference centers respecting the BSL requirements as well as the GMP conditions. It would be very beneficial for industries to collaborate with such reference centers that provide purified pathogens and reagents allowing a prompt start of a vaccine development. What Were the Parameters for the Rapid Development of the SARS Vaccine? The production of a GMP clinical lot of a mono­valent, whole, inactivated, aluminum hydroxide-adjuvanted SARS-CoV vaccine took 12 months. In terms of vaccine development, this is extremely rapid. Several factors contributed to these short timelines. The grants made available by the NIAID for the development of a SARS vaccine completely changed the classical environment, allowing vaccine industries to start almost immediately the development of a new vaccine. Indeed, the development of a new vaccine can only be done to the detriment of other vaccine developments, mobilizing teams and facilities. From a technical point of view, the choice of a classical vaccine development strategy using conventional procedures, such as Vero cell culture for viral propagation and BPL inactivation, was a decisive factor to success. Importantly, we were able to quickly recruit a volunteer workforce that was both familiar with the technology and trained to work in a BSL3-plus environment. A close collaboration with the reference laboratory, the CDC's Influenza Branch, where the SARS-CoV was isolated, was essential. We provided certified Vero cells to the CDC, which allowed us, upon receipt of the purified strain from the CDC, to re-isolate the SARS-CoV under conditions making the prompt start of a vaccine development possible. When initiating vaccine development against a new emerging infectious agent, the problem of availability of reagents and routine tests to perform biological and molecular studies must be addressed. It is obvious, that at the beginning of such development, there are no such reagents or commercial kits available. As a consequence, the first step in the SARS-CoV project was to prepare the different reagents (antisera and monoclonal antibodies) and the appropriate tests (viral titration, PCR, ELISA, immunofluorescence assay, etc.). Finally, a series of preliminary experiments on monkeys (three months after the start of the project) had given guidance whether it was appropriate to use an inactivated vaccine, as well as to the choice of the adjuvant. Constant communication with regulatory authorities has allowed the validation of this strategy from the beginning of the project. This communication was also very important for the qualification of the viral seed lots. The qualification of the Vero cells was not an issue as several vaccines are already produced in Vero cells, but this was obviously not the case for the viral seeds. Two major obstacles had to be overcome: (1) the realization that the animal testing had to be done in BSL3 facilities by BSL3-trained personnel, and (2) the search for adventitious agents using general classical tests (search for adventitious viruses on cells and in animals) and specific tests (PCR). For the latter, there was no list available and the final testing to be performed was under the responsibility of health authorities. This resulted in a rather exhaustive list of PCR testing. Can We Shorten the Timelines Even More When Facing an Emerging Pathogen? It is likely that epidemics will emerge in the future from unrecognized sources and some of these will be highly pathogenic for humans. These pathogens will be categorized as BSL3 or BSL4 pathogens needing high security level laboratories as well as specialized personnel. How to manipulate these pathogens that are highly pathogenic, in large quantities? To face the emergence of new pathogens, dedicated structures are needed with the right equipment and trained personnel. From an industrial perspective, this seems not compatible with the need and use of trained personnel and facilities that do have a constant activity to assure the production of existing vaccines and the development of new vaccines. Such emergency structures could be set up and maintained by national reference centers respecting the BSL requirements as well as the GMP conditions. It would be very beneficial for industries to collaborate with such reference centers that provide purified pathogens and reagents allowing a prompt start of a vaccine development.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9630844/

Hypoxia activates the unfolded protein response signaling network: An adaptive mechanism for endometriosis

Endometriosis (EMS) is a chronic gynecological disease that affects women of childbearing age. However, the exact cause remains unclear. The uterus is a highly vascularized organ that continuously exposes endometrial cells to high oxygen concentrations. According to the "planting theory" of EMS pathogenesis, when endometrial cells fall from the uterine cavity and retrograde to the peritoneal cavity, they will face severe hypoxic stress. Hypoxic stress remains a key issue even if successfully implanted into the ovaries or peritoneum. In recent years, increasing evidence has confirmed that hypoxia is closely related to the occurrence and development of EMS. Hypoxia-inducible factor-1α (HIF-1α) can play an essential role in the pathological process of EMS by regulating carbohydrate metabolism, angiogenesis, and energy conversion of ectopic endometrial cells. However, HIF-1α alone is insufficient to achieve the complete program of adaptive changes required for cell survival under hypoxic stress, while the unfolded protein response (UPR) responding to endoplasmic reticulum stress plays an essential supplementary role in promoting cell survival. The formation of a complex signal regulation network by hypoxia-driven UPR may be the cytoprotective adaptation mechanism of ectopic endometrial cells in unfavorable microenvironments. Introduction Endometriosis is a common benign gynecological disorder characterized by the presence and growth of functional endometrial glands and stroma outside the uterine cavity, usually accompanied by reactive fibrosis and muscular metaplasia of affected organs ( 1 , 2 ). Unfortunately, only a few studies estimated the prevalence and incidence of endometriosis in the general population, reporting that the prevalence of symptomatic endometriosis is about 10%, and approximately 2-7/1000 women are diagnosed with endometriosis every year. However, 11% of cases remain undiagnosed ( 3 , 4 ). EMS is closely related to dysmenorrhea, painful intercourse, painful defecation, and infertility in women of reproductive age and may seriously affect their quality of life ( 5 , 6 ). In addition, patients and physicians face the great challenge of high recurrence rates after conservative EMS surgery ( 7 , 8 ). Lesions are most commonly found in the pelvic cavity, including the ovaries, uterus, sacral ligaments, vaginal septum, bladder, rectum, and ureters. EMS may also invade organs other than the pelvic cavity, such as abdominal incision ( 9 ), diaphragm, and even lungs ( 10 ). Theoretical basis of hypoxia in EMS The exact pathogenesis of EMS is still unclear. However, the implant theory is still widely accepted as the mainstream theory ( 11 ). The hypothesis is based on the concept that EMS lesions' matrix and glands are derived from ectopic endometrium. Thus, EMS is considered a benign metastasis of ectopic endometrium, which is transferred from the uterine cavity to another position in the body through different pathways. The theory of menstrual blood reflux, based on clinical and anatomical observations, was first proposed by Sampson in 1927 ( 12 ). It is believed that most EMS were induced by endometrial fragments entering the pelvic cavity through fallopian tube reflux during menstruation, the transition from the endometrium to ectopic endometrium, and growth on peritoneum as well as ovary. Although menstrual reflux theory cannot explain all forms of EMS, it remains the most accepted hypothesis of EMS. Some scientists found that 76~90% of women have the phenomenon of menstrual blood regurgitation of fallopian tubes ( 13 ), and laparoscopy has revealed that endometrial epithelial cells can be isolated from abdominal fluid at the early stage of endometrial hyperplasia ( 14 ). In addition, some researchers have identified viable single endometrial cells and glandular structures from the shedding of menstrual blood ( 15 ). The "eutopic endometrium determinism", proposed by Lang ( 16 ), believes that the onset of EMS depends on the characteristics of eutopic endometrial cells in the uterine, so eutopic endometrial cells of patients with EMS may have stronger survival and proliferation ability after falling off. Thus, they are more likely to become ectopic lesions. Among this process, retrograde menstruation is the only bridge from the potential pathogenicity of endometrial cells to the onset of EMS. Recently, based on the physiological phenomenon of periodic stripping and regeneration of endometrium, it is speculated that endometrial stem cells with differentiation potential may exist in menstrual blood. Therefore, some scientists proposed the "stem cell theory" of EMS pathogenesis ( 17 ). Subsequently, relevant studies also confirmed that after menstrual blood enters the pelvic cavity from the fallopian tube, endometrial cell fragments with stem cell characteristics can adhere to mesothelial cells and promote the growth of EMS lesions, leading to the occurrence and development of EMS ( 18 , 19 ). These theories enriched the theoretical basis of planting theory. The anatomical characteristics of the uterine vessels are that the body branches of the uterine arteries vertically discharge the arch artery along with the muscle layer of the lateral wall of the uterus. The uterine spiral artery then extends vertically into the endometrium, finally forming a blood-rich capillary network in the endometrium (as shown in Figure 1 ). During menstruation, endometrial vasospasm causes acute hypoxia of the endometrium, leading to necrosis and endometrial dissection. Finally, blood vessels rupture and a mixture of blood and exfoliated endometrium fragments are formed. The main pathogenesis of EMS, including implant theory, menstrual blood reflux theory, endometrial determinism, and stem cell theory, all began with the shedding of endometrial fragments into the pelvic cavity. Under normal circumstances, the shedding endometrial cells immediately convert into a state of severe hypoxia when the blood supply of the endometrial capillaries is lost. Then relevant apoptotic signals are activated to induce apoptosis and endometrial cells are finally cleared by the immune cells in the pelvic. At the same time, this transient physiological hypoxia could promote the timely repair of the exfoliated endometrial surface and prevent excessive menstrual bleeding, which is mostly meditated by hypoxia-induced high expression of vascular endothelial growth factor (VEGF) ( 20 ). However, in patients with EMS, various changes of cell biological functions ( 21 ) may occur in the shedding endometrial cells under hypoxic stress, such as increased adhesive and invasive capacity, enhanced angiogenesis, and dysregulated immuno-clearance system, thus improving their ability to resist hypoxia or activating certain functions to resist apoptosis, adapt to the hypoxic environment, and survive. Eventually, they will develop into EMS lesions. Therefore, hypoxia can also be the driving factor for EMS, but the relevant pathological mechanism remains unclear. Figure 1 Possible adaptive mechanism for endometriosis under hypoxia. During menstruation, the exfoliated endometrium fragments flow into the pelvic cavity with retrograde menstruation. Under normal circumstances (as shown in the left part), the shedding endometrial cells will face acute hypoxic stress, activating apoptotic signals and being cleared by the immune cells. Conversely, those endometrial cells of endometriosis (EMS) patients (as shown in the right part), possess with altered biological functions, may resist hypoxia or apoptosis and finally develop into EMS lesions. Created with BioRender.com . The role of hypoxia-inducible factors in endometriosis Hypoxia ( 22 ) generally refers to the pathological process in which oxygen is not adequately supplied or consumed excessively, resulting in insufficient oxygen concentration, reduced availability, and the inability to maintain normal cellular functions. If hypoxia persists, it will lead to a metabolic crisis and ultimately threaten the cell's survival. The concentration of oxygen in the atmosphere is approximately 20%, but the content of oxygen in human tissues is much lower. Generally, the normal oxygen content of human tissues is approximately 3%~5% (as shown in Figure 2 ) ( 23 ), which is essential for the maintenance of cells' normal life. When the intracellular oxygen content ranges in 1%~3%, it is called mild hypoxia. In this condition, molecular O2 will play a role as a critical signal to regulate cell fate, activating various adaptive mechanisms to promote cell survival and proliferation. Figure 2 Grading of hypoxia. The normal oxygen content in human cell is 3%~5%; mild hypoxia refers to the oxygen concentration raging in 1%-3%; Oxygen content less than 1% is defined as severe hypoxia. Created with BioRender.com . However, in a state of moderate hypoxia, which means that the oxygen content is less than 1%, many cells can still survive. To survive in a moderate hypoxic environment, the expression of various genes is needed to allow cells to adapt to the stress response induced by hypoxia. This process is heavily controlled by the hypoxia-inducible factors (HIFs) family. HIFs, first discovered by Semenza et al. ( 24 ) in 1992, are activated through a transcription mechanism under hypoxic conditions and then promote the expression of many hypoxia-regulated gene products to form an adaptive mechanism for cell protection. Acting as the master switch in the body in response to hypoxia, the downstream mechanisms of HIFs are multifaceted ( 25 ). For example, HIFs promotes increased secretion of erythropoietin to accelerate red blood cell production, enhancing tissue's ability to transport oxygen, ensuring that cells have an adequate supply of oxygen ( 26 ). In addition, switching of metabolic patterns during hypoxia is essential to reduce oxygen consumption. HIFs can activate glycolytic genes and inhibit tricarboxylic acid cycle metabolism, thereby reducing oxygen consumption and maintaining cell survival in hypoxia condition ( 27 ). HIFs can also induce increased secretion of vascular endothelial growth factor (VEGF) in cells and increase the blood supply of tissues by promoting angiogenesis, thus protecting cells from ischemic damage ( 28 ). Hypoxia can also promote the synthesis of some glucose transporters and maintain the production of high-energy molecular ATP in cells ( 29 ). Overall, HIFs are the most sensitive and important nuclear transcription regulators response to hypoxia. They are prevalent in various mammalian cells even in the simplest animal ( 30 ). HIFs regulate the expression of various genes, and they are widely involved in regulating oxygen homeostasis in response to hypoxia and other changes in the cell's internal environment of the cell. Thus, HIF-1 is a key oxygen sensor and a hypoxic adaptive response regulator. Regarding EMS, when endometrial cells enter the pelvic cavity with retrograde menstrual blood and have not established effective blood circulation, continuous hypoxia can induce the overexpression of HIF-1 in endometrial cells, thus inducing the secretion of various factors within the endometrial environment and prompting the development of endometriosis through multiple mechanisms ( 31 ). Angiogenesis is the primary challenge for the survival of endometrial cells flowed into the pelvis, and it is also a major prerequisite for the initiation and progression of endometriosis ( 32 ). In this process, HIFs can induce the expression of numerous downstream factors to promote the angiogenesis of endometrial cells. Among them, VEGF family are the most classical factors which were found up-regulated both in vivo and in vitro ( 33 , 34 ). Other cytokines and chemokines, such as IL-8, leptin, CYR61 and osteopontin also participant in the HIF-induced angiogenesis process ( 35 – 37 ). In addition, HIFs could also enhance cell adhesive ability through the regulation of Transforming growth factor β1 (TGF-β1), Enhancer of zeste homolog 2 (EZH2), and anthrax toxin receptor 2 (ANTXR2) ( 34 , 38 ). Hypoxia also participants in the regulation of inflammation and immune system via mediating the expression of IL-6, DUPS2, and COX-2 in endometrial cells ( 39 ). Furthermore, although EMS is a benign disorder, it displays some pathogenic characteristics of malignant diseases ( 40 ), such as the tendency to invade and relapse, and adaptation to hypoxia ( 41 ). A latest research showed that the expression of early growth response 1 (EGR1) and its downstream target carbonic anhydrase 9 (CA9), which were up-regulated in hypoxic cancers, are significantly increased in ectopic lesions ( 42 ). In addition, malignant cells possess a unique energy metabolism feature which is called the "Warburg effect" ( 43 ), also known as aerobic glycolysis. Specifically, even in anoxic conditions, malignant tumor cells could remain powered by glycolysis, increasing glucose consumption and lactic acid production. This progress also provides abundant energy for the growth of malignant cells, ensuring their ability to proliferate rapidly, and HIF-1α plays a vital role in this energy metabolic pathway and may protect cells from hypoxia ( 44 ). Interestingly, Young et al. ( 45 ) found that the "Warburg effect" also existed in the EMS lesion, and HIF-1α also showed same effect on the aerobic glycolysis of EMS. Hypoxia activates the unfolded protein response Recent evidence suggests that HIFs alone cannot achieve the whole program of adaptive changes required for cell survival under hypoxic stress and the unfolded protein response (UPR) under endoplasmic reticulum stress (ERS) plays an essential complementary role in this process ( 46 , 47 ). UPR and HIF pathway interacts with HIF-independent pathways, forming a highly correlated regulatory network under hypoxia stress. The endoplasmic reticulum (ER) is an extensive intracellular membranous network extending to the entire cytoplasm. The key site for lipid and glucose metabolism, calcium homeostasis, detoxifying drugs, and metabolisms is the central processing unit responsible for protein translation, folding, and modification ( 48 ). In protein synthesis, folding the protein spatial structure depends on the oxygen content, which is also called oxidizing protein folding ( 49 ). When intracellular oxygen availability is reduced (hypoxia), protein folding will be disturbed, leading to accumulation of misfolded or unfolded proteins. These changes break the protein dynamics in the endoplasmic reticulum and activate the UPR signaling network, thus inducing the production of self-protection mechanisms in cells. This process is called endoplasmic reticulum stress (ERS), which aims to restore homeostasis and function of the intracellular environment ( 50 – 52 ). UPR is mediated by a partner molecule specific for the endoplasmic reticulum, namely glucose-regulated protein78(GRP78), and three transmembrane protein stress sensors ( 53 ), namely protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring protein 1α (IRE1α)(as shown in Figure 3 ). UPR can avoid the ERS-caused damage by reducing the accumulation of unfolded proteins and improving the correct folding of proteins, and restoring normal physiological functions of cells. However, if ERS becomes persistent or damage is too severe, the signal will change from pro-survival to pro-apoptosis ( 54 ). Under normal physiological conditions, these three UPR-related transmembrane proteins all bind to GRP78 and remain in an inactive state. When ERS damage occurs, GRP78 will dissociate from these transmembrane proteins ( 55 ). Activated PERK phosphorylates the α subunit of eukaryotic initiation factor 2 (eIF2), initiating selective translation of activating transcription factor 4 (ATF4) under stress conditions. ATF4 is mainly involved in regulating peptide chain biosynthesis, antioxidant action, protein folding, and gene expression related to the maintenance of redox homeostasis ( 56 ). If the stress persists, ATF4 can also promote the transcription of C/EBP homologous protein (CHOP), a pro-apoptotic protein, to induce cell apoptosis ( 57 ). Activated ATF6 can also initiate related transcriptional procedures to restore endoplasmic reticulum homeostasis, including inducing GRP78 expression, promoting protein chaperone and lipid synthesis, stimulating endoplasmic reticulum degradation, and improving N-glycosylation ( 58 , 59 ), and ATF6 also induces CHOP expression, leading to UPR-related apoptosis ( 60 , 61 ). Figure 3 Hypoxia activates the UPR signaling network to mediate apoptosis. When ERS damage occurs, GRP78 dissociates from PERK, ATF6 and IRE1α. Activated ATF6 can be transported to Golgi apparatus. After modification, it could induce CHOP expression and lead to UPR-related apoptosis via the inhibition of BCL-2 protein family and the activation of multiple genes including GADD34, DR5, and DOC. Similarly, activated PERK phosphorylates the α subunit of eIF2 and initiates translation of ATF4, then promoting the transcription of CHOP. Additionally, activated IRE1α stimulates the expression of TRAF2, then activate ASK1 and JNK to promote apoptosis. TRAF2 also promotes clustering of procaspase-12, and it will be activated into caspase 12 by calpains under ER stress. Created with BioRender.com . Activated IRE1α regulates gene expression by increasing ER protein folding capacity via TRAF2. It also promotes the expression of proteins related to disulfide bond formation and molecular chaperones and proteins involved in ER degradation and vesicle transport ( 62 ). TRAF2 also promotes clustering of procaspase-12. Under ER stress, sustained Ca2+ release from the ER and activate calpains, which may induce the activation of caspase 12 to mediate apoptosis ( 63 ). In addition, IRE1α kinase can up-regulated the expression of apoptosis signal regulated kinase 1(ASK1) and then activate c-Jun N-terminal kinase (JNK) to promote apoptosis through the inhibition of BCL-2 protein family ( 64 , 65 ). CHOP, a member of C/EBP transcription factor family, is an ERS-specific effector molecule representing an important cell transition signal to apoptosis ( 66 ). Under normal physiological conditions, CHOP expression remains at a very low level, while in the ERS state, activation of PERK, ATF6, and IRE1α can induce CHOP transcription, significantly increasing its expression and migration into the nucleus, promoting cell apoptosis via the inhibition of BCL-2 protein family ( 67 , 68 ). CHOP also prompt the expression of GADD34 ( 69 ), which complexes with protein phosphatase 1 (PP1) to induce the dephosphorylation of eIF2α, thus forming a negative feedback loop. Other downstream target genes, including death receptor 5 (DR5) and downstream of CHOP (DOC) have also been proposed to lead to apoptosis ( 70 ). Since protein synthesis and oxygen-dependent protein folding are energy-intensive processes, and hypoxia significantly reduces the level of intracellular ATP, regulating mRNA translation is an important cellular response to hypoxia ( 27 ). Hypoxia activates PERK, leading to the inhibition of eIF2α phosphorylation and overall translation, while ATF4 translation increases after PERK/eIF2α is activated ( 71 , 72 ). This is a rapid response independent of HIF-1α and usually occurs within minutes of exposure to hypoxic conditions in cells. The phosphorylation of eIF2α is transient, attributed to the negative feedback caused by GADD34 dependent on ATF4 upregulation. Dephosphorylation of eIF2α will increase the production of various intracellular reactive oxygen species (ROS), thus stimulating various biological reactions, while mitochondria are the main source of oxygen-deficient ROS ( 73 ). Mitochondrial hypoxic ROS activates and integrates the stress response to maintain energy and REDOX homeostasis and then constitutes an early adaptive response to hypoxia. Some enzyme antioxidants, such as catalase and glutathione peroxidase, can reduce eIF2α phosphorylation due to hypoxia. In contrast, ATF4 can promote cell survival by enhancing the upregulation of HIF-1α-mediated downstream targets ( 74 ). Brief exposure to ERS can modulate cells and enable them to survive in more severe stress. Survival-promoting genes may induce this pre-adaptation, and integration of stress response is also a sufficient survival-promoting mechanism under hypoxia. Cells with impaired PERK-eIF2a-ATF4 signal transduction are more sensitive to hypoxic stress in vitro , indicating that the PERK-eIF2a-ATF4 pathway provides a survival advantage for cells under hypoxic conditions ( 75 , 76 ), which is crucial for resistance to intracellular hypoxia, metabolic stress, and starvation. Scientists have analyzed variations of gene expression under hypoxia conditions, but UPR-related genes can be excessively induced in the state of "extreme" hypoxia or "moderate" hypoxia. For example, X-box binding protein 1 (XBP1), a transcription factor containing zinc finger structure, is critical for UPR signaling network and acts on the folding of multiple proteins regulated by downstream target molecules. Under normal conditions, XBP1 exists in XBP1 unspliced (XBP1u). In the hypoxia state, XBP1u is activated into XBP1 spliced (XBP1s) by IRE1, enabling effective transcription of multiple target genes in the nucleus. Meanwhile, hypoxia can also induce XBP1 expression and activate its mRNA splicing in a HIF-1α dependent pathway, leading to increased XBP1s ( 77 ). When XBP1s were co-localized in tumors with hypoxia markers, the loss of XBP1 increased the sensitivity of transformed cells to hypoxia-induced apoptosis and inhibited tumor growth ( 78 ). Studies have demonstrated that XBP1 is critical for carcinogenicity and progression of triple-negative breast cancer (TNBC). TNBC is a type of breast cancer that lacks estrogen receptors, progesterone, and HER2, in which HIF-1α is overactivated. However, XBP1 splicing is not directly regulated by HIF-1α. XBP1 mainly enhances the transcriptional activity of HIF-1α and regulates its transcriptional program by binding to HIF-1α and forming the transcription complex to drive the carcinogenicity of TNBC. In contrast, XBP1 knockout can reduce the formation of breast cancer lesions under hypoxia conditions. The characteristics of XBP1 gene expression in TNBC patients are closely related to those in the state of hypoxia, indicating a poor prognosis. The role of UPR in EMS UPR is a protection mechanism to maintain the stability of the intracellular environment under hypoxia circumstances, and its activation will promote cell survival. To determine the role of UPR in the pathogenesis of EMS, Guzel ( 79 ) et al. detected GRP78 expression in normal and ectopic endometrial cells, finding that the level of GRP78 in ectopic endometrial cells is significantly higher than that of normal endometrial cells. This indicated that the UPR cascade reaction was activated in EMS, and the upregulated expression of GRP78 significantly reduced the sensitivity of ectopic endometrial cells to apoptosis and increased their anti-apoptosis ability, which was beneficial for the survival of ectopic endometrial cells. Other UPR proteins, p-IRE1 and p-PERK, were also found increased in endometrial cells when they were treated with peritoneal fluid obtained from women with endometriosis ( 80 ). Taylor ( 81 ) et al. also reported that UPR induced the increased expression of IL-8, indicating that UPR may be involved in the pathogenesis of EMS by promoting neovascularization and cell survival via IL-8 related pathways. The protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway plays an essential role in enhancing the invasiveness of cells, and inhibition of this pathway can effectively reduce the invasiveness of various cancer cells ( 82 ). Several studies ( 83 , 84 ) have displayed that UPR can inhibit AKT/mTOR pathway through CCAAT/CHOP/(Tribbles Homolog 3, TRIB3) signal transduction and regulate the invasiveness of ectopic endometrial cells. EMS is an estrogen-dependent disease and is related to progesterone resistance ( 85 ). UPR-induced apoptosis can be inhibited by estrogen, thus promoting endometrial cell survival ( 86 ). In contrast, in the secretory phase, due to the antagonistic effect of progesterone, the inhibition effect of estrogen on the UPR-mediated apoptotic pathway can be reduced. Then, UPR upregulation may help reduce the invasiveness of endometrial cells ( 87 ). However, progesterone resistance exists in ectopic and endometrial stromal cells of women with EMS, and in the secretory phase, progesterone resistance in ectopic endometrium stromal cells may alter the effect induced by UPR as described above ( 88 , 89 ). In vitro studies on UPR regulating endometrial cell invasiveness also suggest that ( 90 ), in endometrial cells with estrogen added alone in the proliferative phase, the expressions of GRP78, CHOP, and TRIB3 increased significantly with increased progesterone during the secretory phase. In contrast, endometrial cell invasiveness was significantly suppressed when AKT and mTOR activity was inhibited. Thus, progesterone can upregulate the expression of UPR-related CHOP and TRIB3 by antagonizing estrogen and inhibiting the AKT/mTOR pathway, reducing the invasiveness of endometrial cells. These progesterone-induced signal regulations can occur in normal endometrial cells. In ectopic cells, progesterone has no significant effect on CHOP/TRIB3 or AKT/mTOR signaling, so it does not play a role in the invasiveness of endometrial cells. Discussion EMS severely affects the physical and mental health and quality of life of women of reproductive age. The implantation theory on the pathogenesis of EMS indicates the key role of hypoxia in the occurrence and development of EMS, which involves cell biological processes such as apoptosis, adhesion, proliferation, invasion, and metastasis of ectopic endometrial cells. The mechanism of EMS is complex, and its etiology and pathology cannot be clearly explained with a onefold theory. Hypoxia-induced UPR may be one of the potential mechanisms by which ectopic endometrium cells could resist apoptosis and develop into endometriotic lesions, and some drugs ( 91 , 92 ) targeting this mechanism may become potential effective therapeutic agents for the treatment of the disease. But the exact mechanism of hypoxia-stimulated UPR in EMS remains to be explored, and we look forward to new break-throughs in this field. Author contributions YZ and YJ contributed equally to the manuscript. YZ contributed to the conception and design of the review. YZ and YJ drafted the manuscript. YJ performed the descriptive figures. YW and RW revised the manuscript and provided critical advice on the content of the manuscript. All authors read and approved the final manuscript. Funding This work was supported by National Natural Science foundation of China (82101723), Department of Education of Zhejiang Province (Y202045691), and Zhejiang Province Key R&D Program (2021C03095). Acknowledgments We gratefully acknowledge the assistance of Home for Researchers for language modification. Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher's note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9268905/

Ethnoveterinary Practices and Ethnobotanical Knowledge on Plants Used against Cattle Diseases among Two Communities in South Africa

Ethnoveterinary practices and ethnobotanical knowledge serve as potential therapeutic approaches used to manage and prevent cattle diseases within poor communities in developing nations. Most of the knowledge and practices remain inadequately documented and threatened with extinction in the future. This study aimed to explore the ethnoveterinary practices and knowledge on plants used to treat cattle diseases in two communities of the Ramotshere Moiloa local municipality, South Africa. A semi-structured interview guide, snowball, and purposive technique were used to collect data and recruit 90 participants. Three ethnobotanical indices (informant consensus factor (Fic), use-value (UV), and relative frequency of citation (RFC) were used for quantitative analysis. A total of 64 medicinal plants from 32 families (dominated by Compositae, Fabaceae, and Asparagaceae) were used to treat 27 cattle diseases. The plants with a high frequency of citation and RFC were Gomphocarpus fruticosus (75, 0.83), Opuntia ficus-indica (74, 0.82), Schkuhria pinnata and Portulaca oleracea (73, 0.81), Solanum lichtensteinii (70, 0.77), and Senna italica. In addition, Schkuhria pinnata and Aloe greatheadii (0.077) had the highest UV. About 28.13% of 64 identified plants were documented as ethnoveterinary medicine for treating cattle ailments, for the first time. The remedies were mainly either prepared as a decoction (52.04%), ground, or prepared as an infusion (16.33%). The plants were administered either orally (69.79%) or topically (30.2%). The ailments with a high frequency of citations were: wounds and constipation (76); arthralgia and retained placenta (69); and lumpy skin disease (68). The categories with the highest number of plants used were gastrointestinal problems (53), skin problems (33), respiratory problems (25), and fertility/reproduction disorders (21). The highest Fic score was cited for tick-borne diseases (1), followed by musculoskeletal systems (Fic = 0.89), and general system infection (Fic = 0.88). The current findings contribute to the documentation and preservation of valuable knowledge from indigenous communities for extensive use. Additionally, ethnoveterinary uses of Portulaca oleracea , Securidaca longipedunculata, and Plumbago zeylanica were recorded for the first time. Further scientific evaluation of the most cited and indigenous/native plants is recommended to establish their therapeutic potential and possible integration into the conventional veterinary sector for the welfare of cattle. 1. Introduction Cattle production plays a key role in the rural economies of developing countries in terms of food security, poverty alleviation, and diverse cultural activities, particularly in rural communities [ 1 , 2 ]. Due to their use as draft animals and their ability to convert low-quality forage into energy-dense muscle and milk, cattle provide a significant source of food and nutrition, much-needed income, and nitrogen-rich manure for replenishing soils and other uses [ 3 , 4 ]. They also fulfil a wide variety of socio-cultural roles. However, cattle in rural areas are often susceptible to various diseases [ 5 ]. Changes in population and climate, technology, lifestyles, consumer demands, markets, and other factors are driving rapid change in cattle production. These factors are influencing the way cattle are being produced, improving the livelihoods of people, and sometimes threatening cattle diversity at the local, national, and regional herd levels [ 6 , 7 , 8 ]. Healthy, well-cared-for, and productive cattle contribute to the sustainable, healthier, and inclusive future livelihood of the communities. The clinical service of the public veterinary service is believed to be inefficient and seen to have minimal effect on animal health [ 9 ]. Therefore, maintaining and restoring the health and well-being of the cattle is a critical responsibility for the community members who depend on them. Farmers and cattle herders in rural communities rely on ethnoveterinary medicine (EVM) as a sustainable alternative to western veterinary practices. Ethnoveterinary medicine encompasses a variety of systems and knowledge of maintaining animal health that is based on beliefs, traditional knowledge, skills, methods, medicinal plants, metaphysics, surgical procedures, technologies, and teachings that are used in healing livestock [ 10 ]. The popularity of EVM is often attributed to its ability to improve folk pharmacotherapy which is locally available, economically feasible, accessible, and culturally appropriate [ 11 ]. Furthermore, the practice includes a set of empirical observations of the environment and self-management [ 9 ]. According to McGaw and Eloff [ 8 ], studies in EVM are necessary because plants contain a wide range of phytochemicals. These plants can provide the lead candidates for drug discovery and development of active products, which are useful in managing the health of livestock. In South Africa, the rich and unique flora have been well-utilised in traditional medicine, thereby creating more interest in the potential of medicinal plants [ 12 , 13 ]. As a megadiverse country with a rapidly growing population, the incessant loss of biodiversity justifies the need to document the plant resources, especially the native plants which can be considered as endemic or indigenous [ 14 ]. Cattle diseases are major veterinary health problems, which are experienced by livestock farmers in developing countries. Recently, the Conventional Veterinary Services and Drug Resistance reported a rise in the number of cattle diseases that are affecting cattle production [ 15 ]. The situation has been compounded by the inaccessibility of veterinary services by rural cattle breeders and the escalating cost of veterinary services. Despite the existing efforts [ 1 , 8 , 16 , 17 , 18 ], there is a paucity of documentation and scientific data regarding the knowledge and practices of ethnoveterinary medicine among different ethnic groups globally. The majority of indigenous diagnostic and ethnobotanical knowledge methods used in cattle healthcare have been passed down from generation to generation, mainly by word of mouth and apprenticeship [ 19 , 20 ]. Currently, such indigenous knowledge is held by the community's elders and the limited young members with interest in learning how to use it [ 21 ]. Furthermore, EVM is often locally and culturally specific due to differences in disease epidemiology, culture, and biodiversity. Therefore, if not documented, the immense knowledge, skills, and experience accumulated over generations may become extinct in developing countries because of migrations, urbanisation, and technological development [ 22 , 23 ]. Thus, this study explored the indigenous diagnostic and ethnobotanical skills, methods, and processes used to treat cattle diseases and other husbandry indications among Batswana in two communities in the Ramotshere Moiloa local municipality, South Africa. 2. Results and Discussion 2.1. Socio-Demographic Data of Participants in the Study A total of 90 community members with ages ranging from 18 to 95 years participated in this study ( Table 1 ). The dominant age group, who constituted about 40% of the participants, were aged 61 and above and are regarded as elders in the communities. Generally, indigenous knowledge on the use of EVM is mostly limited to older people in the communities [ 24 , 25 , 26 , 27 , 28 ]. In the current study, all (100%) of the participants acquired knowledge about indigenous diagnostic skills and ethnobotanical knowledge about diseases from elders. This indicates the relative transmission of indigenous practices from one generation to the next one. The environment and experience of others remain two of the most active means to transmit knowledge about the medicinal values of plants [ 29 , 30 ]. This also indicates that the knowledge is facing a threat, which has a negative impact on the use of ethnoveterinary medicine. Likewise, Giday and Teklehaymanot [ 31 ] acknowledged that indigenous knowledge is declining among the younger generation in Africa. However, ethnoveterinary medicine is still prevalent in remote villages of the Ramotshere Moiloa local municipality. The need to preserve the indigenous knowledge, which is at risk of being lost due to the modern lifestyle, remains pertinent [ 23 ]. Gender plays a significant part in ethnoveterinary medicine, and the distribution of the participants by gender was 83% male and 17% female. A similar and common pattern indicated that livestock remain mostly in the care of males rather than females [ 16 , 32 , 33 ]. In Ethiopia, Assefa, and Bahiru [ 34 ] indicated that cattle rearing is under the command of males, which influences the imbalanced gender ratio in the practice of ethnoveterinary healthcare. On the other hand, the dominance of females was evidenced in a few studies from countries such as India [ 35 ] and China [ 28 ]. About 27% of the participants had extensive (more than 40 years) experience in cattle production ( Table 1 ). In the Alaknanda catchment of Uttarakhand in India and the Buyi people of Southwest Guizhou in China, elderly people and male participants were more experienced and had more traditional knowledge of cattle production [ 28 , 35 ]. In the current study, 93% of participants were practicing subsistence farming, whereas a few (7%) engaged in commercial systems. The participants treated the cattle diseases/conditions using medicinal plants (44.4%) or the combination of medicinal plants and conventional medicine (55.6%). Similar results were reported in Eastern Cape, South Africa [ 36 , 37 ]. The use of both methods depended on the availability of funds to procure the conventional medicine, availability of veterinary services, knowledge of indigenous diagnostic methods and plants, value of the animal, and seriousness of the condition [ 38 ]. 2.2. Common Cattle Diseases Treated Using Ethnoveterinary Medicine Based on the Ruminant Veterinary Association of South Africa [ 39 ] and the classification in a previous study [ 40 ], the identified cattle diseases were classified into nine categories ( Table 2 ). An inventory of disease conditions identified by the participants was recorded in a generated database with descriptions of disease categories, names in English and Setswana (local name), signs, and symptoms, causes, affected sex and age, and seasonality of outbreak. When compared to the western veterinary medical system, the naming of ailments by indigenous people did not always discriminate between ailments and symptoms of diseases. This is due to the fact that indigenous ailment nomenclature focuses on symptoms, but diseases in western veterinary science are based on aetiological knowledge [ 9 ]. The participants identified 27 cattle diseases prevalent in the study area ( Table 2 ). The most often cited ailments were wounds and constipation (76); arthralgia and retained placenta (69); and lumpy skin disease (68). The categories with the highest plant species used were gastrointestinal problems (53), skin problem (33), respiratory problem (25), and fertility/reproduction disorders (21). The informant consensus factor (Fic) is determined by the availability of plants in the study area for ailments treatment. The Fic values in this study varied from 0.56 to 1, with an average of 0.80, indicating a high level of agreement among the participants on the use of plants to treat cattle ailments. Tick-borne diseases had the highest Fic (1), followed by musculoskeletal systems (Fic = 0.89), and general system infection (Fic = 0.88). The high Fic values observed in this study show reasonably reliable knowledge of medicinal plants among the participants. A high Fic value is commonly associated with a few specific plants that have high use reports for treating a single disease category, whereas low values are associated with plants that have almost equal or high UR, implying a lower level of agreement among participants on the use of these plants to treat a specific disease category. 2.3. Diagnostic Skills, Treatment Methods, and Endpoint Determination Participants reported signs and symptoms of cattle diseases/conditions, which they use for diagnosis. Seventy-five distinct clinical signs and symptoms of disease were reported by participants in this study. The most common ones were weight loss, loss of appetite, swelling, weakness and tiredness, breathing problem, distress, restless and discomfort, and blood in the faeces ( Table 2 ). Different clinical signs and symptoms were based on the identified diseases. The current findings suggest a high degree of common perception between ethnoveterinary medicine and conventional veterinary systems. The descriptions of cattle ailments were mostly not like that of the conventional veterinary system, as the participants used signs and symptoms. In some cases, there were some similarities. For example, in terms of tick infestation, participants identified six distinct clinical signs and symptoms corresponding closely to conventional veterinary system concepts of external parasites conditions. 2.4. Plants identified as Ethnoveterinary Medicine for Treating Cattle Diseases In this study, 64 plants were documented as medicine used against cattle diseases ( Table 3 ). The current inventory was higher when compared to those documented in previous studies conducted in the North West Province [ 25 , 33 , 40 ]. This study reports on new plants that were not documented in earlier studies [ 25 , 33 ]. Particularly, 18 plants (28.13%) were described as ethnoveterinary medicine in treating cattle for the first-time ( Table 3 ). The RFC indicates the local importance of plant species with reference to the participants, who cited the uses of these plants [ 41 ]. In the study, the RFC ranged from 0.12 to 0.83. Based on the RFC values, the most cited plant species were Gomphocarpus fruticosus (L.) W.T.Aiton (0.83), Opuntia ficus-indica (L.) Mill. (0.82), Schkuhria pinnata (Lam.) Kuntze ex Thell. and Portulaca oleracea L. (0.81), and Solanum lichtensteinii Willd. (0.77). The use-value (UV) is a measure of the types of uses attributed to a particular plant species. In the present study, Schkuhria pinnata (Lam.) Kuntze ex Thell., Senna italica Mill., and Aloe greatheadii Schönland had the highest UV (0.077) followed by Cleome gynandra L., Harpagophytum procumbens (Burch.) DC. ex Meisn., Ximenia caffra Sond. (0.066), and Ziziphus mucronata Willd., Senna italica Mill., Portulaca oleracea L., and Opuntia ficus-indica (L) (0.055) ( Table 3 ). The extent to which a species may be employed is determined by its UV; hence, plantss with a high UV are more exploited in the research area to treat more diseases than those with a low UV. Plants with a higher number of use reports (UR) had higher UV, whereas plants with fewer UR by participants had lower UVs, whereas plants with fewer Ui reported by participants had lower UV. Generally, plants that are utilised repeatedly are more likely to be physiologically active [ 42 ]. Given that UV and RFC values are dynamic and alter with location and people's awareness, UV and RFC values may vary from area to area and even within the same study area [ 43 ]. Plants with lower UV and RFC values are not necessarily unimportant, but their low values may indicate that the participants are unaware of the uses of these plants and, as a result, that understanding of their use is at risk of not being passed down to future generations, and thus this knowledge may eventually disappear. Some of the documented plants are indigenous to the study area and are well-known to the participants. As a result, their specialised qualities for healing various ailments have become well-known and well-established among the participants. Plants with higher UV and RFC are likely to be good candidates for future research. It will be essential to subject these plants to pharmacological, phytochemical, and biological investigation to establish their therapeutic potential and the potential development of low-cost products [ 44 ]. In the current study, the recorded medicinal plants consisted of indigenous (native) and non-endemic plants used against various cattle diseases by the participants of Ramotshere Moloa local municipality. Among the plants, 50 (78.1%) were indigenous/native while 14 (21.8%) were introduced/cultivated/naturalised ( Table 3 ). High levels of usage of these indigenous/native species, particularly those with high use categories, might be of conservation concern if conducted in an unsustainable manner [ 14 ]. Thus, there is a great need to discover new biologically active compounds from herbal plants and develop novel drugs. Few studies are available about EVM plants and their constituents with antimicrobial activities [ 1 , 8 ], and these indigenous plants may contain pharmaceutically essential compounds. To further understand the uniqueness of this EVM, a more in-depth study of how these indigenous plants is used and selected, as well as a comparison study with other sites/locations within South Africa is required. In addition, a closer look at the local conservation status is required to build a sustainable use plan for these valuable plant resources [ 23 ]. 2.5. Therapeutic Uses of Combined Medicinal Plants Participants in the current study reported nine (14.1%) plants from the inventory that had numerous indications (uses), as poly-plant remedies. These results reflect the diversity of ethnic knowledge and heterogeneity in cultural practices. For example, participants reported using a combination of the leaves of Artemisia afra Jacq. ex Willd., Mentha aquatica L., Dicoma macrocephala DC., and Lippia scaberrima Sond., and mixing them with donkey milk to cure cough, intestinal worms, and joints pain. A decoction of Drimia sanguinea (Schinz) Jessop bulb and the roots of Elephantorrhiza burkei Benth. and Senna italica Mill. was administered orally to treat intestinal worms. To treat constipation, a decoction of the roots of Elephantorrhiza burkei Benth., Peltrophorum africanum Sond., and Jatropha zeyheri Sond. is administered orally. The potency of using a combination of different plants or plant parts increased compared to using a single plant to cure a disease is well-recognised [ 25 , 40 , 45 ]. Validation and transmission of this knowledge to livestock raising farmers all over the world so that they know the best plant material near them for the specific ailment will benefit people not only in impoverished nations but also in the developed world [ 46 ]. The use of two or more plants exemplifies the notion of synergy, which highlights that the combination of plants might result in higher therapeutic efficacy [ 32 , 47 ]. 2.6. Plant Families Used to Treat Cattle Diseases In terms of family diversity, 32 families were used to treat and manage cattle diseases in the study area ( Figure 1 ). The families with the largest number of plant species used to treat cattle were Compositae with seven species and Fabaceae and Asparagaceae (five species). Compositae and Fabaceae are the most widely used families in ethnoveterinary studies [ 18 , 48 , 49 , 50 ]. Similar studies have been reported where participants mostly used the members of Compositae for the preparation of EVM for the treatment of different livestock diseases [ 40 , 51 , 52 ]. Furthermore, the widespread use of plants from these dominant families might be attributed to strong traditional beliefs, availability, ease of harvesting, and storage, as well as the evidence of bioactive compounds with therapeutic effect against cattle ailments. However, the trend for plant families utilised to cure cattle ailments in the selected communities differs from those used in other locations in South Africa [ 33 , 53 ]. 2.7. Distribution of Plant Parts Used to Treat Cattle Diseases In the study area, different plant parts were used for the preparation of remedies for treating cattle diseases ( Figure 2 ). The most frequently used plant parts were the leaves (34.55%), roots (25.45%), and whole plant (19.09%). The preference of leaves in treating cattle ailments is due to their easy availability, easy harvesting, and simplicity in remedy preparation. Leaves are the storage site of diverse pools of phytochemicals, the renewable parts of plants, and for a conservation perspective, their collection may not result in the fatality of the mother plants [ 54 ]. A similar trend whereby the leaves were the dominant plant part used in medicine preparation for treating cattle diseases was reported in other studies [ 51 , 55 , 56 , 57 , 58 ]. However, in some cases, the roots were identified as the commonly used plant parts [ 9 , 33 , 40 ]. Roots were the second most commonly utilised plant part, which might be attributed to the fact that roots remain in the soil and are easily accessible even during extended dry seasons in arid and semi-arid environments [ 59 ]. However, this is frequently not suggested because it is harmful and unsustainable, putting plant species at risk of extinction [ 60 ]. 2.8. Method of Preparation and Route of Administration of Medicinal Plants Used to Treat Cattle Diseases As depicted in Figure 3 , the herbal remedies were mainly prepared as decoction (52.04%), ground, and infusion (16.33%). Other ways of preparation were maceration, poulticing, and burning which cumulatively accounted for 31.63%. Decoction is the process of boiling plant components in water and then allowing the liquid to cool before administering. Decoction is a popularpreparation methodthat has been mentioned in several different research studies [ 48 , 61 , 62 ]. The preparation method differs from other study areas including Karamoja in Uganda [ 63 ], the Mana Angetu district of south-eastern Ethiopia [ 64 ], and Yalo Woreda in the Afar regional state, Ethiopia [ 65 ], where crushing and pounding were the most common methods. The widespread usage of decoction might be attributed to the fact that boiling can accelerate biological processes, resulting in the greater availability of several active compounds [ 66 ]. However, preparation procedures vary based on the type of sickness being treated, the location of the condition, and the medicinal components to be extracted [ 67 ]. Furthermore, the local communities use a variety of methods to administer the plants when treating cattle diseases ( Figure 4 ). The route of administration for the plants was oral (69.79%) and topical (30.2%). Oral administration is a simple and non-invasive form of systemic treatment. The route allows for the rapid absorption and distribution of the prepared medicines and allowing for sufficient curative power to be delivered [ 68 ]. Across many African cultures, oral administration of medicinal plants is the most common route used to treat disease in cattle, as this ensures fast and direct interaction with different plant compounds at the site of action [ 69 , 70 , 71 ]. 2.1. Socio-Demographic Data of Participants in the Study A total of 90 community members with ages ranging from 18 to 95 years participated in this study ( Table 1 ). The dominant age group, who constituted about 40% of the participants, were aged 61 and above and are regarded as elders in the communities. Generally, indigenous knowledge on the use of EVM is mostly limited to older people in the communities [ 24 , 25 , 26 , 27 , 28 ]. In the current study, all (100%) of the participants acquired knowledge about indigenous diagnostic skills and ethnobotanical knowledge about diseases from elders. This indicates the relative transmission of indigenous practices from one generation to the next one. The environment and experience of others remain two of the most active means to transmit knowledge about the medicinal values of plants [ 29 , 30 ]. This also indicates that the knowledge is facing a threat, which has a negative impact on the use of ethnoveterinary medicine. Likewise, Giday and Teklehaymanot [ 31 ] acknowledged that indigenous knowledge is declining among the younger generation in Africa. However, ethnoveterinary medicine is still prevalent in remote villages of the Ramotshere Moiloa local municipality. The need to preserve the indigenous knowledge, which is at risk of being lost due to the modern lifestyle, remains pertinent [ 23 ]. Gender plays a significant part in ethnoveterinary medicine, and the distribution of the participants by gender was 83% male and 17% female. A similar and common pattern indicated that livestock remain mostly in the care of males rather than females [ 16 , 32 , 33 ]. In Ethiopia, Assefa, and Bahiru [ 34 ] indicated that cattle rearing is under the command of males, which influences the imbalanced gender ratio in the practice of ethnoveterinary healthcare. On the other hand, the dominance of females was evidenced in a few studies from countries such as India [ 35 ] and China [ 28 ]. About 27% of the participants had extensive (more than 40 years) experience in cattle production ( Table 1 ). In the Alaknanda catchment of Uttarakhand in India and the Buyi people of Southwest Guizhou in China, elderly people and male participants were more experienced and had more traditional knowledge of cattle production [ 28 , 35 ]. In the current study, 93% of participants were practicing subsistence farming, whereas a few (7%) engaged in commercial systems. The participants treated the cattle diseases/conditions using medicinal plants (44.4%) or the combination of medicinal plants and conventional medicine (55.6%). Similar results were reported in Eastern Cape, South Africa [ 36 , 37 ]. The use of both methods depended on the availability of funds to procure the conventional medicine, availability of veterinary services, knowledge of indigenous diagnostic methods and plants, value of the animal, and seriousness of the condition [ 38 ]. 2.2. Common Cattle Diseases Treated Using Ethnoveterinary Medicine Based on the Ruminant Veterinary Association of South Africa [ 39 ] and the classification in a previous study [ 40 ], the identified cattle diseases were classified into nine categories ( Table 2 ). An inventory of disease conditions identified by the participants was recorded in a generated database with descriptions of disease categories, names in English and Setswana (local name), signs, and symptoms, causes, affected sex and age, and seasonality of outbreak. When compared to the western veterinary medical system, the naming of ailments by indigenous people did not always discriminate between ailments and symptoms of diseases. This is due to the fact that indigenous ailment nomenclature focuses on symptoms, but diseases in western veterinary science are based on aetiological knowledge [ 9 ]. The participants identified 27 cattle diseases prevalent in the study area ( Table 2 ). The most often cited ailments were wounds and constipation (76); arthralgia and retained placenta (69); and lumpy skin disease (68). The categories with the highest plant species used were gastrointestinal problems (53), skin problem (33), respiratory problem (25), and fertility/reproduction disorders (21). The informant consensus factor (Fic) is determined by the availability of plants in the study area for ailments treatment. The Fic values in this study varied from 0.56 to 1, with an average of 0.80, indicating a high level of agreement among the participants on the use of plants to treat cattle ailments. Tick-borne diseases had the highest Fic (1), followed by musculoskeletal systems (Fic = 0.89), and general system infection (Fic = 0.88). The high Fic values observed in this study show reasonably reliable knowledge of medicinal plants among the participants. A high Fic value is commonly associated with a few specific plants that have high use reports for treating a single disease category, whereas low values are associated with plants that have almost equal or high UR, implying a lower level of agreement among participants on the use of these plants to treat a specific disease category. 2.3. Diagnostic Skills, Treatment Methods, and Endpoint Determination Participants reported signs and symptoms of cattle diseases/conditions, which they use for diagnosis. Seventy-five distinct clinical signs and symptoms of disease were reported by participants in this study. The most common ones were weight loss, loss of appetite, swelling, weakness and tiredness, breathing problem, distress, restless and discomfort, and blood in the faeces ( Table 2 ). Different clinical signs and symptoms were based on the identified diseases. The current findings suggest a high degree of common perception between ethnoveterinary medicine and conventional veterinary systems. The descriptions of cattle ailments were mostly not like that of the conventional veterinary system, as the participants used signs and symptoms. In some cases, there were some similarities. For example, in terms of tick infestation, participants identified six distinct clinical signs and symptoms corresponding closely to conventional veterinary system concepts of external parasites conditions. 2.4. Plants identified as Ethnoveterinary Medicine for Treating Cattle Diseases In this study, 64 plants were documented as medicine used against cattle diseases ( Table 3 ). The current inventory was higher when compared to those documented in previous studies conducted in the North West Province [ 25 , 33 , 40 ]. This study reports on new plants that were not documented in earlier studies [ 25 , 33 ]. Particularly, 18 plants (28.13%) were described as ethnoveterinary medicine in treating cattle for the first-time ( Table 3 ). The RFC indicates the local importance of plant species with reference to the participants, who cited the uses of these plants [ 41 ]. In the study, the RFC ranged from 0.12 to 0.83. Based on the RFC values, the most cited plant species were Gomphocarpus fruticosus (L.) W.T.Aiton (0.83), Opuntia ficus-indica (L.) Mill. (0.82), Schkuhria pinnata (Lam.) Kuntze ex Thell. and Portulaca oleracea L. (0.81), and Solanum lichtensteinii Willd. (0.77). The use-value (UV) is a measure of the types of uses attributed to a particular plant species. In the present study, Schkuhria pinnata (Lam.) Kuntze ex Thell., Senna italica Mill., and Aloe greatheadii Schönland had the highest UV (0.077) followed by Cleome gynandra L., Harpagophytum procumbens (Burch.) DC. ex Meisn., Ximenia caffra Sond. (0.066), and Ziziphus mucronata Willd., Senna italica Mill., Portulaca oleracea L., and Opuntia ficus-indica (L) (0.055) ( Table 3 ). The extent to which a species may be employed is determined by its UV; hence, plantss with a high UV are more exploited in the research area to treat more diseases than those with a low UV. Plants with a higher number of use reports (UR) had higher UV, whereas plants with fewer UR by participants had lower UVs, whereas plants with fewer Ui reported by participants had lower UV. Generally, plants that are utilised repeatedly are more likely to be physiologically active [ 42 ]. Given that UV and RFC values are dynamic and alter with location and people's awareness, UV and RFC values may vary from area to area and even within the same study area [ 43 ]. Plants with lower UV and RFC values are not necessarily unimportant, but their low values may indicate that the participants are unaware of the uses of these plants and, as a result, that understanding of their use is at risk of not being passed down to future generations, and thus this knowledge may eventually disappear. Some of the documented plants are indigenous to the study area and are well-known to the participants. As a result, their specialised qualities for healing various ailments have become well-known and well-established among the participants. Plants with higher UV and RFC are likely to be good candidates for future research. It will be essential to subject these plants to pharmacological, phytochemical, and biological investigation to establish their therapeutic potential and the potential development of low-cost products [ 44 ]. In the current study, the recorded medicinal plants consisted of indigenous (native) and non-endemic plants used against various cattle diseases by the participants of Ramotshere Moloa local municipality. Among the plants, 50 (78.1%) were indigenous/native while 14 (21.8%) were introduced/cultivated/naturalised ( Table 3 ). High levels of usage of these indigenous/native species, particularly those with high use categories, might be of conservation concern if conducted in an unsustainable manner [ 14 ]. Thus, there is a great need to discover new biologically active compounds from herbal plants and develop novel drugs. Few studies are available about EVM plants and their constituents with antimicrobial activities [ 1 , 8 ], and these indigenous plants may contain pharmaceutically essential compounds. To further understand the uniqueness of this EVM, a more in-depth study of how these indigenous plants is used and selected, as well as a comparison study with other sites/locations within South Africa is required. In addition, a closer look at the local conservation status is required to build a sustainable use plan for these valuable plant resources [ 23 ]. 2.5. Therapeutic Uses of Combined Medicinal Plants Participants in the current study reported nine (14.1%) plants from the inventory that had numerous indications (uses), as poly-plant remedies. These results reflect the diversity of ethnic knowledge and heterogeneity in cultural practices. For example, participants reported using a combination of the leaves of Artemisia afra Jacq. ex Willd., Mentha aquatica L., Dicoma macrocephala DC., and Lippia scaberrima Sond., and mixing them with donkey milk to cure cough, intestinal worms, and joints pain. A decoction of Drimia sanguinea (Schinz) Jessop bulb and the roots of Elephantorrhiza burkei Benth. and Senna italica Mill. was administered orally to treat intestinal worms. To treat constipation, a decoction of the roots of Elephantorrhiza burkei Benth., Peltrophorum africanum Sond., and Jatropha zeyheri Sond. is administered orally. The potency of using a combination of different plants or plant parts increased compared to using a single plant to cure a disease is well-recognised [ 25 , 40 , 45 ]. Validation and transmission of this knowledge to livestock raising farmers all over the world so that they know the best plant material near them for the specific ailment will benefit people not only in impoverished nations but also in the developed world [ 46 ]. The use of two or more plants exemplifies the notion of synergy, which highlights that the combination of plants might result in higher therapeutic efficacy [ 32 , 47 ]. 2.6. Plant Families Used to Treat Cattle Diseases In terms of family diversity, 32 families were used to treat and manage cattle diseases in the study area ( Figure 1 ). The families with the largest number of plant species used to treat cattle were Compositae with seven species and Fabaceae and Asparagaceae (five species). Compositae and Fabaceae are the most widely used families in ethnoveterinary studies [ 18 , 48 , 49 , 50 ]. Similar studies have been reported where participants mostly used the members of Compositae for the preparation of EVM for the treatment of different livestock diseases [ 40 , 51 , 52 ]. Furthermore, the widespread use of plants from these dominant families might be attributed to strong traditional beliefs, availability, ease of harvesting, and storage, as well as the evidence of bioactive compounds with therapeutic effect against cattle ailments. However, the trend for plant families utilised to cure cattle ailments in the selected communities differs from those used in other locations in South Africa [ 33 , 53 ]. 2.7. Distribution of Plant Parts Used to Treat Cattle Diseases In the study area, different plant parts were used for the preparation of remedies for treating cattle diseases ( Figure 2 ). The most frequently used plant parts were the leaves (34.55%), roots (25.45%), and whole plant (19.09%). The preference of leaves in treating cattle ailments is due to their easy availability, easy harvesting, and simplicity in remedy preparation. Leaves are the storage site of diverse pools of phytochemicals, the renewable parts of plants, and for a conservation perspective, their collection may not result in the fatality of the mother plants [ 54 ]. A similar trend whereby the leaves were the dominant plant part used in medicine preparation for treating cattle diseases was reported in other studies [ 51 , 55 , 56 , 57 , 58 ]. However, in some cases, the roots were identified as the commonly used plant parts [ 9 , 33 , 40 ]. Roots were the second most commonly utilised plant part, which might be attributed to the fact that roots remain in the soil and are easily accessible even during extended dry seasons in arid and semi-arid environments [ 59 ]. However, this is frequently not suggested because it is harmful and unsustainable, putting plant species at risk of extinction [ 60 ]. 2.8. Method of Preparation and Route of Administration of Medicinal Plants Used to Treat Cattle Diseases As depicted in Figure 3 , the herbal remedies were mainly prepared as decoction (52.04%), ground, and infusion (16.33%). Other ways of preparation were maceration, poulticing, and burning which cumulatively accounted for 31.63%. Decoction is the process of boiling plant components in water and then allowing the liquid to cool before administering. Decoction is a popularpreparation methodthat has been mentioned in several different research studies [ 48 , 61 , 62 ]. The preparation method differs from other study areas including Karamoja in Uganda [ 63 ], the Mana Angetu district of south-eastern Ethiopia [ 64 ], and Yalo Woreda in the Afar regional state, Ethiopia [ 65 ], where crushing and pounding were the most common methods. The widespread usage of decoction might be attributed to the fact that boiling can accelerate biological processes, resulting in the greater availability of several active compounds [ 66 ]. However, preparation procedures vary based on the type of sickness being treated, the location of the condition, and the medicinal components to be extracted [ 67 ]. Furthermore, the local communities use a variety of methods to administer the plants when treating cattle diseases ( Figure 4 ). The route of administration for the plants was oral (69.79%) and topical (30.2%). Oral administration is a simple and non-invasive form of systemic treatment. The route allows for the rapid absorption and distribution of the prepared medicines and allowing for sufficient curative power to be delivered [ 68 ]. Across many African cultures, oral administration of medicinal plants is the most common route used to treat disease in cattle, as this ensures fast and direct interaction with different plant compounds at the site of action [ 69 , 70 , 71 ]. 3. Materials and Methods 3.1. Study Area This study was conducted in the Gopane (25.3175° S, 25.8231° E) and Dinokana (25.456° S, 25.8799° E) villages of the Ramotshere Moiloa local municipality (25.5623° S, 26.1001° E) located in the Ngaka Modiri Molema district municipality, North West Province of South Africa ( Table S1 and Figure 5 ). Dinokana and Gopane are bordered to the north by Botswana, to the east by Moses Kotane and Kgetleng River local municipalities, and to the south by Ditsobotla and Mafikeng local municipalities [ 72 ]. The two communities are dominantly rural area under the leadership of traditional leader and municipality councillors. The area is rich in floras with potential diverse applications [ 73 ]. The total surface of both communities is 104,882 km 2 . Livestock and agricultural productions provide a significant contribution to the rural economy in the study region, and most rural farming systems transport and generate money both directly and indirectly. The households are active in agricultural activities such as livestock (16,443) and vegetable (1110) production. Particularly, cattle production, consisting of 11,892 households, remains the highest agricultural activity in these communities. The annual income category of agricultural household heads starts from R1 to above R1 228,800 [ 72 ], and according to the Ruminant Veterinary Association of South Africa [ 39 ], the most prevalent animal diseases in the study are internal parasites, external parasites, tick-borne diseases, insect transmittable diseases, venereal diseases, bacterial diseases, and protozoal diseases. The two communities were selected due to the rich plant biodiversity which serves as an important medicinal resource [ 74 ]. Increased population growth, cultural changes such as the rapid shift toward allopathic medicine, and the spread of modern education contribute to the destruction of medicinal plant habitats and the increasing loss of indigenous knowledge due to changes in community inhabitants. Previously, Van der Merwe, Swan, and Botha [ 33 ] documented the ethnoveterinary medicine knowledge of the Madikwe community and Ndou [ 40 ] focused on the EVM in Mahikeng, whereas another study focused on the medicinal plants used for retained placenta [ 25 ]. 3.2. Ethnobotanical Survey An ethnobotanical field survey was conducted from August 2019 to October 2020 (Spring until Summer) in the Dinokana and Gopane villages of the Ramotshere Moiloa local municipality, South Africa ( Figure 5 ). Snowballing was used to recruit and screen eligible participants [ 75 , 76 ]. Ninety participants (83% were male and 17% female) were purposively selected to participate in the study. The age of participants ranged from 18 to 95 and the participants consisted of indigenous knowledge holders, farmers, and cattle herders. The experience and knowledge of participants on the theme of the study, and their interest in participating, were applied as the inclusion criteria [ 77 ]. A face-to-face interview using a semi-structured interview guide prepared in English and translated to Setswana (local language) was used to collect data, after presenting the purpose of the study to the participants, and data was subsequently translated to English. The semi-structured interview guide yielded insightful knowledge for the researcher to develop and generate a rich understanding of the knowledge and skills related to ethnoveterinary [ 78 ]. The data collection questionnaire was divided into three sections to obtain required information. Two phases were followed to collect data. The first phase was interviews, and the second involved a field walk and the collection of plants. Following the Alexiades and Sheldon [ 79 ] technique, responses of participants that contradicted each other were not considered for analysis. The data generated from individual interviews were cross-checked with other participants in the same villages to obtain reliable information in the study area [ 80 ]. The Faculty of Natural and Agricultural Sciences Research Ethics Committee (FNASREC) at the North-West University reviewed and approved the study (Ethics approval number: NWU-01228-19-S9). Traditional authorities in the local municipality granted permission and access to conduct the study in the communities. Prospective participants were approached to seek their consent to participate in the study following detailed and clear explanation on the purpose of the research. The North West Department of Rural, Environmental, and Agricultural Development (NW-READ) granted authorisation for plants collection in the two villages (Permit number HQ 26/01/18-006 NW). Field observations were conducted in study areas to collect the plants mentioned during interviews. Plants were identified by participants and collected by researchers during field walks. Plants used to treat cattle diseases were collected using standard procedures/techniques [ 81 ]. Voucher specimens for the plants were prepared and deposited in the SD Phalatse (UNWH) and AP Goossens Herbarium (PUC) at the North-West University. The nomenclature of all the collected plants was verified using The World Flora Online ( http://www.worldfloraonline.org/ , accessed on 22 March 2022). 3.3. Data Analysis Thematic content and ethnobotanical indices were used to analyse the data collected on indigenous diagnostic skills and ethnobotanical knowledge provided by participants. Thematic content analysis was used to analyse qualitative data [ 82 ]. Following the interviews, the data was transcribed and verified for coherence and saturation. The information from various participants was compared to each other to uncover trends and themes. The emergent themes were linked to data sections with corresponding codes such as participant socio-demographic information, frequently identified cattle ailments, diagnostic procedure, and medicinal plant usage process. When no new data, codes, or themes came from the material, it was considered that saturation had been reached. The ethnobotanical knowledge data were analysed using informant consensus factor (Fic), use-value (UV), and relative frequency of citation (RFC) as described below: Informant consensus factor (Fic): relevant for the categories of diseases to identify the agreement of participants on the reported cures for the group of diseases [ 83 ], which was calculated as follows: Fic = Nur − Nt Nur − 1 where Nur denotes the number of usage reports for a certain ailments category and Nt denotes the number of plants listed for the treatment of that ailment category. Use value (UV): denotes the relative significance of species recognized locally [ 84 ]. The UV was used to identify the plants with the highest utilisation translating to the most frequently mentioned in the treatment of cattle disease [ 85 ]. It was calculated as follows: UV = Ui N where Ui: is the number of uses stated by each participant for a specific species and N: denotes the total number of participants. If a plant secures a high UV score, that indicates that there are many use reports for that plant, whereas a low score indicates fewer use reports cited by the participants. Relative frequency of citation (RFC): as described by Tardío and Pardo-de-Santayana [ 86 ], this measures the agreement among participants on the reported plants. This index calculates the local relevance of each species by dividing the number of participants who mention the species' use, also known as frequency of citation (FC), by the number of target participants included in the study (N). The RFC index was calculated using the following formula: RFC = FC N ( 0 < RFC < 1 ) where FC is the number of participants who reported using a certain species and N denotes the total number of participants in the research. The factor has a value range of 0 to 1, with a high value indicating a high rate of participant consensus. 3.1. Study Area This study was conducted in the Gopane (25.3175° S, 25.8231° E) and Dinokana (25.456° S, 25.8799° E) villages of the Ramotshere Moiloa local municipality (25.5623° S, 26.1001° E) located in the Ngaka Modiri Molema district municipality, North West Province of South Africa ( Table S1 and Figure 5 ). Dinokana and Gopane are bordered to the north by Botswana, to the east by Moses Kotane and Kgetleng River local municipalities, and to the south by Ditsobotla and Mafikeng local municipalities [ 72 ]. The two communities are dominantly rural area under the leadership of traditional leader and municipality councillors. The area is rich in floras with potential diverse applications [ 73 ]. The total surface of both communities is 104,882 km 2 . Livestock and agricultural productions provide a significant contribution to the rural economy in the study region, and most rural farming systems transport and generate money both directly and indirectly. The households are active in agricultural activities such as livestock (16,443) and vegetable (1110) production. Particularly, cattle production, consisting of 11,892 households, remains the highest agricultural activity in these communities. The annual income category of agricultural household heads starts from R1 to above R1 228,800 [ 72 ], and according to the Ruminant Veterinary Association of South Africa [ 39 ], the most prevalent animal diseases in the study are internal parasites, external parasites, tick-borne diseases, insect transmittable diseases, venereal diseases, bacterial diseases, and protozoal diseases. The two communities were selected due to the rich plant biodiversity which serves as an important medicinal resource [ 74 ]. Increased population growth, cultural changes such as the rapid shift toward allopathic medicine, and the spread of modern education contribute to the destruction of medicinal plant habitats and the increasing loss of indigenous knowledge due to changes in community inhabitants. Previously, Van der Merwe, Swan, and Botha [ 33 ] documented the ethnoveterinary medicine knowledge of the Madikwe community and Ndou [ 40 ] focused on the EVM in Mahikeng, whereas another study focused on the medicinal plants used for retained placenta [ 25 ]. 3.2. Ethnobotanical Survey An ethnobotanical field survey was conducted from August 2019 to October 2020 (Spring until Summer) in the Dinokana and Gopane villages of the Ramotshere Moiloa local municipality, South Africa ( Figure 5 ). Snowballing was used to recruit and screen eligible participants [ 75 , 76 ]. Ninety participants (83% were male and 17% female) were purposively selected to participate in the study. The age of participants ranged from 18 to 95 and the participants consisted of indigenous knowledge holders, farmers, and cattle herders. The experience and knowledge of participants on the theme of the study, and their interest in participating, were applied as the inclusion criteria [ 77 ]. A face-to-face interview using a semi-structured interview guide prepared in English and translated to Setswana (local language) was used to collect data, after presenting the purpose of the study to the participants, and data was subsequently translated to English. The semi-structured interview guide yielded insightful knowledge for the researcher to develop and generate a rich understanding of the knowledge and skills related to ethnoveterinary [ 78 ]. The data collection questionnaire was divided into three sections to obtain required information. Two phases were followed to collect data. The first phase was interviews, and the second involved a field walk and the collection of plants. Following the Alexiades and Sheldon [ 79 ] technique, responses of participants that contradicted each other were not considered for analysis. The data generated from individual interviews were cross-checked with other participants in the same villages to obtain reliable information in the study area [ 80 ]. The Faculty of Natural and Agricultural Sciences Research Ethics Committee (FNASREC) at the North-West University reviewed and approved the study (Ethics approval number: NWU-01228-19-S9). Traditional authorities in the local municipality granted permission and access to conduct the study in the communities. Prospective participants were approached to seek their consent to participate in the study following detailed and clear explanation on the purpose of the research. The North West Department of Rural, Environmental, and Agricultural Development (NW-READ) granted authorisation for plants collection in the two villages (Permit number HQ 26/01/18-006 NW). Field observations were conducted in study areas to collect the plants mentioned during interviews. Plants were identified by participants and collected by researchers during field walks. Plants used to treat cattle diseases were collected using standard procedures/techniques [ 81 ]. Voucher specimens for the plants were prepared and deposited in the SD Phalatse (UNWH) and AP Goossens Herbarium (PUC) at the North-West University. The nomenclature of all the collected plants was verified using The World Flora Online ( http://www.worldfloraonline.org/ , accessed on 22 March 2022). 3.3. Data Analysis Thematic content and ethnobotanical indices were used to analyse the data collected on indigenous diagnostic skills and ethnobotanical knowledge provided by participants. Thematic content analysis was used to analyse qualitative data [ 82 ]. Following the interviews, the data was transcribed and verified for coherence and saturation. The information from various participants was compared to each other to uncover trends and themes. The emergent themes were linked to data sections with corresponding codes such as participant socio-demographic information, frequently identified cattle ailments, diagnostic procedure, and medicinal plant usage process. When no new data, codes, or themes came from the material, it was considered that saturation had been reached. The ethnobotanical knowledge data were analysed using informant consensus factor (Fic), use-value (UV), and relative frequency of citation (RFC) as described below: Informant consensus factor (Fic): relevant for the categories of diseases to identify the agreement of participants on the reported cures for the group of diseases [ 83 ], which was calculated as follows: Fic = Nur − Nt Nur − 1 where Nur denotes the number of usage reports for a certain ailments category and Nt denotes the number of plants listed for the treatment of that ailment category. Use value (UV): denotes the relative significance of species recognized locally [ 84 ]. The UV was used to identify the plants with the highest utilisation translating to the most frequently mentioned in the treatment of cattle disease [ 85 ]. It was calculated as follows: UV = Ui N where Ui: is the number of uses stated by each participant for a specific species and N: denotes the total number of participants. If a plant secures a high UV score, that indicates that there are many use reports for that plant, whereas a low score indicates fewer use reports cited by the participants. Relative frequency of citation (RFC): as described by Tardío and Pardo-de-Santayana [ 86 ], this measures the agreement among participants on the reported plants. This index calculates the local relevance of each species by dividing the number of participants who mention the species' use, also known as frequency of citation (FC), by the number of target participants included in the study (N). The RFC index was calculated using the following formula: RFC = FC N ( 0 < RFC < 1 ) where FC is the number of participants who reported using a certain species and N denotes the total number of participants in the research. The factor has a value range of 0 to 1, with a high value indicating a high rate of participant consensus. 4. Conclusions The selected communities are primarily rural in nature, and cattle farmers are exploring their biodiversity and indigenous knowledge practices for meeting the animal health needs and productivity. Based on the current findings, an inventory of 64 medicinal plants from 32 families with a specific indigenous/native rate of 78.1% used to treat 27 cattle ailments from nine categories was documented, with 18 new plants. Three diagnostic skills, 75 distinct clinical signs and symptoms of disease, and two endpoint determinations were reported to understand cattle diseases. Leaves as a plant part, decoction as a preparation method, and oral as an administration route were found to be the most frequently used systems in treating cattle diseases. The plants were prepared as monotherapy and combination. Even though the research area in the Ramotshere Moiloa local municipality was shown to be rich in medicinal plant variety, efforts to study the plants and the indigenous knowledge connected with them are currently limited. To avert additional losses, local communities and responsible entities must conserve therapeutic plants. Furthermore, plants with a high potential based on the applicable ethnobotanical indices should be selected for additional research, such as phytochemical analysis and pharmacological and toxicological studies.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814401/

Progress and Prospects for Genetic Modification of Nonhuman Primate Models in Biomedical Research

The growing interest of modeling human diseases using genetically modified (transgenic) nonhuman primates (NHPs) is a direct result of NHPs (rhesus macaque, etc.) close relation to humans. NHPs share similar developmental paths with humans in their anatomy, physiology, genetics, and neural functions; and in their cognition, emotion, and social behavior. The NHP model within biomedical research has played an important role in the development of vaccines, assisted reproductive technologies, and new therapies for many diseases. Biomedical research has not been the primary role of NHPs. They have mainly been used for safety evaluation and pharmacokinetics studies, rather than determining therapeutic efficacy. The development of the first transgenic rhesus macaque (2001) revolutionized the role of NHP models in biomedicine. Development of the transgenic NHP model of Huntington's disease (2008), with distinctive clinical features, further suggested the uniqueness of the model system; and the potential role of the NHP model for human genetic disorders. Modeling human genetic diseases using NHPs will continue to thrive because of the latest advances in molecular, genetic, and embryo technologies. NHPs rising role in biomedical research, specifically pre-clinical studies, is foreseeable. The path toward the development of transgenic NHPs and the prospect of transgenic NHPs in their new role in future biomedicine needs to be reviewed. This article will focus on the advancement of transgenic NHPs in the past decade, including transgenic technologies and disease modeling. It will outline new technologies that may have significant impact in future NHP modeling and will conclude with a discussion of the future prospects of the transgenic NHP model.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8693014/

Whole genome sequence of bacteremic Clostridium tertium in a World War I soldier, 1914

Highlights • This original article is the first report of isolate and culture of a bacterium from ancient human samples and dental pulp in particular. • The dental pulp is a mirror of the individual's infectious state at the time of death. • Ancient dental pulp culture yielded to the identification and isolation of the bacterium Clostridium tertium responsible for septicaemia during World War I,. Background Dental pulp, encapsulating a blood drop, could be used to diagnose pathogen bacteraemia in archaeological materials using DNA-based techniques. We questioned the viability of such ancient pathogens preserved in ancient dental pulp. Methods After meticulous decontamination of 32 teeth collected from 31 World War I soldiers exhumed in Spincourt, France, dental pulps were extracted and cultured under strict anaerobiosis. Colonies were identified by mass spectrometry and whole genome sequencing. Fluorescent in situ hybridisation (FISH) was used for the direct microscopic detection of pathogens of interest in the dental pulp. All the experimental procedures included negative controls, notably sediments in contact with individual SQ517 to ensure that results did not arise from contamination. Findings Clostridium tertium was detected by FISH in two dental pulp specimens taken from a 1914 soldier. After a two-day incubation period, both dental pulp samples grew colonies identified by mass spectrometry and genome sequencing as C. tertium; whereas negative controls remained free of C. tertium in all the observations, and no C. tertium was founded in sediments. Skeletal remains of this soldier exhibited two notches in the left tibia evocative of a cold steel wound, and a probably fatal unhealed bullet impact in the hip bone. Interpretation Data indicated the presence of C. tertium in the dental pulp at the time of the death of one World War I soldier, in 1914. This observation diagnosed C. tertium bacteraemia, with war wounds as the probable portal of entry for C. tertium. Our C. tertium strains ante-dated by three years, the princeps description of this deadly opportunistic pathogen. 1 Materials and methods 1.1 Archaeological investigations After war declaration between France and Germany on August 3rd 1914, the first weeks of conflict involved territories close to the village of Spincourt, Great-East France (latitude: 49.3333; longitude: 5.6667), regarded as the heart of the « Border battle ». After German troops defeated at Mangiennes, a frontal battle against French troops retreating towards Verdun, took place in Spincourt between August 20th and August 25th. Fights raged and losses were heavy (27,000 deaths only for August 22nd). Fights ended-up with Spincourt firing and its occupation by German troops, until October 1918 ( Verna et al., 2020 ). Few days after August fights, German troops assured the funeral gestion of deads including the French ones into a provisional cemetery. In the aftermath of war during the 1920′s, numerous bodies were unearthed from the provisional cemetery to be returned to their families and be reinsulated in military necropolis. Starting February 2017, the provisional military cemetery was investigated by Institut National Recherche Archéologique Préventive, disclosing testifying of an organised gestion of corpses (graves rigorously aligned, coffin inhumation) and 31 remaining skeletons by October 2018 ( Verna et al., 2021 ). Examination of these skeletons testified of the violence of fightings (multiple traumas on skeleton, supernumerary anatomical part in coffin). Skeleton were examined using the Diagnose Sexuelle Probabiliste v2 (DSP2) software method for sex estimation and Lovejoy's quotation for age ( Lovejoy et al., 1985 ; Brůžek et al., 2017 ). A total of 32 teeth were collected from the 31 skeletons for palaeomicrobiological investigations. 1.2 Paleomicrobiological investigations Thirty-two teeth were investigated for palaeomicrobiology, one tooth for each one of the 31 soldiers and two teeth (45 and 34) for individual SQ517. The external surface of each tooth was cleaned with sterile gauze soaked with pure ethanol and bleach for 30 s ( Fig. 1 ). Pre-opening, fracture and pulp extirpation were all carried out under anaerobiosis to avoid any direct exposure of the dental pulp to atmospheric oxygen. Accordingly, all instruments necessary for opening the teeth and extracting the pulp, including a small circular diamond saw and its motor or excavator, were placed under an anaerobic hood (Don Whitley, Bingley, UK) before the manipulation. Dental pulps were extracted with specific tools (an excavator) following a previously described protocol ( Drancourt et al., 1998 ). Pulps were rehydrated for one minute with 10 µL sterile phosphate buffered saline (PBS) and 0.5 µL of rehydrated dental pulp were inoculated onto a 5% sheep blood agar Petri plate (Becton Dickinson GmbH, Heidelberg, Germany) ( Fig. 1 ). A negative control culture medium was opened in the anaerobiosis hood at the beginning of the manipulation to assess the hood's sterility. A piece of sterile gauze soaked with sterile water was placed under each culture plate to keep humidity, and plates were placed into a microaerophilic bag (BD GasPak, EZ Pouch Systems, Becton-Dickinson, Franklin Lakes, NJ, US) incubated at 37 °C under a 5% CO 2 atmosphere ( Fig. 1 ). Each microaerophilic bag also contained one negative control inoculate with 10 µL sterile PBS. Sediments in direct contacts with individual SQ517's tooth 45, tooth 34 and left tibia were put in culture before decontamination, following the same protocol described above. Fig. 1 Workflow summarizing culture of ancient dental pulps: Step 1: Teeth 45 and 34 were disinfected with pure ethanol and bleach. After the teeth were fractured, the dental pulp was extirpated under anaerobiosis; Step 1a: Sediment from the non-disinfected teeth of individual SQ517 and surrounding the tibia were used; Step 2: Dental pulp was mixed with 10uL of PBS; Step 2a: Sediment from the outer teeth and tibia were respectively mixed with 10μl and 150μl of PBS; Step 3: The rehydrated pulp was placed onto an agar plate with 5% sheep blood (Becton Dickinson GmbH, Heidelberg, Germany); Step 3a: Rehydrated sediments were placed onto an agar plate with 5% sheep blood (Becton Dickinson GmbH, Heidelberg, Germany); Step 4: Agar plates were incubated at 37° under a 5% CO2 atmosphere in a microaerophilic bag and were inspected daily. Fig. 1: 1.3 C. tertium identification Colonies observed by daily naked eye inspection were stained by Gram-staining (bioMérieux, Craponne, France) and further identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) ( Seng et al., 2009 ). The minimum inhibitory concentration (MIC) of amoxicillin–clavulanic acid (30 mg/L), erythromycin (15 mg/L), metronidazole (4 mg/L), and clindamycin (2 mg/L) ( Vanderhofstadt et al., 2010 ; Salvador et al., 2013 ) was determined for each C. tertium isolate by Scan 1200® (Interscience, Saint-Nom-la-Bretèche, France). The C. tertium isolate Q5690 was further investigated by whole genome sequencing (WGS) as previously described ( Muñoz et al., 2019 ). Briefly, DNA was extracted using the standard EZ1 protocol from isolate Q5690, from strain SP2622 initially isolated in 1923 ( Johnson and Francis, 1975 ) (DSMZ 2485, Leibniz Institute, Braunschweig, Germany), and from a previous C. tertium (Q6181) cultivated in our laboratory. DNAs were sequenced on Illumina Miseq and sequencing reads were assembled using Spades software ( Bankevich et al., 2012 ). In silico DNA–DNA hybridization (DDH) was used to delineate bacterial species ( Rossello-Mora, 2006 ) and C. tertium Q5690 genomic sequence was compared to those retrieved in public databases including C. tertium MGYG HGUT 01328 reference genome. Mugsy software was used for genome alignments ( Angiuoli and Salzberg, 2011 ) and a phylogenetic tree was done using Raxml software ( Stamatakis, 2014 ) incorporating the maximum likelihood method (ML) algorithm, with 1,00 bootstrap replicates. Genes related to pathogenicity and virulence factors were searched in the literature and sequences available in the Virulence Factor Database (VFDB) ( Chen, 2004 ). Antibiotic resistance profiling was achieved by using Abricate pipeline comparaison with CARD ( McArthur et al., 2013 ), ARG-ANNOT ( Gupta et al., 2014 ), Restfinder ( Zankari et al., 2012 ) and Bacterial Antimicrobial Resistance Reference Gene Database. SNPs analyses were performed with SNP-sites on ( Angiuoli and Salzberg, 2011 ; Page et al., 2016 ). 1.4 Fluorescence in situ hybridization (FISH) C. tertium was specifically detected in the dental pulp specimen by fluorescent in situ hybridisation (FISH), incorporating probe 3′-CTCCAACCCTAGTAAACCCCT-5′ labelled with Alexa fluor-488 and targeting the specific C. tertium gene fur coding for a transcriptional regulator following the MGYG HGUT 01328 reference genome annotation on Dfast software. The gene sequence was blasted on NCBI against the Clostridium genus (taxid 1485) database yielded 100% identity and 87% coverage with C. tertium and 99.76% identity and 87% coverage with Clostridium perfringens. Then, a specific probe was designed from this gene using NCBI primers and yielded 100% identity and coverage only with C. tertium on NCBI blast against the Clostridium genus (taxid 1485) database. Briefly, the dental pulp of individual SQ517 was fixed for three hours in Sandison's rehydration solution (aqueous formaldehyde 1%, 96% ethanol and 5% aqueous sodium carbonate) ( Collini et al., 2014 ) and smeared on microscopic slade using Cytospin (ThermoFisher, Illkirch, France). In situ hybridization was performed with a hybridiser (Dako, Les Ulis, France) at 65 °C for 10 min and then at 37° overnight, following a previously reported protocol ( Millogo et al., 2020 ). Dental pulp of the individual SQ536, which remained negative in culture for C. tertium was used as a negative control to assess the probe specificity. 1.1 Archaeological investigations After war declaration between France and Germany on August 3rd 1914, the first weeks of conflict involved territories close to the village of Spincourt, Great-East France (latitude: 49.3333; longitude: 5.6667), regarded as the heart of the « Border battle ». After German troops defeated at Mangiennes, a frontal battle against French troops retreating towards Verdun, took place in Spincourt between August 20th and August 25th. Fights raged and losses were heavy (27,000 deaths only for August 22nd). Fights ended-up with Spincourt firing and its occupation by German troops, until October 1918 ( Verna et al., 2020 ). Few days after August fights, German troops assured the funeral gestion of deads including the French ones into a provisional cemetery. In the aftermath of war during the 1920′s, numerous bodies were unearthed from the provisional cemetery to be returned to their families and be reinsulated in military necropolis. Starting February 2017, the provisional military cemetery was investigated by Institut National Recherche Archéologique Préventive, disclosing testifying of an organised gestion of corpses (graves rigorously aligned, coffin inhumation) and 31 remaining skeletons by October 2018 ( Verna et al., 2021 ). Examination of these skeletons testified of the violence of fightings (multiple traumas on skeleton, supernumerary anatomical part in coffin). Skeleton were examined using the Diagnose Sexuelle Probabiliste v2 (DSP2) software method for sex estimation and Lovejoy's quotation for age ( Lovejoy et al., 1985 ; Brůžek et al., 2017 ). A total of 32 teeth were collected from the 31 skeletons for palaeomicrobiological investigations. 1.2 Paleomicrobiological investigations Thirty-two teeth were investigated for palaeomicrobiology, one tooth for each one of the 31 soldiers and two teeth (45 and 34) for individual SQ517. The external surface of each tooth was cleaned with sterile gauze soaked with pure ethanol and bleach for 30 s ( Fig. 1 ). Pre-opening, fracture and pulp extirpation were all carried out under anaerobiosis to avoid any direct exposure of the dental pulp to atmospheric oxygen. Accordingly, all instruments necessary for opening the teeth and extracting the pulp, including a small circular diamond saw and its motor or excavator, were placed under an anaerobic hood (Don Whitley, Bingley, UK) before the manipulation. Dental pulps were extracted with specific tools (an excavator) following a previously described protocol ( Drancourt et al., 1998 ). Pulps were rehydrated for one minute with 10 µL sterile phosphate buffered saline (PBS) and 0.5 µL of rehydrated dental pulp were inoculated onto a 5% sheep blood agar Petri plate (Becton Dickinson GmbH, Heidelberg, Germany) ( Fig. 1 ). A negative control culture medium was opened in the anaerobiosis hood at the beginning of the manipulation to assess the hood's sterility. A piece of sterile gauze soaked with sterile water was placed under each culture plate to keep humidity, and plates were placed into a microaerophilic bag (BD GasPak, EZ Pouch Systems, Becton-Dickinson, Franklin Lakes, NJ, US) incubated at 37 °C under a 5% CO 2 atmosphere ( Fig. 1 ). Each microaerophilic bag also contained one negative control inoculate with 10 µL sterile PBS. Sediments in direct contacts with individual SQ517's tooth 45, tooth 34 and left tibia were put in culture before decontamination, following the same protocol described above. Fig. 1 Workflow summarizing culture of ancient dental pulps: Step 1: Teeth 45 and 34 were disinfected with pure ethanol and bleach. After the teeth were fractured, the dental pulp was extirpated under anaerobiosis; Step 1a: Sediment from the non-disinfected teeth of individual SQ517 and surrounding the tibia were used; Step 2: Dental pulp was mixed with 10uL of PBS; Step 2a: Sediment from the outer teeth and tibia were respectively mixed with 10μl and 150μl of PBS; Step 3: The rehydrated pulp was placed onto an agar plate with 5% sheep blood (Becton Dickinson GmbH, Heidelberg, Germany); Step 3a: Rehydrated sediments were placed onto an agar plate with 5% sheep blood (Becton Dickinson GmbH, Heidelberg, Germany); Step 4: Agar plates were incubated at 37° under a 5% CO2 atmosphere in a microaerophilic bag and were inspected daily. Fig. 1: 1.3 C. tertium identification Colonies observed by daily naked eye inspection were stained by Gram-staining (bioMérieux, Craponne, France) and further identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) ( Seng et al., 2009 ). The minimum inhibitory concentration (MIC) of amoxicillin–clavulanic acid (30 mg/L), erythromycin (15 mg/L), metronidazole (4 mg/L), and clindamycin (2 mg/L) ( Vanderhofstadt et al., 2010 ; Salvador et al., 2013 ) was determined for each C. tertium isolate by Scan 1200® (Interscience, Saint-Nom-la-Bretèche, France). The C. tertium isolate Q5690 was further investigated by whole genome sequencing (WGS) as previously described ( Muñoz et al., 2019 ). Briefly, DNA was extracted using the standard EZ1 protocol from isolate Q5690, from strain SP2622 initially isolated in 1923 ( Johnson and Francis, 1975 ) (DSMZ 2485, Leibniz Institute, Braunschweig, Germany), and from a previous C. tertium (Q6181) cultivated in our laboratory. DNAs were sequenced on Illumina Miseq and sequencing reads were assembled using Spades software ( Bankevich et al., 2012 ). In silico DNA–DNA hybridization (DDH) was used to delineate bacterial species ( Rossello-Mora, 2006 ) and C. tertium Q5690 genomic sequence was compared to those retrieved in public databases including C. tertium MGYG HGUT 01328 reference genome. Mugsy software was used for genome alignments ( Angiuoli and Salzberg, 2011 ) and a phylogenetic tree was done using Raxml software ( Stamatakis, 2014 ) incorporating the maximum likelihood method (ML) algorithm, with 1,00 bootstrap replicates. Genes related to pathogenicity and virulence factors were searched in the literature and sequences available in the Virulence Factor Database (VFDB) ( Chen, 2004 ). Antibiotic resistance profiling was achieved by using Abricate pipeline comparaison with CARD ( McArthur et al., 2013 ), ARG-ANNOT ( Gupta et al., 2014 ), Restfinder ( Zankari et al., 2012 ) and Bacterial Antimicrobial Resistance Reference Gene Database. SNPs analyses were performed with SNP-sites on ( Angiuoli and Salzberg, 2011 ; Page et al., 2016 ). 1.4 Fluorescence in situ hybridization (FISH) C. tertium was specifically detected in the dental pulp specimen by fluorescent in situ hybridisation (FISH), incorporating probe 3′-CTCCAACCCTAGTAAACCCCT-5′ labelled with Alexa fluor-488 and targeting the specific C. tertium gene fur coding for a transcriptional regulator following the MGYG HGUT 01328 reference genome annotation on Dfast software. The gene sequence was blasted on NCBI against the Clostridium genus (taxid 1485) database yielded 100% identity and 87% coverage with C. tertium and 99.76% identity and 87% coverage with Clostridium perfringens. Then, a specific probe was designed from this gene using NCBI primers and yielded 100% identity and coverage only with C. tertium on NCBI blast against the Clostridium genus (taxid 1485) database. Briefly, the dental pulp of individual SQ517 was fixed for three hours in Sandison's rehydration solution (aqueous formaldehyde 1%, 96% ethanol and 5% aqueous sodium carbonate) ( Collini et al., 2014 ) and smeared on microscopic slade using Cytospin (ThermoFisher, Illkirch, France). In situ hybridization was performed with a hybridiser (Dako, Les Ulis, France) at 65 °C for 10 min and then at 37° overnight, following a previously reported protocol ( Millogo et al., 2020 ). Dental pulp of the individual SQ536, which remained negative in culture for C. tertium was used as a negative control to assess the probe specificity. 2 Results 2.1 Archaeological investigations After meticulous inspection and bacterial identification of the 32 dental pulps, only teeth from the individual SQ517 yielded C. tertium as reported below. He was a man aged 30–35 years at the time of his death. The anthropological examination of the skeleton of individual SQ517 revealed two notches in the left tibia, evocative of the impact of a cold steel weapon (knife or bayonet), surrounded by a periosteal reaction indicating that the wounds occurred before death; a fracture to the right zygomatic branch and the right condyle of the mandible; left and right rib fractures which may have occurred at the time of death or post-mortem. A bullet impact was also observed with an entry point in the anterior part of the ilium and the exit point in the posterior part with significant bone loss and no sign of healing ( Fig. 2 ). Fig. 2 Osteological traumas on individual SQ517; A (I), (II) and (III): Marks on the left tibia resulting from a cold steel weapon; B: Bullet exit hole on the ilium of the left hip bone; C: Bullet entry hole on the ilium of the left hip bone; D and E: Perimortem fracture to the right zygomatic branch of the skull and to the mandibular condyle. Fig. 2: 2.2 Culture of ancient dental pulp The negative control culture medium remained free of any visible colonies for five days, but the plate inoculated with the dental pulp of the second right premolar tooth (45) and first left premolar tooth (34), collected directly from individual SQ517's mandible, yielded colonies after three days of incubation ( Fig. 3 ). These colonies were stained purple by Gram staining and were identified as C. tertium by MALDI-TOF mass spectrometry with index of 2.18 for tooth 45 and 2.45 for tooth 34 ( Fig. 4 ). Both C. tertium isolates were deposited in the Collection de Souches de l'Unité des Rickettsies (IHU Méditerranée Infection, Marseille, France) under reference numbers, CSUR Q5690 and CSUR Q5873, respectively. No C. tertium was cultured from sample sediments collected around the individual SQ517, neither from the negative controls manipulated under the anaerobic hood and Phosphate Buffered Saline (PBS) ( Table 1 ). Likewise, no C. tertium isolates was retrieved from any of the 30 dental pulp samples. The antibiogram indicated C. tertium strains CSUR Q5690 and Q5873 have in-vitro a MIC inferior to 30 mg/L for amoxicillin-clavulanic acidwith an inhibitory zone of 30 mm, and inferior to 15 mg/L for erythromycin with an inhibitory zone of 20,2 mm. The strains also have a MIC superior to 2 mg/L for clindamycin and superior to 4 mg/L for metronidazole with both of their inhibitory zone at 0,0 mm ( Fig. 5 ). Fig. 3 C. tertium colonies yield from SQ517 individual after three days of incubation. Fig. 3: Fig. 4 Gram staining from Q5690 C. tertium strain under 100x optical microscopic lenses. Fig. 4: Table 1 Bacteria founded in negative control cultures: Bacterial species isolated from the sediment samples around SQ517, from the anaerobic hood and from the culture media inoculated with PBS. Table 1: Species Score Value 1 (MALDI-TOF) Score Value 2 (MALDI-TOF) Clostridium sordellii 2.28 2.31 Clostridium sporogène 2.00 2.22 Clostridium novyi 2.00 1.92 Bacillus megaterium 2.15 2.24 Bacillus cereus 2.28 2.39 Bacillus simplex 2.00 1.76 Bacillus licheniformis 2.42 2.30 Bacillus pumilus 2.15 2.24 Paenibacillus glucanolyticus 2.13 2.21 Clostridium tetani 2.14 2.13 Bacillus toyonensis 2.19 2.09 Staphylococcus homini 2.19 2.19 Fig. 5 Antibiogram: amoxicillin-clavulanic acid (AMC) 30 mg/L, erythromycin (E) 15 mg/L, metronidazole (MET) 4 mg/L and clindamycin (DA) 2 mg/L on Q5690. - Scan 1200®. Fig. 5: 2.3 C. tertium identification Isolate Q5690 genome sequence (obtained with a 13.7 X coverage for a 3776,523 base pairs (bp) size) yielded DDH values ranging from 19.5% with Clostridium celatum and Clostridium saudiense, 19.9% with Clostridium gasigenes, 28.8% with Clostridium septicum and Clostridium chauvoei up to 89.4% with C. tertium strain Q5690 ( Table 2 ). Accordingly, DDH values between isolate strain Q5690 and C. tertium MGYG HGUT 01328 reference genome were greater than the 70% threshold acknowledged to delimit bacterial species, are support the strain Q5690 is the same species as C. tertium ( Wayne, 1988 ; Tindall et al., 2010 ). Phylogenic tree indicated that isolate Q5690 was closer C. tertium MGYG HGUT 01328 reference genome than to C. tertium strain Q6181 previously cultivated in our laboratory ( Fig. 6 ). Noteworthy, isolate Q5690 genome sequence was closer to that of strain SP2622 isolated in 1923, than to recent isolate genome sequence ( Fig. 7 ). However, no specific difference in the repertoire of virulence and resistance genes was observed between C. tertium strain Q5690 and all the other C. tertium genomes (Supplementary tables A-D). Regarding SNPs analysis, we founded 18,107 SNPs between isolate Q5690 and isolate Q6181, and 15,657 SNPs between isolate Q5690 and C. tertium reference genome MGYG HGUT 01328. Further analysis on SNP profile indicated 36, 615 SNPs differences between isolates Q5690 and SP2622, and 37, 402 SNPs difference between SP2622 and the reference genome MGYG HGUT 01328. Table 2 Pairwise genomic comparison of strain Q5690 with other species using the GGDC software, formula 2 (dDDH estimates based on identities over HSP length). The confidence intervals indicate the inherent uncertainty in estimating dDDH values from intergenomic distances based on models derived from empirical test data sets. Table 2: Q5690 Clostridium tertium Clostridium chauvoei Clostridium isatidis Clostridium sartagoforme Clostridium septicum Clostridium jeddahitimonense Clostridium saudiense Clostridium celatum Clostridium disporicum Clostridium gasigenes Q5690 100% Clostridium tertium 89.4 100% [87 - 92] Clostridium chauvoei 21.7 21.7 100% [19.4–24] [19.4–24] Clostridium isatidis 21.6 21.7 21 100% [19.4–24] [19.4–24] [18.8–23] Clostridium sartagoforme 23.8 23.6 21.6 21 100% [21.5–26] [21.3–26] [19.4–24] [18.8–24] Clostridium 21.9 21.8 28.8 21.1 21.9 100% septicum [19–24] [19.6–24] [26.4–31] [18.9–24] [19.6–24] Clostridium jeddahitimonense 20.5 20.7 20.3 21.1 21.2 21.3 100% [18.2–23] [18.4–23] [18.1–23] [18.9–24] [19–24] [19–24] Clostridium saudiense 20.4 20.4 20.7 20.3 20.7 21.2 21.8 100% [18.2–23] [18.2–23] [18.4–23] [18.1–23] [18.5–23] [19–24] [19.5–24] Clostridium celatum 20. 21.2 20.6 21.2 20.7 21 21.8 19.5 100% [18.2–23] [18.9–24] [18.4–23] [19–24] [18.5–23] [18.7–23] [19.5–24] [17.3–22] Clostridium disporicum 20 20.3 20.3 20.1 20.4 20.7 33.2 22.2 21.9 100% [17.8–22] [18.1–23] [18.1–23] [17.9–23] [18.2–23] [18.5–23] [30.7–36] [19.9–25] [19.7–24] Clostridium gasigenes 19.9 20.7 21.7 21.6 21.2 21.6 21 20.7 20.7 20.6 100% [17.7–22] [18.5–23] [19.5–24] [19.3–24] [19–24] [19.3–24] [18.7–23] [18.4–23] [18.4–23] [18.4–23] Fig. 6 Phylogenic tree of 20 Clostridium species: The tree underlines the proximity between 22 genomes from different Clostridium species. Q5690 appears closer to the reference genome MGYG-HGUT-01,328 than from Q6181 (modern diagnostic strain). The closest relative to C. tertium is Clostridium sartagoforme. All genomes can be found on NCBI. Fig. 6: Fig. 7 Phylogenic tree including all C. tertium genomes available on NCBI database: The tree underlines the proximity between several genomes from different C. tertium strain. Q5690 appears closer to the reference strain from 1923 (SP2622) than from the reference genome MGYG-HGUT-01,328. The closest relative to C. tertium Q5690 is strain BSD2780120874_150,323_E10. All genomes can be found on NCBI. Fig. 7: 2.4 Microscopy FISH investigation showed that the SQ517 dental pulp was positive for C. tertium fur gene, whereas the negative control (the SQ536 dental pulp) remained negative ( Fig. 8 ). Fig. 8 FISH on SQ517 dental pulp: 1: filter AF555; 2: filter AF640; 3: Filter AF488; 4: DAPI; 5: MERCH. Fig. 8: 2.1 Archaeological investigations After meticulous inspection and bacterial identification of the 32 dental pulps, only teeth from the individual SQ517 yielded C. tertium as reported below. He was a man aged 30–35 years at the time of his death. The anthropological examination of the skeleton of individual SQ517 revealed two notches in the left tibia, evocative of the impact of a cold steel weapon (knife or bayonet), surrounded by a periosteal reaction indicating that the wounds occurred before death; a fracture to the right zygomatic branch and the right condyle of the mandible; left and right rib fractures which may have occurred at the time of death or post-mortem. A bullet impact was also observed with an entry point in the anterior part of the ilium and the exit point in the posterior part with significant bone loss and no sign of healing ( Fig. 2 ). Fig. 2 Osteological traumas on individual SQ517; A (I), (II) and (III): Marks on the left tibia resulting from a cold steel weapon; B: Bullet exit hole on the ilium of the left hip bone; C: Bullet entry hole on the ilium of the left hip bone; D and E: Perimortem fracture to the right zygomatic branch of the skull and to the mandibular condyle. Fig. 2: 2.2 Culture of ancient dental pulp The negative control culture medium remained free of any visible colonies for five days, but the plate inoculated with the dental pulp of the second right premolar tooth (45) and first left premolar tooth (34), collected directly from individual SQ517's mandible, yielded colonies after three days of incubation ( Fig. 3 ). These colonies were stained purple by Gram staining and were identified as C. tertium by MALDI-TOF mass spectrometry with index of 2.18 for tooth 45 and 2.45 for tooth 34 ( Fig. 4 ). Both C. tertium isolates were deposited in the Collection de Souches de l'Unité des Rickettsies (IHU Méditerranée Infection, Marseille, France) under reference numbers, CSUR Q5690 and CSUR Q5873, respectively. No C. tertium was cultured from sample sediments collected around the individual SQ517, neither from the negative controls manipulated under the anaerobic hood and Phosphate Buffered Saline (PBS) ( Table 1 ). Likewise, no C. tertium isolates was retrieved from any of the 30 dental pulp samples. The antibiogram indicated C. tertium strains CSUR Q5690 and Q5873 have in-vitro a MIC inferior to 30 mg/L for amoxicillin-clavulanic acidwith an inhibitory zone of 30 mm, and inferior to 15 mg/L for erythromycin with an inhibitory zone of 20,2 mm. The strains also have a MIC superior to 2 mg/L for clindamycin and superior to 4 mg/L for metronidazole with both of their inhibitory zone at 0,0 mm ( Fig. 5 ). Fig. 3 C. tertium colonies yield from SQ517 individual after three days of incubation. Fig. 3: Fig. 4 Gram staining from Q5690 C. tertium strain under 100x optical microscopic lenses. Fig. 4: Table 1 Bacteria founded in negative control cultures: Bacterial species isolated from the sediment samples around SQ517, from the anaerobic hood and from the culture media inoculated with PBS. Table 1: Species Score Value 1 (MALDI-TOF) Score Value 2 (MALDI-TOF) Clostridium sordellii 2.28 2.31 Clostridium sporogène 2.00 2.22 Clostridium novyi 2.00 1.92 Bacillus megaterium 2.15 2.24 Bacillus cereus 2.28 2.39 Bacillus simplex 2.00 1.76 Bacillus licheniformis 2.42 2.30 Bacillus pumilus 2.15 2.24 Paenibacillus glucanolyticus 2.13 2.21 Clostridium tetani 2.14 2.13 Bacillus toyonensis 2.19 2.09 Staphylococcus homini 2.19 2.19 Fig. 5 Antibiogram: amoxicillin-clavulanic acid (AMC) 30 mg/L, erythromycin (E) 15 mg/L, metronidazole (MET) 4 mg/L and clindamycin (DA) 2 mg/L on Q5690. - Scan 1200®. Fig. 5: 2.3 C. tertium identification Isolate Q5690 genome sequence (obtained with a 13.7 X coverage for a 3776,523 base pairs (bp) size) yielded DDH values ranging from 19.5% with Clostridium celatum and Clostridium saudiense, 19.9% with Clostridium gasigenes, 28.8% with Clostridium septicum and Clostridium chauvoei up to 89.4% with C. tertium strain Q5690 ( Table 2 ). Accordingly, DDH values between isolate strain Q5690 and C. tertium MGYG HGUT 01328 reference genome were greater than the 70% threshold acknowledged to delimit bacterial species, are support the strain Q5690 is the same species as C. tertium ( Wayne, 1988 ; Tindall et al., 2010 ). Phylogenic tree indicated that isolate Q5690 was closer C. tertium MGYG HGUT 01328 reference genome than to C. tertium strain Q6181 previously cultivated in our laboratory ( Fig. 6 ). Noteworthy, isolate Q5690 genome sequence was closer to that of strain SP2622 isolated in 1923, than to recent isolate genome sequence ( Fig. 7 ). However, no specific difference in the repertoire of virulence and resistance genes was observed between C. tertium strain Q5690 and all the other C. tertium genomes (Supplementary tables A-D). Regarding SNPs analysis, we founded 18,107 SNPs between isolate Q5690 and isolate Q6181, and 15,657 SNPs between isolate Q5690 and C. tertium reference genome MGYG HGUT 01328. Further analysis on SNP profile indicated 36, 615 SNPs differences between isolates Q5690 and SP2622, and 37, 402 SNPs difference between SP2622 and the reference genome MGYG HGUT 01328. Table 2 Pairwise genomic comparison of strain Q5690 with other species using the GGDC software, formula 2 (dDDH estimates based on identities over HSP length). The confidence intervals indicate the inherent uncertainty in estimating dDDH values from intergenomic distances based on models derived from empirical test data sets. Table 2: Q5690 Clostridium tertium Clostridium chauvoei Clostridium isatidis Clostridium sartagoforme Clostridium septicum Clostridium jeddahitimonense Clostridium saudiense Clostridium celatum Clostridium disporicum Clostridium gasigenes Q5690 100% Clostridium tertium 89.4 100% [87 - 92] Clostridium chauvoei 21.7 21.7 100% [19.4–24] [19.4–24] Clostridium isatidis 21.6 21.7 21 100% [19.4–24] [19.4–24] [18.8–23] Clostridium sartagoforme 23.8 23.6 21.6 21 100% [21.5–26] [21.3–26] [19.4–24] [18.8–24] Clostridium 21.9 21.8 28.8 21.1 21.9 100% septicum [19–24] [19.6–24] [26.4–31] [18.9–24] [19.6–24] Clostridium jeddahitimonense 20.5 20.7 20.3 21.1 21.2 21.3 100% [18.2–23] [18.4–23] [18.1–23] [18.9–24] [19–24] [19–24] Clostridium saudiense 20.4 20.4 20.7 20.3 20.7 21.2 21.8 100% [18.2–23] [18.2–23] [18.4–23] [18.1–23] [18.5–23] [19–24] [19.5–24] Clostridium celatum 20. 21.2 20.6 21.2 20.7 21 21.8 19.5 100% [18.2–23] [18.9–24] [18.4–23] [19–24] [18.5–23] [18.7–23] [19.5–24] [17.3–22] Clostridium disporicum 20 20.3 20.3 20.1 20.4 20.7 33.2 22.2 21.9 100% [17.8–22] [18.1–23] [18.1–23] [17.9–23] [18.2–23] [18.5–23] [30.7–36] [19.9–25] [19.7–24] Clostridium gasigenes 19.9 20.7 21.7 21.6 21.2 21.6 21 20.7 20.7 20.6 100% [17.7–22] [18.5–23] [19.5–24] [19.3–24] [19–24] [19.3–24] [18.7–23] [18.4–23] [18.4–23] [18.4–23] Fig. 6 Phylogenic tree of 20 Clostridium species: The tree underlines the proximity between 22 genomes from different Clostridium species. Q5690 appears closer to the reference genome MGYG-HGUT-01,328 than from Q6181 (modern diagnostic strain). The closest relative to C. tertium is Clostridium sartagoforme. All genomes can be found on NCBI. Fig. 6: Fig. 7 Phylogenic tree including all C. tertium genomes available on NCBI database: The tree underlines the proximity between several genomes from different C. tertium strain. Q5690 appears closer to the reference strain from 1923 (SP2622) than from the reference genome MGYG-HGUT-01,328. The closest relative to C. tertium Q5690 is strain BSD2780120874_150,323_E10. All genomes can be found on NCBI. Fig. 7: 2.4 Microscopy FISH investigation showed that the SQ517 dental pulp was positive for C. tertium fur gene, whereas the negative control (the SQ536 dental pulp) remained negative ( Fig. 8 ). Fig. 8 FISH on SQ517 dental pulp: 1: filter AF555; 2: filter AF640; 3: Filter AF488; 4: DAPI; 5: MERCH. Fig. 8: 3 Discussion We interpreted that C. tertium was encapsulated in the dental pulp of soldier SQ517 at the time of his death after highly vascularized dental pulp of soldier SQ517 has preserved a drop of blood ( Barbieri et al., 2020 ). The analyse of such blood drop allowed detection of C. tertium in two dental pulp specimens collected from soldier SQ517, suggesting individual SQ517 suffered C. tertium bacteraemia. This condition is associated with a fatal outcome in modern literature ( Valtonen et al., 1990 ), even if its pathogenicity mechanism still be misunderstood due to its lack of exotoxin ( Muñoz et al., 2019 ). Nevertheless, nagH gene encoded by C. tertium genome and present in isolate Q5690 (Supplementary Table C), could act on connective tissue of gas gangrene, as it was proposed for C. perfringens ( Muñoz et al., 2019 ). This gene was detected in all investigated genomes sequences including the one isolate from 1914 (Q5690) at the exception of the strain Q6181 previously isolated in our laboratory. Indeed, even if 100 years form a long time to execute a bacterial isolation from dental pulp, regarding bacterial evolution one century may be too short to observe any differences between the archaeological sample here investigated and modern genome sequences as illustrated by a lack of significant differences between ancient and modern genomes. The detection in isolate Q5690 of MLS VatB gene and cdeA gene (Supplementary Table D) conferring respectively resistance to streptogramin A and fluoroquinolones ( Dridi et al., 2004 ; Mayers et al., 2017 ), is one more example of antibiotic resistance predating the medical use of antibiotics. Historical sources indicated that soldier SQ517 died between 20 and 30 August 1914. He was, therefore, the index case of C. tertium bacteraemia, as this microorganism had only previously been isolated in 1917 ( Henry, 1918 ). Our present study took us three years back in time from the first discovery and isolation of C. tertium. We are reporting on the first ever culture-based paleomicrobiological diagnosis from the dental pulp of an individual from 1914 who was suffering from a C. tertium bacteraemia. More than that, it is also the first time that ancient dental pulp was used for bacterial culture and led to bacterial isolation. Previous paleomicrobiological detections of pathogens have indeed all been based upon the detection of specific biomolecules culminating in the recovery of entire genome sequences, yet no such pathogen has ever been isolated and cultivated from human remains. Only sporulated Bacillus genus microorganisms have been reported from soil, the record being Bacillus massiliglaciei which was retrieved by culture from Siberian permafrost specimens dating from, on average, 10 million years ago ( Afouda et al., 2016 ). There has also been report of a 1917 Prussian spy carrying a piece of sugar hiding anthrax spores ( Redmond et al., 1998 ). Such isolates could be explained by the spore forming capacity of Clostridium and Bacillus ( Kennedy et al., 1994 ). The spores are known to allow bacteria to survive in extreme environmental conditions, such as cold, heat or anaerobiosis ( Roszac and Colwell, 1987 ; Kennedy et al., 1994 ).In the present study, we can see that Clostridium tertium remains alive through its spores, even 107 years later. The present case report illustrates the effectiveness of an original culture protocol incorporating anaerobiosis, for the isolation by culture of ancient pathogens, opening the door to the possibility of ancient dental pulp. Funding This work was supported by the French Government under the "Investissements d'avenir" (Investments for the Future) programme managed by the 10.13039/501100001665 Agence Nationale de la Recherche (ANR, fr: National Agency for Research) (reference: Méditerranée Infection 612 10-IAHU-03). Data available Raw sequencing read have been deposited on NCBI (Accession number: PRJNA770891) Contributors MM, RB, MD and GA conceived and designed the study. MM performed the experiments. MM, CC, MS, FA, EV, RB, MD, MB and GA analysed the material and the data. MM, MD, RB, MB and GA wrote the manuscript. MB did the bioinformatical analysis. All authors contributed to data interpretation, critically reviewed the manuscript, and approved the final manuscript for submission. Author statements Conceptualization; M. Meucci; R. Barbieri; M. Drancourt; G. Aboudharam Formal analysis: M. Meucci; R. Barbieri; M. Drancourt; G. Aboudharam; M. Beye; C. Costedoat; E. Verna; M. Signoli; F. Adam Investigation; M. Meucci Software; M. Beye Supervision; R. Barbieri; M. Drancourt; G. Aboudharam Validation; All authors contributed to data interpretation, critically reviewed the manuscript, and approved the final manuscript for submission. Roles/Writing - original draft; M. Meucci; R. Barbieri; M. Drancourt; G. Aboudharam Writing - review & editing:; M. Meucci; R. Barbieri; M. Drancourt; G. Aboudharam; M. Beye Declaration of Competing Interest All authors declare no competing interests. Appendix Supplementary materials Image, application 1

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9029013/

Welder’s Anthrax: A Review of an Occupational Disease

Since 1997, nine cases of severe pneumonia, caused by species within the B. cereus group and with a presentation similar to that of inhalation anthrax, were reported in seemingly immunocompetent metalworkers, with most being welders. In seven of the cases, isolates were found to harbor a plasmid similar to the B. anthracis pXO1 that encodes anthrax toxins. In this paper, we review the literature on the B. cereus group spp. pneumonia among welders and other metalworkers, which we term welder's anthrax. We describe the epidemiology, including more information on two cases of welder's anthrax in 2020. We also describe the health risks associated with welding, potential mechanisms of infection and pathological damage, prevention measures according to the hierarchy of controls, and clinical and public health considerations. Considering occupational risk factors and controlling exposure to welding fumes and gases among workers, according to the hierarchy of controls, should help prevent disease transmission in the workplace. 1. Introduction The Bacillus cereus group classically consists of several Bacillus species with closely related phylogeny including Bacillus anthracis , Bacillus cereus , and Bacillus thuringiensis . Recently, the taxonomy of the B. cereus group has been updated and expanded based on genomic analysis, which has resulted in the naming of additional species, including B. tropicus [ 1 ]. B. anthracis is the etiologic agent of anthrax, which can manifest as cutaneous, inhalation, injection, or ingestion anthrax, or as primary anthrax meningitis. Genes encoding the major anthrax toxins and the poly-γ-D-glutamic acid capsule are located on two virulence plasmids, pXO1 and pXO2, respectively, and are required for full virulence [ 2 , 3 , 4 , 5 ]. B. cereus is ubiquitous in the environment and infections are occasionally associated with food-borne illness. Its presence in cultures is often considered to be a contaminant. However, it can cause a variety of infections, e.g., endophthalmitis, bacteremia, cutaneous infection, central nervous system infection, and pneumonia in individuals who have immunocompromising or other underlying conditions or who are recovering from surgery [ 6 ]. In patients with B. cereus pneumonia, hemoptysis is a common presenting symptom and pulmonary infiltrates are typically present [ 7 , 8 , 9 ]. Mediastinal widening, which occurs in most cases of inhalation anthrax, has not been observed with these pneumonias [ 7 , 8 , 9 ]. Both anthrax toxin-producing and non-anthrax-toxin-producing B. cereus can cause pneumonia in welders. Since 1997, nine cases of severe pneumonia, caused by species within the B. cereus group and with a presentation similar to that of inhalation anthrax, were reported in immunocompetent metalworkers, with most being welders [ 2 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. In seven of the cases, isolates were found to harbor a plasmid similar to the B. anthracis pXO1 that encodes anthrax toxins [ 2 , 11 , 12 , 13 , 14 , 15 , 16 ]. This finding of welders being seemingly disproportionately affected with severe Bacillus spp. infections is not limited to B. cereus or to recent years. The 1979 anthrax outbreak in Sverdlovsk in the former Union of Soviet Socialist Republics included 77 patients, of which 66 died [ 17 ]. The outbreak was thought to be due to the spread of aerosolized B. anthracis from a military microbiology facility. Of the 77 patients, 55 were men with a mean age of 42 years. Among the 35 men whose occupations were known, the most common occupation was a welder (n = 7). Few were reported to have had pre-existing medical conditions, but about half were described as moderate or heavy smokers or moderate or heavy drinkers [ 17 ]. Cases among welders with underlying pneumoconioses noted at autopsy were more likely to have hemorrhagic pulmonary consolidation than those without such conditions [ 18 ]. In this paper, we review the literature on the B. cereus group spp. pneumonia among welders and other metalworkers, which we term welder's anthrax. We describe the epidemiology, including more information on two cases of welder's anthrax in 2020. We also describe the health risks associated with welding, potential mechanisms of infection and pathological damage, prevention measures according to the hierarchy of controls, and clinical and public health considerations. 2. Review of Cases of Welder's Anthrax Our case definition for welder's anthrax comprises an infection caused by an anthrax toxin-expressing species within the B. cereus group and manifesting as pneumonia in a metalworker. Seven patients diagnosed with what is now termed welder's anthrax were reported to the Centers for Disease Control and Prevention (CDC) from 1994–2020 ( Table 1 ). Six were welders and one was another metalworker, and all were confirmed to be infected with B. cereus group bacteria containing anthrax toxin genes [ 2 , 11 , 12 , 13 , 14 , 15 , 16 ]. Of the six patients with available data on signs and symptoms, over half presented with each of the following: fever or chills, cough, dyspnea, and hemoptysis. All had abnormal chest radiographs and were diagnosed with pneumonia. All were hospitalized and were admitted to the intensive care unit if they survived past the emergency department. Five of the seven patients died [ 2 , 11 , 12 , 13 , 14 , 15 , 16 ]. All patients received broad-spectrum antibiotic treatment. One of the surviving patients (Patient F) received raxibacumab, a monoclonal anthrax antitoxin [ 16 ]. Of the seven patients, six were male. The median age was 39 years, with a range of 34–56 years. Of the five patients with reported race/ethnicity information, two were white, one was black, and two were Hispanic/Latino. Three had no known co-morbidities or underlying medical conditions. Reported co-morbidities included alcohol use disorder (n = 2), being a current smoker (n = 2), and asthma (n = 1) [ 2 , 11 , 12 , 13 , 14 , 15 , 16 ]. Worksites were reported to be in Louisiana for three patients and in Texas for four [ 2 , 11 , 12 , 13 , 14 , 15 , 16 ]. Additional work information was sparse in the published case reports. Only two patients (B and G) had information on job tenure (10 and 19 years) [ 12 , 16 ]. Information on the type of welding, job activities, and type of workplace, including indoor or outdoor activities, was not available for most of the patients. Investigators collected additional work information for Patient F, who worked on the roof of an oil tank located in an oil refinery for 49 days prior to his illness onset. All activities for this project were conducted outside. The patient was part of an eight-person crew, which included three other welders who did not work on the roof of the oil tank. The patient welded on new A36 mild carbon steel using a shielded metal arc welding (or stick) process. Reported electrodes used were 6010, 7018, and 7024. Patient F reportedly wore a 3M 6000 series half-mask respirator equipped with P-100 particulate cartridges while performing welding duties. Patient F performed additional tasks on and around the oil tank, including scrubbing debris off the roof of the oil tank using a wire metal brush and was present during other activities, including sandblasting the paint off the oil tank walls and metal-grinding. However, Patient F reportedly did not use respiratory protection during these non-welding activities. No other crew members were reported to have been ill during this same time period. Investigators also collected work information for Patient G, who worked as a welder in the wood fabrication shop of a company that manufactures proprietary fixtures for customers in the oil and gas industry. Seven other workers worked in the wood fabrication shop, which had no local exhaust systems but had large bay doors that were usually open. Outside the bay door, on the other side of a paved driveway, was a field with gravel on one side and dirt/grass on the other. Some wood used inside the wood fabrication shop was stored outdoors alongside the gravel field. The patient welded on low-carbon mild steel with no chemical coatings or treatments. The welding process used was Metal Inert Gas (MIG) with solid or flux core wire and 75% argon/25% carbon dioxide shield gas. Patient G welded steel plate end caps to steel tubing in wooden fixtures and performed some pre- and post-welding grinding using a hand-held, AC-powered tool with abrasive disks, and additionally performed flame cutting. The workstation of Patient G was located inside the wood fabrication shop alongside a bay door, and a plasma cutting station was located immediately outside the wood shop on the wall adjacent to the welding station. Compressed air and dry sweeping were routinely used as part of cleanup activities inside the shop. Patient G was reported to have always worn an N95 filtering facepiece respirator and a welding hood when welding but was not fit tested. It is unknown if respiratory protection was used during non-welding activities. It was reported that Patient G and his co-workers ate lunch and took additional breaks outside. Four case investigations included environmental sampling at the worksite (patients B, C, F, and G), and samples from two investigations yielded B. cereus ( Table 2 ) [ 12 , 16 ]. The environmental investigation of patient B's worksite identified a B. cereus isolate from a dust sample that was positive for B. anthracis capsule genes. However, it lacked toxin genes and did not genetically match the patient's clinical isolate [ 11 ]. The environmental investigation for patient F identified a bacterial isolate from one soil sample that genetically matched a clinical isolate from the patient [ 16 ]. Laboratory testing to detect specific Bacillus spp. in environmental samples can be challenging. Its role in epidemiological investigations is limited by its sensitivity; however, focused PCR or culture testing might help confirm a suspected environmental source. A negative result does not necessarily mean that the suspected Bacillus spp. strain was not present or was not present in the past. From 1996–1997, two welders were reported to have rapidly progressive fatal pneumonia caused by B. cereus that were not found to have anthrax toxin genes [ 10 ]. They did not meet the case definition of welder's anthrax and were excluded from Table 1 . Both patients were otherwise healthy males in their 40s who worked as welders in Louisiana. Additional work information was not reported for either case patient, other than that one patient was exposed to "dust and fumes" at work. 3. Welding Processes and Exposures As of May 2020, nearly 400,000 workers were employed as full-time welders, cutters, solderers, and brazers in the United States, of which only 3.8% were women [ 19 ]. Additionally, it is estimated that over 6 million people worldwide have the occupational title of welder either full-time or part-time [ 20 ]. Globally, millions of workers not classified as full-time welders may perform welding duties in their jobs, such as shipbuilders, pipefitters, ironworkers, boilermakers, construction workers, farmers, manufacturers, and automotive workers. Welding provides a powerful industrial tool for the joining of metals. Nearly all metals and alloys can be welded. The American Welding Society has identified over twenty different metal joining processes that are currently being used [ 21 ]. Most of these processes are classified under electric arc welding and include shielded manual metal arc welding (or stick welding), gas metal arc welding (or MIG welding), gas tungsten arc welding (or TIG welding), and flux-cored arc welding. Electric arc welding joins pieces of metal that have been made into a liquid by the application of intense heat [ 22 ]. The heat needed to melt the metal (>5000 °C) is produced by an electric arc between the work to be welded and an electrode that is continuously fed into the joint. After cooling and solidification, a metallurgic bond is produced [ 23 ]. Other types of welding processes include plasma arc welding, submerged arc welding, and oxygas welding. Electric arc welding produces aerosol by-products composed of a mixture of different metal oxides volatilized from the welding electrode or the flux material incorporated within the electrode [ 24 ]. The generated welding fumes are the vaporized metal that has reacted with air to form respirable size particles. The metals most common in welding fumes are iron, chromium, manganese, and nickel. The size distribution of particles generated during electric arc welding has been reported to be multi-modal and dynamically changes with time [ 24 , 25 ]. Three different modes of particle sizes have been observed: (1) nucleation mode (0.01–0.10 mm) of individual primary particles; (2) accumulation mode (0.10–1.0 mm) of agglomerated and coalesced particles formed; and (3) coarse mode (1–20 mm) of non-agglomerated and more spherical particles [ 24 ]. In addition, different potentially toxic gases, such as carbon monoxide, ozone, and nitrogen oxides, are commonly generated during electric arc welding. Each of the welding processes has its own operational and metallurgical advantage, and each may present a different potential health and safety hazard. Due to this, welders are not a homogeneous working group, and their exposure can greatly vary. They work in a variety of settings, such as well-ventilated indoor and outdoor open-air sites (e.g., farms, construction sites, or open-air warehouses) or in confined, poorly ventilated spaces (e.g., ship hulls, boilers, building crawl spaces, underground mines, or pipelines). The health effects of exposure to welding fumes vary depending on the length and intensity of the exposure and the metals involved. Of particular concern are welding processes involving stainless steel, cadmium- or lead-coated steel, and metals such as manganese, nickel, chrome, zinc, and copper. Fumes from these metals are considerably more toxic than those encountered when welding iron or mild steel. Welding constituents may also interact to produce adverse health effects. Epidemiological studies and case reports of employees exposed to welding emissions have shown an excessive incidence of acute and chronic respiratory disease [ 26 ]. These illnesses include metal fume fever, pneumonitis, pulmonary edema, and lung cancer. Exposure to manganese has been associated with Parkinsons-like health effects, such as poor hand-eye coordination, motor slowing, tremor, reduced response speed, mood disturbance, and possible memory and intellectual loss [ 25 , 27 , 28 ]. Airborne fume concentrations vary greatly in workplaces where welding occurs [ 29 , 30 , 31 , 32 ]. Currently, there is no recommended exposure limit (REL) or threshold limit value (TLV) for welding fumes as established by NIOSH and the American Conference of Governmental Industrial Hygienists (ACGIH), respectively. Airborne welding fume concentrations in the workplace are recommended to be kept at the lowest possible levels and to be maintained below exposure limits for the specific metal constituents of the fume that may pose the greatest risk to health (e.g., chromium, nickel, or manganese). 4. Possible Mechanisms of Infection and Disease Several studies have shown an increased risk of pneumonia (defined as bacterial, lobar, and pneumococcal) and mortality among welders and other workers exposed to metal fumes and mineral dusts [ 33 , 34 , 35 , 36 , 37 , 38 ]. A 2019 review demonstrated that workplace exposures contribute substantially to the burden of community-acquired pneumonia (attributable occupational population fraction, 10%). In seven cohort studies that estimated the risk of pneumonia in welders, metal fumes/welding exposures contributed even more to the burden of community-acquired pneumonia (attributable occupational fraction, 52%) [ 39 ]. It was also determined by a scientific panel that the frequency, duration, and severity of upper and lower respiratory tract infections were slightly increased among welders, raising the possibility that exposure to metal fumes might increase susceptibility to lung infection, even with common, relatively harmless infectious agents [ 40 ]. An increased mortality from pneumonia among welders has also been reported [ 41 , 42 ]. Evidence suggests that the inhalation of ferrous and other metal fumes in the workplace may predispose workers to lung infections [ 43 ]. The mechanisms associated with the immunosuppressive effects of metal fumes after inhalation are mostly unknown. Theories have included that metal fumes (or iron) act as a growth nutrient for bacteria, enhance the binding of bacteria to lung tissues, or impair immune responses in the lung through oxidative stress [ 33 , 37 , 38 , 42 ]. Therefore, it is hypothesized that the occupational risk of infection is primarily from occupational exposure to metal fumes. Whether or not occupational activities also result in an increased exposure to these pathogens is not clear. However, the infecting strain of one welder was detected in the environment at his worksite ( Table 2 ). Animal infectivity studies have indicated that inhalation exposure to common welding fumes during electric arc welding reduced animal body weight and significantly slowed the clearance of a bacterial pathogen after inoculation compared to air controls [ 44 , 45 ]. Bacterial challenge after welding fume exposure in rats resulted in an alteration of multiple cytokines linked to both innate and adaptive immunity. Furthermore, welding fume exposure and the accumulation of metals in the lungs attenuated alveolar macrophage function as they were unable to efficiently respond and clear the bacterial pathogen, resulting in an augmented lung inflammatory response. A graded immunosuppressive response was observed when comparing different welding fumes, with chromium-containing stainless steel welding fumes having the greatest effect on lung defenses against bacterial challenge [ 46 , 47 ]. Like all pathogens, B. anthracis and B. cereus need iron to survive and thrive, and they have similar, though not identical, mechanisms for its acquisition. They both produce the siderophores petrobactin and bacillobactin. However, the two pathogens have different surface proteins involved in iron uptake: iron-regulated leucine-rich surface protein (IlsA) for B. cereus and iron-regulated surface determinant (Isd) proteins for B. anthracis [ 48 , 49 ]. Welders may accrue excess iron, become hyperferritinemic, and develop pulmonary siderosis. The appearance of lung opacities on chest x-rays of welders without symptoms of pulmonary illness, a condition now known as siderosis, was reported as early as the 1930s, soon after the introduction of arc welding [ 50 , 51 ]. Siderosis is caused by an excessive accumulation of iron oxide in the lungs, and pulmonary function in welders with siderosis has been observed within normal limits and not different from matched, non-welding controls [ 52 ]. A significant portion of iron oxide that is deposited in the lungs after welding fume inhalation is present in alveolar macrophages [ 53 , 54 ] and has been observed to persist in the lungs for years, even after removal from exposure [ 55 ]. In a Chinese study, 37 arc welders who had been welding 8 h per day for 2–36 years were compared to sex- and age-matched factory workers with no history of metal exposures. The mean serum iron level in welders was almost twice that of the factory workers (300 ± 137 vs. 160 ± 79 µg/L [ p < 0.01]) [ 56 ]. In a study of 241 welders, respirable iron per cubic meter was highly associated ( p = 0.001) with serum ferritin [ 57 ]. Polycythemia and pulmonary siderosis accompanied by fibrosis can also be seen in arc welders [ 58 ]. In our review of the five of seven cases of welder's anthrax from 1994–2020 with reported hematocrits, three patients (Patients B, D, and F) had evidence of iron overload, with hematocrits between 54.5–64.7% [ 12 , 13 ]. Non-occupational risk groups for B. cereus pneumonias, though not necessarily caused by toxin-producing B. cereus group spp., include other patient groups prone to iron overload: patients with alcohol-use disorders, or acute leukemias [ 9 ], and premature infants [ 6 ]. In the Sverdlovsk incident, as half the patients with inhalation pneumonia were described as moderate-to-heavy consumers of alcohol, a significant portion of the welders likely also belonged to this subgroup [ 17 ]. In our review, Patients F and G were reported to have alcohol use disorders, which might have affected disease severity. In a 2004 NHANES study, mean serum ferritin, transferrin, and serum iron all were increased in mild (120 ng/dL), moderate (151 ng/dL), and heavy (197 ng/dL) consumers of alcohol compared to those who abstained (111 ng/dL) [ 59 ]. In a prospective study of 48 patients with acute leukemias or myelodysplastic syndromes, the median serum ferritin was 1549 ng/mL (normal values are 20–250 for males and 10–120 for females). Of these patients with leukemia, 85% had hepatic iron overload, with half of those having severe overload [ 60 ]. In one study of premature infants, a fifth had overload, with the most conspicuous association being receipt of multiple transfusions [ 61 ]. While iron overload might partially explain the increased susceptibility of welders (and patients with leukemias or alcohol-use disorders or premature infants) for B. cereus infections, exposure is still important. A number of authors have noted that soil iron is much higher around welding sites than elsewhere [ 62 , 63 ]. This observation perhaps provides fertile grounds for future research, and measuring soil iron levels may yield useful information. 5. Occupational/Public Health Prevention Measures Occupational health and safety specialists use the hierarchy of controls ( Figure 1 ) to determine how to implement feasible and effective control solutions to occupational hazards [ 64 ]. This framework can be used to prevent exposure to welding fumes and gases, and also soils that may be contaminated with opportunistic B. cereus group spp. in the workplace. Elimination (removing the hazard) and substitution (replacing the hazard) are the most effective ways to reduce occupational hazards. Engineering controls are physical changes to work processes to remove the hazard or place a barrier between workers and hazards. Administrative controls are methods that change the way the work is performed. Finally, personal protective equipment (PPE) provides a physical barrier between the worker and the hazard. PPE is considered the least effective control measure because it requires a comprehensive program and a high level of worker involvement and commitment for proper use [ 64 ]. A key component in occupational safety and health is the workplace hazard assessment, which is a proactive, ongoing process to identify and assess hazards in the workplace [ 65 ]. Employers should conduct a hazard assessment on all welders, other metalworkers, and supervisors at worksites [ 66 ]. This process involves collecting and reviewing information about the hazards present or likely to be present in the workplace, conducting initial and periodic workplace inspections of the workplace to identify new or recurring hazards, investigating injuries, illnesses, and incidents, determining the severity and likelihood of incidents that could result for each hazard identified, and using this information to prioritize corrective actions. Employers can then take steps to help reduce exposure to fume and gases from welding and soils that may be contaminated with opportunistic B. cereus group spp. during welding operations. Elimination and substitution controls include using a less toxic welding type or consumable and ensuring that welding surfaces are free of any coatings, dirt, and dust that may lead to potentially toxic exposures [ 66 ]. Engineering controls can include the use of general and local exhaust ventilation. When welding outdoors or in open areas, it should not be assumed there is adequate general ventilation, even when the welder uses proper positioning and natural drafts. Local exhaust systems should be positioned to draw fume and gases away from the welder and other workers in the area [ 66 ]. Administrative controls include maintaining a clean and dirt-free worksite. Workplaces should be routinely cleaned with a vacuum equipped with a high-efficiency particulate air (HEPA) filter or wet cleaning methods. Compressed air and dry sweeping or brushing should not be used. Dust control programs in outdoor workplaces and near workplaces open to the outdoors can minimize dirt and dust exposure, and activities in the immediate vicinity should be limited to help minimize disturbing dry dust. In surrounding areas, adding moisture to roadways and surfaces that are heavily traveled via the application of water, hydroscopic compounds, or surfactants can help control dirt and dust exposures [ 67 ]. Water, hydroscopic compounds, and surfactants should not be applied in the immediate area where welding occurs as this may cause an electrocution hazard. It is essential that welders and other metalworkers understand their potential occupational health risks and how to protect themselves. OSHA's Hazard Communication Standard requires employers to inform and train workers on potential work hazards and associated safe practices, procedures, and protective measures [ 68 ]. Recommended components of a written hazard communication program include educating workers about the health risks from welding and B. cereus group spp., signs and symptoms, and how to prevent exposures. Welders should use PPE such as coveralls and work boots in the workplace to prevent their skin and clothing from being contaminated and taking contaminants home. In addition, use of NIOSH-approved respirators as part of a written respiratory protection program may be needed when other controls do not reduce exposures to safe levels [ 66 , 69 ]. 6. Clinical Considerations and Medical Countermeasures In recognition of the association between welding and invasive pneumococcal disease, the 23-valent pneumococcal polysaccharide vaccine has been recommended for welders in the United Kingdom and within a large multi-national corporation for several years [ 70 , 71 ]. Anthrax vaccine adsorbed (AVA) (BioThrax) is licensed for pre-exposure prophylaxis (PrEP) for adults aged 18–65 years at high risk for exposure to B. anthracis . AVA induces immunity through the production of antibodies that target the protective antigen component of the anthrax toxins (edema toxin and lethal toxin). The B. cereus group strains in the welder-related cases contain the pXO1 virulence factor that produces the anthrax toxins. Since the disease severity seen in these cases was related to the effects of the anthrax toxins, an anthrax vaccine can blunt their effects [ 72 ]. When used for PrEP, AVA is administered intramuscularly as a priming series at 0, 1, and 6 months, with booster doses at 12 and 18 months and annually thereafter [ 73 ]. Groups considered to be at high risk for exposure to B. anthracis include members of the U.S. military deployed to areas designated by the Department of Defense as high risk for exposure, laboratory workers who work with high concentrations of B. anthracis , and persons such as farmers, veterinarians, and livestock handlers who might handle animals with anthrax or contaminated animal products [ 74 ]. It is currently unknown to what extent environmental species within the B. cereus group carry anthrax toxin genes or whether their geographic range extends beyond the U.S. Gulf Coast. Therefore, the role of AVA for PrEP or postexposure prophylaxis (PEP) of welders is not currently recognized or understood. However, for welders working in areas where these infections have occurred, the benefit of the vaccine might outweigh potential adverse events. Physicians should include anthrax toxin-expressing B. cereus group spp. in the differential for welders who present with pneumonia, particularly those working in U.S. Gulf Coast states. Welders and other metalworkers who present with B. cereus group spp. infections should be treated in a fashion similar to a patient with inhalation anthrax. Clinical guidelines for the treatment of anthrax are available [ 75 ]. Given the severity of these infections, treatment may initially need to be empirical. Patients should receive a minimum of one bactericidal agent plus one protein synthesis inhibitor (e.g., ciprofloxacin and clindamycin) with activity against the B. cereus group. However, it should be noted that B. cereus has different innate susceptibilities than B. anthracis and is usually resistant to penicillins and cephalosporins because of beta lactamase production [ 6 ]. If infection with anthrax toxin-expressing B. cereus group spp. is suspected, it is important to notify the state health department; a consultation with CDC is recommended. Anthrax antitoxins should be considered as adjunctive therapy if the patient's clinical condition suggests systemic illness from a B. cereus group bacterium. Anthrax antitoxin may be obtained through the U.S. Strategic National Stockpile after consultation with the CDC. 7. Public Health Implications Based on current data collection and surveillance, it is possible that cases of welder's anthrax were missed due to limited detection and understanding of the pathogen, underdiagnosis, and under-reporting of the patient's occupation. Discovering risk factors for transmission and assessing hazards in the workplace could help employers plan disease prevention measures according to the hierarchy of controls, such as implementing changes in work practices or an OSHA-compliant respiratory protection program. Including the systematic collection of occupational information as part of infectious disease surveillance might facilitate identifying future workplace-associated cases and outbreaks. Capturing information on both industry and occupation for B. cereus group spp. infection cases can further inform public health officials on those specific job risk factors needing further assessment. To improve data collection in surveillance systems, the NIOSH Surveillance Program at CDC recommends that occupational questions should be standardized, information on both industry and occupation should be collected, and data should be analyzed with standard coding schemes to monitor disease trends in specific industries or occupations and protect workers' health [ 76 , 77 ]. Other helpful information for the investigation of B. cereus group spp. infections includes the employer's name, work location, job duties, and questions about specific types of welding, metals, and other exposures and protective measures taken. In addition, employers should provide employee rosters to public health agencies to assist in identifying additional cases when necessary. Employers are currently required to report work-related illnesses resulting in hospitalizations among workers to OSHA programs, and public health agencies should establish agreements with occupational safety and health agencies to share data for surveillance purposes. Outreach in affected areas can prompt healthcare providers to recognize potential work-associated B. cereus group spp. infections. 8. Conclusions Welder's anthrax has emerged as a rare but important occupational infectious disease. Communication and cooperation between clinicians, employers, and public health practitioners is important to identify work-related cases and identify occupational and personal risk factors. More research is needed to better understand the mechanisms of infection and disease among welders. Considering occupational risk factors and controlling exposures to welding fumes and gases among workers according to the hierarchy of controls should help prevent disease transmission in the workplace. Future research is needed to better understand the interplay between exposure to metal fumes and other welding hazards, and the possible increased susceptibility to and severity of lung infection seen in this occupational group. The effectiveness of interventions to minimize workers' exposure to metal fumes, including engineering controls and respiratory protection, should also be explored.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9206814/

Healthcare seeking and hospital admissions by people who inject drugs in response to symptoms of injection site infections or injuries in three urban areas of England

SUMMARY People who inject drugs (PWID) are vulnerable to infections and injuries at injection sites. The factors associated with reporting symptoms of these, seeking related advice, and hospital admission are examined. PWID were recruited in Birmingham, Bristol and Leeds using respondent-driven sampling ( N = 855). During the preceding year, 48% reported having redness, swelling and tenderness (RST), 19% an abscess, and 10% an open wound at an injection site. Overall, 54% reported ⩾1 symptoms, with 45% of these seeking medical advice (main sources emergency departments and General Practitioners). Advice was often sought ⩾5 days after the symptom first appeared (44% of those seeking advice about an abscess, 45% about an open wound, and 35% for RST); the majority received antibiotics. Overall, 9·5% reported hospital admission during the preceding year. Ever being diagnosed with septicaemia and endocarditis were reported by 8·8% and 2·9%, respectively. Interventions are needed to reduce morbidity, healthcare burden and delays in accessing treatment.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9695245/

Antibody-Based Immunotherapies as a Tool for Tackling Multidrug-Resistant Bacterial Infections

The discovery of antimicrobials is an outstanding achievement of mankind that led to the development of modern medicine. However, increasing antimicrobial resistance observed worldwide is rendering commercially available antimicrobials ineffective. This problem results from the bacterial ability to adapt to selective pressure, leading to the development or acquisition of multiple types of resistance mechanisms that can severely affect the efficacy of antimicrobials. The misuse, over-prescription, and poor treatment adherence by patients are factors strongly aggravating this issue, with an epidemic of infections untreatable by first-line therapies occurring over decades. Alternatives are required to tackle this problem, and immunotherapies are emerging as pathogen-specific and nonresistance-generating alternatives to antimicrobials. In this work, four types of antibody formats and their potential for the development of antibody-based immunotherapies against bacteria are discussed. These antibody isotypes include conventional mammalian polyclonal antibodies that are used for the neutralization of toxins; conventional mammalian monoclonal antibodies that currently have 100 IgG mAbs approved for therapeutic use; immunoglobulin Y found in birds and an excellent source of high-quality polyclonal antibodies able to be purified noninvasively from egg yolks; and single domain antibodies (also known as nanobodies), a recently discovered antibody format (found in camelids and nurse sharks) that allows for a low-cost synthesis in microbial systems, access to hidden or hard-to-reach epitopes, and exhibits a high modularity for the development of complex structures. 1. Introduction The use of antibodies is considered one of the first consistently effective antimicrobial strategies developed. In the 1890s, specific antibodies were found to be able to protect against bacterial toxins, which in turn led to the development of antibody treatments for diverse infectious diseases [ 1 ]. The initial antibody preparations used were derived from the serum of immune human donors or immunized animals. This approach, known as serum therapy, was somewhat effective but due to the extensive quantities of exogenous proteins administrated to the patients, it was also highly prone to side effects such as hypersensitivity reactions and serum sickness, a form of antigen–antibody complex disease [ 2 ]. Improvements to these techniques were consistently being achieved, reducing some of the side effects. However, a rapid decline of this approach occurred with the availability of the first antimicrobials, which completely changed medicine forever and represents one of the most extraordinary accomplishments in this field [ 2 , 3 ]. The use of serum-based approaches for antibacterial treatments was predominantly discarded, retaining a small slot as a treatment for venoms and toxins. The rise of antimicrobials was an incredible feat of mankind that led to the development of modern medicine, an increase in life expectancy, with an important economic impact in terms of hospital and treatment costs [ 4 ]. The employment of antimicrobials is done all throughout modern medicine and has saved numerous lives. However, this great success has led to the development of a new major threat to global public health, known as antimicrobial resistance, which is rendering many commercially available antimicrobials ineffective [ 5 ]. This issue stems from the bacteria's remarkable ability to adapt to the selective pressure presented by antimicrobials. Bacteria can develop or acquire multiple types of resistance mechanisms that can severely hinder the efficacy of the antibiotic. This issue is being exacerbated by their misuse, over-prescription, and poor treatment adherence by patients [ 3 , 6 ]. The antimicrobial-resistant problem has been increasing over decades, leading to the development of an epidemic of infections untreatable by first-line therapies. This crisis is especially centered on the multidrug-resistant "ESKAPE" bacteria ( Enterococcus spp., Staphylococcus aureus , Klebsiella spp., Acinetobacter baumannii , Pseudomonas aeruginosa and Enterobacter spp.) [ 7 ]. In some cases, such as that of A. baumannii , strains resistant to all available antibiotics are already being isolated [ 8 ]. Despite the increasing need for effective antimicrobials with novel modes of action, this development has been extremely scarce with very few new compounds being approved in the past 30 years. In fact, nearly all antibiotics or their derivatives currently being used in clinical situations were discovered in the decades of 1940 to 1960 [ 3 , 9 ]. Obviously, other alternatives are required to quench this increasing problem. Vaccination is an exceptionally effective approach and is often seen as the possible answer to the problem; however, its application to some of the most problematic multidrug-resistant bacteria has been unsuccessful, and methods of active immunization work better as prevention approaches, not having an immediate effect on the infection, which is required in a clinical situation. Opposite to active immunization, passive immunization offers an immediate protection against the infectious agent, although short-lived and lasting from several weeks to months [ 10 ]. 2. Antibody-Based Immunotherapies—An Overview The use of passive immunization approaches has many of the characteristics required to tackle multidrug-resistant bacteria, including specific activity, ability to target resistant bacteria, and unlikeliness to lead to the development of new antimicrobial resistance. Passive immunization can be simply described as the transfer of already formed antibodies to a receptor individual. Antibodies together with B and T cells constitute one of the most important components of the adaptive immune system. Antibodies or immunoglobulins (Igs) are Y-shaped glycoproteins capable of specifically recognizing antigens, attaching and forming a molecular link in the communication network of the remaining elements of the immune system, activating the complement components of the immune response, and leading to neutralization or elimination of foreign threats [ 11 ]. In mammals, immunoglobulin G (IgG) is the most common isotype. IgGs presents a tetrameric structure composed of two identical heavy chains and two identical light chains. The heavy chains are composed of four domains, three constant and one variable. The light chain has two domains, one constant and one variable. The properties that allow IgGs to recognize and bind to antigens are concentrated in short segments within the variable domains that, as the name indicates, have a high degree of variability [ 12 ] ( Figure 1 ). Immunoglobulin M (IgM) is a different antibody isotype in mammals that presents a predominantly pentameric structure, with 10 or 12 antigen-binding sites [ 13 ]. Following exposure to foreign antigens, they are the first antibodies secreted. Typically, IgM show a lower antigen-binding affinity when compared with IgG. However, the polyvalent nature of their structure contributes for a high avidity binding and an efficient engagement of complement-dependent cell lysis. This higher avidity allows for a greater efficiency on the binding to antigens present at lower concentration, and to non-protein antigens such as lipids and carbohydrates [ 13 ]. Another antibody isotype currently being studied due to its specific characteristics is the immunoglobulin A (IgA). IgA has evolved to be secreted and function in mucosal surfaces, making them vital for protection against mucosal pathogens but also for the function of the healthy microbiome. The production and secretion of IgA is highly efficient, with plasma cells producing around 3 to 5 g each day, more than all other isotypes combined, with over one gram being secreted per day. Structurally its composed of four immunoglobulin domains and can be found in monomers, dimers, and as secretory. Dimerization and secretion is aided by the covalent attachment to the J chain, a critical step in transcytosis as it presents a bridge between the antibody and the polymeric immunoglobulin receptor (PIGR). Both IgA and IgM bind to PIGR; however, IgM binds transiently and IgA uses disulfide bonds to be covalently linked, this causes IgA not to be released from PIGR binding upon arrival at mucosal surfaces. PIGR is cleaved by an unknow protease but remains bound to IgA as a secretory factor and creates the secretory IgA (sIgA). sIgA is believed to have increased resistance to proteolytic degradation in the gastrointestinal tract, an integral characteristic given the high concentration of proteases in the lumen. This leads IgA to be more abundant at mucosal surfaces as IgM is more susceptible to proteolysis. sIgAs are considered the first barrier against pathogens in mucosal surfaces, despite lacking effector functions they can cause bacterial agglutination, disturb motility, neutralize toxins and inhibit adherence to epithelium, preventing circulatory dissemination. These potent antipathogen effects have been described against respiratory viruses and multiple gastrointestinal pathogens such as Clostridioides difficile, Salmonella enterica subsp. enterica serovar Typhimurium, and Shigella flexneri. [ 14 , 15 ]. The therapeutic benefits offered by antibodies result from an assortment of mechanisms. Antibodies can bind to antigens in bacteria preventing adherence and other important steps of infection or block receptor-ligand formation of toxins and viruses [ 16 ]. They can also use a different mechanism that is dependent on mediators, where the binding of the antibody to surface receptors leads to agglutination and the immobilization of the target cell. After this, an immune-complex with the antigen is formed, and initiation of antibody-mediated cellular cytotoxicity, complement-dependent cytotoxicity, or opsonization can occur [ 16 , 17 , 18 ] ( Figure 1 ). All these different mechanisms of action cannot be obtained from a single antibody formulation. Monoclonal antibodies, for example, due to their specificity to a single epitope, are generally used to block receptor-toxin ligand formation but can also be used to treat cancer by targeting specific antigens on cancer cells, initiating antibody-mediated cellular cytotoxicity or complement-dependent cytotoxicity [ 16 ]. For the application of antibody therapies in infectious diseases, the suggested mechanisms are blockage of the pathogenic virulence mechanisms, such as the secretion of virulence factors, recruitment of immune mediators and effectors to eliminate the pathogen through phagocytosis, or usage of an established anti-cancer therapy where the antibody directly kills the pathogen by targeted delivery of radionuclides or toxic drugs [ 19 ]. Despite the increasing emergence caused by the worldwide growth of antimicrobial resistance, the relative inefficacy in immunocompromised patients of antimicrobial drugs and the many technological advances in the field of immunoglobulin research, the number of antibodies licensed for clinical use is scarce. Furthermore, the vast majority of these antibodies were developed for the treatment of non-infectious diseases, such as oncology, rheumatology, and transplant medicine [ 20 ]. The use of these approaches for bacterial infections should, in theory, be less problematic than those developed for non-infectious diseases. This assumption results from the fact that the antigens targeted by the antibody are extremely different from those found in the host, opposite to what happens in tumor treatment, where a discrimination between self-antigens is required [ 2 , 16 ]. Despite the low number of approved antibody-based immunotherapies for infectious diseases, there is evidence that antibodies can exert a protective effect against a variety of microorganisms, including intracellular ones [ 2 , 21 ]. Various studies show positive effects of using monoclonal or polyclonal antibodies to treat bacterial, viral, and fungal infections [ 19 ]. Another interesting approach is the use of a combination of both antibody-based immunotherapy and antimicrobials, which can result in a synergetic or additive effect, having a success rate higher than the separate use [ 22 ]. This opens the door to an easier incorporation of immunotherapies into the existing clinical protocols, but also shorter stays in intensive care units and reductions of morbidity, mortality, and health care costs. The characteristics of antibody-based therapies allow them to have some advantages over the typical use of antimicrobials. Antibodies are highly specific, allowing them to target only the microorganism intended, with minimal influence on other microorganisms present in the normal flora, and do not exert a selecting pressure for resistance on the non-targeted flora [ 6 ]. The use of these approaches has effects on the enhancement of bacterial clearance, prevention of colonization and invasion, and reduction of damage caused by cytotoxic or hyperinflammatory factors. Negative effects of existing antimicrobial resistances or the development of new resistances against these treatments are highly unlikely. The possible use of a combination of both antimicrobials and antibodies can result in a reduction of antimicrobials use and increase in their effectiveness, which may result in a lower selective pressure and decrease the prevalence of antimicrobials resistance [ 22 , 23 ]. The response of bacteria against antibodies is more tamed, resulting in less bacterial SOS responses often responsible for several side effects that may include toxin release and increased transfer of resistant genes [ 6 , 24 ]. Despite some exceptions, such as some nanobodies, antibodies are significantly more stable than antimicrobials, having half-lives from weeks to months, exceeding those of the antimicrobials that only maintain their activity in the patient for a few days [ 22 , 25 , 26 ]. Despite the advantages presented, some important limitations are associated with the antibody therapies. As natural products, the production must occur in cell lines or other live expression systems, requiring a purification step, raising concerns about possible contamination of the preparations by prions, viruses, or other infectious agents. Due to the high specificity mentioned previously, the usage of these approaches requires knowledge of the causative agent of infection, meaning that a fast microbiological diagnosis is always required before the initialization of therapy, especially because the highest efficiency of treatment is obtained for infections at the early stages. Fortunately, the development of PCR and other rapid diagnostic techniques provide fast platforms, helping the establishment of antibody-based therapies [ 2 , 4 ]. Indeed, these fast diagnostic methods allow the specific identification of the pathogen in a short time, enabling the accurate selection of a specific antibody-based therapy. Altogether, this combination increases the confidence and reliability of antibody-based therapies, thus contributing to their establishment. The specificity might also be problematic in the case of mixed infections or the case of pathogens with high antigenic variation. The most obvious solution to this question is the use of a cocktail of antibodies able to target different microorganisms or different antigens [ 27 ]. Logistically, these approaches might present some difficulties, namely as the antibodies are proteins, the administration and maintenance must be done in a medical facility, as administration is typically given intravenously, which is unpractical for non-hospitalized patients. However, recently, an increase in the delivery of antibodies through the subcutaneous and intramuscular routes has been registered. In addition, oral immunotherapy with antibodies is a topic being studied using different isotypes and sources of antibodies, and direct delivery with an aerosol to the infected airways has been shown to be effective [ 28 , 29 , 30 , 31 ]. The major drawback of antibody-based immunotherapy is the high cost of production, storage, and administration. The research required to find the best antigen target and the best binding spot for the antibody is also very labor intensive, time consuming, and costly. The scale-up required for the large-scale production is also extremely difficult and financially demanding, despite some improvements in culturing and processing increasing the yields obtained [ 32 ]. These higher costs of production combined with the high specificity indicate that a potential market for any given antibody will be considerably small, meaning that the development of these strategies will be significantly harder for infectious diseases that are not common enough to provide a financial reward. However, the development of an antibody for passive immunotherapy requires a noticeably shorter time and lower cost than that needed to develop a vaccine [ 2 , 33 ]. For immunotherapy purposes, there are different antibodies formats currently being studied for the development of new therapies to deal with bacterial infections ( Figure 2 ). In this review, the potential of four types of antibody formats as alternatives to antimicrobial therapies is explored. 3. Conventional Mammalian Polyclonal Antibodies The first antibody preparations used for passive immunization contained polyclonal antibodies derived from the sera of immunized animals or humans, and in some cases, convalescing patients [ 2 ]. Nowadays, pAbs are produced by injecting an immunogen into an animal to elicit an immune response against a specific antigen. After immunization, the polyclonal antibodies are purified to obtain a solution that is free from other serum proteins. These antibodies can be purified directly from an animal serum after immunization with the antigen of interest or the infectious agent. The term polyclonal is derived from the fact that these molecules are derived from multiple B cell clones and can recognize different epitopes from the same antigen. This occurs because different lymphocyte clones responding against different epitopes of the antigen are formed during the antibody response [ 11 , 34 ]. pAbs preparations are comprised of numerous antibodies differing in primary structure, isotype, specificity, and glycosylation of the constant region, an important factor for interaction with Fc receptors [ 35 ]. The protective effect of pAbs is observed naturally in the passive transference of maternal immunity to protect newborns during the more vulnerable phases of early life [ 36 ]. The preparation of pAbs for clinical uses starts with the immunization of an animal. As higher volumes of blood can be retrieved from larger animals, the traditional sources of these antibodies are horses, sheep, goats, and rabbits, rather than mice [ 34 ]. This production is relatively inexpensive, and the total amount produced is limited by the amount of blood removed from the animal during its lifetime. Once an animal is exhausted, new immunization in a different animal needs to be performed, which will not be identical to the previous, due to the stochastic nature of the adaptive immune response and the polyclonal nature of these antibody preparations [ 34 ]. The method through which pAbs are produced makes them cost-effective, able to be supplied in large quantities, and the usage of larger animals reduces the need for multiple batches to be validated. Presently, pAbs are a low-cost alternative to monoclonal antibodies which are usually used for snake bites and post-exposure prophylaxis of infectious diseases such as rabies, botulism, and diphtheria [ 37 ]. The ability of these antibodies to recognize several epitopes also challenges the pathogen capability to avoid antibodies by mutating the antigen [ 38 ]. The variability inherently present in pAbs preparations increases the biological effector functions, as multiples sub-isotypes of IgG antibodies are present (IgG1, IgG2, IgG3 and IgG4) providing in many cases added benefits and protective advantage [ 39 ]. The use of these pAbs is also associated with some limitations, namely the lack of standardization due to the lot-to-lot variation could lead to deficient efficacy and low content of specific antibodies. The adoption of approaches using pAbs has been greater for tackling virus such as influenza, HIV, MERS, and SARS, with some levels of success [ 19 , 35 , 36 , 39 , 40 ] but also bacterial toxins such as shiga toxin and toxins from Clostridium difficile [ 11 , 19 ]. The use for treatment of other bacterial infections has been less common. Our group recently showed that a pAbs against an OmpA-like protein from Burkholderia cenocepacia greatly impairs the ability of these bacteria to adhere and invade human epithelial cells in vitro [ 41 ]. Both the adhesion and invasion are highly important steps in the infection process. The fact that the antibodies produced, usually, are non-human may lead to exacerbated immune responses in the patients. This last hurdle could be overcome with the production of humanized antibodies in the animals. Beigel et al. used a novel approach with transchromosomic cattle carrying a human artificial chromosome comprising the entire human immunoglobulin gene repertoire, human immunoglobulin heavy chain (IGH) locus from chromosome 14, and human k light chain (IGK) locus from chromosome 2.6 [ 40 ]. These authors reported the production of a highly potent and specific fully human polyclonal IgG using this system [ 40 ]. An example is the human polyclonal immune globulin preparation (Altastaph) that targets capsular polysaccharides of S. aureus and is being developed for S. aureus infections complicated by bacteremia. A phase II double-blind, placebo-controlled trial found that when compared to the placebo the patients had a shorter median time to the resolution of fever and a shorter length of hospital stay. Unfortunately, the study was not powered to show efficacy. Nonetheless, the preliminary findings and safety profile indicate the potential of this therapy as an adjunct to antimicrobials [ 42 ]. A different human polyclonal preparation named Veronate ® against S. aureus derived from donors with high titers of antibodies against a surface adhesin of S. aureus and Staphylococcus epidermidis also went into clinical trials. The phase II trial showed promising results for the highest dose used. However, a phase III trial (NCT00113191) evaluating the prevention of late-onset sepsis in very low birth weight infants showed no effect of the antibody treatment in reduction of S. aureus [ 43 ]. A possible explanation for the disappointing result is the requirement of a humoral, cellular, and phagocytic response for control of these infections, which is not properly provided by the pre-mature infants [ 19 ]. 4. Conventional Mammalian Monoclonal Antibodies The technology for monoclonal antibodies (mAbs) production was introduced in 1975 by Kohler and Milstein [ 44 ]. mABs were immediately recognized as powerful tools for the targeted treatment of various diseases. mAbs can be from different isotypes of antibodies from human or animal IgG, IgM, IgA, avian IgY or single domain antibodies. Their main difference from polyclonal is they are homogenous immunoglobulins that only recognize one epitope. Due to this feature, they have a higher specific activity than polyclonal preparations. In 1986, a decade after the introduction of the technology, the first mAb was approved for human use by the FDA (Muromonab-CD3) [ 45 ]. As the first molecules were produced using mouse cell lines, they would be recognized as foreign molecules by the host immune response leading to mild to harsh immune reactions. The response to this problem was the development of several platform technologies able to produce mouse chimeric, humanized, and human antibodies [ 22 , 45 ]. Chimeric antibodies are a combination of the murine variable domains fused to the human constant domains. These antibodies are produced by cloning the murine variable region heavy and light chains genes, amplified from the hybridoma, together with the constant region genes of human heavy and light chains into a plasmid. This plasmid is then transfected into bacteria where the antibodies are produced as inclusion bodies, and then purified for in vivo use. These molecules are around 70% human and possess a human Fc sequence, reducing the possibility of immunogenicity [ 46 ]. Humanized antibodies contain 85 to 90% of human sequences. Their production is achieved by replacing all the rodent sequences except the complementarity determining regions (CDRs) for human sequences. However, this process of humanization is technically challenging, the insertion of CDRs in a generic framework is insufficient, as antibody affinity sometimes relies on framework regions. This process leads to antibody activity losses. Conservation of a few rodent sequences in the framework is required to restore the binding [ 46 , 47 ]. The production of fully human monoclonal antibodies was achieved with the development of phage display platforms, and also with the advancements in transgenic mouse platforms [ 46 ]. mAbs are the most successful antibodies, being approved and used as a therapy for cancer, autoimmune disorders, cholesterol, and infectious diseases, with the majority of these approved mAbs being chimeric or humanized [ 46 , 47 , 48 ]. Currently, about 100 IgG mAbs are approved by the Food and Drug Administration (FDA) for therapeutic uses [ 20 ]. New techniques using transgenic animals for the isolation of fully human mAbs are consistently being optimized [ 48 ]. Formulations of monoclonal antibodies are superior to polyclonal ones, in terms of specific activity, safety, constancy, and homogeneity. mAbs are also highly stable, especially the immunoglobulin G1 isotype that can reach half-lives of up to 21 days [ 49 ]. However, homogeneity can sometimes be disadvantageous, especially against more complex bacteria, where the targeting of a single epitope is not enough, in terms of expression and conservation, to develop a proper therapeutic response [ 4 , 26 ]. In these cases, the use of multivalent formulations may be required. The use of several mAbs targeting different antigens leads to an even higher cost of treatment. This high cost of treatment is considered the major drawback of mAbs, increasing concerns about the viability for widespread adoption. However, due to the emergency of multidrug-resistance reported for several bacterial pathogens, the use of mAbs therapies as alternatives to antimicrobials is being extensively studied. Thus far, three mAbs licensed by the FDA against bacterial exotoxins have been approved for treatment and prophylaxis. Raxibacumab [ 50 ] and obiltoxaximab [ 51 ] are monoclonal antibodies that target the lethal toxin of Bacillus anthracis and were approved for treatment of anthrax inhalation. The third mAb approved is bexlotoxumab [ 52 ] and targets the enterotoxin B of Clostridioides difficile . Anti-toxin mAbs inhibit virulence, limiting damage to the host without causing a selective pressure. However, their ability to directly tackle acute diseases is limited, and bexlotoxumab is an example of this, as the therapy is not approved for treatment of initial infection or protection against infection. Instead, it is used for reduction of recurrence of infection, a clinical situation that is frequently observed in C. difficile infections. For this reason, much work has been focusing on antibodies against outer membrane proteins of bacteria, involved in adhesion, evasion of immune system, and other bacterial processes [ 53 , 54 , 55 , 56 ]. These proteins have important functions in the bacterium, acting not only as easy targets but also as effective ones likely conserved in different clinical strains [ 57 ]. P. aeruginosa has been one of the most studied organisms for the development of immunotherapies [ 58 ]. One of the most successful examples is the development of a mAb targeting the conserved PcrV protein of this bacterium. This protein is part of the type III secretion system, an important virulence factor aiding the delivery of exotoxins into the target cells. This antibody displays protection in several animal models [ 59 ] and the mode of action involves the neutralization of the T3SS secretion system function. Another mAb, a PEGylated Fab named KB001, went into a randomized, double-blind, placebo-controlled clinical trial and was shown to prevent ventilator-associated pneumonia caused by P. aeruginosa infections [ 60 ]. Unfortunately, the efficacy was low in patients suffering from cystic fibrosis (CF). This low efficiency might be related to the low levels of the T3SS protein found in the sputum of CF patients chronically infected [ 53 ]. The targeting of polysaccharides, such as lipopolysaccharide (LPS) and capsular polysaccharide (CPS), have also attracted the interest of many researchers since the beginning of immunotherapies. These polysaccharides are essential for many bacteria to avoid immune systems, increasing their potential for the development of immunotherapies, with antibodies attaching to the CPS, improving the opsonophagocytosis of evasive bacteria [ 57 ]. IgM antibodies may have ideal characteristics for targeting these molecules and are thought to have a higher cross-reactivity due to the lack of affinity maturation [ 61 ]. These isotypes are multimeric, combining multiple low affinity interactions to accomplish a high functional avidity. This makes IgM larger molecules with higher side effects and shorter half-lives, decreasing their desirability for immunotherapies. Furthermore, the targeting of a polysaccharide antigen might lead to a shift in bacterial population from the targeted polysaccharide, as observed in Streptococcus pneumoniae strains in response to vaccination [ 62 ]. An example of a polysaccharide target against P. aeruginosa used an IgM mAb, denominated Panobacumab (KBPA-101), targeting the O-antigen of serotype O11. Panobacumab mediates protection in several murine models, has bactericidal and opsonophagocytic activities, and was shown to be safe in healthy volunteers [ 63 ]. It later underwent a phase IIa trial with 18 patients with nosocomial pneumonia caused by P. aeruginosa serotype O11. The antibody was safe and well tolerated, with 100% survival, where a 31% mortality was predicted on APACHE II scores. After 9 days, the pneumonia was considered resolved, compared to the standard-of-care of around 15 days [ 64 , 65 ]. Another bacterium also highly studied for the development of immunotherapies is S. aureus . An example is the antibody-antibiotic conjugate DSTA4637S that targets intracellular S. aureus , which can avoid eradication by current standard-of-care antimicrobials. This conjugate is constituted by a human IgG1 monoclonal antibody anti- S. aureus allied with a novel rifamycin-class antimicrobial through a protease cleavable linker. The mAb specifically attaches to the α-N-acetylglucosamine sugar residues of teichoic acid, a major component of S. aureus cell wall. When the phagocytic cells incorporate the S. aureus with the antibody conjugate attached, intracellular cathepsins cleave the linker, releasing the antimicrobial that kills the intracellular bacteria [ 66 ]. This antibody underwent randomized, double-blind, placebo-controlled, single-ascending-dose phase I trial analyzing safety, immunogenicity and pharmacokinetics in healthy volunteers. The participants received single intravenous doses of 5, 15, 50, 100, and 150 mg/kg of DSTA4637S, or placebo, and after 85 days, no serious or severe adverse events occurred [ 66 ]. A different example for S. aureus is the humanized monoclonal antibody Tefibazumab that targets the surface-expressed adhesion protein clumping factor A. This therapy is being developed as an adjunctive therapy for serious S. aureus infections. These mAbs were used in a phase II, randomized, double-blind clinical trial with the objective of S. aureus bacteremia treatment. The sixty patients enrolled in the study received a concentration of 20 mg/kg of body weight in a single infusion of either tefibazumab or a placebo in addition to an antibiotic. The main goal of the study was the determination of safety and pharmacokinetics. The study found no differences in adverse clinical events or alterations in laboratory values between the treatment groups, with four placebo patients showing progression in the severity of sepsis and none of the tefibazumab-treated patients exhibited this progression. Tefibazumab was found to have a safety profile comparable to other monoclonal antibodies. The authors suggest there is sufficient evidence to warrant further study in a larger trial to address the dosing range and efficacy [ 67 ]. A different approach using IgA antibodies was studied to confer protection against multidrug-resistant Mycobacterium tuberculosis infections. This strategy used the human monoclonal IgA 2E9 antibody targeting the alpha-crystallin (Acr, HspX) antigen in combination with the mouse interferon-gamma (IFN- γ ). The studies were performed in mice transgenic for the human IgA receptor, CD89, and found the effect of the combined treatment was strongest when therapy was applied at the time of infection with reductions of 50-fold. Nonetheless, when therapy was initiated with an already established infection, a statistically significant reduction of lung bacterial load was observed [ 68 ]. 5. Avian Immunoglobulin Y Antibodies Immunoglobulin Y (IgY) is a isotype of immunoglobulin that can be found in birds. Antigen-specific IgY can be obtained from eggs laid by hens immunized with the selected antigens and be produced on a large scale. Three isotypes of immunoglobulins are present in these animals, being distinguishable in structure, immunochemical function, and concentration. These isotypes are IgA, IgM, and IgY, with the last one making up to 75% of the total immunoglobulin pool. The IgA and IgM found in birds are similar to mammalians in molecular weight and structure. The IgY was historically called IgG due to similarities in function and serum concentration. However, this is now considered incorrect, especially due to structural differences between the two ( Figure 2 ) [ 69 ]. IgY (~180 kDa) is heavier than the mammalian IgG (~150 kDa). Structurally, IgY comprises two identical heavy chains and two identical light chains, linked by a disulfide bridge. Similar to the mammalian IgG, the IgY light chain is constituted by one variable domain and one constant domain. However, the intra-chain disulfide linkage between these two domains is absent in the IgY, leading to a more unstable structure with weaker intra-molecular forces. Unlike the mammalian IgG, the IgY heavy chain is composed of one variable domain and four constant domains, contrasting with the three constant domains found in IgG [ 70 ]. In 1893, Klemperer demonstrated that an immunized hen was able to transfer specific antibodies from the serum to the egg yolk [ 71 ], giving rise to the idea of the use of egg antibodies for therapies. The immunoglobins are unevenly distributed within the egg, IgA and IgM are incorporated in the egg white and the IgY in the egg yolk. The IgA (~0.7 mg/mL) and IgM (~0.15 mg/mL) have relatively low concentrations in the egg white, while the concentration of IgY in the egg yolk is significantly higher, ranging from 8 to 25 mg/mL [ 72 ]. Currently, the polyclonal antibodies available for usage in immunotherapies are mainly mammalian. The procedure for obtaining these molecules requires the performance of two steps in the IgG donor animal, which cause distress to the animal. This procedure includes immunization and the sacrifice or repeated bleeding. The adoption of polyclonal IgY for the development of antibody-based immunotherapies would lead to an increase of animal welfare, as the process for attaining the antibodies would be replaced by the collection of eggs. A reduction of the total number of animals used would also be accomplished, since the antibody productivity in this approach is 18 times superior to antibody production in rabbits [ 73 ]. The amount of antibodies present in the yolk is so high: 100 mg of antibodies can be obtained from a single egg, with a single hen producing around 20 eggs per month, making the production of IgY highly efficient [ 74 ]. The production of IgY starts by immunizing the hens with a target antigen ( Figure 3 ). The immune response of the hens will be variable and dependent of several factors such as the antigen dose (with too much or not enough antigen inducing suppression, sensitization, or tolerance), the use of an adjuvant, the route of application (with the most common being injection of the breast muscle), the age and breed of the hen, and finally, immunization frequency and the interval between immunizations. The total number of required immunizations is variable but at least two immunizations are required. The interval between these immunizations is also important, being often reported to range from two to four weeks [ 75 , 76 ]. Booster immunization during the laying period can be performed to maintain the levels of specific antibodies up to a year [ 73 ]. The process for the isolation of IgY begins with the separation of lipids and granulate proteins of the egg yolk, obtaining the remaining water-soluble fraction. This fraction is a crude Ig concentrate. To obtain pure IgY, several methods can be performed. IgY can be separated from the water solute fraction by chromatography, filtration, or precipitation with PEG or salts such as ammonium or sodium sulphate [ 75 ]. The chosen method is highly dependent on the scale, quality, and cost effectiveness of the extraction [ 69 , 75 , 76 ]. A very important factor in the widespread use of antibody therapies is the stability of such antibodies. IgY has been studied in this regard and found to be stable during storage and processing. These antibodies maintain their activity after 6 months at room temperature or 1 month at 37 °C. At 4 °C, they can be stored up to a few years with the addition of 0.02% NaN 3 , 0.03% thimerosal, or gentamicin to prevent bacterial growth. Stability is not affected by freezing and freeze-drying if not extensively repeated. IgY are heat stable. However, at very high temperatures, the stability and binding activity decrease. The suggested method for long term storage is −20 °C, as lower temperatures of −70 °C caused loss of activity of around 50% [ 75 , 76 , 77 , 78 ]. As IgY are not mammalian, it is possible to obtain antibodies against highly conserved mammalian proteins or proteins able to evade the mammalian immune system. These antibodies are also capable of interacting with more epitopes on mammalian antigens, reducing the required amount of antibodies for an appropriate immune response [ 79 ]. Despite lacking recognition of mammalian Fc receptors, IgY do not prompt the mammalian complement activation, avoiding adverse inflammatory responses [ 79 , 80 ]. Recombinant humanized IgY are also being developed, where the constant domain of the IgY is substituted with the corresponding human domain. This allows the combination of the advantages of IgY antibodies with antibodies more suitable for in vivo therapeutics in humans. Similarly, monoclonal IgYs have been developed, having a higher affinity and specificity when compared with their polyclonal equivalents [ 81 , 82 ]. The development of therapeutics using IgY antibodies has been developed for a few years and has been successfully employed for prophylaxis and treatment of various enteric infections in animals such as cattle and swine. Using oral administration, specific IgY protects pigs and newborn calves from diarrhea caused by enterotoxigenic E. coli [ 75 ]. In humans, there have been IgY antibodies developed against multidrug-resistant bacteria, which reached clinical trials with different levels of success [ 79 , 83 ]. This is the case of IgY against C. difficile that is at phase II clinical trial (NCT04121169) using IgY polyclonal antibodies in increased doses administered twice a day for 10 to 14 days. Resolution of diarrhea, other symptoms, and fecal test parameters were used to assess clinical effectiveness. The results of this clinical trial are not available; however, previous studies had shown significant clinical improvement with no bacterial relapse [ 79 ]. A different double-blind, phase III clinical trial (NCT01455675) against P. aeruginosa was described, in which an IgY solution was gargled and swallowed every night for two minutes for a total of two years. The subjects were examined every 3 months regarding safety and efficacy. The results showed a good toleration profile for IgY against P. aeruginosa ; however, lacked a clear demonstration of a therapeutic benefit in patients suffering from cystic fibrosis [ 79 ]. Less advanced studies have shown great promise. For example, in A. baumannii , specific IgY antibodies were produced by immunizing hens with formaldehyde inactivated bacteria, followed by purification from yolks using salt precipitation and ultracentrifugation. The antibodies were able to inhibit in vitro bacterial growth and significantly reduced mortality in BALB/c mice with an induced acute pneumonia caused by A. baumannii after intraperitoneal injection with specific IgYs [ 84 ]. Burned mice immunized with egg yolk specific antibodies raised against the P. aeruginosa OprF protein showed survival rates of 87.5% upon infection with the bacterium, compared to 25% for mice immunized with a control IgY [ 85 ]. 6. Single-Domain Antibodies Single-domain antibodies (sdAbs) or nanobodies are antibodies presenting only one monomeric variable domain, making them smaller than normal antibodies but retaining the ability to selectively bind to specific antigens. These immunoglobulins have evolved to attach to specific antigens using only three complementarity-determining region (CDR) loops, alternatively to the six present in conventional antibodies [ 86 ]. The existence of cryptic epitopes, consisting of narrow cavities (canyons) in the surface of antigens of several pathogens, able to bind to target receptors but inaccessible to intact antibodies, renders them generally immune-silent. These features have caused the interest of several researchers and led them to try to develop single domain antibodies. However, these fragments were mainly laboratory curiosities owing to their poor solubility, susceptibility to aggregation, and the fact that they rarely retained affinity [ 87 ]. This changed in 1993 when Raymond Hamers observed, in healthy dromedaries, a smaller isotype of IgG that lacked the light-chain and the first heavy-chain constant domain. This isotype comprised 75% of the total serum IgG. These antibodies, called HCAbs (heavy-chain only antibodies), were later found in other camelid species (llama and alpaca), albeit at lower concentrations (25% to 50%) [ 88 ]. The variable domain region of HCAbs presents unique sequences and was designated as VhH to distinguish them from the conventional VH domains. A few years after this discovery, a similar immunoglobulin was discovered as being part of the immune system of nurse sharks labeled IgNAR. These antibodies have a variable domain designated vNAR, followed by five constant domains, contrasting with the two constant domains of both HCAbs and normal antibodies, as depicted in Figure 2 . Further studies revealed that vNAR have biophysical and chemical properties similar to VhH, such as high affinities and specificities, small size, and high thermal stability [ 89 ]. Both these variable domains have approximately 12 to 15 kDa, can be recombinantly produced, and are capable of recognizing antigens in the absence of the remainder of its heavy chain [ 86 ]. VhH are the smallest natural antigen binding entities, with a 2.5 nm diameter and 4 nm length [ 90 ]. Early structural studies of these fragments indicated that the interaction with antigens used mechanisms distinct from conventional antibodies. The specific mechanism used by sdAbs to bind to antigens is still not fully understood, and the most accepted general function is protein cleft recognition [ 91 , 92 ]. The paratopes of conventional antibodies against folded proteins present flat or concave structures, with convex binding sites being hard to achieve by murine and human antibodies. Synthetic antibodies can be engineered to present convex structures. Contrarily, sdAbs can adopt both flat and convex topologies, with concave being only inefficiently obtained [ 86 , 91 , 93 ]. In terms of amino acid content, no differences are observed between sdAbs and conventional antibodies, with the sdAbs paratopes having smaller molecular surface areas and smaller diameters. This difference leads to smaller footprints on the antigens targeted by sdAbs [ 86 ]. The binding between the antibodies and antigens is similar between the two immunoglobulin isotypes, using non-covalent interactions. These are present at a higher concentration in sdAbs due to the smaller paratopes, allowing for high-affinity interactions [ 86 ]. Notwithstanding, one of the major advantages of sdAbs is not how they bind to antigens but the accessibility to conserved cleft regions and pockets, such as binding sites and enzymes active sites, not available to traditional antibodies. This access is granted by their compact paratope diameter and long surface loops, usually larger than in conventional antibodies [ 94 ]. In addition, unlike mouse Vh domais, VhH and vNAR are stable, soluble, and easy to produce in vitro, rendering them as great resources for development of sensitive diagnostic platforms and sensors [ 94 , 95 ]. From a manufacturing perspective, sdAbs are inexpensive and simple to produce. The lack of post-translational modification allows their synthesis by a microbial system and the generation of a homogeneous product [ 96 ]. These antibodies have the combined advantages of mAbs therapeutics with the targeting potential of nanoscale delivery, as their sizes and structures allow the access to hidden or hard to reach epitopes, with their high modularity allowing the development of more complex constructs [ 86 , 94 , 95 ]. The modularity of sdAbs enables effective generation of bispecific or multispecific recombinant antibodies, which might include the fusion of sdAbs to form dimers, trimers, or tetramers. There is also the possibility to make sdAbs-mAb hybrid fusions [ 48 ]. It is important to notice some limiting factors, namely nanobodies are more prone to a rapid renal clearance, limiting their therapeutic lifetime. This occurs as their size is below renal filtration molecular mass cut-off, highly reducing their half-life within the organism [ 48 , 95 ]. The lack of a Fc region, besides having an impact on reducing half-live, also means these antibodies cannot directly initiate the Fc-mediated immune response. Due to the high similarity with the human VH domain, small size and low agglutination, these antibodies tend to present low immunogenicity [ 97 ]. Nonetheless, to reduce this immunogenicity, sdAbs can be humanized or fully human [ 98 , 99 ]. This process may, however, decrease their activity and solubility [ 100 ]. A different approach for the use of these antibodies is through engineered probiotic bacteria secreting or displaying the recombinant antibody fragment. As these bacteria can reside within the microbiota of the intestine, the antibody can be present and administered during long periods of time [ 101 , 102 ]. All the characteristics of these antibodies render them great candidates for use in microchip technologies for detection and diagnosis of infections or cancer. Nevertheless, their therapeutical potential is also very promising, as it offers new binding specificities, especially to target antigen binding sites inaccessible to conventional antibodies, such as enzyme active sites, G protein-coupled receptors, and viral surface canyons [ 94 ]. The first VhH-based therapy was approved in 2018 by the European Medicines Agency (EMA), called caplacizumab and is used for a rare blood-clotting disease [ 103 ]. The majority of sdAbs currently being developed target cancer cells or human viruses [ 104 ]. The few targeting bacterial infections target their toxins. This is the case for C. difficile , where the toxin pair A and B is targeted. Yang et al. [ 105 ] isolated TcdA- and TcdB-specific alpaca VhHs and constructed a tetravalent and bispecific tandem linked molecule of four VhH. The termini of this molecule targeted TcdA and the middle targets TcdB. This antibody was able to neutralize both toxins from clinical C. difficile isolates to protect mice from a lethal systemic challenge of a mixture of both toxins at an antibody concentration of 3.2 μg/kg and to reverse C. difficile infection in mice after a single injection. A different study by Andersen et al. [ 102 ] isolated four VhHs from lamas against TcdB RBD after immunization with the whole toxin. As monomers, three were able to neutralize the toxin effect on MA-104 cells. When combined in doublet or triplets, no additive effect was observed. The antibodies were then expressed on the surface of Lactobacillus and the previous three retained their effect and the fourth became neutralizing. Lactobacillus expressing the antibodies were then used in a prophylactic oral treatment of a hamster model of C difficile infection, and a combination of two strains showed a delayed death of the challenged hamsters [ 48 , 102 ]. 7. Conclusions and Future Perspectives Antimicrobials are a cornerstone of modern medicine that allow for tremendous increases in life expectancy and quality of humanity. However, resistance to antimicrobials is challenging this development and is becoming an ever-increasing problem that is estimated to surpass the combined deaths of cancer and heart disease by the year 2050, with an estimated 10 million deaths per year [ 22 ]. Every passing year, more antimicrobial agents are becoming ineffective. This problem is exacerbated by the ESKAPE group of bacteria, that, in some cases, are resistant to all clinically available antimicrobials [ 8 ]. This is a problem that requires the utmost attention, as well as new, innovative, and effective approaches, to be surpassed. Immunotherapies are seen by many as the optimal solution, and several approaches being studied are summarized in Table 1 . Antibody characteristics allow for the targeting of only the bacteria of interest, not affecting the commensal bacteria of the host, while the development of resistance to antimicrobials is extremely unlikely. Antibody-based therapies have the advantage over vaccines of having an immediate response, allowing their use when the patient is already suffering from infection. One of the major drawbacks in the development and widespread use of antibody-based therapies is the upfront cost and research time required, combined with the high cost and difficulty of scaling up production [ 32 ]. Despite having a smaller cost of development when compared to vaccines, the upfront cost is still large. Nonetheless, the hospital stay and treatment offered to solve multidrug-resistant (MDR) infections is estimated to cost USD 55 billion (20 billion in health service cost and 35 billion in lost productivity) per year in the United States alone [ 109 ], adding some perspective on the economic problem that MDR is and will cause. Another problem raised against antibody-based therapies is the narrow spectrum they offer, only targeting one species of bacteria, requiring the knowledge of the agent causative of infection. However, the improvements in diagnostic methods, such as PCR or diagnostic antibodies, have proven that rapid diagnostic is possible and effective. The interplay between constant development and advances in research, and the many possibilities of antibody formats available with their advantages and disadvantages ( Figure 4 ), will help to overcome the high cost and other obstacles required for the widespread use of these approaches. In the future, the MDR problem will not disappear, and without an upfront investment in the development of new immunotherapies and structures for their progress, humanity is risking going back to the medicine before the rise of antimicrobials. Notwithstanding, the growing clinical need, the increasing number of antibodies approved for therapeutic use, and the many technological advances in the field of immunoglobulin research, allows to envision a future with a widespread use of antibody-based therapies for bacterial infections.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7088356/

Synergistic China–US Ecological Research is Essential for Global Emerging Infectious Disease Preparedness

The risk of a zoonotic pandemic disease threatens hundreds of millions of people. Emerging infectious diseases also threaten livestock and wildlife populations around the world and can lead to devastating economic damages. China and the USA—due to their unparalleled resources, widespread engagement in activities driving emerging infectious diseases and national as well as geopolitical imperatives to contribute to global health security—play an essential role in our understanding of pandemic threats. Critical to efforts to mitigate risk is building upon existing investments in global capacity to develop training and research focused on the ecological factors driving infectious disease spillover from animals to humans. International cooperation, particularly between China and the USA, is essential to fully engage the resources and scientific strengths necessary to add this ecological emphasis to the pandemic preparedness strategy. Here, we review the world's current state of emerging infectious disease preparedness, the ecological and evolutionary knowledge needed to anticipate disease emergence, the roles that China and the USA currently play as sources and solutions to mitigating risk, and the next steps needed to better protect the global community from zoonotic disease. Electronic supplementary material The online version of this article (10.1007/s10393-020-01471-2) contains supplementary material, which is available to authorized users. Electronic supplementary material The online version of this article (10.1007/s10393-020-01471-2) contains supplementary material, which is available to authorized users. Introduction The catastrophic health and economic impacts of recent human and agricultural epidemics and the continued risk of novel infectious disease emergence, stemming from the alteration of our planet, are clear (Daszak et al. 2000 ; Altizer et al. 2011 ; Allen et al. 2017 ; Johnson et al. 2015 ; Carroll et al. 2018 ). The global nature of emerging and re-emerging infectious disease threats indicates the critical role of international cooperation, particularly spearheaded by China and the USA, in emerging infectious disease (EID) preparedness. China and the USA are well positioned as leaders in the field of EIDs. They are motivated to act out of both national and geopolitical interest and have the resources and instruments to do so: Together they produce over 40% of the world's livestock (Food and Agriculture Organization of the United Nations 2019 ; Beef2Live 2019 ; National Hog Farmer 2019 ), are the largest legal export/import countries for mammals in the global wildlife trade (Can et al. 2019 ) and constitute a quarter of the world's population (Worldometers 2018 ), and over 30% of the world's purchasing-power-parity gross domestic product (GDP-PPP) (World Bank 2019 ) [Fig. 1 (Guha-Sapir 2019 ; Ritchie and Roser 2019 )]. The world would be much better equipped for curbing the next pandemic, if China and the USA provided a united front for research and progress toward EID preparedness. At recent meetings in Shenzhen China and Berkeley California, Chinese and US researchers in the fields of disease ecology, virology, epidemiology, veterinary medicine and public health united to discuss the opportunities for collaboration on infectious disease risk assessment studies that are strongly embedded in ecological and evolutionary principles. What resulted, as discussed herein, is the central role that cooperation between these two highly populous, economic powerhouses is likely to play in understanding the ecological and evolutionary drivers of disease emergence relative to anticipating and managing zoonotic spillovers in China, the USA and worldwide. Figure 1 Factors contributing to China, USA and rest of the world's stake in emerging infectious disease preparedness. (Since values vary with sources and each source updates its estimates from time to time, the actual numbers reported here are not definitive, but should be treated with circumspection.) EID Preparedness is Needed Two hundred years ago, fewer than one billion people inhabited the earth. Today there are over 7.6 billion (Worldometers 2018 ). This population explosion has necessitated extensive conversion of natural to urban landscapes and agricultural and livestock intensification to support growing human populations. These conversion activities bring wildlife, domestic animals and humans into closer and intensified contact with each other, increasing the likelihood of pathogen evolution, interspecies transmission and spillover of wild and domestic animal pathogens to humans (Daszak et al. 2000 ; Karesh et al. 2005 ; Jones et al. 2013 ). Changes wrought by global warming also play a significant role in pathogen evolution, transmission and spillover, as does expanding socio-economic connectivity—both macro-scale impacts of expanding human populations and resource use. As a result of these global changes, EIDs are on the rise, threatening far-reaching populations (Jones et al. 2008 ). The economic cost of an EID pandemic today, even in the absence of significant global mortality, is estimated to exceed tens of billions of US dollars (Pike et al. 2014 ). Understanding the underlying human activities and ecological changes driving EIDs is paramount to preparing for outbreaks and reducing their economic and public health impacts (Barnosky et al. 2012 ). The incidence of disease epidemics in wildlife, and pathogens spilling over from wildlife into humans, is increasing in countries where urbanization has recently or is currently occurring (Liu et al. 2018 ; Hassell et al. 2017 ). Human urbanization extends city boundaries and alters wildlife host community compositions, leading to biodiversity shifts and loss (Keesing et al. 2010 ), disturbing the delicate balance between microbes and their natural wild animal reservoir hosts. Human urbanization also brings domesticated animals into closer contact with wild animals, providing opportunities for cross-species pathogen transmission, as evidenced by recent outbreaks of canine distemper virus in giant pandas in China (Feng et al. 2015 ; Jin et al. 2017 ) and the introduction of feline leukemia virus in the Florida puma in the USA (Cunningham et al. 2008 ). Changes in wildlife populations due to infectious disease outbreaks can have long-term serious consequences for ecosystem resilience. Disease outbreaks in wildlife may also result in direct infection of nearby human populations or changes in wildlife host population dynamics, leading to spillover of other pathogens to humans or domestic livestock (Daszak et al. 2000 ). A prime example is the recent emergence of a novel coronavirus, SADS-CoV, from bats to pigs, resulting from pig farms encroaching on bat habitat, devastating several pig farms in Southern China and posing a major biosecurity risk to the USA (Luo et al. 2018 ). The rise in intensive farming, particularly in China and the USA, also potentially creates conditions for selection of more virulent pathogens and greater opportunities for cross-species transmission (Mennerat et al. 2010 ). China has recently experienced a rapid transformation from small-scale farms to large-scale livestock production enterprises. During 2018, pork production throughout China was estimated at 5404 tons, with the number of individual slaughtered hogs estimated at 693,820,000 (The National Bureau of Statistics 2018a ). The number of slaughtered poultry in 2016 was estimated to be greater than 12.3 trillion (The National Bureau of Statistics 2018b ). Such intensive production practices in China have contributed to outbreaks of influenza H5N1, H1N1, H7N9, and African Swine Fever viruses (Wei et al. 2016 ; Wang et al. 2018 ). In the USA, due to high-density livestock production methods, there has been identification of methicillin-resistant Staphylococcus aureus (MRSA) strains in people living in proximity to these farms (Casey et al. 2014 ). China is the largest producer and user of antibiotics in the world (Qiao et al. 2018 ), increasing the likelihood of AMR pathogen development in food-borne illness. China was estimated to use 162,000 tons of antibiotics in 2013 (48% by humans and 52% by animals, respectively) which was 9 times that used in the USA in 2011–2012 (Zhang et al. 2015 ). Approximately 46% of the antibiotics used in China were ultimately released into rivers through sewage effluent with the remainder distributed on land through manure and sludge spreading (Zhang et al. 2015 ). AMR is particularly acute in China because of its over-prescription and self-administration practices, as well as its widespread misuse of sub-therapeutic doses of antibiotics in the livestock industry (Yezli and Li 2012 ; Yu et al. 2014 ). Antimicrobial-resistant Escherichia coli (plasmid-mediated colistin resistance mechanism, MCR-1) originating from overcrowding and high-intensity farming of pigs in China (Liu et al. 2016 ) can at present be found in countries far from China, including the USA (Hu et al. 2016 ; Skov and Monnet 2016 ; Sun et al. 2018 ). China and US societies are now integrally connected, with intensive regional and international human movement (Tatem et al. 2006 ), trade of domestic and wild animals (Marano et al. 2007 ), and other economic activities facilitating the spread of infectious pathogens from high-density farming operations to other geographic locations. Increasingly complex and robust global trade systems are also fueling the legal and illegal wildlife trade markets. The USA is the largest importer, and China is the largest exporter of legally traded wild mammals (Can et al. 2019 ) and while challenging to quantify, China is considered the leading country in the consumption and illegal trade of wildlife (Karesh et al. 2005 ; Patel et al. 2015 ). Southern China is a hub for domestically and internationally imported and exported wildlife given its strategic geographic location near major ports of trade, dense human population and increasing human mobility. Local tradition also fuels consumption of wild animals in this region. In Guangdong province alone, there are more than 1300 enterprises engaged in wildlife breeding, of which approximately 600 breed wild animals regulated by CITES Appendix II. During 2001–2004, a total of 21 bird species (around 56 thousand individuals), 21 mammal species (~5400 individuals), 41 amphibious and reptile species (up to 346 tons) were raised and traded in Guangzhou, the most populous city in Guangdong Province. Communities engaged in this concentrated wildlife production and trade enterprise represent a valuable resource at the front lines of pathogen spillover and can be leveraged to understand and control the spread of zoonotic diseases. At a macro-scale, anthropogenic activities have influenced microbial transmission dynamics, particularly for vector-borne pathogens (Jones et al. 2008 ; Goklany 2009 ; Woodward et al. 2014 ; Murray and Daszak 2013 ; Morse 1995 ). For example, climate variation has been documented to drive transmission of dengue virus dynamics in Guangdong, China (Liu et al. 2018 ; Xu et al. 2017 ; Sang et al. 2014 , 2015 ; Xiang et al. 2017 ). This region experienced no outbreaks of dengue-like illness from the period of 1950–1977, followed by a relatively low level of incidence until a large-scale outbreak occurred in 2014, infecting over 45,000 people (Liu et al. 2018 ). Similarly, climate change is projected to impact the distribution of vector-borne disease in the USA, with the environment being more suitable for the introduction of Zika virus in certain regions of the southeastern USA (Carlson et al. 2018 ). The combined US–China resource capacity is critical to better understand and mitigate anthropogenic EID drivers, such as urbanization, biodiversity loss, landscape conversion, intensive farming and climate change that are contributing to the spread of disease across the wildlife–livestock–human health continuum. Required Ecological and Evolutionary Perspective A holistic ecological and evolutionary process perspective is required to understand the risk of spillover and spread of pathogens in humans and animals (Alexander et al. 2018 ). Pathogens are not fixed entities, and some pathogens carry a greater innate ability to evolve and spillover into new hosts than others (Johnson et al. 2015 ; Olival et al. 2017 ). Evaluation of pathogen evolution from initial spillover to establishment in the human population (e.g., simian immunodeficiency virus chimpanzee to human immunodeficiency virus; HIV-1) (Gao et al. 1999 ) is critical to understanding why certain pathogens can establish and others cannot. Viral surveillance in "real-time" is required to examine and track pathogen evolution. For example, the Global Influenza Surveillance and Response System, designed to monitor the quickly evolving and recombining influenza virus in a timely manner, has served for over half a century as a global alert mechanism for the emergence of influenza viruses with pandemic potential (World Health Organization 2019a ). Knowledge of ecological scenarios surrounding the accelerated viral evolution of highly pathogenic avian influenza (HPAI) is vital to preparing for future outbreaks of the disease in humans and worthy of significant investment in China and elsewhere (Gao 2018 ). Monitoring of viral evolution in similarly relevant time scales for other quickly evolving pathogens, such as coronaviruses, lags further behind and is an area of research with much needed additional attention. For example, recent evolutionary analyses of Middle East Respiratory Syndrome (MERS) coronavirus have helped to elucidate viral transmission dynamics between camels and people (Dudas et al. 2018 ). We also now know that viruses sharing diverse vertebrate hosts are "worth watching" for potential emergence in humans, because host breadth (i.e., infecting a taxonomically diverse range of hosts) is a key factor associated with a virus' likelihood of spillover and secondary human-to-human transmission and geographic spread (Johnson et al. 2015 ; Olival et al. 2017 ). Thus, expanding "real-time" surveillance of such pathogens would be a worthwhile investment for public health. Analyzing epidemiological data using theory and methods from macroecology allows us to forecast impacts of ecological and environmental drivers on infectious disease incidence. Examples include how land cover and climate changes influence epidemics in wildlife (e.g., chytrid fungus, white-nose syndrome), livestock (e.g., foot-mouth-disease, African swine fever, bluetongue) and human (e.g., schistosomiasis, malaria) populations, as well as impact complete wildlife–livestock–human systems (e.g., avian influenza, tuberculosis) (Estrada-Pena et al. 2014 ; Peterson 2014 ; Purse et al. 2005 ). Large-scale, high-resolution data on environmental and climatic factors and populations of humans and wildlife facilitate the creation of dynamic distribution models for infectious agents and can help prioritize research and control efforts (Cohen et al. 2016 ; Carlson et al. 2017 ). Collaborative research between China and the USA could greatly advance spatiotemporal disease prediction schemes because both countries encompass large climatic gradients, have high capacity for ecological and climatic data collection, and share some overlapping vectors, and zoonotic pathogens (Liu et al. 2018 ; Estrada-Pena et al. 2012 ; Springer et al. 2015 ; Wu et al. 2013 ; Centers for Disease Control 2018 ). A complete understanding of host–pathogen interactions and how and where to intervene requires an eco-system or "One Health" viewpoint that accounts for processes occurring at both macro- and micro-scales, including at the pathogen, host and environmental levels, as well as an integration of the effects of processes across these scales (Alexander et al. 2018 ; Forst 2010 ; Blackburn et al. 2019 ). Such multiscale "One Health" research requires the incorporation of many disciplines including, but not limited to, human medicine, veterinary medicine, public health, environmental science, ecology, conservation biology, nursing, social sciences, the humanities, engineering, economics, education and public policy (Lu et al. 2016 ; Carlson et al. 2018 ). China and the USA have led response activities for several epidemic and pandemic outbreaks impacting humans and animals which have required a One Health perspective. Three relevant case studies which are expanded upon below include: the West Africa Ebola outbreak, the SARS pandemic, and the emergence of amphibian chytridiomycosis. The first outbreak of Ebola virus disease (EVD) outside of Central Africa demonstrated the importance of focusing on wildlife host and human ecological risk factors in advance of major disease outbreaks and the need for international collaboration in outbreak response (Fig. 2 ). Despite nearly 40 years of research since the first outbreak in 1976, including public investment of US$ 1.035 billion between 1997 and 2015 (Fitchett et al. 2016 ), national and international public health agencies were caught off guard by the 2014 outbreak in West Africa (Kamradt-Scott 2016 ) that resulted in 28,600 cases with more than 11,300 deaths (WHO Ebola Response Team et al. 2016 ). Evidence from humans and wildlife indicating the distribution of Ebola virus (species Zaire ebolavirus ) in West Africa existed prior to this health crisis. Distribution and migration patterns of the hammer-headed fruit bat ( Hypsignathus monstrosus ), little collared fruit bat ( Myoncycteris torquata ), straw-colored fruit bat ( Eidolon helvum ) (Leroy et al. 2005 ), Franquet's epauletted fruit bat ( Epomops franqueti ) (Olival and Hayman 2014 ) and the greater long-fingered bat ( Miniopterus inflatus ) (Kupferschmidt 2018 ), species implicated as reservoir hosts for Ebola virus, were known to extend into West Africa with opportunities for spread of the virus. Human serological exposure also indicated a wider geographical range for ebolaviruses including Guinea (Boiro et al. 1987 ), Liberia (Van der Waals et al. 1986 ) and Sierra Leone (Schoepp et al. 2014 ) well in advance of 2014. During this outbreak, China and the USA collaborated together for the first time in an international health emergency outside of their borders, which constituted China's largest ever humanitarian mission in addressing a public health emergency of international concern (Huang 2017 ). Twenty-four of the Chinese public health experts who were deployed to Africa were graduates of, or residents in, the Chinese Field Epidemiology Training Program (CFETP) established by the US Centers for Disease Control and Prevention (CDC) (Centers for Disease Control 2016 ). Also, the Chinese government sent 115 military medical professionals to Sierra Leone to work along with US medical personnel to assist with infection prevention and control, clinical care and health promotion and training (Lu et al. 2016 ). Further investment from China and the USA in working together on response efforts will undoubtedly be mutually beneficial for pathogens of importation concern. Efforts toward unraveling the disease ecology of ebolaviruses—including a better understanding of the ecology of reservoir host(s), the role of secondary spillover hosts, as well as human behaviors surrounding exposure—is also needed and would benefit exponentially from a China–US collaborative effort. Figure 2 One Health concepts impacting emerging infectious diseases. Climate Change: With the introduction of Zika virus into the Americas, changes in maximum occurrence of mosquito vectors in the USA, due to a changing climate, impact risk of Zika virus distribution. Human Ecology: High-risk human behaviors involving contact with farmed wild animals contributed to the emergence of SARS. Biodiversity: Alteration of wild animal reservoir host populations impacts spillover risk for zoonotic infectious diseases. Animal Host Ecology: Distribution of the bat reservoir hosts for Ebola virus (species Zaire ebolavirus ) likely caused the first human outbreak in West Africa. Together with the impact of global trade and travel, these case examples of the interconnectedness of humans, animals and the environment demonstrate how human and animal ecology influence the global spread of disease. The Severe Acute Respiratory Syndrome (SARS) pandemic caused by a novel zoonotic coronavirus (SARS-CoV) was the first pandemic of the twenty-first century and spread to more than 30 countries (Fig. 2 ). Initial isolation of SARS-related coronavirus (SARSr-CoV) from masked palm civets and the detection of SARS-CoV infection, in humans working at wet markets selling these animals in Guangdong Province, suggested that masked palm civets could serve as a source of human infection (Guan et al. 2003 ). Subsequently, SARSr-CoVs were detected in Chinese horseshoe bats ( Rhinolophus sinicus ) and provided strong evidence that bats are the natural reservoir of SARS-CoV (Ge et al. 2013 ; Li et al. 2005 ; Yang et al. 2015 ). Through long-term human and wildlife surveillance, investigators from China and the USA subsequently found that bats carry a diverse range of SARSr-CoVs (Ge et al. 2013 ; Li et al. 2005 ; Yang et al. 2015 ; Lau et al. 2005 ; Drexler et al. 2010 ; Yuan et al. 2010 ; He et al. 2014 ; Wu et al. 2016 ; Hu et al. 2017 ), extending as far as Yunnan Province, and some of them can directly infect humans without intermediate hosts (Wang et al. 2018 ). While it is unknown whether the SARS outbreak could have been preempted with this knowledge, the joint efforts of China and the USA to rapidly determine where, how and when the virus was spilling over and what human behaviors and populations were at greatest risk for infection may have reduced the severity of the outbreak and will help in mitigating future spillover events. The SARS outbreak was a prime example of the importance of contextualizing epidemiologically notable human behaviors in social, economic and cultural systems in order to decipher causality of an EID. Pandemic diseases in wild animals (epizootics) can also result in devastating impacts to a country's biodiversity and natural resources. For example, amphibian chytridiomycosis, caused by the novel pathogen Batrachochytrium dendrobatidis , is responsible for massive losses of biodiversity to an entire Class of organisms (Amphibians). The global pandemic lineage of the pathogen originated in Eastern Asia (Ostfeld and Keesing 2012 ) and disseminated through human trade and transportation (O'Hanlon et al. 2018 ) into the biodiverse areas of Australia and the Neotropics. The lack of demographic studies, combined with limited population estimates in the IUCN Red List prior to this pandemic, made it difficult to understand the scope of the disease. A better understanding of wildlife population dynamics before massive declines occur is essential to better understanding biodiversity's impact on EIDs and what elements of biodiversity disease theory (Fig. 2 ) apply (Jones et al. 2008 ; Keesing et al. 2010 ; Murray and Daszak 2013 ; Morse 1995 ; Ostfeld and Keesing 2012 ; Civitello et al. 2015 ). Interestingly, another fungal pathogen, white-nose syndrome (WNS), which has decimated bat populations in the USA, may also have an Asian or Eurasian origin because it has been found on bats in Northern China (Hoyt et al. 2016 ). Recent studies suggest that the systemic effects of WNS may down-regulate anti-viral responses in bats persistently infected with coronaviruses and increase the potential of virus shedding (Davy et al. 2018 ). Thus, a pathogen predominantly infecting wildlife may additionally have cascading effects on spillover of other pathogens of significance for human health. Current State of Readiness Global-scale, government-sponsored EID preparedness efforts, initiated or supported to date by joint China–US partnerships, have focused on improving early-warning capabilities for known and novel human pathogens in both humans and animal reservoirs. These initiatives have included the US CDC Global Disease Detection program (CDC-GDDP), which has collaborated with China CDC and 10 other nations to develop international centers that help countries prevent, detect and respond to public health threats (Centers for Disease Control 2018 ); the US Agency for International Development's (USAID) Emerging Pandemic Threats (EPT) Program, which has collaborated with China CDC and Wuhan Institute of Virology and 29 other nations to strengthen EID preparedness through pathogen surveillance in wildlife, domestic animals and humans, risk characterization for pathogen spillover and One Health training and outreach; the WHO, OIE and FAO's collaborative global early-warning system for animal diseases transmissible to humans (GLEWS) (World Health Organization 2018 ); the China National Global Virome Initiative (CNGVI; part of the Global Virome Project (GVP Carroll et al. 2018 ), a pathogen discovery project proposed to identify a large portion of the remaining undiscovered viruses (Carroll et al. 2018 ; Mora et al. 2011 ; Geoghegan et al. 2016 ); and joint research supported by the US National Institutes of Health (NIH) to define the origin of SARS- and MERS-like coronaviruses (Luo et al. 2018 ; Hu et al. 2017 ) and identify other SARS coronavirus mammalian infections in China (Zhou et al. 2018 ). Over the past ten years, the USA has made a significant investment in spear-heading an international network of government collaborative laboratories and surveillance mechanisms for EID preparedness through the USAID EPT program, CDC-GDDP and the Department of Defense's Overseas Research Laboratories. For example, the PREDICT project, a part of the EPT program, was operational in 30 countries, increasing capacity in over 60 laboratories located in EID hot spots and training over 6200 health professionals in laboratory diagnostics, field epidemiology, surveillance and biosafety (PREDICT Consortium 2019 ). Such a large-scale investment by the USA in global disease surveillance, targeting both humans and animals, has laid the foundation and built the networks and infrastructure necessary for implementing future training and research in the underlying disease ecology of EIDs. China, with its much larger population, has historically taken a more nationalist perspective toward EID research and preparedness. Following the outbreak of SARS in China, the Government enhanced infectious disease surveillance, building the web-based Nationwide Notifiable Infectious Disease Reporting Information System (NIDRIS) (Yang et al. 2017 ). Based on experience detecting and responding to national epidemics such as SARS, and influenza H5N1, H1N9 and H7N9, China has expanded their efforts to assist with diagnostics and surveillance in the region; they are a participant in the Mekong Basin Disease Surveillance Network (Phommasack et al. 2013 ) and committed to promoting the prevention and control of communicable diseases and public health emergency response through ASEAN (Association of Southeast Asian Nations)–China health cooperation (Association of Southeast Asian Nations 2018 ). The China International Development Cooperation Agency (CIDCA) (Chinese International Development Cooperative Agency 2019 ) has historically invested in infrastructure projects but is now increasingly supporting global health initiatives. China–US Leadership China and the USA are well placed to lead efforts in EID preparedness both from a national interest standpoint, resource availability and a global health interconnectedness perspective. China and the USA have a long history of collaboration, have the two largest economies in the world with significant resources for investment in global health, the largest current combined investment in infrastructure for infectious disease research, and have the skill sets necessary for advancing disease prevention and response. China and the USA first signed a Protocol for Cooperation in the Science and Technology of Medicine and Public Health in the 1970s (Obamawhitehouse.gov. 2018 ). Today, more than 40% of publications from Chinese scientists are co-authored with scientists from the USA (Wang et al. 2013 ). With successful poverty reduction in China and transformation from a recipient to a provider of aid, China has taken a more active role in global health initiatives, signing an MOU with the USA in 2017 to designate funding toward cooperation on international development, focusing on food security, public health, humanitarian assistance and disaster response (Carnegie-Tsinghua Center for Global Policy 2018 ). China has also made efforts to streamline international scientific collaborations, issuing from the General Office of the State Council in March 2018, the regulatory document entitled, " Measures for the Management of Scientific Data " (People's Republic of China 2018 ; Uga et al. 2001 ), expected to standardize the data sharing process with the goal of encouraging collaboration. Since the end of the nineteenth century, the USA has maintained the largest economy in the world and has been the preeminent international influence on global trade and foreign direct investment (International Monetary Fund 2018 ). China has recently become the second largest economy and is increasingly playing an important and influential role in global trade and infrastructure investment, particularly since the initiation of the Belt and Road Initiative in 2013 (National Development and Reform Commission (NDRC) 2018 ). In 2018, the World Bank ranked China 1st and the USA 2nd for GDP-PPP (gross domestic product taking into account purchasing power parity) (World Bank 2019 ). Further, China accounts for 18.5% of the world's population and the USA an additional 4.3% (over one-fifth combined). Companies, products and employees from both countries are distributed across the world presenting opportunities for both importation and exportation of infectious diseases through animal products, human travel and wildlife trade, and as major drivers of ecological change responsible for the emergence of new diseases. China and the USA thus have imperative moral and fiscal responsibility to invest in global health security, and their cooperation is key for preparedness and control of global EIDs in the future. The USA has several key government agencies which are actively contributing to emerging infectious disease research around the world including the Department of Defense, Health and Human Services, the President's Emergency Plan for AIDS Relief, the President's Malaria Initiative, the State Department and USAID [see program compilation available at the NIH Fogarty International Center website (National Institutes of Health Fogarty International Center 2019 )]. The USA is currently the largest donor to global health in the world (approximately $11 billion in 2019); however, the current US administration has proposed significantly reducing global health funding for the fiscal year 2020 (to approximately $8 Billion) (Henry J Kaiser Family Foundation 2019 ). The USA also benefits from significant private investment in global health, and this trend is likely to continue, with efforts from private foundations such as the Gates Foundation and the Chan Zuckerberg Biohub poised to accompany US government funding of global health-related projects (Reubi 2018 ). China has also recently expanded its national infrastructure for EID preparedness and a country-wide network of laboratories including 11 national technology platforms, 11 national research centers and 6 national key laboratories. China also has several WHO collaborating centers with research focused on: tuberculosis, schistosomiasis, infectious disease surveillance, EIDs, management of HIV, influenza, vector surveillance, infectious disease epidemiology, echinococcosis, tropical diseases, malaria, and emerging and re-emerging infectious diseases (World Health Organization 2019b ), and two OIE collaborating centers with research focused on food-borne parasites and zoonoses (World Organization for Animal Health 2019 ). A strong emphasis on leading edge technology for molecular diagnostics and pathogen characterization has made China a leader in the field of virology and biotechnology (Ellis 2018 ), including investment in the state-of-the-art pathogen isolation and identification technologies such as high-throughput sequencing. In January 2018, for the first time, China overtook the USA in terms of total number of scientific publications, according to statistics compiled by the US National Science Foundation (NSF) (Tollefson 2018 ). Historically, China and the USA have placed different levels of emphasis on the ecological and evolutionary components of infectious diseases research. A search of the Web of Science (August 15, 2018: see supplementary information for details) revealed that from 2000–2007, 43% of publications on ecology/environment that included disease/pathogens were authored by US researchers exclusive of Chinese participation, while only 2.2% were authored by Chinese researchers exclusive of US participation. These figures changed to 36% and 8%, respectively, from 2010 to 2017. If this trend continues into the next decade (2020–2027), then under a linear extrapolation we can expect the USA and China to publish around 30% and 15% of ecological and evolutionary infectious disease-related research (Fig. 3 ). Together, the USA and China have published half of the world's ecological and evolutionary infectious disease-related research. Despite these trends, and while around 45% of all published disease research is undertaken within the USA and China, integrated scientific studies with strong ecological and evolutionary components are largely missing. Figure 3 Current and projected future contributions to research involving the ecology and evolution of infectious diseases from China, the USA and the rest of the world based on the published literature. Proposed Next Steps While current investments from the USA and China have built a foundation for better disease surveillance in humans and animals around the world, an investment in training in disease ecology with an emphasis on critical thinking remains a missing link. A key observation identified by us during the recent US–China workshops was that Chinese infectious disease research has a strong emphasis on biology, biotechnology, genomics, virology and public health surveillance at the expense of research into the spatial/geographic, social/cultural, transmission and ecological components needed to develop models to guide policies for controlling zoonoses and other outbreaks. This technology-oriented bias in research at the cost of a broader social and ecological understanding of disease systems can result in an intellectual trap where developing countries continue to depend on external ecological and environmental systems-level expertise critical to managing outbreaks. In addition, with the explosion of bioinformatics related research in the USA, it is important that the USA not lose sight of the fundamental importance of foundational training in ecological understanding. Future investments in training in the fields of disease ecology, eco-evolutionary dynamics, study design in natural systems, disease modeling and complex data analysis are essential for China—as it is no less for other Asian and sub-Saharan Africa countries—to take a lead role in their own disease investigations and to contribute toward global health security as equal partners. An investment in human capacity for critical thinking regarding infectious diseases involves extending ecological and analytical training in less developed countries, where pathogens are most likely to emerge—moving beyond efforts limited to field sampling and pathogen detection. The UN China One Health Event held in 2011 emphasized the importance of problem-based as opposed to disciplinary learning to achieve these goals (Fearnley et al. 2019 ). This is the critical next step toward fully collaborative EID preparedness programs, so that capacity strengthening can be focused on preparing in-country teams for designing their own outbreak investigations, conducting their own human and wildlife disease ecology surveys, and taking a more active role at every step of the scientific process involved with understanding EIDs and responding to outbreaks. Significant challenges exist to accomplishing this holistic training approach, when in-country tertiary science education has not produced sufficient research groups able to carry out the needed research. China is the logical next step for the global health community's major investment in disease ecology training, given its well-developed scientific infrastructure, current investments in basic science, virology and biotechnology, and national interest to contribute to global EID initiatives. New approaches are needed to integrate ecological training and accelerate EID preparedness. These include broadening data collection to reduce uncertainties and improving analytical techniques to identify regions at highest risk for EIDs, as well as strengthening public health infrastructures in these locations to reduce the number of outbreaks. The application of novel analytical approaches to address these critical needs has been conflated by academic debate on whether or not EID prediction is possible (Geoghegan and Holmes 2017 ). While we currently do not have the ability to accurately forecast the time and location of the next EID outbreak, existing predictive models have been instrumental in prioritizing efforts. These include focusing on: sub-national regions (Allen et al. 2017 ; Jones et al. 2008 ); underlying environmental drivers (Allen et al. 2017 ; Johnson et al. 2015 ); specific reservoir host species (Olival et al. 2017 ); and pathogen traits (Olival et al. 2017 ; Fearnley et al. 2019 ) that facilitate spillover (Carlson et al. 2018 ). With a larger investment in disease ecology training in countries likely to be hot spots for EIDs, we can create the next generation of modeling and analytical techniques that incorporate more robust input from EID source nations. Substantial decadal-long US–China joint funding mechanisms for integrated multidisciplinary EEID research projects are also needed to accomplish cross-training and meet research objectives discussed above. Efforts toward this mission have been limited because of unwillingness of traditional funders to redirect resources across sectors and expand out of siloed missions (Mazet et al. 2016 ). Disease-related research focused on medical or genetic components at the point of human infection and spread has received much more funding than that with an ecological health focus, aiming to describe the environmental or host community scenarios facilitating initial spillover of a pathogen to humans, or the impact of biodiversity loss on human health (Ostfeld 2017 ; Cardinale et al. 2012 ). An important step in the right direction was the Ecology and Evolution of Infectious Diseases (EEID) program through the NSF's announcement in August 2018, of the addition of the National Natural Science Foundation of China as a new international collaborative partner. Through a relatively modest government investment of $275 million USD (including contributions from foreign partners) since 2000, this program has funded over 150 individual projects and led to some key discoveries that have greatly advanced our understanding and prediction of EID spillover, amplification and spread (Lloyd-Smith et al. 2005 ; Kilpatrick et al. 2006 ; Gilbert et al. 2008 ; Chiu et al. 2019 ; Lee et al. 2017 ; Carver et al. 2016 ; Coffey et al. 2008 ). Continuation of this program and further collaborative funding efforts between the USA and China are needed. With the burgeoning world population and dramatically increased movements of individuals, the potential for a disease outbreak to cause the death of hundreds of millions of individuals is now a reality. Only a deep understanding of disease from an ecological systems point of view, taking into account every scale of a pathogen's life cycle, can avert the increasing number of catastrophes in waiting. Very often it is information on disease ecology that is missing from programs purporting to take a One Health approach. Without the scientific and funding support of China and the USA in addressing the ecological components of disease systems through engagement of researchers and health practitioners from every part of the globe, we will continue to remain dangerously naive of how best to confront the threat of pandemic disease. Electronic Supplementary Material Below is the link to the electronic supplementary material. Supplementary material 1 (DOCX 18 kb) Below is the link to the electronic supplementary material. Supplementary material 1 (DOCX 18 kb)

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7149987/

Seeds of Destruction

This chapter provides the reader with an understanding and appreciation for the scope and importance of biological threats and the opportunity to see where they may be and have become the desire of terrorist groups and the makings of weapons of mass destruction. The history of biological warfare is covered in depth. These major events are important in helping us understand the issues related to using biological substances against an adversary. The difference between biosecurity and biodefense are explained and then related to homeland security and homeland defense, respectively. This chapter also details how expensive these programs are, with nearly $80 billion having been spent on civilian biodefense since fiscal year 2001 in the United States alone. As discussed herein, there is a significant difference in the reality and the potential of bioterrorism. Bioterrorism on a large scale is a low-probability event. Bioterrorism on a small scale is a fairly routine occurrence with little potential. Biological threats remain very much in the news. Recent examples, such as laboratory incidents, the Ebola outbreak of 2014, and other emerging threats, are covered in this chapter. Introduction The dawning of the 21st century will be characterized as the Age of Terrorism. Terrorism has affected most of us in one way or another. The shocking images of the September 11, 2001, attacks remind us of just how dramatic and devastating terrorism can be. In most developed countries, the concept of bioterrorism and many of the words associated with it are widely recognized. In the United States, bioterrorism became a household word in October 2001, when Bacillus anthracis (the causative agent of anthrax) spores were introduced into the US Postal Service system by several letters dropped into a mailbox in Trenton, New Jersey (see Fig. 1.1 ). These letters resulted in 5 deaths from pulmonary anthrax and 17 other cases of inhalation and cutaneous anthrax ( Thompson, 2003 ). In the weeks and months that followed, first responders were called to the scene of thousands of "white powder" incidents that came as a result of numerous hoaxes, mysterious powdery substances, and just plain paranoia ( Beecher, 2006 ). Public health laboratories all over the United States were inundated with samples collected from the scene of these incidents. Testing of postal facilities, US Senate office buildings, and news-gathering organizations' offices occurred. Between October and December 2001 the Centers for Disease Control and Prevention (CDC) laboratories successfully and accurately tested more than 125,000 samples, which amounted to more than 1 million separate bioanalytical tests ( CDC, 2015 ). Henceforth there has been a national sense of urgency in preparedness and response activities for a potential act of bioterrorism. Figure 1.1 This letter, postmarked October 5, 2001, was dropped into a mailbox near Princeton University in Trenton, New Jersey. It was addressed to Senator Tom Daschle with a return address indicating a fourth-grade class from Greendale School in Franklin Park, New Jersey (note that there is no such school). A scientist, Dr. John Ezell, at USAMRIID, Fort Detrick, Maryland, is pictured here holding up the letter and the note it contained. Courtesy of the FBI. Humankind has been faced with biological threats since we first learned to walk upright. In his thought-provoking book Guns, Germs and Steel , Dr. Jared Diamond points out the epidemiological transitions we have faced since we were hunters and gatherers. More than 10,000 years ago the human experience with biological peril was mostly parasitic diseases that only affected individuals. After that, human societies began to herd and domesticate animals. The development of agriculture allowed for population growth and a shift from small tribal bands to a concentration of people into villages. Larger groups of people could stand up to smaller elements, thereby enabling them to successfully compete for resources and better defend the ground that they held. Agriculture also brought some deadly gifts : animal diseases that also affected man ( zoonotic diseases ), outbreaks of disease due to massing of people and lack of innate immunity, and a growing reliance on animal protein ( Diamond, 1999 ). For ages human societies and cultures have been looking for a competitive advantage over their adversaries. Advances in weapons of all types and explosives allowed military forces to defeat their enemies overtly on the battlefield and covertly behind the lines. Technologies leading to nuclear, biological, and chemical weapons have also been exploited. Indeed, each has been used legitimately and illegitimately on different scales to bring about a change in the tactics, the military situation, or the political will to face an enemy in battle. Biological agents are no exception to this rule. As such, biowarfare (biological warfare) has a historical aspect to it that must be considered here because advances in the use of biological agents over the last century are one of the main reasons why bioterrorism exists today. When President Richard M. Nixon said, in November 1969, that "Mankind already holds in its hands too many of the seeds of its own destruction," he was signing an Executive Order putting an end to the United States' offensive capabilities for waging biowarfare. It is arguable that this statement foretold the potential doom we might all face when then state-of-the-art technologies became commonplace techniques in laboratories all over the world today. This chapter accordingly derives its name from the preceding quote and should serve to remind the reader that the seeds we sowed so long ago have now sprouted. The question remains: How shall they be reaped? The Reality Versus the Potential Bioterrorism is the intentional use of microorganisms or toxins derived from living organisms to cause death or disease in humans or the animals and plants on which we depend. Biosecurity and biodefense programs exist largely because of the potential devastation that could result from a large-scale act of bioterrorism. Civilian biodefense funding (CBF) reached an all-time high after the anthrax attacks of 2001. Conversely, the reality of the situation is that these well-intended programs cost taxpayers billions of dollars each year. Rapid detection biothreat pathogen tools are available to assist responders with on-site identification of a suspicious substance. In addition, biosecurity and biodefense are "big business" in the private sector. Security measures to protect agriculture and certain vulnerable industries from acts of bioterrorism and natural biological threats are also in place. Detailed reports published in the journal Biosecurity and Bioterrorism ( Schuler, 2005 , Lam et al., 2006 , Sell and Watson, 2013 ) show that US government CBF between fiscal year (FY) 2001 and FY2014 amounted to more than $78 billion. Comparing FY2001 to FY2005, there was an increase in CBF from $420 million to $7.6 billion. The Departments of Health and Human Services and Homeland Security, which together account for approximately 88% of the FY2006 request, have remained relatively constant in their funding. Other agencies, most notably the Department of Agriculture and the Environmental Protection Agency, have been more variable. These two agencies saw increased budget requests in FY2006, focusing on programs that protect the nation's food and water supplies. Civilian biodefense spending, not including special allocations for project BioShield, reached a consistent level of approximately $6 billion from FY2003 to FY2013 ( Sell and Watson, 2013 ). Refer to Table 1.1 for a summary of the CBF budget for FY2010–14. Table 1.1 Civilian biodefense funding (in $ millions) for US government agencies by fiscal year Agency/year FY2010 FY2011 FY2012 FY2013 FY2014 Department of Health and Human Services 4068 4150 3924 3986 4100 Department of Defense 675 789 923 1129 1155 Department of Homeland Security 478 390 335 358 1046 Department of Agriculture 92 84 92 92 94 Environmental Protection Agency 150 128 96 103 102 Department of Commerce 100 103 101 102 112 Department of State 74 74 73 73 68 National Science Foundation 15 15 15 15 15 Department of Veteran Affairs 1 1 1 1 1 Total CBF 5653 5734 5560 5859 6693 FY , fiscal year; CBF , civilian biodefense funding. Amounts are rounded to the nearest whole number. Data from Sell, T., Watson, M., 2013. Federal agency biodefense funding, FY2013–FY2014. Biosecurity and Bioterrorism: Biodefense Strategy, Practice, and Science 11, 196–216. BioShield is a program that was designed to give the United States new medical interventions (eg, vaccines, treatments) for diseases caused by several biothreat pathogens. When BioShield was conceived, it cost US taxpayers a total of $5.6 billion, which was metered out to the Department of Health and Human Services over a 10-year period. Reports surfaced that suggest BioShield funds were being squandered and that few useful products were realized ( Fonda, 2006 ). However, biothreat pathogen research and product development for unusual or rare diseases is fraught with numerous hurdles. This program will be addressed in chapter Biosecurity Programs and Assets. The US Postal Service spent more than $800 million developing and deploying its Biohazard Detection System (BDS). At the peak of its utilization, the US Postal Service was spending more than $70 million each year to operate and maintain the system. The BDS is used only to provide early warning for the presence of a single biothreat pathogen, anthrax. Furthermore, the system screens letter mail that comes from sources such as mailboxes and drops, which accounts for approximately 17% of all letter mail volume ( Schmid, 2006 ). This model program and the technology it uses will be covered extensively in chapter Consequence Management and a Model Program. All of this seems rather incredible when comparing the level of funding given to one of the greatest biological threats of our time, the human immunodeficiency virus (HIV), which causes AIDS. An estimated 1.8 million people are currently living with HIV in the United States, with approximately 50,000 new infections occurring each year. Currently in the United States, approximately 75% of the new infections in women are transmitted heterosexually. Half of all new infections in the United States occur in people 25 years of age or younger. However, the budget of the National Institutes of Health for AIDS research is approximately $3 billion per year ( NIH, 2015 ) compared with the $1.6 billion level of funding it receives for biodefense ( Sell and Watson, 2013 ). The History of Biowarfare Before delving into the subtleties of biosecurity and biodefense, one should explore the historical aspects of the use of biological agents in warfare and terrorism. The history presented here is not all inclusive. Rather, it is a fair assessment of key events and characterizations that can be examined in other more comprehensive documents. Pathogens and biological toxins have been used as weapons throughout history. Some would argue that biological warfare began when medieval armies used festering corpses to contaminate water supplies. Over several centuries this evolved into the development of sophisticated biological munitions for battlefield and covert use. These developments parallel advances in microbiology and include the identification of virulent pathogens suitable for aerosol delivery and large-scale fermentation processes to produce large quantities of pathogens and toxins. However, the history of biological warfare is shrouded by several confounding factors. First, it is difficult to verify alleged or attempted biological attacks. These allegations might have been part of a propaganda campaign, or they may have been due to rumor. Regardless, some of the examples we have been given cannot be supported by microbiological or epidemiologic data. In addition, the incidence of naturally occurring endemic or epidemic diseases during that time complicates the picture so that attribution is impossible ( Christopher et al., 1997 ). More important, our awareness that infectious diseases are caused by microbes does not go back very far in human history. Germ theory, or the fact that infectious diseases are related to and caused by microorganisms, emerged after 1860 through the independent works of Pasteur, Lister, and Koch ( Tortora et al., 1995 ). Therefore how could the attacking or defending commander know that the festering corpses might cause disease when people at that time thought that epidemics were related to "miasmas," the smell of decomposition, or heavenly "influences"? One need only consider the origin of certain disease names to appreciate this confusion. For instance, malaria gets its name from malaria , or "bad air" (ie, swamp gases; Desowitz, 1991 ). It was not until 1880 that we learned that the etiologic agents of malaria are protozoans in the genus Plasmodium. The name influenza refers to the ancient belief that the disease was caused by a misalignment of the stars because of some unknown supernatural or cosmic influence (Latin influentia ) . It was not until 1933 that we learned the flu was caused by the influenza virus ( Potter, 2001 ). Regardless of the lack of awareness of germs at the time, a few of the historic reports about the use of biological weapons in battle are worth noting here: • In the 6th century BC, Assyrians poisoned enemy wells with rye ergot, a fungus. • In the 4th century BC, Scythian archers tipped their arrows with blood, manure, and tissues from decomposing bodies. • In AD 1340, attackers hurled dead horses and other animals by catapult at the castle of Thun L'Eveque in Hainault (northern France). Castle defenders reported that "the stink and the air were so abominable…they could not long endure" and negotiated a truce. • In AD 1422 at Karlstein in Bohemia, attacking forces launched the decaying cadavers of men killed in battle over the castle walls. They also stockpiled animal manure in the hope of spreading illness. However, the defense held fast, and the siege was abandoned after 5 months. Russian troops may have used the same tactic using the corpses of plague victims against the Swedes in 1710. • In AD 1495 the Spanish contaminated French wine with the blood of lepers. • In the mid-1600s a Polish military general reportedly put saliva from rabid dogs into hollow artillery spheres for use against his enemies. • Francisco Pizarro reportedly gave smallpox virus–contaminated clothing to South American natives in the 15th century. • In a letter dated July 16, 1763, General Jeffrey Amherst, a British officer, approved the plan to spread smallpox to Delaware Indians ( Robertson, 2001 ). Amherst suggested the deliberate use of smallpox to "reduce" Native American tribes hostile to the British ( Parkman, 1901 ). An outbreak of smallpox at Fort Pitt resulted in the generation of smallpox-contaminated materials and an opportunity to carry out Amherst's plan. On June 24, 1763, one of Amherst's subordinates gave blankets and a handkerchief from the smallpox hospital to the Native Americans and recorded in his journal, "I hope it will have the desired effect" ( Sipe, 1929 ). • The same tactic was used during the Civil War by Dr. Luke Blackburn, the future governor of Kentucky. Dr. Blackburn infected clothing with smallpox and yellow fever virus, which he then sold to Union troops. One Union officer's obituary stated that he died of smallpox contracted from his infected clothing ( Guillemin, 2006 ). As previously mentioned, scientists discovered microorganisms and made advances toward understanding that a specific agent causes a specific disease, that some are foodborne or waterborne, that an agent can cycle through more than one species, and that insects and ticks are the vectors of disease. Furthermore, medical professionals established that wars, famines, and poverty opened populations to the risk of epidemics. Once these links were established, we learned that we could apply control and intervention methods. Scientific knowledge about disease transmission coupled with social stability and active public health campaigns aided human survival. It subsequently became possible for advanced populations to protect their citizens from the burden of some of the most insidious infectious diseases, such as plague, cholera, diphtheria, smallpox, influenza, and malaria. These epidemics swept across nations in previous centuries, hitting hardest in crowded urban centers and affecting mostly the poor ( Guillemin, 2006 ). At the opening of the Industrial Revolution, public health in cities had improved, water and food sources were monitored by the state, and vaccines and drug therapies were being invented as further protection. With many childhood diseases conquered, more people were living longer, and they were now dying of more "civilized" diseases such as cancer, heart disease, and stroke ( Diamond, 1999 ). In underdeveloped nations, public health did not develop; hence, epidemics were prevalent and continued to be devastating. The dichotomy between developed and developing nations remains marked by generally good health versus widespread, preventable epidemics ( Guillemin, 2006 ). As Western nations were taking advantage of innovations in public health and medicine to mitigate epidemics, their governments invented biological weapons as a means of achieving advantage in warfare ( Diamond, 1999 ). The German military has the dubious honor of being the first example of using biological weapons following a state-sponsored program. However, during World War I, they used disease-causing organisms against animals, not people. The goal of their program was to interrupt the flow of supplies to the Allied frontlines. To do this they targeted the packhorses and mules shipped from Norway, Spain, Romania, and the United States. In 1915, Dr. Anton Dilger, a German-American physician, developed a microbiology facility in Washington, DC. Dilger produced large quantities of anthrax and glanders bacteria using seed cultures provided by the imperial German government. At the loading docks, German agents inoculated more than 3000 animals that were destined for the Allied Forces in Europe ( Wheelis, 1999 ). From the German perspective, these attacks violated no international law. In addition, these activities were dwarfed by the atrocities of chemical warfare that was being waged on both sides of the line. To counter the German threat and explore the potential of air warfare the French sought to improve their integration of aerosols and bombs. At the same time as the French were signing the 1925 Geneva Protocol, they were developing a biological warfare program to complement the one they had established for chemical weapons during World War I ( Rosebury and Kabat, 1947 ). After World War I the Japanese formed a "special weapons" section within their army. The section was designated Unit 731. The unit's leaders set out to exploit chemical and biological agents. In 1936 they expanded their territory into Manchuria, which made available "an endless supply of human experiment materials" (prisoners of war) for Unit 731. Biological weapon experiments in Harbin, Manchuria, directed by Japanese General Shiro Ishii, continued until 1945. A post-World War II autopsy investigation of 1000 victims revealed that most were exposed to aerosolized anthrax. More than 3000 prisoners and Chinese nationals may have died in Unit 731 facilities. In 1939 the Japanese military poisoned Soviet water sources with intestinal typhoid bacteria at the former Mongolian border. During an infamous biowarfare attack in 1941, the Japanese military released millions of plague-infected fleas from airplanes over villages in China and Manchuria, resulting in several plague outbreaks in those villages. The Japanese program had stockpiled 400 kg of anthrax to be used in specially designed fragmentation bombs. In 1942, shortly before the battle of Stalingrad, on the German–Soviet front, a large outbreak of tularemia occurred. Several thousand Soviets and Germans contracted the illness. Some estimate that more than 70% of the victims had inhalation tularemia, which is rare and considered to be evidence of an intentional release. It was determined later that the Soviets had developed a tularemia weapon the prior year ( Alibek and Handelman, 2000 ). During World War II the Allies had great fear of German and Japanese biological weapons programs. Their fears were sparked by sketchy reports that the Japanese had an ongoing effort, and British intelligence suggested that Germany might soon target Britain with a bomb packed with biological agents. On the basis of these fears, Great Britain began its own bioweapons program and urged officials in the United States to create a large-scale biological warfare program. On December 9, 1942, the US government convened a secret meeting at the National Academy of Sciences in Washington, DC. The meeting was called to respond to Great Britain's request. Army officers had urgent questions for an elite group of scientists. Only a few months before, the President of the United States had grappled with the issue of biological weapons. President Franklin D. Roosevelt stated that "I have been loath to believe that any nation, even our present enemies, would be willing to loose upon mankind such terrible and inhumane weapons." Secretary of War, General Henry Stimson, thought differently: "Biological warfare is…dirty business," he wrote to Roosevelt, "but…I think we must be prepared." President Roosevelt approved the launch of the United States' biological warfare program. For the first time US researchers would be trying to make weapons from the deadliest germs known to science. In spring 1943 the United States initiated its bioweapons program at Camp Detrick (now Fort Detrick), Maryland. The program focused primarily on the use of the agents that cause anthrax, botulism, plague, tularemia, Q fever, Venezuelan equine encephalitis, and brucellosis. Production of these agents occurred at Camp Detrick, Maryland, and other sites in Arkansas, Colorado, and Indiana. The British had made two primary requests of us: (1) to mass produce anthrax spores so that they could be placed in bomblets and stored for later deployment against the Germans in retaliation for any future strike and (2) the British supplied us with the recipe to make botulinum toxin and wanted to see if we could mass produce it. Naturally the entire program was wrapped in a cloak of secrecy. Fig. 1.2 is a collage of some important facilities built at Camp Detrick to produce and test bioweapons formulations. Figure 1.2 (A) The "Black Maria" was the first laboratory facility built at Camp Detrick to conduct top-secret bioweapons research. The purpose of this tarpaper building was to produce Agent X (botulinum toxin) for the British. (B) A Camp Detrick researcher works with an aerobiology chamber to conduct a study with microbial aerosols, a biological weapons formulation. (C) This is the old Pilot Plant (Building 470) at Fort Detrick. Here, experimental formulations of anthrax spores were made. The building had a reputation for mystery. Despite three decontamination procedures, it was never certified 100% clean. (D) Pictured here is a 1,000,000-L metal sphere that workers called the Eight Ball. The largest aerobiology chamber ever constructed, it was used to test experimental bioweapons formulations at Fort Detrick. The last experiment in the Eight Ball was in 1969. Courtesy of the US Army, Fort Detrick. The British program focused on the use of B. anthracis (anthrax) spores and their viability and dissemination when delivered with a conventional bomb. Gruinard Island, off of the coast of Scotland, was used as the testing site for formulations. At the time British scientists believed that the testing site was far enough from the coast to not cause any contamination of the mainland. However, in 1943 there was an outbreak of anthrax in sheep and cattle on the coast of Scotland that faced Gruinard. As a result, the British decided to stop the anthrax testing and close down the island site. Despite the cessation of experiments, the island remained contaminated for decades until a deliberate and extensive decontamination program rendered the island inhabitable again. The US bioweapons program continued to grow in scope and sophistication. Much of this was prompted by fear of a new enemy: the threat of communism, the Soviet Union, and its allies. Experiments to test bioweapons formulations were routinely performed on a small scale with research animals. However, more comprehensive field and laboratory studies were performed with human research volunteers exposed to actual live agents and some situational scenarios using surrogate nonpathogenic bacteria to simulate the release of actual pathogens inside of buildings or aimed at cities. In 1949 researchers from Detrick visited the Pentagon on a secret mission. Disguised as maintenance workers, they released noninfectious bacteria into the duct work of the building to assess the vulnerability of people inside large buildings to a bioweapons attack. The Pentagon trial was considered to be a success because it revealed that germs could be formulated and released effectively for a small-scale act of sabotage. However, there was considerable doubt that biological weapons could be effective against a target the size of a city. Accordingly, several tests were conducted on American cities ( Miller et al., 2001 ). In 1977 the US Army admitted that there were 239 intentional releases of noninfectious bacteria in bioweapons experiments ( Cole, 1988 ). One such trial took place in San Francisco in September 1950, when a US Navy ship sailed a course adjacent to the Golden Gate Bridge to release a plume of seemingly nonpathogenic bacteria ( Serratia marcescens ) . This trial was intended to simulate the dispersion of anthrax spores on a large city. On the basis of results from monitoring equipment at 43 locations around the city, the Army determined that San Francisco had received enough of a dose for nearly all of the city's 800,000 residents to inhale at least 5000 of the particles. Although the researchers believed that what they were releasing was harmless, one report shows that 11 people reported to area hospitals with severe infections because of the release of this agent, 1 of which was fatal ( Cole, 1988 ). Three years later, bioweapons experts took their secret exercises to St. Louis and Minneapolis, two cities that resembled potential Soviet targets, where sprayers hidden in cars dispersed invisible clouds of harmless Bacillus spores. In 1966 nonpathogenic Bacillus globigii spores were released into the New York subway system using a broken light bulb to demonstrate the ability of a specific formulation to make its way from a central point source to both ends of the system in less than an hour. Revelations of these experiments became known in 1977 when a Senate Subcommittee panel heard testimony from Pentagon officials (US Department of the Army, DTIC B193427 L, 1977 ). Until that point, neither US citizens nor their representatives in Washington knew anything about the American germ program. After nearly 3 decades of secret research aimed at producing the ultimate biological weapons and stockpiling them for use against our enemies, President Richard Nixon surprised the world by signing an executive order that stopped all offensive biological agent and toxin weapon research and ordered all stockpiles of biological agents and munitions from the US program be destroyed. Accordingly, on November 25, 1969, he uttered these historic words in a speech to the nation on …Biological warfare—which is commonly called "germ warfare." This has massive unpredictable and potentially uncontrollable consequences. It may produce global epidemics and profoundly affect the health of future generations. Therefore, I have decided that the United States of America will renounce the use of any form of deadly biological weapons that either kill or incapacitate. Mankind already carries in its own hands too many of the seeds of its own destruction. Subsequently, in 1972 the United States and many other countries were signatories to the Convention on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction, commonly called the Biological Weapons Convention. This treaty prohibits the stockpiling of biological agents for offensive military purposes and forbids research into offensive use of biological agents. Although the former Soviet Union was a signatory to the Biological Weapons Convention, its development of biological weapons intensified dramatically after the accord and continued well into the 1990s. In late April 1979, an outbreak of pulmonary anthrax occurred in Sverdlovsk (now Yekaterinburg) in the former Soviet Union. Soviet officials explained that the outbreak was due to ingestion of infected meat. However, it was later discovered that the cause was from an accidental release of anthrax in aerosol form from the Soviet Military Compound 19, a Soviet bioweapons facility. (This event is examined thoroughly in chapter Case Studies as a case study to demonstrate the potential of weaponized anthrax.) The robust bioweapons program of the Soviet Union employed more than 60,000 people. Building 15 at Koltsovo was capable of manufacturing tons of smallpox virus each year. In Kirov, the Soviets maintained an arsenal of 20 tons of weaponized plague bacterium. By 1987 Soviet anthrax production capacity reached nearly 5000 tons a year. In the later part of the 1990s the Russians disassembled their awesome bioweapons production capacity and reportedly destroyed their stocks. As the Soviet Union dissolved, it appeared that the threat of biowarfare would diminish. However, the Age of Bioterrorism emerged with the anthrax attacks of 2001. In addition, the US Department of State published a report in 2004 that affirmed that six countries had active bioweapons programs. Table 1.2 summarizes some of these events. Table 1.2 Seminal moments in the history of biowarfare and bioterrorism. Some data in this table taken from Eitzen and Takafuji, 1997 Date Event Significance 6th century, BC Assyrians poisoned enemy wells with rye ergot. First known use of a biological toxin. 1763 British soldiers give blankets infected with the smallpox virus to American Indians. Notable and documented use of virus against combatants. 1915 Anton Dilger produces anthrax and glanders bacterium to infect horses intended for the warfront. Notable and documented use of bacteria against animals. June 17, 1925 Delegates in Switzerland create a Geneva Protocol banning the use of chemical and bacteriological methods of warfare. First international effort to limit use of biologicals in warfare. 1932 The Japanese army gives General Ishii control of three biological research centers, including one in Manchuria. Most despicable character in bioweapons history gets his start. 1934 Great Britain begins taking steps toward establishing its own biological weapons research project. Allies start to develop a program. July 15, 1942 Anthrax tested on Gruinard Island against sheep. Allies' first field test of bioweapon. November 1942 British implore the United States to lead bioweapons production efforts; negotiations commence and President Roosevelt approves the program. Beginning of US bioweapons program. Spring 1943 US bioweapons program begins its activities at Camp Detrick, Maryland. Implementation of plans to begin US bioweapons program. May 1949 The US Army Chemical Corps sets up a Special Operations Division at Camp Detrick to perform field tests with bioweapons formulations. Tests conducted at the Pentagon show that biological weapons formulations are feasible for sabotage. 1950 Navy warships spray the cities of Norfolk, Hampton, Newport News, and San Francisco. Tests show that large-scale deployment of a bioweapon from the sea is feasible. 1953 Conduct of the St. Jo Program stages mock anthrax attacks on St. Louis, Minneapolis, and Winnipeg using aerosol generators placed on top of cars. Tests show that large-scale deployment of a bioweapon from the land is feasible. 1955 Operation Whitecoat uses human research volunteers to study the effects of biological agents on human volunteers. The operation will continue for the next 18 years and involve some 2200 people. 1957 Operation Large Area Concept kicks off to test the release of aerosols from airplanes; the first experiment involves a swath from South Dakota to Minnesota and further tests cover areas from Ohio to Texas and Michigan to Kansas. Tests show that large-scale deployment of a bioweapon from the air is feasible; some of the test particles travel 1200 miles. November 25, 1969 Nixon announces that the United States will renounce the use of any form of deadly biological weapons that either kill or incapacitate. The end of an era in US offensive biological weapons research, production, and storage. April 10, 1972 The Biological Weapons Convention, which bans all bioweapons, is completed and opened for signature. Seventy-nine nations signed the treaty, including the Soviet Union. March 26, 1975 The Biological Weapons Convention officially goes into force; the US Senate also finally ratifies the 1925 Geneva Protocol. Political will to ban biological weapons on the international front. April 1979 Nearly 70 people die from an accidental release of anthrax spores in the Soviet city of Sverdlovsk. The United States suspects that anthrax bacterial spores were accidentally released from a Soviet military biological facility. 1984 The Rajneeshees contaminate food with Salmonella bacterium in a small town in Oregon to influence local elections. The first significant act of bioterrorism in the United States. 1989 A Soviet defector from Biopreparat, Vladimir Pasechnik, reveals the existence of a continuing offensive biological weapons program in the Soviet Union. Evidence that the Soviet Union is violating the Biological Weapons Convention. April 1992 Russian president Boris Yeltsin admits the 1979 outbreak was caused by the Soviet military but gives few details. An admonition that the Soviet Union operated an offensive biological warfare program in violation of the Biological Weapons Convention. Fall 2001 Envelopes filled with anthrax spores are sent to various media and political figures in the United States; 22 people, from Florida to Connecticut, are infected; 5 die. A national movement begins to prepare a citizenry against the threat of bioterrorism, which has now become a household word. 2003–present Letters containing ricin have been mailed to public officials from various people and places. Many perpetrators have been caught and convicted. Others remain at large. These small-scale incidents keep us mindful that some biological agents are easy to acquire and utilize in crimes and small-scale acts of terrorism. Modern-Day Bioterrorism Biodefense programs and initiatives come out of a sense of vulnerability to biowarfare potentials. Bioterrorism is deeply founded in what has been gained from active biowarfare programs ( Miller et al., 2001 ). In the early 1970s the leftist terrorist group, the Weather Underground, reportedly attempted to blackmail an Army officer at Fort Detrick working in the Research Institute of Infectious Diseases (USAMRIID). The group's goal was to get him to supply organisms that would be used to contaminate municipal water supplies in the United States. The plot was discovered when the officer attempted to acquire several items that were "unrelated to his work." Several other attempts are worth mentioning here: • In 1972 members of the right-wing group Order of the Rising Sun were found in possession of 30–40 kg of typhoid bacteria cultures that were allegedly to be used to contaminate the water supplies of several Midwestern cities. • In 1975 the Symbionese Liberation Army was found in possession of technical manuals on how to produce bioweapons. • In 1980 a Red Army Faction safe house reportedly discovered in Paris included a laboratory containing quantities of botulinum toxin. • In 1983 the Federal Bureau of Investigations (FBI) arrested two brothers in the northeastern United States for possession of an ounce of nearly pure ricin. • In 1984 followers of the Bhagwan Shree Rajneesh contaminated salad bars with Salmonella bacteria in a small town in Oregon. It was the largest scale act of bioterrorism in US history. More than 750 cases of salmonellosis resulted from the salad bar contamination. It was later discovered that the Rajneeshees wanted to influence the local county elections. Cult members obtained the Salmonella strain through the mail from American Type Culture Collection and propagated the liquid cultures in their compound's medical clinic. • In 1989 a home laboratory producing botulinum toxin was discovered in Paris. This laboratory was linked to a cell of the German-based Bäder Meinhof Gang. • In Minnesota, four members of the Patriots Council, an antigovernment extremist group, were arrested in 1991 for plotting to kill a US marshal with ricin. The group planned to mix the homemade ricin with a chemical that speeds absorption (dimethylsulfoxide) and then smear it on the door handles of the marshal's car. The plan was discovered and all four men were arrested and the first to be prosecuted under the US Biological Weapons Anti-Terrorism Act of 1989. • In 1995 Aum Shinrikyo, a Japanese doomsday cult, became infamous for an act of chemical terrorism when members released sarin gas into the Tokyo subway. What many people do not know about the group is that it developed and attempted to use biological agents (anthrax, Q fever, Ebola virus, and botulinum toxin) on at least 10 other occasions. Despite several releases, it was unsuccessful in its use of biologicals. This program is examined more thoroughly in chapter Case Studies. • Several small-scale incidents involving the biological poison ricin (refer to Fig. 1.3 ) have occurred since the Amerithrax incident. Here are the more notable ones: • In 2003 several letters containing ricin were recovered from a mail-sorting center in Greenville, South Carolina. A note from someone calling themselves the "Fallen Angel" accompanied those letters. • In 2004 ricin was sent to the office of Senator Bill Frist. Some federal investigators believe that this instance may be tied to the Fallen Angel, but no one has been identified for this biocrime or the 2003 incident. • In 2013 ricin was sent to US President Barack Obama and New York City Mayor Michael Bloomberg. A woman from Shreveport, Louisiana, was arrested for this biocrime and later convicted on several charges. • Also in 2013 a letter containing ricin was sent to President Barack Obama, Mississippi Senator Roger Wicker, and Mississippi judge Sadie Holland. A Tupelo, Mississippi man was convicted of crimes related to these incidents and sentenced to 25 years in prison. • In 2014 a Philadelphia man sent a romantic rival a scratch-and-sniff birthday card laced with ricin. In 2015 he was convicted on several charges related to the incident and subsequently received a sentence of 20–40 years in prison. Figure 1.3 A letter addressed to the White House sent in October 2003. The letter contained ricin and a note from the Fallen Angel. Courtesy of the FBI. The Public Health Security and Bioterrorism Response Act of 2002 On June 12, 2002, President George W. Bush uttered these remarks from the White House at the signing of HR 3448, the Public Health Security and Bioterrorism Response Act of 2002: Bioterrorism is a real threat to our country. It's a threat to every nation that loves freedom. Terrorist groups seek biological weapons; we know some rogue states already have them…It's important that we confront these real threats to our country and prepare for future emergencies. It is clear that September 11 and the anthrax attacks of 2001 sent the country to war and sparked several initiatives against all forms of terrorism. Weaponization Biological agents have some unique characteristics that make weaponizing them attractive to the would-be terrorist. Most biological weapons are made up of living microorganisms, which means that they can replicate once disseminated. This possibility amplifies the problem and the effect of the weapon in several ways. First, some agents are capable of surviving in various different hosts. The target might be humans, but the disease may manifest in other animal hosts, such as companion animals (pets). In doing so, the problem may be more difficult to control. Second, when people become infected with a disease-causing organism, there is an incubation period before signs of illness are apparent. During this incubation period and the periods of illness and recovery, the pathogen may be shed from the victim, causing the contagion to spread (a possibility only with diseases that are transmitted from person to person). There is no rule of thumb for how many people might be infected from a single patient. However, the nature of contagion clearly compounds the problem well beyond the initial release of the agent. In this instance the initial victims from the intentional outbreak become more weapons for the perpetrator, spreading the problem with every step they take. As Grigg et al. (2006) stated so precisely in their paper, "when the threat comes from the infected population, self-defense becomes self-mutilation." The would-be terrorist could surely derive great pleasure from watching government officials and responders tread on the civil liberties of such victims as they attempt to limit the problem from spreading among the population. Making an effective biological weapon is no easy undertaking. The process and complexity depends largely on the pathogen selected to be "weaponized." If the pathogen is a spore-forming bacteria, such as B. anthracis (the causative agent of anthrax), there are five essential steps: germination, vegetation, sporulation, separation, and weaponization. The first three steps are designed to get small quantities of seed stock to propagate into a starter culture, grow them to a significant stage of growth in the proper volume, and turn those active cells into spores. The goal of the last two steps is to separate the spores from the dead vegetative cells and spent media. All five steps have dozens of secondary steps. In addition, each of the five steps requires a fairly sophisticated and well-equipped laboratory if the goal is to develop a sizable quantity of refined materials. Weaponization is a term that applies to the processes necessary to purify, properly size, stabilize, and make biological agents ideally suited for dissemination. Stabilization and dissemination are important issues because of the susceptibility of the biological agents to environmental degradation, not only in storage but also in application. These issues are problems whether the end use is for biological weapons, pharmaceuticals, cosmetics, pesticides, or food-related purposes. The susceptibility of the organisms to inactivation by the environment varies with the agent. As an example, anthrax spores released into the environment may remain viable for decades, whereas plague bacterium may survive for only a few hours. Loss of viability or bioactivity is likely to result from exposure to physical and chemical stressors, such as exposure to ultraviolet radiation (sunlight), high surface area at air–water interfaces (frothing), extreme temperature or pressure, high salt concentration, dilution, or exposure to specific inactivating agents. This requirement of stabilization also extends to the methods of delivery because the organisms are very susceptible to degradation in the environments associated with delivery systems. The primary means of stabilization for storage or packaging are concentration; freeze drying (lyophilization); spray drying; formulation into a stabilizing solid, liquid, or gaseous solution; and deep freezing. Methods of concentration include vacuum filtration, ultrafiltration, precipitation, and centrifugation. Freeze drying is the preferred method for long-term storage of bacterial cultures because freeze-dried cultures can be easily dehydrated and cultured via conventional means. Freeze-dried cultures may remain viable for more than 30 years. Deep freezing of biological products is another long-term storage technique for species and materials not amenable to freeze drying. The method involves storage of the contained products in liquid nitrogen freezers (−196°C/−325°F) or ultralow-temperature mechanical freezers (−70°C/−94°F). Culturing viruses is a more costly and tenuous process because host cells are required for viral propagation. This means that cultures of host cells must be kept alive, often in an oxygen-deficient and temperature-stable atmosphere. In some cases, viruses may be more fragile when deployed as weapons, some becoming inactive on drying. Biological toxins can be difficult to produce and purify, each requiring its own special set of circumstances. Two specific examples are covered in subsequent chapters when those agents are discussed in detail. However, past bioweapons programs have determined that these agents are most effective when prepared as a freeze-dried powder and encapsulated. A Question of Scale Biological attacks by a terrorist group are apparently not easy to conduct or a practical option. If they were easy or practical, then many terrorist groups and hostile states would have done so long ago and frequently. Our experience today with acts of biological terrorism has to do mainly with small-scale, limited attacks. However, if one were to acquire the means to produce the weapons, as described here, or purchase viable, sophisticated materials on the black market, a small group of persons could bring about the infection of a large percentage of targeted persons. Clinical illness could develop within a day of dispersal and last for as long as 2–3 weeks. In a civil situation, major subway systems in a densely populated urban area could be targeted for a biological agent strike, resulting in massive political and social disorganization. It would take little weaponized material to bring about the desired effect. Looking at this potential comparatively on a weight-to-weight basis, approximately 10 g of B. anthracis (anthrax) spores could kill as many people as a ton of the nerve agent sarin. With bioweapons in hand, small countries or terrorist groups might develop the capability to deliver small quantities of agents to a specific target. Under appropriate weather conditions and with an aerosol generator delivering 1- to 10-μm particle-sized droplets, a single aircraft could disperse 100 kg (220 lb) of anthrax over a 300-km 2 area (74,000 acres) and theoretically cause 3 million deaths in a population density of 10,000 people/km 2 (US DOD, ADA 330102, 1998 ). Much has been made of the potential of aerosolized powders and respiratory droplets in factual and fictitious biothreat scenarios. The largest infectious disease outbreak in the history of the United States occurred in April 1993. The event was caused by an accidental waterborne contamination. The outbreak of cryptosporidiosis, which occurred in the greater Milwaukee area, was estimated to have caused more than 430,000 people to become ill with gastroenteritis among a population of 1.6 million ( MacKenzie et al., 1994 ). Approximately 4400 people were hospitalized and about 100 people died as a result of the outbreak. The Milwaukee outbreak was attributed to failure of filtration processes at one of the two water treatment plants that served the city. Several deficiencies were found at the plant, including problems relating to a change in the type of water treatment chemicals used for coagulation of contaminants before the filtration step. Weather conditions at the time were unusual, with a heavy spring snowmelt leading to high source water turbidity and wind patterns that may have changed normal flow patterns in Lake Michigan, the raw water source for the city. Critical Thinking Describe the fundamental difference between biodefense and biosecurity. The Genesis of Biosecurity and Biodefense The secrecy of bioweapons programs of the previous century has been uncloaked. Some of the most insidious disease agents ever to afflict humans, animals, and plants have been mass produced and perfected for maximum effectiveness. Terrorist groups and rogue states may be seeking to develop bioweapons capabilities. These significant developments in bioweapons gave military leaders and politicians cause for great concern over the past few decades. The military necessity to protect the force and defend the homeland is the goal of a good biodefense program. Simply put, biodefense is the need for improved national defenses against biological attacks. These are national programs, mostly planned and carried out by military forces and other government agencies. Initially, biodefense programs require an intelligence-gathering capability that strives to determine what may be in the biological weapons arsenal of an aggressor. Intelligence is needed to guide biodefense research and development efforts aimed at producing and testing effective countermeasures (ie, vaccines, therapeutic drugs, and detection methods). In addition, a real-time reporting system should be developed so that officials can be informed about an emerging threat before an agent has a chance to affect armed forces and millions of people in the homeland. The development of integrated systems for detecting and monitoring biological agents is instrumental to this goal. Although most biodefense initiatives rest with the military, civilian government agencies contribute greatly to the biodefense posture. This is evident by the increases in CBF over the past few years and will be discussed in great detail in Part IV of this book. On the other hand, biosecurity refers to the policies and measures taken for protecting a nation's food supply and agricultural resources from accidental contamination and deliberate attacks of bioterrorism. Biological Threats Today and in the Future As I sit here today writing the second edition of this book, I am reflecting on the most recent concerns that we have for biological threats in modern society. For what it is worth, we seem to be much less concerned about acts of bioterrorism and/or biowarfare than we were 10–50 years ago. Instead I see a great deal of concern, and rightfully so, for emerging infectious diseases and reemerging biological threats. We are also keenly aware of the accidental release of biological agents from research and reference laboratories. To illustrate these points we will briefly discuss four items of international interest that have been emphasized in the media: accidental shipment of live anthrax-positive controls samples, the 2014/2015 Ebola outbreak in West Africa, cases of Middle East respiratory syndrome coronavirus (MERS-CoV) in South Korea and Saudi Arabia, and a massive outbreak of highly pathogenic avian influenza (HPAI). Laboratory Mishaps As previously mentioned, concerns for biological threat led to a wellspring of funding (nearly $80 billion in 15 years) for civilian biodefense programs in the United States. With all of this money the United States was able to build tremendous capabilities to detect and diagnose the agents and the diseases, respectively. With this money a few medical countermeasures (vaccines and treatments) were developed and produced. Centers of Excellence were funded and highly secure containment (biosafety level 4) laboratories were built. With these new programs, testing modalities, and laboratories came the need to provide a ready supply of positive control agents and contracting opportunities for private biotechnology firms. As one very recent example, the US Army laboratory in Dugway Proving Grounds, Utah, provided positive control samples of anthrax ( B. anthracis ) spores to public and private laboratories. Before shipment, the spores had been propagated in the Army laboratory and were exposed to gamma radiation to ensure no living spores were in the vials being provided. Upon receipt of the samples, one laboratory in Maryland questioned the integrity of the contents of the vial they received because there was no "death certificate" accompanying the samples. Out of an abundance of caution they removed a small portion of the vial and streaked it onto sheep blood agar plates. To their amazement, several days later the plates showed growth and tested positive for anthrax. They immediately notified the CDC and the Army. The CDC initiated an investigation and notified the media of the incident. The investigation showed that the living anthrax samples had been shipped to 69 laboratories in 19 US states and 5 other countries ( USA Today, 2015 ). Once again the seeds of our destruction are sprouting, and some are of the opinion that we are our own worst enemy. More than 1100 laboratory incidents involving potential bioterror germs were reported to federal regulators during 2008 through 2012. USA Today (2014) Ebola Ebola virus was first discovered in 1976 in the Sudan and Zaire. Ebola virus exists naturally in fruit bats, with sylvatic transmission to other mammals and sometimes humans when they consume raw or undercooked meat from an infected animal. Infection with Ebola virus in humans leads to severe viral hemorrhagic fever (VHF), which is often fatal ( CDC, 2015 ). In March 2014 an outbreak of Ebola virus disease (EVD) began in Guinea, a Western African nation. Public health agencies at all levels failed to react quickly to the outbreak and it quickly spread to urban areas in Liberia and Sierra Leone. Subsequently, EVD spread to Nigeria and Senegal. International air travel brought EVD to the United States and Europe, although the number of cases was very small and the threat was stamped out with ample infection control procedures in health-care facilities and aggressive public health measures for those exposed to actual case patients ( CDC, 2015 ). This is the largest outbreak of EVD in history. At the time of this writing, the outbreak has been quelled by a "better late than never" effort. Volunteers and medical relief groups from the United States and other countries received special training and deployed to West Africa to help identify cases and treat the victims (see Fig. 1.4 ). However, new cases continue to be reported from Guinea and Sierra Leone. As of June 26, 2015, there have been 16,801 EVD cases (suspect, probable, and confirmed) worldwide with approximately 6411 deaths; this equates to a 38% mortality rate ( WHO, 2015a ). Figure 1.4 This image shows two students in the CDC's Ebola Treatment Unit training course for health-care workers (2014). The program had been designed to educate participants who would be deployed as members of the West African Ebola Response team as to the proper protocols to be followed when treating EVD patients. The two participants were displaying the personal protective equipment worn by treatment specialists who would be interacting with EVD patients. Courtesy of the CDC/Nahid Bhadelia, MD. To most the threat of Ebola virus remains distant and out of mind. However, the stark reality is that international travel can interject EVD into any populace on any continent within a matter of days. No country, person, or organization is immune to this threat. What makes EVD such a great concern? First, Ebola virus is a US Health and Human Services Category A agent. It meets all of the criteria for such a designation. EVD results in high morbidity and mortality. EVD requires special preparedness measures for public health and health care. EVD is spread from person to person. EVD can lead to panic and social disruption ( CDC, 2015 ). With this outbreak in particular, we are seeing all four criteria fulfilled. To make things worse, there is no Food and Drug Administration (FDA)-approved vaccine for humans and no FDA-approved drug for treating VHF case patients. In a health-care setting, EVD patients receive supportive care (hydration therapy) and rarely experimental drugs ( CDC, 2015 ). Perhaps the only good thing to come from this outbreak is the development of a vaccine for Ebola virus. There are currently three vaccine candidates undergoing Phase III clinical trials in West Africa ( WHO, 2015b ). A case study on this outbreak is offered in chapter Case Studies of this book. Critical Thinking How have international and national attitudes toward the biological threat changed since the early post–9/11 era? Include some discussion about the reality of versus the potential for biological threats. Laboratory Mishaps As previously mentioned, concerns for biological threat led to a wellspring of funding (nearly $80 billion in 15 years) for civilian biodefense programs in the United States. With all of this money the United States was able to build tremendous capabilities to detect and diagnose the agents and the diseases, respectively. With this money a few medical countermeasures (vaccines and treatments) were developed and produced. Centers of Excellence were funded and highly secure containment (biosafety level 4) laboratories were built. With these new programs, testing modalities, and laboratories came the need to provide a ready supply of positive control agents and contracting opportunities for private biotechnology firms. As one very recent example, the US Army laboratory in Dugway Proving Grounds, Utah, provided positive control samples of anthrax ( B. anthracis ) spores to public and private laboratories. Before shipment, the spores had been propagated in the Army laboratory and were exposed to gamma radiation to ensure no living spores were in the vials being provided. Upon receipt of the samples, one laboratory in Maryland questioned the integrity of the contents of the vial they received because there was no "death certificate" accompanying the samples. Out of an abundance of caution they removed a small portion of the vial and streaked it onto sheep blood agar plates. To their amazement, several days later the plates showed growth and tested positive for anthrax. They immediately notified the CDC and the Army. The CDC initiated an investigation and notified the media of the incident. The investigation showed that the living anthrax samples had been shipped to 69 laboratories in 19 US states and 5 other countries ( USA Today, 2015 ). Once again the seeds of our destruction are sprouting, and some are of the opinion that we are our own worst enemy. More than 1100 laboratory incidents involving potential bioterror germs were reported to federal regulators during 2008 through 2012. USA Today (2014) Ebola Ebola virus was first discovered in 1976 in the Sudan and Zaire. Ebola virus exists naturally in fruit bats, with sylvatic transmission to other mammals and sometimes humans when they consume raw or undercooked meat from an infected animal. Infection with Ebola virus in humans leads to severe viral hemorrhagic fever (VHF), which is often fatal ( CDC, 2015 ). In March 2014 an outbreak of Ebola virus disease (EVD) began in Guinea, a Western African nation. Public health agencies at all levels failed to react quickly to the outbreak and it quickly spread to urban areas in Liberia and Sierra Leone. Subsequently, EVD spread to Nigeria and Senegal. International air travel brought EVD to the United States and Europe, although the number of cases was very small and the threat was stamped out with ample infection control procedures in health-care facilities and aggressive public health measures for those exposed to actual case patients ( CDC, 2015 ). This is the largest outbreak of EVD in history. At the time of this writing, the outbreak has been quelled by a "better late than never" effort. Volunteers and medical relief groups from the United States and other countries received special training and deployed to West Africa to help identify cases and treat the victims (see Fig. 1.4 ). However, new cases continue to be reported from Guinea and Sierra Leone. As of June 26, 2015, there have been 16,801 EVD cases (suspect, probable, and confirmed) worldwide with approximately 6411 deaths; this equates to a 38% mortality rate ( WHO, 2015a ). Figure 1.4 This image shows two students in the CDC's Ebola Treatment Unit training course for health-care workers (2014). The program had been designed to educate participants who would be deployed as members of the West African Ebola Response team as to the proper protocols to be followed when treating EVD patients. The two participants were displaying the personal protective equipment worn by treatment specialists who would be interacting with EVD patients. Courtesy of the CDC/Nahid Bhadelia, MD. To most the threat of Ebola virus remains distant and out of mind. However, the stark reality is that international travel can interject EVD into any populace on any continent within a matter of days. No country, person, or organization is immune to this threat. What makes EVD such a great concern? First, Ebola virus is a US Health and Human Services Category A agent. It meets all of the criteria for such a designation. EVD results in high morbidity and mortality. EVD requires special preparedness measures for public health and health care. EVD is spread from person to person. EVD can lead to panic and social disruption ( CDC, 2015 ). With this outbreak in particular, we are seeing all four criteria fulfilled. To make things worse, there is no Food and Drug Administration (FDA)-approved vaccine for humans and no FDA-approved drug for treating VHF case patients. In a health-care setting, EVD patients receive supportive care (hydration therapy) and rarely experimental drugs ( CDC, 2015 ). Perhaps the only good thing to come from this outbreak is the development of a vaccine for Ebola virus. There are currently three vaccine candidates undergoing Phase III clinical trials in West Africa ( WHO, 2015b ). A case study on this outbreak is offered in chapter Case Studies of this book. Critical Thinking How have international and national attitudes toward the biological threat changed since the early post–9/11 era? Include some discussion about the reality of versus the potential for biological threats. Middle East Respiratory Syndrome Coronavirus MERS-CoV (see Fig. 1.5 ) was recognized by the World Health Organization (WHO) as a newly emerging pathogen in 2012 ( Berry et al., 2015 ). The initial case where virus isolation and characterization came from occurred in Jeddah, Saudi Arabia. Subsequent infections were reported in Middle Eastern countries (Jordan, Qatar, and the United Arab Emirates), with a few cases also identified in Europe, North Africa, and the United States. MERS-CoV leads to severe respiratory illness in susceptible patients and is spread through person-to-person contact. Figure 1.5 This is a highly magnified, digitally colorized transmission electron micrograph of the MERS-CoV virus. Courtesy of the National Institute of Allergy and Infectious Diseases. South Korea has recently been the epicenter of the largest outbreak of MERS-CoV outside of the Middle East, reporting 180 cases and 29 deaths ( WHO, 2015c ). The outbreak in South Korea was traced to a single infected traveler. Once again, this demonstrates the vulnerability to unexpected outbreaks of unusual diseases that all countries share in this highly mobile world. A report from a joint WHO–South Korean investigation of this outbreak identified several reasons for the severity of the outbreak in South Korea. These include a lack of awareness among health-care workers and the general public about MERS-CoV, the practice of "doctor shopping" (seeking care at multiple hospitals), people visiting infected patients in multibed hospital rooms, substandard infection control and prevention measures in health-care facilities, and contact of infected MERS-CoV patients in crowded emergency rooms. Nearly all of the country's confirmed MERS-CoV patients were infected while seeking care or visiting hospital patients ( Boston Globe, 2015 ). More about MERS-CoV and other emerging pathogens is in chapter Category C Diseases and Agents. Avian Influenza HPAI has been very much in the news since 1997 when the novel strain H5N1 jumped from domestic bird populations (poultry) to humans in South East Asia ( Ryan, 2008 ). H5N1 was very much feared by public health and government officials for its pandemic potential. Since 2003 there have only been approximately 650 cases of H5N1 infection in humans, with a mortality rate of approximately 60% ( HHS, 2015 ). Since that time, numerous other novel strains have emerged. In fact, a novel H1N1 arose out of swine in 2009 and was the cause of a mild pandemic in humans. More recently, the novel strains H5N2 and H5N8 have been found to be the cause of major morbidity and mortality in poultry operations (chicken and turkey) in the United States, with 223 detections affecting more than 48 million birds ( USDA, 2015 ). Refer to Fig. 1.6 for a graphic representation of this outbreak. The financial impact on the poultry growers and the egg and meat industry has been enormous. More can be found on this topic in chapter Recent Animal Disease Outbreaks and Lessons Learned. Figure 1.6 This map of the United States shows the states where there have been cases of HPAI in a recent outbreak. Note that the number of birds that have been affected in a 7-month period exceeds 48 million. Courtesy of the US Department of Agriculture. Avian Influenza HPAI has been very much in the news since 1997 when the novel strain H5N1 jumped from domestic bird populations (poultry) to humans in South East Asia ( Ryan, 2008 ). H5N1 was very much feared by public health and government officials for its pandemic potential. Since 2003 there have only been approximately 650 cases of H5N1 infection in humans, with a mortality rate of approximately 60% ( HHS, 2015 ). Since that time, numerous other novel strains have emerged. In fact, a novel H1N1 arose out of swine in 2009 and was the cause of a mild pandemic in humans. More recently, the novel strains H5N2 and H5N8 have been found to be the cause of major morbidity and mortality in poultry operations (chicken and turkey) in the United States, with 223 detections affecting more than 48 million birds ( USDA, 2015 ). Refer to Fig. 1.6 for a graphic representation of this outbreak. The financial impact on the poultry growers and the egg and meat industry has been enormous. More can be found on this topic in chapter Recent Animal Disease Outbreaks and Lessons Learned. Figure 1.6 This map of the United States shows the states where there have been cases of HPAI in a recent outbreak. Note that the number of birds that have been affected in a 7-month period exceeds 48 million. Courtesy of the US Department of Agriculture. Conclusion From this first chapter we can now understand and appreciate the scope and importance of biological threats and see where they may be and have become the desire of terrorist groups and the makings of weapons of mass destruction. Biowarfare has a history. The major events are important in helping us understand the issues related to using biological substances against an adversary. We now know the difference between biosecurity and biodefense and can relate them to homeland security and homeland defense, respectively. We also know how expensive these programs are because nearly $80 billion has been spent on civilian biodefense since FY2001 in the United States alone. As discussed herein, there is a significant difference in the reality and the potential of bioterrorism. Bioterrorism on a large scale is a low-probability event. Bioterrorism and biocrimes on a small scale (eg, small amount of ricin directed at one or a few individuals) are fairly routine occurrences with little potential. Biological threats remain very much in the news. Recent examples, such as laboratory incidents, the Ebola outbreak of 2014/2015, the outbreak of MERS-CoV in South Korea, and the HPAI outbreak affecting poultry in the United States, make us aware that we must remain vigilant and utilize the biosecurity and biodefense programs to help us identify and respond to these accidental exposures and emerging threats. Essential Terminology • Biodefense . The collective efforts of a nation aimed at improving defenses against biological attacks. Within these efforts are programs and agencies working toward increasing data collection, analysis, and intelligence gathering. The intelligence is applied to programs aimed at mitigating the effects of bioweapons by developing vaccines, therapeutics, and detection methods to increase the defensive posture. Ultimately, biodefense initiatives protect the military forces and the citizens from the effects of biological attack. • Biosecurity . The policies and measures taken for protecting a nation's food supply and agricultural resources from accidental contamination and deliberate attacks of bioterrorism. • Bioterrorism . The intentional use of microorganisms or toxins derived from living organisms to cause death or disease in humans or the animals and plants on which we depend. Bioterrorism might include such deliberate acts as introducing pests intended to kill US food crops; spreading a virulent disease among animal production facilities; and poisoning water, food, and blood supplies. • Biowarfare , also known as germ warfare. The use of any organism (bacteria, virus, or other disease-causing organism) or toxin found in nature as a weapon of war. It is meant to incapacitate or kill an adversary. • Pathogen . A specific causative agent of disease, mostly thought of as being an infectious organism (eg, bacteria, virus, rickettsia, protozoa). • Weaponization . When applied to biologicals, the term implies a process of taking something natural and making it harmful through enhancing the negative characteristics of it. With biological agents, one might weaponize the agent by making more lethal, more stable, and more easily delivered or disseminated against an intended target. There is considerable debate about the use of this term. • Zoonotic disease . An animal disease that may be transmitted to humans. Discussion Questions • How was the decision made to begin the US biological weapons program? • What are the significant events in the history of biowarfare? What makes them significant? • When President Nixon said that "Mankind already holds in its hands too many of the seeds of its own destruction" in November 1969, what did he mean by that? • Weaponizing a biological agent is easy to do, right? • No one knows exactly who perpetrated the Anthrax attacks of 2001, and there has been no repeat of them since. Why do you think we have seen no repeat of the anthrax attacks since 2001? Websites The Center for Arms Control and Nonproliferation has an online course in biosecurity. Type the URL that follows into your Internet browser and click on View Course and select Unit 2 : "The History of Biological Weapons." The six sections in this unit provide an excellent overview and reinforce the material presented in the subheading about the History of Biowarfare: www.armscontrolcenter.org/resources/biosecurity_course . The CDC's Emergency and Preparedness website offers a segmented video short lesson on the history of bioterrorism. The seven sections give a general overview on bioterrorism and separate vignettes on anthrax, plague, tularemia, VHFs, smallpox, and botulism: www.bt.cdc.gov/training/historyofbt .

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8487065/

Titratable transmembrane residues and a hydrophobic plug are essential for manganese import via the Bacillus anthracis ABC transporter MntBC-A

All extant life forms require trace transition metals ( e.g. , Fe 2/3+ , Cu 1/2+ , and Mn 2+ ) to survive. However, as these are environmentally scarce, organisms have evolved sophisticated metal uptake machineries. In bacteria, high-affinity import of transition metals is predominantly mediated by ABC transporters. During bacterial infection, sequestration of metal by the host further limits the availability of these ions, and accordingly, bacterial ABC transporters (importers) of metals are key virulence determinants. However, the structure–function relationships of these metal transporters have not been fully elucidated. Here, we used metal-sensitivity assays, advanced structural modeling, and enzymatic assays to study the ABC transporter MntBC-A, a virulence determinant of the bacterial human pathogen Bacillus anthracis . We find that despite its broad metal-recognition profile, MntBC-A imports only manganese, whereas zinc can function as a high-affinity inhibitor of MntBC-A. Computational analysis shows that the transmembrane metal permeation pathway is lined with six titratable residues that can coordinate the positively charged metal, and mutagenesis studies show that they are essential for manganese transport. Modeling suggests that access to these titratable residues is blocked by a ladder of hydrophobic residues, and ATP-driven conformational changes open and close this hydrophobic seal to permit metal binding and release. The conservation of this arrangement of titratable and hydrophobic residues among ABC transporters of transition metals suggests a common mechanism. These findings advance our understanding of transmembrane metal recognition and permeation and may aid the design and development of novel antibacterial agents. Results Transport specificity of baMntBC-A In a previous study, we determined the metal-binding spectrum of baMntA of B. anthracis ( 24 ), the cognate SBP of the baMntBC transporter. We found that baMntA binds several transition metals with affinities ranging from 10 −6 to 10 −8 M. The highest affinity was toward Co 2+ ( K D ∼ 5 × 10 −8 M) and the lowest toward Ni 2+ ( K D ∼ 4 × 10 −6 M), and intermediate binding affinities ( K D 3–5 × 10 −7 M) were measured for Mn 2+ , Zn 2+ , and Cd 2+ . Although substrate binding by the SBP is essential for transport ( 22 , 23 , 29 , 30 ), it is not sufficient: several studies have now established that the SBP may bind ligands that are not transported by the transporter ( 31 , 32 , 33 ). To investigate the transport specificity of baMntBC-A, we cloned the coding region of the B. anthracis mntBCA transporter and inserted it into an IPTG-inducible Bacillus subtilis chromosomal integration vector ( 34 ). We then transformed WT B. subtilis with either the empty chromosomal-integration plasmid or the same plasmid harboring the baMntBC-A transporter. Various metals were added to midexponential phase cultures, and intracellular metal content was then measured by inductively coupled plasma MS (ICP-MS). We first tested the accumulation of Co 2+ (added as CoSO 4 ) because it is the metal that is bound with the highest affinity by baMntA ( 24 ), the SBP of the system. We found that the intracellular concentration of Co 2+ was independent of the expression of baMntBC-A. Similarly, the intracellular accumulation of Ni 2+ , Zn 2+ , and Cd 2+ was also unchanged upon expression of baMntBC-A ( Fig. 1 A ). In contrast, when MnSO 4 was added to the growth media, the intracellular accumulation of Mn 2+ in cells expressing baMntBC-A was ∼2.5-fold higher than in control cells ( Fig. 1 A ). Dose-dependent experiments revealed that this fold difference was maintained over a relatively broad range (1–250 μM) of manganese concentrations ( Fig. S1 ). Figure 1 baMntBC-A imports only manganese. A , midexponential phase cultures of Bacillus subtilis cells transformed with either a control plasmid ( blue bars ) or a plasmid encoding WT ( black bars ) or E163A ( red bars ) baMntBC-A were incubated for 15 min with 50 μM of the sulfate salts of the indicated metals. Cells were then harvested and washed with PBS-EDTA buffer, and their intracellular metal content was determined by ICP-MS. Data are the mean ∓ standard deviation of the mean of replicates (n = 5). Normal distribution of the data was verified by the Shapiro–Wilk test (α = 0.05), and statistics were calculated using one-way ANOVA. ∗∗∗ p < 0.001. B , shown is the time-dependent accumulation of manganese (added at 50 μM) in control cells ( blue circles ) or in cells expressing WT ( black circles ) or E163A ( red circles ) baMntBC-A. ICP-MS, inductively coupled plasma MS; ns, not significant. These results suggest that despite the broad metal-recognition spectrum of the SBP (MntA) baMntBC-A seems to transport (import) only Mn 2+ . To confirm that the increased accumulation of manganese is specific to the transport (import) activity of baMntBC-A, we repeated the experiments with a baMntBC-A variant that carries a mutation in the glutamate of the Walker B motif (E163A). In all ABC transporters, this conserved glutamate serves as the catalytic base for cleavage of the γ-phosphate bond and is therefore absolutely essential for ATP hydrolysis and transport ( 20 , 35 , 36 , 37 , 38 ). As shown, despite WT-like expression levels of this mutant, the intracellular manganese content of cells expressing baMntBC-A (E163A) was identical to that of cells transformed with the control plasmid ( Fig. 1 A and Fig. S2 A ). These results suggest that the increased accumulation of manganese in cells expressing WT baMntBC-A is indeed due to the ATPase-dependent transport (import) activity of the transporter. Time-dependent metal uptake assays revealed that the transport is quite rapid, and significant baMntBC-A-mediated accumulation of manganese was observed in the expressing cells already after 5 min of activity ( Fig. 1 B ). To complement the Mn 2+ transport assays described above, we performed metal-sensitivity growth assays using the previously described manganese-sensitive ΔmntR B. subtilis strain ( 8 , 39 ). We reasoned that if indeed baMntBC-A functions as a manganese importer its activity will be readily detected in this manganese-sensitive strain. We therefore transformed ΔmntR cells with either an empty chromosomal-integration plasmid (pDR111) or with the same plasmid that contains the complete baMntBC-A operon under control of the lac promoter. As shown in Figure 2 A , in the absence of exogenously added manganese, the growth of ΔmntR B. subtilis was not affected by the expression of baMntBC-A, suggesting that the plasmid-driven expression of baMntBC-A is in itself not inhibitory to cell growth. However, even if low concentrations of manganese were added (5 μM MnSO 4 ), cells that expressed WT baMntBC-A showed a dramatic growth attenuation ( Fig. 2 A ). Importantly, cells expressing the inactive variant (E163A) did not show increased manganese sensitivity. This baMntBC-A-conferred manganese hypersensitivity was observed over a broad range of concentrations (2–160 μM, Fig. 2 B ). In line with their increased manganese sensitivity, ΔmntR cells expressing WT baMntBC-A accumulated more manganese than control cells or baMntBC-A (E163A)-expressing cells, whereas their intracellular zinc concentration remained unchanged ( Fig. 2 C ). Figure 2 Expression of baMntBC-A leads to increased manganese sensitivity. A , cultures of ΔmntR cells transformed with the pDR111 control plasmid ( blue curves ) or with the same plasmid encoding WT baMntBC-A ( black curves ) or the ATPase-deficient mutant baMntBC-A (E163A, red curves ) were grown in LB media supplemented with IPTG in the absence ( solid lines ) or presence ( dotted lines ) of 5 μM MnSO 4 . Shown are representative results of experiments conducted at least three times. B , cells were grown for 12 h in the presence of the indicated concentration of MnSO 4 and their final absorbance is expressed as percentage growth relative to the growth observed in the absence of MnSO 4 . Shown are the averages of biological triplicates, and the error bars (shown unless smaller than icons) represent the SDs of the mean. C , midexponential phase cultures of ΔmntR cells transformed with either a control plasmid ( blue bars ) or a plasmid encoding WT ( black bars ) or E163A ( red bars ) baMntBC-A were incubated for 15 min with 50 μM of either MnSO 4 or ZnSO 4 as indicated. Cells were then harvested and washed with PBS-EDTA buffer, and their intracellular metal content was determined by ICP-MS. Data are the mean ∓ standard deviation of the mean of replicates (n = 4). Normal distribution of the data was verified by the Shapiro–Wilk test (α = 0.05), and statistics were calculated using one-way ANOVA. ∗∗∗ p < 0.001. D , ΔmntR cells were transformed with a pDR111 control plasmid or with the same plasmid harboring the complete baMntBC-A operon, as indicated. Cultures were grown to the midexponential phase and then diluted to A 600 of 0.01. Drops of 2.5 μl were spotted in serial 10-fold dilutions from left to right onto LB-agar plates supplemented with IPTG in the presence of the indicated concentration of MnSO 4 . Shown are representative results of experiments conducted at least three times. The dashed line represents a crop from a single unprocessed image. ICP-MS, inductively coupled plasma MS; ns, not significant. To complement these studies that were conducted in liquid media, we performed drop-dilution tests in solid media. In line with the results obtained in liquid media, expression of baMntBC-A in the ΔmntR strain led to increased manganese sensitivity ( Fig. 2 D ). In a previous study ( 24 ), we identified three residues that comprise the metal-binding site of baMntA: mutations in either H141 or E207 completely abolished manganese recognition, whereas a mutation in H69 led to ∼10-fold reduction in binding affinity. To further substantiate the link between the observed manganese-sensitivity phenotype and expression of functional baMntBC-A, we tested the manganese sensitivity of mutants H141A, E207A, and H69A. In the absence of manganese, the growth of ΔmntR cells expressing these mutants was indiscernible from that of cells expressing WT baMntBC-A or from that of cells transformed with the control plasmid ( Fig. 3 A ). In line with their complete inability to bind manganese ( 24 ), the manganese sensitivity of the fully inactive mutants (H141A and E207A) was identical to that of the cells that did not express baMntBC-A ( Fig. 3 , B and C ), and, as expected, these cells did not overaccumulate manganese ( Fig. 3 D ). This lack of activity of mutants H141A and E207A could not be attributed to changes in expression levels caused by the point mutations ( Fig. S2 A ). Notably, in line with its in vitro ∼10-fold reduced manganese-binding affinity, expression of mutant H69A conferred modest manganese sensitivity, which was significantly different from that of the two fully inactive mutants and from that of the WT protein. ICP-MS analysis revealed that the intracellular manganese concentration of cells expressing mutants H141A and E207A was comparable with that of cells that did not express baMntBC-A, whereas the intracellular manganese concentration found in cells expressing mutant H69A was slightly higher ( Fig. 3 D ). Figure 3 Mutations of the metal-binding residues of baMntA abolish the manganese sensitivity. Cultures of ΔmntR cells transformed with the pDR111 control plasmid or with the same plasmid encoding WT or mutant baMntBC-A (as color indicated) were grown in LB media supplemented with IPTG in the absence ( A ) or presence ( B ) of 5 μM MnSO 4 . Shown are representative results of experiments conducted at least three times. C , ΔmntR cells were grown for 12 h in the presence of the indicated concentration of MnSO 4 , and their final absorbance is expressed as percentage growth relative to the growth observed in the absence of MnSO 4 . Shown are the averages of biological triplicates, and the error bars (shown unless smaller than icons) represent the SDs of the mean. D , midexponential phase cultures of Bacillus subtilis cells transformed with either a control plasmid or the same plasmid encoding WT or mutant baMntBC-A (as color indicated) were incubated for 15 min with 50 μM of MnSO 4 . Cells were then harvested and washed with PBS-EDTA buffer, and their intracellular metal content was determined by ICP-MS. Data are the mean ∓ standard deviation of the mean of replicates (n = 3). Normal distribution of the data was verified by the Shapiro–Wilk test (α = 0.05), and statistics were calculated using one-way ANOVA. ∗∗∗ p < 0.001; ∗∗ p < 0.05. ICP-MS, inductively coupled plasma MS; ns, not significant. Taken together, the results with the four mutations that compromise either the ATPase activity of baMntBC ( Fig. 2 ) or manganese binding by baMntA ( Fig. 3 ) support the conclusion that the manganese hypersensitivity observed in cells that express WT baMntBC-A is indeed due to active import of manganese by baMntBC-A. Zn 2+ , but not other metals, inhibits transport of Mn 2+ In a previous work, we observed that Mn 2+ and Zn 2+ compete for binding to the same metal-binding site in baMntA ( 24 ). In the same work, we also observed that the SBP–Zn +2 complex is very stable and that Zn 2+ is released from the SBP at a very slow rate. Although these experiments were conducted in the absence of the transporter, this suggested that Zn 2+ is a nontransportable metal. Indeed, as shown above ( Fig. 1 A ), Mn 2+ is transported by baMntBC-A, whereas Zn 2+ is apparently not. Very similar findings were originally reported for PsaA, the Mn 2+ SBP of Streptococcus pneumoniae , and therefore, the susceptibility of this bacterium to Zn 2+ was attributed to the Zn 2+ -mediated inhibition of Mn 2+ uptake ( 11 , 40 ). Based on these observations, it was suggested that Zn 2+ may be used as an inhibitor of bacterial manganese ABC importers such as baMntBC-A. To test this, we measured the intracellular accumulation of Mn 2+ in cells that were preincubated with Zn 2+ . As shown in Figure 4 A , preincubating baMntBC-A-expressing cells with Zn 2+ reduced their intracellular Mn 2+ content to levels that were similar to those observed in control cells that did not express baMntBC-A. These results suggest that Zn 2+ inhibits the Mn 2+ uptake activity of baMntBC-A and therefore may alleviate the manganese hypersensitivity of baMntBC-A-expressing cells. Figure 4 Zinc inhibits baMntBC-A-mediated transport of manganese. A , midexponential-phase cultures of WT Bacillus subtilis 168 cells transformed with either a control plasmid ( blue bars ) or a plasmid harboring the complete baMntBC-A operon ( black bars ) were incubated for 15 min with 100 μM of the indicated metals. Cells were then harvested and washed with PBS-EDTA buffer, and the intracellular metal content was determined by ICP-MS. Data are the mean ∓ standard deviation of the mean of replicates (n = 5). Normal distribution of the data was verified by the Shapiro–Wilk test (α = 0.05), and statistics were calculated using one-way ANOVA. ∗∗∗ p < 0.001. B , cultures of Bacillus subtilis ΔmntR cells transformed with a control plasmid ( open symbols ) or a plasmid harboring the complete baMntBC-A operon ( full symbols ) were grown in LB media ( orange symbols ) or LB media supplemented with 10 μM MnSO 4 ( black symbols ), 50 μM ZnSO 4 ( blue symbols ), or their combination ( red symbols ). Shown are averages of biological triplicates, and the error bars (shown unless smaller than the symbols) represent SDs of the mean. ICP-MS, inductively coupled plasma MS; ns, not significant. To test this, we grew control and baMntBC-A-expressing cells in the presence of Mn 2+ , Zn 2+ , and combinations thereof. As shown, addition of 50 μM Zn 2+ was only marginally inhibitory to growth, and this effect was identical in control and baMntBC-A-expressing cells ( Fig. 4 B , compare orange and blue traces). This is not surprising in light of the lack of Zn 2+ import activity of baMntBC-A ( Fig. 1 A ). As expected, cells expressing baMntBC-A were much more sensitive to the addition of 10 μM Mn 2+ than control cells ( Fig. 4 B , black symbols). In cells that did not express baMntBC-A, the addition of 50 μM Zn 2+ on top of 10 μM Mn 2+ led to a modest (∼10%) yet statistically significant greater inhibition ( Fig. 4 B , open red symbols). In contrast, in baMntBC-A-expressing cells, the addition of 50 μM Zn 2+ on top of 10 μM Mn 2+ led to ∼4-fold higher growth ( Fig. 4 B , full red symbols). These results support the notion that as reported for PsaBCA of Streptococcus pneumonia ( 11 , 41 ), Zn 2+ inhibits the Mn 2+ import activity of baMntBC-A. In addition to Mn 2+ and Zn 2+ , MntA also binds Ni 2+ and Co 2+ ( 24 ). We therefore tested whether these metals could also serve as MntBC-A inhibitors. Surprisingly, despite its higher binding affinity (∼10-fold higher than toward either Mn 2+ or Zn 2+ , ( 24 )), Co 2+ had no inhibitory effect, and similar lack of inhibition was also observed with Ni 2+ ( Fig. S3 ). The implications and source of the specificity of Zn 2+ inhibition is discussed later in the article (see Conclusions ). Model structure of baMntBC and identification of a potential TM metal-coordination site To gain insight on the structure of baMntBC and its possible transport mechanism, we attempted to model it, which proved to be nontrivial. The baMntBC transporter is comprised of two identical subunits of MntC that form the TMDs, in complex with two identical subunits of MntB that form the NBDs. The structure of the NBDs is highly conserved among the superfamily of ABC transporters ( 20 , 42 , 43 ), and with query-template sequence identity close to 30%, we could use standard homology modeling to model MntB. Unlike the NBDs, the structure and sequence of the TMDs of ABC transporters can vary significantly ( 42 , 44 ). Indeed, the modeling of MntC proved to be challenging as only remote homologs are available as templates. Another problem with these templates is that they all transport substrates that are much larger than manganese, making the modeling of the translocation pathway particularly challenging. With sequence identity of only 13% to 15% to the available templates, there is uncertainty even regarding the exact number of TM helices. To overcome these challenges, we combined various computational tools to produce multiple model structures of MntC (see Experimental procedures ). The result of these efforts was three models based on three different templates: (1) Haemophilus influenza's putative molybdate/tungstate importer MolBC in an inward-facing conformation (PDB ID: 2NQ2 ; chains A and B) ( 45 ) that resulted in a semiopen MntC model; (2) Yersinia pestis's heme importer HmuUV in an outward-apo conformation (PDB ID: 4G1U ; chains A and B) ( 46 ) that resulted in an occluded state model; and (3) E. coli's vitamin B 12 importer in an outward-facing adenylyl imidodiphosphate-bound conformation (PDB ID: 4R9U chains A and B) ( 47 ) that produced an open conformation model. To further assess MntC's model we also produced a template-independent model using trRosetta ( 48 ) that predicts inter-residue distance and orientation distributions based on neural networks. Both methods (trRosetta and homology modeling) converged to a similar structure with almost identical assignment of TM helices ( Figs. S4 and S5 ). Furthermore, all models were consistent with the expected evolutionary conservation pattern, where the protein's core is composed almost exclusively of conserved residues while its periphery is enriched with variable residues ( Figs. S4 and S5 ). Nevertheless, owing to the low query-to-template sequence identity and the ensuing model uncertainty, in the following structural analysis, we considered only features that were observed in all three models irrespective of the template used. The passage of a charged molecule through a membrane protein often requires coordination by membrane-embedded charged residues that neutralize the translocated charge (chapter 7 in ( 49 )). We hypothesized that this is especially true for transport of manganese, which has one of the highest charge densities of the biologically active divalent metals. Indeed, the model shows six titratable residues that line the transmembrane translocation cavity ( Fig. 5 A , red spheres), comprised of two triads of Asp 47, His 51, and Asp 94 (DHD), where each triad is contributed by a different monomer of MntC. These three residues are highly conserved among the 430 baMntC homologs we analyzed ( Fig. S6 ), with a score of 9 in ConSurf's evolutionary conservation scale (1 being the most variable and 9 the most conserved, ( 50 )). Interestingly, none of these residues is conserved in any of the templates used for the modeling and, to the best of our knowledge, in any other subgroup of ABC transporters. In addition, Asp and His residues are among the most common manganese-coordinating residues ( 51 ). Considering the preferred coordination number of 5 to 6 for manganese ( 51 ), the two DHD triads could provide a possible manganese coordination site. Of note, His and Asp residues were demonstrated to directly coordinate binding of Mn 2+ by baMntA, the system's SBP ( 24 ). Figure 5 Essential titratable residues line the translocation cavity of baMntBC. A , shown are side ( left ) and top (all other) views of semitransparent cartoon representations of the baMntBC models. The two images on the left are of the model that is based on the structure of HmuUV, depicting an occluded conformation. BtuD is colored lime , BtuC in light yellow , and the C-terminal helical extension in salmon pink . The two images on the right are based on the structures of MolBC and BtuCD and depict a semiopen and open conformations, respectively. Residues of interest are shown as either spheres (full-size images) or sticks (zoomed images). The twin charged triads, each composed of Asp 47, His 51, and Asp 94, are shown in red and the "hydrophobic ladder" residues (Ile97, F101, F105, Ile109, and Ile112) are in blue . B – E , cultures of ΔmntR cells were grown in the absence ( B and D ) or presence ( C and E ) of 10 μM MnSO 4 . In the absence of manganese, WT baMntBC-A and all tested mutants grew similarly. In the presence of manganese mutations in the putative metal-binding residues Asp 47, His 51, and Asp 94 ( C ) or in hydrophobic seal residues I97, F101, F105, I109, I112, or truncation of the C-terminal helix ( E ) abolished the activity of baMntBC-A and the growth of these mutants was similar to that of controls cells that did not express baMntBC-A or cells expressing the inactive mutant E163A. To test if residues DHD have a role in manganese transport, we generated mutants with alanine substitutions of these three putative metal-binding residues. Of note, because MntBC is a heterodimer of homodimers, a single substitution at the gene level results in two identical amino acid mutations at the protein level. Next, we compared the activity of these single alanine mutants with that of WT baMntBC using the Mn 2+ sensitivity assay described above. As shown in Figure 5 B , in the absence of manganese, the growth of control cells, cells expressing WT baMntBC, or the tested mutants was indistinguishable. In the presence of manganese ( Fig. 5 C ), mutations D47A, H51A, and D94A completely abolished the baMntBC-A-mediated Mn 2+ sensitivity, and the growth of these mutants was identical to that of the vector control and to that of the inactive E163A mutant (which carries a mutation in the essential glutamate of the Walker B motif). The lack of activity of the mutants was not due to improper expression, as their membrane-fraction expression was similar to that of the WT protein ( Fig. S2 A ). These results demonstrate that the titratable residues are essential for Mn 2+ transport and support the suggestion that they may be involved in TM metal coordination. The model shown in Figure 5 A is based on the structure of the heme transporter HmuUV ( 46 ), depicting baMntBC in an occluded conformation. In this conformation, hydrophobic residues (Ile97, F101, F105, Ile109, and Ile112) from TM helices 5 and 5′ form a "hydrophobic ladder" that effectively seals the metal-binding site to the extracellular side of the membrane ( Fig. 5 A , blue spheres). Interestingly, the other two models (based on apo MolBC and AMP-PNP-bound BtuCD) depict baMntBC in intermediate stages of opening toward the extracellular environment, where the width of the narrowest point in the hydrophobic seal increases from 3.6 à (occluded) to 5.4 à (semiopen), and finally to 11.3 à (fully open, top views in Fig. 5 A , as indicated). To test if the bulky residues that comprise the hydrophobic seal are important for transport, we generated single point glycine substitutions. Glycine was chosen because it is the smallest amino acid. Remarkably, replacement of any of the bulky hydrophobic seal residue with glycine completely abolished the activity of baMntBC ( Fig. 5 , D and E ), despite normal membrane-fraction expression of the mutants ( Fig. S2 B ). The relative motions of the residues that comprise the hydrophobic seal and of those that comprise the charged triads are expected to play an important role in the transport mechanism, as will be discussed later. Of note, according to all 3D models, the TMDs of baMntBC are comprised of nine TM helices per monomer ( Fig. 5 A and Fig. S4 ), in contrast to the ten TM helices observed in all crystal structures of baMntBC homologs ( 45 , 46 , 47 ). The suggestion that the number of baMntBC TM helices differs from ten is also supported by all of the TM prediction tools we tested ( Fig. S6 ). Furthermore, all three homology models of baMntBC, as well as the trRosetta template-free model, show an elongated C-terminal TM helix that extends well into the cytoplasm ( Fig. 5 A ). Given that both the N and C termini were modeled using an ab initio approach (see Experimental procedures ), the quality of the model in these regions is questionable. Nevertheless, the amino acid composition of the C terminus is not amphipathic ( Fig. S7 ) and therefore not expected to lie at the membrane–cytosol interface. In comparison, the N-terminal TM helix is clearly amphipathic ( Fig. S7 ) and as depicted in the model is expected to lie at the membrane–extracellular interface ( Fig. 5 A and Figs. S4–S7 ). Further support for the cytosolic location of the C-terminus helical extension is provided by the fact that out of the 21 amino acids that comprise it, six are titratable and six are polar. The net charge of this helical extension is +5, and its cytoplasmic location would be in line with the "positive inside rule" ( 52 , 53 ). This charge distribution is very different from that observed in all other predicted TM helices of MntC that are heavily enriched in hydrophobic residues ( Figs. S6 and S7 ). To test if the cytoplasmic extension of this C-terminal TM helix was important for function, we truncated it at the membrane–cytosol interface to generate the MntBC-ΔC mutant. Surprisingly, the truncation did not affect the membrane-expression of MntBC ( Fig. S2 B ). However, as judged by manganese-sensitivity assays, the truncated variant was completely inactive ( Fig. 5 , D and E , green traces). Uncoupled ATPase activity of baMntBC ABC transporters that function as importers are divided into two subgroups: type I importers typically import sugars, amino acids, and peptides, whereas type II systems import organometallic complexes such as heme, siderophores, and vitamin B 12 ( 20 , 42 , 54 , 55 ). These two subclasses have been shown to operate by very distinct mechanisms. One central distinctive mechanistic feature is the basal rate of ATPase activity and its modulation by the SBP. In type I systems, the basal rates of ATP hydrolysis are very low, and these are greatly stimulated (10- to 30-fold) upon docking of the substrate-loaded SBP ( 33 , 56 ). In contrast, type-II systems have very high rates of basal ATP hydrolysis that are largely insensitive to substrate loading ( 46 , 57 , 58 ). The extent of substrate modulation of ATPase activity in transition metal ABC importers such as baMntBC is unknown. To study this phenomenon, we codon-optimized the nucleotide sequence of baMntBC for expression in E. coli and synthetically generated codon-optimized coding regions for mntBC (GenScript). We then prepared inverted membrane vesicles ( 59 ) from cells transformed with an empty control plasmid or plasmids encoding WT or mutant (E163A) baMntBC. We reasoned that owing to the high content of baMntBC in these vesicles (inset in Fig. 6 A ), we may be able to measure its ATPase activity without the need of detergent-mediated extraction/purification with its possible pitfalls ( 60 ). Indeed, as shown in Figure 6 A , the ATPase activity of baMntBC-inverted membrane vesicles was ∼5-fold higher than that of vesicles prepared from control cells that were transformed with an empty control plasmid. To verify that this activity is indeed specific to baMntBC, we repeated these experiments with the ATPase-deficient mutant baMntBC (E163A). Despite the high protein content of baMntBC (E163A) vesicles (inset in Fig. 6 A ), their ATPase activity was identical to that of the control vesicles that did not contain any baMntBC ( Fig. 6 A ). Figure 6 Uncoupled ATP hydrolysis by baMntBC. A , 15 μg of inverted membrane vesicles were incubated for 2 min with 1 mM ATP, and to initiate hydrolysis, 2 mM MgSO 4 was injected at 2 min. The rate of release of inorganic phosphate was determined by continuous monitoring of the 360 nm absorbance of the solution using the EnzChek kit. Shown is the activity measured for vesicles containing WT baMntBC ( black ), WT baMntBC+ baMntA ( red ), WT baMntBC+ 10 μM baMntA+ 50 μM MnSO 4 ( blue ), mutant E163A ( magenta ), or control vesicles ( gray ). Results are representative of experiments conducted at least three times. The inset shows an immunoblot of SDS page of the vesicles, and BtuCD (used as benchmark for high expression level) and WT or mutant E163A are shown as indicated. B and C , initial rates of hydrolysis of 15 to 1000 μM ATP were measured (in the presence of baMntA and MnSO 4 ) for control ( B ) and WT baMntBC ( C ) vesicles. D , the initial rates of ATP hydrolysis measured in panel B for the control vesicles were subtracted from the rates measured for WT baMntBC in panel C . The net values were plotted as a function of the ATP concentration, and the data were then fit using the Michaelis–Menten equation ( dashed line ). Also shown are the mean values (n = 3) of the kinetic rate constants, and the error bars represent SDs of the mean. We next used the vesicles system to investigate the effects of the SBP on ATP hydrolysis by the transporter. For this, codon-optimized baMntA was overexpressed and purified in E. coli as previously described ( 24 ) and incorporated into the vesicles' lumen at approximately ∼10-fold molar excess over baMntBC (see Experimental procedures for details). The SBP did not stimulate the ATPase activity of the transporter, and its incorporation led to a mild inhibitory effect ( Fig. 6 A ). We then repeated these experiments incorporating also manganese into the vesicles' lumen at a 50:10:1 manganese-baMntA-baMntBC molar ratio and observed a similarly modest inhibitory effect ( Fig. 6 A ). Collectively, these results suggest that similar to the type II ABC importer BtuCD, yet unlike the type I ABC importers MalFGK, YecSC, and HisPQM, baMntBC has high basal ATPase activity that is not further stimulated by the SBP–substrate complex ( 33 , 36 , 56 , 57 , 61 ). To determine the kinetic rate constants of ATP hydrolysis, we measured the initial rates of activity under a range of ATP concentrations. As shown, at ATP concentrations of 15 to 1000 μM, the initial rates of ATP hydrolysis were linear for at least 2 min ( Fig. 6 , B and C ). To determine the specific ATPase activity of baMntBC, we subtracted the corresponding activity measured in the control vesicles. The net rates of ATP hydrolysis were then plotted as a function of ATP concentration, and the data were fit using the Michaelis–Menten equation ( Fig. 6 D ). Adding the term for the Hill coefficient yielded an n HILL = 1.08, indicating that the two ATP-binding sites of baMntBC do not hydrolyze ATP cooperatively. This differs from the cooperative ATP hydrolysis that was reported for both type I and type II ABC importers ( e.g. , MetNI, HisPQM, MalFGK, YecSC, and BtuCD) ( 33 , 36 , 61 , 62 , 63 ). Notably, even in a membrane environment, baMntBC has very high rates of uncoupled (absence of the substrate and SBP) ATPase activity ( k cat = 1.85 s −1 ), similar to the SBP–substrate–stimulated ATPase rates reported for type I importers ( 56 , 61 ). Transport specificity of baMntBC-A In a previous study, we determined the metal-binding spectrum of baMntA of B. anthracis ( 24 ), the cognate SBP of the baMntBC transporter. We found that baMntA binds several transition metals with affinities ranging from 10 −6 to 10 −8 M. The highest affinity was toward Co 2+ ( K D ∼ 5 × 10 −8 M) and the lowest toward Ni 2+ ( K D ∼ 4 × 10 −6 M), and intermediate binding affinities ( K D 3–5 × 10 −7 M) were measured for Mn 2+ , Zn 2+ , and Cd 2+ . Although substrate binding by the SBP is essential for transport ( 22 , 23 , 29 , 30 ), it is not sufficient: several studies have now established that the SBP may bind ligands that are not transported by the transporter ( 31 , 32 , 33 ). To investigate the transport specificity of baMntBC-A, we cloned the coding region of the B. anthracis mntBCA transporter and inserted it into an IPTG-inducible Bacillus subtilis chromosomal integration vector ( 34 ). We then transformed WT B. subtilis with either the empty chromosomal-integration plasmid or the same plasmid harboring the baMntBC-A transporter. Various metals were added to midexponential phase cultures, and intracellular metal content was then measured by inductively coupled plasma MS (ICP-MS). We first tested the accumulation of Co 2+ (added as CoSO 4 ) because it is the metal that is bound with the highest affinity by baMntA ( 24 ), the SBP of the system. We found that the intracellular concentration of Co 2+ was independent of the expression of baMntBC-A. Similarly, the intracellular accumulation of Ni 2+ , Zn 2+ , and Cd 2+ was also unchanged upon expression of baMntBC-A ( Fig. 1 A ). In contrast, when MnSO 4 was added to the growth media, the intracellular accumulation of Mn 2+ in cells expressing baMntBC-A was ∼2.5-fold higher than in control cells ( Fig. 1 A ). Dose-dependent experiments revealed that this fold difference was maintained over a relatively broad range (1–250 μM) of manganese concentrations ( Fig. S1 ). Figure 1 baMntBC-A imports only manganese. A , midexponential phase cultures of Bacillus subtilis cells transformed with either a control plasmid ( blue bars ) or a plasmid encoding WT ( black bars ) or E163A ( red bars ) baMntBC-A were incubated for 15 min with 50 μM of the sulfate salts of the indicated metals. Cells were then harvested and washed with PBS-EDTA buffer, and their intracellular metal content was determined by ICP-MS. Data are the mean ∓ standard deviation of the mean of replicates (n = 5). Normal distribution of the data was verified by the Shapiro–Wilk test (α = 0.05), and statistics were calculated using one-way ANOVA. ∗∗∗ p < 0.001. B , shown is the time-dependent accumulation of manganese (added at 50 μM) in control cells ( blue circles ) or in cells expressing WT ( black circles ) or E163A ( red circles ) baMntBC-A. ICP-MS, inductively coupled plasma MS; ns, not significant. These results suggest that despite the broad metal-recognition spectrum of the SBP (MntA) baMntBC-A seems to transport (import) only Mn 2+ . To confirm that the increased accumulation of manganese is specific to the transport (import) activity of baMntBC-A, we repeated the experiments with a baMntBC-A variant that carries a mutation in the glutamate of the Walker B motif (E163A). In all ABC transporters, this conserved glutamate serves as the catalytic base for cleavage of the γ-phosphate bond and is therefore absolutely essential for ATP hydrolysis and transport ( 20 , 35 , 36 , 37 , 38 ). As shown, despite WT-like expression levels of this mutant, the intracellular manganese content of cells expressing baMntBC-A (E163A) was identical to that of cells transformed with the control plasmid ( Fig. 1 A and Fig. S2 A ). These results suggest that the increased accumulation of manganese in cells expressing WT baMntBC-A is indeed due to the ATPase-dependent transport (import) activity of the transporter. Time-dependent metal uptake assays revealed that the transport is quite rapid, and significant baMntBC-A-mediated accumulation of manganese was observed in the expressing cells already after 5 min of activity ( Fig. 1 B ). To complement the Mn 2+ transport assays described above, we performed metal-sensitivity growth assays using the previously described manganese-sensitive ΔmntR B. subtilis strain ( 8 , 39 ). We reasoned that if indeed baMntBC-A functions as a manganese importer its activity will be readily detected in this manganese-sensitive strain. We therefore transformed ΔmntR cells with either an empty chromosomal-integration plasmid (pDR111) or with the same plasmid that contains the complete baMntBC-A operon under control of the lac promoter. As shown in Figure 2 A , in the absence of exogenously added manganese, the growth of ΔmntR B. subtilis was not affected by the expression of baMntBC-A, suggesting that the plasmid-driven expression of baMntBC-A is in itself not inhibitory to cell growth. However, even if low concentrations of manganese were added (5 μM MnSO 4 ), cells that expressed WT baMntBC-A showed a dramatic growth attenuation ( Fig. 2 A ). Importantly, cells expressing the inactive variant (E163A) did not show increased manganese sensitivity. This baMntBC-A-conferred manganese hypersensitivity was observed over a broad range of concentrations (2–160 μM, Fig. 2 B ). In line with their increased manganese sensitivity, ΔmntR cells expressing WT baMntBC-A accumulated more manganese than control cells or baMntBC-A (E163A)-expressing cells, whereas their intracellular zinc concentration remained unchanged ( Fig. 2 C ). Figure 2 Expression of baMntBC-A leads to increased manganese sensitivity. A , cultures of ΔmntR cells transformed with the pDR111 control plasmid ( blue curves ) or with the same plasmid encoding WT baMntBC-A ( black curves ) or the ATPase-deficient mutant baMntBC-A (E163A, red curves ) were grown in LB media supplemented with IPTG in the absence ( solid lines ) or presence ( dotted lines ) of 5 μM MnSO 4 . Shown are representative results of experiments conducted at least three times. B , cells were grown for 12 h in the presence of the indicated concentration of MnSO 4 and their final absorbance is expressed as percentage growth relative to the growth observed in the absence of MnSO 4 . Shown are the averages of biological triplicates, and the error bars (shown unless smaller than icons) represent the SDs of the mean. C , midexponential phase cultures of ΔmntR cells transformed with either a control plasmid ( blue bars ) or a plasmid encoding WT ( black bars ) or E163A ( red bars ) baMntBC-A were incubated for 15 min with 50 μM of either MnSO 4 or ZnSO 4 as indicated. Cells were then harvested and washed with PBS-EDTA buffer, and their intracellular metal content was determined by ICP-MS. Data are the mean ∓ standard deviation of the mean of replicates (n = 4). Normal distribution of the data was verified by the Shapiro–Wilk test (α = 0.05), and statistics were calculated using one-way ANOVA. ∗∗∗ p < 0.001. D , ΔmntR cells were transformed with a pDR111 control plasmid or with the same plasmid harboring the complete baMntBC-A operon, as indicated. Cultures were grown to the midexponential phase and then diluted to A 600 of 0.01. Drops of 2.5 μl were spotted in serial 10-fold dilutions from left to right onto LB-agar plates supplemented with IPTG in the presence of the indicated concentration of MnSO 4 . Shown are representative results of experiments conducted at least three times. The dashed line represents a crop from a single unprocessed image. ICP-MS, inductively coupled plasma MS; ns, not significant. To complement these studies that were conducted in liquid media, we performed drop-dilution tests in solid media. In line with the results obtained in liquid media, expression of baMntBC-A in the ΔmntR strain led to increased manganese sensitivity ( Fig. 2 D ). In a previous study ( 24 ), we identified three residues that comprise the metal-binding site of baMntA: mutations in either H141 or E207 completely abolished manganese recognition, whereas a mutation in H69 led to ∼10-fold reduction in binding affinity. To further substantiate the link between the observed manganese-sensitivity phenotype and expression of functional baMntBC-A, we tested the manganese sensitivity of mutants H141A, E207A, and H69A. In the absence of manganese, the growth of ΔmntR cells expressing these mutants was indiscernible from that of cells expressing WT baMntBC-A or from that of cells transformed with the control plasmid ( Fig. 3 A ). In line with their complete inability to bind manganese ( 24 ), the manganese sensitivity of the fully inactive mutants (H141A and E207A) was identical to that of the cells that did not express baMntBC-A ( Fig. 3 , B and C ), and, as expected, these cells did not overaccumulate manganese ( Fig. 3 D ). This lack of activity of mutants H141A and E207A could not be attributed to changes in expression levels caused by the point mutations ( Fig. S2 A ). Notably, in line with its in vitro ∼10-fold reduced manganese-binding affinity, expression of mutant H69A conferred modest manganese sensitivity, which was significantly different from that of the two fully inactive mutants and from that of the WT protein. ICP-MS analysis revealed that the intracellular manganese concentration of cells expressing mutants H141A and E207A was comparable with that of cells that did not express baMntBC-A, whereas the intracellular manganese concentration found in cells expressing mutant H69A was slightly higher ( Fig. 3 D ). Figure 3 Mutations of the metal-binding residues of baMntA abolish the manganese sensitivity. Cultures of ΔmntR cells transformed with the pDR111 control plasmid or with the same plasmid encoding WT or mutant baMntBC-A (as color indicated) were grown in LB media supplemented with IPTG in the absence ( A ) or presence ( B ) of 5 μM MnSO 4 . Shown are representative results of experiments conducted at least three times. C , ΔmntR cells were grown for 12 h in the presence of the indicated concentration of MnSO 4 , and their final absorbance is expressed as percentage growth relative to the growth observed in the absence of MnSO 4 . Shown are the averages of biological triplicates, and the error bars (shown unless smaller than icons) represent the SDs of the mean. D , midexponential phase cultures of Bacillus subtilis cells transformed with either a control plasmid or the same plasmid encoding WT or mutant baMntBC-A (as color indicated) were incubated for 15 min with 50 μM of MnSO 4 . Cells were then harvested and washed with PBS-EDTA buffer, and their intracellular metal content was determined by ICP-MS. Data are the mean ∓ standard deviation of the mean of replicates (n = 3). Normal distribution of the data was verified by the Shapiro–Wilk test (α = 0.05), and statistics were calculated using one-way ANOVA. ∗∗∗ p < 0.001; ∗∗ p < 0.05. ICP-MS, inductively coupled plasma MS; ns, not significant. Taken together, the results with the four mutations that compromise either the ATPase activity of baMntBC ( Fig. 2 ) or manganese binding by baMntA ( Fig. 3 ) support the conclusion that the manganese hypersensitivity observed in cells that express WT baMntBC-A is indeed due to active import of manganese by baMntBC-A. Zn 2+ , but not other metals, inhibits transport of Mn 2+ In a previous work, we observed that Mn 2+ and Zn 2+ compete for binding to the same metal-binding site in baMntA ( 24 ). In the same work, we also observed that the SBP–Zn +2 complex is very stable and that Zn 2+ is released from the SBP at a very slow rate. Although these experiments were conducted in the absence of the transporter, this suggested that Zn 2+ is a nontransportable metal. Indeed, as shown above ( Fig. 1 A ), Mn 2+ is transported by baMntBC-A, whereas Zn 2+ is apparently not. Very similar findings were originally reported for PsaA, the Mn 2+ SBP of Streptococcus pneumoniae , and therefore, the susceptibility of this bacterium to Zn 2+ was attributed to the Zn 2+ -mediated inhibition of Mn 2+ uptake ( 11 , 40 ). Based on these observations, it was suggested that Zn 2+ may be used as an inhibitor of bacterial manganese ABC importers such as baMntBC-A. To test this, we measured the intracellular accumulation of Mn 2+ in cells that were preincubated with Zn 2+ . As shown in Figure 4 A , preincubating baMntBC-A-expressing cells with Zn 2+ reduced their intracellular Mn 2+ content to levels that were similar to those observed in control cells that did not express baMntBC-A. These results suggest that Zn 2+ inhibits the Mn 2+ uptake activity of baMntBC-A and therefore may alleviate the manganese hypersensitivity of baMntBC-A-expressing cells. Figure 4 Zinc inhibits baMntBC-A-mediated transport of manganese. A , midexponential-phase cultures of WT Bacillus subtilis 168 cells transformed with either a control plasmid ( blue bars ) or a plasmid harboring the complete baMntBC-A operon ( black bars ) were incubated for 15 min with 100 μM of the indicated metals. Cells were then harvested and washed with PBS-EDTA buffer, and the intracellular metal content was determined by ICP-MS. Data are the mean ∓ standard deviation of the mean of replicates (n = 5). Normal distribution of the data was verified by the Shapiro–Wilk test (α = 0.05), and statistics were calculated using one-way ANOVA. ∗∗∗ p < 0.001. B , cultures of Bacillus subtilis ΔmntR cells transformed with a control plasmid ( open symbols ) or a plasmid harboring the complete baMntBC-A operon ( full symbols ) were grown in LB media ( orange symbols ) or LB media supplemented with 10 μM MnSO 4 ( black symbols ), 50 μM ZnSO 4 ( blue symbols ), or their combination ( red symbols ). Shown are averages of biological triplicates, and the error bars (shown unless smaller than the symbols) represent SDs of the mean. ICP-MS, inductively coupled plasma MS; ns, not significant. To test this, we grew control and baMntBC-A-expressing cells in the presence of Mn 2+ , Zn 2+ , and combinations thereof. As shown, addition of 50 μM Zn 2+ was only marginally inhibitory to growth, and this effect was identical in control and baMntBC-A-expressing cells ( Fig. 4 B , compare orange and blue traces). This is not surprising in light of the lack of Zn 2+ import activity of baMntBC-A ( Fig. 1 A ). As expected, cells expressing baMntBC-A were much more sensitive to the addition of 10 μM Mn 2+ than control cells ( Fig. 4 B , black symbols). In cells that did not express baMntBC-A, the addition of 50 μM Zn 2+ on top of 10 μM Mn 2+ led to a modest (∼10%) yet statistically significant greater inhibition ( Fig. 4 B , open red symbols). In contrast, in baMntBC-A-expressing cells, the addition of 50 μM Zn 2+ on top of 10 μM Mn 2+ led to ∼4-fold higher growth ( Fig. 4 B , full red symbols). These results support the notion that as reported for PsaBCA of Streptococcus pneumonia ( 11 , 41 ), Zn 2+ inhibits the Mn 2+ import activity of baMntBC-A. In addition to Mn 2+ and Zn 2+ , MntA also binds Ni 2+ and Co 2+ ( 24 ). We therefore tested whether these metals could also serve as MntBC-A inhibitors. Surprisingly, despite its higher binding affinity (∼10-fold higher than toward either Mn 2+ or Zn 2+ , ( 24 )), Co 2+ had no inhibitory effect, and similar lack of inhibition was also observed with Ni 2+ ( Fig. S3 ). The implications and source of the specificity of Zn 2+ inhibition is discussed later in the article (see Conclusions ). Model structure of baMntBC and identification of a potential TM metal-coordination site To gain insight on the structure of baMntBC and its possible transport mechanism, we attempted to model it, which proved to be nontrivial. The baMntBC transporter is comprised of two identical subunits of MntC that form the TMDs, in complex with two identical subunits of MntB that form the NBDs. The structure of the NBDs is highly conserved among the superfamily of ABC transporters ( 20 , 42 , 43 ), and with query-template sequence identity close to 30%, we could use standard homology modeling to model MntB. Unlike the NBDs, the structure and sequence of the TMDs of ABC transporters can vary significantly ( 42 , 44 ). Indeed, the modeling of MntC proved to be challenging as only remote homologs are available as templates. Another problem with these templates is that they all transport substrates that are much larger than manganese, making the modeling of the translocation pathway particularly challenging. With sequence identity of only 13% to 15% to the available templates, there is uncertainty even regarding the exact number of TM helices. To overcome these challenges, we combined various computational tools to produce multiple model structures of MntC (see Experimental procedures ). The result of these efforts was three models based on three different templates: (1) Haemophilus influenza's putative molybdate/tungstate importer MolBC in an inward-facing conformation (PDB ID: 2NQ2 ; chains A and B) ( 45 ) that resulted in a semiopen MntC model; (2) Yersinia pestis's heme importer HmuUV in an outward-apo conformation (PDB ID: 4G1U ; chains A and B) ( 46 ) that resulted in an occluded state model; and (3) E. coli's vitamin B 12 importer in an outward-facing adenylyl imidodiphosphate-bound conformation (PDB ID: 4R9U chains A and B) ( 47 ) that produced an open conformation model. To further assess MntC's model we also produced a template-independent model using trRosetta ( 48 ) that predicts inter-residue distance and orientation distributions based on neural networks. Both methods (trRosetta and homology modeling) converged to a similar structure with almost identical assignment of TM helices ( Figs. S4 and S5 ). Furthermore, all models were consistent with the expected evolutionary conservation pattern, where the protein's core is composed almost exclusively of conserved residues while its periphery is enriched with variable residues ( Figs. S4 and S5 ). Nevertheless, owing to the low query-to-template sequence identity and the ensuing model uncertainty, in the following structural analysis, we considered only features that were observed in all three models irrespective of the template used. The passage of a charged molecule through a membrane protein often requires coordination by membrane-embedded charged residues that neutralize the translocated charge (chapter 7 in ( 49 )). We hypothesized that this is especially true for transport of manganese, which has one of the highest charge densities of the biologically active divalent metals. Indeed, the model shows six titratable residues that line the transmembrane translocation cavity ( Fig. 5 A , red spheres), comprised of two triads of Asp 47, His 51, and Asp 94 (DHD), where each triad is contributed by a different monomer of MntC. These three residues are highly conserved among the 430 baMntC homologs we analyzed ( Fig. S6 ), with a score of 9 in ConSurf's evolutionary conservation scale (1 being the most variable and 9 the most conserved, ( 50 )). Interestingly, none of these residues is conserved in any of the templates used for the modeling and, to the best of our knowledge, in any other subgroup of ABC transporters. In addition, Asp and His residues are among the most common manganese-coordinating residues ( 51 ). Considering the preferred coordination number of 5 to 6 for manganese ( 51 ), the two DHD triads could provide a possible manganese coordination site. Of note, His and Asp residues were demonstrated to directly coordinate binding of Mn 2+ by baMntA, the system's SBP ( 24 ). Figure 5 Essential titratable residues line the translocation cavity of baMntBC. A , shown are side ( left ) and top (all other) views of semitransparent cartoon representations of the baMntBC models. The two images on the left are of the model that is based on the structure of HmuUV, depicting an occluded conformation. BtuD is colored lime , BtuC in light yellow , and the C-terminal helical extension in salmon pink . The two images on the right are based on the structures of MolBC and BtuCD and depict a semiopen and open conformations, respectively. Residues of interest are shown as either spheres (full-size images) or sticks (zoomed images). The twin charged triads, each composed of Asp 47, His 51, and Asp 94, are shown in red and the "hydrophobic ladder" residues (Ile97, F101, F105, Ile109, and Ile112) are in blue . B – E , cultures of ΔmntR cells were grown in the absence ( B and D ) or presence ( C and E ) of 10 μM MnSO 4 . In the absence of manganese, WT baMntBC-A and all tested mutants grew similarly. In the presence of manganese mutations in the putative metal-binding residues Asp 47, His 51, and Asp 94 ( C ) or in hydrophobic seal residues I97, F101, F105, I109, I112, or truncation of the C-terminal helix ( E ) abolished the activity of baMntBC-A and the growth of these mutants was similar to that of controls cells that did not express baMntBC-A or cells expressing the inactive mutant E163A. To test if residues DHD have a role in manganese transport, we generated mutants with alanine substitutions of these three putative metal-binding residues. Of note, because MntBC is a heterodimer of homodimers, a single substitution at the gene level results in two identical amino acid mutations at the protein level. Next, we compared the activity of these single alanine mutants with that of WT baMntBC using the Mn 2+ sensitivity assay described above. As shown in Figure 5 B , in the absence of manganese, the growth of control cells, cells expressing WT baMntBC, or the tested mutants was indistinguishable. In the presence of manganese ( Fig. 5 C ), mutations D47A, H51A, and D94A completely abolished the baMntBC-A-mediated Mn 2+ sensitivity, and the growth of these mutants was identical to that of the vector control and to that of the inactive E163A mutant (which carries a mutation in the essential glutamate of the Walker B motif). The lack of activity of the mutants was not due to improper expression, as their membrane-fraction expression was similar to that of the WT protein ( Fig. S2 A ). These results demonstrate that the titratable residues are essential for Mn 2+ transport and support the suggestion that they may be involved in TM metal coordination. The model shown in Figure 5 A is based on the structure of the heme transporter HmuUV ( 46 ), depicting baMntBC in an occluded conformation. In this conformation, hydrophobic residues (Ile97, F101, F105, Ile109, and Ile112) from TM helices 5 and 5′ form a "hydrophobic ladder" that effectively seals the metal-binding site to the extracellular side of the membrane ( Fig. 5 A , blue spheres). Interestingly, the other two models (based on apo MolBC and AMP-PNP-bound BtuCD) depict baMntBC in intermediate stages of opening toward the extracellular environment, where the width of the narrowest point in the hydrophobic seal increases from 3.6 à (occluded) to 5.4 à (semiopen), and finally to 11.3 à (fully open, top views in Fig. 5 A , as indicated). To test if the bulky residues that comprise the hydrophobic seal are important for transport, we generated single point glycine substitutions. Glycine was chosen because it is the smallest amino acid. Remarkably, replacement of any of the bulky hydrophobic seal residue with glycine completely abolished the activity of baMntBC ( Fig. 5 , D and E ), despite normal membrane-fraction expression of the mutants ( Fig. S2 B ). The relative motions of the residues that comprise the hydrophobic seal and of those that comprise the charged triads are expected to play an important role in the transport mechanism, as will be discussed later. Of note, according to all 3D models, the TMDs of baMntBC are comprised of nine TM helices per monomer ( Fig. 5 A and Fig. S4 ), in contrast to the ten TM helices observed in all crystal structures of baMntBC homologs ( 45 , 46 , 47 ). The suggestion that the number of baMntBC TM helices differs from ten is also supported by all of the TM prediction tools we tested ( Fig. S6 ). Furthermore, all three homology models of baMntBC, as well as the trRosetta template-free model, show an elongated C-terminal TM helix that extends well into the cytoplasm ( Fig. 5 A ). Given that both the N and C termini were modeled using an ab initio approach (see Experimental procedures ), the quality of the model in these regions is questionable. Nevertheless, the amino acid composition of the C terminus is not amphipathic ( Fig. S7 ) and therefore not expected to lie at the membrane–cytosol interface. In comparison, the N-terminal TM helix is clearly amphipathic ( Fig. S7 ) and as depicted in the model is expected to lie at the membrane–extracellular interface ( Fig. 5 A and Figs. S4–S7 ). Further support for the cytosolic location of the C-terminus helical extension is provided by the fact that out of the 21 amino acids that comprise it, six are titratable and six are polar. The net charge of this helical extension is +5, and its cytoplasmic location would be in line with the "positive inside rule" ( 52 , 53 ). This charge distribution is very different from that observed in all other predicted TM helices of MntC that are heavily enriched in hydrophobic residues ( Figs. S6 and S7 ). To test if the cytoplasmic extension of this C-terminal TM helix was important for function, we truncated it at the membrane–cytosol interface to generate the MntBC-ΔC mutant. Surprisingly, the truncation did not affect the membrane-expression of MntBC ( Fig. S2 B ). However, as judged by manganese-sensitivity assays, the truncated variant was completely inactive ( Fig. 5 , D and E , green traces). Uncoupled ATPase activity of baMntBC ABC transporters that function as importers are divided into two subgroups: type I importers typically import sugars, amino acids, and peptides, whereas type II systems import organometallic complexes such as heme, siderophores, and vitamin B 12 ( 20 , 42 , 54 , 55 ). These two subclasses have been shown to operate by very distinct mechanisms. One central distinctive mechanistic feature is the basal rate of ATPase activity and its modulation by the SBP. In type I systems, the basal rates of ATP hydrolysis are very low, and these are greatly stimulated (10- to 30-fold) upon docking of the substrate-loaded SBP ( 33 , 56 ). In contrast, type-II systems have very high rates of basal ATP hydrolysis that are largely insensitive to substrate loading ( 46 , 57 , 58 ). The extent of substrate modulation of ATPase activity in transition metal ABC importers such as baMntBC is unknown. To study this phenomenon, we codon-optimized the nucleotide sequence of baMntBC for expression in E. coli and synthetically generated codon-optimized coding regions for mntBC (GenScript). We then prepared inverted membrane vesicles ( 59 ) from cells transformed with an empty control plasmid or plasmids encoding WT or mutant (E163A) baMntBC. We reasoned that owing to the high content of baMntBC in these vesicles (inset in Fig. 6 A ), we may be able to measure its ATPase activity without the need of detergent-mediated extraction/purification with its possible pitfalls ( 60 ). Indeed, as shown in Figure 6 A , the ATPase activity of baMntBC-inverted membrane vesicles was ∼5-fold higher than that of vesicles prepared from control cells that were transformed with an empty control plasmid. To verify that this activity is indeed specific to baMntBC, we repeated these experiments with the ATPase-deficient mutant baMntBC (E163A). Despite the high protein content of baMntBC (E163A) vesicles (inset in Fig. 6 A ), their ATPase activity was identical to that of the control vesicles that did not contain any baMntBC ( Fig. 6 A ). Figure 6 Uncoupled ATP hydrolysis by baMntBC. A , 15 μg of inverted membrane vesicles were incubated for 2 min with 1 mM ATP, and to initiate hydrolysis, 2 mM MgSO 4 was injected at 2 min. The rate of release of inorganic phosphate was determined by continuous monitoring of the 360 nm absorbance of the solution using the EnzChek kit. Shown is the activity measured for vesicles containing WT baMntBC ( black ), WT baMntBC+ baMntA ( red ), WT baMntBC+ 10 μM baMntA+ 50 μM MnSO 4 ( blue ), mutant E163A ( magenta ), or control vesicles ( gray ). Results are representative of experiments conducted at least three times. The inset shows an immunoblot of SDS page of the vesicles, and BtuCD (used as benchmark for high expression level) and WT or mutant E163A are shown as indicated. B and C , initial rates of hydrolysis of 15 to 1000 μM ATP were measured (in the presence of baMntA and MnSO 4 ) for control ( B ) and WT baMntBC ( C ) vesicles. D , the initial rates of ATP hydrolysis measured in panel B for the control vesicles were subtracted from the rates measured for WT baMntBC in panel C . The net values were plotted as a function of the ATP concentration, and the data were then fit using the Michaelis–Menten equation ( dashed line ). Also shown are the mean values (n = 3) of the kinetic rate constants, and the error bars represent SDs of the mean. We next used the vesicles system to investigate the effects of the SBP on ATP hydrolysis by the transporter. For this, codon-optimized baMntA was overexpressed and purified in E. coli as previously described ( 24 ) and incorporated into the vesicles' lumen at approximately ∼10-fold molar excess over baMntBC (see Experimental procedures for details). The SBP did not stimulate the ATPase activity of the transporter, and its incorporation led to a mild inhibitory effect ( Fig. 6 A ). We then repeated these experiments incorporating also manganese into the vesicles' lumen at a 50:10:1 manganese-baMntA-baMntBC molar ratio and observed a similarly modest inhibitory effect ( Fig. 6 A ). Collectively, these results suggest that similar to the type II ABC importer BtuCD, yet unlike the type I ABC importers MalFGK, YecSC, and HisPQM, baMntBC has high basal ATPase activity that is not further stimulated by the SBP–substrate complex ( 33 , 36 , 56 , 57 , 61 ). To determine the kinetic rate constants of ATP hydrolysis, we measured the initial rates of activity under a range of ATP concentrations. As shown, at ATP concentrations of 15 to 1000 μM, the initial rates of ATP hydrolysis were linear for at least 2 min ( Fig. 6 , B and C ). To determine the specific ATPase activity of baMntBC, we subtracted the corresponding activity measured in the control vesicles. The net rates of ATP hydrolysis were then plotted as a function of ATP concentration, and the data were fit using the Michaelis–Menten equation ( Fig. 6 D ). Adding the term for the Hill coefficient yielded an n HILL = 1.08, indicating that the two ATP-binding sites of baMntBC do not hydrolyze ATP cooperatively. This differs from the cooperative ATP hydrolysis that was reported for both type I and type II ABC importers ( e.g. , MetNI, HisPQM, MalFGK, YecSC, and BtuCD) ( 33 , 36 , 61 , 62 , 63 ). Notably, even in a membrane environment, baMntBC has very high rates of uncoupled (absence of the substrate and SBP) ATPase activity ( k cat = 1.85 s −1 ), similar to the SBP–substrate–stimulated ATPase rates reported for type I importers ( 56 , 61 ). Conclusions In recent years, it is becoming increasingly clear that manganese plays an important role in host–pathogen interactions. Transport and homeostasis of manganese was shown to be essential for the virulence of key bacterial pathogens, such as Enterococcus faecalis , Staphylococcus aureus , group A Streptococcus , Acinetobacter baumannii , Mycobacterium tuberculosis , S. pneumonia , and many others ( 14 , 15 , 16 , 25 , 64 , 65 , 66 , 67 , 68 , 69 , 70 ). Herein, we determined that baMntBC-A transports only manganese and not any other tested metals. Considering that baMntA is absolutely essential for the virulence of B. anthracis ( 10 ) means that the high-affinity acquisition of manganese is a major rate-limiting step in the progression of anthrax. This observation may have interventional potential as had been reported for PsaA of S. pneumonia ( 11 ). In this respect, it is noteworthy that the release kinetics of the metal from the SBP appear to play an essential role in determining the transport specificity on the one hand and the inhibitory potential on the other: for both S. pneumonia PsaA and baMntA, it was found that zinc is recognized with a similar affinity as manganese yet is released from the SBP at a much slower rate ( 24 , 40 ). In both cases, zinc is not transported and acts as an inhibitor. This strategy of using slow-releasing cognate binders of SBPs as inhibitors may be generally applicable also to inhibition of other ABC transporters. Nickel and cobalt are also recognized by baMntA, the latter with ∼10-fold higher affinity than zinc or manganese. Despite that, neither nickel nor cobalt inhibits manganese import by baMntBC. This difference likely stems from the preferred coordination number of the metals, as has been elegantly demonstrated for the PsaA of S. pneumonia ( 40 ). Manganese, cobalt, and nickel all prefer hexa-coordinate ligation, whereas zinc's preferred coordination number is 4 ( 71 , 72 , 73 ). The tetrahedral coordination provided by the metal-ligating residues of baMntA optimally satisfies zinc's preferred coordination, whereas binding of manganese, nickel, and cobalt is less favorable. This leads to an almost irreversible binding of zinc, yet reversible binding of manganese, nickel, and cobalt. This is perhaps also the reason why throughout evolution, amoeba, protozoa, and macrophages use zinc to kill phagocytosed bacteria rather than cobalt or nickel ( 74 , 75 ). On the other side, bacteria use multiple approaches for evading zinc inhibition of manganese import. For example, MntR, the manganese homeostasis master regulator, responds only to manganese, and not to zinc ( 76 ). This means that MntA will not be unnecessarily exposed to zinc. In addition, bacteria do not live in a thermodynamic equilibrium reality but rather under constant pre-equilibrium conditions. In such a shifting reality, the faster k on of manganese binding to MntA relative to zinc ( 24 ) may provide selectivity favoring the former. The closest available structural templates for modeling of baMntBC were all type II ABC importers, suggesting that baMntBC adopts the type II fold. Similar conclusions can be drawn from the trRosetta template-independent modeling, which yielded a very similar structure. Additional observations also support the notion that ABC importers of transition metals are related to type II ABC importers. These include the similarity in their cognate SBPs structures, and the fact that unlike SBPs of type I systems, SBPs of transition metals do not undergo large conformational changes upon ligand binding ( 77 , 78 , 79 ). The high uncoupled ATPase activity of baMntBC-A ( Fig. 6 ) also suggests a similarity to type II ABC importers ( 46 , 57 , 58 ). However, these results need to be interpreted with caution as this ATPase activity was measured in E. coli membranes that may lack unknown B. subtilis components. In conclusion, our findings suggest that baMntBC-A and likely other ABC importers of transition metals contain unique motifs of two triads of titratable residues and a hydrophobic plug. We propose that these motifs have evolved specifically for the TM translocation of high-density charges. Considering their direct relevance to bacterial virulence and pathogenesis, and the emerging power of single-particle cryo-EM analysis, we propose that their structure–function analysis is timely. Experimental procedures Plasmids The baMntBC-A operon was amplified by PCR from the genome of B. anthracis Vollum strain and cloned into the pDR111 (BGSC) vector for expression in B. subtilis , downstream to the IPTG-inducible promoter. Point mutations were introduced into WT baMntBC-A by using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). Mutations were confirmed by sequencing. B. subtilis strains B. subtilis Genotype and description Origin 168 trpC2 (WT strain) BGSC ΔmntR mntR::erm trpC2 (KO of strain 168 locus BSU24520) BGSC Transformation to B. subtilis To construct B. subtilis strains expressing baMntBC-A, a transformation protocol for genomic integration was used. Briefly, a single colony of WT or ΔmntR B. subtilis was picked from the LB plate into transformation buffer containing 1× MC buffer and 1 mM MgSO 4 (10× MC buffer containing 0.62 M K 2 HPO 4 , 0.38 M KH 2 PO 4 , 20% (w/v) glucose, 30 mM trisodium citrate, 0.022 mg/ml ferric ammonium citrate, 1% (w/v) casein hydrolysate, and 2% (w/v) potassium glutamate) and incubated at 37 °C for 4.5 h with shaking. One microgram of DNA was added, and the sample was further incubated for 1.5 h at 37 °C. Samples were plated on LB plates supplemented with the 150 μg/ml spectinomycin, and plates were incubated at 37 °C overnight. Single colonies were picked, baMntBC-A insertion was verified by PCR, and positive colonies were kept in glycerol stock at −80 °C until use. Manganese sensitivity assays B. subtilis ΔmntR transformed with control (pDR111) or baMntBC-A strains were grown in LB supplemented with 150 μg/ml spectinomycin at 37 °C. Cells were diluted to A 600 of 0.05, and 0.15 ml cultures were grown with 1 mM IPTG in the absence or presence of the indicated metal in an automated plate reader (Infinite M200 Pro; Tecan). All metals were added as sulfate salts unless otherwise indicated. The absorbance of the cultures was measured every 10 min for 12 h. All assays were performed in triplicates. For metal sensitivity assays on solid media, cells were grown overnight on LB supplemented with 150 μg/ml spectinomycin at 37 °C. Five dilutions were performed and applied dropwise (1.5 μl) on top of LB plates with the indicated metal concentrations. ICP-MS Metals were added to midexponential phase cultures for 15 min at final concentrations of 50 to 1000 μM, as indicated (all metals were added as sulfate salts unless otherwise indicated). Cells were then harvested and washed with 1× PBS in the presence of 5 mM EDTA. The pellets were resuspended in 69% HNO 3 and incubated in 100 °C dry block until complete evaporation. The remaining dry biomass was resuspended in 3% HNO 3 to yield 5 to 250 ppm, and the final read was corrected for the dilution factor. All ICP-MS measurements were performed in biological triplicates using Agilent 7500cx ICP-MS with a dynamic range of 0.1 ppt to 2500 ppm. Preparation of inverted membrane vesicles Cell pellets were washed once with 50 mM Tris HCl, pH 7.5, and 0.5 M NaCl and resuspended (20% w/v) in 50 mM Tris HCl, pH 7.5, 0.5 M NaCl, 30 μg/ml DNase (Worthington), one EDTA-free protease inhibitor cocktail tablet (Roche), 1 mM CaCl 2 , and 1 mM MgCl 2 and ruptured first by tip-sonication and then using an EmulsiFlex-C3 homogenizer (Avestin) at 12,000 psi external pressure. Debris and unbroken cells were removed by centrifugation (10,000 g for 20 min), and the membranes were collected by ultracentrifugation at 135,000 g for 30 min, washed once in 25 mM Tris HCl, pH 7.5, 0.1 M NaCl, and resuspended in the same buffer to ∼1 mg/ml. ATPase assays ATP hydrolysis was measured using Molecular Probes EnzChek kit, at 37 °C, in a 96-well format, according to the manufacturer's specifications. To initiate hydrolysis, 5 mM MgCl 2 was injected to a solution containing 15 μg of inverted membrane vesicles in 25 mM Tris HCl, pH 7.5, 0.1 M NaCl, 50 μM EDTA, and the indicated ATP concentration. Data were fitted using either the Michaelis–Menten equation or its expanded version, which includes also a term for the Hill coefficient: V = V max [ S ] n [ S ] n + K m Where V is the observed hydrolysis rate, V max is the maximal hydrolysis rate, K m is the Michaelis–Menten constant, [ S ] is the concentration of ATP, and n is the Hill coefficient. baMntBC modeling Searching for structural templates The amino acid sequences of baMntB and baMntC were used as queries in two independent HHpred ( 80 ) searches. For baMntB, the best scoring templates included the NBDs of H. influenza's putative molybdate/tungstate importer MolBC in an inward-facing conformation (PDB ID 2NQ2 ); Y. pestis's heme importer HmuUV in an outward-apo conformation (PDB ID: 4G1U ); and E. coli's vitamin B 12 importer BtuCD (PDB ID: 4R9U ). All three templates showed a 28% sequence identity to baMntB. The best scoring templates for baMntC included the same three templates collected for baMntB, only with much lower sequence identity, specifically 14%, 15%, and 13% for 2NQ2, 4G1U, and 4R9U, respectively. Sequence search and multiple sequence alignment ConSurf ( 50 ) was used to collect independent sets of homolog sequences for baMntB and baMntC and each of the two domains (NBD and TMD) of the three structural templates. Homolog searching was conducted using HMMER ( 81 ), with an E-value of 0.0001, maximal sequence identity of 95%, and minimal sequence identity of 15% against the clean UniProt database. 500 sequences that sample the list of homologs were chosen automatically by ConSurf and were aligned using MAFFT ( 82 ), with default parameters. Fragmented sequences and sequences that introduced large gaps in the multiple sequence alignment (MSA) were then manually removed, and the remaining sequences were realigned using MAFFT. The final MSAs contained 410 homologs for baMntB; 430 homologs for baMntC; 374 homologs for 2NQ2's NBD and 434 for its TMD; 355 homologs for 4G1U's NBD and 355 for its TMD; and 370 homologs for 4R9U's NBD and 392 for its TMD. Next, MAFFT was used to perform profile-to-profile MSA between baMntB/baMntC and each of their three templates. From these MSAs, pairwise alignments between baMntB/baMntC and each template were deduced. Improving the pairwise alignment between baMntC and its templates Given the low query-to-template sequence identity between baMntC and its templates, we used evolutionary conservation analysis alongside multiple computational tools to independently assign TM helices and manually improve our pairwise alignments. Specifically, we used HMMTOP, Phobius, TMHMM, TopGraph ( 83 ), MEMSAT-SVM ( 84 ), TOPCONS ( 85 ), RaptorX ( 86 , 87 ), and ConSurf ( 50 ) ( Figs. S4 and S6 ). Adjustments were made mainly in the regions of the second and fourth predicted helices (amino acids 40-to-67 and 89-to-115, respectively). The improved pairwise alignments were then used for the modeling process. Constructing the 3D models MODELLER 9.18 ( 88 ) with default settings was used to produce the 3D models of baMntB. Each model was built using one template. For constructing the 3D models of baMntC, each of the three templates identified by HHpred was used alongside an ab initio model produced by RaptorX. The latter was mainly used to model gapped regions such as the N and C termini. For each model, 100 different structures were produced. A short energy minimization was conducted using GROMACS 5.1 ( 89 ) and the AMBER99SB-ILDN force field ( 90 ). The model with the predicted lowest energy was then selected. An additional template-independent model was constructed using trRosetta ( 48 ) that models a protein's structure using neural network to predict inter-residue distance and orientation distributions based on coevolution data. Default parameters were used with the no-template option. The resulting model was subjected to a short energy minimization similar to the homology models. Overall, both trRosetta and the homology modeling converged to similar structures. The main difference between the models was the position of TM-4 relative to the other helices ( Fig. S5 A ). As such, two (F105 and I109) of the four residues suggested to compose MntC's hydrophobic ladder face the core of each monomer rather than the interface ( Fig. S5 B ). The overall RMSD when superimposing trRosetta's model to the three homology models ranged between 4.83 and 5.6 à ( Table S1 ). Importantly, the trRosetta calculations account for evolutionary coupling only within the chain. Thus, coupling between the two monomers in MntC's structure is not taken into account. This means that any constrains that relate to the translocation pathway situated in the interface between the two monomers are ignored by the trRosetta calculations. Note added in proof After this article was accepted for publication, the X-ray crystal structure of a close homolog of baMntBC was published, namely, Streptococcus pneumoniae PsaBC manganese transporter (PDB entry: 7KYP; PMID: 34362732). Notably, the three baMntBC models presented in this work, which were constructed based on remote homologs, show remarkable resemblance to this new structure. Specifically, the membrane domains superimpose with an RMSD of 3.4 to 3.8 à and the ATPase domains with an RMSD of 1.8 to 2.0 à . Importantly, the orientation of residues consisting the hydrophobic ladder and those proposed to mediate ion coordination was predicted with high accuracy. Thus, the Streptococcus pneumoniae PsaBC structure further consolidates the hypotheses and conclusions presented in this work. Plasmids The baMntBC-A operon was amplified by PCR from the genome of B. anthracis Vollum strain and cloned into the pDR111 (BGSC) vector for expression in B. subtilis , downstream to the IPTG-inducible promoter. Point mutations were introduced into WT baMntBC-A by using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies). Mutations were confirmed by sequencing. B. subtilis strains B. subtilis Genotype and description Origin 168 trpC2 (WT strain) BGSC ΔmntR mntR::erm trpC2 (KO of strain 168 locus BSU24520) BGSC Transformation to B. subtilis To construct B. subtilis strains expressing baMntBC-A, a transformation protocol for genomic integration was used. Briefly, a single colony of WT or ΔmntR B. subtilis was picked from the LB plate into transformation buffer containing 1× MC buffer and 1 mM MgSO 4 (10× MC buffer containing 0.62 M K 2 HPO 4 , 0.38 M KH 2 PO 4 , 20% (w/v) glucose, 30 mM trisodium citrate, 0.022 mg/ml ferric ammonium citrate, 1% (w/v) casein hydrolysate, and 2% (w/v) potassium glutamate) and incubated at 37 °C for 4.5 h with shaking. One microgram of DNA was added, and the sample was further incubated for 1.5 h at 37 °C. Samples were plated on LB plates supplemented with the 150 μg/ml spectinomycin, and plates were incubated at 37 °C overnight. Single colonies were picked, baMntBC-A insertion was verified by PCR, and positive colonies were kept in glycerol stock at −80 °C until use. Manganese sensitivity assays B. subtilis ΔmntR transformed with control (pDR111) or baMntBC-A strains were grown in LB supplemented with 150 μg/ml spectinomycin at 37 °C. Cells were diluted to A 600 of 0.05, and 0.15 ml cultures were grown with 1 mM IPTG in the absence or presence of the indicated metal in an automated plate reader (Infinite M200 Pro; Tecan). All metals were added as sulfate salts unless otherwise indicated. The absorbance of the cultures was measured every 10 min for 12 h. All assays were performed in triplicates. For metal sensitivity assays on solid media, cells were grown overnight on LB supplemented with 150 μg/ml spectinomycin at 37 °C. Five dilutions were performed and applied dropwise (1.5 μl) on top of LB plates with the indicated metal concentrations. ICP-MS Metals were added to midexponential phase cultures for 15 min at final concentrations of 50 to 1000 μM, as indicated (all metals were added as sulfate salts unless otherwise indicated). Cells were then harvested and washed with 1× PBS in the presence of 5 mM EDTA. The pellets were resuspended in 69% HNO 3 and incubated in 100 °C dry block until complete evaporation. The remaining dry biomass was resuspended in 3% HNO 3 to yield 5 to 250 ppm, and the final read was corrected for the dilution factor. All ICP-MS measurements were performed in biological triplicates using Agilent 7500cx ICP-MS with a dynamic range of 0.1 ppt to 2500 ppm. Preparation of inverted membrane vesicles Cell pellets were washed once with 50 mM Tris HCl, pH 7.5, and 0.5 M NaCl and resuspended (20% w/v) in 50 mM Tris HCl, pH 7.5, 0.5 M NaCl, 30 μg/ml DNase (Worthington), one EDTA-free protease inhibitor cocktail tablet (Roche), 1 mM CaCl 2 , and 1 mM MgCl 2 and ruptured first by tip-sonication and then using an EmulsiFlex-C3 homogenizer (Avestin) at 12,000 psi external pressure. Debris and unbroken cells were removed by centrifugation (10,000 g for 20 min), and the membranes were collected by ultracentrifugation at 135,000 g for 30 min, washed once in 25 mM Tris HCl, pH 7.5, 0.1 M NaCl, and resuspended in the same buffer to ∼1 mg/ml. ATPase assays ATP hydrolysis was measured using Molecular Probes EnzChek kit, at 37 °C, in a 96-well format, according to the manufacturer's specifications. To initiate hydrolysis, 5 mM MgCl 2 was injected to a solution containing 15 μg of inverted membrane vesicles in 25 mM Tris HCl, pH 7.5, 0.1 M NaCl, 50 μM EDTA, and the indicated ATP concentration. Data were fitted using either the Michaelis–Menten equation or its expanded version, which includes also a term for the Hill coefficient: V = V max [ S ] n [ S ] n + K m Where V is the observed hydrolysis rate, V max is the maximal hydrolysis rate, K m is the Michaelis–Menten constant, [ S ] is the concentration of ATP, and n is the Hill coefficient. baMntBC modeling Searching for structural templates The amino acid sequences of baMntB and baMntC were used as queries in two independent HHpred ( 80 ) searches. For baMntB, the best scoring templates included the NBDs of H. influenza's putative molybdate/tungstate importer MolBC in an inward-facing conformation (PDB ID 2NQ2 ); Y. pestis's heme importer HmuUV in an outward-apo conformation (PDB ID: 4G1U ); and E. coli's vitamin B 12 importer BtuCD (PDB ID: 4R9U ). All three templates showed a 28% sequence identity to baMntB. The best scoring templates for baMntC included the same three templates collected for baMntB, only with much lower sequence identity, specifically 14%, 15%, and 13% for 2NQ2, 4G1U, and 4R9U, respectively. Sequence search and multiple sequence alignment ConSurf ( 50 ) was used to collect independent sets of homolog sequences for baMntB and baMntC and each of the two domains (NBD and TMD) of the three structural templates. Homolog searching was conducted using HMMER ( 81 ), with an E-value of 0.0001, maximal sequence identity of 95%, and minimal sequence identity of 15% against the clean UniProt database. 500 sequences that sample the list of homologs were chosen automatically by ConSurf and were aligned using MAFFT ( 82 ), with default parameters. Fragmented sequences and sequences that introduced large gaps in the multiple sequence alignment (MSA) were then manually removed, and the remaining sequences were realigned using MAFFT. The final MSAs contained 410 homologs for baMntB; 430 homologs for baMntC; 374 homologs for 2NQ2's NBD and 434 for its TMD; 355 homologs for 4G1U's NBD and 355 for its TMD; and 370 homologs for 4R9U's NBD and 392 for its TMD. Next, MAFFT was used to perform profile-to-profile MSA between baMntB/baMntC and each of their three templates. From these MSAs, pairwise alignments between baMntB/baMntC and each template were deduced. Improving the pairwise alignment between baMntC and its templates Given the low query-to-template sequence identity between baMntC and its templates, we used evolutionary conservation analysis alongside multiple computational tools to independently assign TM helices and manually improve our pairwise alignments. Specifically, we used HMMTOP, Phobius, TMHMM, TopGraph ( 83 ), MEMSAT-SVM ( 84 ), TOPCONS ( 85 ), RaptorX ( 86 , 87 ), and ConSurf ( 50 ) ( Figs. S4 and S6 ). Adjustments were made mainly in the regions of the second and fourth predicted helices (amino acids 40-to-67 and 89-to-115, respectively). The improved pairwise alignments were then used for the modeling process. Constructing the 3D models MODELLER 9.18 ( 88 ) with default settings was used to produce the 3D models of baMntB. Each model was built using one template. For constructing the 3D models of baMntC, each of the three templates identified by HHpred was used alongside an ab initio model produced by RaptorX. The latter was mainly used to model gapped regions such as the N and C termini. For each model, 100 different structures were produced. A short energy minimization was conducted using GROMACS 5.1 ( 89 ) and the AMBER99SB-ILDN force field ( 90 ). The model with the predicted lowest energy was then selected. An additional template-independent model was constructed using trRosetta ( 48 ) that models a protein's structure using neural network to predict inter-residue distance and orientation distributions based on coevolution data. Default parameters were used with the no-template option. The resulting model was subjected to a short energy minimization similar to the homology models. Overall, both trRosetta and the homology modeling converged to similar structures. The main difference between the models was the position of TM-4 relative to the other helices ( Fig. S5 A ). As such, two (F105 and I109) of the four residues suggested to compose MntC's hydrophobic ladder face the core of each monomer rather than the interface ( Fig. S5 B ). The overall RMSD when superimposing trRosetta's model to the three homology models ranged between 4.83 and 5.6 à ( Table S1 ). Importantly, the trRosetta calculations account for evolutionary coupling only within the chain. Thus, coupling between the two monomers in MntC's structure is not taken into account. This means that any constrains that relate to the translocation pathway situated in the interface between the two monomers are ignored by the trRosetta calculations. Searching for structural templates The amino acid sequences of baMntB and baMntC were used as queries in two independent HHpred ( 80 ) searches. For baMntB, the best scoring templates included the NBDs of H. influenza's putative molybdate/tungstate importer MolBC in an inward-facing conformation (PDB ID 2NQ2 ); Y. pestis's heme importer HmuUV in an outward-apo conformation (PDB ID: 4G1U ); and E. coli's vitamin B 12 importer BtuCD (PDB ID: 4R9U ). All three templates showed a 28% sequence identity to baMntB. The best scoring templates for baMntC included the same three templates collected for baMntB, only with much lower sequence identity, specifically 14%, 15%, and 13% for 2NQ2, 4G1U, and 4R9U, respectively. Sequence search and multiple sequence alignment ConSurf ( 50 ) was used to collect independent sets of homolog sequences for baMntB and baMntC and each of the two domains (NBD and TMD) of the three structural templates. Homolog searching was conducted using HMMER ( 81 ), with an E-value of 0.0001, maximal sequence identity of 95%, and minimal sequence identity of 15% against the clean UniProt database. 500 sequences that sample the list of homologs were chosen automatically by ConSurf and were aligned using MAFFT ( 82 ), with default parameters. Fragmented sequences and sequences that introduced large gaps in the multiple sequence alignment (MSA) were then manually removed, and the remaining sequences were realigned using MAFFT. The final MSAs contained 410 homologs for baMntB; 430 homologs for baMntC; 374 homologs for 2NQ2's NBD and 434 for its TMD; 355 homologs for 4G1U's NBD and 355 for its TMD; and 370 homologs for 4R9U's NBD and 392 for its TMD. Next, MAFFT was used to perform profile-to-profile MSA between baMntB/baMntC and each of their three templates. From these MSAs, pairwise alignments between baMntB/baMntC and each template were deduced. Improving the pairwise alignment between baMntC and its templates Given the low query-to-template sequence identity between baMntC and its templates, we used evolutionary conservation analysis alongside multiple computational tools to independently assign TM helices and manually improve our pairwise alignments. Specifically, we used HMMTOP, Phobius, TMHMM, TopGraph ( 83 ), MEMSAT-SVM ( 84 ), TOPCONS ( 85 ), RaptorX ( 86 , 87 ), and ConSurf ( 50 ) ( Figs. S4 and S6 ). Adjustments were made mainly in the regions of the second and fourth predicted helices (amino acids 40-to-67 and 89-to-115, respectively). The improved pairwise alignments were then used for the modeling process. Constructing the 3D models MODELLER 9.18 ( 88 ) with default settings was used to produce the 3D models of baMntB. Each model was built using one template. For constructing the 3D models of baMntC, each of the three templates identified by HHpred was used alongside an ab initio model produced by RaptorX. The latter was mainly used to model gapped regions such as the N and C termini. For each model, 100 different structures were produced. A short energy minimization was conducted using GROMACS 5.1 ( 89 ) and the AMBER99SB-ILDN force field ( 90 ). The model with the predicted lowest energy was then selected. An additional template-independent model was constructed using trRosetta ( 48 ) that models a protein's structure using neural network to predict inter-residue distance and orientation distributions based on coevolution data. Default parameters were used with the no-template option. The resulting model was subjected to a short energy minimization similar to the homology models. Overall, both trRosetta and the homology modeling converged to similar structures. The main difference between the models was the position of TM-4 relative to the other helices ( Fig. S5 A ). As such, two (F105 and I109) of the four residues suggested to compose MntC's hydrophobic ladder face the core of each monomer rather than the interface ( Fig. S5 B ). The overall RMSD when superimposing trRosetta's model to the three homology models ranged between 4.83 and 5.6 à ( Table S1 ). Importantly, the trRosetta calculations account for evolutionary coupling only within the chain. Thus, coupling between the two monomers in MntC's structure is not taken into account. This means that any constrains that relate to the translocation pathway situated in the interface between the two monomers are ignored by the trRosetta calculations. Note added in proof After this article was accepted for publication, the X-ray crystal structure of a close homolog of baMntBC was published, namely, Streptococcus pneumoniae PsaBC manganese transporter (PDB entry: 7KYP; PMID: 34362732). Notably, the three baMntBC models presented in this work, which were constructed based on remote homologs, show remarkable resemblance to this new structure. Specifically, the membrane domains superimpose with an RMSD of 3.4 to 3.8 à and the ATPase domains with an RMSD of 1.8 to 2.0 à . Importantly, the orientation of residues consisting the hydrophobic ladder and those proposed to mediate ion coordination was predicted with high accuracy. Thus, the Streptococcus pneumoniae PsaBC structure further consolidates the hypotheses and conclusions presented in this work. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Supporting information This article contains supporting information Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article. Supporting information Supplemental Figures S1–S7 and Table S1 Author contributions A. K., E. V., N. L.-L., J. R., M. G., A. B., and O. L. investigation; A. K., J. R., M. G., A. B., and O. L. visualization; G. M. data curation; G. M. formal analysis; G. M., E. V., N. L.-L., and O. L. methodology; N. L.-L., J. G. Y., D. C. R., N. B.-T., and O. L. supervision; J. G. Y., D. C. R., N. B.-T., and O. L. project administration; D. C. R., N. B.-T., and O. L. funding acquisition; O. L. conceptualization; O. L. and N. B.-T. validation; O. L. writing–original draft; O. L. and N. B.-T. writing–review and editing. Funding and additional information This work was supported by grants from the NATO Science for Peace and Security Program (SPS 625 Project G4622, to O. L. and N. B.-T.), the US-Israel Binational Science Foundation (BSF grant 2015102 to O. L. and D. C. R.), and the Israel Academy of Sciences and Humanities project 1006/18 (to O. L.).

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2680498/

Strategies toward vaccines against Burkholderia mallei and Burkholderia pseudomallei

Burkholderia mallei and Burkholderia pseudomallei are Gram-negative, rod-shaped bacteria, and are the causative agents of the diseases glanders and melioidosis, respectively. These bacteria have been recognized as important pathogens for over 100 years, yet a relative dearth of available information exists regarding their virulence determinants and immunopathology. Infection with either of these bacteria presents with nonspecific symptoms and can be either acute or chronic, impeding rapid diagnosis. The lack of a vaccine for either bacterium also makes them potential candidates for bioweaponization. Together with their high rate of infectivity via aerosols and resistance to many common antibiotics, both bacteria have been classified as category B priority pathogens by the US NIH and US CDC, which has spurred a dramatic increase in interest in these microorganisms. Attempts have been made to develop vaccines for these infections, which would not only benefit military personnel, a group most likely to be targeted in an intentional release, but also individuals who may come in contact with glanders-infected animals or live in areas where melioidosis is endemic. This review highlights some recent attempts of vaccine development for these infections and the strategies used to improve the efficacy of vaccine approaches.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103945/

Redefining syndromic surveillance

With growing concerns about international spread of disease and expanding use of early disease detection surveillance methods, the field of syndromic surveillance has received increased attention over the last decade. The purpose of this article is to clarify the various meanings that have been assigned to the term syndromic surveillance and to propose a refined categorization of the characteristics of these systems. Existing literature and conference proceedings were examined on syndromic surveillance from 1998 to 2010, focusing on low- and middle-income settings. Based on the 36 unique definitions of syndromic surveillance found in the literature, five commonly accepted principles of syndromic surveillance systems were identified, as well as two fundamental categories: specific and non-specific disease detection. Ultimately, the proposed categorization of syndromic surveillance distinguishes between systems that focus on detecting defined syndromes or outcomes of interest and those that aim to uncover non-specific trends that suggest an outbreak may be occurring. By providing an accurate and comprehensive picture of this field's capabilities, and differentiating among system types, a unified understanding of the syndromic surveillance field can be developed, encouraging the adoption, investment in, and implementation of these systems in settings that need bolstered surveillance capacity, particularly low- and middle-income countries. 1. Introduction The field of syndromic surveillance is best understood through the context of global efforts to respond and adapt to modern-day surveillance challenges and disease threats. Globalization and the ease of international spread of disease require improved global surveillance capacity in order to rapidly detect and contain public health emergencies. Recognition of this need has led to increased efforts to enhance disease surveillance and demands examination of all available tools—one of which is syndromic surveillance. Such thinking is exemplified by the World Health Organization's (WHO) decision to revise the International Health Regulations (IHR). As part of the 10-year IHR revision process, WHO sponsored a pilot study in 22 countries from 1997 to 1999 to evaluate syndromic reporting. It was concluded that "syndromic reporting, although valuable within a national system, was not appropriate for use in the context of a regulatory framework" [ 1 ]. The final negotiated IHR (2005) regulates detection, reporting and response within a more adaptive category of "events that may constitute a public health emergency of international concern" [ 2 ]. The IHR (2005) also obligates every member state of the WHO to build national core competency for disease surveillance. However, the regulations do not prescribe exactly how nations are to meet this core capacity. Certain low- and middle-income countries—particularly those facing the need to rapidly strengthen disease surveillance and overall public health infrastructure to meet their IHR (2005) obligations—may be looking to syndromic surveillance options and opportunities. A report from the IHR (2005) negotiations stresses this point: "Because areas with the highest needs for surveillance of communicable diseases have often the poorest surveillance systems, new surveillance approaches, such as the surveillance of syndromes, adapted to poor laboratory infrastructure should be developed to respond to the challenge of development gaps" [ 3 ]. In addition to the increased attention to syndromic surveillance in the negotiations of IHR (2005), syndromic surveillance has gained importance for national governments and has become widely used at the country level, particularly in high-income countries. Examples include a syndromic surveillance system in the United Kingdom based on data from the national telehealth system (NHS Direct) and a system in Denmark that utilizes ambulance dispatch records [ 4 ]. In the United States (US), state and local syndromic surveillance systems are widespread [ 5 ], as evidenced by a recent survey that concluded, "populations covered by health departments that reported conducting syndromic surveillance account for 72% of the US population" [ 6 ]. Spurred by a series of reports from the US Government Accountability Office [ 7 ] and other organizations [ 8 ], the US federal government has recently begun re-examining how to best ensure effective and efficient disease surveillance capacity. In this context, the Centers for Disease Control and Prevention (CDC) must re-evaluate biosurveillance for human health [ 9 ]. The United States Agency for International Development's (USAID) PREDICT program aims to integrate human and animal disease surveillance, primarily focused on the implementation of programs in developing nations [ 10 ]. Researchers in the field of syndromic surveillance have similarly turned toward translating syndromic surveillance for use in lower resource settings [ 11 – 13 ]. A 2007 Disease Surveillance Workshop held in Bangkok, Thailand, sponsored by the Department of Defense Global Emerging Infections Surveillance and Response System (DOD-GEIS) and Johns Hopkins University/Applied Physics Laboratory (JHU/APL), focused on the adaptation of electronic surveillance tools to low- and middle-income settings [ 11 ]. In order to reenergize these discussions (which were begun by 13 countries and other stakeholders during the 2007 workshop), clarification of the definition, functions, and challenges of syndromic surveillance is needed. Doing so would help ensure that syndromic surveillance will be adopted in all places where it would be of benefit. Since syndromic surveillance systems began being used in the 1990s and became widespread in early 2000, vast applications of these systems have demonstrated many capabilities and uses. There are numerous variations among syndromic surveillance system definitions, objectives, and surveillance methodologies, which is why there is a need for a comprehensive characterization of the breadth of the term "syndromic surveillance." Common themes across the literature have emerged, suggesting general agreement among those in the field. The commonly accepted principles of syndromic surveillance include: • Early detection and response: Most articles on syndromic surveillance discuss the value of these systems in signaling the presence of an abnormal trend with "sufficient probability" to warrant further investigation (without necessarily providing definitive detection) [ 14 – 20 ]. • Use of "continuously acquired" pre-diagnostic information: By focusing on data collected prior to clinical diagnosis or laboratory confirmation, syndromic surveillance uses non-traditional health indicator data [ 17 , 21 , 22 ]. • Possible situational awareness use: Chretien and his co-authors describe situational awareness as "monitoring the effectiveness of epidemic responses and characterizing affected populations" [ 12 ]. By providing a tool for following the course of an outbreak, syndromic surveillance has value besides merely initial detection in augmenting public health surveillance as well as outbreak response [ 6 , 11 , 15 , 23 – 25 ]. • Providing reassurance that an outbreak is not actually occurring: By monitoring outbreak thresholds, as well as collecting data from a variety of sources, a syndromic surveillance system can provide information to public health authorities confirming or refuting the occurrence of an outbreak [ 17 , 19 , 24 , 26 ]. • Augments traditional public health surveillance: In order to improve outbreak detection [ 13 , 17 , 19 ], several definitions of syndromic surveillance emphasize that its goal is to "enhance, rather than replace, traditional approaches to epidemic detection" [ 14 ]. Despite these points of agreement and the popularity of syndromic surveillance systems in many countries, there is still little or no consensus regarding a standard definition encompassing the full scope of the term "syndromic surveillance." According to Mostashari and Hartman, the term syndromic surveillance is "imprecise and potentially misleading" [ 27 ]. The first point of confusion is the fact that "many of the systems under discussion do not monitor well-defined constellations of signs and symptoms (syndromes), but instead target non-specific indicators of health, such as a patient with a chief complaint of 'cough' or the sale of over-the-counter cold medication; conversely, many systems that do monitor syndromes (e.g., acute flaccid paralysis, Reye's syndrome, or carpal tunnel syndrome) are not included in these discussions" [ 27 ]. Second, some refer to the term "syndrome" as a specific, clinically defined phenomenon, such as severe acute respiratory syndrome (SARS) or acquired immune deficiency syndrome (AIDS), and others use it more loosely and non-specifically as simply a group of symptoms [ 28 ]. Several researchers have proposed alternative names to differentiate the forms of syndromic surveillance; however, these suggestions for clarified terminology have not yet taken hold. Ten other names that have been proposed in the literature include: outbreak detection systems, early warning systems, health indicator surveillance, prodromal/ic surveillance, information system-based sentinel surveillance, pre-diagnosis surveillance, nontraditional surveillance, enhanced surveillance, drop-in surveillance, and biosurveillance [ 12 , 19 , 27 , 28 ]. Problematically, these terms overlap, contradict, or are inconsistently applied, maintaining the terminological confusion. Further, several terms frequently applied to syndromic surveillance are not adequately descriptive to convey the type of system being referred to or do not distinguish between types of systems, and thus may not be appropriate as general, overarching terms. Given the challenge of developing clearer terminology, the potentially confusing term "syndromic surveillance" is still being used [ 27 ]. Due to the significant increase in applications of syndromic surveillance, and the new technologies and expanded potential of the tool—demonstrated by the evolution of the proceedings of the International Society for Disease Surveillance (ISDS) conferences on syndromic surveillance [ 29 ]—it is evident that the field has expanded over the last decade. Thus, there is now an even greater need for a consensus about what syndromic surveillance means. Additionally, as plans to translate syndromic surveillance systems to lower resource settings proceed, a proper conceptualization of the systems could increase their acceptance and efficient use. The development and communication of a unified understanding of the field may encourage governments or localities to adopt, invest in, and implement syndromic surveillance, where appropriate, and thus enhance compliance with IHR (2005); this adoption in middle- and low-income nations is being explored in the ongoing work to identify exemplary case studies of successful utilization of syndromic surveillance [ 30 ]. The purpose of this article is to clarify the various meanings that have been assigned to the term syndromic surveillance and to propose a refined categorization of the characteristics of the systems. 2. Materials and methods In an effort to capture the variety of definitions and explanations of syndromic surveillance in the literature, MEDLINE, Scopus, Google Scholar, proceedings from all ISDS conferences, and previous literature reviews and reference lists related to this topic were searched from 1998 to 2010. Search terms included "syndromic surveillance" and the 10 other terms mentioned above, which are considered synonymous with syndromic surveillance. In addition to general overview articles, the set of articles pertaining to country- and region-specific systems were narrowed by including the terms "low-income" and "developing country" to fit the emphasis on the translation of these systems to lower resource settings. Through a review of titles, abstracts, and full-length articles, 81 general articles were identified describing and evaluating syndromic surveillance systems along with several hundred articles delineating surveillance systems implemented in specific countries or regions. Within the articles collected, those that defined syndromic surveillance or provided a description of fundamental aspects of these systems were selected and compiled for comparison. In total, 43 separate articles defined syndromic surveillance, of which 36 provided unique definitions. A majority of the unique definitions found came from overview of articles of syndromic surveillance, rather than country-specific articles. However, the country-specific articles provided distinguishing examples of systems being implemented in various settings. 3. Results Table 1 contains the 36 unique definitions of syndromic surveillance found in the literature. The five general points of agreement among those in the field and mentioned above are frequently noted in the definitions and are designated in the set of columns on the far right. The two shaded columns indicate that there are two fundamental categories of syndromic surveillance systems conveyed by the collection of definitions. These two categories involve the same investigational approach, and may even be components of the same system, but monitor two distinct outcome types: specific and non-specific outcomes. Within the two fundamental categories of syndromic surveillance (defined further below), all five principles are relevant, with the principles of early detection and response and the use of pre-diagnostic information being the most commonly referred to across the definitions. Table 1 Unique definitions of syndromic surveillance found in the literature, 1998–2010. Article Article makes reference to "Specific Disease" category "Non-specific Disease" category Early detection and response Pre-diagnostic information Situational awareness Outbreak re-assurance Supplement to traditional surveillance World Health Organization [ 1 ] X World Health Organization [ 32 ] X Buehler et al. [ 14 ] X X X X X X Centers for Disease Control and Prevention [ 33 ] X X X Pavlin [ 21 ] X X X Reingold [ 34 ] X X X Smolinski et al. [ 28 ] X X X X Sosin [ 18 ] X X X X Sosin [ 19 ] X X X X X X Buehler [ 35 ] X X X X MMWR Editors [ 36 ] X X Henning [ 26 ] X X Lombardo et al. [ 37 ] X X Mandl et al. [ 22 ] X X X Stoto et al. [ 16 ] X X X Ang et al. [ 38 ] Berger et al. [ 17 ] X X X X Stoto et al. [ 39 ] X X X Chaves and Pascual [ 40 ] X Morse [ 41 ] X X Buehler et al. [ 6 ] X X X X X Centers for Disease Control and Prevention [ 20 ] X X Chretien et al. [ 12 ] X X X X X Chretien et al. [ 11 ] X X X Fearnley [ 42 ] X X Fricker [ 43 ] X X X Fricker et al. [ 15 ] X X X X X Jefferson et al. [ 44 ] X X X Nordin et al. [ 39 ] Tsui et al. [ 46 ] X Buehler et al. [ 23 ] X X X Gault et al. [ 47 ] X May et al. [ 13 ] X X Sintchenko and Gallego [ 24 ] X X X Zhang et al. [ 48 ] X X X Josseran et al. [ 49 ] X 3.1. Previous research The literature review yielded multiple articles that noted the distinction between specific and non-specific surveillance systems. In a foundational article introducing the field of syndromic surveillance to a wider audience, Sosin explains that indicators of a disease outbreak can either be suggestive of "highly specific syndrome[s]" or "non-specific expressions of the target diseases that occur before a diagnosis would routinely be made" [ 18 ]. The following year, an article based on recommendations from a CDC Working Group contrasted surveillance of a syndrome that "is relatively specific for the condition of interest" (such as acute flaccid paralysis as "a syndromic marker" in the detection of poliomyelitis) to surveillance with a broader purpose, such as "sexually transmitted disease detection and control" [ 35 ]. More recently, Fricker differentiates "well-defined" data that are "linked to specific types of outbreaks" to data that are "vaguely defined and perhaps only weakly linked to specific types of outbreaks, such as over-the-counter sales of cough and cold medication or absenteeism rates" [ 43 ]. Fricker continues his summary of the types of syndromic surveillance by observing that the meaning of the term "syndrome" has evolved in the context of syndromic surveillance: "A syndrome is 'a set of symptoms or conditions that occur together and suggest the presence of a certain disease or an increased chance of developing the disease'. In the context of syndromic surveillance, a syndrome is a set of non-specific pre-diagnosis medical and other information that may indicate the release of a bioterrorism agent or natural disease outbreak" [emphasis ours] [ 43 ]. Clearly this term "syndromic" as it is narrowly defined imperfectly captures the full range of these systems. An adjustment of the term, while maintaining the root of its meaning to ensure continuity in the field, can help strengthen and expand understanding of this field. 3.2. Proposed categorization Table 2 outlines the two categories of syndromic surveillance system types—specific and non-specific. As explained above and demonstrated in the literature, the purposes of each syndromic surveillance category are different. Whereas the "specific disease/syndrome detection" category focuses on detecting defined syndromes or a defined outcome of interest, the "non-specific disease detection" category aims to monitor or uncover non-specific indicators/trends that suggest an outbreak may be occurring. Based on this distinction, an alteration of the term "syndromic surveillance" to "syndrome-based" surveillance (SBS) referring to the more specific type of surveillance and "syndromic-non-specific" surveillance (SNS) referring to the now more common, non-specific category of disease detection is proposed. The categorization of SBS and SNS is confirmed by the examined literature and will be used throughout the remainder of the article to refer to these categories of surveillance. Table 2 Specific vs. non-specific syndromic surveillance categories. "Syndrome-based" surveillance (SBS): specific disease/syndrome detection "Syndromic-non-specific" surveillance (SNS): non-specific disease detection Under-lying purpose "Case detection and management of diseases when the condition is infrequent and the syndrome is relatively specific for the condition of interest" [ 35 ] To answer the question: Is there anything unusual or unexpected that public health officials need to investigate? This category focuses on detection of signals that "do not have a specific risk event focus" [ 43 ] System aims A developed syndrome (such as SARS, AIDS, acute flaccid paralysis, or influenza-like illness) Early symptoms (such as gastrointestinal complaints, influenza-like illness) Example: an increase in gastrointestinal illness cases indicating a water-borne outbreak Data sources "Constellations of medical signs and symptoms in persons seen in various clinical settings" [ 35 ], such as ICD-9 codes In addition to the types of data sources at left, unusual patterns in health-related behaviors (e.g., over-the-counter and health product purchases, such as cough medicine; and absenteeism from work or school) Example "The syndromes to be notified where an outbreak is of urgent international public health importance are: acute hemorrhagic fever syndrome, acute respiratory syndrome, acute diarrheal syndrome, acute jaundice syndrome, [and] acute neurological syndrome. In addition, any other syndrome of severe illness not included in the above should be notified if an outbreak is of urgent international public health importance" [ 31 ] "[In developing countries,] syndromic surveillance can identify outbreaks that do not fall into pre-established diagnostic categories, a capability essential for prompt control of new or changing diseases" [ 12 ] Southeast Asia's Early Warning Outbreak Recognition System (EWORS) provides surveillance of 29 non-specific signs and symptoms, which are not grouped into specific syndromes [ 50 ] Because of the terminological confusion with syndromic surveillance, this proposed categorization is necessary. Newcomers to the field of syndromic surveillance frequently have a narrow perspective of what these systems entail. Initial misconceptions include the false belief that it is surveillance based solely on syndromes (such as acute flaccid paralysis) or that this type of surveillance requires significant information technology (IT) capacity. The former point is immediately clarified by separating the definition of syndromic surveillance into two separate terms: SBS and SNS. The latter point concerning technological capacity will be discussed later in the text where the characteristic of technological dependence is described as a gradation within the two larger categories. Other attempts to categorize syndromic surveillance systems have also divided the field into two separate components; however, these categorizations do not encompass the full range of systems and most critical distinctions evident in the literature. One attempt separated systems into those based on a "data collection system that is dedicated to the purpose of this public health surveillance" (e.g., during a specific event) and those for the day-to-day monitoring of "data that are routinely collected for other purposes" [ 27 ]. Another categorization divided systems by data source: those data sources based on the use of health-care services and those based on health-related behaviors [ 6 ]. While these are useful distinctions to make when examining syndromic surveillance systems, they are not the most fundamental. The proposed categorization within the scope of this research addresses the most critical distinction among syndromic surveillance systems, which is the level of specificity of the outcome under surveillance. The distinction between data sources as sub-categories within each of the two larger categories (specific and non-specific detection) is taken into consideration because there is a fair amount of overlap. As noted in Table 2 , the data sources used in SNS systems can include those data sources used in SBS systems, but frequently focus primarily on pre-clinical data. 4. Data sources Data sources for public health surveillance have been traditionally divided into three levels: "pre-clinical data, clinical pre-diagnostic data, and diagnostic data. Syndromic surveillance usually uses two types of data sources: pre-clinical and clinical pre-diagnostic data; traditional surveillance generally focuses on diagnostic data" [ 17 ]. These levels can be further divided among the two categories of syndromic surveillance, with the SNS category being largely comprised of pre-clinical data, whereas the SBS category is focused on clinical pre-diagnostic data. The 36 definitions are rife with examples of each level of data source. Frequently cited clinical pre-diagnostic data sources for the SBS systems include: patient chief complaints or ICD-9 coded health information from clinical records (outpatient, emergency department, and hospital), billing databases, and emergency department triage and discharge data. Though ICD-9 coded health information is a form of diagnostic information, experts in the field of syndromic surveillance have suggested that general groupings of ICD-9 codes can be considered early diagnostic data [ 33 , 35 , 38 , 43 ]. Pre-clinical data used in SNS systems are often pulled from existing databases intended for other purposes [ 21 , 22 , 24 , 27 ] and are therefore "weakly linked" to the target disease [ 43 ]. Examples include "… 'indicator' (pre-diagnostic) data (e.g., syndromes, medication sales, absenteeism, patient chief complaints)" [ 12 ], pharmacy records, telephone health advice/consultation, poison control centers, 911 calls, "phone calls to or Internet use of a health-care information site" [ 18 ], laboratory test requests/orders [ 35 ], veterinary health records, health department requests for influenza testing [ 48 ], health care utilization patterns [ 24 ], ambulance services, and number of hospital admissions. Environmental data sources and water utility complaint lines [ 17 ] are similarly non-specific. 4.1. Classifications within SBS and SNS Within the two overarching categories of SBS and SNS, three additional sub-classifications became evident through the literature review, specifically surveillance to detect influenza-like illness and possible bioterrorism events, as well as the gradations in technological capacity. 4.1.1. Influenza-like illness One common use of syndromic surveillance is for the monitoring and detection of influenza-like illness (ILI), which is unique in that it falls within both specific and non-specific surveillance categories. As a specific syndrome, ILI can be used for monitoring known strains: the ILI syndrome is useful for "clarify[ing] the timing and characteristics of annual influenza outbreaks" [ 18 ]. Conversely, in SNS, an increased number of cases of respiratory symptoms and fever could indicate a bioterrorism-related event or a new strain of a virus with pandemic potential. 4.1.2. Bioterrorism As previously mentioned, syndromic surveillance has evolved from specific disease detection to encompassing more non-specific disease detection. In line with this change, and in response to the anthrax event in 2001, syndromic surveillance came to be seen as potentially useful for the detection of bioterrorism outbreaks. Its utilization of data from a variety of sources makes it a valuable addition to traditional surveillance methods [ 22 ]. While bioterrorism detection is not typically the primary use of syndromic surveillance today, non-specific disease detection continues to be thought of and investigated as a biosecurity tool [ 20 ]. Those in the bioterrorism field explain that syndromic surveillance, when applied to large, concentrated events, can "detect the early manifestations of illness that may occur during a bioterrorism-related epidemic… [such as] the prodromes of bioterrorism-related disease… [but] other uses of syndromic surveillance include detecting naturally occurring epidemics" [ 14 ], and it is more widely applicable than as merely a tool for bioterrorism detection [ 18 ]. The incorporation of biosecurity concepts into the syndromic surveillance field has contributed to the broadening of the five syndrome categories defined by WHO in 1998 ("acute hemorrhagic fever syndrome, acute respiratory syndrome, acute diarrheal syndrome, acute jaundice syndrome, and acute neurological syndrome") [ 31 , 51 ] to include additional categories of symptoms that can be monitored through non-specific surveillance. A CDC-led, multi-agency workgroup identified 11 "syndrome categories to be monitored that were indicative of the clinical presentations of several critical bioterrorism-associated conditions," including several of the WHO syndrome categories as well as more non-specific symptoms such as "rash" and "fever" [ 33 , 5 , 6 ]. 4.1.3. Electronic capabilities Within each of the two categories—specific (SBS) and non-specific (SNS)—there are varying degrees of technology that can be used for the syndromic surveillance system. Depending on the data sources available and the outcome of interest, some systems require significant IT and electronic capabilities [ 6 ]. However, there are also examples of less IT-dependent systems that monitor specific syndromes and/or non-specific disease indicators [ 44 ]. Thus, the distinction between high and low IT dependence is considered a sub-category within each of the two larger categories. The literature review revealed that highly automated systems tend to be used in more developed countries, for large catchment areas, and when there is a focus on bioterrorism. Typically, these systems involve electronic collection and analysis of data [ 49 ], potentially utilizing the "automated extraction of data from electronic medical records" [ 38 ]. On the other hand, less automated, less IT-dependent systems are more frequently seen in developing countries and often incorporate some element of manual data entry, extraction, or analysis, or the involvement of fax or mobile technology [ 52 ], resulting in detection that is "near real-time" as opposed to "real-time" [ 53 ]. In a basic form, syndromic surveillance "is a feasible and effective tool for surveillance in developing countries" and should be supported [ 13 ]. Given the significant applicability of syndromic surveillance systems to low- and medium-resource countries, it is critical that the definition of syndromic surveillance not be limited to highly IT-dependent, strictly automated systems. Clearly, though, where infrastructure allows, "automation of the full cycle of surveillance" allows for more real-time results [ 6 ]. 3.1. Previous research The literature review yielded multiple articles that noted the distinction between specific and non-specific surveillance systems. In a foundational article introducing the field of syndromic surveillance to a wider audience, Sosin explains that indicators of a disease outbreak can either be suggestive of "highly specific syndrome[s]" or "non-specific expressions of the target diseases that occur before a diagnosis would routinely be made" [ 18 ]. The following year, an article based on recommendations from a CDC Working Group contrasted surveillance of a syndrome that "is relatively specific for the condition of interest" (such as acute flaccid paralysis as "a syndromic marker" in the detection of poliomyelitis) to surveillance with a broader purpose, such as "sexually transmitted disease detection and control" [ 35 ]. More recently, Fricker differentiates "well-defined" data that are "linked to specific types of outbreaks" to data that are "vaguely defined and perhaps only weakly linked to specific types of outbreaks, such as over-the-counter sales of cough and cold medication or absenteeism rates" [ 43 ]. Fricker continues his summary of the types of syndromic surveillance by observing that the meaning of the term "syndrome" has evolved in the context of syndromic surveillance: "A syndrome is 'a set of symptoms or conditions that occur together and suggest the presence of a certain disease or an increased chance of developing the disease'. In the context of syndromic surveillance, a syndrome is a set of non-specific pre-diagnosis medical and other information that may indicate the release of a bioterrorism agent or natural disease outbreak" [emphasis ours] [ 43 ]. Clearly this term "syndromic" as it is narrowly defined imperfectly captures the full range of these systems. An adjustment of the term, while maintaining the root of its meaning to ensure continuity in the field, can help strengthen and expand understanding of this field. 3.2. Proposed categorization Table 2 outlines the two categories of syndromic surveillance system types—specific and non-specific. As explained above and demonstrated in the literature, the purposes of each syndromic surveillance category are different. Whereas the "specific disease/syndrome detection" category focuses on detecting defined syndromes or a defined outcome of interest, the "non-specific disease detection" category aims to monitor or uncover non-specific indicators/trends that suggest an outbreak may be occurring. Based on this distinction, an alteration of the term "syndromic surveillance" to "syndrome-based" surveillance (SBS) referring to the more specific type of surveillance and "syndromic-non-specific" surveillance (SNS) referring to the now more common, non-specific category of disease detection is proposed. The categorization of SBS and SNS is confirmed by the examined literature and will be used throughout the remainder of the article to refer to these categories of surveillance. Table 2 Specific vs. non-specific syndromic surveillance categories. "Syndrome-based" surveillance (SBS): specific disease/syndrome detection "Syndromic-non-specific" surveillance (SNS): non-specific disease detection Under-lying purpose "Case detection and management of diseases when the condition is infrequent and the syndrome is relatively specific for the condition of interest" [ 35 ] To answer the question: Is there anything unusual or unexpected that public health officials need to investigate? This category focuses on detection of signals that "do not have a specific risk event focus" [ 43 ] System aims A developed syndrome (such as SARS, AIDS, acute flaccid paralysis, or influenza-like illness) Early symptoms (such as gastrointestinal complaints, influenza-like illness) Example: an increase in gastrointestinal illness cases indicating a water-borne outbreak Data sources "Constellations of medical signs and symptoms in persons seen in various clinical settings" [ 35 ], such as ICD-9 codes In addition to the types of data sources at left, unusual patterns in health-related behaviors (e.g., over-the-counter and health product purchases, such as cough medicine; and absenteeism from work or school) Example "The syndromes to be notified where an outbreak is of urgent international public health importance are: acute hemorrhagic fever syndrome, acute respiratory syndrome, acute diarrheal syndrome, acute jaundice syndrome, [and] acute neurological syndrome. In addition, any other syndrome of severe illness not included in the above should be notified if an outbreak is of urgent international public health importance" [ 31 ] "[In developing countries,] syndromic surveillance can identify outbreaks that do not fall into pre-established diagnostic categories, a capability essential for prompt control of new or changing diseases" [ 12 ] Southeast Asia's Early Warning Outbreak Recognition System (EWORS) provides surveillance of 29 non-specific signs and symptoms, which are not grouped into specific syndromes [ 50 ] Because of the terminological confusion with syndromic surveillance, this proposed categorization is necessary. Newcomers to the field of syndromic surveillance frequently have a narrow perspective of what these systems entail. Initial misconceptions include the false belief that it is surveillance based solely on syndromes (such as acute flaccid paralysis) or that this type of surveillance requires significant information technology (IT) capacity. The former point is immediately clarified by separating the definition of syndromic surveillance into two separate terms: SBS and SNS. The latter point concerning technological capacity will be discussed later in the text where the characteristic of technological dependence is described as a gradation within the two larger categories. Other attempts to categorize syndromic surveillance systems have also divided the field into two separate components; however, these categorizations do not encompass the full range of systems and most critical distinctions evident in the literature. One attempt separated systems into those based on a "data collection system that is dedicated to the purpose of this public health surveillance" (e.g., during a specific event) and those for the day-to-day monitoring of "data that are routinely collected for other purposes" [ 27 ]. Another categorization divided systems by data source: those data sources based on the use of health-care services and those based on health-related behaviors [ 6 ]. While these are useful distinctions to make when examining syndromic surveillance systems, they are not the most fundamental. The proposed categorization within the scope of this research addresses the most critical distinction among syndromic surveillance systems, which is the level of specificity of the outcome under surveillance. The distinction between data sources as sub-categories within each of the two larger categories (specific and non-specific detection) is taken into consideration because there is a fair amount of overlap. As noted in Table 2 , the data sources used in SNS systems can include those data sources used in SBS systems, but frequently focus primarily on pre-clinical data. 4. Data sources Data sources for public health surveillance have been traditionally divided into three levels: "pre-clinical data, clinical pre-diagnostic data, and diagnostic data. Syndromic surveillance usually uses two types of data sources: pre-clinical and clinical pre-diagnostic data; traditional surveillance generally focuses on diagnostic data" [ 17 ]. These levels can be further divided among the two categories of syndromic surveillance, with the SNS category being largely comprised of pre-clinical data, whereas the SBS category is focused on clinical pre-diagnostic data. The 36 definitions are rife with examples of each level of data source. Frequently cited clinical pre-diagnostic data sources for the SBS systems include: patient chief complaints or ICD-9 coded health information from clinical records (outpatient, emergency department, and hospital), billing databases, and emergency department triage and discharge data. Though ICD-9 coded health information is a form of diagnostic information, experts in the field of syndromic surveillance have suggested that general groupings of ICD-9 codes can be considered early diagnostic data [ 33 , 35 , 38 , 43 ]. Pre-clinical data used in SNS systems are often pulled from existing databases intended for other purposes [ 21 , 22 , 24 , 27 ] and are therefore "weakly linked" to the target disease [ 43 ]. Examples include "… 'indicator' (pre-diagnostic) data (e.g., syndromes, medication sales, absenteeism, patient chief complaints)" [ 12 ], pharmacy records, telephone health advice/consultation, poison control centers, 911 calls, "phone calls to or Internet use of a health-care information site" [ 18 ], laboratory test requests/orders [ 35 ], veterinary health records, health department requests for influenza testing [ 48 ], health care utilization patterns [ 24 ], ambulance services, and number of hospital admissions. Environmental data sources and water utility complaint lines [ 17 ] are similarly non-specific. 4. Data sources Data sources for public health surveillance have been traditionally divided into three levels: "pre-clinical data, clinical pre-diagnostic data, and diagnostic data. Syndromic surveillance usually uses two types of data sources: pre-clinical and clinical pre-diagnostic data; traditional surveillance generally focuses on diagnostic data" [ 17 ]. These levels can be further divided among the two categories of syndromic surveillance, with the SNS category being largely comprised of pre-clinical data, whereas the SBS category is focused on clinical pre-diagnostic data. The 36 definitions are rife with examples of each level of data source. Frequently cited clinical pre-diagnostic data sources for the SBS systems include: patient chief complaints or ICD-9 coded health information from clinical records (outpatient, emergency department, and hospital), billing databases, and emergency department triage and discharge data. Though ICD-9 coded health information is a form of diagnostic information, experts in the field of syndromic surveillance have suggested that general groupings of ICD-9 codes can be considered early diagnostic data [ 33 , 35 , 38 , 43 ]. Pre-clinical data used in SNS systems are often pulled from existing databases intended for other purposes [ 21 , 22 , 24 , 27 ] and are therefore "weakly linked" to the target disease [ 43 ]. Examples include "… 'indicator' (pre-diagnostic) data (e.g., syndromes, medication sales, absenteeism, patient chief complaints)" [ 12 ], pharmacy records, telephone health advice/consultation, poison control centers, 911 calls, "phone calls to or Internet use of a health-care information site" [ 18 ], laboratory test requests/orders [ 35 ], veterinary health records, health department requests for influenza testing [ 48 ], health care utilization patterns [ 24 ], ambulance services, and number of hospital admissions. Environmental data sources and water utility complaint lines [ 17 ] are similarly non-specific. 4.1. Classifications within SBS and SNS Within the two overarching categories of SBS and SNS, three additional sub-classifications became evident through the literature review, specifically surveillance to detect influenza-like illness and possible bioterrorism events, as well as the gradations in technological capacity. 4.1.1. Influenza-like illness One common use of syndromic surveillance is for the monitoring and detection of influenza-like illness (ILI), which is unique in that it falls within both specific and non-specific surveillance categories. As a specific syndrome, ILI can be used for monitoring known strains: the ILI syndrome is useful for "clarify[ing] the timing and characteristics of annual influenza outbreaks" [ 18 ]. Conversely, in SNS, an increased number of cases of respiratory symptoms and fever could indicate a bioterrorism-related event or a new strain of a virus with pandemic potential. 4.1.2. Bioterrorism As previously mentioned, syndromic surveillance has evolved from specific disease detection to encompassing more non-specific disease detection. In line with this change, and in response to the anthrax event in 2001, syndromic surveillance came to be seen as potentially useful for the detection of bioterrorism outbreaks. Its utilization of data from a variety of sources makes it a valuable addition to traditional surveillance methods [ 22 ]. While bioterrorism detection is not typically the primary use of syndromic surveillance today, non-specific disease detection continues to be thought of and investigated as a biosecurity tool [ 20 ]. Those in the bioterrorism field explain that syndromic surveillance, when applied to large, concentrated events, can "detect the early manifestations of illness that may occur during a bioterrorism-related epidemic… [such as] the prodromes of bioterrorism-related disease… [but] other uses of syndromic surveillance include detecting naturally occurring epidemics" [ 14 ], and it is more widely applicable than as merely a tool for bioterrorism detection [ 18 ]. The incorporation of biosecurity concepts into the syndromic surveillance field has contributed to the broadening of the five syndrome categories defined by WHO in 1998 ("acute hemorrhagic fever syndrome, acute respiratory syndrome, acute diarrheal syndrome, acute jaundice syndrome, and acute neurological syndrome") [ 31 , 51 ] to include additional categories of symptoms that can be monitored through non-specific surveillance. A CDC-led, multi-agency workgroup identified 11 "syndrome categories to be monitored that were indicative of the clinical presentations of several critical bioterrorism-associated conditions," including several of the WHO syndrome categories as well as more non-specific symptoms such as "rash" and "fever" [ 33 , 5 , 6 ]. 4.1.3. Electronic capabilities Within each of the two categories—specific (SBS) and non-specific (SNS)—there are varying degrees of technology that can be used for the syndromic surveillance system. Depending on the data sources available and the outcome of interest, some systems require significant IT and electronic capabilities [ 6 ]. However, there are also examples of less IT-dependent systems that monitor specific syndromes and/or non-specific disease indicators [ 44 ]. Thus, the distinction between high and low IT dependence is considered a sub-category within each of the two larger categories. The literature review revealed that highly automated systems tend to be used in more developed countries, for large catchment areas, and when there is a focus on bioterrorism. Typically, these systems involve electronic collection and analysis of data [ 49 ], potentially utilizing the "automated extraction of data from electronic medical records" [ 38 ]. On the other hand, less automated, less IT-dependent systems are more frequently seen in developing countries and often incorporate some element of manual data entry, extraction, or analysis, or the involvement of fax or mobile technology [ 52 ], resulting in detection that is "near real-time" as opposed to "real-time" [ 53 ]. In a basic form, syndromic surveillance "is a feasible and effective tool for surveillance in developing countries" and should be supported [ 13 ]. Given the significant applicability of syndromic surveillance systems to low- and medium-resource countries, it is critical that the definition of syndromic surveillance not be limited to highly IT-dependent, strictly automated systems. Clearly, though, where infrastructure allows, "automation of the full cycle of surveillance" allows for more real-time results [ 6 ]. 4.1.1. Influenza-like illness One common use of syndromic surveillance is for the monitoring and detection of influenza-like illness (ILI), which is unique in that it falls within both specific and non-specific surveillance categories. As a specific syndrome, ILI can be used for monitoring known strains: the ILI syndrome is useful for "clarify[ing] the timing and characteristics of annual influenza outbreaks" [ 18 ]. Conversely, in SNS, an increased number of cases of respiratory symptoms and fever could indicate a bioterrorism-related event or a new strain of a virus with pandemic potential. 4.1.2. Bioterrorism As previously mentioned, syndromic surveillance has evolved from specific disease detection to encompassing more non-specific disease detection. In line with this change, and in response to the anthrax event in 2001, syndromic surveillance came to be seen as potentially useful for the detection of bioterrorism outbreaks. Its utilization of data from a variety of sources makes it a valuable addition to traditional surveillance methods [ 22 ]. While bioterrorism detection is not typically the primary use of syndromic surveillance today, non-specific disease detection continues to be thought of and investigated as a biosecurity tool [ 20 ]. Those in the bioterrorism field explain that syndromic surveillance, when applied to large, concentrated events, can "detect the early manifestations of illness that may occur during a bioterrorism-related epidemic… [such as] the prodromes of bioterrorism-related disease… [but] other uses of syndromic surveillance include detecting naturally occurring epidemics" [ 14 ], and it is more widely applicable than as merely a tool for bioterrorism detection [ 18 ]. The incorporation of biosecurity concepts into the syndromic surveillance field has contributed to the broadening of the five syndrome categories defined by WHO in 1998 ("acute hemorrhagic fever syndrome, acute respiratory syndrome, acute diarrheal syndrome, acute jaundice syndrome, and acute neurological syndrome") [ 31 , 51 ] to include additional categories of symptoms that can be monitored through non-specific surveillance. A CDC-led, multi-agency workgroup identified 11 "syndrome categories to be monitored that were indicative of the clinical presentations of several critical bioterrorism-associated conditions," including several of the WHO syndrome categories as well as more non-specific symptoms such as "rash" and "fever" [ 33 , 5 , 6 ]. 4.1.3. Electronic capabilities Within each of the two categories—specific (SBS) and non-specific (SNS)—there are varying degrees of technology that can be used for the syndromic surveillance system. Depending on the data sources available and the outcome of interest, some systems require significant IT and electronic capabilities [ 6 ]. However, there are also examples of less IT-dependent systems that monitor specific syndromes and/or non-specific disease indicators [ 44 ]. Thus, the distinction between high and low IT dependence is considered a sub-category within each of the two larger categories. The literature review revealed that highly automated systems tend to be used in more developed countries, for large catchment areas, and when there is a focus on bioterrorism. Typically, these systems involve electronic collection and analysis of data [ 49 ], potentially utilizing the "automated extraction of data from electronic medical records" [ 38 ]. On the other hand, less automated, less IT-dependent systems are more frequently seen in developing countries and often incorporate some element of manual data entry, extraction, or analysis, or the involvement of fax or mobile technology [ 52 ], resulting in detection that is "near real-time" as opposed to "real-time" [ 53 ]. In a basic form, syndromic surveillance "is a feasible and effective tool for surveillance in developing countries" and should be supported [ 13 ]. Given the significant applicability of syndromic surveillance systems to low- and medium-resource countries, it is critical that the definition of syndromic surveillance not be limited to highly IT-dependent, strictly automated systems. Clearly, though, where infrastructure allows, "automation of the full cycle of surveillance" allows for more real-time results [ 6 ]. 5. Discussion Early syndromic surveillance systems, including those part of the 1997–1999 WHO pilot study and as described in the 1998 Update on the Revision of the IHR, were largely focused on monitoring health events "for which the case definition is based on a syndrome… e.g., acute hemorrhagic fever syndrome, acute respiratory syndrome" [ 32 ]. Over time, the systems have transitioned to monitoring less specific outcomes [ 43 ]. Morse summarized this transition well: syndromic surveillance "once referred to the use of clinical syndromes as criteria for reporting. Now, it usually means data collected from automated non-diagnostic systems such as pharmacy records, ambulance call categories, personnel absences, or emergency department chief complaints" [ 41 ]. Of the 36 unique definitions found in the literature review, several appeared to be overly narrow and might contribute to the confusion ascribed to this term. Most of these narrow definitions suggested that syndromic surveillance is limited to highly IT-dependent systems, require automation or immediate analysis, or are limited to one function, such as bioterrorism [ 16 , 26 , 39 , 45 ]. The literature review makes evident that many systems that are considered "syndromic surveillance" are less IT-dependent, may include some manual component, and have much broader applicability. The importance of taking an all-inclusive view to the field of syndromic surveillance is put best by Fricker: "a myopic focus only on early event detection for bioterrorism in syndromic surveillance systems misses other important benefits electronic biosurveillance can provide, particularly the potential to significantly advance and modernize the practice of public health surveillance" [ 43 ]. 5.1. Broadening the applicability of syndromic surveillance systems An important contribution of a syndromic surveillance system is that it can be established in countries of any resource level. A broad definition, accounting for all of the purposes of syndromic surveillance—and acknowledging the flexibility of the infrastructure requirements—will facilitate its introduction and use in a variety of settings. A recent review described 10 syndromic surveillance systems in developing countries, demonstrating the "feasibility of 'low-tech' syndromic surveillance in low resource countries" [ 13 ], EWORS being one commonly cited example in Southeast Asia [ 52 ]. In developing countries, data sources not traditionally employed in surveillance can be useful, such as environmental sources assisting the detection of vector-borne and neglected tropical diseases, monitoring indoor resting densities of vectors, climate and land use data, and satellite imagery [ 40 ]. Surveillance of sexually transmitted infections could also be augmented by syndromic surveillance. According to WHO, syndromic surveillance of non-specific symptoms, including "urethral discharge and genital ulcer, are potentially useful for monitoring trends in STD incidence" [ 54 ]. 5.2. Purpose of terminological clarifications As the field has expanded, the truly broad nature of these surveillance systems has become apparent. Today, the term "syndromic surveillance" imperfectly describes all forms of syndromic surveillance systems. Fearnley suggests, "This terminological instability reflects an underlying ontological and normative instability," and without a generalized consensus of the definition of syndromic surveillance, "designers and users [may] continue to dispute what syndromic systems can and should do" [ 42 ]. Based on the results of the literature review, and in order to improve the conceptualization of this term, it was necessary to categorize syndromic surveillance into SBS and SNS, based on the fundamentals of specific and non-specific disease detection. The sub-categorization of the systems by data source and IT-capacity required is based on the broad range of features that constitute syndromic surveillance systems. Prior categorizations, described above, have not sufficiently encapsulated all that syndromic surveillance can entail. Recognizing that syndromic surveillance systems comprise these two categories with two different purposes helps clarify the added value of this kind of surveillance and may reduce ontological instability. It is recognized, as mentioned above, that in practice, the distinction between specific and non-specific syndromic surveillance categories can be lost, since many of these systems—particularly those in the United States—incorporate both categories within the same system. These dual-function systems (specific and non-specific detection) collect data from several sources—both the pre-clinical and clinical non-diagnostic types. Nevertheless, the acceptance and application of improved terminology regarding these systems can reduce ambiguity in the field and increase adoption of syndromic surveillance systems where appropriate. Future research must explore the combination of the SBS and SNS systems in more depth. These ongoing questions highlight the importance of incorporating robust system evaluation into future syndromic surveillance implementation efforts. Empirical, quantifiable evidence about the utility of these systems for improved surveillance and detection must be established. Such evidence will be essential for decision makers contemplating investing in syndromic surveillance to help meet IHR (2005) obligations. 5.1. Broadening the applicability of syndromic surveillance systems An important contribution of a syndromic surveillance system is that it can be established in countries of any resource level. A broad definition, accounting for all of the purposes of syndromic surveillance—and acknowledging the flexibility of the infrastructure requirements—will facilitate its introduction and use in a variety of settings. A recent review described 10 syndromic surveillance systems in developing countries, demonstrating the "feasibility of 'low-tech' syndromic surveillance in low resource countries" [ 13 ], EWORS being one commonly cited example in Southeast Asia [ 52 ]. In developing countries, data sources not traditionally employed in surveillance can be useful, such as environmental sources assisting the detection of vector-borne and neglected tropical diseases, monitoring indoor resting densities of vectors, climate and land use data, and satellite imagery [ 40 ]. Surveillance of sexually transmitted infections could also be augmented by syndromic surveillance. According to WHO, syndromic surveillance of non-specific symptoms, including "urethral discharge and genital ulcer, are potentially useful for monitoring trends in STD incidence" [ 54 ]. 5.2. Purpose of terminological clarifications As the field has expanded, the truly broad nature of these surveillance systems has become apparent. Today, the term "syndromic surveillance" imperfectly describes all forms of syndromic surveillance systems. Fearnley suggests, "This terminological instability reflects an underlying ontological and normative instability," and without a generalized consensus of the definition of syndromic surveillance, "designers and users [may] continue to dispute what syndromic systems can and should do" [ 42 ]. Based on the results of the literature review, and in order to improve the conceptualization of this term, it was necessary to categorize syndromic surveillance into SBS and SNS, based on the fundamentals of specific and non-specific disease detection. The sub-categorization of the systems by data source and IT-capacity required is based on the broad range of features that constitute syndromic surveillance systems. Prior categorizations, described above, have not sufficiently encapsulated all that syndromic surveillance can entail. Recognizing that syndromic surveillance systems comprise these two categories with two different purposes helps clarify the added value of this kind of surveillance and may reduce ontological instability. It is recognized, as mentioned above, that in practice, the distinction between specific and non-specific syndromic surveillance categories can be lost, since many of these systems—particularly those in the United States—incorporate both categories within the same system. These dual-function systems (specific and non-specific detection) collect data from several sources—both the pre-clinical and clinical non-diagnostic types. Nevertheless, the acceptance and application of improved terminology regarding these systems can reduce ambiguity in the field and increase adoption of syndromic surveillance systems where appropriate. Future research must explore the combination of the SBS and SNS systems in more depth. These ongoing questions highlight the importance of incorporating robust system evaluation into future syndromic surveillance implementation efforts. Empirical, quantifiable evidence about the utility of these systems for improved surveillance and detection must be established. Such evidence will be essential for decision makers contemplating investing in syndromic surveillance to help meet IHR (2005) obligations. 6. Conclusion Despite early concerns about the benefits of the syndromic approach to surveillance [ 34 , 42 ] and the continued need for further research, this approach has been proven successful in a wide variety of settings [ 5 , 17 , 18 , 30 ]. This paper has attempted to take a broad perspective on the field of syndromic surveillance, acknowledging the numerous syndromic surveillance systems that have been making important contributions to public health for over a decade, and summarizes the term's many definitions. By providing an accurate and comprehensive picture of this field's capabilities, and differentiating between SBS and SNS, it is hoped that syndromic surveillance will be seen more widely as a tool that can help any nation (high, middle, or low income) build comprehensive disease surveillance capacity. Contributors All authors contributed to this manuscript. Rebecca Katz and Larissa May conceptualized the project, directed the research, and reviewed and edited drafts of the manuscript. Elisa Test and Julia Baker conducted the literature review and drafted the manuscript. All authors approved the final article. Funding This work was supported by a grant from the George Washington University Research Facilitating Funds. The sponsor had no role in the study beyond providing financial support for researchers' time. Conflict of interest None declared.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6289300/

Stabilizing Displayed Proteins on Vegetative Bacillus subtilis Cells

Microbes engineered to display heterologous proteins could be useful biotechnological tools for protein engineering, lignocellulose degradation, biocatalysis, bioremediation and biosensing. Bacillus subtilis is a promising host to display proteins, as this model Gram-positive bacterium is genetically tractable and already used industrially to produce enzymes. To gain insight into the factors that affect displayed protein stability and copy-number, we systematically compared the ability of different protease-deficient B. subtilis strains (WB800, BRB07, BRB08 and BRB14) to display a Cel8A-LysM reporter protein in which the Clostridium thermocellum Cel8A endoglucanase is fused to LysM cell wall binding modules. Whole-cell cellulase measurements and fractionation experiments demonstrate that genetically eliminating extracytoplasmic bacterial proteases improves Cel8A-LysM displays levels. However, upon entering stationary phase, for all protease-deficient strains, the amount of displayed reporter dramatically decreases, presumably as a result of cellular autolysis. This problem can be partially overcome by adding chemical protease inhibitors, which significantly increase protein display levels. We conclude that strain BRB08 is well-suited for stably displaying our reporter protein, as genetic removal of its extracellular and cell wall-associated proteases leads to the highest levels of surface accumulated Cel8A-LysM without causing secretion stress or impairing growth. A two-step procedure is presented that enables the construction of enzyme coated vegetative B. subtilis cells that retain stable cell-associated enzyme activity for nearly 3 days. The results of this work could aid the development of whole cell display systems that have useful biotechnological applications.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803740/

Ocular side effects of novel anti-cancer biological therapies

To examine the ocular side effects of selected biological anti-cancer therapies and the ocular and systemic prognosis of patients receiving them. We retrospectively reviewed all medical records of patients who received biological anti-cancer treatment from 1/2012 to 12/2017 and who were treated at our ocular oncology service. The following data was retrieved: primary malignancy, metastasis, type of biological therapy, ocular side effects, ophthalmic treatment, non-ocular side effects, and ocular and systemic disease prognoses. Twenty-two patients received biological therapies and reported ocular side effects. Eighteen patients (81.8%) had bilateral ocular side effects, including uveitis (40.9%), dry eye (22.7%), and central serous retinopathy (22.7%). One patient (4.5%) had central retinal artery occlusion (CRAO), and one patient (4.5%) had branch retinal vein occlusion (BRVO). At the end of follow-up, 6 patients (27.27%) had resolution of the ocular disease, 13 patients (59.09%) had stable ocular disease, and 3 patients (13.64%) had progression of the ocular disease. Visual acuity improved significantly at the end of follow-up compared to initial values. Eighteen patients (81.8%) were alive at study closure. Biological therapies can cause a wide range of ocular side effects ranging from dry eye symptoms to severe pathologies that may cause ocular morbidity and vision loss, such as uveitis, CRAO and BRVO. All patients receiving biological treatments should be screened by ophthalmologists before treatment, re-screened every 4–6 months during treatment, and again at the end of treatment. Patients on biological treatment who have ocular complaints should be urgently referred to ocular consultation for early identification and early intervention. Introduction Cancer is the leading cause of death in the developed world with a mortality case of over 10 million mortality cases annually 1 . The traditional non-surgical treatments for cancer are radiation and chemotherapeutic drugs. However, those treatments also affect healthy cells, causing numerous side effects, some of which lead to severe morbidity 2 . Therefore, the current trend is focused on finding targeted therapies that eliminate specifically cancerous cells only. In the last 2 decades, studies on the molecular basis, epigenetic changes, and gene expression in cancer, as well as new diagnostic technologies have led to advances in understanding the mechanism of cancer development and the discovery of new modalities of therapy 3 , 4 . One of these novel modalities used for various cancer lines is biological therapy. Biological therapy stimulates the body's own immune system to act against cancer cells or interfere with tumor growth and progression by specific molecules or antibodies 5 – 7 . The different types of biological therapies include immune checkpoint inhibitors, immune cell therapy, therapeutic antibodies/immune system molecules, therapeutic vaccines, and immune system modulators 8 . Although those treatments are targeted and may effectively control tumor growth, they still may have side effects in the digestive system, liver, skin, nervous system, heart, and more 9 . Very few studies examined the ocular side effects of those treatments, and most of them were conducted on small groups and focused on a specific medication 10 – 12 . The purposes of the current study is to examine the ocular side effects of various biological therapies as well as to examine ocular and systemic prognoses of the patients receiving them. This knowledge may help individualize patient management and lead to improved vision and quality of life. Methods Patients The medical records of all consecutive patients with ocular side effects while receiving biological anti-cancer treatment who presented to the Ocular Oncology Service of the Goldschleger Eye Institute from January 2012 to December 2017 were retrospectively reviewed. The retrieved data included demographics, primary malignancy, metastasis status, type of biological therapy, laterality of the ocular side effects, ocular side effects, ophthalmic examination, ophthalmic treatment, non-ocular side effects, and both the ocular and systemic disease prognoses. The biological treatments were divided into the following groups according to their mechanisms of action: Group 1 Molecularly targeted therapies—BRAF inhibitors (Vemurafenib) + MEK inhibitors (Trametinib, Pimasertib) Group 2 Immune checkpoint inhibitor—Cytotoxic T-lymphocyte antigen-4 (CTLA-4, Ipilimumab), Programmed death protein 1 (PD-1, Pembrolizumab, Nivolumab), and Programmed death ligand-1 (PD-L1, Durvalumab) Group 3 Therapeutic Antibodies/Immune System Molecule—anti-epidermal growth factor receptor (EGFR inhibitor) + anaplastic lymphoma kinase (ALK, Alectinib) Group 4 Other—Bacillus Calmette-Guerin, Ibrutinib, Ixazomib, Pemetrexed The visual acuity (VA) was examined with Snellen VA charts and converted to log minimum angle of resolution (Log MAR) values at the beginning and end of follow-up. The ocular examination included slit-lamp and fundus examinations. Ancillary imaging testing [ultrasound (US), optical coherence tomography (OCT), fundus photos, and others] were performed as indicated. The treatment modalities varied according to the ocular pathology—for example: for dry eye patients were treated by lubricants, for anterior uveitis by topical steroids and pupil dilator and for posterior and panuveitis by topical steroids or steroid injection depend on the severity of the disease. This retrospective interventional cohort study was approved by the local institutional review board (IRB) of Sheba Medical Center, which waived informed consent. All methods were performed in accordance with to National Institutes of Health guidelines of Israel. Statistical analysis Quantitative variables were described as mean, range, and standard deviation. Categorical variables were described as absolute and relative frequencies. Paired t-test analyses compared VA at presentation and at the end of follow-up. The overall significance level was set to an alpha of 0.05. The statistical analysis was carried out with Microsoft Excel 2017 (Microsoft Corporation, Redmond, WA) and IBM SPSS software version 24.0 (SPSS, Inc., Chicago, IL, USA). Patients The medical records of all consecutive patients with ocular side effects while receiving biological anti-cancer treatment who presented to the Ocular Oncology Service of the Goldschleger Eye Institute from January 2012 to December 2017 were retrospectively reviewed. The retrieved data included demographics, primary malignancy, metastasis status, type of biological therapy, laterality of the ocular side effects, ocular side effects, ophthalmic examination, ophthalmic treatment, non-ocular side effects, and both the ocular and systemic disease prognoses. The biological treatments were divided into the following groups according to their mechanisms of action: Group 1 Molecularly targeted therapies—BRAF inhibitors (Vemurafenib) + MEK inhibitors (Trametinib, Pimasertib) Group 2 Immune checkpoint inhibitor—Cytotoxic T-lymphocyte antigen-4 (CTLA-4, Ipilimumab), Programmed death protein 1 (PD-1, Pembrolizumab, Nivolumab), and Programmed death ligand-1 (PD-L1, Durvalumab) Group 3 Therapeutic Antibodies/Immune System Molecule—anti-epidermal growth factor receptor (EGFR inhibitor) + anaplastic lymphoma kinase (ALK, Alectinib) Group 4 Other—Bacillus Calmette-Guerin, Ibrutinib, Ixazomib, Pemetrexed The visual acuity (VA) was examined with Snellen VA charts and converted to log minimum angle of resolution (Log MAR) values at the beginning and end of follow-up. The ocular examination included slit-lamp and fundus examinations. Ancillary imaging testing [ultrasound (US), optical coherence tomography (OCT), fundus photos, and others] were performed as indicated. The treatment modalities varied according to the ocular pathology—for example: for dry eye patients were treated by lubricants, for anterior uveitis by topical steroids and pupil dilator and for posterior and panuveitis by topical steroids or steroid injection depend on the severity of the disease. This retrospective interventional cohort study was approved by the local institutional review board (IRB) of Sheba Medical Center, which waived informed consent. All methods were performed in accordance with to National Institutes of Health guidelines of Israel. Statistical analysis Quantitative variables were described as mean, range, and standard deviation. Categorical variables were described as absolute and relative frequencies. Paired t-test analyses compared VA at presentation and at the end of follow-up. The overall significance level was set to an alpha of 0.05. The statistical analysis was carried out with Microsoft Excel 2017 (Microsoft Corporation, Redmond, WA) and IBM SPSS software version 24.0 (SPSS, Inc., Chicago, IL, USA). Results Demographics Between January 2012 and December 2017, a total of 22 patients (11 men and 11 women) were treated by biological therapies and reported ocular side effects. Their mean age at diagnosis was 63.32 ± 15.17 years (range 29–89 years). Ocular history Eleven patients (50%) had undergone cataract surgery, one patient (4.5%) had central serous retinopathy (CSR), and one patient (4.5%) had an epiretinal membrane (ERM) before embarking upon the biological treatment. All those patients had the ocular surgeries or problems at least one year before starting the biological treatment. None of the patients had a history of uveitis. Characteristics of the primary malignancy Ten patients (45.5%) had skin melanoma, 4 patients (18.2%) had non-small-cell lung carcinoma, 2 patients (2.8%) had transitional cell carcinoma, and one patient (1.2%) each had small-cell lung carcinoma, diffuse large B-cell lymphoma, uveal melanoma, cervix uteri adenosquamous carcinoma, chronic lymphocytic leukemia, and multiple myeloma. Nineteen patients (86.36%) had metastatic disease to the bone (10 patients, 45.5%), to the liver (7 patients, 31.8%), to the brain (6 patients, 27.3%), to the lung (6 patients, 27.3%), to the mesenteric fat (2 patients, 10.52%), to the pleura (2 patients,10.52%), and one patient (4.5%) each to the retroperitoneum, adrenal, omental fat, and breast. Three patients (13.64%) had stage 3 disease. The distribution of the biological therapies is summarized in Table 1 . There were no demographic differences between the groups (Table 2 ). Table 1 Biological therapies. Type of treatment Number of cases Percent Name of biological drug Ibrutinib 1 4.5 Alectinib 2 9.1 Bacillus Calmette–Guerin 1 4.5 Durvalumab 1 4.5 EGFR inhibitors 1 4.5 Ipilimumab 1 4.5 Ixazomib 1 4.5 Nivolomab 1 4.5 Pembrolizumab 2 9.1 Pemetrexed 1 4.5 Pimasertib 1 4.5 Trametinib 1 4.5 Vemurafenib 8 36.4 Biological treatment groups Anaplastic lymphoma kinase (ALK) 2 9.1 BRAF inhibitors 8 36.4 Cytotoxic T-lymphocyte antigen-4 (CTLA-4) 1 4.5 Epidermal growth factor receptor (EGFR) 1 MEK1/2 2 4.5 Programmed death ligand-1 (PD-1) 4 9.1 Other 4 18.2 Biological treatment groups by mechanisms Group 1—BRAF + MEK 10 45.5 Group 2—CTLA4 + PD1 5 22.7 Group 3—EGFR + ALK 3 13.6 Group 4—Other 4 18.2 Additional treatments—before biological treatment Chemotherapy 6 27.3 Radiation 6 27.3 Surgery 6 27.3 Systemic side effects Musculoskeletal 6 27.3 Skin 13 59.1 Gastrointestinal 7 31.8 Others 12 54.5 Table 2 Demographic charistricts of the groups. Biological treatment groups by mechanisms Number of patients Gender Male: female Age (mean) Group 1—BRAF + MEK 10 6:4 60.50 Group 2—CTLA4 + PD1 5 1:4 72.60 Group 3—EGFR + ALK 3 2:1 59.67 Group 4—Other 4 2:2 67.00 p value 0.469 0.499 Ocular side effects Eighteen patients (81.8%) had bilateral ocular side effects, 2 patients (9.1%) had side effects only to the right eye, and 2 patients (9.1%) had side effects only to the left eye. The side effects and ocular treatments are summarized in Table 3 . There was no difference in ocular side effects and the various types of biological treatment mechanisms ( p = 0.219 and p = 0.235, respectively, χ 2 ). Table 4 lists the differences in ocular and side effect characteristics of each group. The mean pre-treatment VA of right eyes that had side effects was 0.974 ± 0.194 and 0.754 ± 0.481 (t-test, p = 0.046) post-treatment. The mean pre-treatment VA of left eyes that had side effects was 0.893 ± 0.146 and 0.650 ± 0.031 post-treatment (t-test, p = 0.016). There were no differences in VA at diagnosis or at the end of follow-up between the different groups of treatment (Table 5 ). Table 3 Ocular side effects and treatment. Variable Number of cases Percent Side effects Uveitis (anterior, posterior, panuveitis) 9 (7, 1, 1) 40.9 (77.8, 11.1, 11.1) Dry eye 5 22.7 CSR-like 5 22.7 Vitreitis 2 9.1 CRAO 1 4.5 CME 1 4.5 Trichomegaly 3 13.6 BRVO 1 4.5 Treatment Intravitreal bevacizumab injection 1 4.5 Intravitreal Kenalog injection 2 9.00 Topical Artificial tears 5 22.5 Steroids 9 40.5 Pupil dilator 7 31.5 Oral therapy—prednisone 9 40.5 Surgical—pars plana vitrectomy 1 4.5 CSR, central serous retinopathy; CRAO, central retinal artery occlusion; CME, cystoid macular edema; BRVO, branch retinal vein occlusion. Table 4 Ocular and side effect characteristic of biological treatment mechanism groups. Variable Group 1 N (%) Group 2 N (%) Group 3 N (%) Group 4 N (%) p value (χ 2 ) Past ocular history 0.081 Cataract 4 (40) 5 (100) 1 (33.3) 1 (25) ERM 0 (0) 1 (20) 0 (0) 0 (0) CSR 0 (0) 0 (0) 1 (33.3) 0 (0) Side effects 0.219 posterior, panuveitis) 4 (40) (3, 0, 1) 3 (60) (2, 1, 0) 0 (0) (0, 0, 0) 2 (50) (2, 0, 0) CSR-like 3 (30) (0) 1 (33.3) 1 (25) Dry eye 2 (20) 2 (40) 0 (0) 1 (12.5) Vitreitis 2 (20) 0 (0) 0 (0) 0 (0) CRAO 0 (0) 1 (20) 0 (0) 0 (0) CME 1 (10) 0 (0) 0 (0) 0 (0) Trichomegaly 0 (0) 0 (0) 2 (66.6) 1 (25) BRVO 0 (0 1 (20) 0 (0) 0 (0) Treatment 0.325 Intravitreal injection 2 1 0 0 Topical 5 4 0 3 Medical 1 0 0 0 Surgical 1 0 0 0 CSR, central serous retinopathy; CRAO, central retinal artery occlusion; CME, cystoid macular edema; BRVO, branch retinal vein occlusion. Table 5 Visual acuity at diagnosis versus at the end of follow-up. Variable Group 1 Mean ± SD Group 2 Mean ± SD Group 3 Mean ± SD Group 4 Mean ± SD p value between groups Visual acuity at diagnosis: OD 0.932 ± 0.150 1.00 ± 0.205 0.928 ± 0.212 1.100 ± 0.288 0.497 Visual acuity at the end of follow-up: OD 0.594 ± 0.534 1.00 ± 0.115 0.389 ± 0.550 1.086 ± 0.303 0.268 p value at diagnosis versus at the end of follow-up 0.091 0.981 < 0.01 0.391 Visual acuity at diagnosis: OS 0.843 ± 0.071 1.00 ± 0.240 0.836 ± 0.101 0.945 ± 0.169 0.150 Visual acuity at the end of follow-up: OS 0.539 ± 0.484 0.846 ± 0.085 0.551 ± 0.379 0.912 ± 0.180 0.267 p value at diagnosis versus at the end of follow-up 0.072 0.163 0.064 0.391 Prognosis The mean duration of follow-up was 20.88 ± 40.69 months (range 1–177). The short follow-up of one month was that of a single patient who died of the primary systemic disease. The biological treatment was stopped in 9 patients (40.91%), 4 because of systemic and not ocular side effects and the other 5 because of improvement in their disease. Ocular treatment was not stopped in any of the patients. At the end of follow-up, 6 patients (27.27%) had resolution of the ocular disease, 13 patients (59.09%) had stable ocular disease, and 3 patients (13.64%) had progression of the ocular disease. There was no differences in ocular prognosis between the different groups of the biological therapies ( p = 0.187, χ 2 ). At the end of the study period, 18 patients (81.8%) were alive: 11 of them (50%) had stable systemic disease, and 7 (31.8%) had systemic progressive disease. Demographics Between January 2012 and December 2017, a total of 22 patients (11 men and 11 women) were treated by biological therapies and reported ocular side effects. Their mean age at diagnosis was 63.32 ± 15.17 years (range 29–89 years). Ocular history Eleven patients (50%) had undergone cataract surgery, one patient (4.5%) had central serous retinopathy (CSR), and one patient (4.5%) had an epiretinal membrane (ERM) before embarking upon the biological treatment. All those patients had the ocular surgeries or problems at least one year before starting the biological treatment. None of the patients had a history of uveitis. Characteristics of the primary malignancy Ten patients (45.5%) had skin melanoma, 4 patients (18.2%) had non-small-cell lung carcinoma, 2 patients (2.8%) had transitional cell carcinoma, and one patient (1.2%) each had small-cell lung carcinoma, diffuse large B-cell lymphoma, uveal melanoma, cervix uteri adenosquamous carcinoma, chronic lymphocytic leukemia, and multiple myeloma. Nineteen patients (86.36%) had metastatic disease to the bone (10 patients, 45.5%), to the liver (7 patients, 31.8%), to the brain (6 patients, 27.3%), to the lung (6 patients, 27.3%), to the mesenteric fat (2 patients, 10.52%), to the pleura (2 patients,10.52%), and one patient (4.5%) each to the retroperitoneum, adrenal, omental fat, and breast. Three patients (13.64%) had stage 3 disease. The distribution of the biological therapies is summarized in Table 1 . There were no demographic differences between the groups (Table 2 ). Table 1 Biological therapies. Type of treatment Number of cases Percent Name of biological drug Ibrutinib 1 4.5 Alectinib 2 9.1 Bacillus Calmette–Guerin 1 4.5 Durvalumab 1 4.5 EGFR inhibitors 1 4.5 Ipilimumab 1 4.5 Ixazomib 1 4.5 Nivolomab 1 4.5 Pembrolizumab 2 9.1 Pemetrexed 1 4.5 Pimasertib 1 4.5 Trametinib 1 4.5 Vemurafenib 8 36.4 Biological treatment groups Anaplastic lymphoma kinase (ALK) 2 9.1 BRAF inhibitors 8 36.4 Cytotoxic T-lymphocyte antigen-4 (CTLA-4) 1 4.5 Epidermal growth factor receptor (EGFR) 1 MEK1/2 2 4.5 Programmed death ligand-1 (PD-1) 4 9.1 Other 4 18.2 Biological treatment groups by mechanisms Group 1—BRAF + MEK 10 45.5 Group 2—CTLA4 + PD1 5 22.7 Group 3—EGFR + ALK 3 13.6 Group 4—Other 4 18.2 Additional treatments—before biological treatment Chemotherapy 6 27.3 Radiation 6 27.3 Surgery 6 27.3 Systemic side effects Musculoskeletal 6 27.3 Skin 13 59.1 Gastrointestinal 7 31.8 Others 12 54.5 Table 2 Demographic charistricts of the groups. Biological treatment groups by mechanisms Number of patients Gender Male: female Age (mean) Group 1—BRAF + MEK 10 6:4 60.50 Group 2—CTLA4 + PD1 5 1:4 72.60 Group 3—EGFR + ALK 3 2:1 59.67 Group 4—Other 4 2:2 67.00 p value 0.469 0.499 Ocular side effects Eighteen patients (81.8%) had bilateral ocular side effects, 2 patients (9.1%) had side effects only to the right eye, and 2 patients (9.1%) had side effects only to the left eye. The side effects and ocular treatments are summarized in Table 3 . There was no difference in ocular side effects and the various types of biological treatment mechanisms ( p = 0.219 and p = 0.235, respectively, χ 2 ). Table 4 lists the differences in ocular and side effect characteristics of each group. The mean pre-treatment VA of right eyes that had side effects was 0.974 ± 0.194 and 0.754 ± 0.481 (t-test, p = 0.046) post-treatment. The mean pre-treatment VA of left eyes that had side effects was 0.893 ± 0.146 and 0.650 ± 0.031 post-treatment (t-test, p = 0.016). There were no differences in VA at diagnosis or at the end of follow-up between the different groups of treatment (Table 5 ). Table 3 Ocular side effects and treatment. Variable Number of cases Percent Side effects Uveitis (anterior, posterior, panuveitis) 9 (7, 1, 1) 40.9 (77.8, 11.1, 11.1) Dry eye 5 22.7 CSR-like 5 22.7 Vitreitis 2 9.1 CRAO 1 4.5 CME 1 4.5 Trichomegaly 3 13.6 BRVO 1 4.5 Treatment Intravitreal bevacizumab injection 1 4.5 Intravitreal Kenalog injection 2 9.00 Topical Artificial tears 5 22.5 Steroids 9 40.5 Pupil dilator 7 31.5 Oral therapy—prednisone 9 40.5 Surgical—pars plana vitrectomy 1 4.5 CSR, central serous retinopathy; CRAO, central retinal artery occlusion; CME, cystoid macular edema; BRVO, branch retinal vein occlusion. Table 4 Ocular and side effect characteristic of biological treatment mechanism groups. Variable Group 1 N (%) Group 2 N (%) Group 3 N (%) Group 4 N (%) p value (χ 2 ) Past ocular history 0.081 Cataract 4 (40) 5 (100) 1 (33.3) 1 (25) ERM 0 (0) 1 (20) 0 (0) 0 (0) CSR 0 (0) 0 (0) 1 (33.3) 0 (0) Side effects 0.219 posterior, panuveitis) 4 (40) (3, 0, 1) 3 (60) (2, 1, 0) 0 (0) (0, 0, 0) 2 (50) (2, 0, 0) CSR-like 3 (30) (0) 1 (33.3) 1 (25) Dry eye 2 (20) 2 (40) 0 (0) 1 (12.5) Vitreitis 2 (20) 0 (0) 0 (0) 0 (0) CRAO 0 (0) 1 (20) 0 (0) 0 (0) CME 1 (10) 0 (0) 0 (0) 0 (0) Trichomegaly 0 (0) 0 (0) 2 (66.6) 1 (25) BRVO 0 (0 1 (20) 0 (0) 0 (0) Treatment 0.325 Intravitreal injection 2 1 0 0 Topical 5 4 0 3 Medical 1 0 0 0 Surgical 1 0 0 0 CSR, central serous retinopathy; CRAO, central retinal artery occlusion; CME, cystoid macular edema; BRVO, branch retinal vein occlusion. Table 5 Visual acuity at diagnosis versus at the end of follow-up. Variable Group 1 Mean ± SD Group 2 Mean ± SD Group 3 Mean ± SD Group 4 Mean ± SD p value between groups Visual acuity at diagnosis: OD 0.932 ± 0.150 1.00 ± 0.205 0.928 ± 0.212 1.100 ± 0.288 0.497 Visual acuity at the end of follow-up: OD 0.594 ± 0.534 1.00 ± 0.115 0.389 ± 0.550 1.086 ± 0.303 0.268 p value at diagnosis versus at the end of follow-up 0.091 0.981 < 0.01 0.391 Visual acuity at diagnosis: OS 0.843 ± 0.071 1.00 ± 0.240 0.836 ± 0.101 0.945 ± 0.169 0.150 Visual acuity at the end of follow-up: OS 0.539 ± 0.484 0.846 ± 0.085 0.551 ± 0.379 0.912 ± 0.180 0.267 p value at diagnosis versus at the end of follow-up 0.072 0.163 0.064 0.391 Prognosis The mean duration of follow-up was 20.88 ± 40.69 months (range 1–177). The short follow-up of one month was that of a single patient who died of the primary systemic disease. The biological treatment was stopped in 9 patients (40.91%), 4 because of systemic and not ocular side effects and the other 5 because of improvement in their disease. Ocular treatment was not stopped in any of the patients. At the end of follow-up, 6 patients (27.27%) had resolution of the ocular disease, 13 patients (59.09%) had stable ocular disease, and 3 patients (13.64%) had progression of the ocular disease. There was no differences in ocular prognosis between the different groups of the biological therapies ( p = 0.187, χ 2 ). At the end of the study period, 18 patients (81.8%) were alive: 11 of them (50%) had stable systemic disease, and 7 (31.8%) had systemic progressive disease. Discussion Novel types of biological therapies, particularly immunotherapy, have had a marked impact on cancer patient survival, and they are now more commonly implemented for various types of cancer 13 . Although biological therapy is generally less toxic to normal cells compared to chemotherapy, biological treatments can lead to numerous systemic side effects to the skin, joints, heart, lungs, liver, kidneys, central nervous system, etc. The most common side effects are: fatigue (26–53%), skin rash (1–50%), lymphocytopenia (10–49%), and increased pathological liver function tests (1–46%) 14 – 16 . Ocular side effects of immunotherapy are considered uncommon, occurring in approximately 1% of patients while from chemotherapy in various reports the ocular side effects are higher than 1% 17 – 19 . They can affect various parts of the eye and orbit 20 , 21 . The most commonly reported ocular side effects of immunotherapy are dry eye (1–24%), inflammatory uveitis (1%), and myasthenia gravis with ocular involvement 10 , 20 . In this study, we report 22 patients that were treated with biological therapy for advanced or metastatic cancer and referred to our ocular oncology service due to ocular complaints. The most common side effect was inflammatory uveitis (40.9%), and 13 of the affected patients (59%) were treated with topical or systemic corticosteroid without cessation of the biological treatment. All 22 patients showed improvement in their inflammation reaction as well as improvement of their complaints and vision. One patient with posterior uveitis and one patient with panuveitis who were treated with injections of corticosteroid also showed clinical improvement. Other common side effects were dry eye syndrome and CSR-like reaction. Dry eye is considered the most common side effect of biological treatments on the ocular anterior segment 22 , 23 . The reported severity of the symptoms were variable. Nguyen et al. reported one case of corneal perforation due to dry eye after biological treatment 23 . The patients with dry eyes in our cohort were treated locally with artificial preservative free tears and topical cyclosporine (1%), with improvement in their symptoms and none with severity that led to corneal perforation. Another common side effect was CSR-like reaction with subretinal fluid formation. This reaction had also been described after biological therapy with anthrax vaccination 24 . In our cohort, none of the patients required treatment for CSR-like reaction. The subretinal fluid was absorbed and the condition resolved without intervention in all cases. Two patients in our cohort developed severe ocular side effects after biological treatment, specifically, CRAO and BRVO. Those patients were 67 and 90 years old, respectively with positive medical history for vascular risk factors (hypertension and hyperlipidemia) without history of diabetic mellites or stroke. In general, since patients with CRAO experience severe painless loss of vision and are also at increased risk for stroke 25 , it is recommended that treatment with anti-thrombotic drugs be considered 26 . Patients with BRVO are at increased risk of developing macular edema and vision loss 27 . Although CRAO and BRVO are uncommon side effects, their grave impact on vision warrants heightened awareness and urgent screening in patients with ocular side effects during or after biological treatments. To the best of our knowledge, this is the first report on comparative ocular side effects in various biological treatment groups. Analysis of our findings failed to reveal any such differences among our study patients, although that may be attributed to the small number of patients in this study. To date, there are no published guidelines for ophthalmic examinations before, during, and after biological therapies. The current study results demonstrated that biological therapies can cause ocular discomfort due to dry eye symptoms. Moreover, sever pathologies like uveitis, CRVO and BRVO can occur and lead to severe eye morbidity. Therefore, we believe that all patients who start biological treatments should be screened by ophthalmologists before treatment, re-screened every 4–6 months during the treatments, and again at the end of the treatment. Any patient on biological treatment who presents with ocular complaints should be urgently referred to ocular consultation. Early identification of the ocular side effects of cancer therapy may lead to better visual prognosis. The limitations of this study are its retrospective nature, the small number of patients and no standardized diagnosis and periodical follow up protocol. We speculate that ocular side effects after biological treatment are more common than our findings, and that the unreported cases may have less severe symptoms for which they are treated at community clinics. This may explain our small number of patients and may cause a referral bias of only the more severe cases to our ocular oncology service. A larger study with planned screening of all patients that are treated with biological treatment at one center with a periodical ocular follow-up protocol (every 4–6 months) should be performed to better assess the prevalence of ocular side effects of biological treatment. To note in our cohort in 20/22 patients (90.9%) the ocular side effects were successfully controlled and in none of the cases the ocular side effects lead to biological treatment cessation. This fact is of most importance since these treatments can substantially improve patient's survivor. In summary, we present a cohort of 22 patients who received various biological treatments for advanced or metastatic cancer and developed ocular side effects. The most common side effects were uveitis, dry eye syndrome and CSR-like reaction,. Some severe side effects, such as BRVO and CRAO, were also reported. The patients were treated according to their ocular diagnosis, with improvement in their VA and ocular symptoms. There was no case of treatment cessation because of ocular side effects. Larger studies are required in order to examine the prevalence of ocular side effects and the differences of their occurrence between the various groups of biological treatments as well as to compare those side effects to chemotherapy and radiotherapy.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4671295/

Biochemical and Structural Analysis of an Eis Family Aminoglycoside Acetyltransferase from Bacillus anthracis

Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis_ Mtb ) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis_ Ban ). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis_ Ban displays significant differences from Eis_ Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was also different for several AGs, indicating a distinct regiospecificity of Eis_ Ban . Furthermore, most recently identified inhibitors of Eis_ Mtb did not inhibit Eis_ Ban , underscoring the differences between these two enzymes. To explain these differences, we determined an Eis_ Ban crystal structure. The comparison of the crystal structures of Eis_ Ban and Eis_ Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3739536/

The New Global Health

Global health reflects the realities of globalization, including worldwide dissemination of infectious and noninfectious public health risks. Global health architecture is complex and better coordination is needed between multiple organizations. Three overlapping themes determine global health action and prioritization: development, security, and public health. These themes play out against a background of demographic change, socioeconomic development, and urbanization. Infectious diseases remain critical factors, but are no longer the major cause of global illness and death. Traditional indicators of public health, such as maternal and infant mortality rates no longer describe the health status of whole societies; this change highlights the need for investment in vital registration and disease-specific reporting. Noncommunicable diseases, injuries, and mental health will require greater attention from the world in the future. The new global health requires broader engagement by health organizations and all countries for the objectives of health equity, access, and coverage as priorities beyond the Millennium Development Goals are set. Global Health The term global health has replaced tropical medicine and international health, disciplines linked to the history of colonialism, the post-independence era of the former European colonies, and the experience of development assistance ( 2 , 3 ). Global health is multidisciplinary, encompasses many elements besides development, and requires coordination of multiple parties, rather than direction by one organization or discipline. The increased technical and political complexity of global health, with many actors, including philanthropic and faith-based organizations, is reflected in its breadth, which covers diverse diseases but deals also with health systems issues and financing. Global health reflects the realities of globalization, especially the increased movement of persons and goods, and the global dissemination of infectious and noninfectious public health risks. Global health is concerned with protecting the entire global community, not just its poorest segments, against threats to health and with delivering essential and cost-effective public health and clinical services to the world's population. A fundamental tenet is that no country can ensure the health of its population in isolation from the rest of the world, as articulated in the Global Health Strategy of the United States Department of Health and Human Services ( 4 ). This vision reflects today's health realities but was arrived at through milestones such as the 1993 World Development Report (Investing in Health) ( 5 ), the 2000 report of the Commission on Macroeconomics and Health ( 6 ), and the tremendous investment in HIV/AIDS begun earlier this century ( 7 ). Development, Security, and Public Health Three overlapping themes determine global health action: development, security, and public health. These themes provide the humanitarian and political bases for engagement by high-income countries in health matters internationally: for development, to promote health for stability, prosperity, and better international relationships; for security, to protect their populations against internal and external health threats; and for public health, to save lives worldwide and at home. Despite different requirements, organizations and agencies involved must adapt to global trends in socioeconomic development, fertility, population, and urbanization. Development Of 214 countries categorized by the World Bank, only 36 (17%) were classified as low-income countries (gross national income per capita in 2011 10 million persons, including many in Africa ( 9 ), all challenged by the need for basic infrastructure and services. A welcome trend has been renewed attention to reducing avoidable deaths among children. The worldwide reduction in childhood mortality rates means that since the 1980s, deaths among adults have exceeded deaths among children. Recently published estimates of mortality rates among children 200,000 deaths annually among children 50% of deaths in children occur in sub-Saharan Africa. Because of their large populations, India and China contribute substantially to these deaths, as do large countries with poor health indicators, such as Nigeria, the Democratic Republic of Congo, Pakistan, and Ethiopia. Seven countries with >10,000 maternal deaths/year account for >50% of the world's maternal mortality rate. The highest maternal mortality rates are in sub-Saharan Africa, especially western Africa, a finding that is consistent with distribution of adverse rates of child survival. Pakistan and Afghanistan stand out for unfavorable indicators in their region. Further reduction in maternal and child mortality rates globally will require special focus on countries with the greatest absolute numbers of maternal and child deaths. Health Security Drawing on earlier United Nations perspectives that characterized poor health as one of several threats to human security and well-being, health security captures the need for collective action and preparedness to reduce vulnerabilities to public health threats that transcend borders ( 13 ). Earlier optimism predicting the end of infectious diseases was replaced by recognition of the threat to global health from emerging infectious diseases and widespread antimicrobial drug resistance ( 14 ). The pandemic of HIV/AIDS, repeated outbreaks of Ebola and Marburg virus infections, rapid international dissemination of severe acute respiratory syndrome and pandemic influenza, international spread of several foodborne pathogens, and the intentional transmission of anthrax all convincingly illustrated global vulnerability. Other aspects of globalization negatively affecting health security include the trafficking of drugs and persons and population movement consequent to conflict and instability. The global framework for health security is embodied in the International Health Regulations that were revised in 2005 and adopted by the World Health Assembly, but whose implementation is lagging behind the 2012 target date ( 15 ). The diversity of health threats results in involvement of other sectors, such as defense and diplomacy, and linkage with other international agreements, such as those relating to control of chemical, biological, and nuclear weapons. Surveillance and laboratory capacity through strong national public health institutes are essential components of functioning health systems that provide the basis for health security. Ensuring ability to detect, investigate, diagnose, and rapidly contain public health events of concern wherever they occur requires commitment to global health capacity development in all countries and widespread and supportive public health networks ( 16 ). Public Health The scale-up of programs for HIV/AIDS, malaria, and tuberculosis over the past decade through initiatives such as the Global Fund, the United States President's Emergency Plan for AIDS Relief, and the President's Malaria Initiative led to substantial disease-specific progress. The Global Alliance for Vaccines and Immunisation has positively affected vaccine access. However, these experiences also highlighted the relative neglect of other priority areas and led to criticism that vertical, targeted programs failed to strengthen health systems overall ( 17 ). As a result, there has been renewed focus on the other health-related Millennium Development Goals (MDGs), especially relating to children's and maternal health (MDGs 4 and 5, respectively). These perceptions contributed to the establishment of the United States government's Global Health Initiative in 2009 ( 18 ) that addresses all health MDGs and some neglected tropical diseases in a more integrated manner. The longstanding tension between vertical and horizontal approaches is now better understood, and there is greater emphasis on integration of efforts ( 19 ). Initiatives to strengthen general health systems have lacked specificity and agreed upon indicators, and they have had more difficulty showing measurable effects than disease-specific interventions that emphasize integration and linkage to other services. Public health agencies have a major role in strengthening specific areas of health systems, such as health information systems and surveillance, laboratory capacity, workforce skills, operational research and evaluation, and capacity for preparedness and program implementation ( 20 ). National public health institutes and strong ministries have the core responsibility for defining policies, goals and targets, and assuring technical guidance, supervision, program implementation, evaluation, and accountability ( 21 ). Although epidemiology remains at the core of such work, the increased complexity of combinations of interventions in public health has highlighted the utility of mathematical modeling for assisting in decision making and policy setting. Modern public health agencies have to be global in outlook to fulfill their domestic mandates. Because of the credibility emanating from their technical expertise, these agencies play an essential role in health diplomacy and development of public health capacity. Although development agencies concentrate on the needs of the poor, public health agencies potentially interact with all countries to address common challenges. Health systems strengthening, communicable and noncommunicable disease threats, safety and quality of medicines and commodities, and health access and equity are universally challenging to ministries of health, public health institutes and multilateral organizations, which all need to function in a close global network. Development Of 214 countries categorized by the World Bank, only 36 (17%) were classified as low-income countries (gross national income per capita in 2011 10 million persons, including many in Africa ( 9 ), all challenged by the need for basic infrastructure and services. A welcome trend has been renewed attention to reducing avoidable deaths among children. The worldwide reduction in childhood mortality rates means that since the 1980s, deaths among adults have exceeded deaths among children. Recently published estimates of mortality rates among children 200,000 deaths annually among children 50% of deaths in children occur in sub-Saharan Africa. Because of their large populations, India and China contribute substantially to these deaths, as do large countries with poor health indicators, such as Nigeria, the Democratic Republic of Congo, Pakistan, and Ethiopia. Seven countries with >10,000 maternal deaths/year account for >50% of the world's maternal mortality rate. The highest maternal mortality rates are in sub-Saharan Africa, especially western Africa, a finding that is consistent with distribution of adverse rates of child survival. Pakistan and Afghanistan stand out for unfavorable indicators in their region. Further reduction in maternal and child mortality rates globally will require special focus on countries with the greatest absolute numbers of maternal and child deaths. Health Security Drawing on earlier United Nations perspectives that characterized poor health as one of several threats to human security and well-being, health security captures the need for collective action and preparedness to reduce vulnerabilities to public health threats that transcend borders ( 13 ). Earlier optimism predicting the end of infectious diseases was replaced by recognition of the threat to global health from emerging infectious diseases and widespread antimicrobial drug resistance ( 14 ). The pandemic of HIV/AIDS, repeated outbreaks of Ebola and Marburg virus infections, rapid international dissemination of severe acute respiratory syndrome and pandemic influenza, international spread of several foodborne pathogens, and the intentional transmission of anthrax all convincingly illustrated global vulnerability. Other aspects of globalization negatively affecting health security include the trafficking of drugs and persons and population movement consequent to conflict and instability. The global framework for health security is embodied in the International Health Regulations that were revised in 2005 and adopted by the World Health Assembly, but whose implementation is lagging behind the 2012 target date ( 15 ). The diversity of health threats results in involvement of other sectors, such as defense and diplomacy, and linkage with other international agreements, such as those relating to control of chemical, biological, and nuclear weapons. Surveillance and laboratory capacity through strong national public health institutes are essential components of functioning health systems that provide the basis for health security. Ensuring ability to detect, investigate, diagnose, and rapidly contain public health events of concern wherever they occur requires commitment to global health capacity development in all countries and widespread and supportive public health networks ( 16 ). Public Health The scale-up of programs for HIV/AIDS, malaria, and tuberculosis over the past decade through initiatives such as the Global Fund, the United States President's Emergency Plan for AIDS Relief, and the President's Malaria Initiative led to substantial disease-specific progress. The Global Alliance for Vaccines and Immunisation has positively affected vaccine access. However, these experiences also highlighted the relative neglect of other priority areas and led to criticism that vertical, targeted programs failed to strengthen health systems overall ( 17 ). As a result, there has been renewed focus on the other health-related Millennium Development Goals (MDGs), especially relating to children's and maternal health (MDGs 4 and 5, respectively). These perceptions contributed to the establishment of the United States government's Global Health Initiative in 2009 ( 18 ) that addresses all health MDGs and some neglected tropical diseases in a more integrated manner. The longstanding tension between vertical and horizontal approaches is now better understood, and there is greater emphasis on integration of efforts ( 19 ). Initiatives to strengthen general health systems have lacked specificity and agreed upon indicators, and they have had more difficulty showing measurable effects than disease-specific interventions that emphasize integration and linkage to other services. Public health agencies have a major role in strengthening specific areas of health systems, such as health information systems and surveillance, laboratory capacity, workforce skills, operational research and evaluation, and capacity for preparedness and program implementation ( 20 ). National public health institutes and strong ministries have the core responsibility for defining policies, goals and targets, and assuring technical guidance, supervision, program implementation, evaluation, and accountability ( 21 ). Although epidemiology remains at the core of such work, the increased complexity of combinations of interventions in public health has highlighted the utility of mathematical modeling for assisting in decision making and policy setting. Modern public health agencies have to be global in outlook to fulfill their domestic mandates. Because of the credibility emanating from their technical expertise, these agencies play an essential role in health diplomacy and development of public health capacity. Although development agencies concentrate on the needs of the poor, public health agencies potentially interact with all countries to address common challenges. Health systems strengthening, communicable and noncommunicable disease threats, safety and quality of medicines and commodities, and health access and equity are universally challenging to ministries of health, public health institutes and multilateral organizations, which all need to function in a close global network. Unfinished Business: Infectious Disease Priorities Recent estimates of the global incidence of disease suggest that communicable diseases account for ≈19% of global deaths ( 22 ). In Africa, 76% of deaths are still attributable to communicable, maternal, neonatal, or nutritional causes, compared with 25% in the entire world; conditions relevant to MDGs 4, 5, and 6 are responsible for 42% of years of life lost. Focus on infectious diseases remains necessary to prevent their global spread or recrudescence, save lives, enhance economic development, and increase health equity. Major and persistent infectious disease threats, their global incidence, and some of the global health commitments made to address them are shown in the Technical Appendix . The 1993 World Bank report Investing in Health first highlighted the overwhelming role of HIV/AIDS, tuberculosis, and malaria in Africa ( 5 ), but only in the past decade have substantially increased investment and effort enabled measurable progress in these major infectious disease challenges. The world needs to maintain momentum to achieve ambitious health targets and implement recent scientific advances while simultaneously coping with economic austerity. There is increasing pressure to use resources for biomedical interventions with the strongest evidence of efficacy. Efforts toward achieving an AIDS-free generation are centered around HIV treatment scale-up, prevention of mother-to-child transmission (including through immediate and life-long antiretroviral therapy for all HIV-infected pregnant women), medical male circumcision, HIV testing and counseling, and focus on key populations in which HIV infection is concentrated ( 23 ). The primary current research question in HIV/AIDS is how best to use antiretroviral therapy for individual health and for population-based prevention, and more specifically, whether immediate therapy upon early diagnosis would confer the greatest benefit ( 24 , 25 ). The commitment to virtual elimination of HIV disease in children ( 26 ) could usefully link new initiatives to traditional maternal and child health programs delivered through development funding. Tuberculosis is decreasing in incidence in all regions of the world, although more slowly than expected in some regions ( 27 ). In the United States, 63% of all tuberculosis cases now occur in foreign-born persons, indicating likely acquisition of the infection outside the United States ( 28 ). The spread of drug-resistant tuberculosis and extensively drug-resistant tuberculosis (resistant to rifampin, isoniazid, quinolones and injectable antituberculous drugs) highlights global vulnerability and interrelatedness of health systems and challenges health equity. Key scientific advances concern better understanding of the role and use of antiretroviral therapy for persons with tuberculosis co-infected with HIV, new diagnostics with the potential to make case finding more effective, and less strikingly, new drugs. The tools for combating malaria (insecticide-impregnated bed nets, indoor residual spraying of insecticide, artemisinin-based combination therapies, and intermittent preventive therapy for pregnant women) need further scale-up, but such tools are susceptible to development of resistance on the part of the vector or parasite, and evidence is accumulating that nets may be less durable than assumed ( 26 , 29 ). Despite the challenges, malaria elimination has risen up the global agenda in recent time. Poverty-related diseases such as the 17 conditions categorized as neglected tropical diseases have also received increased investment, especially those for which mass drug administration offers a control strategy ( 30 , 31 ). A concern must be that some major causes of illness and death, such as visceral leishmaniasis and African human trypanosomiasis, remain overshadowed and unaddressed. Two groups of diseases meriting global health attention are those that are epidemic prone or vaccine preventable, including influenza. The 2009 pandemic of influenza A(H1N1) demonstrated the global nature of the threat, as well as the need to consider strategies for provision of vaccine for all countries ( 32 ). Dengue and yellow fever are the major mosquito-borne viral infections, and both illustrate the concept of emerging infections promoted by diverse factors, such as urbanization, population growth, inadequate environmental hygiene, and vector resistance to insecticide. In recent years, large outbreaks involving a specific arbovirus, chikungunya virus, have affected the east coast of Africa and islands in the Indian Ocean with importation into Europe. The second decade of this century has been designated as the decade of vaccines ( 33 ). The opportunity exists for a notable effect on the 2.5 million deaths of children annually from vaccine-preventable diseases, including through use of new vaccines for prevention of rotavirus and pneumococcal infection, and by strengthening routine services. Vaccination against type A meningococcal meningitis in the Sahel and against hepatitis B virus and human papillomavirus illustrate the unrivaled possibilities in terms of controlling previously deadly epidemics or virus-induced cancers. A major unfinished priority is polio eradication; this goal is particularly threatened by funding shortfalls and ongoing transmission in Pakistan, Afghanistan, and Nigeria, which have seeded infection in other countries in which polio had been eliminated ( 34 ). Lack of access to water and sanitation highlights some of the greatest inequities in global health. Approximately 1 billion persons worldwide do not have clean drinking water, and ≈2.5 billion persons have to openly defecate, which is an affront to human dignity ( 35 ). Large epidemics of waterborne diseases continue to occur, as exemplified by ongoing cholera transmission in Haiti ( 36 ). It is difficult to explain why investment in separating human drinking water from human feces, the basis of the nineteenth century public health revolution in Europe and North America, has not been a higher political or development priority in resource-poor settings. Noncommunicable Diseases The high-level meeting on noncommunicable diseases at the General Assembly of the United Nations in 2011, only the second such meeting devoted to health, emphasized how these diseases now dominate health worldwide ( 37 ). More than 60% of preventable deaths worldwide are now attributable to noncommunicable diseases (cardiovascular diseases, cancers, diabetes, and chronic respiratory diseases); in low-income and middle-income countries, 48% of such deaths occur in persons 5 million deaths worldwide result from injuries and violence ( 39 ), and ≈1.3 million are caused by road traffic injuries. Mental and behavioral disorders are considered the largest contributor to years lived with disability ( 22 ). Global funding for noncommunicable diseases is minimal and coordination is limited, although opportunities exist for integrating approaches to communicable and noncommunicable diseases. Implementation of surveillance to assess incidence and needs along with selected policy interventions to address them will have the greatest immediate effect for the least cost. Examples of such policies include restricting tobacco sales and access, raising tobacco taxes, limiting unsafe use of alcohol, enacting motorcycle helmet and seat belt laws, and reducing salt and trans fats in commercial food products. To encourage countries to take action, WHO is defining population-level targets for noncommunicable diseases and associated risk factors for program implementation ( 37 ). Experience with HIV/AIDS treatment scale-up ( 40 ) could provide useful lessons for a standardized approach to management of hypertension and diabetes, thereby enhancing cost-effectiveness; facilitating supervision, monitoring, and evaluation; and ensuring accountability. Conclusions Population growth, increased life expectancy of the world's citizens, and decreased age-specific mortality rates in children and young adults, especially those for infectious diseases, have contributed to the altered global health landscape. The New Global Health concerns health in all countries and encompasses poverty alleviation, universal health security, and delivery of appropriate public health and clinical services, including for the increasing prevalence of noncommunicable diseases. Equity, universal health coverage and access, and fairness in health financing are global aspirations likely to feature prominently in discussions about what comes after the 2015 MDG target date. The unfinished infectious disease agenda will remain a priority, but common approaches will have to address noncommunicable diseases, regulation of commerce in medical technologies and pharmaceuticals, health financing, and systems strengthening. An emerging topic will be surveillance for and mitigation of effects of environmental and climate change. Surveillance will have to be strengthened globally to track exposure to risk factors for the major causes of disability and death, disease outcomes, and health systems responses. The past and on-going epidemiologic transitions mean that in many countries, the classic health indicators of international health (infant, under-5, and maternal mortality rates) no longer provide insight into population health. In addition, there is an urgent need for robust vital registration systems and accurate reporting of cause-specific mortality rates across all life stages. We must not forget the current challenges facing the lowest-income countries, the needs of disenfranchised or displaced populations, societies threatened by conflict and humanitarian emergencies, and the urban and rural poor living conditions in the midst of plenty. Nonetheless, global health practice must adapt to globalization and the rapid evolution in health underway worldwide. For donor countries, this will require clear definition of expectations of development assistance and how this differs from other forms of global health engagement, especially for health security and noncommunicable diseases. How to provide appropriate coordination, the kind of leadership desired, and how to ensure the shared responsibility of funding beyond the traditional donors will all feature prominently. Global interconnectedness requires us to address the health of the planet's entire population, irrespective of national borders. Engagement in global health is not simply a humanitarian concern but a priority for our collective well-being, efficient use of resources, and safeguarding our future. Technical Appendix Infectious disease priorities, incidence, and commitments, The New Global Health.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9396156/

Community-based surveillance of infectious diseases: a systematic review of drivers of success

Introduction Community-based surveillance may improve early detection and response to disease outbreaks by leveraging the capacity of community members to carry out surveillance activities within their communities. In 2021, the WHO published a report detailing the evidence gaps and research priorities around community-centred approaches to health emergencies. In response, we carried out a systematic review and narrative synthesis of the evidence describing the drivers of success of community-based surveillance systems. Methods We included grey literature and peer-reviewed sources presenting empirical findings of the drivers of success of community-based surveillance systems for the detection and reporting of infectious disease-related events. We searched for peer-reviewed literature via MEDLINE, EMBASE, Global Health, SCOPUS and ReliefWeb. We carried out grey literature searches using Google Search and DuckDuckGo. We used an evaluation quality checklist to assess quality. Results Nineteen sources (17 peer-reviewed and 2 grey literature) met our inclusion criteria. Included sources reported on community-based surveillance for the detection and reporting of a variety of diseases in 15 countries (including three conflict settings). The drivers of success were grouped based on factors relating to: (1) surveillance workers, (2) the community, (3) case detection and reporting, (4) and integration. Discussion The drivers of success were found to map closely to principles of participatory community engagement with success factors reflecting high levels of acceptability, collaboration, communication, local ownership, and trust. Other factors included: strong supervision and training, a strong sense of responsibility for community health, effective engagement of community informants, close proximity of surveillance workers to communities, the use of simple and adaptable case definitions, quality assurance, effective use of technology, and the use of data for real-time decision-making. Our findings highlight strategies for improving the design and implementation of community-based surveillance. We suggest that investment in participatory community engagement more broadly may be a key surveillance preparedness activity. PROSPERO registration number CRD42022303971. Introduction Community-based surveillance may improve early detection and response to disease outbreaks by leveraging the capacity of community members to carry out surveillance activities within their communities. In 2021, the WHO published a report detailing the evidence gaps and research priorities around community-centred approaches to health emergencies. In response, we carried out a systematic review and narrative synthesis of the evidence describing the drivers of success of community-based surveillance systems. Methods We included grey literature and peer-reviewed sources presenting empirical findings of the drivers of success of community-based surveillance systems for the detection and reporting of infectious disease-related events. We searched for peer-reviewed literature via MEDLINE, EMBASE, Global Health, SCOPUS and ReliefWeb. We carried out grey literature searches using Google Search and DuckDuckGo. We used an evaluation quality checklist to assess quality. Results Nineteen sources (17 peer-reviewed and 2 grey literature) met our inclusion criteria. Included sources reported on community-based surveillance for the detection and reporting of a variety of diseases in 15 countries (including three conflict settings). The drivers of success were grouped based on factors relating to: (1) surveillance workers, (2) the community, (3) case detection and reporting, (4) and integration. Discussion The drivers of success were found to map closely to principles of participatory community engagement with success factors reflecting high levels of acceptability, collaboration, communication, local ownership, and trust. Other factors included: strong supervision and training, a strong sense of responsibility for community health, effective engagement of community informants, close proximity of surveillance workers to communities, the use of simple and adaptable case definitions, quality assurance, effective use of technology, and the use of data for real-time decision-making. Our findings highlight strategies for improving the design and implementation of community-based surveillance. We suggest that investment in participatory community engagement more broadly may be a key surveillance preparedness activity. PROSPERO registration number CRD42022303971. WHAT IS ALREADY KNOWN ON THIS TOPIC Community-based surveillance can provide an essential complement to facility-based surveillance systems, which may have limited effectiveness in some settings due to poor facility attendance. Previous reviews of community-based surveillance (in crisis-affected settings) and event-based surveillance (in low and middle-income settings) have concluded that challenges to community-based surveillance often involve the failure to address operational requirements. WHAT THIS STUDY ADDS Our findings highlight the alignment between many of the identified success factors and principles of participatory community engagement including high levels of community acceptability, meaningful and ongoing collaboration, effective communication, local ownership, and trust. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY Given the close alignment between success factors and the principles of participatory community engagement, we suggest that the emphasis of community-based surveillance preparedness should be on investing in community participation approaches more broadly such that these may be leveraged in an emergency. Developing and deploying a community-based surveillance system based on known drivers of success may improve their efficiency and effectiveness; however, it is important to balance the burdens of community-based surveillance (particularly in resource-limited settings) against the potential benefits. Introduction Community-based surveillance (CBS) is defined by the WHO as: '…the systematic detection and reporting of events of public health significance within a community by community members'. 1 Though CBS is often designed for the routine detection and reporting of infectious diseases, it is a potentially versatile and scalable intervention and has been used for the detection and reporting of non-communicable diseases 2–4 , for monitoring births and deaths 5 6 , for carrying out verbal autopsies 7 8 , and more recently, for containing outbreaks of COVID-19. 9–12 A CBS system can: provide early case detection and reporting during disease outbreaks; monitor events of public health importance in humanitarian emergencies; and supplement non-existent or limited surveillance coverage in other complex settings. 13 In addition, CBS is one of the few suitable options for supporting OneHealth surveillance activities given its proximity to the interface between humans and animals. 14 15 Given its potential to enhance the early warning and containment function of national surveillance systems, CBS is increasingly framed as a promising surveillance modality in the discourse around global health security. 16 Case identification and reporting are often carried out at health facilities. Health facilities are able to perform these functions for various reasons: (1) they are typically staffed by healthcare workers who are able to carry out case identification based on standardised case definitions, (2) they are part of a network capable of centralised communication and reporting using an established data collection system, (3) they may provide allied health services (eg, laboratory services) that enable case detection and confirmation, and (4) they attract people who are seeking care for diseases or conditions under surveillance. The effectiveness of facility-based surveillance systems is largely dependent on context-specific healthcare-seeking behaviours. Health facilities may be difficult to access and may require people to weigh the challenges of accessing a facility against more proximal and practical concerns. Limited access to health facilities may encourage and inculcate community preference for informal care, particularly in remote areas with limited transportation options, and in countries that require out-of-pocket payments for health services. Community distrust of healthcare actors and/or a lack of confidence in the quality of health services also erode willingness to engage with services. Even in settings with strong facility-based surveillance systems, late presentation of patients with an infectious disease is common and often results in over-representation of late-stage infections that may be difficult and costly to manage. Delayed health seeking may increase community transmission, complicate case investigation and contact-tracing, and limit the impact of public health measures including health education and behaviour change initiatives, vaccination, and antimicrobial prophylaxis. CBS—which involves engaging community members to carry out specific surveillance functions within their own communities—is intended to complement facility-based systems by addressing these challenges, particularly in rural areas within low-resource settings. In July 2021, the WHO published the findings of an ad hoc consultation on community-centred approaches to health emergencies with the aim of identifying evidence gaps and research priorities. 17 The consultation proposed that a review of the evidence was required to determine, 'what methodologies and approaches are being used for community surveillance, and what are the fundamental drivers of success?' 17 (p. 32). A 2019 scoping review by Guerra et al 18 presents a thorough description of CBS methods and approaches; thus, the aim of this review is to synthesise the empirical evidence of the key success factors of CBS systems. Our review expands beyond the scope of Ratnayake et al 19 (which was limited to the use of CBS in humanitarian crises) and Kuehne et al 20 (which focused exclusively on event-based surveillance), and incorporates learning from recent studies evaluating the use of CBS for the detection and reporting of COVID-19. This review is reported against the updated Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 guidelines including the extension for abstract reporting. 21 22 The review was published in the PROSPERO prospective review database (CRD42022303971). 23 Methods Eligibility criteria We defined eligibility as any empirical source that included a significant evaluative component and that described CBS in a manner consistent with the definition proposed by the technical contributors to the 2018 WHO global technical meeting. 1 We included peer-reviewed or grey literature sources reporting on the use of CBS for event-based or indicator-based surveillance. We limited sources to those that described CBS in the context of infectious diseases (including parasitic infection) in humans. We included sources that identified drivers of success (regardless of how the authors defined success), provided there was a clear empirical basis for such assertions. We included sources in any language, employing any method (ie, qualitative or quantitative), or reporting on the use of CBS in any setting. Sources that focused exclusively on the use of CBS for vector control or non-human animal surveillance were excluded, as were sources focusing exclusively on the evaluation of a technological solution. In addition, we excluded sources that did not aim to evaluate CBS, that were not substantially focused on CBS, or that focused exclusively on one specific aspect of CBS (eg, training). We excluded reviews and conference abstracts. Information sources and search strategy We searched for peer-reviewed literature via MEDLINE, EMBASE and Global Health databases (via Ovid). We also searched SCOPUS and ReliefWeb. All searches were carried out using proximity and controlled vocabulary searches. We limited our search strategy to the last 10 years (beginning 1 January 2012) assuming: (a) that there would be very little published evidence before this time period (the evidence gap was identified in 2021 17 ), and (b) that CBS approaches have been refined over time and that it would therefore be sensible to focus on current iterations of CBS approaches in the context of diseases of current importance to public health. All database searches were carried out on 7 February 2022. The complete search strategy is included in online supplemental appendix 1 . 10.1136/bmjgh-2022-009934.supp1 Supplementary data Two members of the review team carried out grey literature searches using both the Google and DuckDuckGo search engines, and by searching relevant websites (eg, Médecins Sans Frontières's (MSF) Science Portal, WHO's Publications website) using 'community-based surveillance' as the primary search term. The grey literature searches were carried out during the first week of February 2022. Data extraction and synthesis Two authors independently reviewed all peer-reviewed sources; disagreements were resolved by a third author. One author reviewed grey literature sources. One author manually extracted all data by coding sources in NVivo V.1.0 (Melbourne, Australia: QSR International). Data were extracted based on the following domains: (1) the CBS system (ie, description of the CBS system, the problem CBS was intended to address, the disease(s)/condition(s) under surveillance, the data that were collected, and the setting in which the CBS system was implemented, performance indicators and evaluation methods), and (2) the reported challenges and drivers of success. We undertook a narrative synthesis of the literature with an emphasis on the evidence of drivers of success. Quality assessment We used the evaluation quality checklist created by Warsame et al to simplify the quality assessment ( online supplemental appendix 2 ). 24 This method allowed us to focus on the quality of the evaluative aspects of the included sources and to allocate weighted scores based, in part, on the degree to which the challenges and drivers of success were substantiated by the empirical evidence. One author completed the scorecard for all sources; scores were compared with those of a second author for two sources before the remainder of the scorecards were completed. We did not assess risk of bias. 10.1136/bmjgh-2022-009934.supp2 Supplementary data Patient and public involvement Humanitarian health professionals have been involved in every stage of this review including in its design, conduct, and write-up. Our team includes humanitarians with direct experience designing, implementing, and/or otherwise supporting community-based and facility-based surveillance systems. The entire review team has experience working in conflict settings and/or in infectious disease outbreak response. Eligibility criteria We defined eligibility as any empirical source that included a significant evaluative component and that described CBS in a manner consistent with the definition proposed by the technical contributors to the 2018 WHO global technical meeting. 1 We included peer-reviewed or grey literature sources reporting on the use of CBS for event-based or indicator-based surveillance. We limited sources to those that described CBS in the context of infectious diseases (including parasitic infection) in humans. We included sources that identified drivers of success (regardless of how the authors defined success), provided there was a clear empirical basis for such assertions. We included sources in any language, employing any method (ie, qualitative or quantitative), or reporting on the use of CBS in any setting. Sources that focused exclusively on the use of CBS for vector control or non-human animal surveillance were excluded, as were sources focusing exclusively on the evaluation of a technological solution. In addition, we excluded sources that did not aim to evaluate CBS, that were not substantially focused on CBS, or that focused exclusively on one specific aspect of CBS (eg, training). We excluded reviews and conference abstracts. Information sources and search strategy We searched for peer-reviewed literature via MEDLINE, EMBASE and Global Health databases (via Ovid). We also searched SCOPUS and ReliefWeb. All searches were carried out using proximity and controlled vocabulary searches. We limited our search strategy to the last 10 years (beginning 1 January 2012) assuming: (a) that there would be very little published evidence before this time period (the evidence gap was identified in 2021 17 ), and (b) that CBS approaches have been refined over time and that it would therefore be sensible to focus on current iterations of CBS approaches in the context of diseases of current importance to public health. All database searches were carried out on 7 February 2022. The complete search strategy is included in online supplemental appendix 1 . 10.1136/bmjgh-2022-009934.supp1 Supplementary data Two members of the review team carried out grey literature searches using both the Google and DuckDuckGo search engines, and by searching relevant websites (eg, Médecins Sans Frontières's (MSF) Science Portal, WHO's Publications website) using 'community-based surveillance' as the primary search term. The grey literature searches were carried out during the first week of February 2022. Data extraction and synthesis Two authors independently reviewed all peer-reviewed sources; disagreements were resolved by a third author. One author reviewed grey literature sources. One author manually extracted all data by coding sources in NVivo V.1.0 (Melbourne, Australia: QSR International). Data were extracted based on the following domains: (1) the CBS system (ie, description of the CBS system, the problem CBS was intended to address, the disease(s)/condition(s) under surveillance, the data that were collected, and the setting in which the CBS system was implemented, performance indicators and evaluation methods), and (2) the reported challenges and drivers of success. We undertook a narrative synthesis of the literature with an emphasis on the evidence of drivers of success. Quality assessment We used the evaluation quality checklist created by Warsame et al to simplify the quality assessment ( online supplemental appendix 2 ). 24 This method allowed us to focus on the quality of the evaluative aspects of the included sources and to allocate weighted scores based, in part, on the degree to which the challenges and drivers of success were substantiated by the empirical evidence. One author completed the scorecard for all sources; scores were compared with those of a second author for two sources before the remainder of the scorecards were completed. We did not assess risk of bias. 10.1136/bmjgh-2022-009934.supp2 Supplementary data Patient and public involvement Humanitarian health professionals have been involved in every stage of this review including in its design, conduct, and write-up. Our team includes humanitarians with direct experience designing, implementing, and/or otherwise supporting community-based and facility-based surveillance systems. The entire review team has experience working in conflict settings and/or in infectious disease outbreak response. Results Study selection Our initial database search resulted in 1274 records published between 2012 and 2022. Removal of duplicate records was carried out using EndNote V.20 (Philadelphia, Pennsylvania, USA: Clarivate) and resulted in 881 unique records. Sixty-eight records remained following the initial screening on title and abstract; the full-text was retrieved for all included sources. Two authors reviewed the 68 full-text sources. Full-text review resulted in the exclusion of 51 sources owing to: (1) an unsubstantial focus on CBS, (2) a disproportionate focus on a specific aspect of CBS (eg, training), (3) no evaluative component and/or lack of empirical evidence, (4) a lack of focus on the use of CBS for infectious disease detection and reporting, (5) exclusive focus on vector control or animal surveillance (eg, diseases in pigs/dogs only), (6) exclusive focus on evaluating the effectiveness of a technological solution and (7) no mention of success factors. Other systematic reviews and conference abstracts were also excluded. The grey literature search identified 20 sources; the full text for all 20 sources was retrieved. Of these, only two met the inclusion criteria; the remaining 18 sources did not include any evaluation. Nineteen sources were included in the final synthesis. The PRISMA flow chart is included in figure 1 . Figure 1 PRISMA flow diagram. CBS, community-based surveillance; PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Study characteristics The study characteristics are included in table 1 . The included sources reported on CBS in: Bangladesh 25 , Cambodia 26 , Cameroon 27 , Côte d'Ivoire 28 , the Dominican Republic 29 , the Democratic Republic of the Congo (DRC) 30 , Ethiopia 31 32 , Ghana 33 34 , Niger 11 , Nigeria 35 , Senegal 36 , Sierra Leone 37 38 , South Sudan 39 40 , Vietnam 41 , and Yemen. 12 Two CBS systems were described in the context of refugee/internally displaced person camps (Bangladesh and Yemen) and three were deployed into an active conflict setting (Cameroon, South Sudan, and Yemen). No studies of the use of CBS in high-income settings were identified. Table 1 List of sources and CBS characteristics Author Year of publication Peer reviewed or grey literature Country Disease Conflict setting Data collection Population under surveillance Successes Ahorlu et al 33 2018 Peer reviewed Ghana Buruli ulcer (BU) N Suspect BU cases (physical examination) Ga West Municipality (10 communities: Kojo-Ashong, Onyansana, Otuaplam, Yahoman, Okushibiade, Adeyman, Kramo, Domsampaman, Kwashikuma, Odumtia/Akwakyere) CBS workers Acceptance of CBS workers Community nominates CBS workers Motivation of CBS workers Sense of contributing to health within the community Sense of community ownership Material incentives Baaees et al 12 2021 Peer reviewed Yemen COVID-19 Y Suspect COVID-19 cases (adapted case definition) IDP camps in Aden, Abyan, Lahj and Taiz; Hadramout (urban setting) Case detection and reporting Effective use of technology Simplicity Badara et al 36 2018 Grey literature Senegal Multiple N Measles, bloody diarrhoea, neonatal tetanus, meningitis, yellow fever, AFP, cholera and haemorrhagic fever Tambacounda region (Tambacounda and Koumpentoum districts) and Saint Louis (Pété and Podor districts) Integration Vertical Clara et al 41 2018 Peer reviewed Vietnam Multiple N Rabies, avian influenza, vaccine-preventable diseases, cholera and emerging new diseases (symptoms) Quang Ninh, Nam Dinh, Ba-Ria Vung Tau and An Giang provinces CBS workers Proximity to communities Supervision and training Training increased motivation and quality Community Communication and engagement Increased community involvement and innovative communication strategies Engagement with community leaders Recruitment of community informants Integration Vertical Clara et al 28 2020 Peer reviewed Côte d'Ivoire Multiple N Polio, cholera, measles, meningitis and yellow fever (and illness in a healthcare worker, death of a healthcare worker, unexpected animal or fish deaths, a sudden or unexplained death in the community, and arrival in the community of any person coming from a country or region experiencing an epidemic) (symptoms) Odienne', Touba and Minignan districts of the Kabadougou-Bafing-Folon health region CBS workers Supervision and training Training increased motivation and quality Case detection and reporting Effective use of technology Simplicity Cox et al 26 2014 Peer reviewed Cambodia Malaria N Malaria (RDT confirmed) Pailin, Battambang and Pursat provinces in western Cambodia CBS workers Motivation of CBS workers Sense of service to the community Training opportunities and opportunities to increase knowledge Supervision and training Strong supervision Training increased motivation and quality Curry et al 31 2013 Peer reviewed Ethiopia Polio N Symptoms of AFP (and measles and neonatal tetanus) Rural Ethiopia including pastoralist and semipastoralist populations CBS workers Acceptance of CBS workers Community nominates CBS workers Proximity to communities Community Communication and engagement Community events Trust Integration Vertical Ezenyeaku et al 35 2020 Peer reviewed Nigeria Multiple N Epidemic-prone and other diseases of public health importance through the Integrated Disease Surveillance and Response system Anambra State Community Communication and engagement Feedback provided to communities Recruitment of community informants Hemingway-Foday et al 30 2020 Peer reviewed DRC EVD N Suspect EVD (case definition) case detection and contact-tracing Likati district of the Bas-Ue'le' province Case detection and reporting Quality assurance Real-time use of data for decision-making Effective use of technology JICA AmRids Project 32 2014 Grey literature Ethiopia Multiple N Polio, anthrax, cholera, measles, neonatal tetanus, rabies, meningococcal meningitis, any other public health emergency, diarrhoea and pneumonia (under 5 years), malaria Mecha Woreda West Gojam, Dembia Woreda North Gondar, Ebinat Woreda South Gondar in the Amhara National Regional State and Southern Nations, Nationalities and People's Region CBS workers Supervision and training Strong supervision Case detection and reporting Initiatives to improve record keeping Integration Vertical Kisanga et al 39 2019 Peer reviewed South Sudan Polio Y AFP 34 counties within Unity State, Jonglei, Upper Nile and Kapoeta East CBS workers Supervision and training Strong supervision Training increased motivation and quality Community Communication and engagement Strong engagement Recruitment of community informants Integration Vertical Ladoa et al 40 2012 Peer reviewed South Sudan Guinea worm Y Presence of guinea worm blister and emerging guinea worm All states in South Sudan with the exceptions of areas in Upper Nile, Jongeli and Eastern Equatoria CBS workers Motivation of CBS workers Sense of service to the community Desire to increase ties and trust within the community Case detection and reporting Real-time use of data for decision-making Simplicity Maazou et al 11 2021 Peer reviewed Niger COVID-19 N COVID-19 (adapted case definition) The work was conducted across 37 health districts throughout six regions in Niger Integration Vertical Mejdell Larsen et al 38 2017 Peer reviewed Sierra Leone Multiple N EVD (case definitions), epidemic-prone diseases and community deaths and flood/wildfire): viral haemorrhagic fevers, including Ebola and Lassa fever; acute watery diarrhoea; measles and community deaths Port Loko, Koinadugu and Bonthe CBS workers Acceptance of CBS workers Recruiting CBS workers from within communities under surveillance Motivation of CBS workers Sense of service to the community Satisfaction about knowing lives saved Community Communication and engagement Trust Merali et al 34 2020 Peer reviewed Ghana Multiple N OneHealth Signals (combination of signals to identify animal-related events, vaccine preventable diseases, food-borne illnesses, infectious diseases—acute haemorrhagic conjunctivitis, malaria, skin diseases, suspected cholera, infectious arthritis) and AEFI Phase 1 (Ketu South, a periurban district bordering Togo, and Kassena Nankana West, a rural district bordering Burkina Faso) and phase 2 (Ghana Health Service selected 28 more districts where modified CBS would be implemented, prioritising rural districts with hard-to-reach communities whose residents might face difficulty in accessing healthcare CBS workers Supervision and training Training increased motivation and quality Case detection and reporting Dynamic case definitions Simple case definitions Integration Lateral Metuge et al 27 2021 Peer reviewed Cameroon Multiple Y OPD: AFP, measles, cholera, BU/TU, meningitis (case definition); ED: SAM, displacement, uncomplicated malaria, ARI, AWD, neonatal tetanus (base definition) Ekondo-Titi health district CBS workers Acceptance of CBS workers Recruiting CBS workers from within communities under surveillance Nesting CBS within an existing system that itself had good acceptance Proximity to communities Community Communication and engagement Feedback provided to communities Integration Vertical Stone et al 37 2016 Peer reviewed Sierra Leone EVD N Suspect EVD case Districts of: Bo, Bombali, Kailahun, Kambia, Kenema, Kono, Moyamba, Pujehun, Tonkolili CBS workers Supervision and training Strong supervision Community Communication and engagement Strong engagement Valdez et al 29 2020 Peer reviewed Dominican Republic Malaria N Malaria (RDT confirmed) Los Tres Brazos neighbourhood of urban Santo Domingo CBS workers Motivation of CBS workers Sense of service to the community Desire to increase ties and trust within the community Sense of camaraderie and shared sense of responsibility Proximity to communities Community Communication and engagement Strong engagement Trust Van Boetzelaer et al 25 2020 Peer reviewed Bangladesh Multiple N AWD, acute jaundice syndrome, AFP dengue, diphtheria, measles and meningitis Cox's Bazar CBS workers Acceptance of CBS workers Recruiting CBS workers from within communities under surveillance Community Communication and engagement Trust Case detection and reporting Real-time use of data for decision-making Integration Lateral AEFI, adverse event following immunisation; AFP, acute flaccid paralysis; ARI, acute respiratory infections; AWD, acute watery diarrhoea; BU, Buruli ulcer; CBS, community-based surveillance; DRC, Democratic Republic of the Congo; ED, endemic diseases; ED, emergency department; EVD, Ebola virus disease; IDP, internally displaced persons; OPD, outbreak prone diseases; RDT, rapid diagnostic test; SAM, severe acute malnutrition; TU, tropical ulcer. Sources evaluated CBS for detection and reporting of: buruli ulcer 33 , COVID-19 11 12 , Ebola virus disease 30 37 , guinea worm 40 , malaria 26 29 , polio 31 39 , and multiple infectious diseases. 25 27 28 32 34–36 38 41 The CBS systems were designed specifically to address the following problems: Limitations to the effectiveness of facility-based surveillance systems These limitations included: delayed care seeking resulting in advanced clinical presentation and/or complicated and costly treatment 33 ; delayed care seeking resulting in poor prognosis including comorbidities, permanent disability or death 12 33 ; cases not presenting to health facility, low health service uptake, and/or poor access to health facilities 11 12 27 30 31 35 38 ; and healthcare facilities lacked event-based reporting, had poor indicator reporting (eg, no established alert threshold), or are not required to report. 11 27 34 41 Heightened risk/vulnerability within communities These included: an outbreak or spike in cases detected and/or high community transmission 26 29 30 38 39 , low levels of vaccination 39 , high population movement 39 , insecurity 27 39 , and endemic and/or high numbers of cases 40 and/or the need for urgent containment. 12 26 37 Health system factors These included: existing surveillance system slow to identify cases 36 ; community health workers underused, not engaged in case detection and/or reporting 41 ; insufficient health system capacity to contain outbreak 11 12 30 ; poor integration of OneHealth 34 ; disease or condition is low incidence (requiring expanded case detection) and high priority for early detection 31 39 ; and CBS established in response to a formal review/assessment/technical consultation of surveillance capacity. 34–36 39 41 Three CBS systems were implemented out of a desire/opportunity to scale up an existing community-based function to include CBS, or to scale up an existing CBS system to include surveillance of infectious diseases. 28 32 37 Additionally, one was developed as part of a global elimination effort (ie, guinea worm) 40 ; another was developed as part of a system of standard epidemiological tools/functions to monitor the health of the refugee population in Cox's Bazar, Bangladesh. 25 Included sources described CBS systems that involved the recruitment of community members (eg, community health workers, community health volunteers) to carry out active surveillance, the scale up of an existing surveillance system (to include new infectious diseases), or the scale up of an existing community health worker programme (to involve disease surveillance). The CBS systems included both event-based surveillance, indicator-based surveillance, and a mixture of both. Quality The quality of the included sources ranged from 0.35 (out of 1) to 0.925 ( table 2 ). We considered five sources very high quality (between 0.9 and 1.0) 12 33 35 39 41 , six high quality (between 0.8 and 0.89), 25–28 34 37 three moderate quality (between 0.7 and 0.79), 29 30 38 and five of lower quality (below 0.69). 11 31 32 36 40 Most sources contained a clear rationale, a clear objective, and a thorough description of the operational context. Few sources used or reported using a formal evaluation framework or assessment tool despite the fact that such resources are available. 42–44 Table 2 Quality assessment Author Scope (0.25) Methodology (0.25) Findings (0.25) Recommendations (0.25) Total (1.0) Ahorlu et al 33 0.2 0.2 0.25 0.25 0.9 Baaees et al 12 0.225 0.2 0.25 0.25 0.925 Badara et al 36 0.075 0.025 0.125 0.125 0.35 Clara et al 41 0.225 0.25 0.25 0.25 0.975 Clara et al 28 0.2 0.175 0.25 0.25 0.875 Cox et al 26 0.225 0.125 0.25 0.25 0.85 Curry et al 31 0.175 0.1 0.125 0.125 0.525 Ezenyeaku et al 35 0.225 0.2 0.25 0.25 0.925 Hemingway-Foday et al 30 0.2 0.075 0.25 0.25 0.775 JICA AmRids Project 32 0.175 0.025 0.1875 0.125 0.5125 Kisanga et al 39 0.225 0.2 0.25 0.25 0.925 Ladoa et al 40 0.175 0.15 0.1875 0.125 0.6375 Maazou et al 11 0.2 0.075 0.25 0.125 0.65 Mejdell Larsen et al 38 0.175 0.1 0.1875 0.25 0.7125 Merali et al 34 0.175 0.2 0.25 0.25 0.875 Metuge et al 27 0.225 0.125 0.25 0.25 0.85 Stone et al 37 0.2 0.175 0.1875 0.25 0.8125 Valdez et al 29 0.15 0.175 0.1875 0.25 0.7625 Van Boetzelaer et al 25 0.225 0.15 0.25 0.25 0.875 Results of synthesis We report below a narrative synthesis of our findings. Our emphasis, in line with the WHO call for evidence, is on identifying drivers of success. 17 Some of the challenges we identified whilst carrying out this review have been reported elsewhere 19 20 ; we have included a brief summary of the challenges we identified in online supplemental appendix 3 . 10.1136/bmjgh-2022-009934.supp3 Supplementary data Drivers of success Success factors fell broadly into four categories: (1) CBS workers (often community health workers), (2) community, (3) case detection and reporting, and (4) integration. CBS workers The CBS workers were described as those responsible for active case detection. Additionally, CBS workers were often responsible for a number of additional tasks including: reporting, referral, follow-up, case management, health promotion, physical examinations (eg, for buruli ulcer 33 ), and testing (eg, taking blood slides for malaria parasites 26 ). Acceptance of CBS workers Successful CBS was believed to be associated with community acceptance of the CBS workers that was, in turn, associated with their recruitment (ie, having the community nominate the CBS workers 31 33 or recruiting CBS workers from within the community 25 27 38 ), or with having nested CBS within an existing emergency response system that itself had good acceptability (which, in turn, was attributed to active participation and collaboration with communities). 27 Trust between CBS workers and the community was also described as a key success factor. 25 29 31 38 Motivation of CBS workers Success was also attributed to high motivation of CBS workers who described their motivation in terms of: 'contributing to bringing good health to the people' 33 (p. 10), feeling a sense of service to the community 26 29 38 41 , and a desire to increase ties and trust with other community members. 29 41 Training opportunities and the opportunity to increase knowledge were also described as motivating factors, even when the programme lacked material incentives. 26 Success in achieving high performance and acceptance of CBS workers in Ghana was, in part, ascribed to efforts made to 'follow all community protocols and encourage the people to see the project as their own' 33 (p. 9). A sense of camaraderie amongst CBS workers and a shared sense of responsibility for bringing an outbreak of malaria under control were also felt to be associated with the success of a CBS programme in the Dominican Republic. 29 Volunteer CBS workers in Ghana were provided with material incentives (in the form of a token to cover travel costs, plus a bicycle), which were described as motivating factors in addition to a sense of service and ownership of the CBS programme. 33 Finally, CBS workers in Sierra Leone derived satisfaction from knowing that they had saved lives of people with Ebola; 'volunteers gave examples of cases they had reported which had received response and treatment […] many of the volunteers were confident that the death rate had decreased due to [CBS workers] influencing people to seek medical attention earlier" 38 (p. 4). CBS worker proximity to communities The close proximity of CBS workers to their communities was felt to have contributed to increased detection of disease clusters in Vietnam 41 and better overall case detection in the Dominican Republic. 29 The CBS programme amongst conflict-affected populations in Cameroon succeeded in collecting data from populations despite 'pendular displacements'; this was attributed to the fact that CBS workers were travelling with their communities and were thus able to continue surveillance. 27 Similarly, CBS workers in Ethiopia were able to spend time at locations where pastoralist communities congregate (eg. wells or water collection areas, mosques, marketplaces), allowing CBS workers to carry out polio surveillance among a nomadic population. 31 Supervision and training Strong supervision was believed to have influenced the success of CBS programmes for malaria in Cambodia 26 , acute flaccid paralysis (AFP)—a proxy for polio—in South Sudan 39 , Ebola in Sierra Leone 37 , and eight high-priority diseases in Senegal. 32 Training was also frequently mentioned as a key factor in the success of CBS, either due to its effect on motivation of CBS workers or improving the quality of their work (in terms of quantity of reports and the specificity of case detection). 26 28 34 39 41 Training was felt to be particularly important in low transmission settings where CBS workers have few opportunities to practise their skills. 26 Availability of refresher training was also felt to be important and resulted in an increase in the quality of blood sample slides in a CBS system for malaria in western Cambodia. 26 Community Several success factors related to the interaction between the CBS system and the community: effective communication and engagement strategies, and the recruitment of community informants were both described as influencing the success of CBS programmes. Communication and engagement Communication and engagement were the most frequently cited success factors of CBS. For example, increased community involvement and 'innovative communication strategies' (ie, a text-based reporting system to enable real-time reporting of signals) were felt to improve signal detection in Côte d'Ivoire 28 (p. S-32). Providing feedback to communities was associated with completeness of reporting in Nigeria 35 , and increased community reporting in Cameroon. 27 In Ethiopia, CBS workers organised village coffee ceremonies at which they were able to ask for reports of AFP and discuss signs, symptoms and reporting. 31 Engagement with community leaders was positively associated with programme uptake in Vietnam. 41 Finally, strong community engagement was believed to have been key to the success of an AFP programme in South Sudan 39 , a malaria surveillance and control programme in the Dominican Republic 29 , and an Ebola surveillance system in Sierra Leone. 37 Recruitment of community informants Several CBS systems relied on the recruitment of community informants who would report suspect cases to CBS workers. The recruitment of a diverse team (including money lenders, insurance agents, veterinary health staff, landlords, factory managers, community leaders and others) of informants with strong community ties in Vietnam, '…broadened the sources of reporting and resulted in the reporting of numerous signals that otherwise would have been missed, such as school absenteeism reported by teachers and the resulting multiple detections of vaccine-preventable disease (eg, mumps and chickenpox)' 41 (p. 1656). In Nigeria, there was a significant positive association between informant satisfaction and completeness of disease notification 35 , and field interviews in South Sudan indicated that having a network of community informants in every village contributed to the effective functioning of the CBS system. 39 Case detection and reporting Successes relating to data collection included: dynamic (ie, adapted and improved) use of case definitions 34 ; the implementation of quality assurance procedures (ie, data were regularly reviewed for accuracy and completeness) 30 ; and engaging in rapid, real-time data-driven decision-making. 25 30 41 Efforts to improve the office environment (eg, by implementing a filing system) in the health posts supporting a CBS pilot in Ethiopia were found to improve record keeping and reporting. 32 The use of technology for data collection and reporting was generally reported as a challenge, though two sources referenced successful use of technological solutions which 'removed reporting obstacles and may account for the increase in the number of notifications' (in the case of a text-based reporting system in Côte d'Ivoire) 28 (p. S-31) and was cited by WHO as 'a critical factor for improved early detection of suspected cases' (in the case of a voice and SMS-based alert system in DRC) 30 (p. S-89). It should be noted that the text and voice messaging solutions in Côte d'Ivoire and DRC were described as 'simple' and yet both systems received considerable technological support from the International Rescue Committee (in Côte d'Ivoire), and RT International, MSF and the WHO (in DRC). 28 30 Ultimately, the most often observed driver of success was simplicity with respect to the design of data collection and reporting tools (eg, 'tools should aim to collect a minimum set of data that can provide usable information, should be clear and simple, and minimise burden to implementers' 34 (p. 10)). Simplifying data collection by limiting the number of reportable diseases 28 , and by simplifying signals 28 and case definitions 12 40 , was associated with ease of reporting, case identification and reducing the proportion of false alerts. Integration with the wider surveillance system Effective vertical integration of CBS with different actors along the reporting pathway (eg, from communities, to the health facility, to the regional/national surveillance system), and lateral integration between the CBS system and other components of the surveillance system at, or close to, the same operational level (eg, laboratory services, operational partners) were identified as success factors. Vertical integration Clear reporting pathways from communities through the various levels within the wider surveillance system were felt to have: improved timeliness of reporting and response in Cameroon 27 ; enabled regular reporting and rapid case confirmation in Ethiopia 32 , Vietnam 41 , Senegal 36 , and Niger 11 ; and increased community engagement in polio eradication in South Sudan. 39 Widespread mobile phone coverage, coupled with the close proximity of health posts to communities (most were within a 30-minute walk), ensured regular reporting of suspect cases of communicable diseases from CBS volunteers and health extension workers (who were responsible for case confirmation and reporting to the cluster health centre) in southern Ethiopia. 32 In Niger, an extensive polio surveillance system was scaled to include active case finding and reporting of COVID-19. 11 The polio surveillance system used established reporting pathways (from community health workers up to the Central Supervisory Directorate of Public Health); thus, the COVID-19 CBS capitalised on pre-existing vertical integration and avoided the, 'structural challenges [of] establishing a de novo CBS to respond to an emerging public health crisis' 11 (p. 5). Lateral integration A CBS system for OneHealth surveillance was deemed to have been successful due to the close collaboration between the Ghana Health Service and the Veterinary Services Directorate. 34 Additionally, members of the wider surveillance system (at the regional and district levels) received targeted training on multiagency coordination. 34 The simplicity of the CBS system in Cox's Bazar, Bangladesh was felt to have enabled easy integration with other aspects of the surveillance system (eg, WHO's Early Warning, Alert and Response System). 25 System integration was also managed by a focal point who was appointed to submit reports and coordinate between the WHO and MSF. 25 Study selection Our initial database search resulted in 1274 records published between 2012 and 2022. Removal of duplicate records was carried out using EndNote V.20 (Philadelphia, Pennsylvania, USA: Clarivate) and resulted in 881 unique records. Sixty-eight records remained following the initial screening on title and abstract; the full-text was retrieved for all included sources. Two authors reviewed the 68 full-text sources. Full-text review resulted in the exclusion of 51 sources owing to: (1) an unsubstantial focus on CBS, (2) a disproportionate focus on a specific aspect of CBS (eg, training), (3) no evaluative component and/or lack of empirical evidence, (4) a lack of focus on the use of CBS for infectious disease detection and reporting, (5) exclusive focus on vector control or animal surveillance (eg, diseases in pigs/dogs only), (6) exclusive focus on evaluating the effectiveness of a technological solution and (7) no mention of success factors. Other systematic reviews and conference abstracts were also excluded. The grey literature search identified 20 sources; the full text for all 20 sources was retrieved. Of these, only two met the inclusion criteria; the remaining 18 sources did not include any evaluation. Nineteen sources were included in the final synthesis. The PRISMA flow chart is included in figure 1 . Figure 1 PRISMA flow diagram. CBS, community-based surveillance; PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses. Study characteristics The study characteristics are included in table 1 . The included sources reported on CBS in: Bangladesh 25 , Cambodia 26 , Cameroon 27 , Côte d'Ivoire 28 , the Dominican Republic 29 , the Democratic Republic of the Congo (DRC) 30 , Ethiopia 31 32 , Ghana 33 34 , Niger 11 , Nigeria 35 , Senegal 36 , Sierra Leone 37 38 , South Sudan 39 40 , Vietnam 41 , and Yemen. 12 Two CBS systems were described in the context of refugee/internally displaced person camps (Bangladesh and Yemen) and three were deployed into an active conflict setting (Cameroon, South Sudan, and Yemen). No studies of the use of CBS in high-income settings were identified. Table 1 List of sources and CBS characteristics Author Year of publication Peer reviewed or grey literature Country Disease Conflict setting Data collection Population under surveillance Successes Ahorlu et al 33 2018 Peer reviewed Ghana Buruli ulcer (BU) N Suspect BU cases (physical examination) Ga West Municipality (10 communities: Kojo-Ashong, Onyansana, Otuaplam, Yahoman, Okushibiade, Adeyman, Kramo, Domsampaman, Kwashikuma, Odumtia/Akwakyere) CBS workers Acceptance of CBS workers Community nominates CBS workers Motivation of CBS workers Sense of contributing to health within the community Sense of community ownership Material incentives Baaees et al 12 2021 Peer reviewed Yemen COVID-19 Y Suspect COVID-19 cases (adapted case definition) IDP camps in Aden, Abyan, Lahj and Taiz; Hadramout (urban setting) Case detection and reporting Effective use of technology Simplicity Badara et al 36 2018 Grey literature Senegal Multiple N Measles, bloody diarrhoea, neonatal tetanus, meningitis, yellow fever, AFP, cholera and haemorrhagic fever Tambacounda region (Tambacounda and Koumpentoum districts) and Saint Louis (Pété and Podor districts) Integration Vertical Clara et al 41 2018 Peer reviewed Vietnam Multiple N Rabies, avian influenza, vaccine-preventable diseases, cholera and emerging new diseases (symptoms) Quang Ninh, Nam Dinh, Ba-Ria Vung Tau and An Giang provinces CBS workers Proximity to communities Supervision and training Training increased motivation and quality Community Communication and engagement Increased community involvement and innovative communication strategies Engagement with community leaders Recruitment of community informants Integration Vertical Clara et al 28 2020 Peer reviewed Côte d'Ivoire Multiple N Polio, cholera, measles, meningitis and yellow fever (and illness in a healthcare worker, death of a healthcare worker, unexpected animal or fish deaths, a sudden or unexplained death in the community, and arrival in the community of any person coming from a country or region experiencing an epidemic) (symptoms) Odienne', Touba and Minignan districts of the Kabadougou-Bafing-Folon health region CBS workers Supervision and training Training increased motivation and quality Case detection and reporting Effective use of technology Simplicity Cox et al 26 2014 Peer reviewed Cambodia Malaria N Malaria (RDT confirmed) Pailin, Battambang and Pursat provinces in western Cambodia CBS workers Motivation of CBS workers Sense of service to the community Training opportunities and opportunities to increase knowledge Supervision and training Strong supervision Training increased motivation and quality Curry et al 31 2013 Peer reviewed Ethiopia Polio N Symptoms of AFP (and measles and neonatal tetanus) Rural Ethiopia including pastoralist and semipastoralist populations CBS workers Acceptance of CBS workers Community nominates CBS workers Proximity to communities Community Communication and engagement Community events Trust Integration Vertical Ezenyeaku et al 35 2020 Peer reviewed Nigeria Multiple N Epidemic-prone and other diseases of public health importance through the Integrated Disease Surveillance and Response system Anambra State Community Communication and engagement Feedback provided to communities Recruitment of community informants Hemingway-Foday et al 30 2020 Peer reviewed DRC EVD N Suspect EVD (case definition) case detection and contact-tracing Likati district of the Bas-Ue'le' province Case detection and reporting Quality assurance Real-time use of data for decision-making Effective use of technology JICA AmRids Project 32 2014 Grey literature Ethiopia Multiple N Polio, anthrax, cholera, measles, neonatal tetanus, rabies, meningococcal meningitis, any other public health emergency, diarrhoea and pneumonia (under 5 years), malaria Mecha Woreda West Gojam, Dembia Woreda North Gondar, Ebinat Woreda South Gondar in the Amhara National Regional State and Southern Nations, Nationalities and People's Region CBS workers Supervision and training Strong supervision Case detection and reporting Initiatives to improve record keeping Integration Vertical Kisanga et al 39 2019 Peer reviewed South Sudan Polio Y AFP 34 counties within Unity State, Jonglei, Upper Nile and Kapoeta East CBS workers Supervision and training Strong supervision Training increased motivation and quality Community Communication and engagement Strong engagement Recruitment of community informants Integration Vertical Ladoa et al 40 2012 Peer reviewed South Sudan Guinea worm Y Presence of guinea worm blister and emerging guinea worm All states in South Sudan with the exceptions of areas in Upper Nile, Jongeli and Eastern Equatoria CBS workers Motivation of CBS workers Sense of service to the community Desire to increase ties and trust within the community Case detection and reporting Real-time use of data for decision-making Simplicity Maazou et al 11 2021 Peer reviewed Niger COVID-19 N COVID-19 (adapted case definition) The work was conducted across 37 health districts throughout six regions in Niger Integration Vertical Mejdell Larsen et al 38 2017 Peer reviewed Sierra Leone Multiple N EVD (case definitions), epidemic-prone diseases and community deaths and flood/wildfire): viral haemorrhagic fevers, including Ebola and Lassa fever; acute watery diarrhoea; measles and community deaths Port Loko, Koinadugu and Bonthe CBS workers Acceptance of CBS workers Recruiting CBS workers from within communities under surveillance Motivation of CBS workers Sense of service to the community Satisfaction about knowing lives saved Community Communication and engagement Trust Merali et al 34 2020 Peer reviewed Ghana Multiple N OneHealth Signals (combination of signals to identify animal-related events, vaccine preventable diseases, food-borne illnesses, infectious diseases—acute haemorrhagic conjunctivitis, malaria, skin diseases, suspected cholera, infectious arthritis) and AEFI Phase 1 (Ketu South, a periurban district bordering Togo, and Kassena Nankana West, a rural district bordering Burkina Faso) and phase 2 (Ghana Health Service selected 28 more districts where modified CBS would be implemented, prioritising rural districts with hard-to-reach communities whose residents might face difficulty in accessing healthcare CBS workers Supervision and training Training increased motivation and quality Case detection and reporting Dynamic case definitions Simple case definitions Integration Lateral Metuge et al 27 2021 Peer reviewed Cameroon Multiple Y OPD: AFP, measles, cholera, BU/TU, meningitis (case definition); ED: SAM, displacement, uncomplicated malaria, ARI, AWD, neonatal tetanus (base definition) Ekondo-Titi health district CBS workers Acceptance of CBS workers Recruiting CBS workers from within communities under surveillance Nesting CBS within an existing system that itself had good acceptance Proximity to communities Community Communication and engagement Feedback provided to communities Integration Vertical Stone et al 37 2016 Peer reviewed Sierra Leone EVD N Suspect EVD case Districts of: Bo, Bombali, Kailahun, Kambia, Kenema, Kono, Moyamba, Pujehun, Tonkolili CBS workers Supervision and training Strong supervision Community Communication and engagement Strong engagement Valdez et al 29 2020 Peer reviewed Dominican Republic Malaria N Malaria (RDT confirmed) Los Tres Brazos neighbourhood of urban Santo Domingo CBS workers Motivation of CBS workers Sense of service to the community Desire to increase ties and trust within the community Sense of camaraderie and shared sense of responsibility Proximity to communities Community Communication and engagement Strong engagement Trust Van Boetzelaer et al 25 2020 Peer reviewed Bangladesh Multiple N AWD, acute jaundice syndrome, AFP dengue, diphtheria, measles and meningitis Cox's Bazar CBS workers Acceptance of CBS workers Recruiting CBS workers from within communities under surveillance Community Communication and engagement Trust Case detection and reporting Real-time use of data for decision-making Integration Lateral AEFI, adverse event following immunisation; AFP, acute flaccid paralysis; ARI, acute respiratory infections; AWD, acute watery diarrhoea; BU, Buruli ulcer; CBS, community-based surveillance; DRC, Democratic Republic of the Congo; ED, endemic diseases; ED, emergency department; EVD, Ebola virus disease; IDP, internally displaced persons; OPD, outbreak prone diseases; RDT, rapid diagnostic test; SAM, severe acute malnutrition; TU, tropical ulcer. Sources evaluated CBS for detection and reporting of: buruli ulcer 33 , COVID-19 11 12 , Ebola virus disease 30 37 , guinea worm 40 , malaria 26 29 , polio 31 39 , and multiple infectious diseases. 25 27 28 32 34–36 38 41 The CBS systems were designed specifically to address the following problems: Limitations to the effectiveness of facility-based surveillance systems These limitations included: delayed care seeking resulting in advanced clinical presentation and/or complicated and costly treatment 33 ; delayed care seeking resulting in poor prognosis including comorbidities, permanent disability or death 12 33 ; cases not presenting to health facility, low health service uptake, and/or poor access to health facilities 11 12 27 30 31 35 38 ; and healthcare facilities lacked event-based reporting, had poor indicator reporting (eg, no established alert threshold), or are not required to report. 11 27 34 41 Heightened risk/vulnerability within communities These included: an outbreak or spike in cases detected and/or high community transmission 26 29 30 38 39 , low levels of vaccination 39 , high population movement 39 , insecurity 27 39 , and endemic and/or high numbers of cases 40 and/or the need for urgent containment. 12 26 37 Health system factors These included: existing surveillance system slow to identify cases 36 ; community health workers underused, not engaged in case detection and/or reporting 41 ; insufficient health system capacity to contain outbreak 11 12 30 ; poor integration of OneHealth 34 ; disease or condition is low incidence (requiring expanded case detection) and high priority for early detection 31 39 ; and CBS established in response to a formal review/assessment/technical consultation of surveillance capacity. 34–36 39 41 Three CBS systems were implemented out of a desire/opportunity to scale up an existing community-based function to include CBS, or to scale up an existing CBS system to include surveillance of infectious diseases. 28 32 37 Additionally, one was developed as part of a global elimination effort (ie, guinea worm) 40 ; another was developed as part of a system of standard epidemiological tools/functions to monitor the health of the refugee population in Cox's Bazar, Bangladesh. 25 Included sources described CBS systems that involved the recruitment of community members (eg, community health workers, community health volunteers) to carry out active surveillance, the scale up of an existing surveillance system (to include new infectious diseases), or the scale up of an existing community health worker programme (to involve disease surveillance). The CBS systems included both event-based surveillance, indicator-based surveillance, and a mixture of both. Quality The quality of the included sources ranged from 0.35 (out of 1) to 0.925 ( table 2 ). We considered five sources very high quality (between 0.9 and 1.0) 12 33 35 39 41 , six high quality (between 0.8 and 0.89), 25–28 34 37 three moderate quality (between 0.7 and 0.79), 29 30 38 and five of lower quality (below 0.69). 11 31 32 36 40 Most sources contained a clear rationale, a clear objective, and a thorough description of the operational context. Few sources used or reported using a formal evaluation framework or assessment tool despite the fact that such resources are available. 42–44 Table 2 Quality assessment Author Scope (0.25) Methodology (0.25) Findings (0.25) Recommendations (0.25) Total (1.0) Ahorlu et al 33 0.2 0.2 0.25 0.25 0.9 Baaees et al 12 0.225 0.2 0.25 0.25 0.925 Badara et al 36 0.075 0.025 0.125 0.125 0.35 Clara et al 41 0.225 0.25 0.25 0.25 0.975 Clara et al 28 0.2 0.175 0.25 0.25 0.875 Cox et al 26 0.225 0.125 0.25 0.25 0.85 Curry et al 31 0.175 0.1 0.125 0.125 0.525 Ezenyeaku et al 35 0.225 0.2 0.25 0.25 0.925 Hemingway-Foday et al 30 0.2 0.075 0.25 0.25 0.775 JICA AmRids Project 32 0.175 0.025 0.1875 0.125 0.5125 Kisanga et al 39 0.225 0.2 0.25 0.25 0.925 Ladoa et al 40 0.175 0.15 0.1875 0.125 0.6375 Maazou et al 11 0.2 0.075 0.25 0.125 0.65 Mejdell Larsen et al 38 0.175 0.1 0.1875 0.25 0.7125 Merali et al 34 0.175 0.2 0.25 0.25 0.875 Metuge et al 27 0.225 0.125 0.25 0.25 0.85 Stone et al 37 0.2 0.175 0.1875 0.25 0.8125 Valdez et al 29 0.15 0.175 0.1875 0.25 0.7625 Van Boetzelaer et al 25 0.225 0.15 0.25 0.25 0.875 Results of synthesis We report below a narrative synthesis of our findings. Our emphasis, in line with the WHO call for evidence, is on identifying drivers of success. 17 Some of the challenges we identified whilst carrying out this review have been reported elsewhere 19 20 ; we have included a brief summary of the challenges we identified in online supplemental appendix 3 . 10.1136/bmjgh-2022-009934.supp3 Supplementary data Drivers of success Success factors fell broadly into four categories: (1) CBS workers (often community health workers), (2) community, (3) case detection and reporting, and (4) integration. CBS workers The CBS workers were described as those responsible for active case detection. Additionally, CBS workers were often responsible for a number of additional tasks including: reporting, referral, follow-up, case management, health promotion, physical examinations (eg, for buruli ulcer 33 ), and testing (eg, taking blood slides for malaria parasites 26 ). Acceptance of CBS workers Successful CBS was believed to be associated with community acceptance of the CBS workers that was, in turn, associated with their recruitment (ie, having the community nominate the CBS workers 31 33 or recruiting CBS workers from within the community 25 27 38 ), or with having nested CBS within an existing emergency response system that itself had good acceptability (which, in turn, was attributed to active participation and collaboration with communities). 27 Trust between CBS workers and the community was also described as a key success factor. 25 29 31 38 Motivation of CBS workers Success was also attributed to high motivation of CBS workers who described their motivation in terms of: 'contributing to bringing good health to the people' 33 (p. 10), feeling a sense of service to the community 26 29 38 41 , and a desire to increase ties and trust with other community members. 29 41 Training opportunities and the opportunity to increase knowledge were also described as motivating factors, even when the programme lacked material incentives. 26 Success in achieving high performance and acceptance of CBS workers in Ghana was, in part, ascribed to efforts made to 'follow all community protocols and encourage the people to see the project as their own' 33 (p. 9). A sense of camaraderie amongst CBS workers and a shared sense of responsibility for bringing an outbreak of malaria under control were also felt to be associated with the success of a CBS programme in the Dominican Republic. 29 Volunteer CBS workers in Ghana were provided with material incentives (in the form of a token to cover travel costs, plus a bicycle), which were described as motivating factors in addition to a sense of service and ownership of the CBS programme. 33 Finally, CBS workers in Sierra Leone derived satisfaction from knowing that they had saved lives of people with Ebola; 'volunteers gave examples of cases they had reported which had received response and treatment […] many of the volunteers were confident that the death rate had decreased due to [CBS workers] influencing people to seek medical attention earlier" 38 (p. 4). CBS worker proximity to communities The close proximity of CBS workers to their communities was felt to have contributed to increased detection of disease clusters in Vietnam 41 and better overall case detection in the Dominican Republic. 29 The CBS programme amongst conflict-affected populations in Cameroon succeeded in collecting data from populations despite 'pendular displacements'; this was attributed to the fact that CBS workers were travelling with their communities and were thus able to continue surveillance. 27 Similarly, CBS workers in Ethiopia were able to spend time at locations where pastoralist communities congregate (eg. wells or water collection areas, mosques, marketplaces), allowing CBS workers to carry out polio surveillance among a nomadic population. 31 Supervision and training Strong supervision was believed to have influenced the success of CBS programmes for malaria in Cambodia 26 , acute flaccid paralysis (AFP)—a proxy for polio—in South Sudan 39 , Ebola in Sierra Leone 37 , and eight high-priority diseases in Senegal. 32 Training was also frequently mentioned as a key factor in the success of CBS, either due to its effect on motivation of CBS workers or improving the quality of their work (in terms of quantity of reports and the specificity of case detection). 26 28 34 39 41 Training was felt to be particularly important in low transmission settings where CBS workers have few opportunities to practise their skills. 26 Availability of refresher training was also felt to be important and resulted in an increase in the quality of blood sample slides in a CBS system for malaria in western Cambodia. 26 Community Several success factors related to the interaction between the CBS system and the community: effective communication and engagement strategies, and the recruitment of community informants were both described as influencing the success of CBS programmes. Communication and engagement Communication and engagement were the most frequently cited success factors of CBS. For example, increased community involvement and 'innovative communication strategies' (ie, a text-based reporting system to enable real-time reporting of signals) were felt to improve signal detection in Côte d'Ivoire 28 (p. S-32). Providing feedback to communities was associated with completeness of reporting in Nigeria 35 , and increased community reporting in Cameroon. 27 In Ethiopia, CBS workers organised village coffee ceremonies at which they were able to ask for reports of AFP and discuss signs, symptoms and reporting. 31 Engagement with community leaders was positively associated with programme uptake in Vietnam. 41 Finally, strong community engagement was believed to have been key to the success of an AFP programme in South Sudan 39 , a malaria surveillance and control programme in the Dominican Republic 29 , and an Ebola surveillance system in Sierra Leone. 37 Recruitment of community informants Several CBS systems relied on the recruitment of community informants who would report suspect cases to CBS workers. The recruitment of a diverse team (including money lenders, insurance agents, veterinary health staff, landlords, factory managers, community leaders and others) of informants with strong community ties in Vietnam, '…broadened the sources of reporting and resulted in the reporting of numerous signals that otherwise would have been missed, such as school absenteeism reported by teachers and the resulting multiple detections of vaccine-preventable disease (eg, mumps and chickenpox)' 41 (p. 1656). In Nigeria, there was a significant positive association between informant satisfaction and completeness of disease notification 35 , and field interviews in South Sudan indicated that having a network of community informants in every village contributed to the effective functioning of the CBS system. 39 Case detection and reporting Successes relating to data collection included: dynamic (ie, adapted and improved) use of case definitions 34 ; the implementation of quality assurance procedures (ie, data were regularly reviewed for accuracy and completeness) 30 ; and engaging in rapid, real-time data-driven decision-making. 25 30 41 Efforts to improve the office environment (eg, by implementing a filing system) in the health posts supporting a CBS pilot in Ethiopia were found to improve record keeping and reporting. 32 The use of technology for data collection and reporting was generally reported as a challenge, though two sources referenced successful use of technological solutions which 'removed reporting obstacles and may account for the increase in the number of notifications' (in the case of a text-based reporting system in Côte d'Ivoire) 28 (p. S-31) and was cited by WHO as 'a critical factor for improved early detection of suspected cases' (in the case of a voice and SMS-based alert system in DRC) 30 (p. S-89). It should be noted that the text and voice messaging solutions in Côte d'Ivoire and DRC were described as 'simple' and yet both systems received considerable technological support from the International Rescue Committee (in Côte d'Ivoire), and RT International, MSF and the WHO (in DRC). 28 30 Ultimately, the most often observed driver of success was simplicity with respect to the design of data collection and reporting tools (eg, 'tools should aim to collect a minimum set of data that can provide usable information, should be clear and simple, and minimise burden to implementers' 34 (p. 10)). Simplifying data collection by limiting the number of reportable diseases 28 , and by simplifying signals 28 and case definitions 12 40 , was associated with ease of reporting, case identification and reducing the proportion of false alerts. Integration with the wider surveillance system Effective vertical integration of CBS with different actors along the reporting pathway (eg, from communities, to the health facility, to the regional/national surveillance system), and lateral integration between the CBS system and other components of the surveillance system at, or close to, the same operational level (eg, laboratory services, operational partners) were identified as success factors. Vertical integration Clear reporting pathways from communities through the various levels within the wider surveillance system were felt to have: improved timeliness of reporting and response in Cameroon 27 ; enabled regular reporting and rapid case confirmation in Ethiopia 32 , Vietnam 41 , Senegal 36 , and Niger 11 ; and increased community engagement in polio eradication in South Sudan. 39 Widespread mobile phone coverage, coupled with the close proximity of health posts to communities (most were within a 30-minute walk), ensured regular reporting of suspect cases of communicable diseases from CBS volunteers and health extension workers (who were responsible for case confirmation and reporting to the cluster health centre) in southern Ethiopia. 32 In Niger, an extensive polio surveillance system was scaled to include active case finding and reporting of COVID-19. 11 The polio surveillance system used established reporting pathways (from community health workers up to the Central Supervisory Directorate of Public Health); thus, the COVID-19 CBS capitalised on pre-existing vertical integration and avoided the, 'structural challenges [of] establishing a de novo CBS to respond to an emerging public health crisis' 11 (p. 5). Lateral integration A CBS system for OneHealth surveillance was deemed to have been successful due to the close collaboration between the Ghana Health Service and the Veterinary Services Directorate. 34 Additionally, members of the wider surveillance system (at the regional and district levels) received targeted training on multiagency coordination. 34 The simplicity of the CBS system in Cox's Bazar, Bangladesh was felt to have enabled easy integration with other aspects of the surveillance system (eg, WHO's Early Warning, Alert and Response System). 25 System integration was also managed by a focal point who was appointed to submit reports and coordinate between the WHO and MSF. 25 CBS workers The CBS workers were described as those responsible for active case detection. Additionally, CBS workers were often responsible for a number of additional tasks including: reporting, referral, follow-up, case management, health promotion, physical examinations (eg, for buruli ulcer 33 ), and testing (eg, taking blood slides for malaria parasites 26 ). Acceptance of CBS workers Successful CBS was believed to be associated with community acceptance of the CBS workers that was, in turn, associated with their recruitment (ie, having the community nominate the CBS workers 31 33 or recruiting CBS workers from within the community 25 27 38 ), or with having nested CBS within an existing emergency response system that itself had good acceptability (which, in turn, was attributed to active participation and collaboration with communities). 27 Trust between CBS workers and the community was also described as a key success factor. 25 29 31 38 Motivation of CBS workers Success was also attributed to high motivation of CBS workers who described their motivation in terms of: 'contributing to bringing good health to the people' 33 (p. 10), feeling a sense of service to the community 26 29 38 41 , and a desire to increase ties and trust with other community members. 29 41 Training opportunities and the opportunity to increase knowledge were also described as motivating factors, even when the programme lacked material incentives. 26 Success in achieving high performance and acceptance of CBS workers in Ghana was, in part, ascribed to efforts made to 'follow all community protocols and encourage the people to see the project as their own' 33 (p. 9). A sense of camaraderie amongst CBS workers and a shared sense of responsibility for bringing an outbreak of malaria under control were also felt to be associated with the success of a CBS programme in the Dominican Republic. 29 Volunteer CBS workers in Ghana were provided with material incentives (in the form of a token to cover travel costs, plus a bicycle), which were described as motivating factors in addition to a sense of service and ownership of the CBS programme. 33 Finally, CBS workers in Sierra Leone derived satisfaction from knowing that they had saved lives of people with Ebola; 'volunteers gave examples of cases they had reported which had received response and treatment […] many of the volunteers were confident that the death rate had decreased due to [CBS workers] influencing people to seek medical attention earlier" 38 (p. 4). CBS worker proximity to communities The close proximity of CBS workers to their communities was felt to have contributed to increased detection of disease clusters in Vietnam 41 and better overall case detection in the Dominican Republic. 29 The CBS programme amongst conflict-affected populations in Cameroon succeeded in collecting data from populations despite 'pendular displacements'; this was attributed to the fact that CBS workers were travelling with their communities and were thus able to continue surveillance. 27 Similarly, CBS workers in Ethiopia were able to spend time at locations where pastoralist communities congregate (eg. wells or water collection areas, mosques, marketplaces), allowing CBS workers to carry out polio surveillance among a nomadic population. 31 Supervision and training Strong supervision was believed to have influenced the success of CBS programmes for malaria in Cambodia 26 , acute flaccid paralysis (AFP)—a proxy for polio—in South Sudan 39 , Ebola in Sierra Leone 37 , and eight high-priority diseases in Senegal. 32 Training was also frequently mentioned as a key factor in the success of CBS, either due to its effect on motivation of CBS workers or improving the quality of their work (in terms of quantity of reports and the specificity of case detection). 26 28 34 39 41 Training was felt to be particularly important in low transmission settings where CBS workers have few opportunities to practise their skills. 26 Availability of refresher training was also felt to be important and resulted in an increase in the quality of blood sample slides in a CBS system for malaria in western Cambodia. 26 Acceptance of CBS workers Successful CBS was believed to be associated with community acceptance of the CBS workers that was, in turn, associated with their recruitment (ie, having the community nominate the CBS workers 31 33 or recruiting CBS workers from within the community 25 27 38 ), or with having nested CBS within an existing emergency response system that itself had good acceptability (which, in turn, was attributed to active participation and collaboration with communities). 27 Trust between CBS workers and the community was also described as a key success factor. 25 29 31 38 Motivation of CBS workers Success was also attributed to high motivation of CBS workers who described their motivation in terms of: 'contributing to bringing good health to the people' 33 (p. 10), feeling a sense of service to the community 26 29 38 41 , and a desire to increase ties and trust with other community members. 29 41 Training opportunities and the opportunity to increase knowledge were also described as motivating factors, even when the programme lacked material incentives. 26 Success in achieving high performance and acceptance of CBS workers in Ghana was, in part, ascribed to efforts made to 'follow all community protocols and encourage the people to see the project as their own' 33 (p. 9). A sense of camaraderie amongst CBS workers and a shared sense of responsibility for bringing an outbreak of malaria under control were also felt to be associated with the success of a CBS programme in the Dominican Republic. 29 Volunteer CBS workers in Ghana were provided with material incentives (in the form of a token to cover travel costs, plus a bicycle), which were described as motivating factors in addition to a sense of service and ownership of the CBS programme. 33 Finally, CBS workers in Sierra Leone derived satisfaction from knowing that they had saved lives of people with Ebola; 'volunteers gave examples of cases they had reported which had received response and treatment […] many of the volunteers were confident that the death rate had decreased due to [CBS workers] influencing people to seek medical attention earlier" 38 (p. 4). CBS worker proximity to communities The close proximity of CBS workers to their communities was felt to have contributed to increased detection of disease clusters in Vietnam 41 and better overall case detection in the Dominican Republic. 29 The CBS programme amongst conflict-affected populations in Cameroon succeeded in collecting data from populations despite 'pendular displacements'; this was attributed to the fact that CBS workers were travelling with their communities and were thus able to continue surveillance. 27 Similarly, CBS workers in Ethiopia were able to spend time at locations where pastoralist communities congregate (eg. wells or water collection areas, mosques, marketplaces), allowing CBS workers to carry out polio surveillance among a nomadic population. 31 Supervision and training Strong supervision was believed to have influenced the success of CBS programmes for malaria in Cambodia 26 , acute flaccid paralysis (AFP)—a proxy for polio—in South Sudan 39 , Ebola in Sierra Leone 37 , and eight high-priority diseases in Senegal. 32 Training was also frequently mentioned as a key factor in the success of CBS, either due to its effect on motivation of CBS workers or improving the quality of their work (in terms of quantity of reports and the specificity of case detection). 26 28 34 39 41 Training was felt to be particularly important in low transmission settings where CBS workers have few opportunities to practise their skills. 26 Availability of refresher training was also felt to be important and resulted in an increase in the quality of blood sample slides in a CBS system for malaria in western Cambodia. 26 Community Several success factors related to the interaction between the CBS system and the community: effective communication and engagement strategies, and the recruitment of community informants were both described as influencing the success of CBS programmes. Communication and engagement Communication and engagement were the most frequently cited success factors of CBS. For example, increased community involvement and 'innovative communication strategies' (ie, a text-based reporting system to enable real-time reporting of signals) were felt to improve signal detection in Côte d'Ivoire 28 (p. S-32). Providing feedback to communities was associated with completeness of reporting in Nigeria 35 , and increased community reporting in Cameroon. 27 In Ethiopia, CBS workers organised village coffee ceremonies at which they were able to ask for reports of AFP and discuss signs, symptoms and reporting. 31 Engagement with community leaders was positively associated with programme uptake in Vietnam. 41 Finally, strong community engagement was believed to have been key to the success of an AFP programme in South Sudan 39 , a malaria surveillance and control programme in the Dominican Republic 29 , and an Ebola surveillance system in Sierra Leone. 37 Recruitment of community informants Several CBS systems relied on the recruitment of community informants who would report suspect cases to CBS workers. The recruitment of a diverse team (including money lenders, insurance agents, veterinary health staff, landlords, factory managers, community leaders and others) of informants with strong community ties in Vietnam, '…broadened the sources of reporting and resulted in the reporting of numerous signals that otherwise would have been missed, such as school absenteeism reported by teachers and the resulting multiple detections of vaccine-preventable disease (eg, mumps and chickenpox)' 41 (p. 1656). In Nigeria, there was a significant positive association between informant satisfaction and completeness of disease notification 35 , and field interviews in South Sudan indicated that having a network of community informants in every village contributed to the effective functioning of the CBS system. 39 Communication and engagement Communication and engagement were the most frequently cited success factors of CBS. For example, increased community involvement and 'innovative communication strategies' (ie, a text-based reporting system to enable real-time reporting of signals) were felt to improve signal detection in Côte d'Ivoire 28 (p. S-32). Providing feedback to communities was associated with completeness of reporting in Nigeria 35 , and increased community reporting in Cameroon. 27 In Ethiopia, CBS workers organised village coffee ceremonies at which they were able to ask for reports of AFP and discuss signs, symptoms and reporting. 31 Engagement with community leaders was positively associated with programme uptake in Vietnam. 41 Finally, strong community engagement was believed to have been key to the success of an AFP programme in South Sudan 39 , a malaria surveillance and control programme in the Dominican Republic 29 , and an Ebola surveillance system in Sierra Leone. 37 Recruitment of community informants Several CBS systems relied on the recruitment of community informants who would report suspect cases to CBS workers. The recruitment of a diverse team (including money lenders, insurance agents, veterinary health staff, landlords, factory managers, community leaders and others) of informants with strong community ties in Vietnam, '…broadened the sources of reporting and resulted in the reporting of numerous signals that otherwise would have been missed, such as school absenteeism reported by teachers and the resulting multiple detections of vaccine-preventable disease (eg, mumps and chickenpox)' 41 (p. 1656). In Nigeria, there was a significant positive association between informant satisfaction and completeness of disease notification 35 , and field interviews in South Sudan indicated that having a network of community informants in every village contributed to the effective functioning of the CBS system. 39 Case detection and reporting Successes relating to data collection included: dynamic (ie, adapted and improved) use of case definitions 34 ; the implementation of quality assurance procedures (ie, data were regularly reviewed for accuracy and completeness) 30 ; and engaging in rapid, real-time data-driven decision-making. 25 30 41 Efforts to improve the office environment (eg, by implementing a filing system) in the health posts supporting a CBS pilot in Ethiopia were found to improve record keeping and reporting. 32 The use of technology for data collection and reporting was generally reported as a challenge, though two sources referenced successful use of technological solutions which 'removed reporting obstacles and may account for the increase in the number of notifications' (in the case of a text-based reporting system in Côte d'Ivoire) 28 (p. S-31) and was cited by WHO as 'a critical factor for improved early detection of suspected cases' (in the case of a voice and SMS-based alert system in DRC) 30 (p. S-89). It should be noted that the text and voice messaging solutions in Côte d'Ivoire and DRC were described as 'simple' and yet both systems received considerable technological support from the International Rescue Committee (in Côte d'Ivoire), and RT International, MSF and the WHO (in DRC). 28 30 Ultimately, the most often observed driver of success was simplicity with respect to the design of data collection and reporting tools (eg, 'tools should aim to collect a minimum set of data that can provide usable information, should be clear and simple, and minimise burden to implementers' 34 (p. 10)). Simplifying data collection by limiting the number of reportable diseases 28 , and by simplifying signals 28 and case definitions 12 40 , was associated with ease of reporting, case identification and reducing the proportion of false alerts. Integration with the wider surveillance system Effective vertical integration of CBS with different actors along the reporting pathway (eg, from communities, to the health facility, to the regional/national surveillance system), and lateral integration between the CBS system and other components of the surveillance system at, or close to, the same operational level (eg, laboratory services, operational partners) were identified as success factors. Vertical integration Clear reporting pathways from communities through the various levels within the wider surveillance system were felt to have: improved timeliness of reporting and response in Cameroon 27 ; enabled regular reporting and rapid case confirmation in Ethiopia 32 , Vietnam 41 , Senegal 36 , and Niger 11 ; and increased community engagement in polio eradication in South Sudan. 39 Widespread mobile phone coverage, coupled with the close proximity of health posts to communities (most were within a 30-minute walk), ensured regular reporting of suspect cases of communicable diseases from CBS volunteers and health extension workers (who were responsible for case confirmation and reporting to the cluster health centre) in southern Ethiopia. 32 In Niger, an extensive polio surveillance system was scaled to include active case finding and reporting of COVID-19. 11 The polio surveillance system used established reporting pathways (from community health workers up to the Central Supervisory Directorate of Public Health); thus, the COVID-19 CBS capitalised on pre-existing vertical integration and avoided the, 'structural challenges [of] establishing a de novo CBS to respond to an emerging public health crisis' 11 (p. 5). Lateral integration A CBS system for OneHealth surveillance was deemed to have been successful due to the close collaboration between the Ghana Health Service and the Veterinary Services Directorate. 34 Additionally, members of the wider surveillance system (at the regional and district levels) received targeted training on multiagency coordination. 34 The simplicity of the CBS system in Cox's Bazar, Bangladesh was felt to have enabled easy integration with other aspects of the surveillance system (eg, WHO's Early Warning, Alert and Response System). 25 System integration was also managed by a focal point who was appointed to submit reports and coordinate between the WHO and MSF. 25 Vertical integration Clear reporting pathways from communities through the various levels within the wider surveillance system were felt to have: improved timeliness of reporting and response in Cameroon 27 ; enabled regular reporting and rapid case confirmation in Ethiopia 32 , Vietnam 41 , Senegal 36 , and Niger 11 ; and increased community engagement in polio eradication in South Sudan. 39 Widespread mobile phone coverage, coupled with the close proximity of health posts to communities (most were within a 30-minute walk), ensured regular reporting of suspect cases of communicable diseases from CBS volunteers and health extension workers (who were responsible for case confirmation and reporting to the cluster health centre) in southern Ethiopia. 32 In Niger, an extensive polio surveillance system was scaled to include active case finding and reporting of COVID-19. 11 The polio surveillance system used established reporting pathways (from community health workers up to the Central Supervisory Directorate of Public Health); thus, the COVID-19 CBS capitalised on pre-existing vertical integration and avoided the, 'structural challenges [of] establishing a de novo CBS to respond to an emerging public health crisis' 11 (p. 5). Lateral integration A CBS system for OneHealth surveillance was deemed to have been successful due to the close collaboration between the Ghana Health Service and the Veterinary Services Directorate. 34 Additionally, members of the wider surveillance system (at the regional and district levels) received targeted training on multiagency coordination. 34 The simplicity of the CBS system in Cox's Bazar, Bangladesh was felt to have enabled easy integration with other aspects of the surveillance system (eg, WHO's Early Warning, Alert and Response System). 25 System integration was also managed by a focal point who was appointed to submit reports and coordinate between the WHO and MSF. 25 Discussion Success factors largely fell into four categories: 1) CBS workers, 2) community, 3) case detection and reporting, and 4) integration. In addition to individual-level factors (such as motivating and training CBS workers) and system factors (including simplifying data collection systems and coordinating with formal surveillance systems and partner organisations), successes were largely attributed to effective leveraging of community knowledge and capacity. This acknowledgement of the importance of 'bottom-up' solutions speaks to a common sense recognition of the importance of participatory approaches that are now endorsed as essential to the effectiveness of a myriad of health interventions (eg, maternal and newborn care, 45 water and sanitation 46 ). Ultimately, the evidence largely points towards drivers of success that map closely to principles of participatory community engagement. 47 48 There is an expanding body of literature evidencing the importance of community participation and meaningful co-production in the management of infectious disease outbreaks. 49–51 Guidelines on the design and implementation of CBS attempt, in varying degrees, to operationalise principles of community participation in order to enhance the effectiveness of surveillance efforts. 10 13 52–57 Building genuine community participation into the design and implementation of a specific public health function, like infectious disease surveillance, is both time-consuming and resource intensive. However, our review has highlighted that many of the key drivers of success of CBS map to the principles and best practices of community participation, including: enabling and emphasising community ownership 29 33 ; committing to meaningful engagement 27 28 37 39 41 and bilateral information exchange 11 26–28 31 35 ; involving a diverse group of community informants 35 39 41 ; recognising and enabling the desire, and competency, of community members to help themselves 26 29 33 38 41 ; and ensuring that systems are designed to build on the trust and goodwill within communities. 25 29 31 38 These drivers of success were manifested not only in observed community acceptance (evidenced, for example, in 94% of community members agreeing to a physical examination for buruli ulcer) 25–27 33 , but were attributed to the overall success of the CBS programme. 12 27 34 37 A CBS system cannot operate independently of a facility-based system and must be complemented by a reliable and effective system for responding to alerts. 58 It is notable that strategies which were identified as having improved system integration within the wider surveillance system were both intuitive (eg, capitalising on existing reporting pathways, close proximity of health posts to communities, and widespread mobile phone coverage) and straightforward (ie, assigning a focal point, clarifying reporting pathways). However, some of these successes may have relied on serendipitous features of a particular response that may be difficult to reproduce in a more logistically complex setting. This highlights that do-no-harm approaches to CBS require careful consideration of the operational context, and acknowledgment that in some settings CBS may not be feasible or appropriate. Ultimately, the benefits of CBS should be balanced against potential resource requirements (including the opportunity cost of moving limited resources into surveillance activities and the cost to sustain CBS system over time) as well as the burden CBS places on communities. 44 In some settings, a more effective and efficient approach to surveillance preparedness may involve bolstering the capacity of facility-based systems whilst increasing their accessibility and working to develop community confidence and trust. It is also important to consider that the consequences of poor implementation can be considerable and can further erode trust between communities and healthcare providers. Beyond the benefits to case detection and reporting, a well-designed and skilfully implemented CBS system enables the forming of resilient community networks, increases community awareness of infectious diseases, and provides an effective platform with the potential to absorb additional public health functions. Even though effective CBS may be both time and resource intensive, we find that the evidence largely supports the inclusion of CBS as a component of outbreak preparedness and response. Limitations There are several important limitations to our review. Limitations of the evidence Few sources provided a thorough description of the design and deployment of the CBS system, making it difficult to create a descriptive typology of systems as originally planned 23 : it is notable that a lack of sufficient descriptive information has been identified elsewhere. 18–20 Though we accept that the lack of granular description is almost certainly the result of the often restrictive length limits of academic journals, few sources included additional descriptive information in annexes. This lack of granular description has limited the degree to which we are able to associate drivers of success (and challenges) with aspects of system design. There was also an absence of sufficient detail to describe some drivers of success, which complicates their interpretation and potential for improving future CBS systems. None of the published sources presented the results of a comprehensive evaluation with many reporting on only a few specific outcomes and/or performance indicators. Despite this, all included sources made some attempt to evaluate an operationalised CBS system and to present empirically informed learning. Finally, the authors of the included sources were often involved in the design and implementation of the study suggesting a potential bias towards presenting more favourable results. Limitations of the review process We restricted our search to a 10-year period starting 1 January 2012. Though we found evidence of evaluations published prior to 2012, the bulk of the relevant evidence has been published since 2016 (which coincides with the publication delay of research on the West African Ebola outbreak 2013–2016). Despite carrying out our searches in early 2022, we were only able to identify two sources reporting on the use of CBS in the context of COVID-19. 11 12 This suggests that much of the evidence on the current use of CBS for the detection and reporting of COVID-19 may be forthcoming, and that this review should be updated to provide a more substantial response to the WHO's call for evidence. In addition, database search terms for CBS lack precision and generate many irrelevant sources relating to CBS studies (ie, epidemiological research studies carried out within communities). The high number of irrelevant sources retrieved using the search term 'community-based surveillance' has been noted in other reviews. 18 Finally, we suspect that we may have missed sources that describe CBS, but which do not reference it as such. Limitations There are several important limitations to our review. Limitations of the evidence Few sources provided a thorough description of the design and deployment of the CBS system, making it difficult to create a descriptive typology of systems as originally planned 23 : it is notable that a lack of sufficient descriptive information has been identified elsewhere. 18–20 Though we accept that the lack of granular description is almost certainly the result of the often restrictive length limits of academic journals, few sources included additional descriptive information in annexes. This lack of granular description has limited the degree to which we are able to associate drivers of success (and challenges) with aspects of system design. There was also an absence of sufficient detail to describe some drivers of success, which complicates their interpretation and potential for improving future CBS systems. None of the published sources presented the results of a comprehensive evaluation with many reporting on only a few specific outcomes and/or performance indicators. Despite this, all included sources made some attempt to evaluate an operationalised CBS system and to present empirically informed learning. Finally, the authors of the included sources were often involved in the design and implementation of the study suggesting a potential bias towards presenting more favourable results. Limitations of the review process We restricted our search to a 10-year period starting 1 January 2012. Though we found evidence of evaluations published prior to 2012, the bulk of the relevant evidence has been published since 2016 (which coincides with the publication delay of research on the West African Ebola outbreak 2013–2016). Despite carrying out our searches in early 2022, we were only able to identify two sources reporting on the use of CBS in the context of COVID-19. 11 12 This suggests that much of the evidence on the current use of CBS for the detection and reporting of COVID-19 may be forthcoming, and that this review should be updated to provide a more substantial response to the WHO's call for evidence. In addition, database search terms for CBS lack precision and generate many irrelevant sources relating to CBS studies (ie, epidemiological research studies carried out within communities). The high number of irrelevant sources retrieved using the search term 'community-based surveillance' has been noted in other reviews. 18 Finally, we suspect that we may have missed sources that describe CBS, but which do not reference it as such. Limitations of the evidence Few sources provided a thorough description of the design and deployment of the CBS system, making it difficult to create a descriptive typology of systems as originally planned 23 : it is notable that a lack of sufficient descriptive information has been identified elsewhere. 18–20 Though we accept that the lack of granular description is almost certainly the result of the often restrictive length limits of academic journals, few sources included additional descriptive information in annexes. This lack of granular description has limited the degree to which we are able to associate drivers of success (and challenges) with aspects of system design. There was also an absence of sufficient detail to describe some drivers of success, which complicates their interpretation and potential for improving future CBS systems. None of the published sources presented the results of a comprehensive evaluation with many reporting on only a few specific outcomes and/or performance indicators. Despite this, all included sources made some attempt to evaluate an operationalised CBS system and to present empirically informed learning. Finally, the authors of the included sources were often involved in the design and implementation of the study suggesting a potential bias towards presenting more favourable results. Limitations of the review process We restricted our search to a 10-year period starting 1 January 2012. Though we found evidence of evaluations published prior to 2012, the bulk of the relevant evidence has been published since 2016 (which coincides with the publication delay of research on the West African Ebola outbreak 2013–2016). Despite carrying out our searches in early 2022, we were only able to identify two sources reporting on the use of CBS in the context of COVID-19. 11 12 This suggests that much of the evidence on the current use of CBS for the detection and reporting of COVID-19 may be forthcoming, and that this review should be updated to provide a more substantial response to the WHO's call for evidence. In addition, database search terms for CBS lack precision and generate many irrelevant sources relating to CBS studies (ie, epidemiological research studies carried out within communities). The high number of irrelevant sources retrieved using the search term 'community-based surveillance' has been noted in other reviews. 18 Finally, we suspect that we may have missed sources that describe CBS, but which do not reference it as such. Conclusion Though the evidence details numerous challenges to CBS, it also highlights key successes. Ultimately, our findings—insofar as they emphasise the benefits of meaningful community participation—suggest that developing CBS preparedness is more likely to be both successful and sustainable within communities that are actively engaged in designing and implementing a range of co-produced public health solutions. As such, we believe that the emphasis of CBS preparedness should be on investing in community participation approaches in health more broadly—to enable the leveraging of this approach in an emergency—rather than on investing exclusively in siloed public health functions such as CBS. Our database search identified several sources reporting exclusively on the use of CBS for identifying the presence of animal vectors, and for identifying zoonotic diseases in pigs and dogs. Though outside the scope of this review, we would welcome a systematic review focused exclusively on the use of CBS as part of a OneHealth approach. In addition, few of the sources identified in this review reported on community perceptions of CBS, lending force to the suggestion included in the WHO ad hoc consultation report, that collecting community accounts about their experiences with CBS is an important research priority. 17 Finally, we endorse the recommendation that all CBS programmes be subject to rigorous evaluation 19 20 and reassert the suggestion, published elsewhere, that evaluations be published, that they follow an established evaluation framework or assessment tool that contains multiple domains including those that are often overlooked (eg, connectedness, coherence and impact), and that they include performance indicators co-produced with communities themselves. 59 Data availability statement Data sharing not applicable as no datasets generated and/or analysed for this study. Ethics statements Patient consent for publication Not required. Ethics approval Not applicable. Patient consent for publication Not required. Ethics approval Not applicable.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8624164/

Enceladus as a Potential Niche for Methanogens and Estimation of Its Biomass

Enceladus is a potential target for future astrobiological missions. NASA's Cassini spacecraft demonstrated that the Saturnian moon harbors a salty ocean beneath its icy crust and the existence and analysis of the plume suggest water–rock reactions, consistent with the possible presence of hydrothermal vents. Particularly, the plume analysis revealed the presence of molecular hydrogen, which may be used as an energy source by microorganisms ( e.g., methanogens). This could support the possibility that populations of methanogens could establish in such environments if they exist on Enceladus. We took a macroscale approximation using ecological niche modeling to evaluate whether conditions suitable for methanogenic archaea on Earth are expected in Enceladus. In addition, we employed a new approach for computing the biomass using the Monod growth model. The response curves for the environmental variables performed well statistically, indicating that simple correlative models may be used to approximate large-scale distributions of these genera on Earth. We found that the potential hydrothermal conditions on Enceladus fit within the macroscale conditions identified as suitable for methanogens on Earth, and estimated a concentration of 10 10 – 10 11 cells/cm 3 . 1. Introduction In recent years, Saturn's moon Enceladus has gained the attention of astrobiologists due to the presence of a big ocean of salty water beneath an icy crust, the internal sources of energy, and the presence of macromolecules (such as hydrocarbons) identified in the plume, which supports the habitability potential of the moon. The main component of the plume is water. However there are other compounds in low concentration such as carbon dioxide (CO 2 ), molecular hydrogen (H 2 ), carbon monoxide (CO), salts, molecular nitrogen (N 2 ), methane (CH 4 ), and complex hydrocarbons [ 1 , 2 , 3 , 4 ]. Furthermore, analysis of stream particles in the Saturnian system indicate that silica particles (SiO 2 ) found in Saturn's E–ring have their source in Enceladus [ 5 ]. Of these, three compounds strongly suggest the presence of hydrothermal vents in the interior of Enceladus: SiO 2 grains morphology, higher than expected CH 4 concentration and H 2 [ 2 , 6 , 7 ]. Hydrothermal vents (on Earth) are unique sites with a wide variety of extreme environments that are important as they give an insight into the processes connected to the origin of life on Earth [ 8 , 9 , 10 , 11 , 12 ]. These structures form in the benthic zone of the ocean, in the vicinity of volcanoes, where water interacts with magma through the tectonic plates, and after cooling down, the dissolved minerals solidify, forming the structures known as hydrothermal vents [ 13 ]. Only a few organisms can survive in these extreme environments, most of them being endemic [ 14 , 15 ]. Prokaryotes isolated from these environments are mostly hyperthermophilic microorganisms belonging to the domain Archaea [ 16 , 17 ]. Species require a combination of biotic and abiotic conditions to occupy a determined area and grow and reproduce during a certain period of time [ 18 ]. The entire set of conditions to keep its growth rate positive is known as niche [ 19 , 20 , 21 ]. Particularly, the fundamental niche of a focal species (species under study) is the combination of environmental conditions and resources that allow this species to maintain a positive intrinsic growth rate in the absence of competitors, predators, and migration. In this scenario, the species must tolerate all the environmental conditions and be able to acquire resources to grow [ 22 , 23 ]. Ecological Niche Models (ENMs) of species allow identification of suitable environmental conditions for a focal species, and if these are mapped over the Earth's surface, they allow the prediction of geographic locations where those conditions are met. These potential locations are identified by ecologists to predict where a species could occur, for example, to estimate its geographic distribution, what the geographic limits of an invasive species are, or how will species respond to climatic changes [ 21 , 23 , 24 ]. One of the limitations of ENMs is the lack of available environmental data at micro–scales that may impair our ability to recover the niche of microorganisms at fine spatial scales. Nonetheless, we can envision ENMs at multiple spatiotemporal scales, as long as these are recognized and properly interpreted [ 25 ]. For example, the niche of a tick might be assessed within the skin of a host (fine-scale) or across biomes within a continent (coarse scale) [ 26 ], or the spores of Bacillus anthracis , the causative agent of anthrax, may respond to environmental cues occurring at micro scales [ 27 ], yet it also shows associations with environmental variation at coarse scales [ 28 ], which can be characterized and used to predict areas of potential occurrence [ 29 ]. In this study, we apply the same principle to methanogens on the Earth, while characterizing its niche at a coarse-scale using correlative models that look for associations between environmental variables and the occurrence of organisms. These associations should be interpreted in strict adherence to the scale of measurement. For example, an association between the occurrence of a microorganism and its surrounding environment, characterized at a coarse scale, cannot be interpreted as the environment experienced by individuals. The evidence of hydrothermal activity, the presence of oxidants, and the concentration of H 2 in Enceladus plume, enables its potential as a niche for extremophilic organisms, of which methanogens would be the most suitable. Methanogenesis (production of CH 4 ) is a process that can occur through biotic or abiotic conversion, and plays an important role in the cycle of carbon, occurring in most anaerobic environments [ 30 ], including terrestrial hydrothermal vents. Biotic methanogenesis is made by methanogenic organisms, who possess an ancient metabolism [ 12 , 31 ] and appeared on Earth when life was just emerging. Methanogens might have played an important role in the early evolution of life on Earth [ 32 , 33 , 34 ]. Moreover, they are model organisms to evaluate if life can establish in other places beyond Earth [ 35 ]. Methanogens belong exclusively to the domain Archaea [ 30 , 36 ] and grow strictly in anaerobic conditions [ 31 , 37 , 38 , 39 ]. They can use limited substrates such as acetate, formate, CO and CO 2 as electron acceptors [ 40 , 41 , 42 ] and produce up to two-thirds of the CH 4 found in anaerobic environments, coming almost one third from CO 2 reduction [ 40 ]. Of importance for Enceladus exploration are the latter, known as hydrogenotrophic methanogens, who use H 2 as an electron donor and CO 2 as a terminal electron acceptor to synthesize CH 4 [ 31 , 43 , 44 ]: (1) 4 H 2 + CO 2 → CH 4 + 2 H 2 O . Affholder et al. [ 45 ] have shown that combined biotic and abiotic methanogenesis could explain the composition in Enceladus' plume, and different authors have already estimated the possible concentration of cells in the Saturnian moon. Using the geothermal energy flux ratio and scaling it to Earth's, Porco et al. [ 46 ] assumed that the relation of biomass and geothermal flux is the same in both bodies and estimated the biomass in Enceladus to be 10 5 cells/ml. Steel et al. [ 47 ] estimated a concentration of 10 9 cells/ml in the vents, assuming that 10% of the energy is transported by hydrothermal flow, that the concentration of hydrogen is the same as that on Earth (7.8 mM in Lost City vent field), and that microorganisms convert all hydrogen available to biomass through methanogenesis. On the other hand, Taubner et al. [ 48 ] proved that the methanogenic strain Methanothermococcus okinawensis , can grow and reproduce under Enceladus-like physicochemical conditions. Clearly, methanogens earned the attention of astrobiologists as candidates for thriving in Enceladus' ocean [ 46 , 47 , 48 , 49 , 50 ]. Based on the availability of H 2 for metabolic conversion, we report the potential biomass of methanogenic archaea from a new approach, using the Monod growth model, which is an empirical model for calculating the growth of microorganisms mainly on aqueous environments [ 51 ]. Furthermore, we used a correlative macroscale approach to characterize the ecological niches of various genera of methanogens on Earth, and assessed whether the conditions found in Enceladus' ocean were within the range of conditions suitable for methanogens on Earth. Both approaches contribute to support the potential of methanogens to inhabit Enceladus. 2. Materials and Methods 2.1. Niche Model Three genera of methanogenic archaea were evaluated: Methanobacterium , Methanococcus , and Methanomicrobium . We chose the methanogenic genera with more than 30 occurrence localities available. Even though there is not a minimum amount of localities required to perform a niche model, a representative sample of the environmental space occupied by each genera is necessary to develop an accurate model [ 52 ]. Their niche model was built with a correlative approach, in which the algorithm builds a model relating occurrences with environmental variables at coarse spatial scales, identifying the variables associated with their presence and predicting the distribution in different areas of interest so that the predicted areas are ecologically similar to the areas of occurrence [ 53 , 54 , 55 ]. Correlative models aim to identify associations of environmental variables at coarse scales with the occurrence of a species and should not be interpreted as requirements for growth. Occurrence localities to build the models were taken from the GBIF database (accessed on October 2019) [ 56 , 57 , 58 ] and were subsequently filtered to eliminate dubious occurrences and all continental data. This database has been widely used for ecological studies of macro and microorganisms throughout the globe [ 59 , 60 , 61 ]. Duplicated occurrences (with the same coordinates) were also eliminated to ensure independence among occurrences. 75% of the total data points were used to train the niche model and the remaining 25% were used to quantify the performance of the model. For Methanobacterium , 195 occurrence records were used, 38 for Methanococcus and 39 for Methanomicrobium . There is a lack of large databases for occurrences of these organisms, due to the difficulties to sample and register them [ 61 , 62 ]. However, it is also true that these organisms occur only in special sites, usually under extreme environmental conditions. Therefore, if we can establish the relationship between these environments and microorganisms, we can identify other places on Earth (or beyond) where they could be present even though no occurrence has been reported. Finally, it is important to emphasize that associations are established at coarse scales (see below), and thus are different than the conditions experimented at the exact vicinity where organisms are found. Marine data layers were taken (in October 2019) from the benthic zone from www.bio-oracle.org (v. 2.0) [ 63 ]. Although these layers are offered for global-scale applications, in a correlative model (as in this case), important variables at the microscale may or may not be relevant at coarser scales. Besides, the performance and validity of the model are evaluated with statistical significance using a partial Receiver Operating Characteristic (ROC) curve analysis. To build the niche model we reviewed the literature to identify those variables that were available as layers, and that were also relevant directly or indirectly, for the biology of methanogens [ 35 ]. To prevent collinearity among predictors, we performed pairwise correlations among all variables using 10,000 random points (with QGIS [ 64 ] v.3.8.3) and Pearson correlations tests (with R [ 65 ]). Finally, we selected only variables with less than 0.8 correlation coefficients. Because physical factors constrain the distribution in extreme systems like hydrothermal vents [ 10 ], we performed a niche model based on the Grinnellian fundamental niche concept, which only includes abiotic variables that the focal genera cannot affect, known as scenopoetic variables [ 21 , 23 ], instead of the Eltonian niche, which includes resources and abiotic variables that interact dynamically with the species, for example limited feeding resources [ 22 , 66 ]. We chose four scenopoetic variables: mean temperature, mean silicate concentration, mean salinity and mean current velocity [ 35 ]. These variables have been associated with the ecophysiology of methanogens either directly, such as temperature, or indirectly through the effect of silicate concentration and salinity on osmoregulation, and mean current velocity on water pressure. With these variables, we built a Bioclim niche model using the library NicheToolBox [ 67 ] in R [ 65 ]. Bioclim is an "envelope-based" model proposed and developed by Henry Nix [ 68 , 69 ], whose inputs are climate variables and occurrence localities of the focal genus. It creates a multidimensional "envelope" to define the conditions tolerable by a species (assuming the environment is the only factor that constrains distribution) [ 70 , 71 ] and relates species occurrences with environmental variables, predicting possible species distributions [ 69 ]. To evaluate model performance, we used a partial ROC test that does not require species' absence data [ 72 ]. This test compares the area under the ROC curve (AUC) of the predicted geographic distribution with the AUC of a null model that randomizes the positions of a percentage of the occurrence localities. The null hypothesis ( H 0 ) states that there is no difference between the models and the alternative hypothesis ( H a ) states that the niche model prediction outperforms the null model. Model performance is evaluated based on omission (number of presence localities not predicted by the model) and percent of the available area predicted as present. Model outputs include "response curves", that relate the suitability of each environmental condition to the occurrence of a species. We overlayed the likely environmental conditions found in Enceladus's ocean with the response curves for each environmental variable used in the model to assess whether the conditions found in Enceladus fell within the suitable conditions where methanogens are found on Earth. This would be interpreted as evidence that suitable environments for methanogens on Earth at coarse scales are present in Enceladus ocean. 2.2. Growth Model An indirect method to estimate cell mass is based on measurements of substrate consumption or production due to its strong relation with cell growth [ 73 , 74 ]. The growth depends on many factors, from genetics to metabolism, and to model all of them is almost impossible. Consequently, simplified models have been proposed to reduce the parameters needed, such as Yoon, Bley and Babel, Bell and others [ 73 , 75 , 76 ]. Monod kinetics is a common empirical approximation to estimate microorganisms growth, particularly in hydrogen–consuming microorganisms [ 51 , 75 ]: (2) d X d t = − μ m a x S K s + S X , (3) d S d t = − 1 Y μ m a x S K s + S X , where μ m a x is the maximum specific growth rate per year (yr − 1 ), K s is the Monod half-saturation constant (concentration at which half of the growth rate is reached), S is the concentration of the limiting substrate, Y is the biomass growth yield (g dry weight/mol), and X is the concentration of biomass (g dry weight/cm 3 ). Here, hydrogen is considered the limiting substrate ( S ). According to Waite et al. [ 2 ], 1–5 × 10 9 mol·H 2 /yr are released through the plume, which means a total maximum release of 2.28 × 10 19 mol·H 2 during the history of the solar system (4.56 Gyr). However, by aqueous oxidation of reduced minerals, the theoretical maximum yield of production of hydrothermal hydrogen would be ∼ 20 × 10 19 mol·H 2 [ 2 ], this leaves 17.72 × 10 19 mol·H 2 in the ocean. Assuming that it is produced and released at a constant rate in an ocean of 1.70 × 10 22 cm 3 (considering that the ocean layer has an average thickness of ∼28.5 km [ 77 ]), we obtain a constant rate of production of the limiting nutrient d S / d t ≈ 2.31 × 10 − 12 mol·H 2 cm − 3 yr − 1 . Then, using Equations ( 2 ) and ( 3 ), it is possible to estimate the biomass concentration ( X , g biomass/cm 3 ): (4) d X d t = − Y d S d t , (5) X = X 0 + Y S t t where t is time (yr), S t is the rate of substrate consumption (mol·H 2 cm − 3 yr − 1 ) and X 0 is the initial biomass concentration; and then obtain the cell concentration considering the mass of an individual cell ∼2 × 10 − 14 g [ 47 ]. We used the parameter Y H 2 = 0.4 g/mol of Methanobacterium thermoautotrophicum , a species of a genus here evaluated [ 78 ]. It was determined at grown conditions T = 65 °C and pH = 7.0 and with different substrate concentration (H 2 and CO 2 ). It was independent of substrate concentration and did not present high variation at different temperatures of growth [ 78 ]. Because of the likelihood of a strong relationship between the south polar region in Enceladus covering the stripes and the seafloor hydrothermal field, to account for the methanogens embedded only in a hydrothermal volume, we used 9% of the seafloor surface area (∼ 1.3 × 10 11 m 2 [ 46 ]) which is the percentage of the surface area that covers the stripes [ 79 ], and the ocean thickness used above. 2.1. Niche Model Three genera of methanogenic archaea were evaluated: Methanobacterium , Methanococcus , and Methanomicrobium . We chose the methanogenic genera with more than 30 occurrence localities available. Even though there is not a minimum amount of localities required to perform a niche model, a representative sample of the environmental space occupied by each genera is necessary to develop an accurate model [ 52 ]. Their niche model was built with a correlative approach, in which the algorithm builds a model relating occurrences with environmental variables at coarse spatial scales, identifying the variables associated with their presence and predicting the distribution in different areas of interest so that the predicted areas are ecologically similar to the areas of occurrence [ 53 , 54 , 55 ]. Correlative models aim to identify associations of environmental variables at coarse scales with the occurrence of a species and should not be interpreted as requirements for growth. Occurrence localities to build the models were taken from the GBIF database (accessed on October 2019) [ 56 , 57 , 58 ] and were subsequently filtered to eliminate dubious occurrences and all continental data. This database has been widely used for ecological studies of macro and microorganisms throughout the globe [ 59 , 60 , 61 ]. Duplicated occurrences (with the same coordinates) were also eliminated to ensure independence among occurrences. 75% of the total data points were used to train the niche model and the remaining 25% were used to quantify the performance of the model. For Methanobacterium , 195 occurrence records were used, 38 for Methanococcus and 39 for Methanomicrobium . There is a lack of large databases for occurrences of these organisms, due to the difficulties to sample and register them [ 61 , 62 ]. However, it is also true that these organisms occur only in special sites, usually under extreme environmental conditions. Therefore, if we can establish the relationship between these environments and microorganisms, we can identify other places on Earth (or beyond) where they could be present even though no occurrence has been reported. Finally, it is important to emphasize that associations are established at coarse scales (see below), and thus are different than the conditions experimented at the exact vicinity where organisms are found. Marine data layers were taken (in October 2019) from the benthic zone from www.bio-oracle.org (v. 2.0) [ 63 ]. Although these layers are offered for global-scale applications, in a correlative model (as in this case), important variables at the microscale may or may not be relevant at coarser scales. Besides, the performance and validity of the model are evaluated with statistical significance using a partial Receiver Operating Characteristic (ROC) curve analysis. To build the niche model we reviewed the literature to identify those variables that were available as layers, and that were also relevant directly or indirectly, for the biology of methanogens [ 35 ]. To prevent collinearity among predictors, we performed pairwise correlations among all variables using 10,000 random points (with QGIS [ 64 ] v.3.8.3) and Pearson correlations tests (with R [ 65 ]). Finally, we selected only variables with less than 0.8 correlation coefficients. Because physical factors constrain the distribution in extreme systems like hydrothermal vents [ 10 ], we performed a niche model based on the Grinnellian fundamental niche concept, which only includes abiotic variables that the focal genera cannot affect, known as scenopoetic variables [ 21 , 23 ], instead of the Eltonian niche, which includes resources and abiotic variables that interact dynamically with the species, for example limited feeding resources [ 22 , 66 ]. We chose four scenopoetic variables: mean temperature, mean silicate concentration, mean salinity and mean current velocity [ 35 ]. These variables have been associated with the ecophysiology of methanogens either directly, such as temperature, or indirectly through the effect of silicate concentration and salinity on osmoregulation, and mean current velocity on water pressure. With these variables, we built a Bioclim niche model using the library NicheToolBox [ 67 ] in R [ 65 ]. Bioclim is an "envelope-based" model proposed and developed by Henry Nix [ 68 , 69 ], whose inputs are climate variables and occurrence localities of the focal genus. It creates a multidimensional "envelope" to define the conditions tolerable by a species (assuming the environment is the only factor that constrains distribution) [ 70 , 71 ] and relates species occurrences with environmental variables, predicting possible species distributions [ 69 ]. To evaluate model performance, we used a partial ROC test that does not require species' absence data [ 72 ]. This test compares the area under the ROC curve (AUC) of the predicted geographic distribution with the AUC of a null model that randomizes the positions of a percentage of the occurrence localities. The null hypothesis ( H 0 ) states that there is no difference between the models and the alternative hypothesis ( H a ) states that the niche model prediction outperforms the null model. Model performance is evaluated based on omission (number of presence localities not predicted by the model) and percent of the available area predicted as present. Model outputs include "response curves", that relate the suitability of each environmental condition to the occurrence of a species. We overlayed the likely environmental conditions found in Enceladus's ocean with the response curves for each environmental variable used in the model to assess whether the conditions found in Enceladus fell within the suitable conditions where methanogens are found on Earth. This would be interpreted as evidence that suitable environments for methanogens on Earth at coarse scales are present in Enceladus ocean. 2.2. Growth Model An indirect method to estimate cell mass is based on measurements of substrate consumption or production due to its strong relation with cell growth [ 73 , 74 ]. The growth depends on many factors, from genetics to metabolism, and to model all of them is almost impossible. Consequently, simplified models have been proposed to reduce the parameters needed, such as Yoon, Bley and Babel, Bell and others [ 73 , 75 , 76 ]. Monod kinetics is a common empirical approximation to estimate microorganisms growth, particularly in hydrogen–consuming microorganisms [ 51 , 75 ]: (2) d X d t = − μ m a x S K s + S X , (3) d S d t = − 1 Y μ m a x S K s + S X , where μ m a x is the maximum specific growth rate per year (yr − 1 ), K s is the Monod half-saturation constant (concentration at which half of the growth rate is reached), S is the concentration of the limiting substrate, Y is the biomass growth yield (g dry weight/mol), and X is the concentration of biomass (g dry weight/cm 3 ). Here, hydrogen is considered the limiting substrate ( S ). According to Waite et al. [ 2 ], 1–5 × 10 9 mol·H 2 /yr are released through the plume, which means a total maximum release of 2.28 × 10 19 mol·H 2 during the history of the solar system (4.56 Gyr). However, by aqueous oxidation of reduced minerals, the theoretical maximum yield of production of hydrothermal hydrogen would be ∼ 20 × 10 19 mol·H 2 [ 2 ], this leaves 17.72 × 10 19 mol·H 2 in the ocean. Assuming that it is produced and released at a constant rate in an ocean of 1.70 × 10 22 cm 3 (considering that the ocean layer has an average thickness of ∼28.5 km [ 77 ]), we obtain a constant rate of production of the limiting nutrient d S / d t ≈ 2.31 × 10 − 12 mol·H 2 cm − 3 yr − 1 . Then, using Equations ( 2 ) and ( 3 ), it is possible to estimate the biomass concentration ( X , g biomass/cm 3 ): (4) d X d t = − Y d S d t , (5) X = X 0 + Y S t t where t is time (yr), S t is the rate of substrate consumption (mol·H 2 cm − 3 yr − 1 ) and X 0 is the initial biomass concentration; and then obtain the cell concentration considering the mass of an individual cell ∼2 × 10 − 14 g [ 47 ]. We used the parameter Y H 2 = 0.4 g/mol of Methanobacterium thermoautotrophicum , a species of a genus here evaluated [ 78 ]. It was determined at grown conditions T = 65 °C and pH = 7.0 and with different substrate concentration (H 2 and CO 2 ). It was independent of substrate concentration and did not present high variation at different temperatures of growth [ 78 ]. Because of the likelihood of a strong relationship between the south polar region in Enceladus covering the stripes and the seafloor hydrothermal field, to account for the methanogens embedded only in a hydrothermal volume, we used 9% of the seafloor surface area (∼ 1.3 × 10 11 m 2 [ 46 ]) which is the percentage of the surface area that covers the stripes [ 79 ], and the ocean thickness used above. 3. Results 3.1. Niche Estimation Predictions from all niche models were statistically different from the null model and therefore statistically appropriate to describe the fundamental niche at a coarse scale. The AUC ratios obtained for the models were 1.0742, 1.5906, and 1.5298 for Methanobacterium , Methanococcus , and Methanomicrobium , respectively, all with a p –value = 0 . The niche model for Methanococcus showed the largest difference and is considered a relatively good model. The geographic prediction of the niche models of the three genera included the boundaries of tectonic plates, where hydrothermal environments occur. Figure 1 shows the geographic expression of the niche model for Methanobacterium , where the lighter the color the higher the suitability. It shows high suitability along the Mid–Atlantic Ridge, Juan de Fuca Ridge (in California), Gakkel Ridge (in the Arctic), Java Trench (in the Indian ocean), and Manus Basin (in New Guinea). Methanococcus 's potential distribution ( Figure A1 ) does not show a pattern correlated with the Atlantic Ridge like the previous genus. It shows a wider area of possible distribution in that zone. Moreover, it covers the Indian ridges and the Aeolian Arc in the Mediterranean Sea. The potential distribution of Methanomicrobium ( Figure A2 ) shows a more restricted area, also in the Mid–Atlantic Ridge, Gakkel Ridge, and Manus Basin. The response variables of the niche models constrain the range of the conditions associated with the occurrence of methanogens, providing a framework to evaluate, at coarse scales, whether conditions available in Enceladus include those associated with methanogens' presence on Earth. Salinity (measured in Practical Salinity Unit, PSU) in Enceladus has been estimated to be 5–40 PSU, 40 PSU being the upper limit in the locations where hydrothermal processes occur, and 5 PSU the lower limit, found in the plume and its direct source under the icy shell [ 6 , 80 , 81 ]. This salinity range matches with the salinity associated with methanogens occurrences ( Figure 2 ), being ∼30–40 PSU for Methanobacterium and ∼33–41 PSU for Methanococcus and Methanomicrobium . The current velocity in Enceladus' interior at which material is transported from the interior to the surface is calculated to be 0.01–0.05 m/s [ 3 ]. This estimate refers explicitly to the velocity of ascending currents due to hydrothermal activity and is within the limits of the predictions from the niche models for the methanogens ( Figure 2 ). According to the composition of the plume, the temperature inside Enceladus must be at least 50 °C in the places where water–rock interactions occur and warm fluid is expelled [ 82 ]. Methanogens would be expected at a certain distance from the places in the hydrothermal vents structure where the fluid is expelled and the temperature can be within the suitable range: 0–30 °C, but still inhabiting the surroundings of the hydrothermal site. The concentration of silica in Enceladus on the other hand might represent an obstacle for methanogens. According to our niche model, the highest concentration where methanogens are present on Earth is ∼150 μ M ( Figure 2 ), while the concentration in Enceladus, is estimated to be up to 2500 μ M [ 6 ]. 3.2. Biomass Estimation H 2 can be produced from different sources in Enceladus. We only take into account the hydrogen produced by the aqueous oxidation of reduced minerals. Figure 3 shows the estimated cells concentration during the last 3.5 Gyr, comparable to the time of life on Earth. If methanogens were consuming all the hydrogen that can theoretically be produced in the core and is not expelled by the plume, the current concentration would be of the order of ∼10 11 cell/cm 3 , starting with an initial concentration X 0 = 1 cell/cm 3 . However, we expect methanogens to be only in the hydrothermal volume, between the seafloor with hydrothermal processes and the ice layer. In this case, the current concentration of cells would be ∼10 10 cell/cm 3 . 3.1. Niche Estimation Predictions from all niche models were statistically different from the null model and therefore statistically appropriate to describe the fundamental niche at a coarse scale. The AUC ratios obtained for the models were 1.0742, 1.5906, and 1.5298 for Methanobacterium , Methanococcus , and Methanomicrobium , respectively, all with a p –value = 0 . The niche model for Methanococcus showed the largest difference and is considered a relatively good model. The geographic prediction of the niche models of the three genera included the boundaries of tectonic plates, where hydrothermal environments occur. Figure 1 shows the geographic expression of the niche model for Methanobacterium , where the lighter the color the higher the suitability. It shows high suitability along the Mid–Atlantic Ridge, Juan de Fuca Ridge (in California), Gakkel Ridge (in the Arctic), Java Trench (in the Indian ocean), and Manus Basin (in New Guinea). Methanococcus 's potential distribution ( Figure A1 ) does not show a pattern correlated with the Atlantic Ridge like the previous genus. It shows a wider area of possible distribution in that zone. Moreover, it covers the Indian ridges and the Aeolian Arc in the Mediterranean Sea. The potential distribution of Methanomicrobium ( Figure A2 ) shows a more restricted area, also in the Mid–Atlantic Ridge, Gakkel Ridge, and Manus Basin. The response variables of the niche models constrain the range of the conditions associated with the occurrence of methanogens, providing a framework to evaluate, at coarse scales, whether conditions available in Enceladus include those associated with methanogens' presence on Earth. Salinity (measured in Practical Salinity Unit, PSU) in Enceladus has been estimated to be 5–40 PSU, 40 PSU being the upper limit in the locations where hydrothermal processes occur, and 5 PSU the lower limit, found in the plume and its direct source under the icy shell [ 6 , 80 , 81 ]. This salinity range matches with the salinity associated with methanogens occurrences ( Figure 2 ), being ∼30–40 PSU for Methanobacterium and ∼33–41 PSU for Methanococcus and Methanomicrobium . The current velocity in Enceladus' interior at which material is transported from the interior to the surface is calculated to be 0.01–0.05 m/s [ 3 ]. This estimate refers explicitly to the velocity of ascending currents due to hydrothermal activity and is within the limits of the predictions from the niche models for the methanogens ( Figure 2 ). According to the composition of the plume, the temperature inside Enceladus must be at least 50 °C in the places where water–rock interactions occur and warm fluid is expelled [ 82 ]. Methanogens would be expected at a certain distance from the places in the hydrothermal vents structure where the fluid is expelled and the temperature can be within the suitable range: 0–30 °C, but still inhabiting the surroundings of the hydrothermal site. The concentration of silica in Enceladus on the other hand might represent an obstacle for methanogens. According to our niche model, the highest concentration where methanogens are present on Earth is ∼150 μ M ( Figure 2 ), while the concentration in Enceladus, is estimated to be up to 2500 μ M [ 6 ]. 3.2. Biomass Estimation H 2 can be produced from different sources in Enceladus. We only take into account the hydrogen produced by the aqueous oxidation of reduced minerals. Figure 3 shows the estimated cells concentration during the last 3.5 Gyr, comparable to the time of life on Earth. If methanogens were consuming all the hydrogen that can theoretically be produced in the core and is not expelled by the plume, the current concentration would be of the order of ∼10 11 cell/cm 3 , starting with an initial concentration X 0 = 1 cell/cm 3 . However, we expect methanogens to be only in the hydrothermal volume, between the seafloor with hydrothermal processes and the ice layer. In this case, the current concentration of cells would be ∼10 10 cell/cm 3 . 4. Discussion It is hard to find a place on Earth where life is not to be found, especially in the oceans. The solar system hosts different places comparable with Earth's ocean such as the interior of Enceladus. In this study, we took two radically different approaches to evaluate the hypothesis that methanogens could thrive in the Enceladan ocean. First, based on the similarities found at coarse scales between environmental conditions in Enceladus and those associated with the occurrence of Methanogen genera on Earth, we describe the Enceladan ocean as a potential niche for methanogenic archaea. Secondly, based on a simple growth model that rests on hydrogen concentration, we estimate the potential cell number in Enceladus under several assumptions. ENMs of three genera of methanogens on Earth ( Methanobacterium , Methanococcus , and Methanomicrobium ) suggest suitable sites are distributed along tectonic boundaries, where water filters and forms hydrothermal vents. These models exhibited good performance, indicating that the occurrence of methanogens manifests at coarse scales associated with environmental variables such as mean salinity, temperature, current velocity, and mean silicate concentration. Inside Enceladus, the subsurface ocean is in direct contact with a rocky–core and as a result of this interaction it might create similar structures and conditions, creating places where hydrogen is available and therefore hydrogenotrophic methanogens could also occur. It is important to interpret the results of these models as associations at coarse scales and not as the range of environments that methanogens are exposed to directly in their immediate surroundings. The salinity and the temperature that methanogens would tolerate are found in the deep ocean, close to hydrothermal vents. A key characteristic of hydrothermal vent environments is their high heterogeneity presenting steep gradients in relatively short distances. We would not expect the methanogens of this study to thrive in places with temperatures higher than 30 °C, but this could be easily achieved by distancing from the site of material expulsion in the hydrothermal vents. The mean current velocity within the hydrothermal plume could represent a challenge for organisms to access nutrients or due to the pressure changes that this could cause in the hydrothermal vents. Because of the ascending currents that hydrothermal activity could generate in the seafloor in Enceladus, the focal genera could be expected in the surroundings of the hydrothermal field. However, methanogens on earth are not associated with areas with radically higher water velocities ( Figure 2 ), and these conditions might represent a challenge for microorganisms if this was the case on Enceladus. It is also possible that this variable appears as important for the model, because of its association with other variables that are actually important for methanogens but absent from our models since we are using correlative methods that identify any potential associations between occurrence and environmental variables. We acknowledge that some of the results found in our models could be sampling artifacts related to biased sampling of hydrothermal vents in oceans. High concentrations of silicates on Earth were associated to low suitability for methanogens ( Figure 2 ). Silicates concentration on Earth's oceans is one order of magnitude (2000 μ mol/m 3 ), despite hydrothermal vents on Earth being one of the three major inputs of dissolved silica from the lithosphere to the hydrosphere [ 83 ]. If such high concentrations of silicates on Enceladus represent an obstacle for methanogen establishment and growth, we would expect them to occur in specific places where silicate concentration is reduced, which could be used as a constrain for the search of these organisms in Enceladus. We did not find any direct relationship between silicate concentration and methanogens in the literature, so this association should be interpreted with caution. The limiting substrate was a concept introduced by Monod and it is known as a nutrient with a strong relation to cell growth so that in its absence, the growth of cells stops [ 74 , 84 ], and conversely when this nutrient is in high concentration the growth of cells tends to the maximum [ 73 , 84 ]. In the case of Enceladus, we considered that cell growth is limited only by hydrogen concentration and therefore used the quantity available in this moon to estimate cell concentration [ 51 , 84 ]. Should these microorganisms thrive with the energy, water, and substrates available in Enceladus, we estimate the cell concentration in the Enceladan hydrothermal field to be ∼10 11 cells/cm 3 if they consume all the hydrogen available. For a more conservative scenario, we have also estimated the case where methanogens consume only the hydrogen available in the surroundings of the hydrothermal field. In this case, the concentration is ∼10 10 cells/cm 3 . The latter is one order of magnitude higher than that reported by Steel et al. [ 47 ] (∼10 9 cells/cm 3 ) who did the calculations based on the geothermal power available for energy conversion into biomass, and 4 orders of magnitude higher than in Lost City (∼10 6 cells/cm 3 ), a hydrothermal field on Earth [ 47 ]. However, we could consider this as an upper limit of cell concentration since we have considered the hydrogen concentration as the only limiting factor, while other variables would certainly constrain the cell growth, as the pH and concentration of other nutrients. These results support the idea that Enceladus' ocean could support cultures of methanogens from an ecological approach. 5. Conclusions Enceladus' plume has provided strong evidence of water–rock reactions occurring in its interior, between its core and a subsurface ocean. This creates hydrothermal environments where microorganisms such as methanogens could thrive. The ENMs described here show that terrestrial methanogens have a distribution along tectonic boundaries, where hydrothermal environments are present, the Mid-Atlantic ridge and the surroundings of the Manus basin being the most suitable places. According to the response curves, salinity, temperature and current velocity of the Enceladus ocean fit within the limits that methanogens inhabit the Earth; temperatures suggest these organisms would not be close to the hydrothermal fluids but in the surroundings; and because silica concentration on Enceladus is higher than what methanogens are exposed to on Earth, it could possibly constrain the growth of methanogens to places where silica is less concentrated. Even though the marine layers used for building the model are more often used for macro–scale studies, the statistical analysis indicates that there is a relationship between the occurrence of methanogens and specific environments that can describe their niche, with statistical significance. Furthermore, it is worth noticing that this is a correlative model, which means that the variables used may not represent the essential requirements of a species to survive. Nonetheless, these variables expand the knowledge on the ecology of a particular species, which ultimately can help to strengthen the conceptual framework for habitability and benefit future astrobiological exploration [ 85 ], for example, of a potential limitation due to high silica concentration in Enceladus. Having evaluated a coarse-scale environmental similarity between the conditions that methanogens are exposed to in Earth's and Enceladus' oceans, we used the growth model of Monod, a new computing approach, to estimate the cell concentration, obtaining that the current cells concentration would be ∼ 10 10 – 10 11 cells/cm 3 . Future work includes (i ) improving the growth model using more factors that affect the possible growth of microorganisms in Enceladus, (ii) utilizing interactions with other species that can co-exist with these genera, (iii) further analysis of the composition of the plume to constrain the resources that microorganisms could harvest and could be compared with the response variables here reported, and (iv) improving on the limited occurrence records of microorganisms on the ocean.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8610593/

Structures of Foot-and-Mouth Disease Virus with Bovine Neutralizing Antibodies Reveal the Determinant of Intraserotype Cross-Neutralization

ABSTRACT Foot-and-mouth disease virus (FMDV) exhibits broad antigenic diversity with poor intraserotype cross-neutralizing activity. Studies of the determinant involved in this diversity are essential for the development of broadly protective vaccines. In this work, we isolated a bovine antibody, designated R55, that displays cross-reaction with both FMDV A/AF/72 (hereafter named FMDV-AAF) and FMDV A/WH/09 (hereafter named FMDV-AWH) but only has a neutralizing effect on FMDV-AWH. Near-atomic resolution structures of FMDV-AAF-R55 and FMDV-AWH-R55 show that R55 engages the capsids of both FMDV-AAF and FMDV-AWH near the icosahedral 3-fold axis and binds to the βB and BC/HI-loops of VP2 and to the B-B knob of VP3. The common interaction residues are highly conserved, which is the major determinant for cross-reaction with both FMDV-AAF and FMDV-AWH. In addition, the cryo-EM structure of the FMDV-AWH-R55 complex also shows that R55 binds to VP3 E70 located at the VP3 BC-loop in an adjacent pentamer, which enhances the acid and thermal stabilities of the viral capsid. This may prevent capsid dissociation and genome release into host cells, eventually leading to neutralization of the viral infection. In contrast, R55 binds only to the FMDV-AAF capsid within one pentamer due to the VP3 E70G variation, which neither enhances capsid stability nor neutralizes FMDV-AAF infection. The VP3 E70G mutation is the major determinant involved in the neutralizing differences between FMDV-AWH and FMDV-AAF. The crucial amino acid VP3 E70 is a key component of the neutralizing epitopes, which may aid in the development of broadly protective vaccines. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a highly contagious and economically devastating disease in cloven-hoofed animals, and neutralizing antibodies play critical roles in the defense against viral infections. Here, we isolated a bovine antibody (R55) using the single B cell antibody isolation technique. Enzyme-linked immunosorbent assays (ELISA) and virus neutralization tests (VNT) showed that R55 displays cross-reactions with both FMDV-AWH and FMDV-AAF but only has a neutralizing effect on FMDV-AWH. Cryo-EM structures, fluorescence-based thermal stability assays and acid stability assays showed that R55 engages the capsid of FMDV-AWH near the icosahedral 3-fold axis and informs an interpentamer epitope, which overstabilizes virions to hinder capsid dissociation to release the genome, eventually leading to neutralization of viral infection. The crucial amino acid VP3 E70 forms a key component of neutralizing epitopes, and the determination of the VP3 E70G mutation involved in the neutralizing differences between FMDV-AWH and FMDV-AAF could aid in the development of broadly protective vaccines.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2724991/

Exploring the full spectrum of macrophage activation

Macrophages display remarkable plasticity and can change their physiology in response to environmental cues. These changes can give rise to different populations of cells with distinct functions. In this Review we suggest a new grouping of macrophage populations based on three different homeostatic activities—host defence, wound healing and immune regulation. We propose that similarly to primary colours, these three basic macrophage populations can blend into various other 'shades' of activation. We characterize each population and provide examples of macrophages from specific disease states that have the characteristics of one or more of these populations.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7114995/

Nanomaterials-Based (Bio)Sensing Systems for Safety and Security Applications

The development of new nanomaterials and nanotechnologies has ­provided many new opportunities for (bio)sensing systems. The introduction of nanomaterials, such as magnetic nanoparticles, gold nanoparticles, graphene, quantum dots, etc. is bringing advantages in terms of improving the selectivity and sensitivity of these systems. These nanomaterials also offer advantages in biosensors owing to their nanometric size, shape, composition, physical properties, ability to manipulate their surface chemistry and the property that they have in terms of adsorbing biological molecules and the change of their physical properties. In recent years, several ­bacterial pathogens, toxins, viruses, parasites and explosives have been considered as potential threats for bioterrorism, among which can find Escherichia coli, Salmonella, Bacillus anthracis, Clostridium botulinum, Botulinum Neurotoxin , Vaccinia, Plasmodium falciparum , Trinitrotoluene, etc. Bioterrorism is extremely complex to tackle but the science and technology are fundamental ­elements to reduce its threat. For this reason, monitoring systems for quick identification of biomolecules are the core of much of the basic research activities in combating bioterrorism. In this chapter we discuss the research efforts by using nanobiotechnologies with the aim of developing accurate, easy, cheap, portable and ultrasensitive assays for agents that pose a biologic threat. Some ­nanomaterial-based (bio)sensing systems used to detect agents related with bioterrorism for safety and security applications in agriculture, food, forensic, biomedical are also given. General Introduction. Nanomaterials and Bioterrorism The discovery and study of nanomaterials has enabled the development of ultrasensitive (bio)sensing systems. This is due to their high surface area, favorable electronic and optical properties and electrocatalytic activity as well as good biocompatibility induced by nanometer size and specific physicochemical characteristics [ 1 – 5 ]. In recent years, the advent of nanomaterial-based (bio)sensing systems for safety and security applications is offering key researches and developments. In this ­context, of special interest are the 'nanosized' and nanomaterials based biosensors, called also nanobiosensors – a modern and efficient class of detection systems [ 1 , 3 , 6 – 10 ]. The application of these devices in food industry, environmental monitoring and clinic diagnostics could lead great improvements in safety and security against bioterror agents. Nowadays, laboratories and institutions related with the nano-­biotechnology are working together to increase the capabilities to detect and respond to an attack by biological or chemical weapons [ 11 ]. Examples of nanomaterial-based bioterrosist agents for safety and security applications are given in the Table 3.1 . The magnetic nanoparticles (M NPs), graphene (G), quantum dots (QDs), and more extensively gold nanoparticles (Au NPs) are being invaluable nanomaterials to detect bioterrorist analytes in macro- to nano-scale, including bacterial pathogens, toxins, viruses, parasites, explosives, etc. Table 3.1 Examples of nanomaterial-based bioterrorist agents for safety and security applications Agent Analyte Nanomaterial Technique Sample LOD Ref. Bacteria & Escherichia coli O157:H7 Au NPs (13 nm diameter) Electrochemical Milk 50 CFU/strip [ 12 ] Escherichia coli Cu@Au NPs ( ca. 15 nm) Electrochemical Surface water 3 CFU/10 mL [ 13 ] Glyco-NPs Optical PBS 10 4 cells/mL [ 14 ] Silica particles (200 nm) coated with silver shells ( ca . 20 nm) Optical Water 3–5 cells [ 15 ] Salmonella spp. Au NPs (ca. 25 nm) Electrochemical Pork 1.0  ×  10 2 CFU/mL [ 16 ] Salmonella typhi Au NPs ( ca. 15 nm) Electrochemical PBS 98.9 CFU/mL [ 17 ] Spores Bacillus anthracis EAM Electrochemical DNA 0.01 ng/mL [ 18 ] Toxins Anthrax Eu + NPs Optical PBS 10 pg/mL [ 19 ] Botulinum Neurotoxin B Colloidal gold (25 nm in diameter) IC PBS 50 ng/mL [ 20 ] Botulinum neurotoxin D Colloidal gold (40 nm in diameter) IC Horse faeces 50 ng/L [ 21 ] Botulinum neurotoxin A QDs (655 nm) Fluorescence PBS 5 pM [ 22 ] Viruses H9 AIVs Colloidal gold IC Chicken eggs 0.25 units of HA [ 23 ] H1N1 flu Colloidal gold Electrochemical PBS 577 pM L −1 [ 24 ] Parasites Plasmodium falciparum M NPs (1 μm diameter) Electrochemical Spiked serum 0.36 ng mL −1 [ 25 ] Au NPs Electrochemical Serum – [ 26 ] Explosives TNT CdTe QDs Fluorescence Soil 1.1 nM [ 27 ] Graphene Electrochemical 0.5 M NaCl solution 0.5 ppb [ 28 ] 2.4-DNT Graphene Electrochemical 0.5 M NaCl solution 42 nmol L −1 [ 29 ] PBS phosphate buffer solution, EAM electrically active magnetic nanoparticles, QDs quantum dots, M NPs magnetic nanoparticles, 2.4-DNT 2,4-Dinitrotoluene, TNT trinitrotoluene, Cu@Au NPs gold nanoparticles coated Cu, CFU colony forming units, NPs nanoparticles, CdTe QD cadmium telluride quantum dot nanoparticle, H9 AIVs H9 subtype avian influenza viruses, H1N1 flu Influenza A, IC Immunochromatography, LOD limit of detection, Eu + NPs Europium nanoparticles Biological agents such as bacteria, viruses, biological toxins, and genetically altered organisms are contagious, and during this lag time, infected persons could continue to spread the disease, further increasing its reach. Hundreds or even thousands of people could become sick or die if a biological attack were to occur in a major metropolitan area [ 11 ]. For this reason, problems related to the risks of human health and environmental need to be carefully considered, mainly assays that involve a variety of safety and security applications. In this chapter will be given some important strategies used in (bio)sensing systems with different nanomaterials to detect bioterrorist agents. In addition, this chapter will give opinions about the importance that these systems have for safety and security applications in food, environmental and other related fields. Monitoring Systems for Bioterrorist Agents Numerous laboratories and public and private research institutions during decades have been designing and developing technologies to safeguard from biological attacks provoked by terrorists. Nowadays, they are developing monitoring technologies for bioterrorism agents with fast analysis, low limit of detection, and high ­accuracy to prevent false negatives and positives and maintain confidence in the monitoring system [ 30 ]. Among the highest-risk threat agents in bioterrorism can be considered: (1) Bacteria, they are prokaryotes, and their distribution is ubiquitous (humans, animals, and environment). They are the oldest living organisms in the history of this planet and play an important role in maintaining the ecosystem [ 11 ]. Escherichia coli ( E. coli ) is a family of naturally occurring bacteria. Some of these cause sickness and even death. Most E. coli infections come from eating undercooked (i.e. contaminated ground beef). Terrorists could use E. coli 0157:H7 bacteria as a weapon to strike many people at one time, also the Bacillus anthracis ( B. anthracis ) is considered a high pathogenic agent that could deliberate contamination events causing anthrax [ 31 ]; (2) Neurotoxins produced by Clostridium botulinum are among the most known poisonous substances [ 32 ]. For example, botulinum toxin is the most toxic substance known and is extremely poisonous by the oral route (estimated lethal oral dose, 10–70 μg for a 70 kg human) and potentially toxic by inhalation [ 33 ], for this reason it is used as bioterrorism agent (3) Influenza A (H1N1) virus also could be considered a bioterrorist agent, it belongs to the Orthomyxoviridae family and corresponds to the specific combination of glycol-protein hemagglutinin (HA) and neuraminidase (NA) variants, which are present on the surface of the enveloped RNA virus [ 24 ]. (4) Malaria is the most prevalent parasitic disease in the worldcaused by the apicomplex protozoan of the Plasmodium genus . Malaria is present over the tropics, where four species, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale are transmitted to humans by the bites of the female mosquito vector of the Anopheles genus [ 34 ] and although the use of parasites as bioterrorism agents has not received so much attention. Parasites could contribute to the installation of fear in human population upon intentional addition to their food and water supplies, which makes malaria suitable for being used by terrorists (5). In recent years, the detection and quantification of nitroaromatic explosives such as 2,4,6-trinitrotoluene (TNT) have also received considerable attention due to their environmental, security against terrorists and health related concerns [ 35 , 36 ]. Various methods have been developed for the detection of these bioterrorist agents and many more still are in development phase. Most of the assays are based on detecting the (a) whole organism, (b) bacterial antigens, and/or (c) the related nucleic acid. In the Table 3.2 are described some detection methods more ­commonly used for bioterrorist agents with safety and security applications. Table 3.2 Detection methods commonly used in detection of bioterrorist agents Bioterrorist agents Detection methods References Pathogens Culture and colony counting [ 37 – 41 ] Immunology-based methods Polymerase chain reaction (PCR) Lateral flow Enzyme-linked immunoassays Biosensors Fluorescence immunoassay Chemiluminescence assay Electrochemical immunoassay Surface plasmon resonance sensor Fiber optic sensor Microfluidic biochip Viruses Cell culturing [ 42 – 44 ] Polymerase chain reaction (PCR) Enzyme linked immunosorbent assay (ELISA) Flow cytometry Lateral flow Biosensors Explosives Fluorescence [ 27 , 45 ] Raman and mass spectroscopy Nuclear magnetic resonance spectroscopy Surface plasmon resonance spectroscopy Electrochemistry High performance liquid chromatography (HPLC) Gas chromatography–mass spectrometry (GC–MS) Electrogenerated chemiluminescence (ECL) Enzyme- linked immunosorbent assays (ELISA) Spores Culture counting [ 18 , 46 ] Immunological detection Nucleic-acid based assays Ligand-based (Aptamers and Peptides) detection Biosensors Toxins Radioimmunoassay [ 20 , 47 , 48 ] Enzyme-linked immunosorbent assay Lateral flow Electrochemiluminescence (ECL) Biosensors Fluorescence Förster resonance energy transfer (FRET) Parasites Fluorescent microscopy [ 25 , 26 ] Flow cytometry Automated blood cell analysers Serology antibody detection Molecular methods Laser desorption mass spectrometry Enzyme-linked immunosorbent assays (ELISA) Indirect fluorescence antibody test (IFAT) Conventional methods such as the culture and colony counting methods that involve counting of bacteria, immunology-based methods that use antigen-antibody interactions and the polymerase chain reaction (PCR) method which involves DNA analysis are being used. These methods can be sensitive, inexpensive and give qualitative and quantitative information, however, a pre-treatment of the samples is needed; furthermore they are greatly restricted by assay time [ 37 ]. The electrochemical detection methods possess several advantages such as easy operation, low cost, high sensitivity, simple instrument and suitability for portable devices. However, to improve their performance one of the most popular ways is to use nanomaterials with a high surface area to functionalize the electrode [ 4 , 5 , 10 ]. In this context, nanomaterial-based methods have demonstrated improvement of the sensitivity, but due to reproducibility problems, as well as interferences, their application in real samples is still limited. Bacteria and Spores The prospect of nanomaterials is promising for rapid and sensitive pathogen detection [ 12 , 14 – 16 ]. Current literature shows numerous applications of different nanostructures in biosensor devices for the detection of pathogenic microorganisms that are of importance to food and environmental safety, biosecurity, and medical diagnostics. In this section, some aspects related to the detection of Salmonella , E. Coli, B. anthracis and Clostridium botulinum by using metal nanoparticles such as Au NPs, Cu@Au NPs, M NPs and QDs will be described. Nanotechnology gives new approaches in order to detect microorganisms through the use of nanomaterials. This field has been explored by Lin et al. [ 12 ] using screen-printed carbon electrodes (SPCEs) modified with Au NPs (13-nm diameter) and ferrocenedicarboxylic acid (FeDC). The detection method consists of a sensitive detection of horseradish peroxidise (HRP) activity coupled with Au NPs and FeDC to amplify the amperometric effect. This has the potential for further applications in the rapid pathogen detection. One important advantage of these amperometric immunosensing strips is that approximately 50 CFU of E. coli O157:H7 in milk samples can be detected in 1 h. In this same context, Zhang and co-workers [ 13 ] described a sensitive electrochemical immunoassay for rapid detection of E. coli by ASV based on core–shell Cu@Au nanoparticles (NPs) as anti- E. coli antibody labels. M NPs due to their high surface/volume ratio offer more contact surface area for attaching carbohydrates and for capturing pathogens. Based on this, E. coli detection using epifluorescent microscopy has been performed by functionalizing the surface of M NPs with D-manosse sugar (man-M NPs) through an amide linkage [ 14 ], subsequently incubations with fluorescein-labeled concanavalin A (Con A) and E. coli cells in phosphate buffer solution (PBS). After that, a magnetic field was applied for separating M NPs/ E. coli aggregates (see Fig. 3.1a ). The supernatants were removed and the remaining aggregates were washed thoroughly, stained with a fluorescent dye (PicoGreen), transferred to a glass slide, and imaged. Fluorescent microscopic imaging showed that E. coli can be detected (see Fig. 3.1b ) with a limit of detection 10 4 cells/mL by obtaining a high capture of bacteria. Fig. 3.1 E. coli detection using epi-fluorescent microscopy. ( a ) Schematic representation based on the functionalization of the silica coated magnetite nanoparticles, ( b ) ( above ) Increase in the fluorescent emission spectra for increasing concentrations (cells/mL) of E. coli ., ( below ) TEM images of MGNP 3/ E. coli complexes (Adapted from [ 14 ] with permission) Metal nanoshells based nanomaterials that exhibit a surface plasmon resonance (SPR) are also used for E. coli detection. Metal nanoshells are thin coatings (a few tens of nanometers thick) on large particles (a few hundreds of nanometers in diameter) which form the cores. Researchers have demonstrated a rapid and reliable test for the detection of E. coli , by using the SPR band associated with the coupling of the antibodies to the silver nanoshells [ 15 ]. This detection method has shown that the E. coli antibody interaction is extremely specific and that the presence of other microorganisms could not produce changes in the SPR band. Furthermore, it could help to shorten the testing time of drinking water used as sample with interest in possible terrorist attacks beside other applications related to the human health. Electrochemical immunoassays based on Au NPs have also attracted considerable interest for Salmonella determination due to its simplicity, high sensitivity, inexpensive instrumentation, and miniaturization. For example, a highly sensitive strategy based on Au NPs for detecting Salmonella typhi (S. typhi) has been ­studied by Dungchaia et al. [ 17 ]. They immobilized monoclonal antibodies (McAbs) on polystyrene microwells and captured S. typhi bacteria by using a copper-enhanced Au NPs label coupled with anodic stripping voltammetry (ASV). The amount of deposited copper was related to the amount of Au NP tag present, which was ­controlled by the amount of S. typhi attached to the polyclonal antibody–­colloidal gold conjugate. Therefore, the anodic stripping peak current was linearly dependent on the S. typhi concentration with a detection limit as low as 98.9 CFU/mL with interest for real samples analysis with a low detection limit, high accuracy, and fast analysis time. Au NPs modified with Salmonella spp McAbs can also be evaluated using the electrochemical impedance spectroscopy (EIS) technique as an efficient method for fabricating a capacitive immunosensor for the detection of Salmonella spp. in real samples [ 16 ]. B. anthracis is another of the biggest threats special because of its potential use in bioterrorism. B. anthracis spores can be transmitted easily to humans. These spores are highly resistant to normally destructive environmental factors to living cells, such as heat, toxic chemicals, desiccation, and physical damages. These properties make them suitable for a potential biological warfare [ 49 ]. For this reason, the rapid and accurate detection of B. anthracis spores in the environment prior to infection is very important for human safety and national security. However, few technologies have been widely evaluated under field conditions [ 46 ]. Accordingly, nanomaterial-based biosensors are evolving as promising alternatives to meet this challenge in terms of sensitivity, specificity, time- and cost-­efficiency [ 30 ]. For this, label-free (bio)sensing systems based on nanomaterials present certain advantages in detection. Among label-free assays, the quartz crystal microbalance (QCM) piezoelectric sensor has proved to be a useful platform in the efficient detection of pathogens including B. anthracis . Based on this platform, electrically active magnetic (EAM) nanoparticles are being used as concentrator of DNA targets as well as electrochemical transducers for detection of the B. anthracis protective antigen A (pag A) gene [ 18 ]. More details can be seen in the Fig. 3.2 . Fig. 3.2 Electrically active magnetic ( EAM ) nanoparticles in B. anthracis DNA detection. ( a ) TEM image of EAM nanoparticles, ( b ) Schematic representation of the EAM based electrochemical DNA biosensor detection principle, ( c ) Electrochemical response of the biosensor to different target DNA concentrations (mean current  ±  SD, n  =  3) (Adapted from [ 18 ] with permission) Another biosensor based on QCM has been developed by R. Hao and coworkers for B. anthracis spores detection by an anti- B. anthracis monoclonal antibody ­designated to 8 G3 (mAb 8 G3, IgG) functionalized QCM sensor [ 50 ]. Toxins Neurotoxins produced by Clostridium botulinum , serotypes A through G (BoNT A-G), are considered the most potent toxins known to humans who cause neuro­paralytic disease [ 51 ]. The "Class A agents" are listed as one of the six highest risk threat agents for bioterrorism [ 22 ]. Food-borne botulism is the most common intoxication form due to the ingestion of pre-formed Botulimun Neurotoxin (BoNT) in food. For this reason, the development of nanomaterial-based rapid methods that can help to detect terrorist agents, such as anthrax, BoNT, etc. is very important for safety and security purposes. Colloidal gold has emerged as the preferred label [ 52 ] for toxin detection. Alternative rapid methods, as colloidal gold-based immunochromatographic assays (ICA) (also called lateral flow (LF) or strip assay) have been developed for detection of botulinum neurotoxin type B (BoNT/B). This class of assays was based on the sandwich format using polyclonal antibody (Pab). A thiophilic gel purified ­anti-BoNT/B Pab was immobilized in a defined detection zone on a porous nitrocellulose membrane and conjugated to colloidal gold particles as a detection reagent. The BoNT/B-containing sample was added to the membrane to react with Pab-coated particles, thus, a change of colour was given in the detection zone with an intensity of red colour proportional to BoNT/B concentration. The strip assay exhibits potential as a rapid and simple assay method for the detection of toxin in biological fluid that requires no separation steps. Moreover, in function of these characteristics the assay can be considered superior to other immunoassay, such as radioimmunoassay and enzyme-linked immunosorbent assay [ 20 ]. Another test based on ICA has been reported by Klewitz et al. [ 21 ] to detect botulism neurotoxin D (BoNT/D). The test was based on double sandwich format using a gold-anti BoNT/D monoclonal antibody conjugates (detector reagent) and an anti-BoNT/D chicken polyclonal antibody. Feacal samples or standard samples spiked with various concentrations of BoNT/D were treated in gelatine-phosphate buffer, vortexed and stored at temperature ranging 5–8°C overnight. Polyclonal chicken anti-BoNT/D IgG and gold conjugated monoclonal antibody were added to the sample extracts and incubated at 37°C for 3 h. Then, 50 μL of pre-treated ­samples were applied to the test strip. The intensity of colour, of the red test line (signal intensity), is directly related with the concentration of BoNT/D in the standard or spiked horse faecal samples. Thus, toxin concentrations were determined within 3.5 h down to 50 pg/mL. In the last years, several researchers have used luminescence methods instead of colorimetric methods for (bio)sensing applications. A novel class of luminescence nanomaterial is emerging for botulinum toxin assays [ 22 ]. This is the case of colloidal semiconductor QDs, which are single crystals of few nanometers of diameter whose size and shape can be precisely controlled by the duration, temperature, and ligand molecules used in their synthesis [ 53 ]. These nanomaterials have unique optical properties such as high extinction coefficients over a wide wavelength range, size dependent optical emission (due to quantum confinement effects on the electronic structure of the QDs) and relatively high quantum yields, which makes them suitable for biological applications such as fluorescence immunoassays, DNA array technology, fluorescence labeling of cells and tissues, and in the detection of chemical and biological agents. In Fig. 3.3 is shown an example of a fluorescence sandwich immunoassay using high-affinity antibodies and quantum dot (QD) reporters for detection of BoNT serotype A (BoNT/A) using a nontoxic recombinant fragment of the holotoxin (BoNT/A-H C -fragment) as a structurally valid simulant for the full toxin molecule [ 22 ]. Fig. 3.3 Detection of BoNT/A-HC-fragment with bead-based immunoassay. ( a ) Scheme of the sandwich immunoassay: AR4 primary antibody specific for BoNT attached to a solid support as Sepharose bead; RAZ1 secondary antibody attached to the reporter 655 nm quantum dot and bound to the toxin fragment-primary antibody complex, ( b ) Calibration curve for the detection of BoNT/A-H C -fragment in FBS and ( c ) Detection from BoNT/A-H C -fragment in buffer using 655 nm QDs and detection on a flow system (Adapted from [ 22 ] with permission) Recently, a detection method based on the use of semiconductor QD-peptide Förster resonance energy transfer (FRET) assemblies to monitor the activity of the BoNT serotype A light chain protease (LcA) has been reported by Sapsford et al. [ 48 ].They evaluated the ability to self-assemble dye-labeled LcA peptide substrates by using a number of different QD materials displaying charged or PEGylated surface-coatings by monitoring FRET interactions. Furthermore, Grate and co-authors [ 47 ] have developed for botulinum toxin detection an electrochemiluminescence (ECL) method based on a sandwich complex with paramagnetic beads and capture of antibodies bound to the beads by a streptavidin-biotin linkage, and the detection of antibodies labeled with a ruthenium chelate. Historically, nanoparticles have been used as label biomolecules. Their properties coupled with the ability to attach nearly any biologic recognition element to the particle surfaces, facilitates their application in multi-target assays. These nanomaterials can be conjugated to DNA sequences or proteins so that, using size-­dependent properties as just one example, fluorescence or light scattering can be used as an output signal, respectively. For example, a method for analyzing combinatorial DNA arrays using oligonucleotide-modified gold nanoparticle probes and a conventional flatbed scanner has been studied by the Mirkin's group [ 54 ]. Labeling oligonucleotide targets (based on the anthrax lethal factor sequence) with nanoparticles rather than fluorophore probes substantially alters the melting profiles of the targets from an array substrate. This difference permits the discrimination of an oligonucleotide sequence from targets with single nucleotide mismatches with a selectivity that is over three times that observed for fluorophore-labeled targets. In addition, when coupled with a signal amplification method in which silver ions are reduced by hydroquinone to silver metal at the surfaces of the Au NPs, the sensitivity of this scanometric array detection system exceeds that of the analogous fluorophore system by two orders of magnitude. Labeling technology based on highly fluorescent europium (Eu + ) NPs could provide a rapid and sensitive testing platform for sensing bioterrorist agents. Lately a europium-nanoparticle based immunoassay (ENIA) for the sensitive detection of anthrax protective antigen has been reported [ 19 ]. The use of Eu + NPs further permits to the assay to be adapted to an ELISA format that is already in place in testing laboratories because the antibody-antigen sandwich complex bound to Eu + NPs coated with streptavidin (SA) can be directly measured with a fluorescence reader. This ultrasensitive NP- based assay for the detection of anthrax toxin could provide a useful new tool for infectious agents and chemical contaminants. Viruses The detection of infectious viral diseases is very important for the public health. In recent years, a number of viral outbreaks, such as severe acute respiratory syndrome (SARS), influenza A (H1N1 flu) and avian influenza A (H5N1 flu) are emerging. These have raised significant fears due to that could rapidly spread and turn into a pandemic similar to 1918 Spanish flu that killed more than 50 million people [ 55 ]. For this, rapid and sensitive diagnostic techniques using different nanomaterials are being developed for recognizing and controlling future epidemics. An example of this is the recent development of immunochromatographic strip for rapid detection of H9 subtype avian influenza viruses (H9 AIVs) [ 23 ]. The assay is based on a colloidal gold anti-hemagglutin monoclonal antibody conjugate (detection antibody) and an anti-Nucleocapside protein monoclonal antibody used as a precipitation reagent on the test line of a nitrocellulose membrane. This detection method is rapid and easy to operate without the requirement of special skills and equipment, which makes it a strip suitable for field detection. In addition, this generation of ICA allows doing multiple assays that can help to diagnostic of common poultry diseases, such as Newcastle disease, avian infectious bronchitis, avian infectious laryngotracheitis, etc. Another similar study has been reported by the same authors to detect IgG antibodies against the nucleocapsid protein of AIV subtypes (H5, H7 and H9) in chicken sera [ 56 ]. The use of immunosensors based on the identification of virus glycoproteins, [ 57 , 58 ] or genosensors based on the detection of specific DNA sequences ­correlated to the virus RNA [ 59 , 60 ], has been performed as an alternative to more expensive classical methods that consume time. In Mexico and USA, in March-April 2009, there was an outbreak of human H1N1 flu virus that created a pandemic concern [ 61 ]. At the time more than 207 countries worldwide reported cases of pandemic H1N1, including at least 8,768 deaths [ 62 ]. For this reason developing of accurate, rapid and low cost sensing methods for the early detection of this kind of virus is of great importance. An impedimetric detection method of a DNA sequence correlated to H1N1 virus using carbon nanotubes platform and Au NPs amplification could be considered a good possibility for improving the sensitivity and rapidity of analysis [ 24 ]. Such systems are based on the use of colloidal gold for the labelling of DNA oligonucleotides, and the electrochemical signal of Au NPs onto screen printed carbon nanotubes electrode is measured and correlated to the DNA target concentration. Parasites The use of parasites as bioterrorism agents has not received so much attention. Parasites could contribute to the installation of fear in human population upon intentional addition to their food and water supplies. In the last years, biosecurity issues are gaining importance as a consequence of globalization. Surveillance is critical in maintaining biosecurity and early detection of infectious disease agents is essential [ 63 ]. Infectious diseases, like malaria, are being one of the greatest health challenges worldwide. Nanotechnology is one of the promising strategies for malaria treatment. The identification of new Plasmodium or infected cell targets can be used to modify existing drug delivery systems employing nanotechnology to more ­efficiently deliver antimalarial drug molecules to the newly-targeted sites of action. Electrochemical immunosensors offer several advantages compared to alternative detection methods, including the ability to analyze the direct blood samples, high sensitivity, require low sample volume and can be used in field application [ 64 ]. For example, nanosized carriers are receiving special attention with the goal of minimizing the side effects of drug therapy, such as poor bioavailability and the selectivity of drugs. Several nanosized delivery systems have already proved their effectiveness in animal models for the treatment and prophylaxis of malaria [ 34 ]. A number of strategies for the detection of Plasmodium falciparum are being developed by using rapid diagnostic tests based on nanomaterials. Magneto immunoassay-based strategies for the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) related to malaria have been described by using M NPs [ 25 ]. The immunological reaction for the protein HRP2 was performed in a sandwich assay on M NPs by using a second monoclonal antibody labeled with horseradish peroxidase (HRP) enzyme. The modified M NPs were captured on the graphite-epoxy composite electrode surface using a magnet inside of the composite electrode which was used as transducer in the electrochemical detection. This magneto immunoassay based on magnetic nanoparticles has shown a limit of detection (LOD) of 0.36 ng mL −1 , which makes it a suitable method for Plasmodium falciparum ­histidine-rich Protein 2 detection related to malaria. Recently, electrochemical immunosensors based on screen printed electrodes are (SPEs) attracting great interest [ 65 ] due to ease of fabrication, ability to mass produce, disposability and portability. Sharma et al. [ 26 ] have developed an amperometric immunosensor based on Au NPs/alumina sol–gel modified SPEs for antibodies to Plasmodium falciparum histidine rich protein-2 ( Pf HRP-2) by dripping Al 2 O 3 sol–gel on SPE followed by electrochemical deposition of Au NPs. The antibodies in rabbit serum sample were allowed to react with the Pf HRP-2 protein which was immobilized on the modified SPE to form antigen-antibody immune complex ( Pf HRP-2/anti- Pf HRP-2). The bound antibodies were quantified by alkaline phosphatase (AP) enzyme labeled secondary antibodies (anti-rabbit immunoglobulins-AP conjugate). Enzymatic substrate, 1-naphthyl phosphate was converted to 1-naphthol by AP and an electroactive product was quantified using amperometric technique. Figure 3.4 shows some results of this electrochemical detection. Fig. 3.4 Detection of Plasmodium falciparum histidine rich protein-2. ( a ) Schematic representation of the modification and immobilization of biomolecules on screen-printed electrode (SPEs), ( b ) Amperometric signals for rabbit-anti PfHRP-2 (1:20,000 dilution) on (a) Bare SPE, (b) Al 2 O 3 sol–gel/SPE, and (c) AuNPs/Al 2 O 3 -sol–gel/SPE immunosensor in DEA buffer solution at pH 9.8, applied potential 400 mV vs. Ag/AgCl/sat.KCl reference electrode (Adapted from [ 26 ] with permission) Although the electrochemical immunosensors together with the nanotechnology are providing certain advantages, in terms of improving the selectivity and sensitivity of the detection systems in the field applied to parasites detection, it is a domain that is still under development. Explosives The conventional methods to detect explosives are restricted by disadvantages such as expensive instrument usage and time-consuming processes. Therefore, it is important to develop methods for trinitrotoluene (TNT) assay with simplicity, ­sensitivity, rapidity and cost-efficiency. Lately, methods for TNT assay have been developed by using Au NPs and QDs. For example, Jiang and co-workers [ 66 ] reported a simple and sensitive method for colorimetric visualization of TNT at picomolar levels based on color changes of Au NPs in the presence of TNT via the electron-donor–acceptor interaction between TNT and primary amines. New methods have been tested for the determination of TNT based on fluorescence quenching of QDs. Chen et al. [ 27 ] have reported a sensitive method with high selectivity for TNT detection by using water-soluble L-cysteine-capped CdTe QDs as fluorescence probe. L-cysteine is used as stabilizer of QDs and as primary amine provider. Intermediate complexes between TNT and cysteine are formed, resulting in the fluorescence quenching of the QDs. This method can be adapted for the detection of TNT. The LOD was 1.1 nM and specificity detection was achieved. More details are observed in the Fig. 3.5 . Fig. 3.5 L-cysteine-capped CdTe QD-based sensor for trinitrotoluene (TNT) detection. ( a ) HRTEM image of CdTe QDs, ( b ) Schematic representation for the L-cysteine-capped CdTe QD-based sensor for TNT detection, ( c ) FL spectra and the quenching efficiency (inset) of L-cysteine-capped CdTe QDs in the presence of different concentrations of TNT (from (a) to (i) 1.1  ×  10 −9 , 5.5  ×  10 −9 , 2.2  ×  10 −8 , 8.8  ×  10 −8 , 2.2  ×  10 −7 , 8.8  ×  10 −7 , 4.4  ×  10 −6 and 1.1  ×  10 −5 M) (Adapted from [ 27 ] with permission) Other methods based on fluorescence detection have been reported by Wang and co-authors where the fluorescence of oleic-acid-covered CdSe QDs could be ­efficiently quenched by nitro aromatic analytes [ 67 ]. Zhang et al. found that ­aminoligand-modified ZnS-Mn 2+ nanocrystals can be introduced to detect TNT [ 68 ]. Recently, graphene owing to its unique properties, such as remarkable electronic conductivity, incredibly large electroactive surface area, high affinity and electrocatalytic activity [ 69 ] is considered another of the interesting nanomaterials to be used for nitroaromatic explosives detection. Guo et al. [ 28 ] have evaluated a new ionic liquid (IL)-graphene composite prepared by combining IL and graphene with large specific surface area and pronounced mesoporosity for ultratrace explosive trinitrotoluene detection. On the other hand, the determination of 2,4-Dinitrotoluene (2,4-DNT) by means of electrochemically reduced graphene on glassy carbon electrode (GCE) has been analyzed by using electrochemical detection, which gave a low detection limit of 42 nmol L −1 (S/N  =  3) [ 29 ]. Bacteria and Spores The prospect of nanomaterials is promising for rapid and sensitive pathogen detection [ 12 , 14 – 16 ]. Current literature shows numerous applications of different nanostructures in biosensor devices for the detection of pathogenic microorganisms that are of importance to food and environmental safety, biosecurity, and medical diagnostics. In this section, some aspects related to the detection of Salmonella , E. Coli, B. anthracis and Clostridium botulinum by using metal nanoparticles such as Au NPs, Cu@Au NPs, M NPs and QDs will be described. Nanotechnology gives new approaches in order to detect microorganisms through the use of nanomaterials. This field has been explored by Lin et al. [ 12 ] using screen-printed carbon electrodes (SPCEs) modified with Au NPs (13-nm diameter) and ferrocenedicarboxylic acid (FeDC). The detection method consists of a sensitive detection of horseradish peroxidise (HRP) activity coupled with Au NPs and FeDC to amplify the amperometric effect. This has the potential for further applications in the rapid pathogen detection. One important advantage of these amperometric immunosensing strips is that approximately 50 CFU of E. coli O157:H7 in milk samples can be detected in 1 h. In this same context, Zhang and co-workers [ 13 ] described a sensitive electrochemical immunoassay for rapid detection of E. coli by ASV based on core–shell Cu@Au nanoparticles (NPs) as anti- E. coli antibody labels. M NPs due to their high surface/volume ratio offer more contact surface area for attaching carbohydrates and for capturing pathogens. Based on this, E. coli detection using epifluorescent microscopy has been performed by functionalizing the surface of M NPs with D-manosse sugar (man-M NPs) through an amide linkage [ 14 ], subsequently incubations with fluorescein-labeled concanavalin A (Con A) and E. coli cells in phosphate buffer solution (PBS). After that, a magnetic field was applied for separating M NPs/ E. coli aggregates (see Fig. 3.1a ). The supernatants were removed and the remaining aggregates were washed thoroughly, stained with a fluorescent dye (PicoGreen), transferred to a glass slide, and imaged. Fluorescent microscopic imaging showed that E. coli can be detected (see Fig. 3.1b ) with a limit of detection 10 4 cells/mL by obtaining a high capture of bacteria. Fig. 3.1 E. coli detection using epi-fluorescent microscopy. ( a ) Schematic representation based on the functionalization of the silica coated magnetite nanoparticles, ( b ) ( above ) Increase in the fluorescent emission spectra for increasing concentrations (cells/mL) of E. coli ., ( below ) TEM images of MGNP 3/ E. coli complexes (Adapted from [ 14 ] with permission) Metal nanoshells based nanomaterials that exhibit a surface plasmon resonance (SPR) are also used for E. coli detection. Metal nanoshells are thin coatings (a few tens of nanometers thick) on large particles (a few hundreds of nanometers in diameter) which form the cores. Researchers have demonstrated a rapid and reliable test for the detection of E. coli , by using the SPR band associated with the coupling of the antibodies to the silver nanoshells [ 15 ]. This detection method has shown that the E. coli antibody interaction is extremely specific and that the presence of other microorganisms could not produce changes in the SPR band. Furthermore, it could help to shorten the testing time of drinking water used as sample with interest in possible terrorist attacks beside other applications related to the human health. Electrochemical immunoassays based on Au NPs have also attracted considerable interest for Salmonella determination due to its simplicity, high sensitivity, inexpensive instrumentation, and miniaturization. For example, a highly sensitive strategy based on Au NPs for detecting Salmonella typhi (S. typhi) has been ­studied by Dungchaia et al. [ 17 ]. They immobilized monoclonal antibodies (McAbs) on polystyrene microwells and captured S. typhi bacteria by using a copper-enhanced Au NPs label coupled with anodic stripping voltammetry (ASV). The amount of deposited copper was related to the amount of Au NP tag present, which was ­controlled by the amount of S. typhi attached to the polyclonal antibody–­colloidal gold conjugate. Therefore, the anodic stripping peak current was linearly dependent on the S. typhi concentration with a detection limit as low as 98.9 CFU/mL with interest for real samples analysis with a low detection limit, high accuracy, and fast analysis time. Au NPs modified with Salmonella spp McAbs can also be evaluated using the electrochemical impedance spectroscopy (EIS) technique as an efficient method for fabricating a capacitive immunosensor for the detection of Salmonella spp. in real samples [ 16 ]. B. anthracis is another of the biggest threats special because of its potential use in bioterrorism. B. anthracis spores can be transmitted easily to humans. These spores are highly resistant to normally destructive environmental factors to living cells, such as heat, toxic chemicals, desiccation, and physical damages. These properties make them suitable for a potential biological warfare [ 49 ]. For this reason, the rapid and accurate detection of B. anthracis spores in the environment prior to infection is very important for human safety and national security. However, few technologies have been widely evaluated under field conditions [ 46 ]. Accordingly, nanomaterial-based biosensors are evolving as promising alternatives to meet this challenge in terms of sensitivity, specificity, time- and cost-­efficiency [ 30 ]. For this, label-free (bio)sensing systems based on nanomaterials present certain advantages in detection. Among label-free assays, the quartz crystal microbalance (QCM) piezoelectric sensor has proved to be a useful platform in the efficient detection of pathogens including B. anthracis . Based on this platform, electrically active magnetic (EAM) nanoparticles are being used as concentrator of DNA targets as well as electrochemical transducers for detection of the B. anthracis protective antigen A (pag A) gene [ 18 ]. More details can be seen in the Fig. 3.2 . Fig. 3.2 Electrically active magnetic ( EAM ) nanoparticles in B. anthracis DNA detection. ( a ) TEM image of EAM nanoparticles, ( b ) Schematic representation of the EAM based electrochemical DNA biosensor detection principle, ( c ) Electrochemical response of the biosensor to different target DNA concentrations (mean current  ±  SD, n  =  3) (Adapted from [ 18 ] with permission) Another biosensor based on QCM has been developed by R. Hao and coworkers for B. anthracis spores detection by an anti- B. anthracis monoclonal antibody ­designated to 8 G3 (mAb 8 G3, IgG) functionalized QCM sensor [ 50 ]. Toxins Neurotoxins produced by Clostridium botulinum , serotypes A through G (BoNT A-G), are considered the most potent toxins known to humans who cause neuro­paralytic disease [ 51 ]. The "Class A agents" are listed as one of the six highest risk threat agents for bioterrorism [ 22 ]. Food-borne botulism is the most common intoxication form due to the ingestion of pre-formed Botulimun Neurotoxin (BoNT) in food. For this reason, the development of nanomaterial-based rapid methods that can help to detect terrorist agents, such as anthrax, BoNT, etc. is very important for safety and security purposes. Colloidal gold has emerged as the preferred label [ 52 ] for toxin detection. Alternative rapid methods, as colloidal gold-based immunochromatographic assays (ICA) (also called lateral flow (LF) or strip assay) have been developed for detection of botulinum neurotoxin type B (BoNT/B). This class of assays was based on the sandwich format using polyclonal antibody (Pab). A thiophilic gel purified ­anti-BoNT/B Pab was immobilized in a defined detection zone on a porous nitrocellulose membrane and conjugated to colloidal gold particles as a detection reagent. The BoNT/B-containing sample was added to the membrane to react with Pab-coated particles, thus, a change of colour was given in the detection zone with an intensity of red colour proportional to BoNT/B concentration. The strip assay exhibits potential as a rapid and simple assay method for the detection of toxin in biological fluid that requires no separation steps. Moreover, in function of these characteristics the assay can be considered superior to other immunoassay, such as radioimmunoassay and enzyme-linked immunosorbent assay [ 20 ]. Another test based on ICA has been reported by Klewitz et al. [ 21 ] to detect botulism neurotoxin D (BoNT/D). The test was based on double sandwich format using a gold-anti BoNT/D monoclonal antibody conjugates (detector reagent) and an anti-BoNT/D chicken polyclonal antibody. Feacal samples or standard samples spiked with various concentrations of BoNT/D were treated in gelatine-phosphate buffer, vortexed and stored at temperature ranging 5–8°C overnight. Polyclonal chicken anti-BoNT/D IgG and gold conjugated monoclonal antibody were added to the sample extracts and incubated at 37°C for 3 h. Then, 50 μL of pre-treated ­samples were applied to the test strip. The intensity of colour, of the red test line (signal intensity), is directly related with the concentration of BoNT/D in the standard or spiked horse faecal samples. Thus, toxin concentrations were determined within 3.5 h down to 50 pg/mL. In the last years, several researchers have used luminescence methods instead of colorimetric methods for (bio)sensing applications. A novel class of luminescence nanomaterial is emerging for botulinum toxin assays [ 22 ]. This is the case of colloidal semiconductor QDs, which are single crystals of few nanometers of diameter whose size and shape can be precisely controlled by the duration, temperature, and ligand molecules used in their synthesis [ 53 ]. These nanomaterials have unique optical properties such as high extinction coefficients over a wide wavelength range, size dependent optical emission (due to quantum confinement effects on the electronic structure of the QDs) and relatively high quantum yields, which makes them suitable for biological applications such as fluorescence immunoassays, DNA array technology, fluorescence labeling of cells and tissues, and in the detection of chemical and biological agents. In Fig. 3.3 is shown an example of a fluorescence sandwich immunoassay using high-affinity antibodies and quantum dot (QD) reporters for detection of BoNT serotype A (BoNT/A) using a nontoxic recombinant fragment of the holotoxin (BoNT/A-H C -fragment) as a structurally valid simulant for the full toxin molecule [ 22 ]. Fig. 3.3 Detection of BoNT/A-HC-fragment with bead-based immunoassay. ( a ) Scheme of the sandwich immunoassay: AR4 primary antibody specific for BoNT attached to a solid support as Sepharose bead; RAZ1 secondary antibody attached to the reporter 655 nm quantum dot and bound to the toxin fragment-primary antibody complex, ( b ) Calibration curve for the detection of BoNT/A-H C -fragment in FBS and ( c ) Detection from BoNT/A-H C -fragment in buffer using 655 nm QDs and detection on a flow system (Adapted from [ 22 ] with permission) Recently, a detection method based on the use of semiconductor QD-peptide Förster resonance energy transfer (FRET) assemblies to monitor the activity of the BoNT serotype A light chain protease (LcA) has been reported by Sapsford et al. [ 48 ].They evaluated the ability to self-assemble dye-labeled LcA peptide substrates by using a number of different QD materials displaying charged or PEGylated surface-coatings by monitoring FRET interactions. Furthermore, Grate and co-authors [ 47 ] have developed for botulinum toxin detection an electrochemiluminescence (ECL) method based on a sandwich complex with paramagnetic beads and capture of antibodies bound to the beads by a streptavidin-biotin linkage, and the detection of antibodies labeled with a ruthenium chelate. Historically, nanoparticles have been used as label biomolecules. Their properties coupled with the ability to attach nearly any biologic recognition element to the particle surfaces, facilitates their application in multi-target assays. These nanomaterials can be conjugated to DNA sequences or proteins so that, using size-­dependent properties as just one example, fluorescence or light scattering can be used as an output signal, respectively. For example, a method for analyzing combinatorial DNA arrays using oligonucleotide-modified gold nanoparticle probes and a conventional flatbed scanner has been studied by the Mirkin's group [ 54 ]. Labeling oligonucleotide targets (based on the anthrax lethal factor sequence) with nanoparticles rather than fluorophore probes substantially alters the melting profiles of the targets from an array substrate. This difference permits the discrimination of an oligonucleotide sequence from targets with single nucleotide mismatches with a selectivity that is over three times that observed for fluorophore-labeled targets. In addition, when coupled with a signal amplification method in which silver ions are reduced by hydroquinone to silver metal at the surfaces of the Au NPs, the sensitivity of this scanometric array detection system exceeds that of the analogous fluorophore system by two orders of magnitude. Labeling technology based on highly fluorescent europium (Eu + ) NPs could provide a rapid and sensitive testing platform for sensing bioterrorist agents. Lately a europium-nanoparticle based immunoassay (ENIA) for the sensitive detection of anthrax protective antigen has been reported [ 19 ]. The use of Eu + NPs further permits to the assay to be adapted to an ELISA format that is already in place in testing laboratories because the antibody-antigen sandwich complex bound to Eu + NPs coated with streptavidin (SA) can be directly measured with a fluorescence reader. This ultrasensitive NP- based assay for the detection of anthrax toxin could provide a useful new tool for infectious agents and chemical contaminants. Viruses The detection of infectious viral diseases is very important for the public health. In recent years, a number of viral outbreaks, such as severe acute respiratory syndrome (SARS), influenza A (H1N1 flu) and avian influenza A (H5N1 flu) are emerging. These have raised significant fears due to that could rapidly spread and turn into a pandemic similar to 1918 Spanish flu that killed more than 50 million people [ 55 ]. For this, rapid and sensitive diagnostic techniques using different nanomaterials are being developed for recognizing and controlling future epidemics. An example of this is the recent development of immunochromatographic strip for rapid detection of H9 subtype avian influenza viruses (H9 AIVs) [ 23 ]. The assay is based on a colloidal gold anti-hemagglutin monoclonal antibody conjugate (detection antibody) and an anti-Nucleocapside protein monoclonal antibody used as a precipitation reagent on the test line of a nitrocellulose membrane. This detection method is rapid and easy to operate without the requirement of special skills and equipment, which makes it a strip suitable for field detection. In addition, this generation of ICA allows doing multiple assays that can help to diagnostic of common poultry diseases, such as Newcastle disease, avian infectious bronchitis, avian infectious laryngotracheitis, etc. Another similar study has been reported by the same authors to detect IgG antibodies against the nucleocapsid protein of AIV subtypes (H5, H7 and H9) in chicken sera [ 56 ]. The use of immunosensors based on the identification of virus glycoproteins, [ 57 , 58 ] or genosensors based on the detection of specific DNA sequences ­correlated to the virus RNA [ 59 , 60 ], has been performed as an alternative to more expensive classical methods that consume time. In Mexico and USA, in March-April 2009, there was an outbreak of human H1N1 flu virus that created a pandemic concern [ 61 ]. At the time more than 207 countries worldwide reported cases of pandemic H1N1, including at least 8,768 deaths [ 62 ]. For this reason developing of accurate, rapid and low cost sensing methods for the early detection of this kind of virus is of great importance. An impedimetric detection method of a DNA sequence correlated to H1N1 virus using carbon nanotubes platform and Au NPs amplification could be considered a good possibility for improving the sensitivity and rapidity of analysis [ 24 ]. Such systems are based on the use of colloidal gold for the labelling of DNA oligonucleotides, and the electrochemical signal of Au NPs onto screen printed carbon nanotubes electrode is measured and correlated to the DNA target concentration. Parasites The use of parasites as bioterrorism agents has not received so much attention. Parasites could contribute to the installation of fear in human population upon intentional addition to their food and water supplies. In the last years, biosecurity issues are gaining importance as a consequence of globalization. Surveillance is critical in maintaining biosecurity and early detection of infectious disease agents is essential [ 63 ]. Infectious diseases, like malaria, are being one of the greatest health challenges worldwide. Nanotechnology is one of the promising strategies for malaria treatment. The identification of new Plasmodium or infected cell targets can be used to modify existing drug delivery systems employing nanotechnology to more ­efficiently deliver antimalarial drug molecules to the newly-targeted sites of action. Electrochemical immunosensors offer several advantages compared to alternative detection methods, including the ability to analyze the direct blood samples, high sensitivity, require low sample volume and can be used in field application [ 64 ]. For example, nanosized carriers are receiving special attention with the goal of minimizing the side effects of drug therapy, such as poor bioavailability and the selectivity of drugs. Several nanosized delivery systems have already proved their effectiveness in animal models for the treatment and prophylaxis of malaria [ 34 ]. A number of strategies for the detection of Plasmodium falciparum are being developed by using rapid diagnostic tests based on nanomaterials. Magneto immunoassay-based strategies for the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) related to malaria have been described by using M NPs [ 25 ]. The immunological reaction for the protein HRP2 was performed in a sandwich assay on M NPs by using a second monoclonal antibody labeled with horseradish peroxidase (HRP) enzyme. The modified M NPs were captured on the graphite-epoxy composite electrode surface using a magnet inside of the composite electrode which was used as transducer in the electrochemical detection. This magneto immunoassay based on magnetic nanoparticles has shown a limit of detection (LOD) of 0.36 ng mL −1 , which makes it a suitable method for Plasmodium falciparum ­histidine-rich Protein 2 detection related to malaria. Recently, electrochemical immunosensors based on screen printed electrodes are (SPEs) attracting great interest [ 65 ] due to ease of fabrication, ability to mass produce, disposability and portability. Sharma et al. [ 26 ] have developed an amperometric immunosensor based on Au NPs/alumina sol–gel modified SPEs for antibodies to Plasmodium falciparum histidine rich protein-2 ( Pf HRP-2) by dripping Al 2 O 3 sol–gel on SPE followed by electrochemical deposition of Au NPs. The antibodies in rabbit serum sample were allowed to react with the Pf HRP-2 protein which was immobilized on the modified SPE to form antigen-antibody immune complex ( Pf HRP-2/anti- Pf HRP-2). The bound antibodies were quantified by alkaline phosphatase (AP) enzyme labeled secondary antibodies (anti-rabbit immunoglobulins-AP conjugate). Enzymatic substrate, 1-naphthyl phosphate was converted to 1-naphthol by AP and an electroactive product was quantified using amperometric technique. Figure 3.4 shows some results of this electrochemical detection. Fig. 3.4 Detection of Plasmodium falciparum histidine rich protein-2. ( a ) Schematic representation of the modification and immobilization of biomolecules on screen-printed electrode (SPEs), ( b ) Amperometric signals for rabbit-anti PfHRP-2 (1:20,000 dilution) on (a) Bare SPE, (b) Al 2 O 3 sol–gel/SPE, and (c) AuNPs/Al 2 O 3 -sol–gel/SPE immunosensor in DEA buffer solution at pH 9.8, applied potential 400 mV vs. Ag/AgCl/sat.KCl reference electrode (Adapted from [ 26 ] with permission) Although the electrochemical immunosensors together with the nanotechnology are providing certain advantages, in terms of improving the selectivity and sensitivity of the detection systems in the field applied to parasites detection, it is a domain that is still under development. Explosives The conventional methods to detect explosives are restricted by disadvantages such as expensive instrument usage and time-consuming processes. Therefore, it is important to develop methods for trinitrotoluene (TNT) assay with simplicity, ­sensitivity, rapidity and cost-efficiency. Lately, methods for TNT assay have been developed by using Au NPs and QDs. For example, Jiang and co-workers [ 66 ] reported a simple and sensitive method for colorimetric visualization of TNT at picomolar levels based on color changes of Au NPs in the presence of TNT via the electron-donor–acceptor interaction between TNT and primary amines. New methods have been tested for the determination of TNT based on fluorescence quenching of QDs. Chen et al. [ 27 ] have reported a sensitive method with high selectivity for TNT detection by using water-soluble L-cysteine-capped CdTe QDs as fluorescence probe. L-cysteine is used as stabilizer of QDs and as primary amine provider. Intermediate complexes between TNT and cysteine are formed, resulting in the fluorescence quenching of the QDs. This method can be adapted for the detection of TNT. The LOD was 1.1 nM and specificity detection was achieved. More details are observed in the Fig. 3.5 . Fig. 3.5 L-cysteine-capped CdTe QD-based sensor for trinitrotoluene (TNT) detection. ( a ) HRTEM image of CdTe QDs, ( b ) Schematic representation for the L-cysteine-capped CdTe QD-based sensor for TNT detection, ( c ) FL spectra and the quenching efficiency (inset) of L-cysteine-capped CdTe QDs in the presence of different concentrations of TNT (from (a) to (i) 1.1  ×  10 −9 , 5.5  ×  10 −9 , 2.2  ×  10 −8 , 8.8  ×  10 −8 , 2.2  ×  10 −7 , 8.8  ×  10 −7 , 4.4  ×  10 −6 and 1.1  ×  10 −5 M) (Adapted from [ 27 ] with permission) Other methods based on fluorescence detection have been reported by Wang and co-authors where the fluorescence of oleic-acid-covered CdSe QDs could be ­efficiently quenched by nitro aromatic analytes [ 67 ]. Zhang et al. found that ­aminoligand-modified ZnS-Mn 2+ nanocrystals can be introduced to detect TNT [ 68 ]. Recently, graphene owing to its unique properties, such as remarkable electronic conductivity, incredibly large electroactive surface area, high affinity and electrocatalytic activity [ 69 ] is considered another of the interesting nanomaterials to be used for nitroaromatic explosives detection. Guo et al. [ 28 ] have evaluated a new ionic liquid (IL)-graphene composite prepared by combining IL and graphene with large specific surface area and pronounced mesoporosity for ultratrace explosive trinitrotoluene detection. On the other hand, the determination of 2,4-Dinitrotoluene (2,4-DNT) by means of electrochemically reduced graphene on glassy carbon electrode (GCE) has been analyzed by using electrochemical detection, which gave a low detection limit of 42 nmol L −1 (S/N  =  3) [ 29 ]. Conclusions Nanotechnology could provide unlimited opportunities for improving the efficacy of (bio)sensing systems for bioterrorist agents. In this chapter are presented some of the detection methods more commonly used for bioterrorist agents as well as for other safety and security applications. However, the domain that nanotechnology has in the detection of bacteria, toxins, parasites, viruses and explosives is still in development phase. To overcome the challenges of nanomaterial-based (bio)sensing strategies for safety and security applications a more detailed study related to interferences for real sample analysis as well as technological aspects related to final application need to be addressed. Point strategies to overcome the challenges should be (a) In-field applications . In-field applications of nano-biosensing still need a big effort so as to overcome problems related to applications in real samples. Avoidance of interferences coming from sample matrix is the key point for success; (b) Low detection limits. Reaching of low detection limits (detection of few molecules, bacteria, cells, etc.) in a relatively high volume of samples (i.e. 1 molecule or 1 cell in 1 mL food sample) needs the development of fast and efficient preconcentration tools/routes based on nano & microfabrication.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2648976/

Substrate Inhibition Kinetic Model for West Nile Virus NS2B-NS3 Protease †

West Nile virus (WNV) has recently emerged in North America as a significant disease threat to humans and animals. Unfortunately, no approved antiviral drugs exist to combat WNV or other members of the genus Flavivirus in humans. The WNV NS2B-NS3 protease has been one of the primary targets for anti-WNV drug discovery and design since it is required for virus replication. As part of our efforts to develop effective WNV inhibitors, we reexamined the reaction kinetics of the NS2B-NS3 protease and the inhibition mechanisms of newly discovered inhibitors. The WNV protease showed substrate inhibition in assays utilizing fluorophore-linked peptide substrates GRR, GKR, and DFASGKR. Moreover, a substrate inhibition reaction step was required to accurately model kinetic data generated from protease assays with a peptide inhibitor. The substrate inhibition model suggested peptide substrates could bind to two binding sites on the protease. Reaction product analogs also showed inhibition of the protease, demonstrating product inhibition in addition to, and distinct from, substrate inhibition. We propose that small peptide substrates and inhibitors may interact with protease residues that form either the P3-P1 binding surface (i.e., the S3-S1 sites) or the P1′-P3′ interaction surface (i.e., the S1′-S3′ sites). Optimization of substrate analog inhibitors that target these two independent sites may lead to novel anti-WNV drugs.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7107177/

Antibodies: an alternative for antibiotics?

In 1967, the success of vaccination programs, combined with the seemingly unstoppable triumph of antibiotics, prompted the US Surgeon General to declare that "it was time to close the books on infectious diseases." We now know that the prediction was overly optimistic and that the fight against infectious diseases is here to stay. During the last 20 yr, infectious diseases have indeed made a staggering comeback for a variety of reasons, including resistance against existing antibiotics. As a consequence, several alternatives to antibiotics are currently being considered or reconsidered. Passive immunization (i.e., the administration of more or less pathogen-specific antibodies to the patient) prior to or after exposure to the disease-causing agent is one of those alternative strategies that was almost entirely abandoned with the introduction of chemical antibiotics but that is now gaining interest again. This review will discuss the early successes and limitations of passive immunization, formerly referred to as "serum therapy," the current use of antibody administration for prophylaxis or treatment of infectious diseases in agriculture, and, finally, recent developments in the field of antibody engineering and "molecular farming of antibodies in various expression systems. Especially the potential of producing therapeutic antibodies in crops that are routine dietary components of farm animals, such as corn and soy beans, seems to hold promise for future application in the fight against infectious diseases.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306470/

Selective Small Molecule Inhibition of Poly(ADP-Ribose) Glycohydrolase (PARG)

The poly(ADP-ribose) (PAR) post-translational modification is essential for diverse cellular functions, including regulation of transcription, response to DNA damage, and mitosis. Cellular PAR is predominantly synthesized by the enzyme poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a critical node in the DNA damage response pathway, and multiple potent PARP-1 inhibitors have been described, some of which show considerable promise in the clinic for the treatment of certain cancers. Cellular PAR is efficiently degraded by poly(ADP-ribose) glycohydrolase (PARG), an enzyme for which no potent, readily accessible, and specific inhibitors exist. Herein we report the discovery of small molecules that effectively inhibit PARG in vitro and in cellular lysates. These potent PARG inhibitors can be produced in two chemical steps from commercial starting materials and have complete specificity for PARG over the other known PAR glycohydrolase (ADP-ribosylhydrolase 3, ARH3) and over PARP-1, and thus will be useful tools to study the biochemistry of PAR signaling.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2810317/

Monoclonal antibody-based therapies for microbial diseases

The monoclonal antibody (mAb) revolution that currently provides many new options for the treatment of neoplastic and inflammatory diseases has largely bypassed the field of infectious diseases. Only one mAb is licensed for use against an infectious disease, although there are many in various stages of development. This situation is peculiar given that serum therapy was one of the first effective treatments for microbial diseases and that specific antibodies have numerous antimicrobial properties. The underdevelopment and underutilization of mAb therapies for microbial diseases has various complex explanations that include the current availability of antimicrobial drugs, small markets, high costs and microbial antigenic variation. However, there are signs that the climate for mAb therapeutics in infectious diseases is changing given increasing antibiotic drug resistance, the emergence of new pathogenic microbes for which no therapy is available, and development of mAb cocktail formulations. Currently, the major hurdle for the widespread introduction of mAb therapies for microbial diseases is economic, given the high costs of immunoglobulin preparations and relatively small markets. Despite these obstacles there are numerous opportunities for mAb development against microbial diseases and the development of radioimmunotherapy provides new options for enhancing the magic bullet. Hence, there is cautious optimism that the years ahead will see more mAbs in clinical use against microbial diseases. 1 Historical perspective: from the origins of serum therapy to antibody use today The prophylactic and therapeutic potential of immune serum was discovered by Behring and Kitasato, who showed that passive transfer of antibody from the blood of infected animals could provide immunity to diphtheria [9] . Their work led to the first instance of industrial production of protective serum from sheep for human therapy in 1893 [10] and to the first Nobel Prize in Medicine for Behring. Immune animal sera from horses, sheep, and chickens were used to treat diseases where a protective immune response could be induced in the animal host by vaccination. In cases where humans were the only hosts, such as viral diseases, human convalescent sera were successfully used. For example, in the early 1900s, serum from individuals who recovered from measles was used to treat and prevent infection. Until the 1930s, serum from animals or people was collected and pooled to treat a number of infections, from streptococcal infection to toxin-mediated diseases like diphtheria [11] . Overall, serum was effective, and for some diseases like pneumococcal pneumonia and meningococcal meningitis, the prompt administration of serum was associated with significantly improved survival [3] . However, despite these successes, the discovery of antibiotics in the 1930s and 1940s rapidly replaced serum therapy. Antibiotics were easier to manufacture, had less toxicity in patients and produced more consistent results. In contrast to serum therapy, which depended on animal sources that exhibited great lot-to-lot variation, antibiotics were the products of industrial processes and could be formulated in preparations with consistent activity. Furthermore, serum therapy was generally effective only early in the course of infection while antibiotic therapy maintained efficacy even when given late in the course of a microbial disease. Another advantage of antibiotic therapy was that it could be used without a specific diagnosis while the use of antibody therapy required knowledge of the pathogen responsible for disease. Consequently, serum therapy was unable to compete with antibiotics, and the development of new broad-spectrum antibiotic therapy displaced antibody-based therapies ( Table 1 ). Table 1 Comparison of serum therapy, antimicrobial therapy and mAb therapy. Parameter Serum therapy Antimicrobial therapy mAb therapy Cost High Low High Easy to use No Yes Yes Specific diagnosis Yes No Yes Toxicity High Low Low Lot variation High Low Low Source Animals Industrial production Industrial production Damage to microflora No Yes No Despite the general abandonment of serum therapy for bacterial diseases, certain niches developed where it continued to be used, such as the prophylaxis and treatment of a small number of viral and toxin-mediated diseases for which there were no alternative therapeutic options. This point is important because it illustrated that antibody therapy can thrive in certain situations where it lacked competition, such as in the treatment of diseases which have no other effective therapies. For example, antibody preparations continue to be used to prevent rabies and toxicity from snakebite venoms. In developed countries serum therapy was often replaced by hyperimmune serum from pooled human donors. Today, hyperimmune human sera immunoglobulin is used to treat many diseases including those caused by cytomegalovirus (CMV), respiratory syncytial virus (RSV), hepatitis A virus (HAV), hepatitis B virus (HBV), rabies, vaccinia, vesicular stomatitis virus (VZV), and measles, underscoring the fact that antibody therapy remains an effective means of treatment [6] , [12] . Compared to hyperimmune sera, or even to modern antibiotics, mAb therapy has many advantages and some disadvantages ( Table 1 ). mAbs inherently have a high specificity for their target and, since microbes are generally antigenically distinct from humans, the cross-reactivity with host tissues is minimal. In contrast to antibiotics, which target both harmful microbes and the host flora, mAbs will only target a specific microbe and their systemic administration should not affect other resident beneficial microbes. This could prove to be a significant advantage given increasing reports associating certain chronic diseases such as asthma, atopy, and even certain forms of cancer with antimicrobial drug use [13] , [14] . Microbial specificity means that mAbs are unlikely to select for drug-resistant microbes among non-targeted microbes. The ability to specifically target disease-causing microbial populations without selecting for resistance makes mAb therapy potentially superior to current broad-spectrum antibiotics that are generally used in therapy, at least for microbial diseases caused by single microbes. The increasing prevalence and rising cost of treating methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and other resistant infections in both nosocomial and community settings emphasizes the need to develop new strategies for controlling infections. 2 mAbs as therapeutics Serum therapy by definition uses immune sera-derived immunoglobulins that are polyclonal preparations consisting of many types of antibodies of which only a minute fraction is specific for the intended microbe. In contrast, mAb preparations consist of one type of immunoglobulin with a defined specificity and a single isotype. This represents both an advantage and a disadvantage when mAbs are compared to polyclonal preparations. One advantage is that mAbs, by virtue of the fact that they are chemically defined reagents, exhibit relatively low lot-to-lot variability in contrast to polyclonal preparations, which can differ over time and by source of origin since different hosts mount different antibody responses. Another advantage for mAb preparations is a much greater activity per mass of protein since all the immunoglobulin molecules are specific for the desired target. This phenomenon is illustrated by the report that two 0.7 mg doses of two mAbs provided the same protection against tetanus toxin as 100–170 mg of tetanus immune globulin [15] . However, mAb preparations lack variability with regards to epitope and isotype, and consequently polyclonal preparations have potentially greater biological activity by targeting multiple microbial epitopes and providing various effector functions through different isotypes. With the development of human and humanized mAbs, the toxicity of these agents is also relatively low. Current technology makes the production of mAbs relatively easy and effective, requiring only tissue culture or microbial expression systems, as opposed to the live human or animal donors that were required for serum therapy. Hence, the potential toxicity of human and humanized mAbs is comparable to antibiotics and lower than serum therapy, especially heterologous preparations. mAb therapies are also much less likely to inadvertently transmit other infectious diseases. However, antibody therapies remain very costly relative to antimicrobial drugs. Consequently, mAbs are unlikely to successfully compete with antimicrobial drugs against diseases for which cheap effective therapy is available unless a clear superiority is established for the immunoglobulin therapy alone or in combination with conventional antimicrobial therapy. The fact that specific antibodies are often synergistic with conventional antimicrobial therapy suggests that combination therapy with current antimicrobial regimens may confer potential advantages relative to either alone [16] , [17] . Not only can mAbs make antibiotics more effective, but the research driving mAb development can also enhance other areas, such as identifying new targets for vaccine development [18] , [19] . In this regard, efforts to develop mAb-based therapies have the potential of impacting many aspects of infectious disease medicine. Furthermore, some mAbs can be effective in immunocompromised hosts, as evidenced by the efficacy of palivizumab in reducing hospitalizations for RSV-associated disease in preterm infants [20] . Even in the setting of reduced immune response, mAbs can function to replace lost immunity or benefit the host by direct activity, such as neutralization. Consequently, mAbs are an attractive alternative alone or as adjuncts to current antimicrobial therapy that will be effective in hosts with different states of immunity. Despite these strengths, mAb therapy has some inherent limitations. The cost factor has already been alluded to and remains a major obstacle to widespread mAb use. Antibodies are macromolecules that are fragile, perishable and require refrigeration, each of which contributes to their high cost. Furthermore, most mAbs require systemic administration, which complicates their delivery in many clinical settings. Finally, a mAb usually cannot be used until a specific diagnosis is made. In an era when broad-spectrum antibiotics are relied upon so heavily, treatment is often begun before diagnostic identification of a disease-causing microbe is made [5] . However, recent advances in rapid diagnostic techniques provide hope for earlier specific diagnosis, which is essential for making mAb treatments more realizable. Even so, based on experience with serum therapy, one could anticipate that a mAb will only be effective when administered relatively early in the course of infection. A decrease in antibody efficacy over time occurs quickly as the duration of infection increases, highlighting the need for rapid diagnosis and treatment initiation. Another, theoretical drawback to mAb therapy is that these reagents, by definition, target a single epitope, and provide one type of effector function corresponding to their isotype. Although the specificity of mAbs is a strength, a microbe that undergoes rapid antigenic variation poses a significant hurdle for mAb development. For example, the high mutation rate of certain viruses enables them to escape neutralization. There are numerous examples in the literature where experimental mAb therapy has resulted in the emergence of escape variants as a result of microbial mutation and/or microevolution [21] , [22] , [23] . This problem may be counterbalanced by selecting mAbs that target conserved areas of viral particles or by using mixtures of mAbs that target various epitopes. For example, combination therapy with mAb cocktails prevents escape variants for many viruses including influenza [24] , coronavirus [25] , and lymphocytic choriomeningitis virus (LCMV) [26] . Although cocktails are effective in providing protection against the emergence of resistant mutants, the inclusion of multiple immunoglobulins in any therapeutic formulation is fraught with complex regulatory and licensing issues. Nevertheless, progress in overcoming these burdensome regulatory issues is evidenced by the recent development of a mAb mixture for the prophylaxis of rabies that targets several virus types [27] . Indeed, as polyclonal sera may be beneficial due to the presence of multiple protective antibodies, a cocktail of functional mAbs could provide more protection and target more microbial strains than a single one. As experience with mAb cocktails accumulates it may be possible to design therapeutic combinations that include multiple effector functions in the form of different isotypes and epitope specificities. Even in the face of these obstacles, mAb therapy is booming in other areas of medicine, with over 20 in clinical use, representing a market that is expected to reach $20 billion by 2010 [28] . The majority of approved mAbs treat cancer, autoimmune or allergic conditions, where they have shown considerable success and spawned a mAb 'gold rush' [29] . The antigenic differences between the host and the microbe make mAb development for infectious diseases easier than for cancer or immunity fields, where the target is often a self-antigen that is differentially expressed in transformed cells. Additionally, mAbs have direct and indirect antimicrobial mechanisms of action. Direct mechanisms include neutralizing toxins or binding to viruses to prevent host cell entry. Recently mAbs have also been shown to be directly bactericidal [30] . Indirect mechanisms involve Fc-mediated functions, such as modulation of the inflammatory response, promoting opsonic phagocytosis, and enhancing complement-mediated effects. This wide array of functions makes mAbs potentially useful against a variety of infectious diseases. Despite these advantages, there is currently only one licensed mAb for an infectious disease, and understanding its success is instructive for the possibilities inherent in this approach. The humanized mAb palivizumab, brand name Synagis ® , binds the RSV F protein and is effective for the prevention of severe respiratory disease in high-risk infants and immunocompromised adults [31] . It received regulatory approval in 1998. Prior to palivizumab, prophylaxis of RSV disease depended on RespiGam, or RSV-IGIV, a prophylactic polyclonal RSV hyperimmune serum [1] . This prior polyclonal preparation was plagued by low specific activity, and effective dosing required the administration of large volumes of antibody, which was problematic in low weight infants. Palivizumab obviated this problem since it had 50-fold greater potency than the polyclonal serum, and this example shows how greater specific activity can translate into a superior product. The greater potency significantly reduced the volume needed to deliver a therapeutic dose to an infant and has improved treatment while avoiding the side effects of pooled serum. 3 Opportunities for mAb in infectious diseases In our view opportunities for the development of mAbs include niches where their use would bring a large therapeutic benefit relative to existing therapies. Although there are many diseases where mAb therapy could make a major contribution (see below) the economics are not favorable in each instance. In this regard, the exquisite specificity of antibody-based therapies means necessarily smaller markets than broad-spectrum antimicrobial therapy. This combined with the relatively high costs of producing and delivering immunoglobulin therapies can make the market analysis of many reagents not economically viable. However, for microbial diseases with inadequate therapeutic options, antibody-based therapies are likely to be competitive, provided that the disease prevalence is of sufficient size to create a tenable demand. It is noteworthy that even for diseases where there is currently adequate antimicrobial therapy, new developments could make mAb more attractive. For example, consider the case of antibiotic prophylaxis against Group B streptococcus (GBS) neonatal sepsis. Mothers carrying GBS are routinely treated with antibiotics and this has resulted in a dramatic reduction in neonatal disease. However, antibiotic therapy affects the infant microflora and has been epidemiologically linked to the development of atopy and asthma in children [14] . Furthermore, prophylactic antibiotic therapy can select for resistant microbes or disturb the flora in a manner that could make colonization with resistant microbes more likely, could be followed by superinfections, and may even have untoward consequences later in life [5] . Passive antibody therapy against GBS is also effective [32] and not encumbered by the problems of altered flora and its consequences. Hence, the economics of passive antibody and antibiotic prophylaxis are more nuanced than simply valuing the different alternatives. A truer cost comparison must take into account the complications that follow broad-spectrum therapy. 4 Targets of mAb therapy Historically, the major targets for antibody therapy have been diseases caused by encapsulated bacteria (e.g. pneumococcus and meningococcus), toxins (e.g. diphtheria and tetanus) and viruses. In general, most efforts to develop antibody-based therapies have focused on diseases where the humoral immune system was known to make a strong contribution to host defense. For these microbial diseases the efficacy of humoral immunity was implied from demonstration of passive antibody protection and/or correlation of specific antibody with resistance to disease. However, recent work has established that mAbs are effective even against microbes for which the standard studies do not clearly establish a role for humoral immunity [33] . For example, several mAbs have now been generated that are effective against intracellular pathogens and other microbes where natural Ab is not believed to have a primary role in host defense [33] . Overall, diseases which are currently not preventable by vaccination or that target populations with weak immune systems (for example, the very young or old, and immunosuppressed or immunocompromised patients) are the most valuable for which to develop mAb therapies. Here, both viral and bacterial toxin diseases that are the target of mAb development will be discussed, as well as potential targets based on need toward which efforts should be focused in the future. Our goal is to survey this field, with the understanding that cataloguing all ongoing efforts is beyond the scope of this article. 4.1 Viral targets Viral targets are particularly attractive because for most viruses there is no specific therapy. The potential for mAbs to be used as neutralizing antibodies to prevent viral binding and entry to host cells makes them a good platform for prophylaxis, preemptive or acute treatment of viral illnesses [34] . There are several current areas of viral mAb development, and many in clinical trials (see Table 2 ). Table 2 Anti-infective mAb in clinical trial development a . Name (type) Target Clinical trials phase Virus RSV Palivizumab (humanized mAb) Glycoprotein F Approved Motavizumab (humanized mAb) Glycoprotein F I–III HIV CCR5mAb004 (human mAb) CCR5 I PRO 140 (humanized mAb) CCR5 II 3 mAb cocktail I/II F105 (human mAb) gp120 I Ibalizumab (humanized mAb) CD4 II CMV Sevirumab (human mAb) Envelope glycoprotein H II, III HCV Bavituximab (chimeric mAb) Phosphatidylserine Ib MDX1106 (human mAb) PD-1 I Rabies CL184 (mAb cocktail) I WNV MGAWN1 (humanized mAb) Envelope glycoprotein I Bacteria/toxin E. coli Urtoxazumab (humanized mAb) Shiga-like toxin 2B C. difficile GS-CDA1 (human mAb) C. difficile toxin A II MDX-388 (human mAb) C. difficile toxin B II Staphylococcus Pagibaximab (chimeric mAb) LTA II Tefibazumab (humanized mAb) Clumping factor A II B. anthracis Anthim PA I Raxibacumab PA III Fungal C. neoformans 18B7 (murine mAb) Capsular polysaccharide I a Source : http://clinicaltrials.gov/ (not a complete list). While the approved drug palivizumab is effective against RSV disease in susceptible groups, there has been considerable effort to develop 2nd and 3rd generation mAbs: motavizumab (brand name Numax ® ) and Numax-YTE ® [1] . Motavizumab was engineered to have increased affinity through induced somatic hypermutation, with the hopes that increased binding will improve function in humans, and is currently being tested in phase III clinical trials. Numax-YTE is the result of additional efforts to prolong this mAb's serum half-life, another potential avenue of mAb development. Furthermore, clinical trials are underway testing both escalating doses of mAb and intramuscular (i.m.) administration, showing that the improvement of delivery and dosing of mAb drugs are an important next step in advancing therapy. These developments illustrate how technological advances may improve existing successful mAb therapeutics. HIV has always been an area of great interest for mAb therapy [35] , [36] , [37] , [38] . There are several mAbs for HIV in development, designed to inhibit viral entry, reduce viral load in HIV-patients, and potentially to prevent infection in certain cases [39] , [40] . Viral entry inhibitor mAbs target either the cellular receptors, CCR5 and CD4, or the cognate viral protein gp120. Efforts to develop neutralizing mAbs with broader strain specificity have found success targeting the V3 loop of gp120 [41] . As is true with all mAbs designed for infectious disease, the development of a successful vaccine would reduce their need. However, given the slow progress on the front of HIV vaccine development, mAb research in the HIV field is a promising alternative. Although there are numerous antiretroviral drugs available for the treatment of HIV, the availability of effective mAb therapy could complement chemotherapy by slowing the onset of resistance and possibly enhancing therapeutic efficacy. Another target for mAb development is CMV. Sevirumab is currently being assessed for treatment of CMV retinitis in HIV-infected individuals and neonatal congenital CMV. Another important complication of CMV infection is reactivation disease, a distinct burden on transplantation medicine. CMV infects a large portion of the population, at least 60% of adults in the U.S., and the virus can reactivate with devastating consequences during the course of immunosuppression that solid organ or hematologic transplant patients must undergo [42] . The use of mAb to control this reactivation is an area of development that could benefit a large number of patients. Hepatitis B and C virus (HBV, HCV) infections are areas where antibody therapies offer great hope in helping to control disease and improve transplantation success. Hepatitis remains the leading indication for liver transplant, and viral reinfection of the transplanted liver is a major complication. Two different monoclonal antibodies are in clinical trials for the improving transplant success, of which neither are specific for the virus itself. Both bavituximab, specific for phosphatidylserine, a phospholipid exposed on membranes of damaged cells, and MDX-1106, specific for PD-1, an inhibitory T cell costimulation receptor, have been used to control chronic hepatitis infection, particularly in the setting of HIV-coinfection [34] , [43] . The threat of a new pandemic makes influenza virus an important candidate for mAb development, with research currently being done to characterize human neutralizing antibodies and explore their therapeutic potential [24] , [44] , [45] . Historically, influenza virus has been extremely challenging because of its high antigenic variability. However, progress has been made in identifying antibodies that are broadly neutralizing [46] , [47] . In the event of a new pandemic mAbs could provide important options for disease control since they confer immediate immunity and may be used as prophylaxis for individuals who are likely to have been exposed to infection. Considerable excitement followed the discovery of a broadly cross-reactive antibody specific for HA2 of influenza A that allows the neutralization of different viral strains, including avian H5N1 and human H1N1 [48] . Technology now exists where plasma cells secreting influenza-specific antibody can be cloned from human donors and used to generate high affinity mAbs within a matter of weeks [49] . Another promising opportunity for antibody therapy is rabies, where current standard of care depends on administration of immune globulin and vaccination. In this regard, a major innovation was the development of the mAb cocktail CL184 [27] . This cocktail is comprised of two mAbs, specific for two different epitopes of the rabies virus, and shows good neutralizing activity in vitro and was well tolerated in patients. Its success validates the idea that cocktails of multiple mAbs represent an important logistical improvement in mAb therapy, allowing for expanded target coverage, broader specificity and a wider range of effector functions. There is no available vaccine or specific therapeutic agent for human flaviviral infections, and after the outbreak of West Nile encephalitis in 1999, efforts were directed towards developing antibody therapies for prophylactic treatment [50] . A hyperimmune preparation derived from human convalescent sera protected mice from West Nile virus (WNV), both in the setting of induced immunocompromise and after the onset of encephalitis [51] . This observation suggested that antibody was effective at treating disease after dissemination to the brain and spinal cord had occurred. Development of mAb treatment for WNV in elderly or immunocompromised patients might prevent the debilitating disease and paralysis that can occur in these populations [52] . MGAWN1, a humanized mAb to the structural envelope protein, is currently in clinical trials, and could provide a new therapeutic option against WNV. The ability to rapidly identify microbial targets and produce mAbs makes them a feasible tool to combat emergent situations. An excellent example of the rapidity with which mAbs can be generated in response to an outbreak was provided by the response to SARS corona virus (SARS-CoV). In early 2003, respiratory disease due to the virus broke out in human populations, and within a year the cellular receptor [53] and viral glycoprotein [54] responsible for binding were identified. By early 2004, neutralizing human mAbs had been developed and were being tested by laboratories [55] , illustrating the potential speed of bench-to-bedside transition. Human mAbs have been developed to the Hendra and Nipah viruses, which are both paramyxoviruses that can cause fatal hemorrhagic fevers and were responsible for outbreaks in the late 1990s [56] , [57] . Emerging diseases are just one area where research into the specificity of mAb targets and their protective efficacy in animal models should be a priority. Other areas where mAbs targeting a needed pathogen may be in development but have not reached clinical trials yet are listed in Table 3 . Table 3 Examples of potential mAb targets based on need. Category Target Comments/concerns Bioterrorism Anthrax ( B. anthracis ) Need for immediate dispersal upon exposure Small pox (Variola virus) Population of non-immune individuals Ebola virus Highly virulent Emerging diseases Henipavirus Also potential bioterrorist agents SARS-CoV Possibility of recurrence of 2003 pandemic Influenza virus H1N1 and H5N1 are pandemic threats Susceptible populations Parainfluenza virus LRI protection in at-risk pediatric populations Candida spp. Important class of nosocomial infection MDR bacteria MRSA, VRSA Increasing prevelance in community settings Pseudomonas aeruginosa Important class of nosocomial infection 4.2 Bacterial/toxin/fungal targets Bacterial diseases mediated by toxins have historically responded to specific antibody and consequently remain good targets for treatment with toxin-neutralizing mAbs. In addition to toxin-mediated diseases, recent studies have shown that specific mAbs can alter the course of bacterial and fungal infection, and that immunoglobulins can enhance the action of other antimicrobials. The issue of mAb synergy with conventional antimicrobial drugs is of particular importance since development of mAbs as an adjunct to existing therapy is an attractive possibility to improve therapeutic outcomes for bacterial infection. Several mAbs, targeting diseases that are in clinical trials ( Table 2 ) or being developed as needed therapeutics ( Table 3 ), are discussed below. The Shiga-like toxin IIB produced by pathogenic strains of Escherichia coli is responsible for organ damage in the hemorrhagic colitis and hemolytic uremic syndrome (HUS) that develop during infection. Outbreaks of the enterohaemorrhagic E. coli O157:H7 are often linked to contaminated food and can be potentially fatal. Treatment of HUS is complicated, because antibiotics can potentially worsen the disease [58] , [59] . Several mAbs designed to neutralize the systemic toxin have been developed, and one is currently having success in phase I clinical trials [60] , [61] , [62] . Another toxin-mediated disease of growing concern is Clostridium difficile colitis, which usually results from broad-spectrum antibiotic use [63] . Although oral antibiotics can often clear infection, there are indications that new hypervirulent strains are emerging, which increase disease severity [64] . Furthermore, for certain individuals the disease can become chronic [65] . mAbs to toxin A and toxin B are in development and currently being tested in clinical trials. Laboratory research has shown that mAbs may act by more than just direct binding and neutralization of toxin, as Fc receptors have been shown to be essential for mAb protection, presumably through increasing endocytic uptake of the toxin by effector cells [66] . The increasing prevalence and toll of this disease underscores both the dangers of antibiotic use and the potential for mAb as a new therapy platform. mAbs may become essential tools to fight this and many other hospital-acquired infections, where often antibiotics have already failed to improve outcomes [67] . Staphylococcal disease is an example of bacterial disease where both the bacterium and disease-mediating toxins can be targeted by mAbs. Two mAbs in clinical trials recognize staphylococcal virulence factors. Pagibaximab is a chimeric mAb specific for lipoteichoic acid (LTA), which was shown to be protective against both coagulase-negative staphylococci (CoNS) and S. aureus infection [68] . Certain populations are very susceptible to staphylococcal disease, such as very low birth weight (VLBW) newborns, where pagibaximab is currently in phase I/II clinical trials for preventing infection [69] , [70] . Another target is the S. aureus protein clumping factor A (ClfA), targeted by tefibazumab (brand name Aurexis ® ) which is in phase II clinical trials for S. aureus bacteremia [71] . These two mAbs show promise and provide hope for the development of improved mAbs targeting staphylococcal species. Alpha-hemolysin (H1a) is a pore-forming cytotoxin that is an essential virulence factor for the development staphylococcal pneumonia. Researchers have recently shown that passive administration of a mAb to H1a, as well as vaccination to elicit active immunity against this antigen, afforded protection to pneumonia in mouse models [72] . Antibodies neutralizing toxins and viruses can provide immediate defense against many biological weapons [73] . Anthrax is an example of one such disease, where current treatment recommendations are inadequate, as the anthrax vaccine is currently not indicated for post-exposure prophylaxis and antibiotic-resistant strains of Bacillus anthracis are a rising concern [74] . Protective antigen (PA) is the common subunit to the two dimeric anthrax toxins, where PA pairs with edema factor (EF) or lethal factor (LF) to form cellular toxins mediating edema and cell death, respectively. The two mAbs specific for PA currently in clinical trials are anthim, which was just announced to be restarting phase I clinical trials, and raxibacumab, which is currently maintained by the U.S. Strategic National Stockpile. Toxins from Clostridium botulinum are another example of extremely potent neurotoxins that can lead to fatal respiratory disease and are capable of being weaponized. Development of mAbs to neutralize these toxins for post-exposure treatment is an important avenue of research. One area where antibody therapy has long provided the only therapeutic option is the neutralization of snake venoms. Most of the current preparations fall into the category of serum therapy, with immunoglobulins derived from heterologous sources. One group has developed human single chain variable antibody fragments (HuScFv) from a phage display library that are effective in neutralizing the neurotoxin and preventing lethality in mice [75] , illustrating how new mAb technology and antibody formats are able to improve and expand on current treatments. However, economics could pose a formidable problem with developing mAbs venom therapy, since the number of different poisons is great and, fortunately, the number of cases is relatively few. Fungal diseases are a promising area for mAb therapy. Fungal diseases are chronic, difficult to treat, and carry a high mortality and morbidity despite antifungal therapy. For this group of diseases mAb therapy could find a niche because current therapies are suboptimal. To date only one mAb has been studied clinically for the treatment of cryptococcal meningitis [76] . That antibody reduced serum cryptococcal antigen but only at very high doses, which made it economically unfeasible. An antibody fragment to a surface heat shock protein showed promising efficacy against human candidiasis in a phase III trial [16] . Recently a broadly cross-reactive mAb to a fungal cell wall antigen was described that was effective against experimental aspergillosis, candidiasis, and cryptococcosis [77] , [78] . Another example of broadly active antifungal mAbs are those that mimic killer toxin action [79] . 4.1 Viral targets Viral targets are particularly attractive because for most viruses there is no specific therapy. The potential for mAbs to be used as neutralizing antibodies to prevent viral binding and entry to host cells makes them a good platform for prophylaxis, preemptive or acute treatment of viral illnesses [34] . There are several current areas of viral mAb development, and many in clinical trials (see Table 2 ). Table 2 Anti-infective mAb in clinical trial development a . Name (type) Target Clinical trials phase Virus RSV Palivizumab (humanized mAb) Glycoprotein F Approved Motavizumab (humanized mAb) Glycoprotein F I–III HIV CCR5mAb004 (human mAb) CCR5 I PRO 140 (humanized mAb) CCR5 II 3 mAb cocktail I/II F105 (human mAb) gp120 I Ibalizumab (humanized mAb) CD4 II CMV Sevirumab (human mAb) Envelope glycoprotein H II, III HCV Bavituximab (chimeric mAb) Phosphatidylserine Ib MDX1106 (human mAb) PD-1 I Rabies CL184 (mAb cocktail) I WNV MGAWN1 (humanized mAb) Envelope glycoprotein I Bacteria/toxin E. coli Urtoxazumab (humanized mAb) Shiga-like toxin 2B C. difficile GS-CDA1 (human mAb) C. difficile toxin A II MDX-388 (human mAb) C. difficile toxin B II Staphylococcus Pagibaximab (chimeric mAb) LTA II Tefibazumab (humanized mAb) Clumping factor A II B. anthracis Anthim PA I Raxibacumab PA III Fungal C. neoformans 18B7 (murine mAb) Capsular polysaccharide I a Source : http://clinicaltrials.gov/ (not a complete list). While the approved drug palivizumab is effective against RSV disease in susceptible groups, there has been considerable effort to develop 2nd and 3rd generation mAbs: motavizumab (brand name Numax ® ) and Numax-YTE ® [1] . Motavizumab was engineered to have increased affinity through induced somatic hypermutation, with the hopes that increased binding will improve function in humans, and is currently being tested in phase III clinical trials. Numax-YTE is the result of additional efforts to prolong this mAb's serum half-life, another potential avenue of mAb development. Furthermore, clinical trials are underway testing both escalating doses of mAb and intramuscular (i.m.) administration, showing that the improvement of delivery and dosing of mAb drugs are an important next step in advancing therapy. These developments illustrate how technological advances may improve existing successful mAb therapeutics. HIV has always been an area of great interest for mAb therapy [35] , [36] , [37] , [38] . There are several mAbs for HIV in development, designed to inhibit viral entry, reduce viral load in HIV-patients, and potentially to prevent infection in certain cases [39] , [40] . Viral entry inhibitor mAbs target either the cellular receptors, CCR5 and CD4, or the cognate viral protein gp120. Efforts to develop neutralizing mAbs with broader strain specificity have found success targeting the V3 loop of gp120 [41] . As is true with all mAbs designed for infectious disease, the development of a successful vaccine would reduce their need. However, given the slow progress on the front of HIV vaccine development, mAb research in the HIV field is a promising alternative. Although there are numerous antiretroviral drugs available for the treatment of HIV, the availability of effective mAb therapy could complement chemotherapy by slowing the onset of resistance and possibly enhancing therapeutic efficacy. Another target for mAb development is CMV. Sevirumab is currently being assessed for treatment of CMV retinitis in HIV-infected individuals and neonatal congenital CMV. Another important complication of CMV infection is reactivation disease, a distinct burden on transplantation medicine. CMV infects a large portion of the population, at least 60% of adults in the U.S., and the virus can reactivate with devastating consequences during the course of immunosuppression that solid organ or hematologic transplant patients must undergo [42] . The use of mAb to control this reactivation is an area of development that could benefit a large number of patients. Hepatitis B and C virus (HBV, HCV) infections are areas where antibody therapies offer great hope in helping to control disease and improve transplantation success. Hepatitis remains the leading indication for liver transplant, and viral reinfection of the transplanted liver is a major complication. Two different monoclonal antibodies are in clinical trials for the improving transplant success, of which neither are specific for the virus itself. Both bavituximab, specific for phosphatidylserine, a phospholipid exposed on membranes of damaged cells, and MDX-1106, specific for PD-1, an inhibitory T cell costimulation receptor, have been used to control chronic hepatitis infection, particularly in the setting of HIV-coinfection [34] , [43] . The threat of a new pandemic makes influenza virus an important candidate for mAb development, with research currently being done to characterize human neutralizing antibodies and explore their therapeutic potential [24] , [44] , [45] . Historically, influenza virus has been extremely challenging because of its high antigenic variability. However, progress has been made in identifying antibodies that are broadly neutralizing [46] , [47] . In the event of a new pandemic mAbs could provide important options for disease control since they confer immediate immunity and may be used as prophylaxis for individuals who are likely to have been exposed to infection. Considerable excitement followed the discovery of a broadly cross-reactive antibody specific for HA2 of influenza A that allows the neutralization of different viral strains, including avian H5N1 and human H1N1 [48] . Technology now exists where plasma cells secreting influenza-specific antibody can be cloned from human donors and used to generate high affinity mAbs within a matter of weeks [49] . Another promising opportunity for antibody therapy is rabies, where current standard of care depends on administration of immune globulin and vaccination. In this regard, a major innovation was the development of the mAb cocktail CL184 [27] . This cocktail is comprised of two mAbs, specific for two different epitopes of the rabies virus, and shows good neutralizing activity in vitro and was well tolerated in patients. Its success validates the idea that cocktails of multiple mAbs represent an important logistical improvement in mAb therapy, allowing for expanded target coverage, broader specificity and a wider range of effector functions. There is no available vaccine or specific therapeutic agent for human flaviviral infections, and after the outbreak of West Nile encephalitis in 1999, efforts were directed towards developing antibody therapies for prophylactic treatment [50] . A hyperimmune preparation derived from human convalescent sera protected mice from West Nile virus (WNV), both in the setting of induced immunocompromise and after the onset of encephalitis [51] . This observation suggested that antibody was effective at treating disease after dissemination to the brain and spinal cord had occurred. Development of mAb treatment for WNV in elderly or immunocompromised patients might prevent the debilitating disease and paralysis that can occur in these populations [52] . MGAWN1, a humanized mAb to the structural envelope protein, is currently in clinical trials, and could provide a new therapeutic option against WNV. The ability to rapidly identify microbial targets and produce mAbs makes them a feasible tool to combat emergent situations. An excellent example of the rapidity with which mAbs can be generated in response to an outbreak was provided by the response to SARS corona virus (SARS-CoV). In early 2003, respiratory disease due to the virus broke out in human populations, and within a year the cellular receptor [53] and viral glycoprotein [54] responsible for binding were identified. By early 2004, neutralizing human mAbs had been developed and were being tested by laboratories [55] , illustrating the potential speed of bench-to-bedside transition. Human mAbs have been developed to the Hendra and Nipah viruses, which are both paramyxoviruses that can cause fatal hemorrhagic fevers and were responsible for outbreaks in the late 1990s [56] , [57] . Emerging diseases are just one area where research into the specificity of mAb targets and their protective efficacy in animal models should be a priority. Other areas where mAbs targeting a needed pathogen may be in development but have not reached clinical trials yet are listed in Table 3 . Table 3 Examples of potential mAb targets based on need. Category Target Comments/concerns Bioterrorism Anthrax ( B. anthracis ) Need for immediate dispersal upon exposure Small pox (Variola virus) Population of non-immune individuals Ebola virus Highly virulent Emerging diseases Henipavirus Also potential bioterrorist agents SARS-CoV Possibility of recurrence of 2003 pandemic Influenza virus H1N1 and H5N1 are pandemic threats Susceptible populations Parainfluenza virus LRI protection in at-risk pediatric populations Candida spp. Important class of nosocomial infection MDR bacteria MRSA, VRSA Increasing prevelance in community settings Pseudomonas aeruginosa Important class of nosocomial infection 4.2 Bacterial/toxin/fungal targets Bacterial diseases mediated by toxins have historically responded to specific antibody and consequently remain good targets for treatment with toxin-neutralizing mAbs. In addition to toxin-mediated diseases, recent studies have shown that specific mAbs can alter the course of bacterial and fungal infection, and that immunoglobulins can enhance the action of other antimicrobials. The issue of mAb synergy with conventional antimicrobial drugs is of particular importance since development of mAbs as an adjunct to existing therapy is an attractive possibility to improve therapeutic outcomes for bacterial infection. Several mAbs, targeting diseases that are in clinical trials ( Table 2 ) or being developed as needed therapeutics ( Table 3 ), are discussed below. The Shiga-like toxin IIB produced by pathogenic strains of Escherichia coli is responsible for organ damage in the hemorrhagic colitis and hemolytic uremic syndrome (HUS) that develop during infection. Outbreaks of the enterohaemorrhagic E. coli O157:H7 are often linked to contaminated food and can be potentially fatal. Treatment of HUS is complicated, because antibiotics can potentially worsen the disease [58] , [59] . Several mAbs designed to neutralize the systemic toxin have been developed, and one is currently having success in phase I clinical trials [60] , [61] , [62] . Another toxin-mediated disease of growing concern is Clostridium difficile colitis, which usually results from broad-spectrum antibiotic use [63] . Although oral antibiotics can often clear infection, there are indications that new hypervirulent strains are emerging, which increase disease severity [64] . Furthermore, for certain individuals the disease can become chronic [65] . mAbs to toxin A and toxin B are in development and currently being tested in clinical trials. Laboratory research has shown that mAbs may act by more than just direct binding and neutralization of toxin, as Fc receptors have been shown to be essential for mAb protection, presumably through increasing endocytic uptake of the toxin by effector cells [66] . The increasing prevalence and toll of this disease underscores both the dangers of antibiotic use and the potential for mAb as a new therapy platform. mAbs may become essential tools to fight this and many other hospital-acquired infections, where often antibiotics have already failed to improve outcomes [67] . Staphylococcal disease is an example of bacterial disease where both the bacterium and disease-mediating toxins can be targeted by mAbs. Two mAbs in clinical trials recognize staphylococcal virulence factors. Pagibaximab is a chimeric mAb specific for lipoteichoic acid (LTA), which was shown to be protective against both coagulase-negative staphylococci (CoNS) and S. aureus infection [68] . Certain populations are very susceptible to staphylococcal disease, such as very low birth weight (VLBW) newborns, where pagibaximab is currently in phase I/II clinical trials for preventing infection [69] , [70] . Another target is the S. aureus protein clumping factor A (ClfA), targeted by tefibazumab (brand name Aurexis ® ) which is in phase II clinical trials for S. aureus bacteremia [71] . These two mAbs show promise and provide hope for the development of improved mAbs targeting staphylococcal species. Alpha-hemolysin (H1a) is a pore-forming cytotoxin that is an essential virulence factor for the development staphylococcal pneumonia. Researchers have recently shown that passive administration of a mAb to H1a, as well as vaccination to elicit active immunity against this antigen, afforded protection to pneumonia in mouse models [72] . Antibodies neutralizing toxins and viruses can provide immediate defense against many biological weapons [73] . Anthrax is an example of one such disease, where current treatment recommendations are inadequate, as the anthrax vaccine is currently not indicated for post-exposure prophylaxis and antibiotic-resistant strains of Bacillus anthracis are a rising concern [74] . Protective antigen (PA) is the common subunit to the two dimeric anthrax toxins, where PA pairs with edema factor (EF) or lethal factor (LF) to form cellular toxins mediating edema and cell death, respectively. The two mAbs specific for PA currently in clinical trials are anthim, which was just announced to be restarting phase I clinical trials, and raxibacumab, which is currently maintained by the U.S. Strategic National Stockpile. Toxins from Clostridium botulinum are another example of extremely potent neurotoxins that can lead to fatal respiratory disease and are capable of being weaponized. Development of mAbs to neutralize these toxins for post-exposure treatment is an important avenue of research. One area where antibody therapy has long provided the only therapeutic option is the neutralization of snake venoms. Most of the current preparations fall into the category of serum therapy, with immunoglobulins derived from heterologous sources. One group has developed human single chain variable antibody fragments (HuScFv) from a phage display library that are effective in neutralizing the neurotoxin and preventing lethality in mice [75] , illustrating how new mAb technology and antibody formats are able to improve and expand on current treatments. However, economics could pose a formidable problem with developing mAbs venom therapy, since the number of different poisons is great and, fortunately, the number of cases is relatively few. Fungal diseases are a promising area for mAb therapy. Fungal diseases are chronic, difficult to treat, and carry a high mortality and morbidity despite antifungal therapy. For this group of diseases mAb therapy could find a niche because current therapies are suboptimal. To date only one mAb has been studied clinically for the treatment of cryptococcal meningitis [76] . That antibody reduced serum cryptococcal antigen but only at very high doses, which made it economically unfeasible. An antibody fragment to a surface heat shock protein showed promising efficacy against human candidiasis in a phase III trial [16] . Recently a broadly cross-reactive mAb to a fungal cell wall antigen was described that was effective against experimental aspergillosis, candidiasis, and cryptococcosis [77] , [78] . Another example of broadly active antifungal mAbs are those that mimic killer toxin action [79] . 5 Enhancing the magic bullet with radiation The exquisite specificity of antibodies for their targets provides a means of delivering harmful cargo to their targets. This concept has long been recognized in oncology where investigators have tried to enhance the efficacy of anti-tumor antibody therapies by conjugating antibodies to bacterial toxins and radioisotopes. Like use of mAb therapy in general, the field of infectious diseases has been slow in adopting such strategies, although considerable experimental work was done to develop immunotoxins to target HIV-infected cells [80] . Nevertheless, in recent years progress has been made developing radioimmunotherapy for infectious diseases. Radioimmunotherapy (RIT) was first developed for cancer treatment almost three decades ago [81] . RIT uses the antigen–antibody interaction to deliver radionuclides that emanate lethal doses of cytotoxic radiation to cancer cells [82] , [83] , and can provide a valuable alternative to chemotherapy and external radiation beam therapy (EBRT). RIT is a successful therapy for certain cancers as evidenced by the recent approval of mAb-based drugs such as Zevalin ® and Bexxar ® (anti-CD20 mAbs labeled with 90-Yttrium ( 90 Y) and 131-Iodine ( 131 I), respectively) for the treatment of relapsed or refractory B-cell non-Hodgkin's lymphoma. Recent reports on the use of RIT as an initial treatment for follicular lymphoma [84] are encouraging thus making RIT first line therapy in some types of cancer. Until recently the potential of RIT as an antimicrobial strategy had not been explored. The reasons for this are enigmatic but could reflect the lack of awareness of the difficult problems in clinical infectious diseases by the nuclear medicine community and of RIT by the infectious diseases community. During the last 5 years RIT has been successfully adapted in our laboratories for the treatment of experimental fungal, bacterial and viral infections [85] , [86] , [87] , [88] , [89] , [90] . Here we briefly review the data on efficacy and safety of RIT of infectious diseases. The potential efficacy of RIT against an infectious diseases was first established using Cryptococcus neoformans (CN) [85] . CN is a major fungal pathogen that causes life-threatening meningoencephalitis in 6–8% of patients with AIDS. CN provided a good system to study the potential usefulness of RIT because there were excellent animal models available, well characterized mAbs to CN antigens existed, and immunotherapy of CN infection with capsule polysaccharide-binding antibody 18B7 was already in clinical evaluation [76] . In spite of high level of circulating polysaccharide in the blood of infected mice, in both pulmonary and systemic animal models of CN infection radiolabeled mAb preferentially localized to the sites of infection. mAb 18B7 radiolabeled with 213-bismuth ( 213 Bi) or 188-rhenium ( 188 Re) killed CN cells in vitro , thus providing the basis for in vivo experiments in AJ/Cr mice infected systemically with CN. Mice treated with radiolabeled CN-specific mAb 18B7 lived significantly longer than mice given irrelevant labeled IgG1 or PBS. Mice infected with CN and given RIT had significantly reduced fungal burden in lungs and brains 48 h after treatment compared to infected mice in the control groups. The antimicrobial RIT approach was subsequently extended to another human pathogenic fungus, Histoplasma capsulatum (HC) [86] , which is the most common cause of fungal pneumonia in immunocompromised patients [91] , by treating HC in vitro with 188 Re-labeled mAb 9C7 (IgM) which binds to a protein antigen on the surface of the HC cell wall [92] . Ninety percent of HC cells were killed with 32 μCi HC-specific 188 Re-9C7 mAb. In contrast, incubation of HC with a radiolabeled control IgM with the same specific activity produced only minimal killing within the investigated range of doses. Cellular dosimetry calculations based on the mean absorbed dose to the cell showed that RIT with alpha- and beta-emitting radioisotopes was approximately 1000-fold more efficient in killing CN and approximately 100-fold more efficient in killing HC than gamma radiation. The potential of RIT against bacterial infection was established using Streptococcus pneumoniae because this is an important extracellular pathogen, and there are good animal models and mAbs available [88] . A greater percentage of mice treated with 213 Bi-D11 survived relative to the control groups, where mice in control groups succumbed to bacteremia on days 1–3, while mice treated with 80 μCi 213 Bi-D11 mice demonstrated 87–100% survival. Treatment with radiolabeled D11 was very well tolerated as no weight loss was observed in treated animals. RIT could also be potentially effective against chronically infected cells including those with viral infections [6] . In contrast to RIT of fungal and bacterial diseases where the target is the microbe itself, in RIT of viral infections the infected mammalian cells would be targeted thus providing a general strategy for eliminating reservoirs of infected cells and viral cellular factories. This approach could be developed for the treatment of drug-resistant HIV strains [93] . The efficacy of RIT for treatment of HIV infection in vivo was explored with a radiolabeled HIV envelope-specific human anti-gp41 mAb 246-D radiolabeled with 213 Bi and 188 Re [89] . For these studies human peripheral blood mononuclear cells (PBMCs) infected with HIV-1 JR-CSF were injected into the spleens of SCID mice and the mice were treated with RIT. Treatment of mice with 188 Re-labeled mAb 246-D administered either before or after intrasplenic injection with HIV-1 JR-CSF -infected human PBMCs dramatically reduced the number of HIV-1-infected cells. Similar results were obtained after treatment of mice with 213 Bi-246-D. Administration of equivalent amounts of "cold" mAb 246-D or of a radioisotope-coupled irrelevant control mAb did not reduce the average number of infected cells detected in the SCID mouse spleens. The demonstration of efficacy of RIT against HIV provided a proof-of-principle for the concept of treating viral infections by targeting viral-infected cells and this approach could potentially be applied to other chronic viral diseases like hepatitis C. The success of RIT approach in laboratory studies combined with earlier nuclear medicine experience on pre-clinical and clinical studies showing the utility of radiolabeled organism-specific antibodies for imaging of infections (reviewed in [94] ) provides encouragement for the feasibility of therapeutically targeting microbes with labeled antibodies. In fact, the ability of specific antibody to localize to a site of infection provides strong support for the potential usefulness of this technique as a broad antimicrobial strategy. It might be possible to create a so-called "pan-antibody" which would recognize an antigen shared by a particular class of human pathogens. An example of such a "pan-antibody" is mAb 6D2 initially developed against fungal melanin that also binds to synthetic, invertebrate (cuttlefish), murine and human melanins [95] . The availability of such antibodies would eliminate the necessity of having antibodies specific for each particular microorganism and would enormously enhance the development of RIT of infectious diseases. 6 Future of mAb therapy for infectious diseases Considering the breadth of research into new targets for microbial mAbs, as well as the vast need for new therapeutic modalities for infectious diseases, this area of medicine is a growing field. There are many new approaches to improving the efficacy of mAbs, such as using cocktails of mAbs or combining mAbs with existing drugs for synergistic effects. There are also many new research developments that will expand the possibilities for mAb drugs. The next generation of mAbs could be developed against novel targets, such as quorum sensing molecules, which regulate bacterial growth and virulence factor expression [96] , or type III secretion systems, which are used by a subset of microbes to infect host cells [97] . To fight MDR bacteria, some successful approaches have targeted the cellular efflux pumps responsible for antibiotic resistance [98] . In general, more of these types of "broad-spectrum" mAbs will enable a therapeutic mAb to be developed which could be useful against a number of microbes, thus extending its market and increasing its profit potential. Bavituximab is an example of one such mAb, specific for phospholipids on the exposed inner surface of damaged cells, where potential applications include both HIV and HCV [34] . Progress has also been made generating mAbs that have bispecificity, where mAb variants are selected that bind to multiple microbial epitopes [99] . Finally, more direct engineering of mAbs could result in enhanced efficacy, such as Fc region alterations that can extend serum half-life or influence effector function [100] . For example, alterations in Fc region glycosylation can increase antibody-dependent cell-mediated cytotoxicity (ADCC) [101] . Although this review has focused on the use of intact mAbs against microbial diseases, we note that for some microbes the Fc region is not necessary for efficacy, providing numerous additional therapeutic options. For example, mAb fragments which lack Fc regions can be effective in situations where effector cell function is not necessary, for example in inhibiting HIV entry [102] . We are cautiously optimistic that the mAb therapeutic revolution will make a much greater impact against microbial diseases in the years ahead. There is no shortage of targets for mAbs to treat microbial diseases. At this time, the major problem is economic and it is likely that success will come first in niche areas where there is sufficient need, urgency, and market size to support the development of mAb therapies. In the longer term, the field needs to build economic models that are suitable for the development of antibody therapies against a much broader set of microbial targets. Conflict of Interest The authors state that they have no conflict of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5031848/

Acute and Subacute Toxicological Evaluation of the Aerial Extract of Monsonia angustifolia E. Mey. ex. A. Rich in Wistar Rats

The acute and subacute toxicity profile of the aerial extract of Monsonia angustifolia in Wistar rats was evaluated. The Organization for Economic Cooperation and Development (OECD) 420 guideline was adopted in the acute toxicity testing with a single oral dose of 5000 mg/kg (b.w.). For the 28-day daily oral dosing, the extract was administered at 75, 150, and 300 mg/kg b.w.; 1% ethanol in sterile distilled water was used as control. Clinical toxicity signs were subsequently evaluated. At a single dose of 5000 mg/kg b.w. the extract elicited no treatment-related signs of toxicity in the animals during the 14 days of experimental period. In the subacute toxicity, there was no significant difference in hematological, renal, and liver function indices. However, dose-dependent significant increases were observed on the plasma concentrations of white blood cell and platelet counts of the treated animals compared to the control group. While cage observations revealed no treatment-facilitated signs of toxicity, histopathological examinations of the kidneys and liver also showed no obvious lesions and morphological changes. These results suggest that the extract may be labelled and classified as safe and practically nontoxic within the doses and period of investigation in this study. 1. Introduction The genus Monsonia is dispersed over both hemispheres in Africa, America, Europe, Asia, and Australia [ 1 ]. It is widespread in Africa from Nigeria to Somalia and in the south in South Africa and also in Madagascar [ 2 ]. Monsonia angustifolia E. Mey. ex. A. Rich, commonly known as Crane's bill, Alsbos, Angelbossie, or Teebossie, belongs to the family Geraniaceae. It is a suberect annual plant growing in sandy soils, usually on granite, often on rocky areas, and along roadsides. Teebossie is widely distributed in the southern African region, from South Africa to Lesotho, Swaziland, Namibia, and Mozambique. Monsonia angustifolia (MA) is provincially distributed in the Eastern Cape, Free State, Gauteng, KwaZulu-Natal, Limpopo, and Mpumalanga in South Africa [ 3 ]. Its red thick woody based stems are usually about 50 cm high with characteristic short pubescent gland-tipped hairs. The leaves are narrow with irregular tooth of cuneate based oblong or elliptic margins. While its flowers are small and purple-tinted with overlapping toothed petals and the stamens are arranged in an outward spreading pattern from the center, the fruits are held in an erect fashion and are approximately 50–90 mm long. MA is rich in calcium, iron, proteins, and vitamin C and the presence of these constituents in its organs may be attributed to its excellently displayed importance as a medicinal agent [ 4 ]. Ethnomedicinally, MA has therapeutic significance as a blood cleanser and aphrodisiac and enhances libido [ 5 ]. Fouche et al. [ 6 ] have also reported its sexual competence enhancing ability in rats. Its pharmacological relevance in the treatment of heartburn, anthrax, and diarrhoea has also been documented [ 7 ]. The ethnomedicinal application of different formulations of MA to treat various diseases is increasing appreciably. Despite the profound therapeutic significance of MA, there is a paucity of information in the literatures about the safety of its standardized aqueous extract. Accordingly, since nontoxicity is one of the criteria set by the World Health Organization for the use of herbs as medicines, the present study was therefore conceptualized to investigate the effect of oral acute and 28-day repeated dose administration of standardized extract of MA on haematological and kidney and liver function parameters of Wistar rats. 2. Experimental 2.1. Chemicals, Reagents, and Assay Kits The assay kits for the determination of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were supplied by Randox Laboratories Ltd., United Kingdom, while those of alkaline phosphatase (ALP) and gamma glutamyl transferase (GGT) were obtained from Roche Diagnostics GmbH, Mannheim, Germany. All other chemicals and reagents were of analytical grade and prepared in glass-distilled water. 2.2. Plant Collection, Authentication, and Processing Fresh plant materials comprising the aerial parts of Monsonia angustifolia were collected near Chuenespoort, Polokwane area, Limpopo Province, South Africa. The plant was identified at the South African Biodiversity Institute (SANBI), where a voucher specimen (number 582251.0) was prepared and deposited. The identified sample was thoroughly rinsed under running water to remove foliar contaminants, air-dried to constant weight, and subsequently pulverized in a hammer mill. 2.3. Extract Preparation The aqueous extract was prepared by a method similar to making a tea infusion. One liter of deionized water was added to 86 g of the ground material and boiled for an hour. The water solution was left to cool to room temperature. The resulting infusion was filtered and subsequently freeze-dried. This yielded a brownish fluffy powder corresponding to 22.16 g of the crude extract. The crude extract obtained was thereafter standardized by dissolution in 200 mL of distilled water and then successively fractionated on a C18 reverse phase silica gel flash cartridge using 100 mL of each of water, methanol/water (1 : 1 v/v), and methanol, respectively. This yielded 200 mg of the standardized extract. The extract was only readily soluble in 1% ethanol (EtOH) and as such was reconstituted in it (1% EtOH) to give the various concentrations used in this study. 2.4. Experimental Animals Thirty-six healthy male and female Wistar rats weighing 200–250 g were utilized in this study and were obtained from the animal house of the School of Biological and Environmental Sciences, University of Fort Hare, Alice 5700, South Africa. The animals were kept in clean polypropylene cages under standard animal house conditions (temperature: 23 ± 1°C; photoperiod: 12 h natural light and 12 h dark; humidity: 45–50%). A 7-day acclimatization period was observed before dosing and the rats were allowed ad libitum access to feed (Epol Feed Chunks, South Africa) and tap water under hygienic conditions. All treatments were in accordance with the Guide for the Care and Use of Laboratory Animals [ 8 ]. Approval (number AFO022) was granted by the Animal Ethics Committee of the University of Fort Hare, South Africa, prior to commencement of the study. 2.5. Acute Toxicity Test This was performed according to the Organization for Economic Cooperation and Development (OECD) 420 guideline for testing chemicals with slight modification [ 9 ]. Twelve rats used for this study were first fasted for 18 h prior to assignment into two groups of six rats each. Each group contained equal numbers of male and female rats (i.e., 3 males and 3 females). Group A was given 1 mL single oral dose of 5000 mg/kg body weight (b.w.) of the extract, while group B (control) received 1 mL of 1% EtOH in distilled water. Following this treatment, the animals were observed closely for the first 24 h with particular attention on the first 6 h and then once daily for the 14-day experimental period. Initial and 7-day interval body weight changes of all the rats were monitored and recorded. They were also subjected to detailed gross necropsy and signs of toxicity/mortality were monitored and observed throughout the investigation period. Based on the mortality observed in each group, LD 50 was subsequently determined [ 10 ]. 2.6. 28-Day Repeated Dose Toxicity Test Twenty-four Wistar strain rats were randomly divided into four groups of six animals each. Each group was made up of equal numbers of males and females. Animals in group 1 (control) were given 1 mL of 1% EtOH in distilled water. Groups 2–4 comprised animals given 1 mL of the extract at 75, 150, and 300 mg/kg b.w., respectively. All administration instances were done every 24 hours via oral gavage throughout the investigation period. The rats were weighed daily and also subjected to thorough observations for mortality, behavioral changes, and possible symptoms of humane end point during the 28-day experimental period. 2.7. Blood Collection and Organ Isolation On the 29th day of the experiment, the rats were humanely sacrificed under halothane anesthesia and blood samples were collected into plain and EDTA-containing bottles as previously described [ 11 ]. The collected samples were thereafter centrifuged at 1282 ×g for 5 min using Hermie Bench Top Centrifuge (Model Hermie Z300, Hamburg, Germany) and subsequently used for biochemical and haematological analyses, respectively. The rats were immediately dissected and the liver and kidneys were isolated, freed of fat, blotted with clean tissue paper, and weighed. Relative organ-body weight ratios were thereafter evaluated. 2.8. Determination of Haematological Parameters Haematological parameters, including red blood cell (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet, white blood cells (WBC), and lymphocytes, were determined using Automated Haematologic Coulter Analyzer (Beckman Coulter Inc., CA, USA). 2.9. Determination of Biochemical Parameters The serum activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphate (ALP), and gamma glutamyl transferase (GGT) as well as concentrations of total bilirubin, total protein, albumin, electrolytes (calcium and potassium ions), creatinine, uric acid, and urea were evaluated using Piccolo Express Automatic Chemistry Analyzer (Abaxis Inc., Union City, CA 94587, USA). 2.10. Histopathological Examination A portion of each of the excised organs was fixed in 10% (v/v) buffered formaldehyde solution, dehydrated through ascending grades of ethanol (70, 90, and 95% v/v), cleaned in xylene, and embedded in paraffin wax. Tissue sections were then prepared and stained with haematoxylin-eosin [ 12 ]. The photomicrographs of the tissue sections were taken at ×400 using the Leitz DIALUX research microscope. 2.11. Statistical Analysis Statistical analysis was performed using Minitab Student release version 12, Windows 95. Where necessary, data were represented as mean of six replicates ± standard error of mean and were analyzed using one-way analysis of variance (ANOVA) and complemented with Student's t -test. Significant differences between the treatment means were considered at p 0.05) change in the body weight gain of the extract-treated rats throughout the study period ( Table 1 ). 3.2. 28-Day Toxicity Test Daily repeated oral dose treatment with the extract for 28 days did not induce any evident sign of toxicity in the treated animals, including those given 300 mg/kg b.w. dose. No deaths or obvious adverse clinical signs were observed in any of the test groups throughout the treatment period. Note that the data for both males and females in the same group were merged together for presentation in this study as there were no pronounced/significant variations in the parameters measured between the male and female rats. 3.3. Serum Haematological Parameters The effects of 28-day administration of M. angustifolia extract at 75, 150, and 300 mg/kg b.w. doses on the haematological parameters of the animals are represented in Table 2 . With the exception of the significant dose-specific increases in the plasma counts of platelets and WBC in the extract-treated rats, administration of the extract at all the investigated doses had no significant ( p > 0.05) effect on all other parameters evaluated including HGB and RBC, when compared with the control. 3.4. Liver and Kidney Function Indices Data obtained with respect to liver function parameters (AST, ALT, ALP, GGT, total bilirubin, total protein, and albumin) evaluated in this study revealed that the extract caused no significant ( p > 0.05) alterations on these parameters for all the animals when compared with the control ( Table 3 ). Similarly, except for the marginal variations in the serum concentrations of urea and creatinine, the extract at the tested regimens had no significant ( p > 0.05) effect on the serum level of the electrolytes (calcium and potassium ions) and uric acid when compared with the control ( Table 4 ). 3.5. Organ-Body Weight Ratio The respective initial body weights of the treated rats and the control were compared with their final weights. Except for the significantly increased liver-body weight ratio at 300 mg/kg b.w. dose of the extract, normal body weight gains corresponding to a similar pattern of nonsignificant difference in the absolute organ weight of the kidneys and liver were observed in the extract-treated groups during the study period compared to the control group ( Table 5 ). 3.6. Histopathological Examination Detailed microscopic and histoarchitectural examinations of the kidney and liver of the extract-treated animals revealed no abnormalities in overall structural orientation of the organs and there were no observable cellular injuries. The nuclear characteristics, morphological features, and tissue integrity of organs of the treated rats were essentially normal and comparable to the normal control (Figures 1 and 2 ). 3.1. Acute Toxicity The oral administration of the standardized extract of Monsonia angustifolia at 5000 mg/kg b.w. dose had no clinical adverse effect of substance related toxicity and did not cause mortality of any rat during the 14-day observation period. Also, there was no morbidity or behavioral or physiological changes. Compared with the control, there was no significant ( p > 0.05) change in the body weight gain of the extract-treated rats throughout the study period ( Table 1 ). 3.2. 28-Day Toxicity Test Daily repeated oral dose treatment with the extract for 28 days did not induce any evident sign of toxicity in the treated animals, including those given 300 mg/kg b.w. dose. No deaths or obvious adverse clinical signs were observed in any of the test groups throughout the treatment period. Note that the data for both males and females in the same group were merged together for presentation in this study as there were no pronounced/significant variations in the parameters measured between the male and female rats. 3.3. Serum Haematological Parameters The effects of 28-day administration of M. angustifolia extract at 75, 150, and 300 mg/kg b.w. doses on the haematological parameters of the animals are represented in Table 2 . With the exception of the significant dose-specific increases in the plasma counts of platelets and WBC in the extract-treated rats, administration of the extract at all the investigated doses had no significant ( p > 0.05) effect on all other parameters evaluated including HGB and RBC, when compared with the control. 3.4. Liver and Kidney Function Indices Data obtained with respect to liver function parameters (AST, ALT, ALP, GGT, total bilirubin, total protein, and albumin) evaluated in this study revealed that the extract caused no significant ( p > 0.05) alterations on these parameters for all the animals when compared with the control ( Table 3 ). Similarly, except for the marginal variations in the serum concentrations of urea and creatinine, the extract at the tested regimens had no significant ( p > 0.05) effect on the serum level of the electrolytes (calcium and potassium ions) and uric acid when compared with the control ( Table 4 ). 3.5. Organ-Body Weight Ratio The respective initial body weights of the treated rats and the control were compared with their final weights. Except for the significantly increased liver-body weight ratio at 300 mg/kg b.w. dose of the extract, normal body weight gains corresponding to a similar pattern of nonsignificant difference in the absolute organ weight of the kidneys and liver were observed in the extract-treated groups during the study period compared to the control group ( Table 5 ). 3.6. Histopathological Examination Detailed microscopic and histoarchitectural examinations of the kidney and liver of the extract-treated animals revealed no abnormalities in overall structural orientation of the organs and there were no observable cellular injuries. The nuclear characteristics, morphological features, and tissue integrity of organs of the treated rats were essentially normal and comparable to the normal control (Figures 1 and 2 ). 4. Discussion Toxicity studies on herbal extracts are commonly used to evaluate the possible health risk of the intrinsic chemical compounds in the plant which could result in adverse effects from the plant [ 13 ]. Specifically, acute toxicity and LD 50 determination have been described as initial steps in the toxicological evaluations of plant extracts (Oladipipo et al. [ 10 ]), and data from such evaluations provide comprehensive information on the toxicological classification and labelling of such compounds [ 14 ]. According to Lorke [ 15 ], substances with LD 50 values of ≥5000 mg/kg b.w. are said to be safe and practically nontoxic. Hence, extract of M. angustifolia may be considered nontoxic when administered via oral route and could be adjudged to be relatively safe for consumption in rats. Also, that the extract at a single oral dose of 5000 mg/kg had no treatment-related adverse effect on the tested animals up to 14 days of investigation is another supportive fact of its nonacute toxicity effect. This nonacute toxicity observation could also suggest that the LD 50 of the extract is greater than 5000 mg/kg b.w. in rats. The proportionate and nonsignificant weight gained across all the treatment groups relative to the control not only is indicative of the nontoxic potential of the extract but also suggests that growth and developmental mechanisms in the treated animals were not hampered. Since treatment-related toxicity was not evidenced during the acute toxicity evaluation, further testing was conducted to evaluate the 28-day repeated daily dose of the extract on key metabolic markers of rats. This was done with a view to providing comprehensive toxicological data on this emerging botanical. The selected doses (75, 150, and 300 mg/kg b.w.) in this study were informed by the averages of daily consumed regimens and reported pharmacological study on the extract [ 6 ]. Generally, the fact that 28-day daily dose treatment with the extract elicited no clinical signs of toxicity, morbidity, or mortality across all the treatment groups may be a tenable inference that the extract of M. angustifolia is unlikely to be toxic at the tested doses over the observation period. Investigation on the haematological parameters can be used to determine the extent of the deleterious effect of foreign compounds in plant extracts on the blood constituents of an animal [ 16 ]. In this study, the nonsignificant difference in RBC and HGB counts following repeated daily dose treatment with the extract could be an indication that it may not be toxic to the blood. This implies that the morphology and osmotic fragility of the RBC, as well as HGB incorporation into the RBC, were not affected. This may also suggest that the oxygen-carrying capacity of the blood and amount of oxygen delivered to the tissues following treatment with the extract are intact [ 17 ]. Evaluation of indices (HCT, MCV, MCH, MCHC, and RCDW) relating to the status of RBC is imperative to the diagnosis of anaemia in animals [ 17 ]. The nonsignificant effect on these indices for the extract-treated animals relative to control may be an indication that the extract at the tested doses had no overall adverse effect on RBCs' microcytes and HGB weight per RBC. This suggests that the 28-day daily oral dose treatment with the extract does not predispose the animals to anaemic condition. Our submission is consistent with the findings of Ashafa and Olunu [ 18 ], where the administration of ethanolic root extract of M. lucida was nonhaematotoxic to the animals. The plasma level of the WBC counts is a pointer to an organism's defensive potential against infections. The dose-dependent significantly increased WBC counts following M. angustifolia extract administration in the animals may indicate immune system boost. The increase in the white blood cells could also suggest that the effects on cells of the immune system at the tested doses were not adversely affected and further supported the nonhaematotoxic nature of the extract. This agrees with the report of Ashafa and Kazeem [ 19 ], who gave similar submission on administration of Dianthus basuticus on leukocyte status of rats. That the extract also dose-dependently increased platelet counts in the animals over the 28-day investigation period may be informative of its stimulatory effect on thrombopoietin. This is not only suggestive of the extract's capability to considerably manage thrombocytopenia in rats (Geddis [ 20 ]), but also indicative of its unlikely toxicity. Liver and kidney function tests are crucial in toxicological evaluation of plant extracts due to the utmost involvement of these organs in xenobiotic biotransformation [ 6 ]. Significantly increased serum activities of ALP, ALT, AST, and GGT are closely associated with hepatic injury [ 21 ]. The nonsignificant differences in the serum activities of these marker enzymes in the extract-treated rats relative to normal control are informative either of the fact that the extract does not impede hepatocytes function in the rats or of the fact that the integrity of the liver cells was not compromised. Additionally, concentrations of total protein, bilirubin, and albumin in the serum may indicate the state of the liver and the type of damage. The nonsignificant effect of the extract on these parameters further confirms that it is unlikely to be hepatotoxic. Kidney damage may be ascertained by measurements of urea, uric acid, creatinine, and electrolytes, and deviations from normal in their serum concentrations are a tentative pointer to nephrotic injury [ 22 ]. In this study, the nonsignificant difference observed in the kidney function indices in the extract-treated animals is suggestive of normal renal function and further supports the nontoxic tendency of the extract. A drastic change in body weight is a critical evaluator of toxicity and may serve as a sensitive indication of the general wellbeing of animals [ 23 ]. The mean body weight gained by the animals in all the treatment groups may be an indication that the extract did not interfere with their normal metabolism as closely supported by the nonsignificant difference in this parameter when compared with the control group. The increase in body weight could be attributed to the nutritive components in their feed and the palatability of the extract [ 24 ]. Organ-body weight ratio may indicate organ swelling, atrophy, or hypertrophy [ 25 ]. In this study, the nonsignificant changes in the weights of the liver and kidneys suggest that these organs neither were adversely affected nor produced treatment-related/clinical signs of toxicity throughout the treatment period with the extract. However, the observed increase in the absolute liver-body weight ratio in the rats given 300 mg/kg b.w. dose of the extract could be regarded as being toxicologically and biochemically insignificant as it was not corroborated by both the histological examination and other clinical biochemical parameters of liver function evaluated in this study. Besides complementing biochemical investigations, histological examination of organs following exposure to pharmacological agents is an important consideration in assessing the safety of such agents on organ injury [ 17 ]. Hence, the apparently preserved histoarchitectural features as evident from microscopic examinations of the kidneys and liver sections of the extract-treated animals in this study are another supportive fact that the organs were void of injuries and further indicate that M. angustifolia extract was not toxic to them at the tested doses. Furthermore, the no treatment-induced infiltration and inflammation as shown in the microscopic examination of the organs from extract-treated groups are also supportive of its capability to maintain and sustain histoarchitectural integrity of the organs. In light of the foregoing, it may be inferred that the extract of M. angustifolia is unlikely to be toxic (at the investigated doses) to the studied organs in this study and also supported its pharmacological significance. 5. Conclusion Overall, it is evident from the present study that LD 50 of the standardized extract of M. angustifolia is well above 5000 mg/kg b.w. in Wistar rats. Following its 28-day repeated daily oral dose administration in the animals, it may be concluded that it does not elicit any treatment-related adverse effect at the doses investigated and thus may be classified to be relatively safe and practically nontoxic for consumption. Although work is in progress in evaluating its safety tendency on other systemic organs and tissues in animals, the data from this study have supported the safety and therapeutic importance of M. angustifolia in the traditional system of medicine. Competing Interests The authors declare that they have no competing interests regarding the publication of this paper.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5404255/

Taiwan's Experience in Hospital Preparedness and Response for Emerging Infectious Diseases

The Communicable Disease Control Medical Network (CDCMN), established in 2003 after the SARS outbreak in Taiwan, has undergone several phases of modification in structure and activation. The main organizing principles of the CDCMN are centralized isolation of patients with severe highly infectious diseases and centralization of medical resources, as well as a network of designated regional hospitals like those in other countries. The CDCMN is made up of a command system, responding hospitals, and supporting hospitals. It was tested and activated in response to the H1N1 influenza pandemic in 2009-10 and the Ebola outbreak in West Africa in 2014-2016, and it demonstrated high-level functioning and robust capacity. In this article, the history, structure, and operation of the CDCMN is introduced globally for the first time, and the advantages and challenges of this system are discussed. The Taiwanese experience shows an example of a collaboration between the public health system and the medical system that may help other public health authorities plan management and hospital preparedness for highly infectious diseases. Establishment and History During the SARS outbreak in Taiwan, several hospitals were forced to close because of nosocomial clusters of infections. Some hospitals started to refuse to take in patients with similar and suspected syndromes. Meanwhile, the public lost confidence in the medical system, and many people avoided seeking medical attention despite developing symptoms. It is estimated that outpatient visits were reduced by 14% in total and by 30% in public hospitals, 10 indicating that public hospitals bore the brunt of this loss of trust. Furthermore, because of patient referral procedures at that time, the referral system was disorganized, which may have expanded possible transmission to other hospitals, making the situation even worse. The Department of Health (now the Ministry of Health and Welfare) established a SARS Coordination Center to integrate resources and coordinate the academic, medical, and private sectors to combat SARS. 11 Under the command structure, an infection prevention network was organized, comprised of 12 designated SARS treatment hospitals located in northern, central, southern, and eastern regions. A funded infection prevention network with 6 regions (Taipei, north, central, south, Kao-Ping, and east) was approved and incorporated into the plans for Post-SARS Reconstruction and the 2005-2008 Biological Defense against Emerging Infections, establishing a permanent strategy of graded medical treatment. 12 In each region, a commander and a deputy commander were selected to oversee the coordination and operation of responding hospitals (at the time they were called infectious disease hospitals), and a consulting committee composed of epidemiologists, lab researchers, medical workers, hospital managers, and jurists was set up to advise on policy formulation. A command center would be activated depending on the epidemic emergency and would be staffed by the local health bureaus, medical centers, and other coordinating organizations. 12 From 2004 to 2013, the number of responding hospitals varied from 22 to 25 in 6 regions, with another 19 supporting hospitals serving as backup for medical resources and manpower. Nearly 400 negative pressure isolation wards and nearly 200 isolation wards were ready for patients with emerging infectious diseases. In terms of the activation mechanism, reforms were implemented in 3 phases ( Table 1 ). In phase 1 (July 2003-March 2004), 22 responding hospitals—initially based on the 12 designated SARS treatment hospitals and 10 other public health hospitals affiliated with the Department of Health—were divided into 3 categories based on disease and hospital capacity, and they were responsible for patient management of specific communicable diseases. 13 In addition, based on different scenarios of outbreak scale and disease type, some of the 22 hospitals could be activated for additional diseases. Table 1. Summary of 4 Phases of Communicable Disease Control Medical Network from 2003 to 2016 Categories of Hospitals Number of Responding Hospitals Activation Tiers Phase 1 (Jul 2003-Mar 2004) 3 categories of hospitals: 4 levels Special treatment center 2 Category I responding hospitals 7 Category II responding hospitals 13 Phase 2 (Apr 2004-Dec 2004) 3 categories of hospitals: a 3 levels National treatment center 2 Regional responding hospitals 6 Local responding hospitals 17 Phase 3 2005-2008 4 categories of hospitals: a 2005 3 levels National treatment center 2 2006-2008 cross-regional activation Regional responding hospitals 6 Local responding hospitals 18 Supporting hospitals 19 2008-present 2 categories of hospitals Year 2008-2012 Year 2013 Year 2014-Now cross-regional activation Responding hospitals 25 22 6 Supporting hospitals 19 17 6 a In phases 2 and 3, the total numbers of responding hospitals are 24 and 25 separately in 25 administrative districts, with 1 treatment center designated as both national and regional responding hospital. Among 25 administrative districts, 2 districts in phase 2 and 1 district in phase 3 did not have responding hospitals and instead coordinated with neighboring responding hospitals for geographic and traffic factors. During phases 2 and 3, the selection of the responding hospital candidates was handed over to local health authorities, who took into consideration factors such as local geography and traffic when designating responding hospitals. In phase 2 (April 2004-December 2004), the categorization framework was modified to a tiered approach of responding hospitals, including 2 national treatment hospitals, 6 regional hospitals, and 17 local hospitals. In phase 3, starting in 2006 and continuing through the present, the tiered approach was adjusted to incorporate cross-regional cooperation. 13 In accordance with cross-regional activation, during an epidemic regions in the network are activated depending on the outbreak location, and then they coordinate with and support one another with medical resource allocation Since the completion of phase 3, Taiwan has continued to adjust the number and composition of its responding hospitals. In 2008, the responding hospitals were no longer categorized as national, regional, and local, because in the event of cross-regional activation it is more efficient and flexible to launch a responding hospital where an epidemic occurs, as shown by the H1N1 influenza pandemic. In addition, considering the continuously improving healthcare system in Taiwan, all the responding hospitals were able to take in patients with highly infectious diseases and rapidly activate the emergency response plan, regardless of the category level. The tiered system of responding hospitals was determined to be impractical and was abandoned. Soon after a system review in 2012, an expert consultation meeting held by Taiwan CDC decided to gradually adjust the operation and the reimbursement scheme of CDCMN. In 2013, the number of responding hospitals decreased to 22 based on improved hospital capacity and readiness for highly infectious diseases and national-level budget constraints. In 2014, the number of regional responding hospitals was further decreased to 6 (1 per region); however, hospitals that were no longer regional responding hospitals could be designated as local responding hospitals and funded by the local public health bureaus. In 2007, the infection prevention network was formalized and renamed the Communicable Disease Control Medical Network. This ensured that the activation of a command center, the assignment of a commander and a deputy commander, the selection process of responding hospitals (renamed from infectious disease hospitals), the allocation of resources and staffing, and the hospital response and preparedness work are regulated to effectively act to ensure disease control and patient treatment. The current elements of CDCMN are described below. Framework Since 2013, the CDCMN has gradually adjusted the number of responding hospitals. In addition, the operation and reimbursement scheme was decided by a comprehensive review and discussion meeting that included attendees from local health bureaus, commanders and deputy commanders in each region, and policy officers from Taiwan CDC in 2012. Now 3 core elements comprise the medical network: (1) a command system, (2) responding hospitals, and (3) supporting hospitals ( Figure 1 ). The network is divided into 6 regions, and 1 responding hospital and 1 supporting hospital are designated in each region. Figure 1. Conceptual Framework of the Communicable Disease Control Medical Network Command System One commander and 1 deputy commander are assigned by the Ministry of Health and Welfare in each network region. They review relevant plans of communicable disease control, and they supervise and provide consultation to local health bureaus. During an outbreak, the command center leads case investigation; infection control and patient transport in medical facilities; coordination; expropriation; requisition; and allocation of hospitals, hospital beds, and manpower. Responding and Supporting Hospitals At the local level, public health bureaus may designate isolation hospitals based on the specific condition, distribution of medical care facilities, and hospital capacities. From these, 1 local responding hospital is designated for specific disease control needs. Currently there are 134 isolation hospitals, including 21 local responding hospitals. At the national level, Taiwan CDC designates 6 regional responding hospitals in 6 regions based on the isolation hospital list and a SWOT (strengths, weaknesses, opportunities, and threats) analysis of candidates. In addition, 3 other local responding hospitals in off-island areas receive funds from the Taiwan CDC to tackle health discrepancies in demographic distribution and insufficient medical resources. Table 2 shows general groups of the regional responding hospitals, supporting hospitals, and the number of local responding hospitals in each region. The central and local responding hospitals may receive subsidies from the Taiwan CDC and public health bureaus, respectively, on personnel training and drills and procurement and maintenance costs for facilities and equipment of isolation wards. Table 2. Regional Responding Hospitals, Supporting Hospitals, and Local Responding Hospitals in Communicable Disease Control Medical Network Regions Responding Hospital/Supporting Hospital Local Responding Hospital Taipei Metropolitan Heping Hospital a (Taipei City Hospital)/ National Taiwan University Hospital • Heping Branch, Taipei City Hospital a • Sanchung Branch, New Taipei City Hospital • Keelung Hospital b • Yilan Branch, National Yang-Ming University Hospital • Kimen Hospital b • Lienchiang County Hospital Northern Sinwu Branch, Taoyuan Hospital a , b / Linkou Chang Gung Memorial Hospital • Sinwu Branch, Taoyuan Hospital a , b • Hsinchu Branch, National Taiwan University Hospital • Chutung Branch, National Taiwan University Hospital • Miao-Li Hospital b Central Taichung Hospital a , b / China Medical University Hospital 1. Taichung Hospital a , b 2. Changhua Hospital b 3. Nantou Hospital b Southern Tainan Hospital a , b / National Cheng Kung University Hospital 1. Yunlin Branch, National Taiwan University Hospital 2. Chiayi Hospital b 3. Chiayi Chang Gung Memorial Hospital 4. Tainan Hospital a , b Kao-Ping Pingtung Hospital a , b / Kaohsiung Medical University Chung-Ho Memorial Hospital 1. Pingtung Hospital a , b 2. Penghu Branch, Tri-Service General Hospital Eastern Hualien Hospital a , b / Hualien Tzu Chi Hospital 1. Hualien Hospital a , b 2. Taitung Hospital b Appointed/requisitioned places Depending on the outbreak scale and risk assessment, by regulation the commander of the Central Epidemic Command Center shall decide the launch of additional appointed/requisitioned places for quarantine or other public health purposes. a Designated as both regional and local responding hospitals. b Hospital affiliated with the MOHW. The regional and local hospitals are responsible for taking in patients with category I and V communicable diseases (eg, smallpox, plague, rabies, novel influenza, MERS, Ebola, and other hemorrhagic fevers). Each regional responding hospital has qualified negative pressure isolation wards, with 2 to 4 beds per million population and 2 beds in off-island areas. This is standard practice in line with Japan, Singapore, and Hong Kong. Facilities and negative pressure equipment in regional responding hospitals are inspected and validated annually. Every responding hospital is required to formulate an emergency response plan for emerging infectious diseases. Plans should include the structure of the command and response task force, reporting procedures and information management, patient transport and care, medical personnel safety measures, environment maintenance, infection control, and risk assessment. Drills and training courses are held to strengthen knowledge and clinical skills of healthcare workers and disease control personnel. 14 In each region, 1 supporting hospital is designated from regional medical centers. Supporting hospitals are responsible for offering medical consultations to support the responding hospitals and serve as a back-up for manpower and medical resources during the period of outbreak. Healthcare workers in supporting hospitals offer professional consultations during ordinary times, while during an outbreak they are dispatched by the regional commander for medical care assistance. 14 Emergency Response Plan Depending on the scale of the outbreak, a tiered activation process of a regional responding hospital is further defined as the initial launch of isolation wards (including negative pressure wards), the floor evacuation, the building evacuation, and the whole hospital evacuation ( Figure 2 ). Once the evacuation is activated, patients without the outbreak disease will be evacuated and transferred to other hospitals to allow the responding hospital to take in patients with category I and V communicable diseases. If the outbreak expands further, the commander may appoint isolation hospitals or requisition medical facilities at various levels to take in priority patients with emergency or outbreak diseases. The regional commander may request cross-regional assistance as well. Figure 2. Activation Procedure of a Regional Responding Hospital Upon the order of the regional commander, medical facilities transfer infected patients to the regional responding hospital or other appointed isolation hospitals. For off-island areas, patient transport is divided into 2 options to be decided by the commander, based on the evaluation of the case status, the outbreak situation, hospital capacity, risk of transport, and other administrative factors. One option is to dispatch the support workforce to the local responding hospital in off-island areas where a patient could be treated directly. The other option is to transfer the patient by airplane to a regional responding hospital. However, the former option would be costly for the transport of the support team, and the latter option could increase the risk of transmission en route. Therefore, the patient's status and the need for the patient's advanced medical care are 2 major factors that the commander evaluates and decides for best patient care. Workforce In addition to healthcare workers in responding and supporting hospitals, a list of support healthcare workers, including physicians, nurses, respiratory therapists, radiographers, psychiatrists, medical technologists, and pharmacists, is made and updated regularly by local health bureaus. The support workforce in each region should be equivalent to 30% of the healthcare staffing in responding hospitals. 15 This ensures that in the event of an outbreak, surge capacity is available. Furthermore, frontline healthcare workers from local health centers or clinics may be requisitioned to expand workforce capacity. All healthcare workers on the support list are required to participate in training courses, personal protective equipment (PPE) donning and doffing exercises, and annual simulation drills held by responding and supporting hospitals to ensure safety and enhance willingness to serve. Command System One commander and 1 deputy commander are assigned by the Ministry of Health and Welfare in each network region. They review relevant plans of communicable disease control, and they supervise and provide consultation to local health bureaus. During an outbreak, the command center leads case investigation; infection control and patient transport in medical facilities; coordination; expropriation; requisition; and allocation of hospitals, hospital beds, and manpower. Responding and Supporting Hospitals At the local level, public health bureaus may designate isolation hospitals based on the specific condition, distribution of medical care facilities, and hospital capacities. From these, 1 local responding hospital is designated for specific disease control needs. Currently there are 134 isolation hospitals, including 21 local responding hospitals. At the national level, Taiwan CDC designates 6 regional responding hospitals in 6 regions based on the isolation hospital list and a SWOT (strengths, weaknesses, opportunities, and threats) analysis of candidates. In addition, 3 other local responding hospitals in off-island areas receive funds from the Taiwan CDC to tackle health discrepancies in demographic distribution and insufficient medical resources. Table 2 shows general groups of the regional responding hospitals, supporting hospitals, and the number of local responding hospitals in each region. The central and local responding hospitals may receive subsidies from the Taiwan CDC and public health bureaus, respectively, on personnel training and drills and procurement and maintenance costs for facilities and equipment of isolation wards. Table 2. Regional Responding Hospitals, Supporting Hospitals, and Local Responding Hospitals in Communicable Disease Control Medical Network Regions Responding Hospital/Supporting Hospital Local Responding Hospital Taipei Metropolitan Heping Hospital a (Taipei City Hospital)/ National Taiwan University Hospital • Heping Branch, Taipei City Hospital a • Sanchung Branch, New Taipei City Hospital • Keelung Hospital b • Yilan Branch, National Yang-Ming University Hospital • Kimen Hospital b • Lienchiang County Hospital Northern Sinwu Branch, Taoyuan Hospital a , b / Linkou Chang Gung Memorial Hospital • Sinwu Branch, Taoyuan Hospital a , b • Hsinchu Branch, National Taiwan University Hospital • Chutung Branch, National Taiwan University Hospital • Miao-Li Hospital b Central Taichung Hospital a , b / China Medical University Hospital 1. Taichung Hospital a , b 2. Changhua Hospital b 3. Nantou Hospital b Southern Tainan Hospital a , b / National Cheng Kung University Hospital 1. Yunlin Branch, National Taiwan University Hospital 2. Chiayi Hospital b 3. Chiayi Chang Gung Memorial Hospital 4. Tainan Hospital a , b Kao-Ping Pingtung Hospital a , b / Kaohsiung Medical University Chung-Ho Memorial Hospital 1. Pingtung Hospital a , b 2. Penghu Branch, Tri-Service General Hospital Eastern Hualien Hospital a , b / Hualien Tzu Chi Hospital 1. Hualien Hospital a , b 2. Taitung Hospital b Appointed/requisitioned places Depending on the outbreak scale and risk assessment, by regulation the commander of the Central Epidemic Command Center shall decide the launch of additional appointed/requisitioned places for quarantine or other public health purposes. a Designated as both regional and local responding hospitals. b Hospital affiliated with the MOHW. The regional and local hospitals are responsible for taking in patients with category I and V communicable diseases (eg, smallpox, plague, rabies, novel influenza, MERS, Ebola, and other hemorrhagic fevers). Each regional responding hospital has qualified negative pressure isolation wards, with 2 to 4 beds per million population and 2 beds in off-island areas. This is standard practice in line with Japan, Singapore, and Hong Kong. Facilities and negative pressure equipment in regional responding hospitals are inspected and validated annually. Every responding hospital is required to formulate an emergency response plan for emerging infectious diseases. Plans should include the structure of the command and response task force, reporting procedures and information management, patient transport and care, medical personnel safety measures, environment maintenance, infection control, and risk assessment. Drills and training courses are held to strengthen knowledge and clinical skills of healthcare workers and disease control personnel. 14 In each region, 1 supporting hospital is designated from regional medical centers. Supporting hospitals are responsible for offering medical consultations to support the responding hospitals and serve as a back-up for manpower and medical resources during the period of outbreak. Healthcare workers in supporting hospitals offer professional consultations during ordinary times, while during an outbreak they are dispatched by the regional commander for medical care assistance. 14 Emergency Response Plan Depending on the scale of the outbreak, a tiered activation process of a regional responding hospital is further defined as the initial launch of isolation wards (including negative pressure wards), the floor evacuation, the building evacuation, and the whole hospital evacuation ( Figure 2 ). Once the evacuation is activated, patients without the outbreak disease will be evacuated and transferred to other hospitals to allow the responding hospital to take in patients with category I and V communicable diseases. If the outbreak expands further, the commander may appoint isolation hospitals or requisition medical facilities at various levels to take in priority patients with emergency or outbreak diseases. The regional commander may request cross-regional assistance as well. Figure 2. Activation Procedure of a Regional Responding Hospital Upon the order of the regional commander, medical facilities transfer infected patients to the regional responding hospital or other appointed isolation hospitals. For off-island areas, patient transport is divided into 2 options to be decided by the commander, based on the evaluation of the case status, the outbreak situation, hospital capacity, risk of transport, and other administrative factors. One option is to dispatch the support workforce to the local responding hospital in off-island areas where a patient could be treated directly. The other option is to transfer the patient by airplane to a regional responding hospital. However, the former option would be costly for the transport of the support team, and the latter option could increase the risk of transmission en route. Therefore, the patient's status and the need for the patient's advanced medical care are 2 major factors that the commander evaluates and decides for best patient care. Workforce In addition to healthcare workers in responding and supporting hospitals, a list of support healthcare workers, including physicians, nurses, respiratory therapists, radiographers, psychiatrists, medical technologists, and pharmacists, is made and updated regularly by local health bureaus. The support workforce in each region should be equivalent to 30% of the healthcare staffing in responding hospitals. 15 This ensures that in the event of an outbreak, surge capacity is available. Furthermore, frontline healthcare workers from local health centers or clinics may be requisitioned to expand workforce capacity. All healthcare workers on the support list are required to participate in training courses, personal protective equipment (PPE) donning and doffing exercises, and annual simulation drills held by responding and supporting hospitals to ensure safety and enhance willingness to serve. Response to H1N1 Pandemic, 2009-10 As H1N1 influenza emerged in Mexico and the United States in late April 2009 and the WHO announced a phase 4 global influenza pandemic, the Central Epidemic Command Center (CECC) for H1N1 influenza was established at the level of Executive Yuan. 16 , 17 H1N1 novel influenza was listed as a Category I notifiable disease, which meant that all patients under investigation for H1N1 were prioritized to be treated in responding hospitals. 17 , 18 In the early phase, 25 responding hospitals were notified to be "ready for response." These hospitals were required to report back to the CECC on the results of inspections of negative pressure isolation wards, manpower mobilization, training and drill plans, PPE stockpile status, and transport procedures. As the epidemic developed, the regional commanders were authorized to coordinate and organize isolation hospitals and negative pressure isolation wards in regulating regions. In late May 2009, soon after several domestic cases were reported, the CECC commander decided to launch 4 responding hospitals (at the activation level of building evacuation) to admit and treat patients confirmed with H1N1 influenza. 17 , 18 As the WHO announced that the pandemic was of moderate severity, and the major strategy changed from containment to mitigation, the CECC decided to remove H1N1 novel influenza from the notifiable disease list, and patients were no longer placed under compulsory isolation. They could also seek medical attention at clinics or emergency departments and directly receive treatment. In July 2009, the CECC integrated the emergency medical services, the CDCMN, and medical institutions contracted with National Health Insurance. The regional commanders of CDCMN were further authorized to mobilize medical resources in the integrated system and to provide complicated cases with adequate treatment. 16-18 During the H1N1 pandemic, the average utilization rate of negative pressure isolation beds was around 40.6%. 17 The mortality of H1N1 influenza in Taiwan was 1.8 per million. 18 This was the third lowest mortality rate compared to other Organization for Economic Co-operation and Development (OECD) members ( Figure 3 ) and one-fifth of that in the United States, 16 , 18 indicating the epidemic was well controlled and the medical network operated robustly. Figure 3. Mortality Rate of H1N1 Influenza in Taiwan and OECD members (edited and adapted from Taiwan's Response to the H1N1 Influenza Pandemic 15 ) Response to Ebola in West Africa, 2014-2016 In the early phase of the Ebola outbreak in West Africa, regional responding hospitals were instructed to be aware of patients who had a travel history to Ebola-affected countries and to enhance infection prevention and control measures. As soon as the WHO declared the Ebola outbreak a public health emergency of international concern (PHEIC) on August 8, 2014, 19 the Taiwan CDC consulted with regional commanders and deputy commanders to establish an emergency response task force and strengthen 4 areas: health education, quarantine control, hospital preparedness, and international cooperation. 20 , 21 In light of reported Ebola cases among healthcare workers in Spain and the United States, all regional-level hospitals, medical centers, and responding hospitals in the CDCMN were requested to hold training and drills on proper donning and doffing of PPE. 20 Furthermore, a series of conference calls were held by the Taiwan CDC, covering (1) the emergency response plan for managing a patient with Ebola and other response efforts with medical directors in regional responding hospitals; (2) public health measures with local health bureaus; and (3) issues of healthcare personnel safety with relevant medical associations. Meanwhile regional and off-island responding hospitals were prioritized to receive specific types of PPE procured by the Taiwan CDC, in accordance with the WHO and the US CDC's guidance. No-notice inspections were also performed in regional hospitals and medical centers to understand current infection control measures and collection of travel history, occupation, contact history, and cluster information in emergency departments. 22 The inspection showed rapid patient management and transfer flow; good infection control, with physicians and other medical staff dressed in full PPE; and timely reporting to local health authorities. Other areas, such as the waiting time to access isolation wards, and collection of travel history, occupation, contact history, and cluster information in emergency departments, needed improvement. 22 In total, 6 suspected cases (none confirmed) were reported in Taiwan before the WHO announced the Ebola outbreak over. Discussion Commonalities with the US System After 2 American nurses contracted Ebola from an imported case in a hospital in Texas, the US CDC and the secretary for preparedness and response (ASPR) of the Department of Health and Human Services (HHS) recognized that not all hospitals have the same capacity to manage Ebola patients. A 5-year 3-tiered hospital program for Ebola and other highly infectious diseases was announced in December 2014. 23 , 24 In addition, the National Ebola Training and Education Center (NETEC) was established in 2015, which recruits professionals with Ebola experience to develop training courses and assess the preparedness of designated hospitals. 24 , 25 This new system shares many elements with the CDCMN in Taiwan. For instance, both use a centralized patient isolation approach and designate hospitals with stronger capacity, personnel training, and adequate facility and resources. In addition, the Center for Infectious Disease Control and Prevention in Taiwan was established in 2004 as an avenue for training and drills in public health, medicine, disease control, and anti-bioterrorism, which is similar to the NETEC. Strengths The Communicable Disease Control Medical Network has been tested and demonstrated to be flexible and have robust capacity in responding to epidemics over the past several years. The structure and activation mechanism has evolved over time. In 2012, a comprehensive review of the operation of the CDCMN was conducted. This system has several strengths, which should be maintained. The command system as well as the responding and supporting hospitals, are empowered legally to effectively activate and coordinate isolation wards, medical resources, and manpower as needed during the early phase of an outbreak. In addition, the command structure connects public health authorities and the medical system to integrate medical resources and share information with coordinating partners. After the SARS outbreak, awareness of healthcare workers' safety was raised. Currently, most healthcare workers exercise good infection prevention and control practices. Furthermore, emergency response plans in responding hospitals are in place, and the inspection of negative pressure isolation wards is conducted annually. In terms of the enhancement of healthcare workers' knowledge and clinical skills, systematic health personnel training and drills have been regularly performed in responding hospitals. Challenges There are other areas of challenges where improvement is needed. First, the role of the local public health authorities in the CDCMN is ambiguous in the command system, and their responsibilities should be further clarified. Second, the government funds for CDCMN have been reduced, even though facilities and equipment need to be renewed and replaced after 14 years of operation. Also, since there are only a few confirmed cases with highly infectious disease treated in negative pressure isolation wards, the wards have rarely been used. However, maintenance of the negative pressure isolation wards is costly. As a result, economic factors may have reduced responding hospitals' willingness to be in the network. In a post-Ebola time, training programs of comprehensive core clinical and infection control skills, such as hands-on practices while wearing PPE and mental health and behavior changes, should be further developed with reference to the WHO and other countries' guidance in response to future emerging disease epidemics. In addition, strategies are needed to incentivize participation of frontline medical workers to ensure a robust response team. Further, fair risk compensation payments for caring for highly infectious diseases by healthcare workers is still a topic of debate despite existing regulations governing the operation procedures and compensation for requisitioned health personnel. However, these have not been reviewed since the SARS outbreak. Changes As discussed previously, the number of responding hospitals evolved over time. The challenges of identifying a role for local public health bureaus in the CDCMN, using negative pressure isolation wards during nonemergency operations, and operating within a decreasing CDCMN budget resulted in a change in the number of regional responding hospitals, from 25 hospitals in 2012 to 22 hospitals in 2013 to 6 hospitals in 2014. This alteration enhanced the local public health bureaus' role in the hospital preparedness efforts by allowing them to identify and coordinate local responding hospitals and enabled local hospitals to use negative pressure isolation rooms during daily operations, which better justified the investment required to maintain them. In addition, the 6 regional responding hospitals received more CDCMN resources (previously diverted to 25 hospitals) to maximize the effectiveness. The changes create a more cost-effective network that still maintains flexibility and surge capacity during an epidemic. Although changes from 2013 to date showed improvement in the operation and budget allocation, current challenges, such as the cost for renewal and replacement of the medical equipment in responding hospitals, the development of integrated and comprehensive training courses of core skills for healthcare workers, and risk compensation payments, need the Taiwanese government's ongoing commitment and collaboration with medical partners to continue efforts in enhancing a more solid and effective system. Commonalities with the US System After 2 American nurses contracted Ebola from an imported case in a hospital in Texas, the US CDC and the secretary for preparedness and response (ASPR) of the Department of Health and Human Services (HHS) recognized that not all hospitals have the same capacity to manage Ebola patients. A 5-year 3-tiered hospital program for Ebola and other highly infectious diseases was announced in December 2014. 23 , 24 In addition, the National Ebola Training and Education Center (NETEC) was established in 2015, which recruits professionals with Ebola experience to develop training courses and assess the preparedness of designated hospitals. 24 , 25 This new system shares many elements with the CDCMN in Taiwan. For instance, both use a centralized patient isolation approach and designate hospitals with stronger capacity, personnel training, and adequate facility and resources. In addition, the Center for Infectious Disease Control and Prevention in Taiwan was established in 2004 as an avenue for training and drills in public health, medicine, disease control, and anti-bioterrorism, which is similar to the NETEC. Strengths The Communicable Disease Control Medical Network has been tested and demonstrated to be flexible and have robust capacity in responding to epidemics over the past several years. The structure and activation mechanism has evolved over time. In 2012, a comprehensive review of the operation of the CDCMN was conducted. This system has several strengths, which should be maintained. The command system as well as the responding and supporting hospitals, are empowered legally to effectively activate and coordinate isolation wards, medical resources, and manpower as needed during the early phase of an outbreak. In addition, the command structure connects public health authorities and the medical system to integrate medical resources and share information with coordinating partners. After the SARS outbreak, awareness of healthcare workers' safety was raised. Currently, most healthcare workers exercise good infection prevention and control practices. Furthermore, emergency response plans in responding hospitals are in place, and the inspection of negative pressure isolation wards is conducted annually. In terms of the enhancement of healthcare workers' knowledge and clinical skills, systematic health personnel training and drills have been regularly performed in responding hospitals. Challenges There are other areas of challenges where improvement is needed. First, the role of the local public health authorities in the CDCMN is ambiguous in the command system, and their responsibilities should be further clarified. Second, the government funds for CDCMN have been reduced, even though facilities and equipment need to be renewed and replaced after 14 years of operation. Also, since there are only a few confirmed cases with highly infectious disease treated in negative pressure isolation wards, the wards have rarely been used. However, maintenance of the negative pressure isolation wards is costly. As a result, economic factors may have reduced responding hospitals' willingness to be in the network. In a post-Ebola time, training programs of comprehensive core clinical and infection control skills, such as hands-on practices while wearing PPE and mental health and behavior changes, should be further developed with reference to the WHO and other countries' guidance in response to future emerging disease epidemics. In addition, strategies are needed to incentivize participation of frontline medical workers to ensure a robust response team. Further, fair risk compensation payments for caring for highly infectious diseases by healthcare workers is still a topic of debate despite existing regulations governing the operation procedures and compensation for requisitioned health personnel. However, these have not been reviewed since the SARS outbreak. Changes As discussed previously, the number of responding hospitals evolved over time. The challenges of identifying a role for local public health bureaus in the CDCMN, using negative pressure isolation wards during nonemergency operations, and operating within a decreasing CDCMN budget resulted in a change in the number of regional responding hospitals, from 25 hospitals in 2012 to 22 hospitals in 2013 to 6 hospitals in 2014. This alteration enhanced the local public health bureaus' role in the hospital preparedness efforts by allowing them to identify and coordinate local responding hospitals and enabled local hospitals to use negative pressure isolation rooms during daily operations, which better justified the investment required to maintain them. In addition, the 6 regional responding hospitals received more CDCMN resources (previously diverted to 25 hospitals) to maximize the effectiveness. The changes create a more cost-effective network that still maintains flexibility and surge capacity during an epidemic. Although changes from 2013 to date showed improvement in the operation and budget allocation, current challenges, such as the cost for renewal and replacement of the medical equipment in responding hospitals, the development of integrated and comprehensive training courses of core skills for healthcare workers, and risk compensation payments, need the Taiwanese government's ongoing commitment and collaboration with medical partners to continue efforts in enhancing a more solid and effective system. Conclusion This is the first time that the Taiwan CDC has shared its experience in constructing and operating the Communicable Disease Control Medical Network. The framework has been further empowered since its legislation in 2007, with an overarching structure divided into a command system, responding hospitals, and supporting hospitals. Over the past 14 years, the control strategies of the CDCMN have been tested during the H1N1 influenza pandemic, the H7N9 epidemic, Ebola in West Africa, and other outbreaks, demonstrating a high level of functioning and robust capacity. The command system also bridges the public health and medical systems to improve allocation of manpower and resources at the national and local levels. The Taiwan CDC will continue to maintain the key elements of the CDCMN and resolve challenges through continued work with hospitals, local health bureaus, medical associations, and other cooperating partners, in order to protect people from emerging infectious disease threats. As national and global progress is made toward building a safe and secure network to respond to infectious diseases, hospital preparedness work highlights the critical functions needed to identify, isolate, and respond rapidly and coordinate smoothly. At the international level, countries could help each other to strengthen and build a more resilient healthcare system. It would be of great value for the CDCMN partner in global alliance to develop a strong international partnership with other similar designated hospital frameworks in Japan, Europe, and the United States. In this way, information exchange, health and safety issues of medical workers, technical and clinical skills, the response team framework, response workforce and logistics, and other key topics can be discussed and reviewed by experienced professionals across the world. This could help hospital preparedness networks to become more efficient while maintaining core capacities in preparing for the next emerging health threat.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152105/

Infectious Disease Considerations for the Operating Room

Etiologic Agent The infectious vector may be any microorganism capable of causing infection. The pathogenicity is the ability to induce disease, which is characterized by its virulence (infection severity, determined by the germ morbidity and mortality rates) and the level of invasiveness (capacity to invade tissues). No microorganism is completely avirulent. An organism may have a very low level of virulence, but if the host (patient or health care provider) is highly susceptible, infection by the organism may cause disease. The risk of infection increases with the infecting dose (i.e., the number of organisms available to induce disease), the reservoir (i.e., the site where the organisms reside and multiply), and the infection source (i.e., the site from where it is transmitted to a susceptible host either directly or indirectly through an intermediary object). The infection source may be a human (e.g., health care providers, children, visitors, housekeeping personnel) with a symptomatic or an asymptomatic infection during the incubation period. The source may also be temporarily or permanently colonized (the most frequently colonized tissues are the skin and digestive and respiratory tracts). Host The presence of a susceptible host is an increasingly important element in the chain of infection that paradoxically results from advances in current medical therapies and technology (e.g., children undergoing organ transplantation, chemotherapy, or extremely premature neonates) and the presence of children with diseases that compromise their immune systems (e.g., acquired immunodeficiency syndrome [AIDS], tuberculosis, malnutrition, burns). The organism may enter the host through the skin, mucous membranes, lungs, gastrointestinal tract, genitourinary tract, or the bloodstream via intravenous solutions, following laryngoscopy, or from surgical wounds. Organisms may also infect the individual as a result of work accidents with cutting or piercing devices. The development of infection is influenced by the host defense mechanisms that may be classified as either nonspecific or specific: • Nonspecific defense mechanisms include the skin, mucous membranes, secretions, excretions, enzymes, inflammatory responses, genetic factors, hormonal responses, nutritional status, behavior patterns, and the presence of other diseases. • Specific defense mechanisms or immunity may occur as a result of exposure to an infectious agent (antibody formation) or through placental transfer of antibodies; artificial defense may be acquired through vaccines, toxoids, or exogenously administered immunoglobulins. Methods of Transmission Microorganisms are transmitted in the hospital environment through a number of different routes; the same microorganism may also be transmitted via more than one route. In the operating room, three main routes of transmission are possible: air, direct contact, and indirect contact. Air Transmission Airborne infections that may infect susceptible hosts are transmitted via two mechanisms: droplets and droplet nuclei. Droplets Droplet contamination is considered a direct transmission of organisms because there is a direct transfer of microorganisms from the colonized or infected person to the host. This generally occurs with particles whose diameters are greater than 5 μm that are expelled from an individual's mouth or nose, mainly during sneezing, coughing, or talking or during procedures such as suction, laryngoscopy, and bronchoscopy ( Fig. 50-2 ). Transmission occurs when the microorganism-containing droplets, expelled or shed by the infected person (source), are propelled a short distance (usually not exceeding 60 cm or about 3 feet through the air) and deposited on the host's conjunctivae or oral or nasal mucous membranes. When a person coughs, the exhaled air may reach a speed of up to 965 km/hr (600 mph). 5 However, because the droplets are relatively large they tend to descend quickly and remain suspended in the air for a very brief period, thus obviating the need for special handling procedures for the operating room air. Examples of droplet-borne diseases include influenza, respiratory syncytial virus, severe acute respiratory syndrome (SARS), and others commonly found in droplets from the respiratory tract. Figure 50-2 Droplets expelled during sneezing. (From www.vaccineinformation.org/photos/flu ) Droplet Nuclei Droplet nuclei result from the evaporation of droplets while suspended in the air. Unlike droplets, the nuclei have an outer layer of desiccated organic material and a very small diameter (1 to 5 μm) and remain suspended in air indefinitely. The microorganisms contained within these nuclei may be spread by air drafts over great distances, depending on the environmental conditions (dry and cold atmosphere, with limited or nonexistent exposure to sunlight favor the spreading). 6 In contrast to droplets, which are deposited on mucous membranes, the droplet nuclei may enter the susceptible host by inhalation; examples of droplet nuclei-borne diseases include tuberculosis, varicella, and measles. Contact Transmission Direct and indirect contacts are the most significant and frequent methods of hospital infection transmission. Direct Contact This type of disease transmission involves direct physical contact between two individuals. The physical transfer of microorganisms from an infected or colonized person to a susceptible host may occur from child to health care provider or from health care provider to the child during professional practice (e.g., venous cannulation, laryngoscopy, burn care, suction of secretions). Health care providers working in the operating room may be exposed to skin contamination by body fluids. This is an issue of grave concern because of the potential exposure of health care providers to patients with unrecognized infections, especially hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Hepatitis B is a highly infectious virus that requires a small amount of blood (10 −7 to 10 −9 mL) to transmit the disease. The incidence of skin contamination of anesthesiologists and related personnel by blood and saliva is quite high. One study examined 270 anesthetic procedures during 7 consecutive days. The blood of 35 patients (14%) contaminated the skin of 65 anesthesiologists in 46 incidents. Twenty-eight contamination events (61%) occurred during venous cannulation. Five of 65 anesthesiologists who had been contaminated by blood (8%) had cuts in the skin of their hands. 7 The importance of this observation is that seroconversion of health care providers has been reported after skin contamination by infected blood from HIV carriers 8 and HBV infection after blood splashing into health care workers' eyes. 9 Scabies, pediculosis, and herpes simplex are among the diseases most frequently transmitted by direct contact. 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 These studies explain why meticulous hand washing and routine use of barriers such as gloves and eye protection are such an important part of protecting ourselves from such exposures even during routine procedures such as starting an intravenous line or performing laryngoscopy. Indirect Contact Indirect contact involves the transmission of microorganisms from a source (animate or inanimate) to a susceptible host by means of a vehicle (e.g., an intermediary object) contaminated by body fluids. Tables 50-2 and 50-3 provide examples of diseases associated with bodily fluids to which health care workers may be exposed. The vehicle for transmission may be the hands of a health care provider who is not wearing gloves or a provider who fails to wash his or her hands between children. This type of contact can also come from health care providers who touch (with or without gloves) contaminated monitoring or other patient care devices (e.g., blood pressure cuffs, stethoscopes, electrocardiographic cables, ventilation systems [respirators, corrugated tubes, Y pieces, valves]), which are used with several children without proper cleaning or disinfection in between each use. 18 , 19 , 20 Table 50-2 Body Fluids and Diseases They May Transmit Body Fluid Diseased Transmitted Blood HBV, HIV, HCV, CMV, EBV, NANBH Seminal fluid HIV, HBV, CMV Vaginal discharge HIV, HBV, CMV Saliva and sputum HSV, TB, CMV, respiratory diseases Cerebrospinal fluid Encephalopathic organisms (see Table 50-5 ), HIV Breast milk HIV, HBV, CMV Urine CMV, EBV, HBV Feces and intestinal fluid HAV, gastrointestinal diseases (see Table 50-5 ) HBV, hepatitis B virus; HIV, human immunodeficiency virus; HCV, hepatitis C virus; CMV, cytomegalovirus; EBV, Epstein-Barr virus; NANBH, non-A non-B hepatitis; TB, tuberculosis; HAV, hepatitis A virus; HSV, herpes simplex types I and II. Modified with permission from Browne RA, Chenesky MA: Infectious diseases and the anaesthetist. Can J Anesth 1988; 35:655-665. © 2009 2009 Table 50-3 Infectious Agents That May Be Found in the Operating Room Viral Hepatitis Hepatitis A virus Hepatitis B virus Hepatitis C virus Delta hepatitis virus Non-A, non-B hepatitis Human immunodeficiency virus Cytomegalovirus Epstein-Barr virus Herpes simplex virus Respiratory Bacteria Streptococcus Pneumococcus Meningococcus Diphtheria Mycobacterium * Legionella * Fungi Candida * Nocardia * Cryptococcus * Parasites Pneumocystis * Viruses Rhinovirus Influenza Parainfluenza Adenovirus Respiratory syncytial virus Measles Rubella Cytomegalovirus * Gastrointestinal Viruses: hepatitis A virus, rotavirus, adenovirus, enterovirus Bacteria: Giardia, * Cryptosporidium, Isospora * Fungi: Candida * Central Nervous System Viruses: Human immunodeficiency virus, * herpes simplex virus, * Epstein-Barr virus * Parasites: Toxoplasma * Fungi: Cryptococcus * Opportunistic infections in immunocompromised patients, especially those with acquired immunodeficiency. Modified with permission from Browne RA, Chenesky MA: Infectious diseases and the anaesthetist. Can J Anesth 1988; 35:655-665. © 2009 2009 Although no definitive studies have demonstrated a cause-and-effect relationship in the transmission of infections by anesthesiologists or anesthetic staff, there are reports of elements, fomites, and drugs (mainly propofol) that have resulted in hospital-acquired infections. 11 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 However, many of the following situations could potentially cause an infection: • Up to 40% of the anesthetic equipment in the operating room that was in direct or indirect contact with the child (blood pressure cuffs, cables, oximeters, laryngoscopes, monitors, respirator settings and horizontal and vertical surfaces) may be contaminated with blood because of inadequate cleansing procedures between children. 2, 3, 18, 39, 40 • In some institutions, up to 8% of the Bain circuits that were reused without previous sterilization were contaminated. 41 • Contamination of syringe contents has occurred with glass particles during ampule opening, which in turn may compromise the sterility of the contents, presumably due to the passage of bacteria contained on glass particles into the solution. 42 , 43 , 44 • Intravenous tubing has a significant blood contamination rate as well as contamination by blood from syringes used to inject drugs. This can occur with the absence of visible blood reflux in the tubing or syringe. Changing a fresh needle to a syringe that will be reused is useless to prevent cross infection, thus emphasizing the importance of not using the same syringe on multiple patients. 45 • Refilling both glass and plastic syringes several times has been shown to result in contamination of the contents. 45, 46 • Some drug formulations, especially propofol, can sustain bacterial growth under certain conditions. Thus, great care should be given to aseptic technique when transferring drugs from the vial to a syringe and not allowing the syringe to remain unused for more than 4 hours. 47 , 48 , 49 , 50 , 51 • Needles that had been used for spinal or epidural anesthesia were found to be contaminated with coagulase-negative staphylococci (15.7%), yeasts (1.5%), enterococcus (0.8%), pneumococcus (0.8%), and micrococcus (0.8%), suggesting that despite standard skin preparation and cleansing there is a significant rate of needle contamination. 52 It is unclear whether these organisms produce an inoculation that could lead to an infection during neuraxial blockade. • There is a high incidence of skin contamination by blood and saliva of anesthetic personnel that occurs during routine anesthetic practice. 7 • Violations of contemporary guidelines for preventing infections (hand washing, wearing gloves, surgical masks, ocular protection, scrubs, syringe reutilization) by anesthesiologists are frequent. Anesthesia staff are aware of the potentially infectious working environment, but to a great extent (11%-99%) they do not implement protection measures for themselves or their patients. 18 , 53 , 54 , 55 Accidents with Cutting or Piercing Devices Percutaneous contamination as a result of a cutting or piercing accident is the most effective means to transmit blood-borne pathogens. Evidence suggests that this is the main route of HIV, HBV, and HCV 56, 57 infection, especially if injuries are caused by hollow-bore needles that were used to draw blood or introduce an intravenous line. 58, 59 Over 20 other blood-borne pathogens have been transmitted by this means, including those causing herpes, malaria, and tuberculosis. 60 The infectious risk after a percutaneous exposure to blood or body fluids from an HIV-positive person is 0.3%. Among health care providers lacking protective antibodies, the risk of HBV infection after an injury with a cutting or piercing device infected with hepatitis B antigen is approximately 37%; in the case of HCV it is 1.8% (0%-7%). Anesthesia staff lacking HBV protective antibodies are at high risk for acquiring the disease. 61, 62 These infection rates underscore the need for the use of "safe" needles and the need to advocate the use of "needleless" systems even though they are significantly more expensive. This also emphasizes the need for meticulous handling and disposal of needles and other sharp instruments as well as the use of special "sharps boxes" designed to minimize accidental needlesticks (e.g. "mail box" type boxes that do not allow the hand to enter the disposal area). 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 The U.S. Centers for Disease Control and Prevention (CDC) has estimated that in the United States there are approximately 385,000 cutting and piercing accidents annually among health care providers in hospitals; 25% of these occur in the operating room. 60 However, the actual prevalence is believed to be much greater because many of these events are unreported. The distribution of these accidents among anesthesiologists is shown in Figure 50-3A ; the distribution of the elements most frequently associated with cutting and piercing injuries in health care providers is shown in Figure 50-3B . Should such an accident occur (needle puncture, exposure to nonintact skin, mucous membrane exposure) there are now specific recommendations regarding immediate assessment of risk, assessment of the exposure source (chart review, inform the patient that an accident has occurred and ask permission to determine HBV, HCV, and HIV serologic tests) and initiation of appropriate treatment of the health care worker. It is advised to obtain as much information regarding the patient as possible if the patient is known, to obtain a sample of blood from the patient for determination of potential carrier state ( Table 50-4 ), and to report to the health service for immediate institution of prophylaxis and follow-up ( Table 50-5 ), especially for HIV exposure ( Tables 50-6 and 50-7 ). Figure 50-3 A , Percentage distribution of percutaneous injury in anesthesiologists caused by contaminated cutting or piercing devices. B , Percentage distribution of elements associated with percuta-neous injuries in health care work. Table 50-4 Recommendations for the Contents of the Occupational Exposure Report • Date and time of exposure • Details of the procedure being performed, including where and how the exposure occurred; if related to a sharp device, the type and brand of device; and how and when in the course of handling the device the exposure occurred • Details of the exposure, including the type and amount of fluid or material and the severity of the exposure; for example, for a percutaneous exposure, depth of injury and whether fluid was injected and for a skin or mucous membrane exposure, the estimated volume of material and the condition of the skin (e.g., chapped, abraded, intact) • Details about the exposure source (e.g., whether the source material contained hepatitis B virus, hepatitis C virus, or human immunodeficiency virus; if the source is infected with human immunodeficiency virus, the stage of disease, history of antiretroviral therapy, viral load, and antiretroviral resistance information, if known) • Details about the exposed person (e.g., hepatitis B vaccination and vaccine-response status) • Details about counseling, postexposure management, and follow-up http://www.cdc.gov/mmwr/PDF/rr/rr5011.pdf Updated U.S. Public Health Service guidelines for the management of occupational exposures to HBV, HCV, and HIV and recommendations for postexposure prophylaxis. MMWR Recomm Rep 2001; 50:1-52. © 2009 2009 Table 50-5 Factors to Consider in Assessing the Need for Follow-up of Occupational Exposures Type of Exposure Percutaneous injury Mucous membrane exposure Nonintact skin exposure Bites resulting in blood exposure to either person involved Type and Amount of Fluid/Tissue Blood Fluids containing blood Potentially infectious fluid or tissue (semen; vaginal secretions; and cerebrospinal, synovial, pleural, peritoneal, pericardial, and amniotic fluids) Direct contact with concentrated virus Infectious Status of Source Presence of HBsAg Presence of HCV antibody Presence of HIV antibody Susceptibility of Exposed Person Hepatitis B vaccine and vaccine response status HBV, HCV, and HIV immune status http://www.cdc.gov/mmwr/PDF/rr/rr5011.pdf Updated U.S. Public Health Service guidelines for the management of occupational exposures to HBV, HCV, and HIV and recommendations for postexposure prophylaxis. MMWR Recomm Rep 2001; 50:1-52 © 2009 2009 Table 50-6 Recommended HIV Postexposure Prophylaxis (PEP) for Percutaneous Injuries Infection Status of Source Exposure Type HIV-Positive Class 1 * HIV-Positive Class 2 * Source of Unknown HIV Status †Unknown Source ‡ HIV-Negative No PEP Less severe § Recommend basic 2-drug PEP Recommend expanded ≥3-drug PEP Generally, no PEP warranted; however, consider basic 2-drug PEP ‖ for source with HIV risk factors ¶ Generally, no PEP warranted; however, consider basic 2-drug PEP ‖ in settings in which exposure to HIV-infected persons is likely warranted More severe ** Recommend expanded 3-drug PEP Recommend expanded ≥3-drug PEP Generally, no PEP warranted; however, consider basic 2-drug PEP ‖ for source with HIV risk factors ¶ Generally, no PEP warranted; however, consider basic 2-drug PEP ‖ in settings in which exposure to HIV-infected persons is likely No PEP warranted * HIV-positive class 1: asymptomatic HIV infection or known low viral load (e.g., <1500 ribonucleic acid copies/mL). HIV-positive class 2: symptomatic HIV infection, acquired immunodeficiency syndrome, acute seroconversion, or known high viral load. If drug resistance is a concern, obtain expert consultation. Initiation of PEP should be delayed pending expert consultation, and because expert consultation alone cannot substitute for face-to-face counseling, resources should be available to provide immediate evaluation and follow-up care for all exposures. †For example, deceased source person with no samples available for HIV testing. ‡ For example, a needle from a sharps disposal container. § For example, solid needle or superficial injury. ‖ The recommendation "consider PEP" indicates that PEP is optional; a decision to initiate PEP should be based on a discussion between the exposed person and the treating clinician regarding the risks versus benefits of PEP. ¶ If PEP is offered and administered and the source is later determined to be HIV-negative, PEP should be discontinued. ** For example, large-bore hollow needle, deep puncture, visible blood on device, or needle used in patient's artery or vein. Updated U.S. Public Health Service guidelines for the management of occupational exposures to HBV, HCV, and HIV and recommendations for postexposure prophylaxis. MMWR Recomm Rep 2001; 50:1-52. Available at http://www.cdc.gov/mmwr/PDF/rr/rr5011.pdf © 2009 2009 Table 50-7 Recommended HIV Postexposure Prophylaxis (PEP) for Mucous Membrane Exposures and Nonintact Skin * Exposures Infection Status of Source Exposure Type HIV-Positive Class 1 †HIV-Positive Class 2 †Source of Unknown HIV Status ‡ Unknown Source § HIV-Negative Small volume ‖ Consider basic 2-drug PEP ¶ Recommend basic 2-drug PEP Generally, no PEP warranted ** Generally, no PEP warranted No PEP warranted Large volume ††Recommend basic 2-drug PEP Recommend expanded ≥3-drug PEP Generally, no PEP warranted; however, consider basic 2-drug PEP ¶ for source with HIV risk factors ** Generally, no PEP warranted; however, consider basic 2-drug PEP ¶ in settings in which exposure to HIV-infected persons is likely No PEP warranted * For skin exposures, follow-up is indicated only if evidence exists of compromised skin integrity (e.g., dermatitis, abrasion, or open wound). †HIV-positive class 1: asymptomatic HIV infection or known low viral load (e.g., <1500 ribonucleic acid copies/mL). HIV-positive class 2: symptomatic HIV infection, acquired immunodeficiency syndrome, acute seroconversion, or known high viral load. If drug resistance is a concern, obtain expert consultation. Initiation of PEP should be delayed pending expert consultation, and because expert consultation alone cannot substitute for face-to-face counseling, resources should be available to provide immediate evaluation and follow-up care for all exposures. ‡ For example, deceased source person with no samples available for HIV testing. § For example, splash from inappropriately disposed blood. ‖ For example, a few drops. ¶ The recommendation "consider PEP" indicates that PEP is optional; a decision to initiate PEP should be based on a discussion between the exposed person and the treating clinician regarding the risks versus benefits of PEP. ** If PEP is offered and administered and the source is later determined to be HIV-negative, PEP should be discontinued. ††For example, a major blood splash. Updated U.S. Public Health Service guidelines for the management of occupational exposures to HBV, HCV, and HIV and recommendations for postexposure prophylaxis. MMWR Recomm Rep 2001; 50:1-52. Available at http://www.cdc.gov/mmwr/PDF/rr/rr5011.pdf © 2009 2009 Air Transmission Airborne infections that may infect susceptible hosts are transmitted via two mechanisms: droplets and droplet nuclei. Droplets Droplet contamination is considered a direct transmission of organisms because there is a direct transfer of microorganisms from the colonized or infected person to the host. This generally occurs with particles whose diameters are greater than 5 μm that are expelled from an individual's mouth or nose, mainly during sneezing, coughing, or talking or during procedures such as suction, laryngoscopy, and bronchoscopy ( Fig. 50-2 ). Transmission occurs when the microorganism-containing droplets, expelled or shed by the infected person (source), are propelled a short distance (usually not exceeding 60 cm or about 3 feet through the air) and deposited on the host's conjunctivae or oral or nasal mucous membranes. When a person coughs, the exhaled air may reach a speed of up to 965 km/hr (600 mph). 5 However, because the droplets are relatively large they tend to descend quickly and remain suspended in the air for a very brief period, thus obviating the need for special handling procedures for the operating room air. Examples of droplet-borne diseases include influenza, respiratory syncytial virus, severe acute respiratory syndrome (SARS), and others commonly found in droplets from the respiratory tract. Figure 50-2 Droplets expelled during sneezing. (From www.vaccineinformation.org/photos/flu ) Droplet Nuclei Droplet nuclei result from the evaporation of droplets while suspended in the air. Unlike droplets, the nuclei have an outer layer of desiccated organic material and a very small diameter (1 to 5 μm) and remain suspended in air indefinitely. The microorganisms contained within these nuclei may be spread by air drafts over great distances, depending on the environmental conditions (dry and cold atmosphere, with limited or nonexistent exposure to sunlight favor the spreading). 6 In contrast to droplets, which are deposited on mucous membranes, the droplet nuclei may enter the susceptible host by inhalation; examples of droplet nuclei-borne diseases include tuberculosis, varicella, and measles. Droplets Droplet contamination is considered a direct transmission of organisms because there is a direct transfer of microorganisms from the colonized or infected person to the host. This generally occurs with particles whose diameters are greater than 5 μm that are expelled from an individual's mouth or nose, mainly during sneezing, coughing, or talking or during procedures such as suction, laryngoscopy, and bronchoscopy ( Fig. 50-2 ). Transmission occurs when the microorganism-containing droplets, expelled or shed by the infected person (source), are propelled a short distance (usually not exceeding 60 cm or about 3 feet through the air) and deposited on the host's conjunctivae or oral or nasal mucous membranes. When a person coughs, the exhaled air may reach a speed of up to 965 km/hr (600 mph). 5 However, because the droplets are relatively large they tend to descend quickly and remain suspended in the air for a very brief period, thus obviating the need for special handling procedures for the operating room air. Examples of droplet-borne diseases include influenza, respiratory syncytial virus, severe acute respiratory syndrome (SARS), and others commonly found in droplets from the respiratory tract. Figure 50-2 Droplets expelled during sneezing. (From www.vaccineinformation.org/photos/flu ) Droplet Nuclei Droplet nuclei result from the evaporation of droplets while suspended in the air. Unlike droplets, the nuclei have an outer layer of desiccated organic material and a very small diameter (1 to 5 μm) and remain suspended in air indefinitely. The microorganisms contained within these nuclei may be spread by air drafts over great distances, depending on the environmental conditions (dry and cold atmosphere, with limited or nonexistent exposure to sunlight favor the spreading). 6 In contrast to droplets, which are deposited on mucous membranes, the droplet nuclei may enter the susceptible host by inhalation; examples of droplet nuclei-borne diseases include tuberculosis, varicella, and measles. Contact Transmission Direct and indirect contacts are the most significant and frequent methods of hospital infection transmission. Direct Contact This type of disease transmission involves direct physical contact between two individuals. The physical transfer of microorganisms from an infected or colonized person to a susceptible host may occur from child to health care provider or from health care provider to the child during professional practice (e.g., venous cannulation, laryngoscopy, burn care, suction of secretions). Health care providers working in the operating room may be exposed to skin contamination by body fluids. This is an issue of grave concern because of the potential exposure of health care providers to patients with unrecognized infections, especially hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Hepatitis B is a highly infectious virus that requires a small amount of blood (10 −7 to 10 −9 mL) to transmit the disease. The incidence of skin contamination of anesthesiologists and related personnel by blood and saliva is quite high. One study examined 270 anesthetic procedures during 7 consecutive days. The blood of 35 patients (14%) contaminated the skin of 65 anesthesiologists in 46 incidents. Twenty-eight contamination events (61%) occurred during venous cannulation. Five of 65 anesthesiologists who had been contaminated by blood (8%) had cuts in the skin of their hands. 7 The importance of this observation is that seroconversion of health care providers has been reported after skin contamination by infected blood from HIV carriers 8 and HBV infection after blood splashing into health care workers' eyes. 9 Scabies, pediculosis, and herpes simplex are among the diseases most frequently transmitted by direct contact. 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 These studies explain why meticulous hand washing and routine use of barriers such as gloves and eye protection are such an important part of protecting ourselves from such exposures even during routine procedures such as starting an intravenous line or performing laryngoscopy. Indirect Contact Indirect contact involves the transmission of microorganisms from a source (animate or inanimate) to a susceptible host by means of a vehicle (e.g., an intermediary object) contaminated by body fluids. Tables 50-2 and 50-3 provide examples of diseases associated with bodily fluids to which health care workers may be exposed. The vehicle for transmission may be the hands of a health care provider who is not wearing gloves or a provider who fails to wash his or her hands between children. This type of contact can also come from health care providers who touch (with or without gloves) contaminated monitoring or other patient care devices (e.g., blood pressure cuffs, stethoscopes, electrocardiographic cables, ventilation systems [respirators, corrugated tubes, Y pieces, valves]), which are used with several children without proper cleaning or disinfection in between each use. 18 , 19 , 20 Table 50-2 Body Fluids and Diseases They May Transmit Body Fluid Diseased Transmitted Blood HBV, HIV, HCV, CMV, EBV, NANBH Seminal fluid HIV, HBV, CMV Vaginal discharge HIV, HBV, CMV Saliva and sputum HSV, TB, CMV, respiratory diseases Cerebrospinal fluid Encephalopathic organisms (see Table 50-5 ), HIV Breast milk HIV, HBV, CMV Urine CMV, EBV, HBV Feces and intestinal fluid HAV, gastrointestinal diseases (see Table 50-5 ) HBV, hepatitis B virus; HIV, human immunodeficiency virus; HCV, hepatitis C virus; CMV, cytomegalovirus; EBV, Epstein-Barr virus; NANBH, non-A non-B hepatitis; TB, tuberculosis; HAV, hepatitis A virus; HSV, herpes simplex types I and II. Modified with permission from Browne RA, Chenesky MA: Infectious diseases and the anaesthetist. Can J Anesth 1988; 35:655-665. © 2009 2009 Table 50-3 Infectious Agents That May Be Found in the Operating Room Viral Hepatitis Hepatitis A virus Hepatitis B virus Hepatitis C virus Delta hepatitis virus Non-A, non-B hepatitis Human immunodeficiency virus Cytomegalovirus Epstein-Barr virus Herpes simplex virus Respiratory Bacteria Streptococcus Pneumococcus Meningococcus Diphtheria Mycobacterium * Legionella * Fungi Candida * Nocardia * Cryptococcus * Parasites Pneumocystis * Viruses Rhinovirus Influenza Parainfluenza Adenovirus Respiratory syncytial virus Measles Rubella Cytomegalovirus * Gastrointestinal Viruses: hepatitis A virus, rotavirus, adenovirus, enterovirus Bacteria: Giardia, * Cryptosporidium, Isospora * Fungi: Candida * Central Nervous System Viruses: Human immunodeficiency virus, * herpes simplex virus, * Epstein-Barr virus * Parasites: Toxoplasma * Fungi: Cryptococcus * Opportunistic infections in immunocompromised patients, especially those with acquired immunodeficiency. Modified with permission from Browne RA, Chenesky MA: Infectious diseases and the anaesthetist. Can J Anesth 1988; 35:655-665. © 2009 2009 Although no definitive studies have demonstrated a cause-and-effect relationship in the transmission of infections by anesthesiologists or anesthetic staff, there are reports of elements, fomites, and drugs (mainly propofol) that have resulted in hospital-acquired infections. 11 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 However, many of the following situations could potentially cause an infection: • Up to 40% of the anesthetic equipment in the operating room that was in direct or indirect contact with the child (blood pressure cuffs, cables, oximeters, laryngoscopes, monitors, respirator settings and horizontal and vertical surfaces) may be contaminated with blood because of inadequate cleansing procedures between children. 2, 3, 18, 39, 40 • In some institutions, up to 8% of the Bain circuits that were reused without previous sterilization were contaminated. 41 • Contamination of syringe contents has occurred with glass particles during ampule opening, which in turn may compromise the sterility of the contents, presumably due to the passage of bacteria contained on glass particles into the solution. 42 , 43 , 44 • Intravenous tubing has a significant blood contamination rate as well as contamination by blood from syringes used to inject drugs. This can occur with the absence of visible blood reflux in the tubing or syringe. Changing a fresh needle to a syringe that will be reused is useless to prevent cross infection, thus emphasizing the importance of not using the same syringe on multiple patients. 45 • Refilling both glass and plastic syringes several times has been shown to result in contamination of the contents. 45, 46 • Some drug formulations, especially propofol, can sustain bacterial growth under certain conditions. Thus, great care should be given to aseptic technique when transferring drugs from the vial to a syringe and not allowing the syringe to remain unused for more than 4 hours. 47 , 48 , 49 , 50 , 51 • Needles that had been used for spinal or epidural anesthesia were found to be contaminated with coagulase-negative staphylococci (15.7%), yeasts (1.5%), enterococcus (0.8%), pneumococcus (0.8%), and micrococcus (0.8%), suggesting that despite standard skin preparation and cleansing there is a significant rate of needle contamination. 52 It is unclear whether these organisms produce an inoculation that could lead to an infection during neuraxial blockade. • There is a high incidence of skin contamination by blood and saliva of anesthetic personnel that occurs during routine anesthetic practice. 7 • Violations of contemporary guidelines for preventing infections (hand washing, wearing gloves, surgical masks, ocular protection, scrubs, syringe reutilization) by anesthesiologists are frequent. Anesthesia staff are aware of the potentially infectious working environment, but to a great extent (11%-99%) they do not implement protection measures for themselves or their patients. 18 , 53 , 54 , 55 Direct Contact This type of disease transmission involves direct physical contact between two individuals. The physical transfer of microorganisms from an infected or colonized person to a susceptible host may occur from child to health care provider or from health care provider to the child during professional practice (e.g., venous cannulation, laryngoscopy, burn care, suction of secretions). Health care providers working in the operating room may be exposed to skin contamination by body fluids. This is an issue of grave concern because of the potential exposure of health care providers to patients with unrecognized infections, especially hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Hepatitis B is a highly infectious virus that requires a small amount of blood (10 −7 to 10 −9 mL) to transmit the disease. The incidence of skin contamination of anesthesiologists and related personnel by blood and saliva is quite high. One study examined 270 anesthetic procedures during 7 consecutive days. The blood of 35 patients (14%) contaminated the skin of 65 anesthesiologists in 46 incidents. Twenty-eight contamination events (61%) occurred during venous cannulation. Five of 65 anesthesiologists who had been contaminated by blood (8%) had cuts in the skin of their hands. 7 The importance of this observation is that seroconversion of health care providers has been reported after skin contamination by infected blood from HIV carriers 8 and HBV infection after blood splashing into health care workers' eyes. 9 Scabies, pediculosis, and herpes simplex are among the diseases most frequently transmitted by direct contact. 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 These studies explain why meticulous hand washing and routine use of barriers such as gloves and eye protection are such an important part of protecting ourselves from such exposures even during routine procedures such as starting an intravenous line or performing laryngoscopy. Indirect Contact Indirect contact involves the transmission of microorganisms from a source (animate or inanimate) to a susceptible host by means of a vehicle (e.g., an intermediary object) contaminated by body fluids. Tables 50-2 and 50-3 provide examples of diseases associated with bodily fluids to which health care workers may be exposed. The vehicle for transmission may be the hands of a health care provider who is not wearing gloves or a provider who fails to wash his or her hands between children. This type of contact can also come from health care providers who touch (with or without gloves) contaminated monitoring or other patient care devices (e.g., blood pressure cuffs, stethoscopes, electrocardiographic cables, ventilation systems [respirators, corrugated tubes, Y pieces, valves]), which are used with several children without proper cleaning or disinfection in between each use. 18 , 19 , 20 Table 50-2 Body Fluids and Diseases They May Transmit Body Fluid Diseased Transmitted Blood HBV, HIV, HCV, CMV, EBV, NANBH Seminal fluid HIV, HBV, CMV Vaginal discharge HIV, HBV, CMV Saliva and sputum HSV, TB, CMV, respiratory diseases Cerebrospinal fluid Encephalopathic organisms (see Table 50-5 ), HIV Breast milk HIV, HBV, CMV Urine CMV, EBV, HBV Feces and intestinal fluid HAV, gastrointestinal diseases (see Table 50-5 ) HBV, hepatitis B virus; HIV, human immunodeficiency virus; HCV, hepatitis C virus; CMV, cytomegalovirus; EBV, Epstein-Barr virus; NANBH, non-A non-B hepatitis; TB, tuberculosis; HAV, hepatitis A virus; HSV, herpes simplex types I and II. Modified with permission from Browne RA, Chenesky MA: Infectious diseases and the anaesthetist. Can J Anesth 1988; 35:655-665. © 2009 2009 Table 50-3 Infectious Agents That May Be Found in the Operating Room Viral Hepatitis Hepatitis A virus Hepatitis B virus Hepatitis C virus Delta hepatitis virus Non-A, non-B hepatitis Human immunodeficiency virus Cytomegalovirus Epstein-Barr virus Herpes simplex virus Respiratory Bacteria Streptococcus Pneumococcus Meningococcus Diphtheria Mycobacterium * Legionella * Fungi Candida * Nocardia * Cryptococcus * Parasites Pneumocystis * Viruses Rhinovirus Influenza Parainfluenza Adenovirus Respiratory syncytial virus Measles Rubella Cytomegalovirus * Gastrointestinal Viruses: hepatitis A virus, rotavirus, adenovirus, enterovirus Bacteria: Giardia, * Cryptosporidium, Isospora * Fungi: Candida * Central Nervous System Viruses: Human immunodeficiency virus, * herpes simplex virus, * Epstein-Barr virus * Parasites: Toxoplasma * Fungi: Cryptococcus * Opportunistic infections in immunocompromised patients, especially those with acquired immunodeficiency. Modified with permission from Browne RA, Chenesky MA: Infectious diseases and the anaesthetist. Can J Anesth 1988; 35:655-665. © 2009 2009 Although no definitive studies have demonstrated a cause-and-effect relationship in the transmission of infections by anesthesiologists or anesthetic staff, there are reports of elements, fomites, and drugs (mainly propofol) that have resulted in hospital-acquired infections. 11 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 However, many of the following situations could potentially cause an infection: • Up to 40% of the anesthetic equipment in the operating room that was in direct or indirect contact with the child (blood pressure cuffs, cables, oximeters, laryngoscopes, monitors, respirator settings and horizontal and vertical surfaces) may be contaminated with blood because of inadequate cleansing procedures between children. 2, 3, 18, 39, 40 • In some institutions, up to 8% of the Bain circuits that were reused without previous sterilization were contaminated. 41 • Contamination of syringe contents has occurred with glass particles during ampule opening, which in turn may compromise the sterility of the contents, presumably due to the passage of bacteria contained on glass particles into the solution. 42 , 43 , 44 • Intravenous tubing has a significant blood contamination rate as well as contamination by blood from syringes used to inject drugs. This can occur with the absence of visible blood reflux in the tubing or syringe. Changing a fresh needle to a syringe that will be reused is useless to prevent cross infection, thus emphasizing the importance of not using the same syringe on multiple patients. 45 • Refilling both glass and plastic syringes several times has been shown to result in contamination of the contents. 45, 46 • Some drug formulations, especially propofol, can sustain bacterial growth under certain conditions. Thus, great care should be given to aseptic technique when transferring drugs from the vial to a syringe and not allowing the syringe to remain unused for more than 4 hours. 47 , 48 , 49 , 50 , 51 • Needles that had been used for spinal or epidural anesthesia were found to be contaminated with coagulase-negative staphylococci (15.7%), yeasts (1.5%), enterococcus (0.8%), pneumococcus (0.8%), and micrococcus (0.8%), suggesting that despite standard skin preparation and cleansing there is a significant rate of needle contamination. 52 It is unclear whether these organisms produce an inoculation that could lead to an infection during neuraxial blockade. • There is a high incidence of skin contamination by blood and saliva of anesthetic personnel that occurs during routine anesthetic practice. 7 • Violations of contemporary guidelines for preventing infections (hand washing, wearing gloves, surgical masks, ocular protection, scrubs, syringe reutilization) by anesthesiologists are frequent. Anesthesia staff are aware of the potentially infectious working environment, but to a great extent (11%-99%) they do not implement protection measures for themselves or their patients. 18 , 53 , 54 , 55 Accidents with Cutting or Piercing Devices Percutaneous contamination as a result of a cutting or piercing accident is the most effective means to transmit blood-borne pathogens. Evidence suggests that this is the main route of HIV, HBV, and HCV 56, 57 infection, especially if injuries are caused by hollow-bore needles that were used to draw blood or introduce an intravenous line. 58, 59 Over 20 other blood-borne pathogens have been transmitted by this means, including those causing herpes, malaria, and tuberculosis. 60 The infectious risk after a percutaneous exposure to blood or body fluids from an HIV-positive person is 0.3%. Among health care providers lacking protective antibodies, the risk of HBV infection after an injury with a cutting or piercing device infected with hepatitis B antigen is approximately 37%; in the case of HCV it is 1.8% (0%-7%). Anesthesia staff lacking HBV protective antibodies are at high risk for acquiring the disease. 61, 62 These infection rates underscore the need for the use of "safe" needles and the need to advocate the use of "needleless" systems even though they are significantly more expensive. This also emphasizes the need for meticulous handling and disposal of needles and other sharp instruments as well as the use of special "sharps boxes" designed to minimize accidental needlesticks (e.g. "mail box" type boxes that do not allow the hand to enter the disposal area). 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 The U.S. Centers for Disease Control and Prevention (CDC) has estimated that in the United States there are approximately 385,000 cutting and piercing accidents annually among health care providers in hospitals; 25% of these occur in the operating room. 60 However, the actual prevalence is believed to be much greater because many of these events are unreported. The distribution of these accidents among anesthesiologists is shown in Figure 50-3A ; the distribution of the elements most frequently associated with cutting and piercing injuries in health care providers is shown in Figure 50-3B . Should such an accident occur (needle puncture, exposure to nonintact skin, mucous membrane exposure) there are now specific recommendations regarding immediate assessment of risk, assessment of the exposure source (chart review, inform the patient that an accident has occurred and ask permission to determine HBV, HCV, and HIV serologic tests) and initiation of appropriate treatment of the health care worker. It is advised to obtain as much information regarding the patient as possible if the patient is known, to obtain a sample of blood from the patient for determination of potential carrier state ( Table 50-4 ), and to report to the health service for immediate institution of prophylaxis and follow-up ( Table 50-5 ), especially for HIV exposure ( Tables 50-6 and 50-7 ). Figure 50-3 A , Percentage distribution of percutaneous injury in anesthesiologists caused by contaminated cutting or piercing devices. B , Percentage distribution of elements associated with percuta-neous injuries in health care work. Table 50-4 Recommendations for the Contents of the Occupational Exposure Report • Date and time of exposure • Details of the procedure being performed, including where and how the exposure occurred; if related to a sharp device, the type and brand of device; and how and when in the course of handling the device the exposure occurred • Details of the exposure, including the type and amount of fluid or material and the severity of the exposure; for example, for a percutaneous exposure, depth of injury and whether fluid was injected and for a skin or mucous membrane exposure, the estimated volume of material and the condition of the skin (e.g., chapped, abraded, intact) • Details about the exposure source (e.g., whether the source material contained hepatitis B virus, hepatitis C virus, or human immunodeficiency virus; if the source is infected with human immunodeficiency virus, the stage of disease, history of antiretroviral therapy, viral load, and antiretroviral resistance information, if known) • Details about the exposed person (e.g., hepatitis B vaccination and vaccine-response status) • Details about counseling, postexposure management, and follow-up http://www.cdc.gov/mmwr/PDF/rr/rr5011.pdf Updated U.S. Public Health Service guidelines for the management of occupational exposures to HBV, HCV, and HIV and recommendations for postexposure prophylaxis. MMWR Recomm Rep 2001; 50:1-52. © 2009 2009 Table 50-5 Factors to Consider in Assessing the Need for Follow-up of Occupational Exposures Type of Exposure Percutaneous injury Mucous membrane exposure Nonintact skin exposure Bites resulting in blood exposure to either person involved Type and Amount of Fluid/Tissue Blood Fluids containing blood Potentially infectious fluid or tissue (semen; vaginal secretions; and cerebrospinal, synovial, pleural, peritoneal, pericardial, and amniotic fluids) Direct contact with concentrated virus Infectious Status of Source Presence of HBsAg Presence of HCV antibody Presence of HIV antibody Susceptibility of Exposed Person Hepatitis B vaccine and vaccine response status HBV, HCV, and HIV immune status http://www.cdc.gov/mmwr/PDF/rr/rr5011.pdf Updated U.S. Public Health Service guidelines for the management of occupational exposures to HBV, HCV, and HIV and recommendations for postexposure prophylaxis. MMWR Recomm Rep 2001; 50:1-52 © 2009 2009 Table 50-6 Recommended HIV Postexposure Prophylaxis (PEP) for Percutaneous Injuries Infection Status of Source Exposure Type HIV-Positive Class 1 * HIV-Positive Class 2 * Source of Unknown HIV Status †Unknown Source ‡ HIV-Negative No PEP Less severe § Recommend basic 2-drug PEP Recommend expanded ≥3-drug PEP Generally, no PEP warranted; however, consider basic 2-drug PEP ‖ for source with HIV risk factors ¶ Generally, no PEP warranted; however, consider basic 2-drug PEP ‖ in settings in which exposure to HIV-infected persons is likely warranted More severe ** Recommend expanded 3-drug PEP Recommend expanded ≥3-drug PEP Generally, no PEP warranted; however, consider basic 2-drug PEP ‖ for source with HIV risk factors ¶ Generally, no PEP warranted; however, consider basic 2-drug PEP ‖ in settings in which exposure to HIV-infected persons is likely No PEP warranted * HIV-positive class 1: asymptomatic HIV infection or known low viral load (e.g., <1500 ribonucleic acid copies/mL). HIV-positive class 2: symptomatic HIV infection, acquired immunodeficiency syndrome, acute seroconversion, or known high viral load. If drug resistance is a concern, obtain expert consultation. Initiation of PEP should be delayed pending expert consultation, and because expert consultation alone cannot substitute for face-to-face counseling, resources should be available to provide immediate evaluation and follow-up care for all exposures. †For example, deceased source person with no samples available for HIV testing. ‡ For example, a needle from a sharps disposal container. § For example, solid needle or superficial injury. ‖ The recommendation "consider PEP" indicates that PEP is optional; a decision to initiate PEP should be based on a discussion between the exposed person and the treating clinician regarding the risks versus benefits of PEP. ¶ If PEP is offered and administered and the source is later determined to be HIV-negative, PEP should be discontinued. ** For example, large-bore hollow needle, deep puncture, visible blood on device, or needle used in patient's artery or vein. Updated U.S. Public Health Service guidelines for the management of occupational exposures to HBV, HCV, and HIV and recommendations for postexposure prophylaxis. MMWR Recomm Rep 2001; 50:1-52. Available at http://www.cdc.gov/mmwr/PDF/rr/rr5011.pdf © 2009 2009 Table 50-7 Recommended HIV Postexposure Prophylaxis (PEP) for Mucous Membrane Exposures and Nonintact Skin * Exposures Infection Status of Source Exposure Type HIV-Positive Class 1 †HIV-Positive Class 2 †Source of Unknown HIV Status ‡ Unknown Source § HIV-Negative Small volume ‖ Consider basic 2-drug PEP ¶ Recommend basic 2-drug PEP Generally, no PEP warranted ** Generally, no PEP warranted No PEP warranted Large volume ††Recommend basic 2-drug PEP Recommend expanded ≥3-drug PEP Generally, no PEP warranted; however, consider basic 2-drug PEP ¶ for source with HIV risk factors ** Generally, no PEP warranted; however, consider basic 2-drug PEP ¶ in settings in which exposure to HIV-infected persons is likely No PEP warranted * For skin exposures, follow-up is indicated only if evidence exists of compromised skin integrity (e.g., dermatitis, abrasion, or open wound). †HIV-positive class 1: asymptomatic HIV infection or known low viral load (e.g., <1500 ribonucleic acid copies/mL). HIV-positive class 2: symptomatic HIV infection, acquired immunodeficiency syndrome, acute seroconversion, or known high viral load. If drug resistance is a concern, obtain expert consultation. Initiation of PEP should be delayed pending expert consultation, and because expert consultation alone cannot substitute for face-to-face counseling, resources should be available to provide immediate evaluation and follow-up care for all exposures. ‡ For example, deceased source person with no samples available for HIV testing. § For example, splash from inappropriately disposed blood. ‖ For example, a few drops. ¶ The recommendation "consider PEP" indicates that PEP is optional; a decision to initiate PEP should be based on a discussion between the exposed person and the treating clinician regarding the risks versus benefits of PEP. ** If PEP is offered and administered and the source is later determined to be HIV-negative, PEP should be discontinued. ††For example, a major blood splash. Updated U.S. Public Health Service guidelines for the management of occupational exposures to HBV, HCV, and HIV and recommendations for postexposure prophylaxis. MMWR Recomm Rep 2001; 50:1-52. Available at http://www.cdc.gov/mmwr/PDF/rr/rr5011.pdf © 2009 2009 Strategy for Preventing Infection Transmission in Health Care Institutions Institutional administrative measures aimed at developing, implementing, and monitoring specifically designed accident prevention policies and procedures are key factors in reducing and preventing transmission of infectious agents in health care centers. To this end, the center must take the following actions: 60, 79 • Include infection control as a major goal in the organizational mission statement and implement safety programs both for patients and health care workers. • Provide sufficient administrative and financial support to carry out this mission. • Provide sufficient administrative and financial support for the microbiology laboratory and implement an infection surveillance plan, especially for postsurgical infections. • Establish a multidisciplined cross-functional team (e.g., a team manager, an epidemiologist, a representative from industrial health, a person trained in quality control) to identify health and safety issues within the institution, analyze trends, implement interventions, assess outcomes, and make recommendations to other members of the organization. • Provide sufficient administrative and financial support to develop and implement education programs for health care providers, patients, and their families. One positive example of such education is that anesthesiologists who have read the CDC's Universal Precaution Guidelines for the Prevention of Occupational Transmission of HIV and HBV develop better hygienic practices. 55 • Provide health care workers with hepatitis B vaccine and document that an appropriate immunologic response was achieved. Provide hepatitis B immune globulin (HBIG) for those exposed who do not have established immunity. 4 • Provide a health care service for employees for counseling and post-exposure prophylaxis should an exposure to HIV occur. 80 • Provide regular surveillance of health care workers to determine established immunity to infectious diseases such as tuberculosis, measles, mumps, rubella, and chickenpox. Lack of immunity may require immunization; several studies have demonstrated the cost-effectiveness of immunization (for prevention of disease) versus the cost of replacement of health care workers who have become infected. 57 , 81 , 82 , 83 , 84 , 85 , 86 Measures for Prevention of Infection Transmission in the Operating Room Prevention of Air-borne Pathogen Transmission Air-borne pathogens may be transmitted through the operating room heating, ventilation, and air conditioning systems. Thus, it is vital to have in place proper systems to (1) remove contaminated air, (2) facilitate air management requirements to protect susceptible health care providers and children against hospital-related air-borne pathogens, and (3) minimize the risk of air-borne pathogens being transmitted by children. Table 50-8 shows the 2003 HICPAC's (Healthcare Infection Control Practices Advisory Committee) and CDC's general recommendations for ventilation system specifications for the operating room. 87 Children with tuberculosis require special consideration because of the high risk of occupational transmission of Mycobacterium tuberculosis , 88, 89 especially after the emergence of multidrug-resistant strains of M. tuberculosis (MDR-TB) ( Table 50-9 ). An easy preventive measure is to screen all children before coming to the operating room to determine recent exposure to infectious disease such as measles, mumps, rubella, and chickenpox because these diseases can pose a significant risk to health care providers and to patients, especially immunocompromised patients. 57, 82 Another potential source for air-borne spread of pathogens is through the anesthesia circuit that may be reduced by the use of filters within the circuit. However, at present there are no regulatory requirements to use such devices and performance characteristics vary widely. 19, 20, 90, 91 Table 50-8 Ventilation System Specifications for the Operating Room • Minimize the circulation of people during surgeries. It has been proved that the level of microbes in the operating room air is directly proportional to the number of people moving inside the room. • Maintain humidity under 68% and temperature control to prevent environmental conditions that favor the development of germs. • Maintain positive pressure compared with corridors and surrounding areas to prevent microorganisms from entering the operating room. • Provide at least 15 air changes per hour in the operating room, 20% of which should be fresh air. Air should be recirculated through a high-efficiency particulate air (HEPA) filter. • Air should be introduced at ceiling level and disposed of at ground level. Guidelines for environmental infection control in health-care facilities: recommendations of CDC and the Healthcare Infection Control Practices Advisory Committee (HICPAC). 2003. www.cdc.gov/ncidod/dhqp/gl_environinfection.html Table 50-9 Summary of Recommended Tuberculosis Control Guidelines Early diagnosis Availability and access to diagnostic tests Improved tests and reporting of results Source controls Containment of infectious nuclei from coughs and sneezes Patient isolation Personal respirators Engineering controls Negative-pressure patient environments * Minimum of 6 air changes per hour Ultraviolet light sources High-efficiency particulate air (HEPA) filters Personal respirators Decontamination Sterilization and disinfection of equipment Screening/treatment Annual tuberculin testing Availability and compliance with chemoprophylaxis Bacille Calmette-Guérin (BCG) vaccination * Negative-pressure rooms are to prevent the escape of contaminated air to the outside. From Tait A: Occupational transmission of tuberculosis: implications for anesthesiologists. Anesth Analg 1997; 85:444-451. © 2009 2009 Standard Precautions Standard Precautions 92 assume that any person or patient is potentially infected or colonized by microorganisms that could be transmitted and cause an infectious process. Standard Precautions must be implemented with all patients and include: • Universal Precautions—Blood and Body Fluid Precautions, developed to reduce blood-borne pathogen transmission • Body Substance Isolation, designed to reduce the risk of pathogen transmission by moist body substances Standard Precautions are used to reduce the transmission of all infectious agents from one person to another, thus protecting health care providers and children against exposure to the most common microorganisms. Standard Precautions are implemented for any contact with blood and body fluids, secretions, and excretions (except sweat), whether or not they contain visible blood, as well as for any contact with non-intact skin, mucous membranes, and intact skin that is visibly soiled with blood and/or body fluids. Summaries of Standard Precautions, Droplet Precautions, Air-borne Precautions, and Contact Precautions are available on line. 93 , 94 , 95 , 96 Hand Washing Hand washing is considered the most important and cost-effective individual intervention in the prevention of hospital-acquired infections in children and health care providers. 97 Its significance in medical practice had not been universally accepted despite the pioneering work by Oliver Wendell Holmes 98 (1843) and Ignaz Semmelweis 99 (1846), who separately recognized the role of the contaminated hands of physicians performing autopsies in the spread of puerperal fever due to Streptococcus sepsis and how by washing their hands before delivering a baby they could reduce maternal mortality by 90%! Unfortunately, the scientific basis for hand washing was not established until the introduction of the germ theory of disease by Louis Pasteur 100 and the discovery of the microorganism that caused anthrax ( Bacillus anthracis ) by Robert Koch 101 in the late 19th century. More than one and a half centuries later, and with strong evidence that health care providers are one of the most frequent sources of infection transmission among patients, 102 health care providers' compliance with hand hygiene protocols in the hospital environment is generally small (5%-48%) and difficult to change, 103 , 104 , 105 , 106 , 107 , 108 , 109 especially in intensive care areas, operating rooms, and postanesthesia care units. The risk of pathogen transmission through the hands is proportional to the power of the number of times a child is touched. 110 Table 50-10 presents a summary of the indications for hand washing and antisepsis. Table 50-10 Indications for Hand Washing and Antisepsis Hand washing is defined as a process for removal of soil and transient microorganisms from the hands. Hands should be washed with soap and water or disinfected. 1. When hands are visibly dirty or contaminated with proteinaceous material or are visibly soiled with blood or other body fluids, wash hands with either a non-antimicrobial soap and water or an antimicrobial soap and water. 2. If hands are not visibly soiled, use an alcohol-based hand rub for routinely decontaminating hands in all other clinical situations described in items 3 to 10. Alternatively, wash hands with an antimicrobial soap and water in all clinical situations described in items 3 to 10. 3. Decontaminate hands before having direct contact with patients. 4. Decontaminate hands before donning sterile gloves when inserting a central intravascular catheter. 5. Decontaminate hands before inserting indwelling urinary catheters, peripheral vascular catheters, or other invasive devices that do not require a surgical procedure. 6. Decontaminate hands after contact with a patient's intact skin (e.g., when taking a pulse or blood pressure and lifting a patient). 7. Decontaminate hands after contact with body fluids or excretions, mucous membranes, non-intact skin, and wound dressings if hands are not visibly soiled. 8. Decontaminate hands if moving from a contaminated body site to a clean body site during patient care. Decontaminate hands after contact with inanimate objects (including medical equipment) in the immediate vicinity of the patient. 9. Decontaminate hands after removing gloves. 10. Before eating and after using a rest room, wash hands with a non-antimicrobial soap and water or with an antimicrobial soap and water. 11. Antimicrobial-impregnated wipes (i.e., towelettes) may be considered as an alternative to washing hands with non-antimicrobial soap and water. Because they are not as effective as alcohol-based hand rubs or washing hands with an antimicrobial soap and water for reducing bacterial counts on the hands of health care workers, they are not a substitute for using an alcohol-based hand rub or antimicrobial soap. 12. Wash hands with non-antimicrobial soap and water or with antimicrobial soap and water if exposure to Bacillus anthracis is suspected or proven. The physical action of washing and rinsing hands under such circumstances is recommended because alcohols, chlorhexidine, iodophors, and other antiseptic agents have poor activity against spores. Modified from Boyce JM, Pittet D: Guideline for Hand Hygiene in Health-Care Settings. Recommendations of the Healthcare Infection Control Practices Advisory Committee and the HIPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. Am J Infect Control 2002; 30:S1-S46. © 2009 2009 Compared with soap and water, alcohol-based hand rubs are more effective in reducing microbial colonization of hands. 111 The use of alcohol-based hand soaps prompted some authors to change the term hand washing to hand hygiene . An important addition to the 2002 CDC Hand Washing Guide 103 is to use alcohol-based hand rubs, because they work more rapidly (10–20 seconds compared with 90-120 for hand washing) and can be used while ambulating. These advantages preclude the usual objections of health care workers to hand washing that include a lack of time, absence of sinks, and skin damage. 112 Furthermore, the scarcity of water in developing countries would no longer be a constraint against hand hygiene. After hand washing, it is very important to dry them properly with appropriate paper towels, hot air flow, or both, because the level of pathogen transmission from a health care worker's hands to a patient is greatly increased if the hands are wet. 113 Transmission may also occur from patients' wet sites, such as groins or armpits, or when a health care worker gets his or her hands wet when opening parenteral solutions. It is critical for health institutions to establish written procedures and protocols to support adherence to the recommended hand hygiene practices. Gloves Wearing clean or sterile gloves while caring for children is an effective means of reducing hospital-acquired infections. Gloves remain a supplementary barrier to infection that should not replace proper hand hygiene. Gloves protect patients by reducing health care provider hand contamination and the subsequent transmission of pathogens to other children, provided the gloves are changed between children. Additionally, when the use of gloves is combined with CDC Standard Precautions, they protect the health care provider against exposure to blood-borne infections or infections transmitted by any other body fluids, such as excretions, secretions (except sweat), mucous membranes, and non-intact skin. Recommendation for the use of gloves include: 114 , 115 , 116 • Wear gloves in case of contact with blood or any other potentially infecting body fluid such as excretions, secretions (except sweat), mucous membranes, and non-intact skin. • Remove the gloves immediately after providing care to a child. Staff should not wear the same pair of gloves to take care of more than one child, nor should they touch the surfaces of any equipment, monitoring devices, or even light switches. Contaminated gloves can pass blood or other body fluids to working areas and have proved to be a vector for hepatitis transmission. 56 • Change gloves when taking care of a child if you must move from a contaminated to a clean body site. • Apply hand hygiene measures immediately after removing the gloves because, despite the use of gloves, hands may get contaminated through small holes in the gloves. 102, 117, 118 Microbial contamination of hands and possible infection transmission have been reported even with the use of gloves. 119 • Remove the gloves by using an appropriate technique (so as not to contaminate your hands with the contaminated surface of the gloves). • Alcohol-based hand rub dispensers and clean glove boxes (at least two sizes) should be in place near every patient care site (e.g., on top of every anesthesia cart, medication cart, or in the nursing station). • Disposable gloves should not be washed, resterilized, or disinfected. If gloves are reused, appropriate reprocessing methods should be in place to ensure the physical integrity of the gloves and their full decontamination. • Sterile gloves are much more expensive than clean, disposable gloves and should be used only for certain procedures, such as when hands are in contact with normally sterile body areas or when inserting intravascular or urinary catheters. Clean gloves should be used during any other procedure, including wound dressing. • Latex free gloves should be worn when caring for children at risk for latex allergy. Antimicrobial Prophylaxis Surgical antimicrobial prophylaxis is an essential tool to reduce the risk of postoperative infections, and the anesthesia team plays a central role in ensuring the proper timing of drug administration. 120, 121 The aim of the perioperative administration of antibiotics is to obtain plasma and tissue drug levels exceeding the minimal inhibitory concentration of those organisms most likely to cause an infection. This will reduce the microbial load of the intraoperative contamination to a level not exceeding the host defenses; it is not the intent to cover all possible pathogens, because this can lead to the selection of drug-resistant bacteria. Selection of the Antimicrobial Agent Several antimicrobial prophylaxis guidelines have been published ( Table 50-11 ). For most surgical procedures that do not involve chronically colonized organs, the most common pathogens are the skin flora, Streptococcus and Staphylococcus . A first-generation cephalosporin (i.e., cefazolin) can provide cost-effective coverage for these organisms. Surgical procedures that involve contamination from the bowel require antibiotic treatment against gram-negative and anaerobic pathogens. For these procedures, cefoxitin, cefotetan, or a second-generation cephalosporin is appropriate. 122 The selection of antibiotics requires consideration of resistance patterns as determined by local microbiology or health center infectious disease departments. The newer-generation broad-spectrum antibiotics should not be used for routine antibiotic prophylaxis but should be reserved for the treatment of resistant organisms. Moreover, the dose of antibiotic selected should be based on the child's weight or body mass index; administration should be repeated intraoperatively if surgery exceeds more than two half-lives after the first antibiotic administration (see Table 50-11 ), if the duration of surgery exceeds 4 to 8 hours, if blood loss is extreme, or if the drug has a particularly short half-life (e.g., penicillin or cefoxitin) to ensure appropriate tissue concentrations of antibiotic until wound closure. 123 Table 50-11 Suggested Initial Dose and Time to Redosing for Antimicrobials Commonly Used for Surgical Prophylaxis Antimicrobial Half-Life Normal Renal Function (hr) Half-Life End-Stage Renal Disease (hr) Recommended Infusion Time (min) Standard Intravenous Dose (g) Weight-Based Dose Recommendation * (mg) Recommended Dosing Interval †(hr) Aztreonam 1.5-2 6 3-5 ‡ 1–2 Max 2 g (adults) 3–5 Ciprofloxacin 3.5-5 5–9 60 400 mg 400 mg 4–10 Cefazolin 1.2-2.5 40–70 3-5 ‡ 15-60 § 1–2 20-30 mg/kg 2–5 1 g < 80 kg 2 g ≥ 80 kg Cefuroxime 1–2 15–22 3-5 ‡ 15-60 § 1.5 50 mg/kg 3–4 Cefamandole 0.5-2.1 12.3-18 ‖ 3-5 ‡ 15-60 § 1 3–4 Cefoxitin 0.5-1.1 6.5-23 3-5 ‡ 15-60 § 1–2 20-40 mg/kg 2–3 Cefotetan 2.8-4.6 13–25 3-5 ‡ 20-60 § 1–2 20-40 mg/kg 3–6 Clindamycin 2-5.1 3.5-5.0 ¶ 10–60 600–900 <10 kg: at least 3–6 (Do not exceed 30 mg/min) 37.5 mg ≥10 kg: 3-6 mg/kg Erythromycin base 0.8-3 5–6 NA 1 g orally 19, 18, 9 hr before surgery 9-13 mg/kg NA Gentamicin 2–3 50–70 1.5 mg/kg ** See footnote ** 3–6 Neomycin 2-3 hours (3% absorbed under normal GI conditions 12-≥ 24 NA 1 g orally 19, 18, 9 hr before surgery 20 mg/kg NA Metronidazole 6–14 7-21 no change 30–60 0.5-1 15 mg/kg (adult) 7.5 mg/kg on subsequent doses 6–8 Vancomycin 4–6 44.1-406.4 (Cl cr <10 mL/min) 1 g ≥ 60 min (use longer infusion time if dose < 1 g) 1.0 10-15 mg/kg (adult) 6–12 DW, dosing weight; IBW, ideal body weight; NA, not applicable. * Weight-based doses are primarily from published pediatric recommendations. †For procedures of long duration, antimicrobials should be redosed at intervals of 1 to 2 times the half-life of the drug. The intervals in the table were calculated for patients with normal renal function. ‡ Dose injected directly into vein or running intravenous fluids. § Intermittent intravenous infusion. ‖ In patients with a serum creatinine value of 5 to 9 mg/dL. ¶ The half-life of clindamycin is the same or slightly increased in patients with end-stage renal disease compared with patients with normal renal function. ** If the patient's weight is 30% above the ideal body weight, dosing weight can be determined as follows: DW = IBW + 0.4 (total body weight − IBW). The Timing of Antibiotic Prophylaxis A key element in the prevention of surgical site infection is the timely administration of prophylactic antibiotics. For most surgical procedures, a single prophylactic dose of antibiotics should be administered 30 to 60 minutes before the skin incision. This should provide appropriate plasma concentrations of the antibiotic. 124, 125 However, in the case of children, intravenous access is often established after induction of anesthesia. With a brief time interval between establishing intravenous access and skin incision, it is important to administer the antibiotics as soon as possible after intravenous access is established. If vancomycin must be used for prophylaxis, it should be infused slowly over 60 minutes (to minimize the risk of severe hypotension) beginning within 2 hours of skin incision. If a tourniquet is required, the full antibiotic dose should be administered before the tourniquet is pressurized. 126 Postsurgical prophylactic antibiotics are not necessary for most procedures and should generally be stopped within 24 hours after the surgical procedure. 126 Allergy to β-Lactams Several studies have shown that the true incidence of allergy is less than that reflected in medical charts. 127 For surgical procedures where cephalosporins are the prophylaxis of choice, alternative antibiotics should be administered to those children at high risk for serious adverse reactions or allergy, based on their history or diagnostic tests (e.g., skin testing). However, the incidence of adverse reactions to cephalosporins in children with reported allergy to penicillin is rare; further-more, skin testing does not reliably predict the likelihood of adverse reactions to cephalosporins in those with reported allergy to penicillin. 128 , 129 , 130 For the most part, "allergies" to oral antibiotics that appear on children's charts (rash, vomiting, gastrointestinal disturbances) are reactions to the additives in the antibiotic formulation including food dyes, fillers, and other compounds. Intravenous administration of small test doses of the pure antibiotics in a fully monitored (and anesthetized) child with a so-called allergy may be used to establish the child's susceptibility to an allergic reaction to the antibiotic. In the case of surgical procedures where antibiotic prophylaxis is mainly directed at gram-positive cocci, children who are truly allergic to β-lactams (cephalosporins) should receive either vancomycin or clindamycin. 122 Indications for Prophylactic Antibiotics Surgical wounds are classified in four categories ( Table 50-12 ). The use of antibiotic prophylaxis for postoperative infections is well established for clean-contaminated procedures. Within the clean category, prophylaxis has been traditionally reserved for surgical procedures involving a foreign body implantation or for any surgical procedure where a surgical site infection would be catastrophic (e.g., cardiac surgery or neurosurgical procedures). However, there is evidence to demonstrate that postoperative infections resulting from procedures not involving prosthetic elements are underreported; estimates show that over 50% of all complications occur after the patient is discharged from hospital and are unrecognized by the surgical team. Therefore, antibiotic prophylaxis is also recommended for certain procedures such as herniorrhaphy. 131, 132 The direct and indirect costs of these complications will not affect the hospital budget; however, they represent a high cost for the community at large. In the case of contaminated or dirty procedures, bacterial contamination or infection is established before the procedure begins. Accordingly, the perioperative administration of antibiotics is a therapeutic, not a prophylactic, measure. The use of antibiotics in children has implications not only for the response to the current treatment but also to future treatments. Thus, all medical professionals are jointly responsible for the rational use of antibiotics. Table 50-12 Wound Classification System Wound Category Description Class I/clean Uninfected wound with no inflammation and the respiratory, alimentary, genital, or uninfected urinary tract is not entered. Clean wounds primarily are closed and drained, when necessary, with closed drainage. Operative wounds after blunt trauma may be included in this category if they meet criteria. Class II/clean contaminated Operative wound in which the respiratory, alimentary, genital, or urinary tract is entered under controlled conditions and without unusual contamination. Specifically, operations involving the biliary tract, appendix, vagina, and oropharynx are included in the category, provided no evidence of infection or major break in technique is encountered. Class III/contaminated Open, fresh, accidental wounds; operations with major breaks in sterile technique (e.g., open cardiac massage) or gross spillage from the gastrointestinal tract; and incisions in which acute, nonpurulent inflammation is encountered Class IV/dirty-infected Old traumatic wounds with retained devitalized tissue and those that involve existing clinical infection or perforated viscera, suggesting that the organisms causing postoperative infection were present in the operative field before operation. From Neville HL, Lally KP: Pediatric surgical wound infections. Semin Pediatr Infect Dis 2001; 12:124-129. © 2009 2009 Protocols, although effective, require continuous feedback on their acceptance and surgical site infection results. No surgical protocol can replace the judgment of the medical professional; clinical reasoning must be tailored to the individual circumstances. Finally, children with congenital heart disease and many of those with repaired congenital heart disease will require subacute bacterial endocarditis prophylaxis (see also Tables 14-1 and 14-2). 133 Prevention of Air-borne Pathogen Transmission Air-borne pathogens may be transmitted through the operating room heating, ventilation, and air conditioning systems. Thus, it is vital to have in place proper systems to (1) remove contaminated air, (2) facilitate air management requirements to protect susceptible health care providers and children against hospital-related air-borne pathogens, and (3) minimize the risk of air-borne pathogens being transmitted by children. Table 50-8 shows the 2003 HICPAC's (Healthcare Infection Control Practices Advisory Committee) and CDC's general recommendations for ventilation system specifications for the operating room. 87 Children with tuberculosis require special consideration because of the high risk of occupational transmission of Mycobacterium tuberculosis , 88, 89 especially after the emergence of multidrug-resistant strains of M. tuberculosis (MDR-TB) ( Table 50-9 ). An easy preventive measure is to screen all children before coming to the operating room to determine recent exposure to infectious disease such as measles, mumps, rubella, and chickenpox because these diseases can pose a significant risk to health care providers and to patients, especially immunocompromised patients. 57, 82 Another potential source for air-borne spread of pathogens is through the anesthesia circuit that may be reduced by the use of filters within the circuit. However, at present there are no regulatory requirements to use such devices and performance characteristics vary widely. 19, 20, 90, 91 Table 50-8 Ventilation System Specifications for the Operating Room • Minimize the circulation of people during surgeries. It has been proved that the level of microbes in the operating room air is directly proportional to the number of people moving inside the room. • Maintain humidity under 68% and temperature control to prevent environmental conditions that favor the development of germs. • Maintain positive pressure compared with corridors and surrounding areas to prevent microorganisms from entering the operating room. • Provide at least 15 air changes per hour in the operating room, 20% of which should be fresh air. Air should be recirculated through a high-efficiency particulate air (HEPA) filter. • Air should be introduced at ceiling level and disposed of at ground level. Guidelines for environmental infection control in health-care facilities: recommendations of CDC and the Healthcare Infection Control Practices Advisory Committee (HICPAC). 2003. www.cdc.gov/ncidod/dhqp/gl_environinfection.html Table 50-9 Summary of Recommended Tuberculosis Control Guidelines Early diagnosis Availability and access to diagnostic tests Improved tests and reporting of results Source controls Containment of infectious nuclei from coughs and sneezes Patient isolation Personal respirators Engineering controls Negative-pressure patient environments * Minimum of 6 air changes per hour Ultraviolet light sources High-efficiency particulate air (HEPA) filters Personal respirators Decontamination Sterilization and disinfection of equipment Screening/treatment Annual tuberculin testing Availability and compliance with chemoprophylaxis Bacille Calmette-Guérin (BCG) vaccination * Negative-pressure rooms are to prevent the escape of contaminated air to the outside. From Tait A: Occupational transmission of tuberculosis: implications for anesthesiologists. Anesth Analg 1997; 85:444-451. © 2009 2009 Standard Precautions Standard Precautions 92 assume that any person or patient is potentially infected or colonized by microorganisms that could be transmitted and cause an infectious process. Standard Precautions must be implemented with all patients and include: • Universal Precautions—Blood and Body Fluid Precautions, developed to reduce blood-borne pathogen transmission • Body Substance Isolation, designed to reduce the risk of pathogen transmission by moist body substances Standard Precautions are used to reduce the transmission of all infectious agents from one person to another, thus protecting health care providers and children against exposure to the most common microorganisms. Standard Precautions are implemented for any contact with blood and body fluids, secretions, and excretions (except sweat), whether or not they contain visible blood, as well as for any contact with non-intact skin, mucous membranes, and intact skin that is visibly soiled with blood and/or body fluids. Summaries of Standard Precautions, Droplet Precautions, Air-borne Precautions, and Contact Precautions are available on line. 93 , 94 , 95 , 96 Hand Washing Hand washing is considered the most important and cost-effective individual intervention in the prevention of hospital-acquired infections in children and health care providers. 97 Its significance in medical practice had not been universally accepted despite the pioneering work by Oliver Wendell Holmes 98 (1843) and Ignaz Semmelweis 99 (1846), who separately recognized the role of the contaminated hands of physicians performing autopsies in the spread of puerperal fever due to Streptococcus sepsis and how by washing their hands before delivering a baby they could reduce maternal mortality by 90%! Unfortunately, the scientific basis for hand washing was not established until the introduction of the germ theory of disease by Louis Pasteur 100 and the discovery of the microorganism that caused anthrax ( Bacillus anthracis ) by Robert Koch 101 in the late 19th century. More than one and a half centuries later, and with strong evidence that health care providers are one of the most frequent sources of infection transmission among patients, 102 health care providers' compliance with hand hygiene protocols in the hospital environment is generally small (5%-48%) and difficult to change, 103 , 104 , 105 , 106 , 107 , 108 , 109 especially in intensive care areas, operating rooms, and postanesthesia care units. The risk of pathogen transmission through the hands is proportional to the power of the number of times a child is touched. 110 Table 50-10 presents a summary of the indications for hand washing and antisepsis. Table 50-10 Indications for Hand Washing and Antisepsis Hand washing is defined as a process for removal of soil and transient microorganisms from the hands. Hands should be washed with soap and water or disinfected. 1. When hands are visibly dirty or contaminated with proteinaceous material or are visibly soiled with blood or other body fluids, wash hands with either a non-antimicrobial soap and water or an antimicrobial soap and water. 2. If hands are not visibly soiled, use an alcohol-based hand rub for routinely decontaminating hands in all other clinical situations described in items 3 to 10. Alternatively, wash hands with an antimicrobial soap and water in all clinical situations described in items 3 to 10. 3. Decontaminate hands before having direct contact with patients. 4. Decontaminate hands before donning sterile gloves when inserting a central intravascular catheter. 5. Decontaminate hands before inserting indwelling urinary catheters, peripheral vascular catheters, or other invasive devices that do not require a surgical procedure. 6. Decontaminate hands after contact with a patient's intact skin (e.g., when taking a pulse or blood pressure and lifting a patient). 7. Decontaminate hands after contact with body fluids or excretions, mucous membranes, non-intact skin, and wound dressings if hands are not visibly soiled. 8. Decontaminate hands if moving from a contaminated body site to a clean body site during patient care. Decontaminate hands after contact with inanimate objects (including medical equipment) in the immediate vicinity of the patient. 9. Decontaminate hands after removing gloves. 10. Before eating and after using a rest room, wash hands with a non-antimicrobial soap and water or with an antimicrobial soap and water. 11. Antimicrobial-impregnated wipes (i.e., towelettes) may be considered as an alternative to washing hands with non-antimicrobial soap and water. Because they are not as effective as alcohol-based hand rubs or washing hands with an antimicrobial soap and water for reducing bacterial counts on the hands of health care workers, they are not a substitute for using an alcohol-based hand rub or antimicrobial soap. 12. Wash hands with non-antimicrobial soap and water or with antimicrobial soap and water if exposure to Bacillus anthracis is suspected or proven. The physical action of washing and rinsing hands under such circumstances is recommended because alcohols, chlorhexidine, iodophors, and other antiseptic agents have poor activity against spores. Modified from Boyce JM, Pittet D: Guideline for Hand Hygiene in Health-Care Settings. Recommendations of the Healthcare Infection Control Practices Advisory Committee and the HIPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. Am J Infect Control 2002; 30:S1-S46. © 2009 2009 Compared with soap and water, alcohol-based hand rubs are more effective in reducing microbial colonization of hands. 111 The use of alcohol-based hand soaps prompted some authors to change the term hand washing to hand hygiene . An important addition to the 2002 CDC Hand Washing Guide 103 is to use alcohol-based hand rubs, because they work more rapidly (10–20 seconds compared with 90-120 for hand washing) and can be used while ambulating. These advantages preclude the usual objections of health care workers to hand washing that include a lack of time, absence of sinks, and skin damage. 112 Furthermore, the scarcity of water in developing countries would no longer be a constraint against hand hygiene. After hand washing, it is very important to dry them properly with appropriate paper towels, hot air flow, or both, because the level of pathogen transmission from a health care worker's hands to a patient is greatly increased if the hands are wet. 113 Transmission may also occur from patients' wet sites, such as groins or armpits, or when a health care worker gets his or her hands wet when opening parenteral solutions. It is critical for health institutions to establish written procedures and protocols to support adherence to the recommended hand hygiene practices. Gloves Wearing clean or sterile gloves while caring for children is an effective means of reducing hospital-acquired infections. Gloves remain a supplementary barrier to infection that should not replace proper hand hygiene. Gloves protect patients by reducing health care provider hand contamination and the subsequent transmission of pathogens to other children, provided the gloves are changed between children. Additionally, when the use of gloves is combined with CDC Standard Precautions, they protect the health care provider against exposure to blood-borne infections or infections transmitted by any other body fluids, such as excretions, secretions (except sweat), mucous membranes, and non-intact skin. Recommendation for the use of gloves include: 114 , 115 , 116 • Wear gloves in case of contact with blood or any other potentially infecting body fluid such as excretions, secretions (except sweat), mucous membranes, and non-intact skin. • Remove the gloves immediately after providing care to a child. Staff should not wear the same pair of gloves to take care of more than one child, nor should they touch the surfaces of any equipment, monitoring devices, or even light switches. Contaminated gloves can pass blood or other body fluids to working areas and have proved to be a vector for hepatitis transmission. 56 • Change gloves when taking care of a child if you must move from a contaminated to a clean body site. • Apply hand hygiene measures immediately after removing the gloves because, despite the use of gloves, hands may get contaminated through small holes in the gloves. 102, 117, 118 Microbial contamination of hands and possible infection transmission have been reported even with the use of gloves. 119 • Remove the gloves by using an appropriate technique (so as not to contaminate your hands with the contaminated surface of the gloves). • Alcohol-based hand rub dispensers and clean glove boxes (at least two sizes) should be in place near every patient care site (e.g., on top of every anesthesia cart, medication cart, or in the nursing station). • Disposable gloves should not be washed, resterilized, or disinfected. If gloves are reused, appropriate reprocessing methods should be in place to ensure the physical integrity of the gloves and their full decontamination. • Sterile gloves are much more expensive than clean, disposable gloves and should be used only for certain procedures, such as when hands are in contact with normally sterile body areas or when inserting intravascular or urinary catheters. Clean gloves should be used during any other procedure, including wound dressing. • Latex free gloves should be worn when caring for children at risk for latex allergy. Hand Washing Hand washing is considered the most important and cost-effective individual intervention in the prevention of hospital-acquired infections in children and health care providers. 97 Its significance in medical practice had not been universally accepted despite the pioneering work by Oliver Wendell Holmes 98 (1843) and Ignaz Semmelweis 99 (1846), who separately recognized the role of the contaminated hands of physicians performing autopsies in the spread of puerperal fever due to Streptococcus sepsis and how by washing their hands before delivering a baby they could reduce maternal mortality by 90%! Unfortunately, the scientific basis for hand washing was not established until the introduction of the germ theory of disease by Louis Pasteur 100 and the discovery of the microorganism that caused anthrax ( Bacillus anthracis ) by Robert Koch 101 in the late 19th century. More than one and a half centuries later, and with strong evidence that health care providers are one of the most frequent sources of infection transmission among patients, 102 health care providers' compliance with hand hygiene protocols in the hospital environment is generally small (5%-48%) and difficult to change, 103 , 104 , 105 , 106 , 107 , 108 , 109 especially in intensive care areas, operating rooms, and postanesthesia care units. The risk of pathogen transmission through the hands is proportional to the power of the number of times a child is touched. 110 Table 50-10 presents a summary of the indications for hand washing and antisepsis. Table 50-10 Indications for Hand Washing and Antisepsis Hand washing is defined as a process for removal of soil and transient microorganisms from the hands. Hands should be washed with soap and water or disinfected. 1. When hands are visibly dirty or contaminated with proteinaceous material or are visibly soiled with blood or other body fluids, wash hands with either a non-antimicrobial soap and water or an antimicrobial soap and water. 2. If hands are not visibly soiled, use an alcohol-based hand rub for routinely decontaminating hands in all other clinical situations described in items 3 to 10. Alternatively, wash hands with an antimicrobial soap and water in all clinical situations described in items 3 to 10. 3. Decontaminate hands before having direct contact with patients. 4. Decontaminate hands before donning sterile gloves when inserting a central intravascular catheter. 5. Decontaminate hands before inserting indwelling urinary catheters, peripheral vascular catheters, or other invasive devices that do not require a surgical procedure. 6. Decontaminate hands after contact with a patient's intact skin (e.g., when taking a pulse or blood pressure and lifting a patient). 7. Decontaminate hands after contact with body fluids or excretions, mucous membranes, non-intact skin, and wound dressings if hands are not visibly soiled. 8. Decontaminate hands if moving from a contaminated body site to a clean body site during patient care. Decontaminate hands after contact with inanimate objects (including medical equipment) in the immediate vicinity of the patient. 9. Decontaminate hands after removing gloves. 10. Before eating and after using a rest room, wash hands with a non-antimicrobial soap and water or with an antimicrobial soap and water. 11. Antimicrobial-impregnated wipes (i.e., towelettes) may be considered as an alternative to washing hands with non-antimicrobial soap and water. Because they are not as effective as alcohol-based hand rubs or washing hands with an antimicrobial soap and water for reducing bacterial counts on the hands of health care workers, they are not a substitute for using an alcohol-based hand rub or antimicrobial soap. 12. Wash hands with non-antimicrobial soap and water or with antimicrobial soap and water if exposure to Bacillus anthracis is suspected or proven. The physical action of washing and rinsing hands under such circumstances is recommended because alcohols, chlorhexidine, iodophors, and other antiseptic agents have poor activity against spores. Modified from Boyce JM, Pittet D: Guideline for Hand Hygiene in Health-Care Settings. Recommendations of the Healthcare Infection Control Practices Advisory Committee and the HIPAC/SHEA/APIC/IDSA Hand Hygiene Task Force. Am J Infect Control 2002; 30:S1-S46. © 2009 2009 Compared with soap and water, alcohol-based hand rubs are more effective in reducing microbial colonization of hands. 111 The use of alcohol-based hand soaps prompted some authors to change the term hand washing to hand hygiene . An important addition to the 2002 CDC Hand Washing Guide 103 is to use alcohol-based hand rubs, because they work more rapidly (10–20 seconds compared with 90-120 for hand washing) and can be used while ambulating. These advantages preclude the usual objections of health care workers to hand washing that include a lack of time, absence of sinks, and skin damage. 112 Furthermore, the scarcity of water in developing countries would no longer be a constraint against hand hygiene. After hand washing, it is very important to dry them properly with appropriate paper towels, hot air flow, or both, because the level of pathogen transmission from a health care worker's hands to a patient is greatly increased if the hands are wet. 113 Transmission may also occur from patients' wet sites, such as groins or armpits, or when a health care worker gets his or her hands wet when opening parenteral solutions. It is critical for health institutions to establish written procedures and protocols to support adherence to the recommended hand hygiene practices. Gloves Wearing clean or sterile gloves while caring for children is an effective means of reducing hospital-acquired infections. Gloves remain a supplementary barrier to infection that should not replace proper hand hygiene. Gloves protect patients by reducing health care provider hand contamination and the subsequent transmission of pathogens to other children, provided the gloves are changed between children. Additionally, when the use of gloves is combined with CDC Standard Precautions, they protect the health care provider against exposure to blood-borne infections or infections transmitted by any other body fluids, such as excretions, secretions (except sweat), mucous membranes, and non-intact skin. Recommendation for the use of gloves include: 114 , 115 , 116 • Wear gloves in case of contact with blood or any other potentially infecting body fluid such as excretions, secretions (except sweat), mucous membranes, and non-intact skin. • Remove the gloves immediately after providing care to a child. Staff should not wear the same pair of gloves to take care of more than one child, nor should they touch the surfaces of any equipment, monitoring devices, or even light switches. Contaminated gloves can pass blood or other body fluids to working areas and have proved to be a vector for hepatitis transmission. 56 • Change gloves when taking care of a child if you must move from a contaminated to a clean body site. • Apply hand hygiene measures immediately after removing the gloves because, despite the use of gloves, hands may get contaminated through small holes in the gloves. 102, 117, 118 Microbial contamination of hands and possible infection transmission have been reported even with the use of gloves. 119 • Remove the gloves by using an appropriate technique (so as not to contaminate your hands with the contaminated surface of the gloves). • Alcohol-based hand rub dispensers and clean glove boxes (at least two sizes) should be in place near every patient care site (e.g., on top of every anesthesia cart, medication cart, or in the nursing station). • Disposable gloves should not be washed, resterilized, or disinfected. If gloves are reused, appropriate reprocessing methods should be in place to ensure the physical integrity of the gloves and their full decontamination. • Sterile gloves are much more expensive than clean, disposable gloves and should be used only for certain procedures, such as when hands are in contact with normally sterile body areas or when inserting intravascular or urinary catheters. Clean gloves should be used during any other procedure, including wound dressing. • Latex free gloves should be worn when caring for children at risk for latex allergy. Antimicrobial Prophylaxis Surgical antimicrobial prophylaxis is an essential tool to reduce the risk of postoperative infections, and the anesthesia team plays a central role in ensuring the proper timing of drug administration. 120, 121 The aim of the perioperative administration of antibiotics is to obtain plasma and tissue drug levels exceeding the minimal inhibitory concentration of those organisms most likely to cause an infection. This will reduce the microbial load of the intraoperative contamination to a level not exceeding the host defenses; it is not the intent to cover all possible pathogens, because this can lead to the selection of drug-resistant bacteria. Selection of the Antimicrobial Agent Several antimicrobial prophylaxis guidelines have been published ( Table 50-11 ). For most surgical procedures that do not involve chronically colonized organs, the most common pathogens are the skin flora, Streptococcus and Staphylococcus . A first-generation cephalosporin (i.e., cefazolin) can provide cost-effective coverage for these organisms. Surgical procedures that involve contamination from the bowel require antibiotic treatment against gram-negative and anaerobic pathogens. For these procedures, cefoxitin, cefotetan, or a second-generation cephalosporin is appropriate. 122 The selection of antibiotics requires consideration of resistance patterns as determined by local microbiology or health center infectious disease departments. The newer-generation broad-spectrum antibiotics should not be used for routine antibiotic prophylaxis but should be reserved for the treatment of resistant organisms. Moreover, the dose of antibiotic selected should be based on the child's weight or body mass index; administration should be repeated intraoperatively if surgery exceeds more than two half-lives after the first antibiotic administration (see Table 50-11 ), if the duration of surgery exceeds 4 to 8 hours, if blood loss is extreme, or if the drug has a particularly short half-life (e.g., penicillin or cefoxitin) to ensure appropriate tissue concentrations of antibiotic until wound closure. 123 Table 50-11 Suggested Initial Dose and Time to Redosing for Antimicrobials Commonly Used for Surgical Prophylaxis Antimicrobial Half-Life Normal Renal Function (hr) Half-Life End-Stage Renal Disease (hr) Recommended Infusion Time (min) Standard Intravenous Dose (g) Weight-Based Dose Recommendation * (mg) Recommended Dosing Interval †(hr) Aztreonam 1.5-2 6 3-5 ‡ 1–2 Max 2 g (adults) 3–5 Ciprofloxacin 3.5-5 5–9 60 400 mg 400 mg 4–10 Cefazolin 1.2-2.5 40–70 3-5 ‡ 15-60 § 1–2 20-30 mg/kg 2–5 1 g < 80 kg 2 g ≥ 80 kg Cefuroxime 1–2 15–22 3-5 ‡ 15-60 § 1.5 50 mg/kg 3–4 Cefamandole 0.5-2.1 12.3-18 ‖ 3-5 ‡ 15-60 § 1 3–4 Cefoxitin 0.5-1.1 6.5-23 3-5 ‡ 15-60 § 1–2 20-40 mg/kg 2–3 Cefotetan 2.8-4.6 13–25 3-5 ‡ 20-60 § 1–2 20-40 mg/kg 3–6 Clindamycin 2-5.1 3.5-5.0 ¶ 10–60 600–900 <10 kg: at least 3–6 (Do not exceed 30 mg/min) 37.5 mg ≥10 kg: 3-6 mg/kg Erythromycin base 0.8-3 5–6 NA 1 g orally 19, 18, 9 hr before surgery 9-13 mg/kg NA Gentamicin 2–3 50–70 1.5 mg/kg ** See footnote ** 3–6 Neomycin 2-3 hours (3% absorbed under normal GI conditions 12-≥ 24 NA 1 g orally 19, 18, 9 hr before surgery 20 mg/kg NA Metronidazole 6–14 7-21 no change 30–60 0.5-1 15 mg/kg (adult) 7.5 mg/kg on subsequent doses 6–8 Vancomycin 4–6 44.1-406.4 (Cl cr <10 mL/min) 1 g ≥ 60 min (use longer infusion time if dose < 1 g) 1.0 10-15 mg/kg (adult) 6–12 DW, dosing weight; IBW, ideal body weight; NA, not applicable. * Weight-based doses are primarily from published pediatric recommendations. †For procedures of long duration, antimicrobials should be redosed at intervals of 1 to 2 times the half-life of the drug. The intervals in the table were calculated for patients with normal renal function. ‡ Dose injected directly into vein or running intravenous fluids. § Intermittent intravenous infusion. ‖ In patients with a serum creatinine value of 5 to 9 mg/dL. ¶ The half-life of clindamycin is the same or slightly increased in patients with end-stage renal disease compared with patients with normal renal function. ** If the patient's weight is 30% above the ideal body weight, dosing weight can be determined as follows: DW = IBW + 0.4 (total body weight − IBW). The Timing of Antibiotic Prophylaxis A key element in the prevention of surgical site infection is the timely administration of prophylactic antibiotics. For most surgical procedures, a single prophylactic dose of antibiotics should be administered 30 to 60 minutes before the skin incision. This should provide appropriate plasma concentrations of the antibiotic. 124, 125 However, in the case of children, intravenous access is often established after induction of anesthesia. With a brief time interval between establishing intravenous access and skin incision, it is important to administer the antibiotics as soon as possible after intravenous access is established. If vancomycin must be used for prophylaxis, it should be infused slowly over 60 minutes (to minimize the risk of severe hypotension) beginning within 2 hours of skin incision. If a tourniquet is required, the full antibiotic dose should be administered before the tourniquet is pressurized. 126 Postsurgical prophylactic antibiotics are not necessary for most procedures and should generally be stopped within 24 hours after the surgical procedure. 126 Allergy to β-Lactams Several studies have shown that the true incidence of allergy is less than that reflected in medical charts. 127 For surgical procedures where cephalosporins are the prophylaxis of choice, alternative antibiotics should be administered to those children at high risk for serious adverse reactions or allergy, based on their history or diagnostic tests (e.g., skin testing). However, the incidence of adverse reactions to cephalosporins in children with reported allergy to penicillin is rare; further-more, skin testing does not reliably predict the likelihood of adverse reactions to cephalosporins in those with reported allergy to penicillin. 128 , 129 , 130 For the most part, "allergies" to oral antibiotics that appear on children's charts (rash, vomiting, gastrointestinal disturbances) are reactions to the additives in the antibiotic formulation including food dyes, fillers, and other compounds. Intravenous administration of small test doses of the pure antibiotics in a fully monitored (and anesthetized) child with a so-called allergy may be used to establish the child's susceptibility to an allergic reaction to the antibiotic. In the case of surgical procedures where antibiotic prophylaxis is mainly directed at gram-positive cocci, children who are truly allergic to β-lactams (cephalosporins) should receive either vancomycin or clindamycin. 122 Indications for Prophylactic Antibiotics Surgical wounds are classified in four categories ( Table 50-12 ). The use of antibiotic prophylaxis for postoperative infections is well established for clean-contaminated procedures. Within the clean category, prophylaxis has been traditionally reserved for surgical procedures involving a foreign body implantation or for any surgical procedure where a surgical site infection would be catastrophic (e.g., cardiac surgery or neurosurgical procedures). However, there is evidence to demonstrate that postoperative infections resulting from procedures not involving prosthetic elements are underreported; estimates show that over 50% of all complications occur after the patient is discharged from hospital and are unrecognized by the surgical team. Therefore, antibiotic prophylaxis is also recommended for certain procedures such as herniorrhaphy. 131, 132 The direct and indirect costs of these complications will not affect the hospital budget; however, they represent a high cost for the community at large. In the case of contaminated or dirty procedures, bacterial contamination or infection is established before the procedure begins. Accordingly, the perioperative administration of antibiotics is a therapeutic, not a prophylactic, measure. The use of antibiotics in children has implications not only for the response to the current treatment but also to future treatments. Thus, all medical professionals are jointly responsible for the rational use of antibiotics. Table 50-12 Wound Classification System Wound Category Description Class I/clean Uninfected wound with no inflammation and the respiratory, alimentary, genital, or uninfected urinary tract is not entered. Clean wounds primarily are closed and drained, when necessary, with closed drainage. Operative wounds after blunt trauma may be included in this category if they meet criteria. Class II/clean contaminated Operative wound in which the respiratory, alimentary, genital, or urinary tract is entered under controlled conditions and without unusual contamination. Specifically, operations involving the biliary tract, appendix, vagina, and oropharynx are included in the category, provided no evidence of infection or major break in technique is encountered. Class III/contaminated Open, fresh, accidental wounds; operations with major breaks in sterile technique (e.g., open cardiac massage) or gross spillage from the gastrointestinal tract; and incisions in which acute, nonpurulent inflammation is encountered Class IV/dirty-infected Old traumatic wounds with retained devitalized tissue and those that involve existing clinical infection or perforated viscera, suggesting that the organisms causing postoperative infection were present in the operative field before operation. From Neville HL, Lally KP: Pediatric surgical wound infections. Semin Pediatr Infect Dis 2001; 12:124-129. © 2009 2009 Protocols, although effective, require continuous feedback on their acceptance and surgical site infection results. No surgical protocol can replace the judgment of the medical professional; clinical reasoning must be tailored to the individual circumstances. Finally, children with congenital heart disease and many of those with repaired congenital heart disease will require subacute bacterial endocarditis prophylaxis (see also Tables 14-1 and 14-2). 133 Selection of the Antimicrobial Agent Several antimicrobial prophylaxis guidelines have been published ( Table 50-11 ). For most surgical procedures that do not involve chronically colonized organs, the most common pathogens are the skin flora, Streptococcus and Staphylococcus . A first-generation cephalosporin (i.e., cefazolin) can provide cost-effective coverage for these organisms. Surgical procedures that involve contamination from the bowel require antibiotic treatment against gram-negative and anaerobic pathogens. For these procedures, cefoxitin, cefotetan, or a second-generation cephalosporin is appropriate. 122 The selection of antibiotics requires consideration of resistance patterns as determined by local microbiology or health center infectious disease departments. The newer-generation broad-spectrum antibiotics should not be used for routine antibiotic prophylaxis but should be reserved for the treatment of resistant organisms. Moreover, the dose of antibiotic selected should be based on the child's weight or body mass index; administration should be repeated intraoperatively if surgery exceeds more than two half-lives after the first antibiotic administration (see Table 50-11 ), if the duration of surgery exceeds 4 to 8 hours, if blood loss is extreme, or if the drug has a particularly short half-life (e.g., penicillin or cefoxitin) to ensure appropriate tissue concentrations of antibiotic until wound closure. 123 Table 50-11 Suggested Initial Dose and Time to Redosing for Antimicrobials Commonly Used for Surgical Prophylaxis Antimicrobial Half-Life Normal Renal Function (hr) Half-Life End-Stage Renal Disease (hr) Recommended Infusion Time (min) Standard Intravenous Dose (g) Weight-Based Dose Recommendation * (mg) Recommended Dosing Interval †(hr) Aztreonam 1.5-2 6 3-5 ‡ 1–2 Max 2 g (adults) 3–5 Ciprofloxacin 3.5-5 5–9 60 400 mg 400 mg 4–10 Cefazolin 1.2-2.5 40–70 3-5 ‡ 15-60 § 1–2 20-30 mg/kg 2–5 1 g < 80 kg 2 g ≥ 80 kg Cefuroxime 1–2 15–22 3-5 ‡ 15-60 § 1.5 50 mg/kg 3–4 Cefamandole 0.5-2.1 12.3-18 ‖ 3-5 ‡ 15-60 § 1 3–4 Cefoxitin 0.5-1.1 6.5-23 3-5 ‡ 15-60 § 1–2 20-40 mg/kg 2–3 Cefotetan 2.8-4.6 13–25 3-5 ‡ 20-60 § 1–2 20-40 mg/kg 3–6 Clindamycin 2-5.1 3.5-5.0 ¶ 10–60 600–900 <10 kg: at least 3–6 (Do not exceed 30 mg/min) 37.5 mg ≥10 kg: 3-6 mg/kg Erythromycin base 0.8-3 5–6 NA 1 g orally 19, 18, 9 hr before surgery 9-13 mg/kg NA Gentamicin 2–3 50–70 1.5 mg/kg ** See footnote ** 3–6 Neomycin 2-3 hours (3% absorbed under normal GI conditions 12-≥ 24 NA 1 g orally 19, 18, 9 hr before surgery 20 mg/kg NA Metronidazole 6–14 7-21 no change 30–60 0.5-1 15 mg/kg (adult) 7.5 mg/kg on subsequent doses 6–8 Vancomycin 4–6 44.1-406.4 (Cl cr <10 mL/min) 1 g ≥ 60 min (use longer infusion time if dose < 1 g) 1.0 10-15 mg/kg (adult) 6–12 DW, dosing weight; IBW, ideal body weight; NA, not applicable. * Weight-based doses are primarily from published pediatric recommendations. †For procedures of long duration, antimicrobials should be redosed at intervals of 1 to 2 times the half-life of the drug. The intervals in the table were calculated for patients with normal renal function. ‡ Dose injected directly into vein or running intravenous fluids. § Intermittent intravenous infusion. ‖ In patients with a serum creatinine value of 5 to 9 mg/dL. ¶ The half-life of clindamycin is the same or slightly increased in patients with end-stage renal disease compared with patients with normal renal function. ** If the patient's weight is 30% above the ideal body weight, dosing weight can be determined as follows: DW = IBW + 0.4 (total body weight − IBW). The Timing of Antibiotic Prophylaxis A key element in the prevention of surgical site infection is the timely administration of prophylactic antibiotics. For most surgical procedures, a single prophylactic dose of antibiotics should be administered 30 to 60 minutes before the skin incision. This should provide appropriate plasma concentrations of the antibiotic. 124, 125 However, in the case of children, intravenous access is often established after induction of anesthesia. With a brief time interval between establishing intravenous access and skin incision, it is important to administer the antibiotics as soon as possible after intravenous access is established. If vancomycin must be used for prophylaxis, it should be infused slowly over 60 minutes (to minimize the risk of severe hypotension) beginning within 2 hours of skin incision. If a tourniquet is required, the full antibiotic dose should be administered before the tourniquet is pressurized. 126 Postsurgical prophylactic antibiotics are not necessary for most procedures and should generally be stopped within 24 hours after the surgical procedure. 126 Allergy to β-Lactams Several studies have shown that the true incidence of allergy is less than that reflected in medical charts. 127 For surgical procedures where cephalosporins are the prophylaxis of choice, alternative antibiotics should be administered to those children at high risk for serious adverse reactions or allergy, based on their history or diagnostic tests (e.g., skin testing). However, the incidence of adverse reactions to cephalosporins in children with reported allergy to penicillin is rare; further-more, skin testing does not reliably predict the likelihood of adverse reactions to cephalosporins in those with reported allergy to penicillin. 128 , 129 , 130 For the most part, "allergies" to oral antibiotics that appear on children's charts (rash, vomiting, gastrointestinal disturbances) are reactions to the additives in the antibiotic formulation including food dyes, fillers, and other compounds. Intravenous administration of small test doses of the pure antibiotics in a fully monitored (and anesthetized) child with a so-called allergy may be used to establish the child's susceptibility to an allergic reaction to the antibiotic. In the case of surgical procedures where antibiotic prophylaxis is mainly directed at gram-positive cocci, children who are truly allergic to β-lactams (cephalosporins) should receive either vancomycin or clindamycin. 122 Indications for Prophylactic Antibiotics Surgical wounds are classified in four categories ( Table 50-12 ). The use of antibiotic prophylaxis for postoperative infections is well established for clean-contaminated procedures. Within the clean category, prophylaxis has been traditionally reserved for surgical procedures involving a foreign body implantation or for any surgical procedure where a surgical site infection would be catastrophic (e.g., cardiac surgery or neurosurgical procedures). However, there is evidence to demonstrate that postoperative infections resulting from procedures not involving prosthetic elements are underreported; estimates show that over 50% of all complications occur after the patient is discharged from hospital and are unrecognized by the surgical team. Therefore, antibiotic prophylaxis is also recommended for certain procedures such as herniorrhaphy. 131, 132 The direct and indirect costs of these complications will not affect the hospital budget; however, they represent a high cost for the community at large. In the case of contaminated or dirty procedures, bacterial contamination or infection is established before the procedure begins. Accordingly, the perioperative administration of antibiotics is a therapeutic, not a prophylactic, measure. The use of antibiotics in children has implications not only for the response to the current treatment but also to future treatments. Thus, all medical professionals are jointly responsible for the rational use of antibiotics. Table 50-12 Wound Classification System Wound Category Description Class I/clean Uninfected wound with no inflammation and the respiratory, alimentary, genital, or uninfected urinary tract is not entered. Clean wounds primarily are closed and drained, when necessary, with closed drainage. Operative wounds after blunt trauma may be included in this category if they meet criteria. Class II/clean contaminated Operative wound in which the respiratory, alimentary, genital, or urinary tract is entered under controlled conditions and without unusual contamination. Specifically, operations involving the biliary tract, appendix, vagina, and oropharynx are included in the category, provided no evidence of infection or major break in technique is encountered. Class III/contaminated Open, fresh, accidental wounds; operations with major breaks in sterile technique (e.g., open cardiac massage) or gross spillage from the gastrointestinal tract; and incisions in which acute, nonpurulent inflammation is encountered Class IV/dirty-infected Old traumatic wounds with retained devitalized tissue and those that involve existing clinical infection or perforated viscera, suggesting that the organisms causing postoperative infection were present in the operative field before operation. From Neville HL, Lally KP: Pediatric surgical wound infections. Semin Pediatr Infect Dis 2001; 12:124-129. © 2009 2009 Protocols, although effective, require continuous feedback on their acceptance and surgical site infection results. No surgical protocol can replace the judgment of the medical professional; clinical reasoning must be tailored to the individual circumstances. Finally, children with congenital heart disease and many of those with repaired congenital heart disease will require subacute bacterial endocarditis prophylaxis (see also Tables 14-1 and 14-2). 133

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4179248/

Cross-Sectional Study of Malnutrition and Associated Factors among School Aged Children in Rural and Urban Settings of Fogera and Libo Kemkem Districts, Ethiopia

Introduction Little information is available on malnutrition-related factors among school-aged children ≥5 years in Ethiopia. This study describes the prevalence of stunting and thinness and their related factors in Libo Kemkem and Fogera, Amhara Regional State and assesses differences between urban and rural areas. Methods In this cross-sectional study, anthropometrics and individual and household characteristics data were collected from 886 children. Height-for-age z-score for stunting and body-mass-index-for-age z-score for thinness were computed. Dietary data were collected through a 24-hour recall. Bivariate and backward stepwise multivariable statistical methods were employed to assess malnutrition-associated factors in rural and urban communities. Results The prevalence of stunting among school-aged children was 42.7% in rural areas and 29.2% in urban areas, while the corresponding figures for thinness were 21.6% and 20.8%. Age differences were significant in both strata. In the rural setting, fever in the previous 2 weeks (OR: 1.62; 95% CI: 1.23–2.32), consumption of food from animal sources (OR: 0.51; 95% CI: 0.29–0.91) and consumption of the family's own cattle products (OR: 0.50; 95% CI: 0.27–0.93), among others factors were significantly associated with stunting, while in the urban setting, only age (OR: 4.62; 95% CI: 2.09–10.21) and years of schooling of the person in charge of food preparation were significant (OR: 0.88; 95% CI: 0.79–0.97). Thinness was statistically associated with number of children living in the house (OR: 1.28; 95% CI: 1.03–1.60) and family rice cultivation (OR: 0.64; 95% CI: 0.41–0.99) in the rural setting, and with consumption of food from animal sources (OR: 0.26; 95% CI: 0.10–0.67) and literacy of head of household (OR: 0.24; 95% CI: 0.09–0.65) in the urban setting. Conclusion The prevalence of stunting was significantly higher in rural areas, whereas no significant differences were observed for thinness. Various factors were associated with one or both types of malnutrition, and varied by type of setting. To effectively tackle malnutrition, nutritional programs should be oriented to local needs. Introduction Little information is available on malnutrition-related factors among school-aged children ≥5 years in Ethiopia. This study describes the prevalence of stunting and thinness and their related factors in Libo Kemkem and Fogera, Amhara Regional State and assesses differences between urban and rural areas. Methods In this cross-sectional study, anthropometrics and individual and household characteristics data were collected from 886 children. Height-for-age z-score for stunting and body-mass-index-for-age z-score for thinness were computed. Dietary data were collected through a 24-hour recall. Bivariate and backward stepwise multivariable statistical methods were employed to assess malnutrition-associated factors in rural and urban communities. Results The prevalence of stunting among school-aged children was 42.7% in rural areas and 29.2% in urban areas, while the corresponding figures for thinness were 21.6% and 20.8%. Age differences were significant in both strata. In the rural setting, fever in the previous 2 weeks (OR: 1.62; 95% CI: 1.23–2.32), consumption of food from animal sources (OR: 0.51; 95% CI: 0.29–0.91) and consumption of the family's own cattle products (OR: 0.50; 95% CI: 0.27–0.93), among others factors were significantly associated with stunting, while in the urban setting, only age (OR: 4.62; 95% CI: 2.09–10.21) and years of schooling of the person in charge of food preparation were significant (OR: 0.88; 95% CI: 0.79–0.97). Thinness was statistically associated with number of children living in the house (OR: 1.28; 95% CI: 1.03–1.60) and family rice cultivation (OR: 0.64; 95% CI: 0.41–0.99) in the rural setting, and with consumption of food from animal sources (OR: 0.26; 95% CI: 0.10–0.67) and literacy of head of household (OR: 0.24; 95% CI: 0.09–0.65) in the urban setting. Conclusion The prevalence of stunting was significantly higher in rural areas, whereas no significant differences were observed for thinness. Various factors were associated with one or both types of malnutrition, and varied by type of setting. To effectively tackle malnutrition, nutritional programs should be oriented to local needs. Introduction Adequate nutrition is essential during childhood to ensure healthy growth, proper organ formation and function, a strong immune system, and neurological and cognitive development [1] . Nutritional status has a major impact on children's survival mainly due to the synergistic relationships between malnutrition and diseases [2] , [3] . In Eastern and Southern Africa, malnutrition is a major underlying cause of the persistently high child mortality, contributing to more than a third of all deaths among children under age 5 [4] . The two main anthropometric indicators used to define malnutrition– stunting, and wasting or thinness– represent different histories of nutritional insult to the child. Linear growth retardation (chronic malnutrition or stunting) is frequently associated with repeated exposure to adverse economic conditions, poor sanitation, and the interactive effects of poor nutrient intakes and infection. Low weight-for-height or low body mass index (BMI) for age (acute malnutrition, wasting or thinness) is generally associated with recent illness and/or food deprivation [5] . The causes of childhood malnutrition are diverse, multidimensional, and interrelated. An analytical framework suggested by the United Nations Children's Fund (UNICEF) categorizes the causes into (a) immediate causes: inadequate dietary intake and illness, (b) underlying causes: insufficient access to food in a household; inadequate health services and unhealthy environment; and inadequate care for children and women at the household level, and (c) basic causes: insufficient current and potential resources at societal level [6] . In Sub-Saharan Africa, various indicators of social economic status have been associated with children's nutritional status, such as maternal and paternal educational level, parental income, and family assets [7] – [9] . In addition, child nutrition outcomes in developing countries have been characterized by large rural-urban disparities over the last few decades [10] . In Ethiopia, child malnutrition continues to be a major public health problem. According to the Ethiopian National Demographic Health Survey (2011), the prevalence of both wasting and stunting in children under 5 years is very high (10% and 44% respectively) [11] , while the situation in older children is not so well known [12] , [13] . Furthermore, rural-urban disparities in child nutrition, as well as growing urbanization that results in increasing inequalities in urban areas, underlines the need to improve our knowledge of the main drivers of urban-rural differences [14] . The Amhara Region is one of the four primary agricultural regions in Ethiopia [15] , and most households rely upon livestock and crop sales to generate cash income. This region, and especially the Tana Zuria Zone, has a moderate population density, fertile soils and good rainfall. For this reason, it is amongst the most food self-sufficient regions in the country [16] . Despite this good regional profile, other factors may be determining the high prevalence of infant malnutrition in this area [17] . According to the Food and Agriculture Organization of the United Nations (FAO), the four pillars of food security are food availability, stability of the food supply, food access and the utilization of food by the body [18] . In our context, availability is strongly affected by seasonality; many households are only able to produce sufficient food to meet their food requirements for less than six months of the year [19] . Food access may be affected by market conditions, but also by cultural and religious practices. For example, the high number of fasting days commemorated by the Ethiopian Orthodox Church, the main religion in the country, may have repercussions on the nutritional status of the community, particularly in rural Ethiopia [20] . Even if people get enough to eat, good nutrition requires access to a sufficient, supply of varied, safe and nutritious food to meet daily nutritional requirements [21] . Although diet diversity questionnaires are extensively used in Ethiopia to investigate relationships between food intake and nutritional status, there is limited knowledge of nutrition outcomes, dietary practices and socioeconomic factors among school-aged children in this specific context [17] . The present study aimed to [22] describe the prevalence of stunting and thinness, and their related factors, including dietary habits, and [23] document the differences in nutritional status across urban and rural areas accounting for household and individual characteristics in school-aged children in Libo Kemkem and Fogera, Amhara regional State, Ethiopia. Material and Methods Study area and population The study was carried out during May–June 2009 in the districts ( woredas ) of Libo Kemkem and Fogera (Amhara regional state, Ethiopia). Libo Kemkem and Fogera woredas are located in the Tana Zuria Livelihood Zone, within the Amhara Regional State, northwestern Ethiopia at an altitude of 2,000 m above sea level. According to the 2009 census, the population was 198,374 and 226,595 for Libo Kemkem and Fogera, respectively. These two districts are located in a black cotton clay soil flat plain. Temperatures are relatively high, but rainfall is unusually abundant, with a mean of 1173 mm per annum. Agricultural activities are dependent on a single rainy season (from June to September). Maize, barley and millet are the main food crops, while rice, vetch and chickpea are the main cash crops. Livestock holdings in sheep and cattle are relatively modest, but livestock and butter sales make a substantial complement to the predominant crop sales. The major hazards to crop production and livestock are pests, occasional flooding, and zoonosis such as anthrax, trypanosomiasis, pasteurellosis and black leg [16] . Study design This cross-sectional survey was part of a UBS Optimus Foundation funded project called Visceral Leishmaniasis (VL) and Malnutrition in Amhara State, Ethiopia. Among its specific objectives, the project aimed to characterize nutritional, immunological, and parasitological factors in school-aged children in the districts of Fogera and Libo Kemkem. Other methodological aspects have previously been published [24] – [26] . Sampling was carried out by multistage cluster survey. A total of 886 children aged 4 to 15 years were recruited. Primary sampling units were sub-districts ( kebeles ) with a high incidence of VL according to the 2008 register of the Addis Zemen VL Treatment Centre: one urban (Addis Zemen) and the rest rural: Bura, Yifag Akababi and Agita from Libo Kemkem, and Sifatra and Rib Gebriel from Fogera. Secondary sampling units were randomly selected villages ( gotts ) in each of the selected sub-districts. Third-stage sampling units were randomly selected households in each of the villages. All children with reported age between 4 and 15 years living in the household at the time of the survey were recruited. Sample size was calculated according to previous estimates of malnutrition for children under age 5 in the area and taking into account a design effect of 2, corresponding to the complex design. Data collection All children were measured and weighed according to standard WHO procedures [27] . Weight was measured to the nearest 0.1 kg on a battery-powered digital scale (SECA 881©). Standing height was measured to the nearest 0.1 cm using a portable adult/infant measuring unit (PE-AIM-101©). A pre-tested questionnaire translated into Amharic, the local language, was administered to the caretaker/head of household (HH) of each child in the study by trained medical personnel (nurses and health officers). We asked about individual demographic characteristics, health status and behavior determinants. The following household variables were also recorded: household socio-demographic characteristics, person in charge of food preparation (PCFP), house construction material and assets (land and cultivation, domestic animal assets) and community variables. Dietary data was collected through a 24-hour diet recall. Statistical analysis Stunting and thinness were the main outcomes of interest, defined as height-for-age z-score (HAZ) 0.05). Acute malnutrition in rural communities In the bivariate analysis ( Table S2 ), the prevalence of acute malnutrition was higher in children age 10 and over (OR: 4.74; 95% CI: 3.24–6.94) and among boys (OR: 1.58; 95% CI: 1.18–2.12). Children from rural communities who herded the cattle were 2.43 times more likely to be thin (95%CI: 1.63–3.61) than those who did not. The number of children living in the house (p = 0.007) and teff cultivation (OR: 1.53; 95% CI: 1.02–2.30) were positively associated with higher prevalence of thinness, while living with a HH under 40 years old and rice farming showed an inverse association with thinness (OR: 0.75; 95% CI: 0.56–0.99 and 0.63; 95% CI: 0.42–0.95, respectively). After adjusting for socio demographic and household characteristics in the model, sex differences lost significance, and the relationship between thinness and age was slightly weakened (OR: 4.11; 95% CI: 2.74–6.16) ( Table 4 ). Children from rural communities were significantly less likely to be thin if the HH was female (OR: 0.40; 95% CI: 0.16–0.70). The number of children living in the house showed a positive relationship with thinness in this setting (p = 0.027), while children from households that cultivate rice were less likely to be thin (OR: 0.64; 95% CI: 0.41–0.99). 10.1371/journal.pone.0105880.t004 Table 4 Multivariable logistic regression analysis of thinness in school-aged children, stratified by setting in Libo Kemkem and Fogera districts, Ethiopia, May–July 2009. VARIABLES RURAL (N = 711) URBAN (N = 178) n (%) Adjusted OR 95% CI p value n (%) Adjusted OR 95% CI p value CHILD CHARACTERISTICS Sex male 97 (26.22) 1 21 (23.33) 1 female 56 (16.57) 0.69 (0.46–1.03) 0.073 16 (18.18) 0.86 (0.39–1.92) 0.717 Age group 0.05). Acute malnutrition in rural communities In the bivariate analysis ( Table S2 ), the prevalence of acute malnutrition was higher in children age 10 and over (OR: 4.74; 95% CI: 3.24–6.94) and among boys (OR: 1.58; 95% CI: 1.18–2.12). Children from rural communities who herded the cattle were 2.43 times more likely to be thin (95%CI: 1.63–3.61) than those who did not. The number of children living in the house (p = 0.007) and teff cultivation (OR: 1.53; 95% CI: 1.02–2.30) were positively associated with higher prevalence of thinness, while living with a HH under 40 years old and rice farming showed an inverse association with thinness (OR: 0.75; 95% CI: 0.56–0.99 and 0.63; 95% CI: 0.42–0.95, respectively). After adjusting for socio demographic and household characteristics in the model, sex differences lost significance, and the relationship between thinness and age was slightly weakened (OR: 4.11; 95% CI: 2.74–6.16) ( Table 4 ). Children from rural communities were significantly less likely to be thin if the HH was female (OR: 0.40; 95% CI: 0.16–0.70). The number of children living in the house showed a positive relationship with thinness in this setting (p = 0.027), while children from households that cultivate rice were less likely to be thin (OR: 0.64; 95% CI: 0.41–0.99). 10.1371/journal.pone.0105880.t004 Table 4 Multivariable logistic regression analysis of thinness in school-aged children, stratified by setting in Libo Kemkem and Fogera districts, Ethiopia, May–July 2009. VARIABLES RURAL (N = 711) URBAN (N = 178) n (%) Adjusted OR 95% CI p value n (%) Adjusted OR 95% CI p value CHILD CHARACTERISTICS Sex male 97 (26.22) 1 21 (23.33) 1 female 56 (16.57) 0.69 (0.46–1.03) 0.073 16 (18.18) 0.86 (0.39–1.92) 0.717 Age group <10 years 53 (11.83) 1 18 (15.02) 1 ≥10 years 100 (38.84) 4.11 (2.74–6.16) 0.000 19 (32.81) 3.67 (1.63–8.30) 0.002 Does the child herd the cattle? No 40 (13.51) 1 36 (21.05) 1 Yes 113 (27.49) 1.50 (0.96–2.36) 0.076 1 (14.29) 0.55 (0.06–5.03) 0.598 DOES THE CHILD CONSUME… Any food from animal sources No 129 (22.28) 1 18 (28.13) 1 Yes 24 (18.60) 0.83 (0.49–1.41) 0.493 19 (16.67) 0.26 (0.10–0.67) 0.005 HOUSEHOLD AND LAND PRODUCTION Sex of head of household Male 146 (22.26) 1 23 (22.33) 1 Female 7 (13.46) 0.40 (0.16–0.70) 0.043 14 (18.67) 0.67 (0.26–1.72) 0.407 Literacy of head of household (can read and write) No 93 (21.83) 1 25 (25.77) 1 Yes 60 (21.51) 1.39 (0.91–2.11) 0.127 12 (14.81) 0.24 (0.09–0.65) 0.005 Number of people living in the house Mean (sd) AOR 95% CI p value Mean (sd) AOR 95% CI p value 6.45 (1.68) 0.87 (0.75–1.00) 0.054 5.14 (1.72) 0.87 (0.64–1.20) 0.403 Number of children in the house 3.06 (1.14) 1.28 (1.03–1.60) 0.027 2.19 (0.94) 1.02 (0.61–1.70) 0.955 n (%) AOR 95% CI p value n (%) AOR 95% CI p value Does the family cultivate rice? No 115 (24.01) 1 36 (20.45) 1 Yes 38 (16.59) 0.64 (0.41–0.99) 0.045 1 (50.00) 3.26 (0.15–72.94) 0.456 Acute malnutrition in urban communities In the bivariate analysis ( Table S2 ), no significant associations with thinness were found apart from age group (OR: 2.76; 95% CI: 1.13–5.80). After adjusting the analysis, age group remained significantly related to thinness (OR: 3.67; 95% CI: 1.63–8.30). Food consumption from animal sources on the day before the survey was inversely associated with acute malnutrition (OR: 0.26; 95% CI: 0.10–0.67) and thinness prevalence was lower among in households headed by literate persons (OR: 0.24; 95% CI: 0.09–0.65) ( Table 4 ). Acute malnutrition in rural communities In the bivariate analysis ( Table S2 ), the prevalence of acute malnutrition was higher in children age 10 and over (OR: 4.74; 95% CI: 3.24–6.94) and among boys (OR: 1.58; 95% CI: 1.18–2.12). Children from rural communities who herded the cattle were 2.43 times more likely to be thin (95%CI: 1.63–3.61) than those who did not. The number of children living in the house (p = 0.007) and teff cultivation (OR: 1.53; 95% CI: 1.02–2.30) were positively associated with higher prevalence of thinness, while living with a HH under 40 years old and rice farming showed an inverse association with thinness (OR: 0.75; 95% CI: 0.56–0.99 and 0.63; 95% CI: 0.42–0.95, respectively). After adjusting for socio demographic and household characteristics in the model, sex differences lost significance, and the relationship between thinness and age was slightly weakened (OR: 4.11; 95% CI: 2.74–6.16) ( Table 4 ). Children from rural communities were significantly less likely to be thin if the HH was female (OR: 0.40; 95% CI: 0.16–0.70). The number of children living in the house showed a positive relationship with thinness in this setting (p = 0.027), while children from households that cultivate rice were less likely to be thin (OR: 0.64; 95% CI: 0.41–0.99). 10.1371/journal.pone.0105880.t004 Table 4 Multivariable logistic regression analysis of thinness in school-aged children, stratified by setting in Libo Kemkem and Fogera districts, Ethiopia, May–July 2009. VARIABLES RURAL (N = 711) URBAN (N = 178) n (%) Adjusted OR 95% CI p value n (%) Adjusted OR 95% CI p value CHILD CHARACTERISTICS Sex male 97 (26.22) 1 21 (23.33) 1 female 56 (16.57) 0.69 (0.46–1.03) 0.073 16 (18.18) 0.86 (0.39–1.92) 0.717 Age group <10 years 53 (11.83) 1 18 (15.02) 1 ≥10 years 100 (38.84) 4.11 (2.74–6.16) 0.000 19 (32.81) 3.67 (1.63–8.30) 0.002 Does the child herd the cattle? No 40 (13.51) 1 36 (21.05) 1 Yes 113 (27.49) 1.50 (0.96–2.36) 0.076 1 (14.29) 0.55 (0.06–5.03) 0.598 DOES THE CHILD CONSUME… Any food from animal sources No 129 (22.28) 1 18 (28.13) 1 Yes 24 (18.60) 0.83 (0.49–1.41) 0.493 19 (16.67) 0.26 (0.10–0.67) 0.005 HOUSEHOLD AND LAND PRODUCTION Sex of head of household Male 146 (22.26) 1 23 (22.33) 1 Female 7 (13.46) 0.40 (0.16–0.70) 0.043 14 (18.67) 0.67 (0.26–1.72) 0.407 Literacy of head of household (can read and write) No 93 (21.83) 1 25 (25.77) 1 Yes 60 (21.51) 1.39 (0.91–2.11) 0.127 12 (14.81) 0.24 (0.09–0.65) 0.005 Number of people living in the house Mean (sd) AOR 95% CI p value Mean (sd) AOR 95% CI p value 6.45 (1.68) 0.87 (0.75–1.00) 0.054 5.14 (1.72) 0.87 (0.64–1.20) 0.403 Number of children in the house 3.06 (1.14) 1.28 (1.03–1.60) 0.027 2.19 (0.94) 1.02 (0.61–1.70) 0.955 n (%) AOR 95% CI p value n (%) AOR 95% CI p value Does the family cultivate rice? No 115 (24.01) 1 36 (20.45) 1 Yes 38 (16.59) 0.64 (0.41–0.99) 0.045 1 (50.00) 3.26 (0.15–72.94) 0.456 Acute malnutrition in urban communities In the bivariate analysis ( Table S2 ), no significant associations with thinness were found apart from age group (OR: 2.76; 95% CI: 1.13–5.80). After adjusting the analysis, age group remained significantly related to thinness (OR: 3.67; 95% CI: 1.63–8.30). Food consumption from animal sources on the day before the survey was inversely associated with acute malnutrition (OR: 0.26; 95% CI: 0.10–0.67) and thinness prevalence was lower among in households headed by literate persons (OR: 0.24; 95% CI: 0.09–0.65) ( Table 4 ). Discussion Our study shows that there is a high prevalence of stunting (39.8%) and thinness (21.4%) among school-aged children in Libo Kemkem and Fogera regions of Ethiopia. The prevalence of stunting was significantly higher in rural areas (42.7% vs. 29.2%), but no significant differences were observed for thinness. These results are similar to those observed in other developing countries [29] . Various intermediate and distal factors like age, consumption of food from animal sources and family size were associated with both types of malnutrition in one or both settings. Other determinants such as years of school attendance of the PCFP and consumption of the family's own cattle products were related to only one kind of malnutrition. Although malnutrition among pre-school children has been well documented in Ethiopia [5] , [30] , [31] , to our knowledge this is the first research to assess factors related to acute and chronic malnutrition stratified by setting in school aged children. These results may assist stakeholders in planning and undertaking contextual and evidence-based policy initiatives. We found that the probability of a child being malnourished increases with age. Age-group differences were significant in both strata for stunting and thinness. No sex differences were found in either strata. As children mature, household socioeconomic characteristics may emerge in conjunction with behavioral and biological variables as important risk factors [32] . Chronic malnutrition The prevalence of stunting in rural areas in our study was higher (42.7%) than that found in a study conducted in the same age group in rural settings of Fogera in 2012 (30.7%) [33] . Our study was carried out in 2009, which may partially explain this difference due to possible improvements in local conditions; another reason may be that children in the Fogera study had to be enrolled in school in order to participate, which could result in selection bias. In addition, our sampling was done in sub-districts with a high incidence of VL, a characteristic that may be associated with fewer resources and worse health status in children. The prevalence of chronic malnutrition in the urban area (29.2%) could not be compared to previous data due to the lack of research targeting this particular age group in this setting. In rural communities, the setting with the highest stunting prevalence, we found several factors associated with chronic malnutrition: age group, fever in the previous 15 days, herding the cattle, consumption of any food from animal sources, sex of the HH, family size, cultivation of millet and consumption of the family's own cattle products. These factors should be considered when targeting chronic malnutrition in this region. Children who had fever in the previous 15 days were 62% more likely to be stunted than those who did not. Infection and malnutrition are intricately linked through extensive, synergistic, antagonistic, and cyclical interactions [34] , [35] . Although our study area is known to be a low endemic area for malaria and leishmaniasis [25] , [36] , other specific infections associated with malnutrition (such as chronic parasitic infestations) are highly prevalent [37] , [38] . Infectious diseases manifested in the form of fever affect both dietary intake and utilization, which may affect child growth. Not having empirical data on specific infections is a limitation, but we considered fever in the previous 15 days could act as a proxy for non-specific infection. On the other hand, stunted growth and related immunosuppression may lead to intermittent fever [39] . In our research, children who herd the cattle were less likely to be stunted. This could be explained by better activity levels in non-stunted children, given that stunted children show behavioral differences in early childhood including apathy and reduced activity, play and exploration [40] . Finally, children from households with millet farming and from families who consume their own cattle products were less likely to be stunted in rural communities. Although most rural families own land and animals (97.6% and 96.3% respectively), we observed that they do not consume their own products as often. Only 11.1% of the children had eaten any meat or fish over the last 24 hours, while 99.6% and 90.4%, respectively, had consumed basic staples and legumes and/or pulses respectively. In Ethiopia, child diet is based mainly based on plant foods like the traditional Ethiopian staple food called " injera ", a yeast-risen flatbread made of a blend of cereals, usually served with legumes or pulses. This may not provide all the nutritional requirements of children [41] . We are aware that market access in this livelihood zone is poor; moreover what little trade interaction exists is restricted to cash crops that are attractive for their high price (i.e. rice and teff), while other essential crops cultivated mainly for personal consumption face disincentives [16] , [42] . In urban communities, age group and years of school of the PCFP were significantly associated with chronic malnutrition in children. This educational factor may operate indirectly to affect children's nutritional status by determining the quality of the child's diet, care and physical environment [30] . The level of education of the PCFP may have a positive impact on his/her knowledge on food facilities, controlling contamination, time and temperature parameters for controlling pathogens, and advice on good dietary habits [43] . Acute malnutrition The prevalence of acute malnutrition in the rural settings of Fogera and Libo Kemkem was 21.6%. The prevalence of underweight found in the study conducted in Fogera was 37.2% [33] . These results cannot be directly compared as different anthropometric indices were used. And again, we did not find any data on thinness to compare with our results in the urban population. In rural settings, age group, sex of the HH, number of children in the house and rice cultivation were factors associated with thinness. The number of children in a household and the prevalence of thinness were positively associated. Larger family size may put children at higher risk for acute malnutrition, which could be due to the imbalance between family size and resources [44] . Those whose families cultivate rice were less likely to be thin in rural communities. In this zone, rice production might be acting as a proxy for better socioeconomic status, as rice consumption is relatively recent but is one of the main cash crops in the area [16] . The low consumption of animal source foods and its association with acute malnutrition has been previously identified as a major contributing factor to delayed growth in children [45] and suboptimal dietary practices among adolescents in Ethiopia [17] . In urban areas, children with a literate HH were 4 times less likely to be thin than those living in houses headed by illiterate adults. Some studies have shown that parental education is associated with more efficient management of limited household resources, improved utilization of available health care services, and better health-promoting behaviors, all of which are associated with better child nutrition [46] , [47] . This result is similar to what we previously observed for stunting and PCFP years of education in the urban setting. A possible explanation could be the existence of an educational gap in urban but not in rural areas. Stunting versus thinness: associated factors Clear differences among risk factors for stunting ( Table 3 ) and thinness ( Table 4 ) emerged from this study. The literature on the causes of stunting is vast, and conventional thinking is summarized in the Lancet series on maternal and child under-nutrition [48] . Recognized causal factors include prenatal and postnatal periods. Stunting is seen as closely tied to poverty and access to services. Less knowledge is available on risk factors for thinness [1] . In our research, risk factors for chronic malnutrition encompass a wide range of variables.. The relatively consistent pattern of related factors for stunting suggests that continued exposure to adverse conditions retards children's linear growth. Conversely, the greater diversity observed in the factors associated with thinness is consistent with the fact that a relatively short period of risk exposure can precipitate its onset in children [32] . In rural communities, children from male-headed households were more likely to be thin than children from female-headed households (p = 0.043), while stunting was significantly more frequent in female-headed households (p = 0.002). The disparate sample size in rural and urban areas may have influenced these results. However, the result in the rural area is consistent with the study conducted in North Ethiopia by Haidar et al. [49] . This study found a significantly higher proportion of stunted and underweight pre-school children in female headed-households, whereas the prevalence of thinness was similar [48] – [50] . Women who are single HH may be removed from their support structures and may face constraints in accessing services, including food, as a result of insecurity, cultural discrimination and limited mobility [48] , [50] . This situation may have a long-term impact in child nutrition. Rural versus urban: associated factors Ethiopia remains one of the least urbanized countries in the world [42] . Globally, malnutrition is less common in urban areas [2] , [31] . We found that malnutrition in rural communities was associated with food habits and the lack of material resources whereas in the urban area, it was better predicted by socio demographic factors. Inequalities in child health outcomes are known to vary between rural and urban areas, and are often due to unequal allocation of resources [51] . Significant urban-rural differences remained in the multivariable model. This shows that even in the presence of important individual factors and socioeconomic variables, area of residence is still a predictor of children's nutritional status. Our results highlight the need to stratify data when rural and urban communities are targeted in nutritional research in this kind of context. Limitations The present study was conducted in two single districts in Ethiopia, thus, the findings may not be generalizable to a larger population. Additionally, the cross-sectional nature of this data does not allow us to examine causality in the relationship between malnutrition and diverse risk factors. Seasonality should be given special attention; the season of the year has a significant effect not only on food security and nutritional status, but also on patterns and trends of infectious disease incidence. Therefore, consecutive measurements are desirable.. This research is part of a project which aimed to characterize nutritional, immunological, and parasitological aspects in school-aged children from urban and rural villages with a high incidence of VL in 2005–07. However, the VL prevalence found in the study was very low [25] , and no association was found between nutritional status and asymptomatic infection [24] . Therefore, we are confident that this limitation does not alter our general conclusions. Chronic malnutrition The prevalence of stunting in rural areas in our study was higher (42.7%) than that found in a study conducted in the same age group in rural settings of Fogera in 2012 (30.7%) [33] . Our study was carried out in 2009, which may partially explain this difference due to possible improvements in local conditions; another reason may be that children in the Fogera study had to be enrolled in school in order to participate, which could result in selection bias. In addition, our sampling was done in sub-districts with a high incidence of VL, a characteristic that may be associated with fewer resources and worse health status in children. The prevalence of chronic malnutrition in the urban area (29.2%) could not be compared to previous data due to the lack of research targeting this particular age group in this setting. In rural communities, the setting with the highest stunting prevalence, we found several factors associated with chronic malnutrition: age group, fever in the previous 15 days, herding the cattle, consumption of any food from animal sources, sex of the HH, family size, cultivation of millet and consumption of the family's own cattle products. These factors should be considered when targeting chronic malnutrition in this region. Children who had fever in the previous 15 days were 62% more likely to be stunted than those who did not. Infection and malnutrition are intricately linked through extensive, synergistic, antagonistic, and cyclical interactions [34] , [35] . Although our study area is known to be a low endemic area for malaria and leishmaniasis [25] , [36] , other specific infections associated with malnutrition (such as chronic parasitic infestations) are highly prevalent [37] , [38] . Infectious diseases manifested in the form of fever affect both dietary intake and utilization, which may affect child growth. Not having empirical data on specific infections is a limitation, but we considered fever in the previous 15 days could act as a proxy for non-specific infection. On the other hand, stunted growth and related immunosuppression may lead to intermittent fever [39] . In our research, children who herd the cattle were less likely to be stunted. This could be explained by better activity levels in non-stunted children, given that stunted children show behavioral differences in early childhood including apathy and reduced activity, play and exploration [40] . Finally, children from households with millet farming and from families who consume their own cattle products were less likely to be stunted in rural communities. Although most rural families own land and animals (97.6% and 96.3% respectively), we observed that they do not consume their own products as often. Only 11.1% of the children had eaten any meat or fish over the last 24 hours, while 99.6% and 90.4%, respectively, had consumed basic staples and legumes and/or pulses respectively. In Ethiopia, child diet is based mainly based on plant foods like the traditional Ethiopian staple food called " injera ", a yeast-risen flatbread made of a blend of cereals, usually served with legumes or pulses. This may not provide all the nutritional requirements of children [41] . We are aware that market access in this livelihood zone is poor; moreover what little trade interaction exists is restricted to cash crops that are attractive for their high price (i.e. rice and teff), while other essential crops cultivated mainly for personal consumption face disincentives [16] , [42] . In urban communities, age group and years of school of the PCFP were significantly associated with chronic malnutrition in children. This educational factor may operate indirectly to affect children's nutritional status by determining the quality of the child's diet, care and physical environment [30] . The level of education of the PCFP may have a positive impact on his/her knowledge on food facilities, controlling contamination, time and temperature parameters for controlling pathogens, and advice on good dietary habits [43] . Acute malnutrition The prevalence of acute malnutrition in the rural settings of Fogera and Libo Kemkem was 21.6%. The prevalence of underweight found in the study conducted in Fogera was 37.2% [33] . These results cannot be directly compared as different anthropometric indices were used. And again, we did not find any data on thinness to compare with our results in the urban population. In rural settings, age group, sex of the HH, number of children in the house and rice cultivation were factors associated with thinness. The number of children in a household and the prevalence of thinness were positively associated. Larger family size may put children at higher risk for acute malnutrition, which could be due to the imbalance between family size and resources [44] . Those whose families cultivate rice were less likely to be thin in rural communities. In this zone, rice production might be acting as a proxy for better socioeconomic status, as rice consumption is relatively recent but is one of the main cash crops in the area [16] . The low consumption of animal source foods and its association with acute malnutrition has been previously identified as a major contributing factor to delayed growth in children [45] and suboptimal dietary practices among adolescents in Ethiopia [17] . In urban areas, children with a literate HH were 4 times less likely to be thin than those living in houses headed by illiterate adults. Some studies have shown that parental education is associated with more efficient management of limited household resources, improved utilization of available health care services, and better health-promoting behaviors, all of which are associated with better child nutrition [46] , [47] . This result is similar to what we previously observed for stunting and PCFP years of education in the urban setting. A possible explanation could be the existence of an educational gap in urban but not in rural areas. Stunting versus thinness: associated factors Clear differences among risk factors for stunting ( Table 3 ) and thinness ( Table 4 ) emerged from this study. The literature on the causes of stunting is vast, and conventional thinking is summarized in the Lancet series on maternal and child under-nutrition [48] . Recognized causal factors include prenatal and postnatal periods. Stunting is seen as closely tied to poverty and access to services. Less knowledge is available on risk factors for thinness [1] . In our research, risk factors for chronic malnutrition encompass a wide range of variables.. The relatively consistent pattern of related factors for stunting suggests that continued exposure to adverse conditions retards children's linear growth. Conversely, the greater diversity observed in the factors associated with thinness is consistent with the fact that a relatively short period of risk exposure can precipitate its onset in children [32] . In rural communities, children from male-headed households were more likely to be thin than children from female-headed households (p = 0.043), while stunting was significantly more frequent in female-headed households (p = 0.002). The disparate sample size in rural and urban areas may have influenced these results. However, the result in the rural area is consistent with the study conducted in North Ethiopia by Haidar et al. [49] . This study found a significantly higher proportion of stunted and underweight pre-school children in female headed-households, whereas the prevalence of thinness was similar [48] – [50] . Women who are single HH may be removed from their support structures and may face constraints in accessing services, including food, as a result of insecurity, cultural discrimination and limited mobility [48] , [50] . This situation may have a long-term impact in child nutrition. Rural versus urban: associated factors Ethiopia remains one of the least urbanized countries in the world [42] . Globally, malnutrition is less common in urban areas [2] , [31] . We found that malnutrition in rural communities was associated with food habits and the lack of material resources whereas in the urban area, it was better predicted by socio demographic factors. Inequalities in child health outcomes are known to vary between rural and urban areas, and are often due to unequal allocation of resources [51] . Significant urban-rural differences remained in the multivariable model. This shows that even in the presence of important individual factors and socioeconomic variables, area of residence is still a predictor of children's nutritional status. Our results highlight the need to stratify data when rural and urban communities are targeted in nutritional research in this kind of context. Limitations The present study was conducted in two single districts in Ethiopia, thus, the findings may not be generalizable to a larger population. Additionally, the cross-sectional nature of this data does not allow us to examine causality in the relationship between malnutrition and diverse risk factors. Seasonality should be given special attention; the season of the year has a significant effect not only on food security and nutritional status, but also on patterns and trends of infectious disease incidence. Therefore, consecutive measurements are desirable.. This research is part of a project which aimed to characterize nutritional, immunological, and parasitological aspects in school-aged children from urban and rural villages with a high incidence of VL in 2005–07. However, the VL prevalence found in the study was very low [25] , and no association was found between nutritional status and asymptomatic infection [24] . Therefore, we are confident that this limitation does not alter our general conclusions. Conclusions Our findings suggest that improving food availability is a necessary but not sufficient condition to improve the nutritional status of school-aged children in this region. Especially in rural areas, the challenge will be for health and development extension workers to build on this knowledge through educational campaigns when advising households about balanced diet, food production and consumption, and hygienic behavior. It is also important to emphasize that nutritional programs should not be biased towards rural areas at the cost of excluding the urban poor. To effectively tackle malnutrition, nutritional programs should be oriented to the local needs. Our findings can be used to help policy makers plan and undertake regional initiatives to streamline recommendations. Supporting Information Table S1 Factors related to stunting in school-aged children by setting in Libokemkem and Fogera districts, Ethiopia, May–June 2009. Bivariate analysis. (DOCX) Click here for additional data file. Table S2 Factors related to thinness in school-aged children by setting in Libokemkem and Fogera districts, Ethiopia, May–June 2009. Bivariate analysis. (DOCX) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382615/

Artificial intelligence-based conversational agent to support medication prescribing

Abstract Objective This article describes the system architecture, training, initial use, and performance of Watson Assistant (WA), an artificial intelligence-based conversational agent, accessible within Micromedex ® . Materials and methods The number and frequency of intents (target of a user's query) triggered in WA during its initial use were examined; intents triggered over 9 months were compared to the frequency of topics accessed via keyword search of Micromedex. Accuracy of WA intents assigned to 400 queries was compared to assignments by 2 independent subject matter experts (SMEs), with inter-rater reliability measured by Cohen's kappa. Results In over 126 000 conversations with WA, intents most frequently triggered involved dosing ( N = 30 239, 23.9%) and administration ( N = 14 520, 11.5%). SMEs with substantial inter-rater agreement (kappa = 0.71) agreed with intent mapping in 247 of 400 queries (62%), including 16 queries related to content that WA and SMEs agreed was unavailable in WA. SMEs found 57 (14%) of 400 queries incorrectly mapped by WA; 112 (28%) queries unanswerable by WA included queries that were either ambiguous, contained unrecognized typographical errors, or addressed topics unavailable to WA. Of the queries answerable by WA (288), SMEs determined 231 (80%) were correctly linked to an intent. Discussion A conversational agent successfully linked most queries to intents in Micromedex. Ongoing system training seeks to widen the scope of WA and improve matching capabilities. Conclusion WA enabled Micromedex users to obtain answers to many medication-related questions using natural language, with the conversational agent facilitating mapping to a broader distribution of topics than standard keyword searches. Objective This article describes the system architecture, training, initial use, and performance of Watson Assistant (WA), an artificial intelligence-based conversational agent, accessible within Micromedex ® . Materials and methods The number and frequency of intents (target of a user's query) triggered in WA during its initial use were examined; intents triggered over 9 months were compared to the frequency of topics accessed via keyword search of Micromedex. Accuracy of WA intents assigned to 400 queries was compared to assignments by 2 independent subject matter experts (SMEs), with inter-rater reliability measured by Cohen's kappa. Results In over 126 000 conversations with WA, intents most frequently triggered involved dosing ( N = 30 239, 23.9%) and administration ( N = 14 520, 11.5%). SMEs with substantial inter-rater agreement (kappa = 0.71) agreed with intent mapping in 247 of 400 queries (62%), including 16 queries related to content that WA and SMEs agreed was unavailable in WA. SMEs found 57 (14%) of 400 queries incorrectly mapped by WA; 112 (28%) queries unanswerable by WA included queries that were either ambiguous, contained unrecognized typographical errors, or addressed topics unavailable to WA. Of the queries answerable by WA (288), SMEs determined 231 (80%) were correctly linked to an intent. Discussion A conversational agent successfully linked most queries to intents in Micromedex. Ongoing system training seeks to widen the scope of WA and improve matching capabilities. Conclusion WA enabled Micromedex users to obtain answers to many medication-related questions using natural language, with the conversational agent facilitating mapping to a broader distribution of topics than standard keyword searches. INTRODUCTION The prevalence of preventable adverse drug events (ADEs) in hospital populations ranges from 0.6 per 100 hospitalized patients 1 to as many as 1 in 10, 2 with related costs ranging from $6 to $29 billion annually. 2 Pharmacological information systems can improve access to drug information and potentially decrease ADEs by automating content retrieval and providing evidence-based information. 3–6 Micromedex ® is a pharmacological knowledge base supported by evidence from current literature and resources. 6 The core content in Micromedex is developed through curation of pharmacological, regulatory, biomedical, and scientific information by pharmacy specialists, medical librarians, and biostatisticians. Content is evaluated for clinical significance and accuracy by experts in drugs, diseases, toxicology, and patient education, with additional review of critical content areas by editorial board members, outside peer reviewers, academic scientists, and healthcare professionals. Micromedex is used globally by roughly 4500 healthcare organizations and is a key resource for Poison Control Centers 7 as well as Medicare and Medicaid 8 evaluation of off-label drugs in the United States. Micromedex contains a comprehensive listing of drug–drug interactions 9–13 ; many commonly used drug combinations are available for review in Micromedex. 9–11 , 14–16 Micromedex details information on the frequency, severity, and management of drug reactions. 17 It uniquely offers users side-by-side comparisons of drug monographs, 10 as well as natural language search capability through Watson Assistant (WA). WA is an artificial intelligence (AI)-based conversational agent powered by Watson, a supercomputer that relies on IBM's DeepQA to support advanced analytics and information retrieval. WA, integrated with Micromedex, enables users to ask medication-related questions using natural language. With natural language processing (NLP) and machine learning (ML), WA functions as a pharmacological question-answering system. 18 , 19 OBJECTIVE The objective of this article is to describe the architecture of the conversational agent, WA, to describe an initial experience with system use, detailing the types of queries entered and to evaluate system success in mapping queries to appropriate content. System overview The system architecture for Micromedex equipped with WA is depicted in Figure 1 . The combined health internet delivery system (HIDS) consists of components that are proprietary to the Micromedex user interface (UI, Figure 1 , green), including tools to disambiguate lexical variants, acronyms and abbreviations, a Lucene service, Oracle database (DB), a content management system (CMS), and ontology. Keyword search of Micromedex content is mediated by the open-source Lucene service. The lexical service integrates clinical terminology and taxonomies, helping to connect keyword entries with clinical terminology. The CMS contains drug content which is created and maintained by clinical editors; drug content maintained in the Oracle database originates from the CMS. The Oracle database contains Micromedex's Quick Answers, housing summary-level drug information accessed by cloud database 2 (DB2). The ontology is a representation of summary-level information, defined by organization of drug information, relationships between domains and entities (eg, drug or condition), classifications of entities, WA intents (target of a user's query), and metadata elements. The ontology underpinning WA contains the domains of knowledge found in Micromedex's Quick Answers database, drug–drug interactions and IV compatibility. Figure 1. System architecture. CMS, content management system; DB, database; HIDS, health internet delivery system; UI, user interface. Dotted lines, data flow; red lines, representational state transfer application (REST) services; solid lines, data retrieval. Connecting Micromedex's HIDS ( Figure 1 , green) to IBM cloud applications ( Figure 1 , blue) is the representational state transfer application interface (REST API) that maintains interoperability and transfer of data between system components. The Micromedex UI, cloud applications interface, and WA are supported by the conversation manager, based on rules for business logic which establish system awareness of clinical reference content, allowing a query to be interpreted by WA. WA is part of the UI that links user queries to information contained in the Micromedex knowledge base. Other operations found within IBM cloud applications include internal functions for system maintenance controlled from the IBM cloud, as well as a test UI to evaluate new codes and interfaces as they are developed. The updater service handles daily content updates and deployment of all client and server applications that interface between WA and cloud DB2. The summary drug information used by WA is stored in the DB2 database. The DB2 database links WA, the ontology, and the CMS, handling tracking and logging of user queries for quality review and ongoing WA system training. Intents are the intended target of a user's query. For example, user may desire to determine adverse effects of a drug and thus, there is an intent named "adverse effects." An entity represents a drug or condition relevant to a specific intent and provides context for that intent. Intents are represented hierarchically within the ontology. Intents such as "pediatric dosing," "FDA and non-FDA uses," "dose adjustments," "administration," "contraindications," "precautions," "adverse effects," "black box warnings," "risk evaluation," and "mitigation strategy," "drug interactions," "pregnancy and lactation," "monitoring," "mechanism of action," "pharmacokinetics," and "how supplied" are parent nodes in the ontology; "common side effects" and "serious side effects" are examples of child nodes under the parent node of "adverse effects." WA's NLP, ML, and integrated ontology allows WA to retrieve answers to common medication-related questions by matching queries to underlying intents. WA's natural language engine interprets a user's query over its domain of drug knowledge, which assists the conversation service in identification of intents and entities. System overview The system architecture for Micromedex equipped with WA is depicted in Figure 1 . The combined health internet delivery system (HIDS) consists of components that are proprietary to the Micromedex user interface (UI, Figure 1 , green), including tools to disambiguate lexical variants, acronyms and abbreviations, a Lucene service, Oracle database (DB), a content management system (CMS), and ontology. Keyword search of Micromedex content is mediated by the open-source Lucene service. The lexical service integrates clinical terminology and taxonomies, helping to connect keyword entries with clinical terminology. The CMS contains drug content which is created and maintained by clinical editors; drug content maintained in the Oracle database originates from the CMS. The Oracle database contains Micromedex's Quick Answers, housing summary-level drug information accessed by cloud database 2 (DB2). The ontology is a representation of summary-level information, defined by organization of drug information, relationships between domains and entities (eg, drug or condition), classifications of entities, WA intents (target of a user's query), and metadata elements. The ontology underpinning WA contains the domains of knowledge found in Micromedex's Quick Answers database, drug–drug interactions and IV compatibility. Figure 1. System architecture. CMS, content management system; DB, database; HIDS, health internet delivery system; UI, user interface. Dotted lines, data flow; red lines, representational state transfer application (REST) services; solid lines, data retrieval. Connecting Micromedex's HIDS ( Figure 1 , green) to IBM cloud applications ( Figure 1 , blue) is the representational state transfer application interface (REST API) that maintains interoperability and transfer of data between system components. The Micromedex UI, cloud applications interface, and WA are supported by the conversation manager, based on rules for business logic which establish system awareness of clinical reference content, allowing a query to be interpreted by WA. WA is part of the UI that links user queries to information contained in the Micromedex knowledge base. Other operations found within IBM cloud applications include internal functions for system maintenance controlled from the IBM cloud, as well as a test UI to evaluate new codes and interfaces as they are developed. The updater service handles daily content updates and deployment of all client and server applications that interface between WA and cloud DB2. The summary drug information used by WA is stored in the DB2 database. The DB2 database links WA, the ontology, and the CMS, handling tracking and logging of user queries for quality review and ongoing WA system training. Intents are the intended target of a user's query. For example, user may desire to determine adverse effects of a drug and thus, there is an intent named "adverse effects." An entity represents a drug or condition relevant to a specific intent and provides context for that intent. Intents are represented hierarchically within the ontology. Intents such as "pediatric dosing," "FDA and non-FDA uses," "dose adjustments," "administration," "contraindications," "precautions," "adverse effects," "black box warnings," "risk evaluation," and "mitigation strategy," "drug interactions," "pregnancy and lactation," "monitoring," "mechanism of action," "pharmacokinetics," and "how supplied" are parent nodes in the ontology; "common side effects" and "serious side effects" are examples of child nodes under the parent node of "adverse effects." WA's NLP, ML, and integrated ontology allows WA to retrieve answers to common medication-related questions by matching queries to underlying intents. WA's natural language engine interprets a user's query over its domain of drug knowledge, which assists the conversation service in identification of intents and entities. SYSTEM INTERFACES Keyword-based and WA-assisted searches The information contained in Micromedex is accessible to users through the keyword search function available in Micromedex on the upper left of the Micromedex home screen ( Figure 2A , upper left). It allows users to navigate to both detailed and summary level information, including related evidence and the ranking of that evidence. For example, a user searching for serious immunologic side effects of trastuzumab-qyyp can enter trastuzumab-qyyp in the keyword search bar and view "Adverse Effects" followed by "Serious" and "Immunologic" under that heading. Figure 2. User interface. (A) Micromedex home page. Red arrow to "learn more" under "Ask Watson" explains what information Watson can provide (light green box), including examples of real-world questions, the types of information Micromedex with Watson understands (ie, drug information, drug interactions, and IV compatibility) and what Micromedex with Watson does not understand (ie, in-depth answers, NeoFax/pediatrics, toxicology, diseases, laboratory, alternative medicine, reproductive risks and third party content such as Martindale and Index Nominum). (B) WA chat window key features. Alternatively, users can obtain information through WA's natural language conversational search by directly entering questions using natural language into the "Ask Watson" search bar found on the upper right of the Micromedex home page ( Figure 2a , black box). For example, a user can enter "What are serious immunologic side effects of Trazimera?" (Trazimera, trastuzumab-qyyp) in the "Ask Watson" search bar. WA opens a chat window and returns information specific to severe immunologic side effects of the drug (with links to further information) within the same chat window. If WA requires more information to answer a query, it asks the user a question or series of questions to help WA understand the context and intent of the query, using smart filtering which helps the system resolve ambiguities. Smart filtering allows the conversational agent to ask for additional information when, for example, an entity match exists but the intent is unclear to WA. NLP maps entities and relationships related to the query to WA's ontology to identify the intent of a query. 20 Upon matching a query to intent, WA provides clinically actionable answers with evidence links within the chat window, drawing upon the same information and database that is interrogated by a keyword search of Micromedex. Conversational search The conversational search allows clinicians to pose a series of questions on the same topic or on different topics. For example, a user can initiate a conversation with Watson by entering "What's the adult dose of rivaroxaban for DVT?" (DVT, deep vein thrombosis; Figure 2B , right). In the conversation window, WA provides the adult dosing information for rivaroxaban with links to either "Quick Answers" or "In-Depth Answers" for rivaroxaban. When asking a series of questions, WA uses smart filtering to provide answers that include the context of prior questions by maintaining the identified entities over the course of the conversation. For example, a user can append "Are dose adjustments needed?" to the conversation without repeating the drug name. WA then returns "Here are the rivaroxaban dose adjustments," followed by dose adjustments for renal impairment, hepatic impairment, geriatrics, bariatric surgery, and obesity, along with links to more information. If the user changes the topic of the conversation to another drug, WA will overwrite previous drugs and conditions and answer based on the new drug or condition. When WA cannot map a query to an intent, for example, when a user enters "off-label uses of erdafitinib," WA responds with "I did not find adult non-FDA uses for erdafitinib." Keyword-based and WA-assisted searches The information contained in Micromedex is accessible to users through the keyword search function available in Micromedex on the upper left of the Micromedex home screen ( Figure 2A , upper left). It allows users to navigate to both detailed and summary level information, including related evidence and the ranking of that evidence. For example, a user searching for serious immunologic side effects of trastuzumab-qyyp can enter trastuzumab-qyyp in the keyword search bar and view "Adverse Effects" followed by "Serious" and "Immunologic" under that heading. Figure 2. User interface. (A) Micromedex home page. Red arrow to "learn more" under "Ask Watson" explains what information Watson can provide (light green box), including examples of real-world questions, the types of information Micromedex with Watson understands (ie, drug information, drug interactions, and IV compatibility) and what Micromedex with Watson does not understand (ie, in-depth answers, NeoFax/pediatrics, toxicology, diseases, laboratory, alternative medicine, reproductive risks and third party content such as Martindale and Index Nominum). (B) WA chat window key features. Alternatively, users can obtain information through WA's natural language conversational search by directly entering questions using natural language into the "Ask Watson" search bar found on the upper right of the Micromedex home page ( Figure 2a , black box). For example, a user can enter "What are serious immunologic side effects of Trazimera?" (Trazimera, trastuzumab-qyyp) in the "Ask Watson" search bar. WA opens a chat window and returns information specific to severe immunologic side effects of the drug (with links to further information) within the same chat window. If WA requires more information to answer a query, it asks the user a question or series of questions to help WA understand the context and intent of the query, using smart filtering which helps the system resolve ambiguities. Smart filtering allows the conversational agent to ask for additional information when, for example, an entity match exists but the intent is unclear to WA. NLP maps entities and relationships related to the query to WA's ontology to identify the intent of a query. 20 Upon matching a query to intent, WA provides clinically actionable answers with evidence links within the chat window, drawing upon the same information and database that is interrogated by a keyword search of Micromedex. Conversational search The conversational search allows clinicians to pose a series of questions on the same topic or on different topics. For example, a user can initiate a conversation with Watson by entering "What's the adult dose of rivaroxaban for DVT?" (DVT, deep vein thrombosis; Figure 2B , right). In the conversation window, WA provides the adult dosing information for rivaroxaban with links to either "Quick Answers" or "In-Depth Answers" for rivaroxaban. When asking a series of questions, WA uses smart filtering to provide answers that include the context of prior questions by maintaining the identified entities over the course of the conversation. For example, a user can append "Are dose adjustments needed?" to the conversation without repeating the drug name. WA then returns "Here are the rivaroxaban dose adjustments," followed by dose adjustments for renal impairment, hepatic impairment, geriatrics, bariatric surgery, and obesity, along with links to more information. If the user changes the topic of the conversation to another drug, WA will overwrite previous drugs and conditions and answer based on the new drug or condition. When WA cannot map a query to an intent, for example, when a user enters "off-label uses of erdafitinib," WA responds with "I did not find adult non-FDA uses for erdafitinib." MATERIALS AND METHODS System development and training Ground truth An initial ground truth was generated by clinical subject matter experts (SMEs), clinical content technical specialists, IBM research professionals, clinical pharmacists, and pharmacy students. This ground truth included common questions and frequently accessed information in Micromedex, mapped to correct intents and answers as determined by SMEs. To further development of the ground truth, a preliminary concept map of common questions was developed that linked questions to information contained in the Micromedex Drug Information knowledge base. NLP training A supervised learning approach was used to train the NLP components native to WA. The NLP system components disambiguated utterances (questions) by parsing named entities, such as medications, treatments, and conditions, mapping the information to entities modeled in the ontology. The ground truth provided the labeled training data that informed the mapping of system outputs. System inputs are a combination of the original concept map and real-world examples sourced from WA users. Corresponding answers (output) are drawn from Micromedex's Quick Answer content, which is created and maintained by CMS clinical editors and by lexicon content specialists, updated daily in Micromedex, and supported by corresponding monographic content and references from clinical studies. Machine learning components The ML components integrated with WA include methods to predict and classify information and data. Specifically, an ML approach using support vector machines (SVM) 21–24 is used to optimize solutions when users require additional suggestions. Although use of neural networks and other deep learning approaches were becoming more commonplace during development of Watson, the SVM approach used by WA remains one of the most robust methods available to perform the core classification task of matching utterances to intents when only small training sets are available. During system training, the SVM refined suggestions to end-users with inputs that comprised both the syntactic and semantic features obtained from the NLP components that parsed and classified entities and intents from user queries. System outputs were further refined within user queries to optimize search results. The training examples and the SVM native to WA are responsible for the deductive aspects of WA that allow it to function as a semantic reasoner. System optimization The native WA system—integrated with Micromedex—underwent successive rounds of iterative training using labeled examples curated from user utterances that were mapped to intents. Questions posed by pharmacists, nurses, physicians, clinicians, and medical librarians were used for system training during further refinement of the ground truth. Intents and real-user utterances were iteratively added. After each system enhancement, information from chat logs and other user feedback are systematically analyzed to uncover information used to fine-tune the system. Utterances that return unexpected results are reviewed by clinical pharmacists and project developers to determine underlying intents. Results such as these are used to improve system training and performance and are integrated into the ground truth of the system. SMEs periodically review random chat log entries to examine the clinical output, identify defects, review which intent was fired given each utterance, and confirm the clinical accuracy of system-generated matches, informing ongoing system training and enhancements. Frequency of intents and accuracy of matching Intents triggered by users during the initial 4-month use of WA at a mid-size (300 bed) tertiary care facility were recorded. Users were primarily pharmacists, nurses, and other clinical staff. Utterances were grouped by intent type to which they were mapped by WA, and frequency of intents was tabulated. WA uses a proprietary classification and detection method using SVMs to optimize features and match utterances to intents. To examine accuracy of matching queries to intents, 400 sequential user queries collected over a 2-week period were analyzed. Each query was independently reviewed by 2 clinical pharmacists (SMEs). Each SME was blinded to intents assigned by the other SME and by WA. Any disagreements between the first 2 SMEs were arbitrated by a third SME who was also blinded to intents assigned by SMEs and WA. Any additional discrepancies were finalized using a consensus review to generate a final intent. The WA-assigned intents were compared to the ground-truth intents established by SMEs, and the percent of intents correctly assigned by WA was calculated. The beyond-chance agreement between reviewers involved in establishing the ground truth for matching was measured with Cohen's kappa. To examine potential differences in users' approaches to finding information using a standard keyword search versus a conversational agent, we compared the top intents triggered by WA users to analogous drug topics accessed by keyword searches of Micromedex over the course of 9 months. The number of intents or drug topics accessed through WA-assisted versus keyword search was calculated as a percent of total searches by each method. The top intents triggered in WA were compared to the frequency of analogous drug topics accessed through keyword searches of Micromedex. System development and training Ground truth An initial ground truth was generated by clinical subject matter experts (SMEs), clinical content technical specialists, IBM research professionals, clinical pharmacists, and pharmacy students. This ground truth included common questions and frequently accessed information in Micromedex, mapped to correct intents and answers as determined by SMEs. To further development of the ground truth, a preliminary concept map of common questions was developed that linked questions to information contained in the Micromedex Drug Information knowledge base. NLP training A supervised learning approach was used to train the NLP components native to WA. The NLP system components disambiguated utterances (questions) by parsing named entities, such as medications, treatments, and conditions, mapping the information to entities modeled in the ontology. The ground truth provided the labeled training data that informed the mapping of system outputs. System inputs are a combination of the original concept map and real-world examples sourced from WA users. Corresponding answers (output) are drawn from Micromedex's Quick Answer content, which is created and maintained by CMS clinical editors and by lexicon content specialists, updated daily in Micromedex, and supported by corresponding monographic content and references from clinical studies. Machine learning components The ML components integrated with WA include methods to predict and classify information and data. Specifically, an ML approach using support vector machines (SVM) 21–24 is used to optimize solutions when users require additional suggestions. Although use of neural networks and other deep learning approaches were becoming more commonplace during development of Watson, the SVM approach used by WA remains one of the most robust methods available to perform the core classification task of matching utterances to intents when only small training sets are available. During system training, the SVM refined suggestions to end-users with inputs that comprised both the syntactic and semantic features obtained from the NLP components that parsed and classified entities and intents from user queries. System outputs were further refined within user queries to optimize search results. The training examples and the SVM native to WA are responsible for the deductive aspects of WA that allow it to function as a semantic reasoner. System optimization The native WA system—integrated with Micromedex—underwent successive rounds of iterative training using labeled examples curated from user utterances that were mapped to intents. Questions posed by pharmacists, nurses, physicians, clinicians, and medical librarians were used for system training during further refinement of the ground truth. Intents and real-user utterances were iteratively added. After each system enhancement, information from chat logs and other user feedback are systematically analyzed to uncover information used to fine-tune the system. Utterances that return unexpected results are reviewed by clinical pharmacists and project developers to determine underlying intents. Results such as these are used to improve system training and performance and are integrated into the ground truth of the system. SMEs periodically review random chat log entries to examine the clinical output, identify defects, review which intent was fired given each utterance, and confirm the clinical accuracy of system-generated matches, informing ongoing system training and enhancements. Ground truth An initial ground truth was generated by clinical subject matter experts (SMEs), clinical content technical specialists, IBM research professionals, clinical pharmacists, and pharmacy students. This ground truth included common questions and frequently accessed information in Micromedex, mapped to correct intents and answers as determined by SMEs. To further development of the ground truth, a preliminary concept map of common questions was developed that linked questions to information contained in the Micromedex Drug Information knowledge base. NLP training A supervised learning approach was used to train the NLP components native to WA. The NLP system components disambiguated utterances (questions) by parsing named entities, such as medications, treatments, and conditions, mapping the information to entities modeled in the ontology. The ground truth provided the labeled training data that informed the mapping of system outputs. System inputs are a combination of the original concept map and real-world examples sourced from WA users. Corresponding answers (output) are drawn from Micromedex's Quick Answer content, which is created and maintained by CMS clinical editors and by lexicon content specialists, updated daily in Micromedex, and supported by corresponding monographic content and references from clinical studies. Machine learning components The ML components integrated with WA include methods to predict and classify information and data. Specifically, an ML approach using support vector machines (SVM) 21–24 is used to optimize solutions when users require additional suggestions. Although use of neural networks and other deep learning approaches were becoming more commonplace during development of Watson, the SVM approach used by WA remains one of the most robust methods available to perform the core classification task of matching utterances to intents when only small training sets are available. During system training, the SVM refined suggestions to end-users with inputs that comprised both the syntactic and semantic features obtained from the NLP components that parsed and classified entities and intents from user queries. System outputs were further refined within user queries to optimize search results. The training examples and the SVM native to WA are responsible for the deductive aspects of WA that allow it to function as a semantic reasoner. System optimization The native WA system—integrated with Micromedex—underwent successive rounds of iterative training using labeled examples curated from user utterances that were mapped to intents. Questions posed by pharmacists, nurses, physicians, clinicians, and medical librarians were used for system training during further refinement of the ground truth. Intents and real-user utterances were iteratively added. After each system enhancement, information from chat logs and other user feedback are systematically analyzed to uncover information used to fine-tune the system. Utterances that return unexpected results are reviewed by clinical pharmacists and project developers to determine underlying intents. Results such as these are used to improve system training and performance and are integrated into the ground truth of the system. SMEs periodically review random chat log entries to examine the clinical output, identify defects, review which intent was fired given each utterance, and confirm the clinical accuracy of system-generated matches, informing ongoing system training and enhancements. Frequency of intents and accuracy of matching Intents triggered by users during the initial 4-month use of WA at a mid-size (300 bed) tertiary care facility were recorded. Users were primarily pharmacists, nurses, and other clinical staff. Utterances were grouped by intent type to which they were mapped by WA, and frequency of intents was tabulated. WA uses a proprietary classification and detection method using SVMs to optimize features and match utterances to intents. To examine accuracy of matching queries to intents, 400 sequential user queries collected over a 2-week period were analyzed. Each query was independently reviewed by 2 clinical pharmacists (SMEs). Each SME was blinded to intents assigned by the other SME and by WA. Any disagreements between the first 2 SMEs were arbitrated by a third SME who was also blinded to intents assigned by SMEs and WA. Any additional discrepancies were finalized using a consensus review to generate a final intent. The WA-assigned intents were compared to the ground-truth intents established by SMEs, and the percent of intents correctly assigned by WA was calculated. The beyond-chance agreement between reviewers involved in establishing the ground truth for matching was measured with Cohen's kappa. To examine potential differences in users' approaches to finding information using a standard keyword search versus a conversational agent, we compared the top intents triggered by WA users to analogous drug topics accessed by keyword searches of Micromedex over the course of 9 months. The number of intents or drug topics accessed through WA-assisted versus keyword search was calculated as a percent of total searches by each method. The top intents triggered in WA were compared to the frequency of analogous drug topics accessed through keyword searches of Micromedex. RESULTS Intents triggered There was a total of 42 771 conversations logged over the 4-month initial use study in late 2018; 27 768 (64.9%) of these conversations were mapped by WA to an intent, as determined by the system ( Figure 3 ). Questions in the conversation logs ranged from very specific (ie, "What is the t 1/2 of itraconazole?") to general ("Heart attack") to nonsensical ("dsfsdfsdfsdfysdftysdtfy…"). Representative user queries and intents to which they were mapped by the system are shown in Table 1 . Unassigned queries are those that either lack the requisite information needed to map to an intent (ambiguous queries) or the information is unavailable in the Micromedex Quick Answers database (unavailable). Unassigned queries include those such as "Is there a term for increase in white blood cells" (no condition name was provided). Examples of unavailable queries include utterances such as "How to pronounce Lunesta" and "is magnesium phosphate available in South Africa," as well as questions such as "what is 2 + 2" or "how do I print this." Figure 3. Frequency of intents. Utterances that mapped to queries (42 771 total queries) collected between October of 2018 through January 2019, grouped by category or intent to which it was mapped by WA. Table 1. Examples of user queries and intents (as determined by SME) Example query Intent "What is the dose of adalimumab in plaque psoriasis?" Dosing "How fast can I run phenytoin sodium?" Administration "What drugs that treat migraine?" Drugs that treat "What drugs are used for asthma?" "What is the t 1/2 of itraconazole?" Pharmacokinetics "FDA uses for Harvoni?" Uses (FDA, non-FDA, all) "Off label uses of secukinumab?" "What is dronedarone used for?" "Show me serious adverse effects for cyclosporine" Adverse effects "Tacrolimus" Single drug a "Are amiodarone and cimetidine iv compatible?" IV compatibility "Amoxicillin anthrax" Drug and condition b "Dose adjustments for gentamicin" Dose adjustments "Heart attack" Single condition c "What schedule is lorazepam?" Regulatory status "What are the drug interactions for atorvastatin?" Drug–drug interactions "Does prednisone come in capsules?" How supplied "How supplied for diltiazem?" "What is the pregnancy category of captopril?" Pregnancy/lactation "Is abraxane safe in breastfeeding?" "zovirax class" Drug class "What to monitor for carbamazepine?" Monitoring Queries vary Self-correction "Show me mechanism of action of droperidol" Mechanism of action "Contraindications of mesoridazine" Contraindications "Does morphine have black box warnings?" Black box warning "Precautions for trintellix" Precautions "By what route is naloxone given?" Route of administration "What is the brand name of doxazosin?" Brand name "What is the drug class of amphotericin b?" Drug class a Watson returns use information. b Watson currently returns drug and condition but will eventually return a choice of adverse effects, precautions, contraindications, uses, or dosing. c Watson returns drugs used for named condition. Frequency and accuracy of intents The frequency of intents that was triggered by queries during the initial 4-month study period is shown in Figure 3 . Most questions that were mapped to an intent by WA involved dosing and administration, followed by drugs that treat (certain conditions), pharmacokinetics, and on- and off-label usage. The accuracy of WA intents assigned to 400 sequential queries, regardless of availability in Micromedex, was 61.75%, as determined by SMEs displaying substantial inter-rater agreement (Cohen's kappa 0.71, 95% CI 0.66–0.76). 25–27 Of these 400 queries, 38.25% ( N = 153) were not successfully matched to an intent, for reasons as follows: 14.25% ( N = 57) were incorrectly mapped by WA; 13.5% ( N = 54) were unavailable in WA; 5.25% ( N = 21) were too ambiguous to determine accuracy, and 5.25% ( N = 21) contained typographic errors or abbreviations that were not recognized by WA. Mismatches of queries to intents by WA ( N = 57) that SMEs considered to be within WA's domain of knowledge included: IV compatibility, 13; drug–drug interactions, 6; drug dosage for condition, 5; drug case, 5; drugs that treat (specific conditions), 4; precautions, 3; pharmacokinetics, 2; administration of drug, 1; brand name, 1; condition case, 1; mechanism of action, 1; monitoring of drug, 1; pregnancy and lactation, 1; and regulatory status, 1. In addition, WA returned intents for 12 queries that should have returned "unavailable." The accuracy analysis identified 112 queries (28% of 400) that were unanswerable by WA. These included queries that were either ambiguous, contained unrecognizable typographical errors, or related to content unavailable in the Micromedex Quick Answers database. Of the 288 queries that SMEs determined were within WA's knowledge domain and specific enough to be answered by WA, 231 (80% of 288 answerable queries) were determined by SMEs to be mapped to the correct intent. The accuracy analysis triggered all of the intents shown in Figure 3 , as well as queries related to "care notes," "comparative efficacy," "do not crush consult," "drug consult," "drug–alcohol interactions," "drug food interactions," "drugs that cause," "Neofax," and "Redbook." Training for these terms and phrases was added after the initial use data collection to facilitate linkage of these phrases to appropriate intents. WA-assisted search versus keyword search A total of 126 765 conversations with WA were mapped to intents during a 9-month study period. The most common topic accessed using WA was dosing, followed by administration, drugs that treat (specified conditions), uses (including both FDA and non-FDA), pharmacokinetics, IV compatibility, and adverse effects. Although dosing was also the most common drug topic accessed in a keyword search of Micromedex, the frequency of searches differed by search method (dosing, 23.9% with WA vs 43.3% with keyword search); other search topics and frequencies also differed according to search method ( Table 2 ). Table 2. Top 7 topics accessed with WA search versus keyword search Watson assistant intent WA search, N (%) Keyword search, N (%) Watson assistant example query Dosing 30 239 (23.9%) 34 322 058 (43.3%) "What is the dosage of azithromycin?" Administration 14 520 (11.5%) 2 764 488 (3.5%) "Can burosumab be administered SQ in the legs or abdomen?" Drugs that treat a 10 199 (8.0%) 190 217 (0.2%) "Medications used for hypertension" Uses (FDA, non-FDA, all) 8651 (6.8%) 5 641 662 (11.5%) a "Can moxifloxacin be used in UTI?" Pharmacokinetics 8495 (6.7%) 1 544 830 (1.9%) "How is nimbex metabolized?" IV compatibility 7942 (6.3%) 435 461 (0.05%) "Is daptomycin compatible with micafungin" Adverse effects 6676 (5.3%) 5 416 938 (6.8%) "Diazepam side effect" a Sum of on-label and off-label uses. Intents triggered There was a total of 42 771 conversations logged over the 4-month initial use study in late 2018; 27 768 (64.9%) of these conversations were mapped by WA to an intent, as determined by the system ( Figure 3 ). Questions in the conversation logs ranged from very specific (ie, "What is the t 1/2 of itraconazole?") to general ("Heart attack") to nonsensical ("dsfsdfsdfsdfysdftysdtfy…"). Representative user queries and intents to which they were mapped by the system are shown in Table 1 . Unassigned queries are those that either lack the requisite information needed to map to an intent (ambiguous queries) or the information is unavailable in the Micromedex Quick Answers database (unavailable). Unassigned queries include those such as "Is there a term for increase in white blood cells" (no condition name was provided). Examples of unavailable queries include utterances such as "How to pronounce Lunesta" and "is magnesium phosphate available in South Africa," as well as questions such as "what is 2 + 2" or "how do I print this." Figure 3. Frequency of intents. Utterances that mapped to queries (42 771 total queries) collected between October of 2018 through January 2019, grouped by category or intent to which it was mapped by WA. Table 1. Examples of user queries and intents (as determined by SME) Example query Intent "What is the dose of adalimumab in plaque psoriasis?" Dosing "How fast can I run phenytoin sodium?" Administration "What drugs that treat migraine?" Drugs that treat "What drugs are used for asthma?" "What is the t 1/2 of itraconazole?" Pharmacokinetics "FDA uses for Harvoni?" Uses (FDA, non-FDA, all) "Off label uses of secukinumab?" "What is dronedarone used for?" "Show me serious adverse effects for cyclosporine" Adverse effects "Tacrolimus" Single drug a "Are amiodarone and cimetidine iv compatible?" IV compatibility "Amoxicillin anthrax" Drug and condition b "Dose adjustments for gentamicin" Dose adjustments "Heart attack" Single condition c "What schedule is lorazepam?" Regulatory status "What are the drug interactions for atorvastatin?" Drug–drug interactions "Does prednisone come in capsules?" How supplied "How supplied for diltiazem?" "What is the pregnancy category of captopril?" Pregnancy/lactation "Is abraxane safe in breastfeeding?" "zovirax class" Drug class "What to monitor for carbamazepine?" Monitoring Queries vary Self-correction "Show me mechanism of action of droperidol" Mechanism of action "Contraindications of mesoridazine" Contraindications "Does morphine have black box warnings?" Black box warning "Precautions for trintellix" Precautions "By what route is naloxone given?" Route of administration "What is the brand name of doxazosin?" Brand name "What is the drug class of amphotericin b?" Drug class a Watson returns use information. b Watson currently returns drug and condition but will eventually return a choice of adverse effects, precautions, contraindications, uses, or dosing. c Watson returns drugs used for named condition. Frequency and accuracy of intents The frequency of intents that was triggered by queries during the initial 4-month study period is shown in Figure 3 . Most questions that were mapped to an intent by WA involved dosing and administration, followed by drugs that treat (certain conditions), pharmacokinetics, and on- and off-label usage. The accuracy of WA intents assigned to 400 sequential queries, regardless of availability in Micromedex, was 61.75%, as determined by SMEs displaying substantial inter-rater agreement (Cohen's kappa 0.71, 95% CI 0.66–0.76). 25–27 Of these 400 queries, 38.25% ( N = 153) were not successfully matched to an intent, for reasons as follows: 14.25% ( N = 57) were incorrectly mapped by WA; 13.5% ( N = 54) were unavailable in WA; 5.25% ( N = 21) were too ambiguous to determine accuracy, and 5.25% ( N = 21) contained typographic errors or abbreviations that were not recognized by WA. Mismatches of queries to intents by WA ( N = 57) that SMEs considered to be within WA's domain of knowledge included: IV compatibility, 13; drug–drug interactions, 6; drug dosage for condition, 5; drug case, 5; drugs that treat (specific conditions), 4; precautions, 3; pharmacokinetics, 2; administration of drug, 1; brand name, 1; condition case, 1; mechanism of action, 1; monitoring of drug, 1; pregnancy and lactation, 1; and regulatory status, 1. In addition, WA returned intents for 12 queries that should have returned "unavailable." The accuracy analysis identified 112 queries (28% of 400) that were unanswerable by WA. These included queries that were either ambiguous, contained unrecognizable typographical errors, or related to content unavailable in the Micromedex Quick Answers database. Of the 288 queries that SMEs determined were within WA's knowledge domain and specific enough to be answered by WA, 231 (80% of 288 answerable queries) were determined by SMEs to be mapped to the correct intent. The accuracy analysis triggered all of the intents shown in Figure 3 , as well as queries related to "care notes," "comparative efficacy," "do not crush consult," "drug consult," "drug–alcohol interactions," "drug food interactions," "drugs that cause," "Neofax," and "Redbook." Training for these terms and phrases was added after the initial use data collection to facilitate linkage of these phrases to appropriate intents. WA-assisted search versus keyword search A total of 126 765 conversations with WA were mapped to intents during a 9-month study period. The most common topic accessed using WA was dosing, followed by administration, drugs that treat (specified conditions), uses (including both FDA and non-FDA), pharmacokinetics, IV compatibility, and adverse effects. Although dosing was also the most common drug topic accessed in a keyword search of Micromedex, the frequency of searches differed by search method (dosing, 23.9% with WA vs 43.3% with keyword search); other search topics and frequencies also differed according to search method ( Table 2 ). Table 2. Top 7 topics accessed with WA search versus keyword search Watson assistant intent WA search, N (%) Keyword search, N (%) Watson assistant example query Dosing 30 239 (23.9%) 34 322 058 (43.3%) "What is the dosage of azithromycin?" Administration 14 520 (11.5%) 2 764 488 (3.5%) "Can burosumab be administered SQ in the legs or abdomen?" Drugs that treat a 10 199 (8.0%) 190 217 (0.2%) "Medications used for hypertension" Uses (FDA, non-FDA, all) 8651 (6.8%) 5 641 662 (11.5%) a "Can moxifloxacin be used in UTI?" Pharmacokinetics 8495 (6.7%) 1 544 830 (1.9%) "How is nimbex metabolized?" IV compatibility 7942 (6.3%) 435 461 (0.05%) "Is daptomycin compatible with micafungin" Adverse effects 6676 (5.3%) 5 416 938 (6.8%) "Diazepam side effect" a Sum of on-label and off-label uses. DISCUSSION This report describes an AI-enabled conversational agent, WA, linked to a pharmacological database, Micromedex, to identify answers to clinicians' medication-related questions posed using natural language. In this study, 62% of all natural language queries were correctly matched to intent by WA, and 80% of queries within WA's domain of knowledge were correctly matched by WA, according to a gold standard developed by 2 independent SMEs. WA relies on an ontology and NLP to map queries to intents, as compared to keyword searches related to drug topics. The content most frequently accessed by either search method was related to dosing, but the proportion differed by method. Although dosing made up almost half of all topics accessed by keyword search, only about a quarter of intents were linked to dosing using WA. There were 3 times as many queries related to medication administration using WA as compared to keyword search; other topics and intents and their frequencies also differed by search method. These differences may reflect the ability of WA to aid users in articulating questions that are not easily answered with a standard keyword search. Further studies are underway which explore the user experiences associated with each method. Many queries were either too vague to be matched to an intent or were related to information not contained in WA's knowledge domain. In the case of vague queries, WA provides users with a choice of content areas for further consideration to facilitate the matching process. Many of the queries that were either not mapped to an intent or not mapped correctly were related to misspellings or unrecognized abbreviations. During the accuracy analysis, SMEs were able to map some of these to appropriate content, but many could not be interpreted. To assist with this problem, WA's 'fuzzy match' feature can help correct minor misspellings; however, it can also fail to recognize a misspelling, replace a misspelled word with a word may then be incorrectly matched to an intent, or identify a correctly spelled word as a misspelled word. Furthermore, highlighting the types of information that can and cannot be accessed in WA would likely reduce the number of queries related to information not available in WA. An important measure of system performance is whether a user's question was answered appropriately. To determine this, a ground truth for accuracy of intent mapping was developed by SMEs with substantial inter-rater agreement. The accuracy of intent mapping by WA for all queries was 61.75%. Two-thirds of the queries not successfully mapped were either unavailable in WA's knowledge domain or too ambiguous to map. Some of the utterances that WA was unable to assign to an intent contained phrases such as "other names," "how often," "taken together," "ran together," "mix," "IV…hang," and "absorption." For queries that were mapped incorrectly by WA, the greatest number were related to IV compatibility, followed by those that should have been categorized as unavailable in WA but returned an intent. Many of the incorrectly mapped queries were due to the system's difficulty with identification of contextual features, as well as difficulty with making inferences from natural language. There are few rigorous studies of conversational agents in healthcare, and none related to pharmacy. 28 Thus, the current study is the first to report the technical performance of a conversational agent aimed at answering medication-related questions using ground-truth intents established by 2 independent SMEs, underpinned by a curated knowledge base. This work has several limitations. First, the study involved data collected over a relatively short span of time, and only a small subset of queries were independently evaluated by 2 SMEs. A more comprehensive review of system-generated matches is underway, with results informing future system enhancements. Second, this work describes early adopters' experiences with WA and may not reflect the types of queries or system performance after users gain experience with the tool or after optional integration of the tool into an electronic health record. CONCLUSIONS We have described the architecture and early user experiences with WA, an AI-based conversational agent linked to a curated pharmacological knowledge base. WA allowed users to access a broad range of topics and correctly linked most user queries to intents. One-third of queries not mapped were either ambiguous or related to information not available to WA. The distribution of information sought via WA's conversational search versus keyword search differed depending on search method; WA may enable users to articulate different types of questions than what is sought using a standard keyword search of Micromedex. FUNDING This work was funded by IBM. AUTHOR CONTRIBUTIONS AMP, BS, AB, JH, and GPJ contributed to project design and drafting of the manuscript. MB, JH, and AB contributed to data acquisition; AMP and SW performed data analysis. AMP, NK, BB, and GJP provided leadership for the project. All authors contributed to data interpretation, manuscript revision, and approval of the final manuscript. CONFLICT OF INTEREST STATEMENT The authors are employed by IBM Watson Health.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108559/

Issues Related to the Use of Animals in Biocontainment Research Facilities

Abstract The expansion and improvement of high-containment animal facilities has been driven by terrorism, economics, the emergence of new pathogens, and the re-emergence of other pathogens in new areas. Working with highly infectious viral agents requires a team of trained scientists, laboratory technicians, veterinarians, animal care staff, biological safety officers, engineers, and physical plant staff to ensure safety, biocontainment, and the animals' well being, while providing essential scientific data. The challenges of working with infectious disease agents in high levels of containment, and some solutions to these challenges, are described from an animal care point of view. Basic Need for Expansion and Improvement of Present Animal Facilities Since the early 1990s, it has become increasingly apparent that there is a growing need for a wide range of biocontainment facilities that are properly designed to accommodate animals safely for infectious disease research. Since the 1980s, several countries, including Australia, the Netherlands, and Canada, have built state-of-the-art biological containment laboratories ( Murray 1998 ); and the United States has embarked on a major regional construction program for new biocontainment laboratories. The defection of senior Soviet scientists Vladimir Pasechnik and Kanatjan Alibekov, and subsequent revelations of the Soviet research programs on biological weapons, awakened the world to the need for more research into biological defense ( Albeck 1998 ). The events of September 11, 2001, and anthrax-related terrorism have changed the entire focus of many governments, especially the United Kingdom and the United States, as defense and homeland security have become an important focus of government funding agencies ( Baker et al. 2004 ; BRA 2002 ; NIAID 2002 ). These events illustrated many weaknesses within the biological defense policies of governments, including prevention, diagnosis, disinfection, emergency response, and security planning ( BRA 2002 ; NIAID 2002 ). Several nations have drafted reports on the needs and direction for research to meet biosecurity and defense requirements ( BRA 2002 ; NIAID 2002 ), with subsequent large budgetary allotments. The need was accentuated by long-term severe underfunding of biological containment facilities in the United States and around the world, which led to a huge deficiency in biocontainment infrastructure capacity that was incapable of meeting the growing demand. Constructed in the late 20th century, the facilities generally lacked in space and quickly became overwhelmed with projects. The deficit was most marked in the higher levels of biological containment, specifically Canadian containment levels (CLs 1 )-3 and -4 ( BRA 2002 ) and US biosafety containment levels (BSLs 1 )-3 and -4 ( NIAID 2002 ). The increasing occurrence of new or re-emerging pathogens such as bovine spongiform encephalopathy, new variant Creutzfeldt-Jakob disease, West Nile virus (WNV 1 ), severe acute respiratory syndrome (SARS 1 ), foot and mouth disease, and H5 and H7 avian influenza (AI 1 ) has further heightened the need for CL-3 and -4 research space. It is important to note that each agent may require different research and animal facility characteristics. A variety of standards must be met in the design and operation of these facilities, which can be quite challenging and must include current and emerging construction guidelines and regulations. Pressure comes not only from government agencies for more accountability, but also from commercial companies vying for the profits of biodefense products. Facilities must also comply with new, existing, and increasingly rigorous and changing standards to qualify for certificates of good laboratory practices, good manufacturing practices, or quality assurance programs such as the International Organisation for Standards. The public has become more aware of the risks associated with performing research with biohazardous agents. Through their elected officials, the general public has demanded higher standards of security. These demands include more rapid and accurate reporting of incidents, higher levels of containment and security of the agents, and more accountability by the research community. The frontline workers—research facility occupational health and safety committees, research scientists, laboratory technicians, and animal care staff—all demand higher levels of protection when working with these new and emerging diseases. These increasing demands must be met through revised standard operating procedures (SOPs 1 ) and upgrading of existing infrastructure. Basic Needs of the Scientist in the Biological Containment Animal Facility Fulfillment of Legal Requirements Scientists face a number of regulatory barriers before they can perform research on many infectious agents. The legal requirements for permits and acquisition of the newest strain of the organism can be problematic. The research facility and animal housing space must be certified for use of the infectious agent. Permits can take months, if not years, if regulatory agencies are overwhelmed with work or if existing regulations no longer apply. In Canada, for example, it is necessary to fulfill both a Health Standard and a Veterinary Standard, which are similar but not exactly the same. This is also the case in the United States, where there can be differences between, for example, Biosafety in Microbiological and Biomedical Laboratories ( Richmond and McKinney 1999 ) and US Department of Agriculture ( USDA/APHIS 1999 ) standards. These standards and other guidelines (e.g., CCAC 1984 , 1993 ; NRC 1996 ) have been developed over the years as a result of much discussion among scientists, and considering the interests of the public, the scientist, the research staff, and the animals. Naturally, this necessary process may seem cumbersome to the scientist wanting to perform research. The needs of the scientist may be vastly different from those of the research animal model criteria. The basic goal for most scientists is the observation or sample required to achieve the objective of the research. Samples and procedures must be scientifically and statistically justified, reviewed, and approved in the initial planning phases. Animal infectious disease models often require frequent sampling, which can engender debate with institutional animal care and use committees. A thorough knowledge of the laws and guidelines of each country, coupled with in-depth justification for the frequency, nature, and volume of samples, will help with this review process. Facing uncertainty with limited knowledge of a newly emerged organism, as well as the uncertainty of working with agents in new or unproven animal models for which the behavior of the agent may be unpredictable, can also challenge the scientist to devise with a working protocol. A mechanism must also be in place to ensure rapid access to animals in the face of an outbreak of a serious human or animal pathogen. Standing approved protocols can allow an animal care committee to review the document before the animals are required. The route of inoculation can be problematic when dealing with many highly contagious diseases such as tuberculosis, WNV, and AI, as well as less contagious agents such as prions. Research on the agents of tuberculosis, AI, SARS, and other respiratory diseases may require nasal or aerosol challenge; others such as WNV may require intravenous inoculation, and intracranial inoculation is common with agents such as prions. Each route and agent requires different animal care facilities, SOPs, training of personnel, and personal protection equipment. When working with scrapie in hamsters, for example, it is important to develop SOPs before the work is undertaken to satisfy the permitting agencies, local animal care committees, local safety officers, and animal care staff. The detail requirements may include a description of a calibrated injector, gauge of needle, requirement for general gaseous anesthesia (including safety associated with the anesthetic agent), and personal protection. Sampling Procedures Before the project starts, the goals of the scientist must be clearly articulated and understood by all technical and support personnel to achieve success. Most commonly, infectious disease research requires mucosal surface swabs, blood, and tissues. To accomplish these tasks, it is often necessary to develop novel approaches or search the literature for methodology. The "holistic" sampling method—a request for everything from everywhere at every point in time—is common. In CL-3 and -4, the personal safety requirements for protective equipment will affect the ability of the individual to obtain samples. Some sampling procedures may be considered too risky to perform; it is always important to keep the time factor in mind. For example, to take one blood sample from one pig will take about 15 min in a level 2 animal facility, 30 min at level 3 (including the time it takes to don and doff protective equipment and shower out), and 1 hr at level 4 because of the need to check all systems, change, get suited, restrain the animal, have a chemical shower, remove the suit and shower out. Therefore, the general rule is as the CL level increases, the number of procedures and frequency of sampling should decrease. It may be necessary to replace full necropsies with a preselected set of samples. Alternatively, it may be necessary to increase the number of trained staff needed to complete the work. Scientists enjoy overseeing experiments and dropping in to ensure that the project is proceeding well. To do so, the scientist, and even the director, must be adequately trained in high-containment safety procedures to be in the room with the animals. Initial training can take years, and all procedures that will be carried out must be reviewed, practiced, and discussed to ensure that safety is not compromised, even by these scientific observers. Once the sample is taken from the animals, it is important to have planned where the sample will be manipulated and how it will be treated before the samples leave the animal room. First, the scientist must remember that removal of samples must follow the local, regional, and national biological safety regulations, as well as the guidelines for the transport of dangerous goods. Second, it may be necessary for the sample to be treated before it is removed from a higher level of containment to a lower level. For example, serum from CL-3 is frequently gamma irradiated at 2 mega rads to remove any possible contaminating pathogens. The irradiation can alter the protein configuration, which may negate its usefulness for analyzing antibody or antigen in the native configuration. Third, it may be necessary to fix samples in formalin for a length of time before they are eligible for removal, and the analysis will be unavoidably delayed. All staff, including animal care staff, must be aware in advance of how the samples are to be processed. Photographs and video clips of animals exhibiting signs or lesions of infectious diseases are extremely useful and easily accomplished. High-quality publishable digital pictures can be produced within 1 day and can be sent to as many sites as required. The use of any sharp object must be kept to a minimum. If sharps are used, it is critical to know the location of all staff, as well as the sharps container; constant control is vital to maintain a safe working environment. The use of sharps should be limited to a select few highly trained, highly experienced individuals to minimize potential accidents. Exposure can also be decreased by continuous improvement and review of procedures to eliminate the use of any sharp. For example, the elimination of use of a scalpel in favor of blunt scissors during a necropsy procedure greatly enhances safety. However, not all modifications can be adopted. Vanish Point TM needles can be used for injections; the needle retracts into the barrel of the syringe. For small animals that do not move, the use of these needles increases safety. However, if larger animals move, Vanish Point TM needles may bend or break more easily than conventional needles and cannot be retracted, decreasing the safety of the procedure. Vacutainer bleeding systems are used for both large and small animal bleeding techniques. The availability of sharps containers for the removal of the needle sharps will allow the staff member to dispose of the needle without handling the needle hub or fumbling when removing the needle from the holder. Alternatively, the entire needle and holder can be discarded. Fulfillment of Legal Requirements Scientists face a number of regulatory barriers before they can perform research on many infectious agents. The legal requirements for permits and acquisition of the newest strain of the organism can be problematic. The research facility and animal housing space must be certified for use of the infectious agent. Permits can take months, if not years, if regulatory agencies are overwhelmed with work or if existing regulations no longer apply. In Canada, for example, it is necessary to fulfill both a Health Standard and a Veterinary Standard, which are similar but not exactly the same. This is also the case in the United States, where there can be differences between, for example, Biosafety in Microbiological and Biomedical Laboratories ( Richmond and McKinney 1999 ) and US Department of Agriculture ( USDA/APHIS 1999 ) standards. These standards and other guidelines (e.g., CCAC 1984 , 1993 ; NRC 1996 ) have been developed over the years as a result of much discussion among scientists, and considering the interests of the public, the scientist, the research staff, and the animals. Naturally, this necessary process may seem cumbersome to the scientist wanting to perform research. The needs of the scientist may be vastly different from those of the research animal model criteria. The basic goal for most scientists is the observation or sample required to achieve the objective of the research. Samples and procedures must be scientifically and statistically justified, reviewed, and approved in the initial planning phases. Animal infectious disease models often require frequent sampling, which can engender debate with institutional animal care and use committees. A thorough knowledge of the laws and guidelines of each country, coupled with in-depth justification for the frequency, nature, and volume of samples, will help with this review process. Facing uncertainty with limited knowledge of a newly emerged organism, as well as the uncertainty of working with agents in new or unproven animal models for which the behavior of the agent may be unpredictable, can also challenge the scientist to devise with a working protocol. A mechanism must also be in place to ensure rapid access to animals in the face of an outbreak of a serious human or animal pathogen. Standing approved protocols can allow an animal care committee to review the document before the animals are required. The route of inoculation can be problematic when dealing with many highly contagious diseases such as tuberculosis, WNV, and AI, as well as less contagious agents such as prions. Research on the agents of tuberculosis, AI, SARS, and other respiratory diseases may require nasal or aerosol challenge; others such as WNV may require intravenous inoculation, and intracranial inoculation is common with agents such as prions. Each route and agent requires different animal care facilities, SOPs, training of personnel, and personal protection equipment. When working with scrapie in hamsters, for example, it is important to develop SOPs before the work is undertaken to satisfy the permitting agencies, local animal care committees, local safety officers, and animal care staff. The detail requirements may include a description of a calibrated injector, gauge of needle, requirement for general gaseous anesthesia (including safety associated with the anesthetic agent), and personal protection. Sampling Procedures Before the project starts, the goals of the scientist must be clearly articulated and understood by all technical and support personnel to achieve success. Most commonly, infectious disease research requires mucosal surface swabs, blood, and tissues. To accomplish these tasks, it is often necessary to develop novel approaches or search the literature for methodology. The "holistic" sampling method—a request for everything from everywhere at every point in time—is common. In CL-3 and -4, the personal safety requirements for protective equipment will affect the ability of the individual to obtain samples. Some sampling procedures may be considered too risky to perform; it is always important to keep the time factor in mind. For example, to take one blood sample from one pig will take about 15 min in a level 2 animal facility, 30 min at level 3 (including the time it takes to don and doff protective equipment and shower out), and 1 hr at level 4 because of the need to check all systems, change, get suited, restrain the animal, have a chemical shower, remove the suit and shower out. Therefore, the general rule is as the CL level increases, the number of procedures and frequency of sampling should decrease. It may be necessary to replace full necropsies with a preselected set of samples. Alternatively, it may be necessary to increase the number of trained staff needed to complete the work. Scientists enjoy overseeing experiments and dropping in to ensure that the project is proceeding well. To do so, the scientist, and even the director, must be adequately trained in high-containment safety procedures to be in the room with the animals. Initial training can take years, and all procedures that will be carried out must be reviewed, practiced, and discussed to ensure that safety is not compromised, even by these scientific observers. Once the sample is taken from the animals, it is important to have planned where the sample will be manipulated and how it will be treated before the samples leave the animal room. First, the scientist must remember that removal of samples must follow the local, regional, and national biological safety regulations, as well as the guidelines for the transport of dangerous goods. Second, it may be necessary for the sample to be treated before it is removed from a higher level of containment to a lower level. For example, serum from CL-3 is frequently gamma irradiated at 2 mega rads to remove any possible contaminating pathogens. The irradiation can alter the protein configuration, which may negate its usefulness for analyzing antibody or antigen in the native configuration. Third, it may be necessary to fix samples in formalin for a length of time before they are eligible for removal, and the analysis will be unavoidably delayed. All staff, including animal care staff, must be aware in advance of how the samples are to be processed. Photographs and video clips of animals exhibiting signs or lesions of infectious diseases are extremely useful and easily accomplished. High-quality publishable digital pictures can be produced within 1 day and can be sent to as many sites as required. The use of any sharp object must be kept to a minimum. If sharps are used, it is critical to know the location of all staff, as well as the sharps container; constant control is vital to maintain a safe working environment. The use of sharps should be limited to a select few highly trained, highly experienced individuals to minimize potential accidents. Exposure can also be decreased by continuous improvement and review of procedures to eliminate the use of any sharp. For example, the elimination of use of a scalpel in favor of blunt scissors during a necropsy procedure greatly enhances safety. However, not all modifications can be adopted. Vanish Point TM needles can be used for injections; the needle retracts into the barrel of the syringe. For small animals that do not move, the use of these needles increases safety. However, if larger animals move, Vanish Point TM needles may bend or break more easily than conventional needles and cannot be retracted, decreasing the safety of the procedure. Vacutainer bleeding systems are used for both large and small animal bleeding techniques. The availability of sharps containers for the removal of the needle sharps will allow the staff member to dispose of the needle without handling the needle hub or fumbling when removing the needle from the holder. Alternatively, the entire needle and holder can be discarded. Basic Needs of the Animal in the Biological Containment Animal Facility Housing Housing animals within a high-containment laboratory is a difficult task. The building design frequently dictates how the animals can be housed, what kinds of animals can be used, and what kind of environment they can live in. Buildings with inadequate design for certain types of animals simply cannot be used for those animals despite the desire of the scientist. When designing an animal facility, it is important to consult with a wide range of experts, and it is vital to solicit input from the animal care staff. Only animals that are of the highest quality and free of adventitious pathogens should be used in high-containment facilities. The cost of operating a high-containment facility on a daily basis far exceeds the extra cost of quality animals. The facility must always yield the highest quality of results. Rodents, for example, should be selected for infectious disease studies based on a complete health profile, which includes serological panels and analysis for pathogenic parasites, fungi, and bacteria. When possible, it is beneficial to use calm strains that are noted for ease of handling, rather than strains known to bite or that are intractable. All animals must be properly acclimated to the high air flows, room temperature, feed, lighting regimes, and noise. Larger animals such as nonhuman primates, domestic pigs, sheep, goats, and poultry pose unique housing problems. For example, the volume of manure an adult cow generates is not the same as an adult mouse. It may be necessary to increase the frequency of cleaning or to keep the stocking density to a minimum to accommodate the volume of feces produced. The type of infectious disease agent also affects the requirement regarding treatment or sterilization of the waste. In Canada, the effluent human CL-3 from a facility may be treated directly within the laboratory, whereas an agricultural CL-3 pathogen requires secondary effluent treatment ( CSVF 1996 ; Murray 1998 ). Most agricultural animals are governed separately if they are inoculated with CL-3 agricultural agents such as foot and mouth disease, classical swine fever, and capripox. Pretreatment of large volumes of organic material with disinfectants such as sodium hypochlorite, Virkon®, or iodine-containing compounds is widely advocated, but difficult to justify scientifically. It is well known that organic matter decreases or inactivates many disinfectants (Vesley et al. 2000). Heat and pressure in cookers provide one of the best methods of treating organic material ( Edwards et al. 2002 ; Murray 1998 ). Specific pathogen-free (SPF 1 ) large animal species are rare and expensive. One SPF goat, for example, can cost up to $5,000 Canadian, but may be necessary for polyclonal antibody production destined for humans. Prior acclimation in conventional housing or a farm will decrease the amount of time for the animals to be housed in containment. It is possible to decrease certain acclimation periods from 2 to 3 wk to 1 wk, which increases annual program output. For example, nonhuman primates can be tested for tuberculosis while being acclimated to new caging systems, feed, cage mates, and personnel before they enter the high-containment area. Sheep can be shorn, dewormed, vaccinated, acclimated to pelleted feed or hay cubes, and treated for parasites. Furthermore, this period can be used to assess the temperment of larger animals, and thus their suitability, for use in the containment facility. Species, size, sex, and containment level all are factors for consideration. Planning work with infectious agents in animals requires the selection of the correct animal model. Literature searches and web-based articles can provide the scientist with vital information on a wide range of animal models. The most common species used for infectious disease studies are mice, rats, guinea pigs, and rabbits. Many guidelines and SOPs have been written for their well-being. Problems may be encountered when items such as novel caging systems, special bedding, new kinds of food, and environmental enrichment devices are needed. All items must be able to withstand frequent disinfection, if not sterilization, and some environmental enrichment devices may be unworkable or create hazards. Caging Systems Cages must be manipulated within a biological safety cabinet, which requires a great deal of coordination, training, and man power. Often, the cage cleaning equipment is not located near the CL-3 or -4 areas. The cages are cleaned and autoclaved within the CL-3 or -4 area and then sent to the cage wash area for disinfection. Single housing of animals may be required to ensure that animals do not grab, bite, or touch the staff member during the cleaning, feeding, observing, or sampling of the animal. Ethically, group housing must always be considered if personal safety is not compromised. It is easily possible to maintain small animal comfort with the use of bedding, hiding places, proper animal density per cage, and cage change frequency, as is commonly undertaken in level 2. Many different caging systems are available for rodents in CL-3 and -4. Basic rodent microisolators can be used for many agents and can be disassembled, sterilized, and stored very easily. Ventilated racks and cages offer the added advantage of more animal protection, which helps eliminate cross-contamination and increases safety for the animal care staff. Automatic watering decreases the work for staff and means less time in containment safety apparel. However, all parts of the ventilated racks and watering systems must be able to be sterilized and not simply sanitized. Prion caging requires high-temperature plastics that can be autoclaved at or above 132°C ( Prusiner et al. 1984 ). SOPs are required to decrease tracking of the agent as well as to ensure proper bedding and cage sterilization. Procedures address the pretreatment, rinsing, and removal of soiled bedding within a biological safety cabinet. The cage life will be extended if the cage is rinsed before sterilization. Quality assurance testing of every autoclave run is vital to the safety of the staff and helps eliminate cross-contamination. Extended autoclave run times are required to ensure penetration of the steam and heat into the bedding. The SOP must be part of the safety protocol, and test runs are invaluable. Once sterilized, waste may require a second step such as an additional autoclave run, alkaline digestion, or incineration, depending on the local, state, or federal laws. The goal after each cage change is to eliminate the infectious agent from the environment as completely as possible and to work in a manner that eliminates possible contamination of the laboratory. Washing Systems Animal species used in the room, cost, and availability of water treatment dictate the installation of a particular washing system. Three types of systems are used within containment facilities: (1) The most common system uses no floor drains and requires no floor mopping. Cages are sterilized with an autoclave and run through a classical cage wash unit. (2) The high-volume low-pressure washing system is used for large animal facilities. Typically, this system is characterized by a wide-bore 5- to 10-cm-diameter fire hose with a line pressure of 40 to 60 psi. The advantages are ease of cleaning large volumes of feces with few side effects for staff. The disadvantage is the significant amount of water consumption, the necessity of treating large volumes of water, and the potential smell from holding lagoons. (3) The high-pressure (up to 1200 psi) low-volume washing system shaves the manure off the flooring. The advantages of low water consumption and treatment must be weighed against the potential for occupational health hazards. Grasping high-pressure spray guns for extended periods of time can result in repetitive strain injuries. The high-pressure spray can also generate a mist, potentially aerosolizing the infectious agent. Waste Treatment and Disposal In large animal facilities, all waste must be treated by heating under pressure or for extended periods of time. Separate treatment protocols and holding tanks for different agents or containment levels are also in use ( Edwards et al. 2002 ). The large animal enclosure must be easily disinfected and preferably decontaminated with a strong anti-infective agent such as formaldehyde gas. Epoxy finishes are preferred. The pen is cleaned daily to reduce the contamination rate within the room while total decontamination procedures are left to the end of the study. One of the most contentious problems encountered is the cleaning method for the large animal pens. Gang-housed nonhuman primates can be housed on litter if there is a substantial investment in man power and if incineration is available. Also with chickens, bedding (litter) can be removed and sterilized on a regular basis. However, bedding is generally not used within large animal rooms. Most large animal waste treatment systems do not readily allow for the flushing of wood shavings, straw, or hay down the drainage system. The total elimination of bedding poses an ethical problem. Providing soft rubber mats for large animals is common; however, animals tend to defecate on the mat and become soiled and uncomfortable. Rubber inlaid flooring that contains channels will decrease but not eliminate soiling while providing a drier place for the animal to rest. It is important to keep in mind that large adult animals such as pigs can chew through rubber flooring. Tie stalls can successfully restrain the animal while the manure is channeled away, and soft rubber mats can provide comfort for the animals. Tie stalls decrease the animal's mobility, which may cause bed sores and is an ethical concern. Doors Sealed doors and directional air flow maintain the infectious material within the room ( Murray 1998 ). The animal care staff must be consulted on the style of door to be used within the facility. Large heavy doors that require substantial force to open may lead to repetitive strain injuries if staff must open and shut numerous doors during the work day. Increased numbers of staff and mechanical advantage levers may be needed to work within a facility using large door seals. Doors with sills or elevated thresholds should not be used where there is a significant amount of movement of animals or heavy items. Air gaskets can seal efficiently; however, they break down, requiring excessive compressed air capacity, directional airflow, and automated alarms to ensure the safety of the enclosure ( Murray 1998 ). Additional Needs of the Animals Nonhuman primates can easily be maintained with automatic or bottled waterers. However, there is no simple answer for watering other species. Water for large animals can be from wall-mounted automatic waterers as well as rubber water troughs; both are common. Wall-mounted units must be flushed regularly and can rupture or jam, causing flooding of the pen and excessive waste treatment. Fixed troughs are more rigid and decrease the workable space, yet plastic water buckets can be tipped over easily or the water can become contaminated. It is imperative to be able to decontaminate all watering systems, which involves the use of a suitable disinfectant agent and an appropriate contact time. A device to prevent back flow and filters within the line are important to ensure that there is no retrograde movement of the water in the lines ( Edwards et al. 2002 ). Feeding practices for nonhuman primates are well established and must be strictly regulated within the high-containment laboratories. A set schedule of food pellets, fruits, and treats must be established to ensure the health and well-being of the animals, as well as to avoid interfering with the goals of the study. Feeding sheep, pigs, cattle, and other large animal species is easily established by using a complete pelleted ration supplemented with mineral blocks and cubed hay. The ration can be presented in a fixed trough, a rubber trough, or a removable bucket. Food storage, and preparation to the extent possible, must be kept to a minimum within containment. For example, cutting fruit for nonhuman primates, pigs, or cattle within CL-3 or -4 is much more dangerous than in CL-2. The food can be transported into containment in batches in disposable containers such as plastic bags, which provide the added benefit of easily disposing of the original packaging. Environmental enrichment should be maintained within the CL-3 and -4 areas when working with large animals. Boredom in a stark environment will affect the well-being of the animals, which in turn can compromise the study. Nevertheless, devices must not pose a safety hazard. Numerous devices have sharp edges, can clutter the cage, and may be difficult to sterilize between animals. It is essential to maintain a balance between the number of devices used and the safety requirements. Smooth chain, rubber balls, and cleaning equipment make excellent environmental enrichment devices. It is critical to consult both the scientist and the safety personnel before using any environmental enrichment device. Restraint By far, one of the largest differences between the containment levels is how to restrain and manipulate animals. Restraint starts with manipulating the animal within the home cage. As a general rule of thumb, manipulations of all animal species infected with a CL-3 or -4 human pathogen should be kept to a minimum, well thought out in advance, and performed according to SOPs. Animal nails, teeth, beaks, and horns should be considered sharps. Each species must be considered individually and the peculiarities understood. For example, mice can be manipulated with forceps, avoiding any contact with the animal, whereas this procedure would be impossible for Syrian hamsters, which have no tail. It is possible to move hamsters easily from one area to another by using a strainer. Rats and guinea pigs require the animal care staff to pick up the animals physically when moving between areas or cages. Armored ( Figure 1 ) or Kevlar® ( Figure 2 ) gloves can keep the individual staff member safe, but have the disadvantage of being bulky ( Table 1 ). It is also difficult to gauge the pressure required to grasp the animal safely, which can be an ethical problem. Numerous standard commercial restraint devices that are suitable for high containment are available for mice, rats, rabbits, and guinea pigs. In the author's opinion, nonhuman primates should not be handled in CL-4 without chemical restraint. Frequently, in CL-3, leather restraining gloves are worn. Leather is not easily decontaminated, which should lead to serious consideration for the use of chemical restraint or armored gloves when using infectious CL-3 agents. The most common drugs used for restraint in nonhuman primates are Telazol® and ketamine in combination with xylazine (Popilskis and Kohn 1997). The animal can be restrained within the home cage with a squeeze, and then given an injection intramuscularly. Once the animal appears sedate, the door is opened, the animal can be gently prodded or touched with a plastic or metal rod, and then picked up while wearing restraint gloves. Figure 1 Armored gloves provide protection but are bulky and difficult for grasping. Gates and Enclosures With the use of a gating system, larger animals such as cattle and sheep can be coaxed into a head gate and a halter for most procedures. Kicking, head butting, and trampling are the main concerns. Restraint when inoculating a CL-3 or -4 agent into sensitive areas such as the feet is best completed under sedation. Xylazine is the most common and reliable drug for sedation of both of these species. Sedation allows for the accurate and safe placement of the pathogen into the animal. Figure 2 Kevlar® gloves, like armored gloves, provide protection but also are bulky and difficult for grasping. Pigs, however, are more complex. Several good methods for working with small pigs in CL-4 have been published ( Abraham et al. 2002 ). The pig must first be placed in a confined area or a crush to immobilize the animal temporarily. An intramuscular injection using a 20- to 30-cm plastic catheter allows for injection while the pig is moving. When this method is used in CL-3 or -4, it is necessary to wear armored gloves or Kevlar because the needle may spring back toward the person injecting. Catheters made with tubing that has no memory are very useful. Small pigs can be squeezed into a tight chute that allows for easy direct injection with little movement. The animal can also be picked up and placed into a sling. The sling alone can act as the restraint device, or intramuscular sedation with Telazol® or xylazine can be easily accomplished. One last alternative for the pig is to mask the animal down with halothane or isoflurane. Both procedures work quickly and effectively on most strains of pigs, and do not require needles. However, it is important to wear protective gloves to avoid being bitten. Pigs are also commonly restrained with wire or rope snares. Exotic animal handling may require novel enclosures and modifications in common techniques used in domesticated animal species or at zoos. For example, caging and housing of crows for West Nile virus studies have been accomplished using fish netting, poultry isolators, and high-tension wire. Feed has been placed in stainless steel dog food dishes, cattle chutes used as perches, and plastic tubs used as bird baths and as a source of water. Fish nets have been used to capture the birds. Kevlar gloves aided in the handling, and the birds were easily anesthetised using a small animal gas anesthetic machine. Endpoints For animals infected with CL3 or -4 agents, it is extremely important to establish endpoints. In most animal models, animals show clinical signs of infection, and some of the agents cause severe clinical signs, including death. Endpoints must be established to protect the animal from pain and distress while maintaining the integrity of the research. An excellent summary of setting endpoints for infectious disease work has been published ( Bhasin et al. 1998 ). An endpoint can be established using a series of observations or measurements and scoring the animal as the infection progresses. Once a predetermined endpoint is reached, the animal is euthanized. Endpoints other than death allow for high-quality fresh samples to be obtained, to the benefit of the animal. Housing Housing animals within a high-containment laboratory is a difficult task. The building design frequently dictates how the animals can be housed, what kinds of animals can be used, and what kind of environment they can live in. Buildings with inadequate design for certain types of animals simply cannot be used for those animals despite the desire of the scientist. When designing an animal facility, it is important to consult with a wide range of experts, and it is vital to solicit input from the animal care staff. Only animals that are of the highest quality and free of adventitious pathogens should be used in high-containment facilities. The cost of operating a high-containment facility on a daily basis far exceeds the extra cost of quality animals. The facility must always yield the highest quality of results. Rodents, for example, should be selected for infectious disease studies based on a complete health profile, which includes serological panels and analysis for pathogenic parasites, fungi, and bacteria. When possible, it is beneficial to use calm strains that are noted for ease of handling, rather than strains known to bite or that are intractable. All animals must be properly acclimated to the high air flows, room temperature, feed, lighting regimes, and noise. Larger animals such as nonhuman primates, domestic pigs, sheep, goats, and poultry pose unique housing problems. For example, the volume of manure an adult cow generates is not the same as an adult mouse. It may be necessary to increase the frequency of cleaning or to keep the stocking density to a minimum to accommodate the volume of feces produced. The type of infectious disease agent also affects the requirement regarding treatment or sterilization of the waste. In Canada, the effluent human CL-3 from a facility may be treated directly within the laboratory, whereas an agricultural CL-3 pathogen requires secondary effluent treatment ( CSVF 1996 ; Murray 1998 ). Most agricultural animals are governed separately if they are inoculated with CL-3 agricultural agents such as foot and mouth disease, classical swine fever, and capripox. Pretreatment of large volumes of organic material with disinfectants such as sodium hypochlorite, Virkon®, or iodine-containing compounds is widely advocated, but difficult to justify scientifically. It is well known that organic matter decreases or inactivates many disinfectants (Vesley et al. 2000). Heat and pressure in cookers provide one of the best methods of treating organic material ( Edwards et al. 2002 ; Murray 1998 ). Specific pathogen-free (SPF 1 ) large animal species are rare and expensive. One SPF goat, for example, can cost up to $5,000 Canadian, but may be necessary for polyclonal antibody production destined for humans. Prior acclimation in conventional housing or a farm will decrease the amount of time for the animals to be housed in containment. It is possible to decrease certain acclimation periods from 2 to 3 wk to 1 wk, which increases annual program output. For example, nonhuman primates can be tested for tuberculosis while being acclimated to new caging systems, feed, cage mates, and personnel before they enter the high-containment area. Sheep can be shorn, dewormed, vaccinated, acclimated to pelleted feed or hay cubes, and treated for parasites. Furthermore, this period can be used to assess the temperment of larger animals, and thus their suitability, for use in the containment facility. Species, size, sex, and containment level all are factors for consideration. Planning work with infectious agents in animals requires the selection of the correct animal model. Literature searches and web-based articles can provide the scientist with vital information on a wide range of animal models. The most common species used for infectious disease studies are mice, rats, guinea pigs, and rabbits. Many guidelines and SOPs have been written for their well-being. Problems may be encountered when items such as novel caging systems, special bedding, new kinds of food, and environmental enrichment devices are needed. All items must be able to withstand frequent disinfection, if not sterilization, and some environmental enrichment devices may be unworkable or create hazards. Caging Systems Cages must be manipulated within a biological safety cabinet, which requires a great deal of coordination, training, and man power. Often, the cage cleaning equipment is not located near the CL-3 or -4 areas. The cages are cleaned and autoclaved within the CL-3 or -4 area and then sent to the cage wash area for disinfection. Single housing of animals may be required to ensure that animals do not grab, bite, or touch the staff member during the cleaning, feeding, observing, or sampling of the animal. Ethically, group housing must always be considered if personal safety is not compromised. It is easily possible to maintain small animal comfort with the use of bedding, hiding places, proper animal density per cage, and cage change frequency, as is commonly undertaken in level 2. Many different caging systems are available for rodents in CL-3 and -4. Basic rodent microisolators can be used for many agents and can be disassembled, sterilized, and stored very easily. Ventilated racks and cages offer the added advantage of more animal protection, which helps eliminate cross-contamination and increases safety for the animal care staff. Automatic watering decreases the work for staff and means less time in containment safety apparel. However, all parts of the ventilated racks and watering systems must be able to be sterilized and not simply sanitized. Prion caging requires high-temperature plastics that can be autoclaved at or above 132°C ( Prusiner et al. 1984 ). SOPs are required to decrease tracking of the agent as well as to ensure proper bedding and cage sterilization. Procedures address the pretreatment, rinsing, and removal of soiled bedding within a biological safety cabinet. The cage life will be extended if the cage is rinsed before sterilization. Quality assurance testing of every autoclave run is vital to the safety of the staff and helps eliminate cross-contamination. Extended autoclave run times are required to ensure penetration of the steam and heat into the bedding. The SOP must be part of the safety protocol, and test runs are invaluable. Once sterilized, waste may require a second step such as an additional autoclave run, alkaline digestion, or incineration, depending on the local, state, or federal laws. The goal after each cage change is to eliminate the infectious agent from the environment as completely as possible and to work in a manner that eliminates possible contamination of the laboratory. Washing Systems Animal species used in the room, cost, and availability of water treatment dictate the installation of a particular washing system. Three types of systems are used within containment facilities: (1) The most common system uses no floor drains and requires no floor mopping. Cages are sterilized with an autoclave and run through a classical cage wash unit. (2) The high-volume low-pressure washing system is used for large animal facilities. Typically, this system is characterized by a wide-bore 5- to 10-cm-diameter fire hose with a line pressure of 40 to 60 psi. The advantages are ease of cleaning large volumes of feces with few side effects for staff. The disadvantage is the significant amount of water consumption, the necessity of treating large volumes of water, and the potential smell from holding lagoons. (3) The high-pressure (up to 1200 psi) low-volume washing system shaves the manure off the flooring. The advantages of low water consumption and treatment must be weighed against the potential for occupational health hazards. Grasping high-pressure spray guns for extended periods of time can result in repetitive strain injuries. The high-pressure spray can also generate a mist, potentially aerosolizing the infectious agent. Waste Treatment and Disposal In large animal facilities, all waste must be treated by heating under pressure or for extended periods of time. Separate treatment protocols and holding tanks for different agents or containment levels are also in use ( Edwards et al. 2002 ). The large animal enclosure must be easily disinfected and preferably decontaminated with a strong anti-infective agent such as formaldehyde gas. Epoxy finishes are preferred. The pen is cleaned daily to reduce the contamination rate within the room while total decontamination procedures are left to the end of the study. One of the most contentious problems encountered is the cleaning method for the large animal pens. Gang-housed nonhuman primates can be housed on litter if there is a substantial investment in man power and if incineration is available. Also with chickens, bedding (litter) can be removed and sterilized on a regular basis. However, bedding is generally not used within large animal rooms. Most large animal waste treatment systems do not readily allow for the flushing of wood shavings, straw, or hay down the drainage system. The total elimination of bedding poses an ethical problem. Providing soft rubber mats for large animals is common; however, animals tend to defecate on the mat and become soiled and uncomfortable. Rubber inlaid flooring that contains channels will decrease but not eliminate soiling while providing a drier place for the animal to rest. It is important to keep in mind that large adult animals such as pigs can chew through rubber flooring. Tie stalls can successfully restrain the animal while the manure is channeled away, and soft rubber mats can provide comfort for the animals. Tie stalls decrease the animal's mobility, which may cause bed sores and is an ethical concern. Doors Sealed doors and directional air flow maintain the infectious material within the room ( Murray 1998 ). The animal care staff must be consulted on the style of door to be used within the facility. Large heavy doors that require substantial force to open may lead to repetitive strain injuries if staff must open and shut numerous doors during the work day. Increased numbers of staff and mechanical advantage levers may be needed to work within a facility using large door seals. Doors with sills or elevated thresholds should not be used where there is a significant amount of movement of animals or heavy items. Air gaskets can seal efficiently; however, they break down, requiring excessive compressed air capacity, directional airflow, and automated alarms to ensure the safety of the enclosure ( Murray 1998 ). Additional Needs of the Animals Nonhuman primates can easily be maintained with automatic or bottled waterers. However, there is no simple answer for watering other species. Water for large animals can be from wall-mounted automatic waterers as well as rubber water troughs; both are common. Wall-mounted units must be flushed regularly and can rupture or jam, causing flooding of the pen and excessive waste treatment. Fixed troughs are more rigid and decrease the workable space, yet plastic water buckets can be tipped over easily or the water can become contaminated. It is imperative to be able to decontaminate all watering systems, which involves the use of a suitable disinfectant agent and an appropriate contact time. A device to prevent back flow and filters within the line are important to ensure that there is no retrograde movement of the water in the lines ( Edwards et al. 2002 ). Feeding practices for nonhuman primates are well established and must be strictly regulated within the high-containment laboratories. A set schedule of food pellets, fruits, and treats must be established to ensure the health and well-being of the animals, as well as to avoid interfering with the goals of the study. Feeding sheep, pigs, cattle, and other large animal species is easily established by using a complete pelleted ration supplemented with mineral blocks and cubed hay. The ration can be presented in a fixed trough, a rubber trough, or a removable bucket. Food storage, and preparation to the extent possible, must be kept to a minimum within containment. For example, cutting fruit for nonhuman primates, pigs, or cattle within CL-3 or -4 is much more dangerous than in CL-2. The food can be transported into containment in batches in disposable containers such as plastic bags, which provide the added benefit of easily disposing of the original packaging. Environmental enrichment should be maintained within the CL-3 and -4 areas when working with large animals. Boredom in a stark environment will affect the well-being of the animals, which in turn can compromise the study. Nevertheless, devices must not pose a safety hazard. Numerous devices have sharp edges, can clutter the cage, and may be difficult to sterilize between animals. It is essential to maintain a balance between the number of devices used and the safety requirements. Smooth chain, rubber balls, and cleaning equipment make excellent environmental enrichment devices. It is critical to consult both the scientist and the safety personnel before using any environmental enrichment device. Restraint By far, one of the largest differences between the containment levels is how to restrain and manipulate animals. Restraint starts with manipulating the animal within the home cage. As a general rule of thumb, manipulations of all animal species infected with a CL-3 or -4 human pathogen should be kept to a minimum, well thought out in advance, and performed according to SOPs. Animal nails, teeth, beaks, and horns should be considered sharps. Each species must be considered individually and the peculiarities understood. For example, mice can be manipulated with forceps, avoiding any contact with the animal, whereas this procedure would be impossible for Syrian hamsters, which have no tail. It is possible to move hamsters easily from one area to another by using a strainer. Rats and guinea pigs require the animal care staff to pick up the animals physically when moving between areas or cages. Armored ( Figure 1 ) or Kevlar® ( Figure 2 ) gloves can keep the individual staff member safe, but have the disadvantage of being bulky ( Table 1 ). It is also difficult to gauge the pressure required to grasp the animal safely, which can be an ethical problem. Numerous standard commercial restraint devices that are suitable for high containment are available for mice, rats, rabbits, and guinea pigs. In the author's opinion, nonhuman primates should not be handled in CL-4 without chemical restraint. Frequently, in CL-3, leather restraining gloves are worn. Leather is not easily decontaminated, which should lead to serious consideration for the use of chemical restraint or armored gloves when using infectious CL-3 agents. The most common drugs used for restraint in nonhuman primates are Telazol® and ketamine in combination with xylazine (Popilskis and Kohn 1997). The animal can be restrained within the home cage with a squeeze, and then given an injection intramuscularly. Once the animal appears sedate, the door is opened, the animal can be gently prodded or touched with a plastic or metal rod, and then picked up while wearing restraint gloves. Figure 1 Armored gloves provide protection but are bulky and difficult for grasping. Gates and Enclosures With the use of a gating system, larger animals such as cattle and sheep can be coaxed into a head gate and a halter for most procedures. Kicking, head butting, and trampling are the main concerns. Restraint when inoculating a CL-3 or -4 agent into sensitive areas such as the feet is best completed under sedation. Xylazine is the most common and reliable drug for sedation of both of these species. Sedation allows for the accurate and safe placement of the pathogen into the animal. Figure 2 Kevlar® gloves, like armored gloves, provide protection but also are bulky and difficult for grasping. Pigs, however, are more complex. Several good methods for working with small pigs in CL-4 have been published ( Abraham et al. 2002 ). The pig must first be placed in a confined area or a crush to immobilize the animal temporarily. An intramuscular injection using a 20- to 30-cm plastic catheter allows for injection while the pig is moving. When this method is used in CL-3 or -4, it is necessary to wear armored gloves or Kevlar because the needle may spring back toward the person injecting. Catheters made with tubing that has no memory are very useful. Small pigs can be squeezed into a tight chute that allows for easy direct injection with little movement. The animal can also be picked up and placed into a sling. The sling alone can act as the restraint device, or intramuscular sedation with Telazol® or xylazine can be easily accomplished. One last alternative for the pig is to mask the animal down with halothane or isoflurane. Both procedures work quickly and effectively on most strains of pigs, and do not require needles. However, it is important to wear protective gloves to avoid being bitten. Pigs are also commonly restrained with wire or rope snares. Exotic animal handling may require novel enclosures and modifications in common techniques used in domesticated animal species or at zoos. For example, caging and housing of crows for West Nile virus studies have been accomplished using fish netting, poultry isolators, and high-tension wire. Feed has been placed in stainless steel dog food dishes, cattle chutes used as perches, and plastic tubs used as bird baths and as a source of water. Fish nets have been used to capture the birds. Kevlar gloves aided in the handling, and the birds were easily anesthetised using a small animal gas anesthetic machine. Endpoints For animals infected with CL3 or -4 agents, it is extremely important to establish endpoints. In most animal models, animals show clinical signs of infection, and some of the agents cause severe clinical signs, including death. Endpoints must be established to protect the animal from pain and distress while maintaining the integrity of the research. An excellent summary of setting endpoints for infectious disease work has been published ( Bhasin et al. 1998 ). An endpoint can be established using a series of observations or measurements and scoring the animal as the infection progresses. Once a predetermined endpoint is reached, the animal is euthanized. Endpoints other than death allow for high-quality fresh samples to be obtained, to the benefit of the animal. Basic Needs of the Staff Working in the Biological Containment Animal Facility Team Approach The scientist must be trained in all safety and animal care techniques required to obtain the necessary samples from the animals in CL-3 and -4. Alternatively, trained veterinarians, technicians, or veterinary technicians can collect the samples. The best team approach developed in our laboratory is to use a combination of all staff members, which maximizes their training. For example, the scientist completes the animal use document, explains the rationale for the sampling techniques, and assists with sample selection at necropsy. The veterinarian develops the sampling techniques so they can be practiced easily and safely, trains staff in techniques, and possibly completes the necropsy. The laboratory technicians aid with restraint, sample identification, preservation, and decontamination, and conduct the laboratory testing. The animal care staff member takes care of the animals, changes cages, makes daily observations, provides sedation, and takes various samples including swabs, blood, and feces. A means of communication from inside the animal rooms must be available to staff members, particularly if they are working alone. Table 1 Suggested hand protection for mouse procedures a CL-2 infectious CL-3 zoonotic/infectious Prions Procedure Gloves Anesthesia Restraint Gloves Anesthesia Restraint Gloves Anesthesia Restraint IV injection Nitrile – D AG-R – D # – – IM injection AG-R – P AG-R – P # – – IP injection AG-R – P AG-R – P AG-R – P SQ injection AG-R – P AG-R – P AG-R – P ICr injection AG-R X – AG-R X – # – – IN injection Nitrile – P # – – # – – Oral injection AG-R – – AG-R – P AG-R – P Orbital bleed AG-R X – AG-R X P # – – Saphenous bleed – – – # – – # – – IC bleed AG-R X – AG-R X – # – – Ear notch Nitrile – P # – – # – – a –, not required. Gloves types: AG-R, armored; Nitrile, Nitrile. Anesthesia: X, required. Restraint: P, second personnel; D, device. #, procedure not carried out at the facility. Counseling, extensive training, and ongoing education of all staff are essential for success. Mock exercises and training with the live animals before they are infected or in low-containment levels are essential. After the exercise, the entire group will understand and appreciate the time, energy, and effort that is required for each procedure. Slow, methodical, well-planned experiments are much safer than hurried, ill-prepared ventures. All staff must be enrolled and trained in an effective occupational health and safety program. Biological Safety Officers Biological safety officers are pillars for safe work within the animal facility if they understand animal-based research. A biological safety officer can aid with the animal experimentation in many ways. The most important way is to compile a safety document that clearly outlines the following information: agent, dose, route, safety requirements, relevant SOPs, decontamination procedures, signage, relevant material safety data sheets (MSDSs 1 ), verification of training, and finally, a signature indicating informed consent by all involved. MSDSs are available for most infectious pathogens, but they may be outdated. Regular meetings with the animal care attendants will quickly earn trust and foster a safe work ethic. Not all biological safety officers are comfortable working within high containment, and they must be willing to accept guidance from the scientist and animal care staff to keep them safe when they enter the CL-3 and -4 animal facilities. Personal Protection Protective clothing varies with agent, procedure, and local biological safety requirements. In CL-3, most rodent work requires the following wardrobe: long-sleeved scrubs and dedicated footwear covered with booties, high-efficiency particulate air (HEPA 1 )-filtered face protection, face shield or glasses, a long-sleeved laboratory coat with impervious front and arms, and one or two pairs of disposable gloves. All of the equipment is important, but gloves and respiratory protection are the most critical. As stated above, the addition of safety gloves makes animal manipulations much safer. Kevlar and armored gloves are recommended over chain mail. Chain mail can cut or catch, and it is extremely difficult to manipulate objects or animals while wearing the gloves. Kevlar gloves are not needle stick-resistant or -proof but are highly cut-resistant and moderately flexible. Armored gloves are needle stick- and cut-resistant but have poor flexibility. Multiple layers of gloves can increase safety but the benefit is tempered by the decreased ability to complete fine motor skill tasks. Personal respiratory protection must be tailored to the agent and local biological safety requirements ( McCullough 2000 ). Within the animal pen or facility, the use of a powered air-purifying respirator with a hood or face piece is comfortable and provides protection for many hours ( McCullough 2000 ). When using the powered air-purifying respirator, a completely charged, well-maintained spare unit must be readily available. In CL-4, a full supplied-air pressurized suit and chemical showers to clean the outside of the suit are required ( Abraham et al. 2002 ; Wihelmson et al. 2002). Selection of personal protective equipment and clothing must also take comfort into account, to ensure that it is readily used and not circumvented. Clothing in the large animal facility varies with the organism and the potential for human infection. Most CL-3 agricultural agents are not zoonotic and require only rubber boots, coveralls, and gloves. Zoonotic CL-3 and human CL-3 pathogens require extra protection, especially when necropsies are performed. When working in an infected large animal room, all staff should consider themselves to be in the biological safety cabinet with the organism. Commonly, hip waders and plastic rain coats ( Figure 3 ) cover the pathologist so they can be rinsed and disinfected after the procedure. Within large animal facilities, the hoses and HEPA filter unit can be damaged by the pens, animals, and fellow workers. Sampling the animals at the endpoint is probably the most important and can be the most dangerous procedure. Comprehensive necropsy procedures have been published for CL-4 ( Abraham et al. 2002 ). These guidelines can be a starting point for most CL-3 infectious agents. Every project should have a predesigned sampling strategy. For example, in a project involving pigs and chickens infected with SARS, the animal care technician cleaned the room and bled the animals. A veterinary pathologist completed the necropsy and selected the tissue samples. The scientist ensured that all samples were taken and placed in the appropriate containers, and transferred them to the high-containment laboratory. The laboratory technician immediately performed the tests as well as preserved and catalogued the samples. Team Approach The scientist must be trained in all safety and animal care techniques required to obtain the necessary samples from the animals in CL-3 and -4. Alternatively, trained veterinarians, technicians, or veterinary technicians can collect the samples. The best team approach developed in our laboratory is to use a combination of all staff members, which maximizes their training. For example, the scientist completes the animal use document, explains the rationale for the sampling techniques, and assists with sample selection at necropsy. The veterinarian develops the sampling techniques so they can be practiced easily and safely, trains staff in techniques, and possibly completes the necropsy. The laboratory technicians aid with restraint, sample identification, preservation, and decontamination, and conduct the laboratory testing. The animal care staff member takes care of the animals, changes cages, makes daily observations, provides sedation, and takes various samples including swabs, blood, and feces. A means of communication from inside the animal rooms must be available to staff members, particularly if they are working alone. Table 1 Suggested hand protection for mouse procedures a CL-2 infectious CL-3 zoonotic/infectious Prions Procedure Gloves Anesthesia Restraint Gloves Anesthesia Restraint Gloves Anesthesia Restraint IV injection Nitrile – D AG-R – D # – – IM injection AG-R – P AG-R – P # – – IP injection AG-R – P AG-R – P AG-R – P SQ injection AG-R – P AG-R – P AG-R – P ICr injection AG-R X – AG-R X – # – – IN injection Nitrile – P # – – # – – Oral injection AG-R – – AG-R – P AG-R – P Orbital bleed AG-R X – AG-R X P # – – Saphenous bleed – – – # – – # – – IC bleed AG-R X – AG-R X – # – – Ear notch Nitrile – P # – – # – – a –, not required. Gloves types: AG-R, armored; Nitrile, Nitrile. Anesthesia: X, required. Restraint: P, second personnel; D, device. #, procedure not carried out at the facility. Counseling, extensive training, and ongoing education of all staff are essential for success. Mock exercises and training with the live animals before they are infected or in low-containment levels are essential. After the exercise, the entire group will understand and appreciate the time, energy, and effort that is required for each procedure. Slow, methodical, well-planned experiments are much safer than hurried, ill-prepared ventures. All staff must be enrolled and trained in an effective occupational health and safety program. Biological Safety Officers Biological safety officers are pillars for safe work within the animal facility if they understand animal-based research. A biological safety officer can aid with the animal experimentation in many ways. The most important way is to compile a safety document that clearly outlines the following information: agent, dose, route, safety requirements, relevant SOPs, decontamination procedures, signage, relevant material safety data sheets (MSDSs 1 ), verification of training, and finally, a signature indicating informed consent by all involved. MSDSs are available for most infectious pathogens, but they may be outdated. Regular meetings with the animal care attendants will quickly earn trust and foster a safe work ethic. Not all biological safety officers are comfortable working within high containment, and they must be willing to accept guidance from the scientist and animal care staff to keep them safe when they enter the CL-3 and -4 animal facilities. Personal Protection Protective clothing varies with agent, procedure, and local biological safety requirements. In CL-3, most rodent work requires the following wardrobe: long-sleeved scrubs and dedicated footwear covered with booties, high-efficiency particulate air (HEPA 1 )-filtered face protection, face shield or glasses, a long-sleeved laboratory coat with impervious front and arms, and one or two pairs of disposable gloves. All of the equipment is important, but gloves and respiratory protection are the most critical. As stated above, the addition of safety gloves makes animal manipulations much safer. Kevlar and armored gloves are recommended over chain mail. Chain mail can cut or catch, and it is extremely difficult to manipulate objects or animals while wearing the gloves. Kevlar gloves are not needle stick-resistant or -proof but are highly cut-resistant and moderately flexible. Armored gloves are needle stick- and cut-resistant but have poor flexibility. Multiple layers of gloves can increase safety but the benefit is tempered by the decreased ability to complete fine motor skill tasks. Personal respiratory protection must be tailored to the agent and local biological safety requirements ( McCullough 2000 ). Within the animal pen or facility, the use of a powered air-purifying respirator with a hood or face piece is comfortable and provides protection for many hours ( McCullough 2000 ). When using the powered air-purifying respirator, a completely charged, well-maintained spare unit must be readily available. In CL-4, a full supplied-air pressurized suit and chemical showers to clean the outside of the suit are required ( Abraham et al. 2002 ; Wihelmson et al. 2002). Selection of personal protective equipment and clothing must also take comfort into account, to ensure that it is readily used and not circumvented. Clothing in the large animal facility varies with the organism and the potential for human infection. Most CL-3 agricultural agents are not zoonotic and require only rubber boots, coveralls, and gloves. Zoonotic CL-3 and human CL-3 pathogens require extra protection, especially when necropsies are performed. When working in an infected large animal room, all staff should consider themselves to be in the biological safety cabinet with the organism. Commonly, hip waders and plastic rain coats ( Figure 3 ) cover the pathologist so they can be rinsed and disinfected after the procedure. Within large animal facilities, the hoses and HEPA filter unit can be damaged by the pens, animals, and fellow workers. Sampling the animals at the endpoint is probably the most important and can be the most dangerous procedure. Comprehensive necropsy procedures have been published for CL-4 ( Abraham et al. 2002 ). These guidelines can be a starting point for most CL-3 infectious agents. Every project should have a predesigned sampling strategy. For example, in a project involving pigs and chickens infected with SARS, the animal care technician cleaned the room and bled the animals. A veterinary pathologist completed the necropsy and selected the tissue samples. The scientist ensured that all samples were taken and placed in the appropriate containers, and transferred them to the high-containment laboratory. The laboratory technician immediately performed the tests as well as preserved and catalogued the samples. Operational Needs Within the Animal Facility Design Features The "box within a box" classical theory of biological containment is well known, and many programs are in place for various kinds of agents such as viruses, bacteria, mycoplasma, and prions. The paradigm has served well for the basic concept involved in building new animal facilities and working within existing facilities. Many papers, guidelines, and excellent references are available that describe requirements within the animal room. Briefly, the floors, walls, and ceilings must be impervious to water, and easily cleaned and sterilized. Epoxy-covered cement is the most common example. HEPA filtration of the exhaust air is essential, and high flow rates up to 20 air exchanges per hour are common. Frequently, noise can become a problem because the baffling of incoming air is expensive and often omitted unless considered essential by the animal care staff who will work in the area. Ante-rooms provide a high degree of safety and an area to don, doff, and store essential safety equipment. If the ante-room is not big enough for highly infectious agents, adjacent rooms and return hallways can provide large areas for safe decontamination and removal of equipment. Figure 3 Example of protective clothing for Canadian containment level 3 work. The requirement for body showers when entering or leaving a containment area is a very contentious issue. However, from an animal care perspective, they are very important for the following reasons: (1) Body showers help stop fomite spread between animal rooms, which is essential if they are infected with different agents or even different strains of the same organism. In short, it forces staff to change clothing to prevent the spread of organisms. (2) Exit showers provide a mechanism to help remove any pathogen on the surface of the body. (3) The showers prevent quick access, which can lead to circumvention of safety protocols; and finally (4) showers refresh the staff members after a long experimental procedure. Heating, Ventilation, Air Conditioning (HVAC 1 ) The HVAC system is essential to the safe functioning of the high-containment animal care facility. The requirements of modern HVAC systems are more centralized and highly dependent on computers and computer software ( Murray 1998 ). Lighting, heating, and air flow all are governed by staff not directly within the high-containment facility. When the containment areas run smoothly, there are few problems. Minor problems can be analyzed with the data presented to management and regulatory bodies in an efficient manner. However, local input into the system is essential when obvious problems occur at the room level that may even register as normal in the computer. Safety can be compromised if the staff member is not taken seriously or the problem cannot be quickly solved. Containment facilities have been designed where the CL-4 animal holding area is controlled both locally and centrally, adding a second level of confidence in the system while allowing the input of the end user into the HVAC systems functioning ( Abraham et al. 2002 ). It may not be possible for HVAC systems to adjust for every animal species. Chicken feathers and fluff will clog rough filters and prefilters within 18 to 24 hr even though stocking density is low. On the Canadian prairies, the humidity levels in the winter can be extremely low and, coupled with high air flow rates, may be unsuitable for rodents unless humidified. The need for annual recertification of the CL-3 and -4 laboratories can involve checking the functioning of back-up systems, fire alarms, and air flow alarms. Ongoing maintenance of complex animal care facilities will cost program time as well as have the potential to disturb animals severely. Checks, tests, and maintenance must be factored into the total available program time for animal experimentation. Air flow, fire, and breathing air alarms indicate when the HVAC system in not functioning properly or the occurrence of a dangerous incident. All staff entering the CL-3 and -4 must be familiar with the proper course of evacuation or response to the alarms and be tested before entry. Design Features The "box within a box" classical theory of biological containment is well known, and many programs are in place for various kinds of agents such as viruses, bacteria, mycoplasma, and prions. The paradigm has served well for the basic concept involved in building new animal facilities and working within existing facilities. Many papers, guidelines, and excellent references are available that describe requirements within the animal room. Briefly, the floors, walls, and ceilings must be impervious to water, and easily cleaned and sterilized. Epoxy-covered cement is the most common example. HEPA filtration of the exhaust air is essential, and high flow rates up to 20 air exchanges per hour are common. Frequently, noise can become a problem because the baffling of incoming air is expensive and often omitted unless considered essential by the animal care staff who will work in the area. Ante-rooms provide a high degree of safety and an area to don, doff, and store essential safety equipment. If the ante-room is not big enough for highly infectious agents, adjacent rooms and return hallways can provide large areas for safe decontamination and removal of equipment. Figure 3 Example of protective clothing for Canadian containment level 3 work. The requirement for body showers when entering or leaving a containment area is a very contentious issue. However, from an animal care perspective, they are very important for the following reasons: (1) Body showers help stop fomite spread between animal rooms, which is essential if they are infected with different agents or even different strains of the same organism. In short, it forces staff to change clothing to prevent the spread of organisms. (2) Exit showers provide a mechanism to help remove any pathogen on the surface of the body. (3) The showers prevent quick access, which can lead to circumvention of safety protocols; and finally (4) showers refresh the staff members after a long experimental procedure. Heating, Ventilation, Air Conditioning (HVAC 1 ) The HVAC system is essential to the safe functioning of the high-containment animal care facility. The requirements of modern HVAC systems are more centralized and highly dependent on computers and computer software ( Murray 1998 ). Lighting, heating, and air flow all are governed by staff not directly within the high-containment facility. When the containment areas run smoothly, there are few problems. Minor problems can be analyzed with the data presented to management and regulatory bodies in an efficient manner. However, local input into the system is essential when obvious problems occur at the room level that may even register as normal in the computer. Safety can be compromised if the staff member is not taken seriously or the problem cannot be quickly solved. Containment facilities have been designed where the CL-4 animal holding area is controlled both locally and centrally, adding a second level of confidence in the system while allowing the input of the end user into the HVAC systems functioning ( Abraham et al. 2002 ). It may not be possible for HVAC systems to adjust for every animal species. Chicken feathers and fluff will clog rough filters and prefilters within 18 to 24 hr even though stocking density is low. On the Canadian prairies, the humidity levels in the winter can be extremely low and, coupled with high air flow rates, may be unsuitable for rodents unless humidified. The need for annual recertification of the CL-3 and -4 laboratories can involve checking the functioning of back-up systems, fire alarms, and air flow alarms. Ongoing maintenance of complex animal care facilities will cost program time as well as have the potential to disturb animals severely. Checks, tests, and maintenance must be factored into the total available program time for animal experimentation. Air flow, fire, and breathing air alarms indicate when the HVAC system in not functioning properly or the occurrence of a dangerous incident. All staff entering the CL-3 and -4 must be familiar with the proper course of evacuation or response to the alarms and be tested before entry. Summary Work with highly infectious disease organisms in animal model systems will continue to be required in the future. A team of highly motivated individuals can work safely within CL-3 and -4 if they plan ahead, use basic safety principles, and communicate effectively. The scientist, laboratory technician, veterinarian, biological safety officer, animal care staff, engineering staff, and physical plant staff all must understand the ethical use of animals, animal use protocol, and the building design. Acknowledgments I would like to thank the Animal Care staff of Nicole Beausoleil, Christine de Graff, Michelle French, Stacey Halayko, Hilary Holland, Marlee Ritchie, Kevin Tierney, Shannon Toback, and Leigh Weatherburn, along with Biological Safety Officer Dr. Peter Cairns, and Pamela Johnson for her support and editing skills. The views and opinions expressed herein are my own and do not necessarily reflect those of my employers.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047809/

Impact of BRICS’ investment in vaccine development on the global vaccine market

Abstract Brazil, the Russian Federation, India, China and South Africa – the countries known as BRICS – have made considerable progress in vaccine production, regulation and development over the past 20 years. In 1993, all five countries were producing vaccines but the processes used were outdated and non-standardized, there was little relevant research and there was negligible international recognition of the products. By 2014, all five countries had strong initiatives for the development of vaccine technology and had greatly improved their national regulatory capacity. South Africa was then the only BRICS country that was not completely producing vaccines. South Africa is now in the process of re-establishing its own vaccine production and passing beyond the stage of simply importing, formulating and filling vaccine bulks. Changes in the public sector's price per dose of selected vaccines, the global market share represented by products from specific manufacturers, and the attractiveness, for multinational companies, of partnership and investment opportunities in BRICS companies have all been analysed. The results indicate that the BRICS countries have had a major impact on vaccine price and availability, with much of that impact attributable to the output of Indian vaccine manufacturers. China is expected to have a greater impact soon, given the anticipated development of Chinese vaccine manufacturers in the near future. BRICS' accomplishments in the field of vaccine development are expected to reshape the global vaccine market and accelerate access to vaccines in the developing world. The challenge is to turn these expectations into strategic actions and practical outcomes.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5656412/

Modeling the environmental suitability of anthrax in Ghana and estimating populations at risk: Implications for vaccination and control

Anthrax is hyper-endemic in West Africa. Despite the effectiveness of livestock vaccines in controlling anthrax, underreporting, logistics, and limited resources makes implementing vaccination campaigns difficult. To better understand the geographic limits of anthrax, elucidate environmental factors related to its occurrence, and identify human and livestock populations at risk, we developed predictive models of the environmental suitability of anthrax in Ghana. We obtained data on the location and date of livestock anthrax from veterinary and outbreak response records in Ghana during 2005–2016, as well as livestock vaccination registers and population estimates of characteristically high-risk groups. To predict the environmental suitability of anthrax, we used an ensemble of random forest (RF) models built using a combination of climatic and environmental factors. From 2005 through the first six months of 2016, there were 67 anthrax outbreaks (851 cases) in livestock; outbreaks showed a seasonal peak during February through April and primarily involved cattle. There was a median of 19,709 vaccine doses [range: 0–175 thousand] administered annually. Results from the RF model suggest a marked ecological divide separating the broad areas of environmental suitability in northern Ghana from the southern part of the country. Increasing alkaline soil pH was associated with a higher probability of anthrax occurrence. We estimated 2.2 (95% CI: 2.0, 2.5) million livestock and 805 (95% CI: 519, 890) thousand low income rural livestock keepers were located in anthrax risk areas. Based on our estimates, the current anthrax vaccination efforts in Ghana cover a fraction of the livestock potentially at risk, thus control efforts should be focused on improving vaccine coverage among high risk groups. Introduction Anthrax is a soil-borne, zoonotic disease found on nearly every continent (except Antarctica) that primarily infects herbivorous animals while secondarily infecting humans through the handling or ingestion of contaminated meat or animal by-products [ 1 , 2 ]. The geographic distribution of the disease appears to be limited by a combination of climatic (e.g. precipitation and temperature) and environmental (e.g. alkaline soil pH) conditions [ 3 , 4 ]. Under the appropriate ecological conditions, which remain poorly understood, the causative agent of anthrax, Bacillus anthracis , can survive for long-periods of time in the environment, perhaps years [ 1 , 4 ]. Although it has received much attention as a potential agent of bioterrorism, the World Health Organization (WHO) has listed anthrax as a neglected disease [ 5 ]. Poor livestock keepers and their animals often experience a disproportionate burden of anthrax in the hyper-endemic regions of Central Asia and West Africa [ 5 , 6 ]. Despite the effectiveness of regular animal vaccination and proper outbreak response following recommended guidelines in controlling anthrax in humans, underreporting of the disease often skews its true burden and geographic distribution making it difficult to implement adequate vaccination campaigns [ 1 , 7 ]. In Ghana, anthrax outbreaks have been reported annually in humans associated with contact with infected livestock and their contaminated animal by-products (e.g. meat or hides) [ 8 ]. Anthrax vaccine is manufactured locally by the Central Veterinary Laboratory in Pong-Tamale, Ghana and is fully subsidized by the government. Despite this, animal outbreaks are documented annually, and primarily affect cattle. Although both human and animal cases are reported, few human cases are linked to confirmed animal cases [ 9 ]. As a result, surveillance data alone provide limited information to efficiently plan prevention activities. Previous efforts to elucidate the environmental suitability of anthrax in Africa have been focused on southern countries, such as Zimbabwe [ 10 ], or national parks [ 11 ]. A recent study from West Africa also used a machine learning algorithm to map and model the distribution of anthrax and B . anthracis in Cameroon, Chad, and Nigeria, however, that effort was based on limited sample size and no comparable efforts have been carried out in Ghana [ 12 ]. To support Ghana's national anthrax control and assessment, we our study had the following objectives: (1) model the environmental suitability of anthrax; (2) identify environmental and climatic factors associated with the occurrence of anthrax; (3) describe seasonal patterns; and (4) estimate populations at risk. Methods Ethics statement This work was performed on nationally available data on anthrax outbreaks in livestock from the Ministry of Food and Agriculture in Ghana. Anthrax occurrence data We constructed a GIS of livestock anthrax outbreaks using data collected by the Ghana Field Epidemiology and Laboratory Training Program (GFELTP) and the Ministry of Food and Agricultural Veterinary Services. ( Fig 1 ). Outbreaks were mapped using GPS coordinates collected by field personnel responding to outbreaks or the center of the village where the outbreak occurred. We included data on outbreaks from 2005 through the first 6-months of 2016 included information on the geographic coordinates, date, livestock species, and number of individual animals infected (periodically recording mortality and survival status) for each outbreak. However, total livestock populations on affected properties was rarely reported. For this study, an outbreak was defined as any location with one or more anthrax cases in animals. We plotted the seasonality of anthrax outbreaks in relation to the average rainfall during 1991–2015 using data obtained from the Climate Change Knowledge Portal ( http://sdwebx.worldbank.org/climateportal/index.cfm?page=country_historical_climate&ThisCCode=GHA ). We also obtained livestock anthrax vaccine administration data during 2005–2015 from the World Animal health Information Database Interface (OIE; http://www.oie.int/animal-health-in-the-world/the-world-animal-health-information-system/data-after-2004-wahis-interface/ ). Mapping and spatial analysis was performed in Q-GIS version 2.14 ( www.qgis.org ) and the R statistical package ( https://www.r-project.org/ ). Final maps were produced in ArcGIS version 10.3.1 (ESRI, Redlands, CA, USA). 10.1371/journal.pntd.0005885.g001 Fig 1 Spatial setting of Ghana in West Africa (inset A) and the geographic distribution of anthrax outbreaks (January 2005—June 2016) and generated pseudo absence data (inset B). The number of outbreaks by region in Ghana (inset C). Political boundary data were downloaded from www.gadm.org and all maps were produced in ArcGIS ( www.esri.com ; see Methods ). Environmental and climatic data We used a combination of environmental and climatic variables at a spatial resolution of 30-arcseconds (approximately 1km x 1km) that followed, in part, recent studies in West Africa [ 13 ] and Central Asia [ 14 ] ( Table 1 ). Five "bioclimatic" variables describing measures of temperature and precipitation were obtained from the WorldClim database ( www.worldclim.org ) [ 15 ]. WorldClim variables are interpolated monthly measurements recorded at weather stations located worldwide between 1950 and 2000. WorldClim produces bioclimatic variable grids to describe annual trends, seasonality, and ecological parameters such as temperature of the coldest and warmest quarters. We also used a combination of physical (sand content), chemical (soil pH), and taxonomic classifications of soil characteristics (cancerous vertisols and humults). Soil data were obtained from the SoilGrids1km database http://www.isric.org/explore/soilgrids ) [ 16 ]. SoilGrid variables were created using spatial model predictions based on a global database of soil profiles and a combination of environmental covariates. Furthermore, we used two normalized difference vegetation index (NDVI) variables describing average conditions and the amplitude of vegetation greenness, which were obtained from the Trypanosomiasis and Land Use in Africa (TALA) research group (Oxford, United Kingdom) [ 17 ]. TALA variables were derived from temporal Fourier analysed (TFA) time series data of advanced very-high resolution radiometer (AVHRR) satellite measurements taken between 1992 and 1996 [ 17 ]. Mapped variables are shown in S1 Fig . 10.1371/journal.pntd.0005885.t001 Table 1 Environmental and climatic variables used in the random forest models. Environmental variable Variable name Data source Reference Elevation (m) elevation Worldclim [ 6 ] Mean annual temperature (°C) bio 1 Worldclim [ 6 ] Annual temperature range (°C) bio 7 Worldclim [ 6 ] Annual precipitation (mm) bio 12 Worldclim [ 6 ] Precipitation: wettest month (mm) bio 13 Worldclim [ 6 ] Precipitation driest month (mm) bio 14 Worldclim [ 6 ] Average soil pH soil pH SoilGrids1km [ 7 ] Calcerous vertisols (% coverage) vertisol SoilGrids1km [ 7 ] Humult (% coverage) humult SoilGrids1km [ 7 ] Sand (% mass fraction) sand SoilGrids1km [ 7 ] Temporal Fourier mean NDVI wd0114a0 TALA [ 8 ] Temporal Fourier NDVI annual amplitude wd0114a1 TALA [ 8 ] Data analysis Random Forest (RF) modeling [ 18 , 19 ] was used to identify environmental characteristics associated with the occurrence of anthrax outbreaks using the 'randomForest' package for R. Previous studies have used this approach to map and model the distribution of Anopheles spp . mosquito vectors in Africa and Europe [ 20 ] and reservoirs of avian influenza [ 21 ]. RF modeling has been described and compared to other modeling approaches in detail elsewhere [ 18 , 22 ]. Briefly, RF is a non-parametric method derived from classification and regression trees that consists of a combination of trees built using randomly selected bootstrap samples of the training data (used to build the model), with the number of bootstrap samples equal to the number of trees ( ntrees ) selected. Each tree is split by randomly sampling a number of predictor variables to use ( mtry) at each node and then choosing the best split. Model error estimates are obtained by internal splits of the training data (63.2% for model building) and then predicting the data not used to build a tree (out-of-bag or OOB) and aggregating these predictions for each ensemble of trees [ 18 ]. Since internal validation of the OOB data is performed, no external testing data is required to validate the model, but testing splits (external data withheld from the model) of the data are routinely utilized to assess model performance. Partial dependence plots and variable importance of RF models were assessed for covariates in the model. We used an ensemble modeling approach that incorporated information from multiple random splits of our data into training (80%) and testing (20%) sets. Since our data consisted of presence only records of anthrax outbreaks, we generated pseudo-absence data from all available background data. Several studies have either relied on internal derivations of pseudo-absence in species distribution models [ 23 ] or user-defined generations such as in the modeling of the global distribution of dengue virus [ 24 ]. The required number of user-defined background pseudo-absence draws for every presence location is not standardized. It has been suggested that a 1:1 random draw of pseudo-absence to presence data in machine learning algorithms such as RF produces optimal results [ 25 ], although variations of this (2:1 or 3:1 draws) have been adopted successfully [ 24 ]. Similarly, pseudo-absence data creation has been shown to influence results; thus, research has recommended filtering pseudo-absence data from locations that are known to fall within suitable habitat or that occur within a defined proximity threshold [ 25 , 26 ]. We first filtered geo-located anthrax presence data in Ghana (n = 61) using a 5km x 5km proximity threshold in order to improve model performance and avoid overfitting [ 27 ]. We generated background pseudo-absence data (n = 200), from all available background [ 24 ], at a ratio of four absence points to every one filtered presence point (n = 50), restricting pseudo-absence data to exclude landscape within 5km of presence locations. We then generated 10 random draws each of 1:1, 2:1, and 3:1 pseudo-absence to presence data (30 total draws) with replacement. Each randomly generated pseudo-absence to presence draw (n = 30) was randomly divided into training and testing data splits to validate model performance. The final RF models were built using a mtry = 4 at each split and ntrees = 1000 with a combination of variables in which the ensemble list contributed to a mean decrease in accuracy >1%. The 30 individual RF models were then combined into an ensemble prediction at a spatial resolution of ~1km x 1km and scaled from 0 (low suitability) to 1 (high suitability); uncertainty in the model prediction was calculated by taking the range in the 95% confidence intervals of the ensemble model scaled from 0 (low uncertainty) to 1 (high uncertainty) following Deribe et al. [ 28 ]. The resulting output of our ensemble RF model represents the environmental suitability of anthrax in Ghana. To estimate the number of livestock and poor rural livestock keepers at risk in anthrax suitable areas, we dichotomized the modeled environmental suitability into a suitable versus not suitable prediction using a probability threshold that maximized sensitivity and specificity. We then overlaid a database of global livestock density at a spatial resolution of ~1km x 1km ( http://www.livestock.geo-wiki.org/ ) [ 29 ] with the dichotomized anthrax prediction to estimate the livestock populations (cattle, sheep, goats, and swine) at risk. Livestock populations at risk were further stratified to estimate the population at risk within each of the livestock production zones of Ghana using the livestock production systems data version 5 ( http://www.livestock.geo-wiki.org/ ) [ 29 – 31 ]. Furthermore, we estimated the number of low income rural livestock keepers at risk within each livestock production zone by overlaying the dichotomized anthrax suitable areas with estimates of the population of low income rural livestock keepers provided in Robinson et al. [ 31 ] and deriving the fraction of cells that were within our model prediction. Uncertainty in the populations at risk and 95% confidence intervals were calculated by using the 2.5% (lower) and 97.5% (upper) bounds of the ensemble RF model prediction [ 28 ]. Model performance and validation was conducted for each individual RF model and included the internal: OOB error classification, area under the receiver operating characteristics curve (AUC), sensitivity, and specificity. Additionally, we performed accuracy assessments on the external testing data, which consisted of thirty random subsets of 20% of the data sampled with replacement. Mean values and 95% confidence intervals were estimated for each accuracy metric. The AUC has been used extensively in species distribution modeling to measure the discriminatory performance of models [ 32 ]; an AUC value of 1 indicates a perfect discrimination while values of >0.9 are outstanding, 0.8–0.9 excellent, 0.7–0.8 acceptable, and 1%. The 30 individual RF models were then combined into an ensemble prediction at a spatial resolution of ~1km x 1km and scaled from 0 (low suitability) to 1 (high suitability); uncertainty in the model prediction was calculated by taking the range in the 95% confidence intervals of the ensemble model scaled from 0 (low uncertainty) to 1 (high uncertainty) following Deribe et al. [ 28 ]. The resulting output of our ensemble RF model represents the environmental suitability of anthrax in Ghana. To estimate the number of livestock and poor rural livestock keepers at risk in anthrax suitable areas, we dichotomized the modeled environmental suitability into a suitable versus not suitable prediction using a probability threshold that maximized sensitivity and specificity. We then overlaid a database of global livestock density at a spatial resolution of ~1km x 1km ( http://www.livestock.geo-wiki.org/ ) [ 29 ] with the dichotomized anthrax prediction to estimate the livestock populations (cattle, sheep, goats, and swine) at risk. Livestock populations at risk were further stratified to estimate the population at risk within each of the livestock production zones of Ghana using the livestock production systems data version 5 ( http://www.livestock.geo-wiki.org/ ) [ 29 – 31 ]. Furthermore, we estimated the number of low income rural livestock keepers at risk within each livestock production zone by overlaying the dichotomized anthrax suitable areas with estimates of the population of low income rural livestock keepers provided in Robinson et al. [ 31 ] and deriving the fraction of cells that were within our model prediction. Uncertainty in the populations at risk and 95% confidence intervals were calculated by using the 2.5% (lower) and 97.5% (upper) bounds of the ensemble RF model prediction [ 28 ]. Model performance and validation was conducted for each individual RF model and included the internal: OOB error classification, area under the receiver operating characteristics curve (AUC), sensitivity, and specificity. Additionally, we performed accuracy assessments on the external testing data, which consisted of thirty random subsets of 20% of the data sampled with replacement. Mean values and 95% confidence intervals were estimated for each accuracy metric. The AUC has been used extensively in species distribution modeling to measure the discriminatory performance of models [ 32 ]; an AUC value of 1 indicates a perfect discrimination while values of >0.9 are outstanding, 0.8–0.9 excellent, 0.7–0.8 acceptable, and 1.2 (95% CI: 1.1, 1.3) million ( Table 3 ). 10.1371/journal.pntd.0005885.t003 Table 3 Livestock production systems ¥ and the livestock population in the Ghanaian anthrax risk zone. Population at risk [95% CI] (thousands of livestock) Livestock LGA LGH MRA MRH MIA cattle 108.5 [83.1, 123.2] 38.2 [30, 48.8] 283.3 [272.3, 289.7] 49.2 [47.7, 63.8] 1.2 [1.1, 1.2] sheep 166.2 [125.4, 188] 50.6 [41.2, 74] 368 [353.8, 376.6] 63.9 [61.6, 105.5] 1.2 [1.1, 1.2] goats 262.3 [201, 301.7] 47.9 [38, 67.8] 509.9 [491.3, 524.2] 117.8 [115, 164] 1.1 [1.0, 1.1] swine 32.1 [24.7, 36.1] 11.5 [9.8, 17.1] 70.6 [67.6, 72.2] 14.7 [13.9, 24.9] 0.2 [0.2, 0.3] ¥ Livestock production systems ( http://www.fao.org/docrep/014/i2414e/i2414e.pdf ) LGA: Livestock only systems, arid and semi-arid LGH: Livestock only systems, humid and sub-humid MRA: Rainfed mixed crop/livestock system, arid and semi-arid MRH: Rainfed mixed crop/livestock system, humid and sub-humid MIA: Irrigated mixed crop/livestock systems, arid and semi-arid Nationally, there are approximately 3 million low income rural livestock keepers in Ghana ( Table 4 ). Our model suggests that ≈ 805 (95% CI: 519, 890) thousand are located in areas suitable for anthrax, with the majority located in a humid and sub-humid, mixed crop livestock system production zone (MRH). 10.1371/journal.pntd.0005885.t004 Table 4 Rural low income livestock keepers in the Ghanaian anthrax risk zone by livestock production system. Population at risk [95% CI] (thousands of people) Estimates ¥ LGA LGH MRA MRH MIA National 71.1 238.9 411.9 2152.9 1.2 [1.1, 1.2] Modeled 48.6 [34.1, 56.9] 105.4 [65.7, 120.2] 300 [263.6, 325.1] 346.6 [155, 387.5] 0.4 [0.3, 0.4] ¥ Livestock production systems ( http://www.fao.org/docrep/014/i2414e/i2414e.pdf ) LGA: Livestock only systems, arid and semi-arid LGH: Livestock only systems, humid and sub-humid MRA: Rainfed mixed crop/livestock system, arid and semi-arid MRH: Rainfed mixed crop/livestock system, humid and sub-humid MIA: Irrigated mixed crop/livestock systems, arid and semi-arid Anthrax outbreaks From 2005 through the first 6 months of 2016, there were 67 reported anthrax outbreaks in livestock (61 that were geo-located) ( Fig 1 ). Nationally, there was a mean of 6 (95% CI: 4, 7) outbreaks per year with a peak in 2011 (n = 12) and lull in reporting in 2009 (n = 2) ( Fig 2 ). The geography of outbreaks shows a higher frequency of anthrax in northern Ghana in the Upper East and Northern regions. Of the reported outbreaks, 4 (6%) were comprised of two or more livestock types. Domestic cattle were reported in 53% (35) of outbreaks, followed by sheep in 32% (21), goats in 11% (7), and swine in 5% (3). During 2005–2016, cattle anthrax cases were reported every year except in 2009. Sheep cases were ubiquitous annually and were characterized by a large number of deaths in 2012, the same year there was also a large number of swine cases (n = 500) ( Table 2 ). 10.1371/journal.pntd.0005885.g002 Fig 2 Annual number of livestock anthrax outbreaks in Ghana during January 2005- June 2016. 10.1371/journal.pntd.0005885.t002 Table 2 Cases of anthrax by livestock type in Ghana, 2005–2016. Year Cattle Sheep Goats Swine 2005 27 1 0 0 2006 17 15 0 0 2007 15 9 3 0 2008 21 6 11 0 2009 0 2 0 0 2010 2 1 0 0 2011 13 7 0 3 2012 6 66 1 500 2013 50 14 0 0 2014 3 1 0 1 2015 26 1 0 0 2016 4 6 19 0 Totals 184 129 34 504 The seasonality of anthrax outbreaks nationally and regionally are illustrated in Fig 3 . Nationally, outbreaks were reported, on average, across seasons and in every month (except November). There was an increase in outbreaks in the late winter and early spring months, with February through April having the highest reported number of outbreaks. On average, there outbreaks appeared to occur in the dry season before the onset of the rains. 10.1371/journal.pntd.0005885.g003 Fig 3 Seasonal distribution of anthrax outbreaks (black bars) during 2005–2016 with average rainfall totals (red line) in Ghana nationally (A) and by region: Upper West (B), Northern (C), Brong Ahafo (D), and Upper East (E). Anthrax outbreaks by Region. Livestock vaccination Trends in livestock anthrax vaccination among livestock type are shown in Fig 4 . From 2000–20015, there was a median of 17,957 doses [0–175 thousand] of anthrax livestock vaccine administered annually livestock vaccination occurred annually with a median number of doses administered of 19,709 [range: 0–175 thousand doses], followed by a decline in vaccine administration during 2008–2015. No vaccination was administered during the years 2010, 2012, and 2013. During 2008–2015, there was a median of 542 [range: 0–147 thousand doses] doses administered. In response to ongoing outbreaks, there was a vaccination campaign in 2014 that resulted in nearly an 8-fold increase in the number of doses administered compared to the previous six years. Among livestock types, cattle were most frequently administered vaccine, followed by sheep, goats and swine ( Fig 4 ). 10.1371/journal.pntd.0005885.g004 Fig 4 Annual livestock anthrax vaccine doses administered in Ghana during January 2005- June 2016. Environmental suitability of anthrax in Ghana The ensemble RF model suggests a latitudinal gradient in the environmental suitability of anthrax in Ghana ( Fig 5A ). High environmental suitability was identified in the Northern, Upper East, and Upper West regions of Ghana that encompass seasonal livestock migration routes from Burkina Faso in the north. Conversely, low or no environmental suitability was identified in southern Ghana among the more acidic soils in the Western, Ashanti, Central, and Eastern regions. Uncertainty (range: 0–0.20) in the model prediction was scaled from 0 to 1 and showed it was highest in the Upper West and Northern regions ( Fig 5B ). The internal OOB model validation indicated excellent discrimination with an AUC = 0.88 (95% CI: 0.87, 0.89). The external validation of anthrax outbreak locations withheld from the model (testing data) also showed excellent discrimination (AUC = 0.87 [95% CI: 0.85, 0.90]). 10.1371/journal.pntd.0005885.g005 Fig 5 Environmental suitability of anthrax in Ghana as predicted by the ensemble random forest model (inset A). Uncertainty was calculated as the range of the 95% confidence intervals in predicted probability of suitability for each pixel, with areas of highest uncertainty in red, with greener colors representing low uncertainty (inset B). The final list of variables used in the ensemble model are shown in Fig 6 . A combination of bioclimatic, environmental, and soil characteristics had the greatest impact on the OOB prediction errors. The most important variables influencing accuracy were: soil pH, bio7 (annual temperature range), and bio14 (precipitation of the driest month) ( S2 Fig ). The probability of the occurrence of anthrax increased in a step like manner in response to soil pH, increasing as the soil became more alkaline, between 5.5 and 6.5, and again between 6.5 and 7.0. Annual temperature ranges between 16 and 20°C were also related to a greater probability of occurrence. The occurrence of anthrax showed an affinity for low values of precipitation during the driest month (0 to 10 mm) and then dropped off dramatically as precipitation increased from 10 to 40 mm. Furthermore, as average NDVI (wd0114a0) increased from 0.3 to 0.6 the probability of anthrax occurrence decreased linearly, with a more suitable range of vegetation greenness identified in the lower ranges between 0.1 and 0.3 ( Fig 6 ). 10.1371/journal.pntd.0005885.g006 Fig 6 Partial dependency plots of environmental variables used in the random forest. Gray shading represented confidence intervals derived from the model iterations. Estimating populations at risk To estimate livestock and human populations at risk, we dichotomized the environmental suitability prediction (on a continuous probability scale) into suitable versus non-suitable environments for anthrax based on the optimal threshold (0.46) that maximized sensitivity (0.78) plus specificity (0.89) ( Fig 7 ). The dichotomized prediction shows a marked north-south demarcation in the suitability of anthrax, with a majority of northern Ghana predicted as suitable within the accompanying upper (97.5%) and lower (2.5%) confidence bounds. 10.1371/journal.pntd.0005885.g007 Fig 7 Dichotomized prediction of anthrax suitability with lower (2.5%) and upper (97.5%) occurrence limits. Dichotomized prediction was calculated using an optimal probability threshold (0.46) that maximized sensitivity plus specificity. The national livestock population located in areas environmentally suitable for anthrax was estimated to be ≈ 2.2 (95% CI: 2.0, 2.5) million ( Table 3 ). More than 50% of the livestock populations at risk were sheep and cattle (650 [95% CI: 583, 745] thousand and 480 [95% CI: 434, 527] thousand, respectively). Among livestock production systems, semi-arid rain-fed, mixed crop livestock systems (MRA) contained the greatest number of livestock at risk > 1.2 (95% CI: 1.1, 1.3) million ( Table 3 ). 10.1371/journal.pntd.0005885.t003 Table 3 Livestock production systems ¥ and the livestock population in the Ghanaian anthrax risk zone. Population at risk [95% CI] (thousands of livestock) Livestock LGA LGH MRA MRH MIA cattle 108.5 [83.1, 123.2] 38.2 [30, 48.8] 283.3 [272.3, 289.7] 49.2 [47.7, 63.8] 1.2 [1.1, 1.2] sheep 166.2 [125.4, 188] 50.6 [41.2, 74] 368 [353.8, 376.6] 63.9 [61.6, 105.5] 1.2 [1.1, 1.2] goats 262.3 [201, 301.7] 47.9 [38, 67.8] 509.9 [491.3, 524.2] 117.8 [115, 164] 1.1 [1.0, 1.1] swine 32.1 [24.7, 36.1] 11.5 [9.8, 17.1] 70.6 [67.6, 72.2] 14.7 [13.9, 24.9] 0.2 [0.2, 0.3] ¥ Livestock production systems ( http://www.fao.org/docrep/014/i2414e/i2414e.pdf ) LGA: Livestock only systems, arid and semi-arid LGH: Livestock only systems, humid and sub-humid MRA: Rainfed mixed crop/livestock system, arid and semi-arid MRH: Rainfed mixed crop/livestock system, humid and sub-humid MIA: Irrigated mixed crop/livestock systems, arid and semi-arid Nationally, there are approximately 3 million low income rural livestock keepers in Ghana ( Table 4 ). Our model suggests that ≈ 805 (95% CI: 519, 890) thousand are located in areas suitable for anthrax, with the majority located in a humid and sub-humid, mixed crop livestock system production zone (MRH). 10.1371/journal.pntd.0005885.t004 Table 4 Rural low income livestock keepers in the Ghanaian anthrax risk zone by livestock production system. Population at risk [95% CI] (thousands of people) Estimates ¥ LGA LGH MRA MRH MIA National 71.1 238.9 411.9 2152.9 1.2 [1.1, 1.2] Modeled 48.6 [34.1, 56.9] 105.4 [65.7, 120.2] 300 [263.6, 325.1] 346.6 [155, 387.5] 0.4 [0.3, 0.4] ¥ Livestock production systems ( http://www.fao.org/docrep/014/i2414e/i2414e.pdf ) LGA: Livestock only systems, arid and semi-arid LGH: Livestock only systems, humid and sub-humid MRA: Rainfed mixed crop/livestock system, arid and semi-arid MRH: Rainfed mixed crop/livestock system, humid and sub-humid MIA: Irrigated mixed crop/livestock systems, arid and semi-arid Discussion Anthrax is a globally distributed neglected disease that is often underreported, particularly in West Africa where it is hyper-endemic [ 1 , 2 , 6 , 13 ]. Given the reliance of control on the vaccination of livestock, understanding the occurrence of anthrax is crucial for identifying populations at risk in order to disseminate limited resources. Here, we used data on the location of livestock outbreaks to identify seasonal patterns and model the environmental suitability of anthrax in Ghana. In keeping with previous studies, our findings indicate a defined outbreak season with a combination of ecological constraints on the potential geographic distribution of anthrax [ 3 , 34 ]. Our modeled prediction suggests a marked ecological divide separating the broad areas of environmental suitability in northern Ghana from the southern part of the country. Additionally, we estimated that populations characteristically at high risk for anthrax, which included >3 million combined ruminant livestock and poor rural livestock keepers are situated within the predicted anthrax risk zone. Based on our estimates, current anthrax vaccination efforts cover only a fraction of the livestock potentially at risk. Hence, these findings can be used to better direct public health intervention strategies and inform surveillance. Official reports of livestock anthrax in endemic areas often go undocumented for a number of reasons, including the inability or unwillingness to report, limited surveillance capacity, and a lack of local knowledge about the disease [ 1 ]. In Ghana, livestock cases are likely underreported due to the slaughter and consumption of sick or dead animals [ 8 , 35 ], consistent with findings in the Caucasus and elsewhere [ 1 , 6 , 36 , 37 ]. This practice is often undertaken as a means of recouping economic losses from livestock mortality as well as providing food and a readily available source of protein [ 1 , 8 , 35 ]. The livestock anthrax outbreak data we used in this study were concordant with data reported to OIE during the same time frame suggesting Veterinary Services in Ghana are compliant with international reporting requirements ( http://www.oie.int/wahis_2/public/wahid.php/Wahidhome/Home ). Despite the close proximity to the equator, we identified marked seasonality in anthrax reporting; outbreaks increased during the onset of the rainy season from February through April. Similar patterns of anthrax outbreaks associated with the rainy-season have also been reported in Namibia [ 34 ]. One hypothesis suggests that there is greater soil consumption among ruminants during with the rainy season [ 34 ], although soil exposure during the dry season has also been hypothesized as a cause of anthrax outbreaks [ 1 ]. Regardless, these findings suggest vaccination of livestock could be carried out in Ghana ahead of the peak outbreak season (September–November). Livestock anthrax control in Ghana follows a similar trend in many endemic regions of reactively vaccinating in response to anthrax outbreaks [ 1 , 38 ]. In Ghana, the livestock population we identified at risk comprises approximately ≈ 25% of the total national livestock population [ 29 ]. Based on official vaccination reports ( Fig 4 ), our estimates of the livestock populations at risk indicates poor vaccine coverage; this finding is consistent with ongoing outbreaks in endemic communities in Ghana where vaccination has not been officially documented for at least a decade [ 39 ]. Barriers to vaccine uptake such as practices of livestock keepers my also affect coverage [ 1 , 40 ]. However, Ghana faces additional control challenges with the potential presence of B . cereus biovar (bv) anthracis and West Africa strains (D and E Clades, respectively [ 41 ]). The West African strains have been hypothesized to evade the Sterne vaccine, which is the vaccine used in Ghana and throughout much of the world [ 13 , 42 ]. Further research is needed on vaccine efficacy and to understand what proportion of anthrax outbreaks are due to either insufficient application methods or the vaccine itself. Research has suggested that soil pH >6.1 in conjunction with high calcium levels are a crucial component of B . anthracis spore survival [ 1 , 4 , 43 ]. Alkaline soils were also found to be associated with the persistence of anthrax transmission over several years [ 43 , 44 ]. In keeping with these findings, we identified an increasingly higher likelihood of anthrax occurrence in soils as pH increased from 5.5 to 7.0 and with an increasing level of calcareous vertisols. The association of anthrax suitability with lower levels of precipitation in our model is in line with reports that have documented soil nutrient leaching in regions with high precipitation, which may lead to soil acidification [ 45 ]. We predicted an area of environmental suitability for anthrax that encompasses ≈ 36% of Ghana's total area ( Fig 7 ); this is demarcated by a south (largely unsuitable) to north (highly suitable) divide, which closely mirrors the ecotone transitions from southern tropical and deciduous forests to the northern Sudanian and Guinea Savanna. Our study had several limitations. As with all neglected zoonoses, our data likely represent an underestimation of the true burden of disease due to underreporting and limited resources for surveillance and testing. To better address issues with diagnostic testing and reporting we used a more contemporary dataset of anthrax outbreaks recorded during the last decade. Anthrax can also be transmitted from contaminated feed that is imported, and animal mortality may occur from livestock moved across long distances; however, we had no information on any outbreaks arising in these instances [ 1 , 46 ]. The use of machine learning algorithms to model the distribution of environmental pathogens has been well described, but such approaches, by their definition in conjunction with the use of averaged climate data, may over-generalize the landscape that supports the occurrence of anthrax outbreaks. Other factors not included in our models that may influence the occurrence of anthrax include the health and immune status of the livestock [ 47 ]. In conclusion, the current anthrax situation in West Africa, and in particular Ghana, remains a public and veterinary health threat due to challenges with reporting, surveillance, and control. Our findings suggest that broad areas of northern Ghana are environmentally suitable for anthrax. Furthermore, based on recent vaccination efforts, our estimates indicate that only a fraction of livestock at risk are being vaccinated. These findings can be used to help improve differential diagnostics, vaccine coverage estimates, and surveillance efforts. Given the reliance on agriculture and the large population of low income rural livestock keepers at risk in the northern part of the country where predicted suitability was highest, future control efforts should focus on improving livestock vaccination coverage and public awareness of the disease, prioritizing communities in the predicted anthrax zone. Supporting information S1 Fig The spatial distribution of environmental variables used in the random forest models. Variable names are matched to variable descriptions and sources from Table 1 . (TIF) Click here for additional data file. S2 Fig Importance plot of variables included in the final ensemble random forest model prediction. Bars in darker blue represent variables that were more important in discriminating class prediction. (TIF) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3743785/

Protective Efficacy and Immunogenicity of a Combinatory DNA Vaccine against Influenza A Virus and the Respiratory Syncytial Virus

The Respiratory Syncytial Virus (RSV) and Influenza A Virus (IAV) are both two major causative agents of severe respiratory tract infections in humans leading to hospitalization and thousands of deaths each year. In this study, we evaluated the immunogenicity and efficacy of a combinatory DNA vaccine in comparison to the single component vaccines against both diseases in a mouse model. Intramuscular electroporation with plasmids expressing the hemagglutinin (HA) of IAV and the F protein of RSV induced strong humoral immune responses regardless if they were delivered in combination or alone. In consequence, high neutralizing antibody titers were detected, which conferred protection against a lethal challenge with IAV. Furthermore, the viral load in the lungs after a RSV infection could be dramatically reduced in vaccinated mice. Concurrently, substantial amounts of antigen-specific, polyfunctional CD8 + T-cells were measured after vaccination. Interestingly, the cellular response to the hemagglutinin was significantly reduced in the presence of the RSV-F encoding plasmid, but not vice versa. Although these results indicate a suppressive effect of the RSV-F protein, the protective efficacy of the combinatory vaccine was comparable to the efficacy of both single-component vaccines. In conclusion, the novel combinatory vaccine against RSV and IAV may have great potential to reduce the rate of severe respiratory tract infections in humans without increasing the number of necessary vaccinations. Introduction Influenza A Virus and the Respiratory Syncytial Virus are causative agents of severe respiratory tract infection especially in young children and elderly people. The global disease burden is estimated to ∼600 million and ∼60 million cases per year for IAV and RSV, respectively, leading to estimated ∼0.5 million deaths/year worldwide ( www.who.int ). Vaccinations against both viruses would provide therefore a substantial cost reduction in the global health system, as demonstrated by the already licensed vaccines against seasonal IAV [1] . Nevertheless, the production processes of these vaccines (e.g. subunit vaccine, whole inactivated virus vaccine) are very time-consuming and the efficacy is modest and short-lived. Thus, alternative strategies to reduce the production timeline and increase the efficacy are highly appreciable. In addition, there is no prophylactic vaccine against the RSV available so far. Recently, DNA vaccines have demonstrated great potential as an alternative vaccine platform capable of inducing protective immune responses against a variety of infectious diseases in preclinical models (reviewed in [2] ), including RSV [3] , [4] and IAV [5] . The implementation of more effective delivery methods, like electroporation, and the use of codon-optimized expression systems had further boosted the immunogenicity of such vaccines. DNA vaccines for a wide range of disease indications have advanced into human clinical trials and several are approved for use in the field of veterinary medicine (reviewed in [6] , [7] ). In our previous work, we successfully generated DNA vaccines providing protection against RSV or IAV using expression plasmids encoding the viral surface proteins RSV-F or the hemagglutinin of IAV, respectively [3] , [5] . Given the overlap in populations vulnerable to these respiratory infections, assessing the feasibility of combining these DNA vaccines represents a logical strategy. Such combinatory vaccines could reduce the number of immunizations an individual needs, leading to enhanced compliance and improved cost effectiveness. This concept was successfully established for many pediatric vaccines, like the mumps-measles-rubella (MMR) vaccine. Nevertheless, there are also reports on reduced immunogenicity or efficacy of conventionally developed combinatory vaccines in comparison to the respective single-component vaccines, e.g. Hepatitis A and B vaccine [8] . For DNA vaccines, it has already been demonstrated that plasmids encoding different antigens from either the same [9] , [10] or a second pathogen could induce substantial immune responses against both antigens [11] , [12] . However, in other studies, the simple addition of two expression plasmids encoding NP and M2 from IAV reduced the protective capacity of a combinatory DNA vaccine encoding HA and NA from the same virus by 30% [13] . This is not unexpected due to immunological interference within the recipient, thus indicating the need for an extensive immunogenicity analysis for each combinatory DNA vaccine. The mechanisms of such immunological interference are not yet fully understood, but possible explanations include interference in the antigen presenting pathway and/or alterations at the level of transcription/translation leading to changes in antigen expression levels. In the present study, we tested the immunogenicity of a combinatory DNA vaccine encoding the viral surface proteins HA from IAV (IAV-HA) and the F protein from RSV (RSV-F). The vaccines were delivered by intramuscular electroporation and the cellular as well as humoral immune responses to both antigens were analyzed in depth to rule out any form of immunological interference. Furthermore the protective efficacy of the combinatory vaccine was compared to the ones of the single component vaccines. Materials and Methods Plasmids and Vaccines The plasmid pF syn , based on pcDNA3.1, contained the codon-optimized sequence of the full-length RSV-F protein and is described elsewhere [14] . The codon-optimized sequence of the HA of the virus strain A/Puerto Rico/8/34 was synthesized by Geneart (Regensburg, Germany), followed by PCR amplification and cloning into the pVAX backbone. The HA sequence is followed by an ollas-tag [15] for protein detection and the resulting plasmid is referred to as pHA syn . A non-coding pcDNA3.1 plasmid was used as control plasmid. DNA for immunization was prepared using the NucleoBond® Xtra Maxi EF Kit (Macherey-Nagel, Düren, Germany) and tested for endotoxin levels with the LAL quantification assay (Cambrex Bio Science, Verviers, Belgium), confirming that the dose used for immunization of mice contained less than 0.1 endotoxin units (EU). Expression Analysis 293T cells were transiently transfected using PEI (polyethyleneimine) with either 1 µg or 10 µg of plasmid DNA as described elsewhere [16] . The two plasmids pHA syn and pF syn were transfected either in combination with each other or with pcDNA3.1. The influence on the expression levels of both viral proteins were analyzed by Western Blot analyses. Cell lysates were prepared 48 h after transfection. The protocol for the detection of RSV-F by Western blot under non-reducing conditions is described in a previous study [14] . For the detection of IAV-HA, a monoclonal anti-HA antibody was purified from hybridoma culture and used together with an HRP-conjugated anti-mouse IgG (DakoCytomation, Glostrup, Denmark) antibody in Western blots under reducing conditions, as described elsewhere [17] . The housekeeping protein α-tubulin was used as loading control and detected by a polyclonal antibody (Rockland, Gilbertsville, PA, USA) in combination with an HRP-conjugated anti-rabbit antibody (DakoCytomation, Glostrup, Denmark). A semi-quantitative, densitometry analysis was performed with the software Wasabi! (Hamamatsu Photonics Germany GmbH, Herrsching am Ammersee, Germany). Animals and Immunizations 6–8 week old female BALB/cJRj mice were purchased from Janvier (Le Genest-ST-Isle, France) and housed in individually-ventilated cages in accordance with the national law and institutional guidelines. The study was approved by an external ethics committee authorized by the North Rhine-Westphalia State Office for Consumer Protection and Food Safety and performed with the project licenses (AZ 87-51.04.2010.A302). The injection and electroporation procedure was performed in accordance to the manual supplied by the manufacturer (Ichor Medical Inc., San Diego, USA) and is described elsewhere [5] , [18] . The respective amounts of the DNAs ( Table 1 ) were diluted in 100 µl PBS and the animals were immunized twice within a 3-week interval under ketamine (50 mg/kg)/xylazine (10 mg/kg) anesthesia. One week after the second vaccination, the animals were sacrificed by cervical dislocation and the cellular immune responses were analyzed by intracellular cytokine staining of isolated splenocytes. Mice were bled under general anesthesia by exposing them briefly to isofluran (Paragos, Frankfurt, Germany) vapors, and then 15 drops of blood were drawn from the retro-orbital sinus using a glass capillary tube. Blood samples were collected on days 20 and 49 and analyzed for antigen-specific antibodies. The protective efficacies of the vaccines were determined by a challenge infection with either RSV or IAV five weeks after the final immunization. 10.1371/journal.pone.0072217.t001 Table 1 Experimental setup. Group Vaccine (d0+d21) IAV-Challenge (d56) RSV-Challenge (d56) Naïve – + + (HA) low 2 µg pHA syn +2 µg pcDNA3.1 + – (F) low 2 µg pF syn +2 µg pcDNA3.1 – + (HA+F) low 2 µg pHA syn +2 µg pF syn + + (HA) hi 20 µg pHA syn +20 µg pcDNA3.1 + + (F) hi 20 µg pF syn +20 µg pcDNA3.1 + + (HA+F) hi 20 µg pHA syn +20 µg pF syn + + Intracellular Cytokine Staining One week after the second immunization spleen lymphocytes were isolated and re-stimulated as previously described [5] . Briefly, CD8 + T-cells were re-stimulated for 6 h in the presence of monensin (2 µM), αCD107a-FITC (1 µl, BD Bioscience, Germany) and the peptide HA 532–540 (IYSTVASSL), 5 µg/ml) or the combined RSV-F peptides (DKYKNAVTELQLLMQ; VTTPVSTYMLTNSEL; VSTYMLTNSELLSLI, each 5 µg/ml). Non-stimulated splenocytes were used as controls for unspecific cytokine production. After the stimulation, surface staining was performed with αCD8-PerCP or αCD4-PerCP antibodies (BD Bioscience, Heidelberg, Germany). Cells were fixed in 2% paraformaldehyde and permeabilized with 0.5% Saponin in PBS/BSA/azide buffer. Cytokines were detected with αIFN-γ-PE and αIL-2-APC on a FACSCalibur® flow cytometer (BD Bioscience, Germany). Serological Assays To analyze the sera for antibodies recognizing the viral antigens in their membrane-bound conformation, a flow cytometric analysis was performed with transfected 293T cells as previously described [5] . Briefly, 293T cells were transfected with the IAV-HA or RSV-F expressing plasmid two days prior incubation with the sera of vaccinated animals. Antibodies bound to the surface proteins of the transfected cells were detected via FITC-labeled anti mouse IgG antibodies (BD Bioscience, Germany). The mean fluorescence intensities of the cells correspond to the level of antigen-specific antibodies in the sera. To quantify IAV and RSV specific IgG1 and IgG2a antibody levels, ELISA were performed as previously described [19] . Briefly, 96-well plates were coated overnight at 4°C with heat-inactivated Influenza A/PR/8/34 or RSV A/Long (10 6 plaque forming units (PFU)/well). The sera were diluted 1–100 with PBS containing 2% fat dried milk powder and 0.5% Tween20 and incubated for 1 h. Finally, horseradish peroxidase-coupled antibodies against mouse IgG1 or IgG2a antibodies (BD Bioscience) were used for the detection. To determine influenza specific neutralizing antibody titers, mouse sera were 2-fold serially diluted in 100 µl DMEM (Invitrogen) containing 0.6% BSA, 1% penicillin-streptomycin and 0.12% trypsin and pre-incubated with 2000 PFU of Influenza A/PR/8/34 at 37°C for 1 h. In the next step, the serum-virus mix was applied to MDCK cells, which had been seeded at 5×10 4 cells/well the day before, and incubated at 37°C for 1.5 h. Finally, additional 150 µl of medium were added to the cells. After 96 h incubation at 37°C non-infected wells were identified by staining with crystal violet dye. The reciprocal dilution of the sera completely inhibiting infection was considered as neutralizing antibody titer. RSV specific neutralizing antibody assays were performed using rgRSV in accordance with the protocol described previously [3] . Challenge Experiments 5–6 weeks after the second immunization mice were anesthetized with ketamine/xylazine and challenged intranasally with either influenza A/PR/8/34 (250 PFU) or RSV A/Long (10 6 PFU) diluted in 50 µl PBS [3] . The weight loss of the mice was monitored daily after the infection as an indicator of disease progression. Mice were sacrificed six (Influenza) or five (RSV) days after infection and the lungs were removed for the quantification of the viral load. Lungs were homogenized in 2 ml PBS using the GentleMACS Dissociator (Milteny) according to the manufacturer's protocol. Viral RNA was isolated from lung homogenates using the QIAamp® Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions. The quantity of viral RNA was determined by reverse transcription real time PCR (RT-qPCR) using the QuantiTect™ Probe RT-PCR Kit (Qiagen) with SYBR-Green. The following specific primers were used: Influenza ( cttctaaccgaggtcgaaacg + agggcattttggacaaag/tcgtcta) [20] ; RSV ( agatcaacttctgtcatccagcaa + gcacatcataattaggagtatcaat ) [3] . The detection limits were 5 copies/PCR for the Influenza-RT-PCR and 50 copies/PCR for the RSV-RT-PCR, which corresponds to 428 and 4280 copies/ml lung homogenate, respectively. Statistical Analysis Results are expressed as the means ± standard errors of the means (SEM). Statistical comparisons were performed by one-way ANOVA test, followed by a Tukey post test using the Prism 5.0, GraphPad Software. A P value of <0.05 was considered to be statistically significant. Plasmids and Vaccines The plasmid pF syn , based on pcDNA3.1, contained the codon-optimized sequence of the full-length RSV-F protein and is described elsewhere [14] . The codon-optimized sequence of the HA of the virus strain A/Puerto Rico/8/34 was synthesized by Geneart (Regensburg, Germany), followed by PCR amplification and cloning into the pVAX backbone. The HA sequence is followed by an ollas-tag [15] for protein detection and the resulting plasmid is referred to as pHA syn . A non-coding pcDNA3.1 plasmid was used as control plasmid. DNA for immunization was prepared using the NucleoBond® Xtra Maxi EF Kit (Macherey-Nagel, Düren, Germany) and tested for endotoxin levels with the LAL quantification assay (Cambrex Bio Science, Verviers, Belgium), confirming that the dose used for immunization of mice contained less than 0.1 endotoxin units (EU). Expression Analysis 293T cells were transiently transfected using PEI (polyethyleneimine) with either 1 µg or 10 µg of plasmid DNA as described elsewhere [16] . The two plasmids pHA syn and pF syn were transfected either in combination with each other or with pcDNA3.1. The influence on the expression levels of both viral proteins were analyzed by Western Blot analyses. Cell lysates were prepared 48 h after transfection. The protocol for the detection of RSV-F by Western blot under non-reducing conditions is described in a previous study [14] . For the detection of IAV-HA, a monoclonal anti-HA antibody was purified from hybridoma culture and used together with an HRP-conjugated anti-mouse IgG (DakoCytomation, Glostrup, Denmark) antibody in Western blots under reducing conditions, as described elsewhere [17] . The housekeeping protein α-tubulin was used as loading control and detected by a polyclonal antibody (Rockland, Gilbertsville, PA, USA) in combination with an HRP-conjugated anti-rabbit antibody (DakoCytomation, Glostrup, Denmark). A semi-quantitative, densitometry analysis was performed with the software Wasabi! (Hamamatsu Photonics Germany GmbH, Herrsching am Ammersee, Germany). Animals and Immunizations 6–8 week old female BALB/cJRj mice were purchased from Janvier (Le Genest-ST-Isle, France) and housed in individually-ventilated cages in accordance with the national law and institutional guidelines. The study was approved by an external ethics committee authorized by the North Rhine-Westphalia State Office for Consumer Protection and Food Safety and performed with the project licenses (AZ 87-51.04.2010.A302). The injection and electroporation procedure was performed in accordance to the manual supplied by the manufacturer (Ichor Medical Inc., San Diego, USA) and is described elsewhere [5] , [18] . The respective amounts of the DNAs ( Table 1 ) were diluted in 100 µl PBS and the animals were immunized twice within a 3-week interval under ketamine (50 mg/kg)/xylazine (10 mg/kg) anesthesia. One week after the second vaccination, the animals were sacrificed by cervical dislocation and the cellular immune responses were analyzed by intracellular cytokine staining of isolated splenocytes. Mice were bled under general anesthesia by exposing them briefly to isofluran (Paragos, Frankfurt, Germany) vapors, and then 15 drops of blood were drawn from the retro-orbital sinus using a glass capillary tube. Blood samples were collected on days 20 and 49 and analyzed for antigen-specific antibodies. The protective efficacies of the vaccines were determined by a challenge infection with either RSV or IAV five weeks after the final immunization. 10.1371/journal.pone.0072217.t001 Table 1 Experimental setup. Group Vaccine (d0+d21) IAV-Challenge (d56) RSV-Challenge (d56) Naïve – + + (HA) low 2 µg pHA syn +2 µg pcDNA3.1 + – (F) low 2 µg pF syn +2 µg pcDNA3.1 – + (HA+F) low 2 µg pHA syn +2 µg pF syn + + (HA) hi 20 µg pHA syn +20 µg pcDNA3.1 + + (F) hi 20 µg pF syn +20 µg pcDNA3.1 + + (HA+F) hi 20 µg pHA syn +20 µg pF syn + + Intracellular Cytokine Staining One week after the second immunization spleen lymphocytes were isolated and re-stimulated as previously described [5] . Briefly, CD8 + T-cells were re-stimulated for 6 h in the presence of monensin (2 µM), αCD107a-FITC (1 µl, BD Bioscience, Germany) and the peptide HA 532–540 (IYSTVASSL), 5 µg/ml) or the combined RSV-F peptides (DKYKNAVTELQLLMQ; VTTPVSTYMLTNSEL; VSTYMLTNSELLSLI, each 5 µg/ml). Non-stimulated splenocytes were used as controls for unspecific cytokine production. After the stimulation, surface staining was performed with αCD8-PerCP or αCD4-PerCP antibodies (BD Bioscience, Heidelberg, Germany). Cells were fixed in 2% paraformaldehyde and permeabilized with 0.5% Saponin in PBS/BSA/azide buffer. Cytokines were detected with αIFN-γ-PE and αIL-2-APC on a FACSCalibur® flow cytometer (BD Bioscience, Germany). Serological Assays To analyze the sera for antibodies recognizing the viral antigens in their membrane-bound conformation, a flow cytometric analysis was performed with transfected 293T cells as previously described [5] . Briefly, 293T cells were transfected with the IAV-HA or RSV-F expressing plasmid two days prior incubation with the sera of vaccinated animals. Antibodies bound to the surface proteins of the transfected cells were detected via FITC-labeled anti mouse IgG antibodies (BD Bioscience, Germany). The mean fluorescence intensities of the cells correspond to the level of antigen-specific antibodies in the sera. To quantify IAV and RSV specific IgG1 and IgG2a antibody levels, ELISA were performed as previously described [19] . Briefly, 96-well plates were coated overnight at 4°C with heat-inactivated Influenza A/PR/8/34 or RSV A/Long (10 6 plaque forming units (PFU)/well). The sera were diluted 1–100 with PBS containing 2% fat dried milk powder and 0.5% Tween20 and incubated for 1 h. Finally, horseradish peroxidase-coupled antibodies against mouse IgG1 or IgG2a antibodies (BD Bioscience) were used for the detection. To determine influenza specific neutralizing antibody titers, mouse sera were 2-fold serially diluted in 100 µl DMEM (Invitrogen) containing 0.6% BSA, 1% penicillin-streptomycin and 0.12% trypsin and pre-incubated with 2000 PFU of Influenza A/PR/8/34 at 37°C for 1 h. In the next step, the serum-virus mix was applied to MDCK cells, which had been seeded at 5×10 4 cells/well the day before, and incubated at 37°C for 1.5 h. Finally, additional 150 µl of medium were added to the cells. After 96 h incubation at 37°C non-infected wells were identified by staining with crystal violet dye. The reciprocal dilution of the sera completely inhibiting infection was considered as neutralizing antibody titer. RSV specific neutralizing antibody assays were performed using rgRSV in accordance with the protocol described previously [3] . Challenge Experiments 5–6 weeks after the second immunization mice were anesthetized with ketamine/xylazine and challenged intranasally with either influenza A/PR/8/34 (250 PFU) or RSV A/Long (10 6 PFU) diluted in 50 µl PBS [3] . The weight loss of the mice was monitored daily after the infection as an indicator of disease progression. Mice were sacrificed six (Influenza) or five (RSV) days after infection and the lungs were removed for the quantification of the viral load. Lungs were homogenized in 2 ml PBS using the GentleMACS Dissociator (Milteny) according to the manufacturer's protocol. Viral RNA was isolated from lung homogenates using the QIAamp® Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions. The quantity of viral RNA was determined by reverse transcription real time PCR (RT-qPCR) using the QuantiTect™ Probe RT-PCR Kit (Qiagen) with SYBR-Green. The following specific primers were used: Influenza ( cttctaaccgaggtcgaaacg + agggcattttggacaaag/tcgtcta) [20] ; RSV ( agatcaacttctgtcatccagcaa + gcacatcataattaggagtatcaat ) [3] . The detection limits were 5 copies/PCR for the Influenza-RT-PCR and 50 copies/PCR for the RSV-RT-PCR, which corresponds to 428 and 4280 copies/ml lung homogenate, respectively. Statistical Analysis Results are expressed as the means ± standard errors of the means (SEM). Statistical comparisons were performed by one-way ANOVA test, followed by a Tukey post test using the Prism 5.0, GraphPad Software. A P value of <0.05 was considered to be statistically significant. Results Influence of Co-delivery of DNA Vaccines on Antigen Expression Levels In this study, we compared the efficacy of a combinatory DNA vaccine to the efficacy of single component vaccines. Therefore we had to confirm that the simultaneous delivery of both plasmids did not result in a considerable loss of expression of either antigen. 293T cells were transfected with the IAV-HA and RSV-F encoding plasmids either together with each other or with an empty control plasmid (pcDNA3.1). Cells were transfected with 0.05, 0.5 or 5 µg per plasmid and lysed two days later. The proteins were detected by Western blot analyses using HA-, RSV-F- or α-tubulin-specific antibodies and all proteins appeared at the expected size. ( Fig. 1A ). A semi-quantitative, densitometry analysis revealed that the expression of neither HA nor RSV-F was significantly inhibited by the presence of the second expression plasmid ( Fig. 1B ). Although the total amount of HA and RSV-F were slightly lower in the presence of the second antigen encoding plasmid than in the presence of the control plasmid, the ratios to the internal control protein, α-tubulin, were not affected. This indicates that there is no intracellular inhibition by one of these viral proteins on the expression rate of the other one in vitro. 10.1371/journal.pone.0072217.g001 Figure 1 Expression analysis. 293T cells were transiently transfected with the IAV-HA or RSV-F expressing plasmids either alone or in combination. To guarantee same amounts of input DNA, pcDNA3.1 was added to the transfection samples of a single plasmid. The transfections were done with 0.05, 0.5 or 5 µg per plasmid and cell lysates were prepared 48 h after transfection. The expression of the viral surface proteins was detected by Western blot analyses under either denaturing conditions for the detection with an anti-HA antibody or non-reducing conditions for the detection with an anti-RSV-F antibody. The detection of α-tubulin served as a loading control ( A ). A semi-quantitative, densitometry analysis was performed with the software Wasabi! for the lysates obtained after transfecting 5 µg of DNA. Listed are the RLUs detected in the different Western blots ( B ). Cellular Immune Response after Combinatory Immunization To compare the immunogenicity of the combinatory vaccine to that of the single component vaccines, mice were immunized twice with different doses of DNA (hi =  20 µg; low =  2 µg) or left untreated according to Table 1 . The single component vaccines were mixed with an empty pcDNA-vector to keep the total amount of plasmid constant. Additionally, the high-dose (20 µg) of the irrelevant antigen-expressing plasmid were always used as a control for unspecific immune stimulation by the vaccination procedure itself; i.e. (HA) hi serves as a control for all RSV-specifc assays and (F) hi for the IAV-specific ones. The cellular immune responses were analyzed one week after the second immunization by intracellular cytokine staining ( Fig. 2 ). Since two immunizations with low doses of the plasmids (2 µg each) did not result in substantial responses, only the results of the groups, which received high doses of the antigen-expressing plasmid were shown. In accordance with our previous studies, 20 µg of the IAV-HA expressing plasmid, group (HA) hi , induced high numbers of antigen-specific CD8 + T-cells ( Fig. 2A ), mainly characterized by the expression of CD107a and IFN-γ (ca. 1.3% of all CD8 + T-cells). Surprisingly, the immune response was significantly reduced (∼0.5%) if the RSV-F encoding plasmid was co-applied (HA+F) hi . This could be observed for all subpopulations of antigen-specific CD8 + T-cells analyzed in this ICS assay ( Fig. 2A ). Furthermore, the HA-specific CD4 + T-cell responses detected by intracellular cytokine staining for the T H 1 cytokines IFN-α, TNF and IL-2 were also reduced in the presence of the RSV-F encoding plasmid ( Fig. S1A ). We further measured the typical T H 2 cytokines IL-4, IL-5, IL-10 and IL-13 in the supernatant of HA-stimulated splenocytes and could not detect any IL-5 or IL-10 production. Interestingly, the IL-4 production, although at a low level, was comparable for (HA+F) hi and (HA) hi , whereas the IL-13 production was again significantly reduced in the combined vaccine group ( Fig. S1B ). 10.1371/journal.pone.0072217.g002 Figure 2 Antigen-specific CD8 + T-cell responses. Balb/c mice were immunized according to Table 1 . Since the low dose regimen did not result in substantial responses, only the groups which received a total of 40 µg of plasmid DNA were included. IAV-HA ( A )- and RSV-F ( B )-specific CD8 + T-cell responses were analyzed one week after the second immunization by staining for the degranulation marker CD107a and intracellular staining for the inflammatory cytokines IFN-γ and IL-2. The percentages of the different populations among the total CD8 + T-cells are shown. Mean values and standard errors of the means (SEM) represent 8 mice per vaccine group out of two independent experiments and 4 mice for the naïve group. (*** = p<0.001, ** = p<0.01, * = p<0.05; 1 way-ANOVA, Tukey post-test). In contrast to the IAV-HA-specific response, the RSV-F-specific CD8 + T-cell response was not significantly different between the groups (HA+F) hi and (F) hi ( Fig. 2B ). In both groups nearly 1% of all CD8 + T-cells were positive for CD107a and IFN-γ after RSV-F peptide stimulation. Unexpectedly, in the control group treated with the IAV-HA expressing plasmid (HA) hi , some T-cells also reacted specifically to the RSV-F peptides used in the assay, indicating possibly cross-reactive epitopes. Unfortunately, this led to a high variation of reacting CD8 + T-cells in these animals so that the multiple group comparison by 1-way ANOVA did not reach statistical significance for some subpopulation (e.g. IFN-γ+/CD8+ cells: naïve vs. F hi ). However, in all populations analyzed no reduction of the RSV-F-specific response could be observed in the presence of the IAV-HA expressing plasmid. Humoral Immune Response after Combinational Immunization Since virus-specific antibodies are important for protection against the infection with these two viruses, we analyzed the antibodies in the sera of the vaccinated mice in regard to their binding capacity to the natural membrane-bound surface protein, to their IgG-subtype and their neutralization efficacy in vitro ( Fig. 3 and 4 ). Three weeks (day 20) after the first immunization, IAV-HA-specific antibodies could be detected in the groups (HA) hi and (HA+F) hi , but not in the low-dose groups. These antibodies were able to bind the HA protein expressed on the surface of transfected cells and could neutralize the A/PR/8/34 virus in vitro at serum dilutions ranging from 1/10 to 1/640 ( Fig. 3A and C ). These antibody responses were substantially boosted by the second immunization and the mean neutralizing antibody titers increased to 775 and 592 for (HA) hi and (HA+F) hi , respectively. After the second immunization, the animals of the low dose groups (HA) low and (HA+F) low also had neutralizing antibody titers significantly elevated above control animals ( Fig. 3C ). Although the antibody response measured by the FACS-based assay were still significantly lower in these two groups compared to that in the high dose groups ( Fig. 3A ), the differences did not reach statistical significance in the micro-neutralization assay ( Fig. 3C ). To further characterize the IgG subtype of the virus-specific antibodies, virus-coated ELISA plates were incubated with the sera of day 49 after first immunization and bound antibodies were detected by IgG1- and IgG2a-specific antibodies ( Fig. 3B ). All immunized animals showed a balanced IgG1/IgG2a response with no significant differences between the groups, which received one or two antigen expressing plasmids. Overall the IAV-HA-specific humoral immune response was comparable for animals treated with the combinatory or the single-component vaccine. The same kind of analyses were performed to detect RSV-F-specific antibodies and revealed a very similar picture ( Fig. 4 ). Again, the high vaccine dose induced neutralizing antibodies as early as twenty days after the first immunization, whereas the animals of the groups (F) low and the (HA+F) low needed a second immunization to produce comparable levels of RSV-neutralizing antibodies ( Fig. 4C ). Nevertheless, the levels of serum antibodies binding RSV-F on the surface of transfected cells were significantly higher at day 49 in the high dose groups (F) hi and (HA+F) hi than in the respective low dose groups (F) low and (HA+F) low ( Fig. 4A ). Similar to the IAV-HA-specific response, the distribution of RSV-specific antibodies is balanced for the subtypes IgG1 and IgG2a ( Fig. 4B ). Overall, the combinatory vaccine induced antibody responses comparable to the single-component vaccines, including high amounts of binding and neutralizing antibodies specific for both viral surface proteins. Furthermore, no evidence of anti-DNA antibodies were detected in the sera of vaccinated mice (data not shown). 10.1371/journal.pone.0072217.g003 Figure 3 Influenza-specific antibody response. Balb/c mice were immunized according to Table 1 . Sera were collected at days 20 and 49 and IAV-HA-specific antibody responses were analyzed. To analyze antibodies binding IAV-HA in its membrane bound conformation, the sera were incubated with IAV-HA expressing 293T cells and bound antibodies were subsequently detected by FITC-labeled anti-mouse IgG antibodies. The mean fluorescence intensities (MFI) of each group (means+SEM) are shown for sera from day 20 and day 49 ( A ). The distribution of IAV-HA-specific IgG1 and IgG2a were analyzed in an ELISA using IAV coated plates and HRP-conjugated anti-IgG1 and IgG2a antibodies. The means and SEM of the relative light units (RLU) are shown for sera from day 49 ( B ). The neutralizing antibody titer (NT) was analyzed by a microneutralization assay. The reciprocal value of the serum dilution which results in complete protection from infection is given as neutralizing titer. The mean and SEM of each group are indicated for the sera of day 20 and day 49 ( C ). The results for each group represent at least 12 mice out of 2–3 independent experiments with the exception from the results of group (HA) low , which based on a single experiment with 6 mice. (p<0.05): * vs. naïve; # vs. (F) hi ; + vs. (HA+F) low ; ∼ vs. (HA) low (1 way-ANOVA, Tukey post-test). 10.1371/journal.pone.0072217.g004 Figure 4 RSV-specific antibody response. Balb/c mice were immunized according to Table 1 . Sera were collected at days 20 and 49 and RSV-F-specific antibody responses were analyzed. To analyze antibodies binding RSV-F in its membrane bound conformation, the sera were incubated with RSV-F expressing 293T cells and bound antibodies were subsequently detected by FITC-labeled anti-mouse IgG antibodies. The mean fluorescence intensities (MFI) of each group (means+SEM) are shown for sera from day 20 and day 49 ( A ). The distribution of RSV-F-specific IgG1 and IgG2a were analyzed in an ELISA using RSV coated plates and HRP-conjugated anti-IgG1 and IgG2a antibodies. The means and SEM of the relative light units (RLU) are shown for sera from day 49 ( B ). The reciprocal value of the serum dilution which results in 50% inhibition of infection with rgRSV is given as neutralizing antibody titer (NT). The mean and SEM of each group are indicated for the sera of day 20 and day 49 ( C ). The results for each group represent at least 12 mice out of 2–3 independent experiments. (p<0.05): * vs. naïve; # vs. (HA) hi ; + vs. (HA+F) low ; ∼ vs. (F) low (1 way-ANOVA, Tukey post-test). Protective Efficacy Against RSV and IAV Infections Following confirmation of vaccine induced anti-RSV and anti-IAV immune responses, the protective efficacy was assessed by experimental infections via the intranasal route. In accordance with the high neutralizing antibody titers against IAV, all vaccinated animals were fully protected against disease progression indicated by constant weights after IAV challenge infection ( Fig. 5A ). In contrast, all animals of the two control groups, naïve and (F) hi , started to lose weight two days post infection and had to be sacrificed by day 6 due to loss of more than 25% of the initial body weight. To further analyze the protective efficacy achieved by vaccination, the viral loads were measured in the lung homogenates at day 6 post infection ( Fig. 5B ). There is no evidence of non-specific protection conferred by the application of the DNA electroporation as demonstrated by the animals of group (F) hi , which had comparable viral loads as the naïve control animals. The median viral load was reduced at least by 5 logs for all groups, which received the IAV-HA-expressing plasmid ( Fig. 5B ). The best protection was observed in the group (HA+F) hi , in which the viral loads of five out of six animals were below the detection limit of the qRT-PCR and therefore not distinguishable from non-infected controls. Nevertheless, there were neither statistical significant differences in the efficacy between the low and the high dose regimen nor between the single-component and the combinatory vaccines. 10.1371/journal.pone.0072217.g005 Figure 5 Protection from Influenza A Virus challenge. Balb/c mice were immunized according to Table 1 . Five weeks after the second immunization, the mice were challenged with 250 PFU IAV (PR/8/34). The weight loss of each animal was monitored daily and the means and SEM (n = 5–6) are indicated for each group up to day 6 post challenge ( A ). At day 6, viral loads in lung homogenates (vRNA copies/ml) were measured by qRT-PCR. Each animal is represented by one dot and the means of each group are marked by the line. The dotted line indicates the detection limit (428 copies/ml) ( B ). (p<0.05): * vs. naïve; # vs. (F) hi (1 way-ANOVA, Tukey post-test). In contrast to the IAV infection, the infection with the RSV is not lethal in mice and there is only a minor weight loss detectable in non-vaccinated mice. Although there are some variations in the weight loss from animal to animal, there were no significant differences between the groups at any time point ( Fig. 6A ). In contrast, the viral loads in the lung were statistically significant reduced in all animals which received the RSV-F expression plasmid compared to the naïve animals ( Fig. 6B ). Interestingly, the animals of group (HA) hi had also 3-fold lower copy numbers of viral RNA than the naïve animals. This is in line with the observation that in some animals of this group CD8 + T-cells reactive to the RSV-F specific peptides were detected ( Fig. 2B ). Again, there were no differences in the efficacy of the single-component, (F) hi and (F) low , and the combinatory vaccines, (HA+F) hi and (HA+F) low . The median viral load were reduced in the low dose groups by a factor of 83 for (F) low and 136 for (HA+F) low . Although it did not reach statistical significance in the multi-group comparison, the viral load could be further reduced by 1-log if the higher dose of the expression plasmid was used ( Fig. 6B ). 10.1371/journal.pone.0072217.g006 Figure 6 Protection from RSV challenge. Balb/c mice were immunized according to Table 1 . Five weeks after the second immunization, the mice were challenged with 10 6 PFU RSV (A/Long). The weight loss of each animal was monitored daily and the means and SEM (n = 5–6) are indicated for each group up to day 5 post challenge ( A ). At day 5, viral loads in lung homogenates (vRNA copies/ml) were measured by qRT-PCR. Each animal is represented by one dot and the means of each group are marked by the line. The detection limit of the qRT-PCR was 4280 copies/ml ( B ). (p<0.05): * vs. naïve; # vs. (HA) hi (1 way-ANOVA, Tukey post-test). We could demonstrate that the combinatory DNA vaccine protected at least as efficient as the single-component vaccine against infections of both viruses. Furthermore, the protection in both models strongly correlated with the neutralizing antibody titer (data not shown). Influence of Co-delivery of DNA Vaccines on Antigen Expression Levels In this study, we compared the efficacy of a combinatory DNA vaccine to the efficacy of single component vaccines. Therefore we had to confirm that the simultaneous delivery of both plasmids did not result in a considerable loss of expression of either antigen. 293T cells were transfected with the IAV-HA and RSV-F encoding plasmids either together with each other or with an empty control plasmid (pcDNA3.1). Cells were transfected with 0.05, 0.5 or 5 µg per plasmid and lysed two days later. The proteins were detected by Western blot analyses using HA-, RSV-F- or α-tubulin-specific antibodies and all proteins appeared at the expected size. ( Fig. 1A ). A semi-quantitative, densitometry analysis revealed that the expression of neither HA nor RSV-F was significantly inhibited by the presence of the second expression plasmid ( Fig. 1B ). Although the total amount of HA and RSV-F were slightly lower in the presence of the second antigen encoding plasmid than in the presence of the control plasmid, the ratios to the internal control protein, α-tubulin, were not affected. This indicates that there is no intracellular inhibition by one of these viral proteins on the expression rate of the other one in vitro. 10.1371/journal.pone.0072217.g001 Figure 1 Expression analysis. 293T cells were transiently transfected with the IAV-HA or RSV-F expressing plasmids either alone or in combination. To guarantee same amounts of input DNA, pcDNA3.1 was added to the transfection samples of a single plasmid. The transfections were done with 0.05, 0.5 or 5 µg per plasmid and cell lysates were prepared 48 h after transfection. The expression of the viral surface proteins was detected by Western blot analyses under either denaturing conditions for the detection with an anti-HA antibody or non-reducing conditions for the detection with an anti-RSV-F antibody. The detection of α-tubulin served as a loading control ( A ). A semi-quantitative, densitometry analysis was performed with the software Wasabi! for the lysates obtained after transfecting 5 µg of DNA. Listed are the RLUs detected in the different Western blots ( B ). Cellular Immune Response after Combinatory Immunization To compare the immunogenicity of the combinatory vaccine to that of the single component vaccines, mice were immunized twice with different doses of DNA (hi =  20 µg; low =  2 µg) or left untreated according to Table 1 . The single component vaccines were mixed with an empty pcDNA-vector to keep the total amount of plasmid constant. Additionally, the high-dose (20 µg) of the irrelevant antigen-expressing plasmid were always used as a control for unspecific immune stimulation by the vaccination procedure itself; i.e. (HA) hi serves as a control for all RSV-specifc assays and (F) hi for the IAV-specific ones. The cellular immune responses were analyzed one week after the second immunization by intracellular cytokine staining ( Fig. 2 ). Since two immunizations with low doses of the plasmids (2 µg each) did not result in substantial responses, only the results of the groups, which received high doses of the antigen-expressing plasmid were shown. In accordance with our previous studies, 20 µg of the IAV-HA expressing plasmid, group (HA) hi , induced high numbers of antigen-specific CD8 + T-cells ( Fig. 2A ), mainly characterized by the expression of CD107a and IFN-γ (ca. 1.3% of all CD8 + T-cells). Surprisingly, the immune response was significantly reduced (∼0.5%) if the RSV-F encoding plasmid was co-applied (HA+F) hi . This could be observed for all subpopulations of antigen-specific CD8 + T-cells analyzed in this ICS assay ( Fig. 2A ). Furthermore, the HA-specific CD4 + T-cell responses detected by intracellular cytokine staining for the T H 1 cytokines IFN-α, TNF and IL-2 were also reduced in the presence of the RSV-F encoding plasmid ( Fig. S1A ). We further measured the typical T H 2 cytokines IL-4, IL-5, IL-10 and IL-13 in the supernatant of HA-stimulated splenocytes and could not detect any IL-5 or IL-10 production. Interestingly, the IL-4 production, although at a low level, was comparable for (HA+F) hi and (HA) hi , whereas the IL-13 production was again significantly reduced in the combined vaccine group ( Fig. S1B ). 10.1371/journal.pone.0072217.g002 Figure 2 Antigen-specific CD8 + T-cell responses. Balb/c mice were immunized according to Table 1 . Since the low dose regimen did not result in substantial responses, only the groups which received a total of 40 µg of plasmid DNA were included. IAV-HA ( A )- and RSV-F ( B )-specific CD8 + T-cell responses were analyzed one week after the second immunization by staining for the degranulation marker CD107a and intracellular staining for the inflammatory cytokines IFN-γ and IL-2. The percentages of the different populations among the total CD8 + T-cells are shown. Mean values and standard errors of the means (SEM) represent 8 mice per vaccine group out of two independent experiments and 4 mice for the naïve group. (*** = p<0.001, ** = p<0.01, * = p<0.05; 1 way-ANOVA, Tukey post-test). In contrast to the IAV-HA-specific response, the RSV-F-specific CD8 + T-cell response was not significantly different between the groups (HA+F) hi and (F) hi ( Fig. 2B ). In both groups nearly 1% of all CD8 + T-cells were positive for CD107a and IFN-γ after RSV-F peptide stimulation. Unexpectedly, in the control group treated with the IAV-HA expressing plasmid (HA) hi , some T-cells also reacted specifically to the RSV-F peptides used in the assay, indicating possibly cross-reactive epitopes. Unfortunately, this led to a high variation of reacting CD8 + T-cells in these animals so that the multiple group comparison by 1-way ANOVA did not reach statistical significance for some subpopulation (e.g. IFN-γ+/CD8+ cells: naïve vs. F hi ). However, in all populations analyzed no reduction of the RSV-F-specific response could be observed in the presence of the IAV-HA expressing plasmid. Humoral Immune Response after Combinational Immunization Since virus-specific antibodies are important for protection against the infection with these two viruses, we analyzed the antibodies in the sera of the vaccinated mice in regard to their binding capacity to the natural membrane-bound surface protein, to their IgG-subtype and their neutralization efficacy in vitro ( Fig. 3 and 4 ). Three weeks (day 20) after the first immunization, IAV-HA-specific antibodies could be detected in the groups (HA) hi and (HA+F) hi , but not in the low-dose groups. These antibodies were able to bind the HA protein expressed on the surface of transfected cells and could neutralize the A/PR/8/34 virus in vitro at serum dilutions ranging from 1/10 to 1/640 ( Fig. 3A and C ). These antibody responses were substantially boosted by the second immunization and the mean neutralizing antibody titers increased to 775 and 592 for (HA) hi and (HA+F) hi , respectively. After the second immunization, the animals of the low dose groups (HA) low and (HA+F) low also had neutralizing antibody titers significantly elevated above control animals ( Fig. 3C ). Although the antibody response measured by the FACS-based assay were still significantly lower in these two groups compared to that in the high dose groups ( Fig. 3A ), the differences did not reach statistical significance in the micro-neutralization assay ( Fig. 3C ). To further characterize the IgG subtype of the virus-specific antibodies, virus-coated ELISA plates were incubated with the sera of day 49 after first immunization and bound antibodies were detected by IgG1- and IgG2a-specific antibodies ( Fig. 3B ). All immunized animals showed a balanced IgG1/IgG2a response with no significant differences between the groups, which received one or two antigen expressing plasmids. Overall the IAV-HA-specific humoral immune response was comparable for animals treated with the combinatory or the single-component vaccine. The same kind of analyses were performed to detect RSV-F-specific antibodies and revealed a very similar picture ( Fig. 4 ). Again, the high vaccine dose induced neutralizing antibodies as early as twenty days after the first immunization, whereas the animals of the groups (F) low and the (HA+F) low needed a second immunization to produce comparable levels of RSV-neutralizing antibodies ( Fig. 4C ). Nevertheless, the levels of serum antibodies binding RSV-F on the surface of transfected cells were significantly higher at day 49 in the high dose groups (F) hi and (HA+F) hi than in the respective low dose groups (F) low and (HA+F) low ( Fig. 4A ). Similar to the IAV-HA-specific response, the distribution of RSV-specific antibodies is balanced for the subtypes IgG1 and IgG2a ( Fig. 4B ). Overall, the combinatory vaccine induced antibody responses comparable to the single-component vaccines, including high amounts of binding and neutralizing antibodies specific for both viral surface proteins. Furthermore, no evidence of anti-DNA antibodies were detected in the sera of vaccinated mice (data not shown). 10.1371/journal.pone.0072217.g003 Figure 3 Influenza-specific antibody response. Balb/c mice were immunized according to Table 1 . Sera were collected at days 20 and 49 and IAV-HA-specific antibody responses were analyzed. To analyze antibodies binding IAV-HA in its membrane bound conformation, the sera were incubated with IAV-HA expressing 293T cells and bound antibodies were subsequently detected by FITC-labeled anti-mouse IgG antibodies. The mean fluorescence intensities (MFI) of each group (means+SEM) are shown for sera from day 20 and day 49 ( A ). The distribution of IAV-HA-specific IgG1 and IgG2a were analyzed in an ELISA using IAV coated plates and HRP-conjugated anti-IgG1 and IgG2a antibodies. The means and SEM of the relative light units (RLU) are shown for sera from day 49 ( B ). The neutralizing antibody titer (NT) was analyzed by a microneutralization assay. The reciprocal value of the serum dilution which results in complete protection from infection is given as neutralizing titer. The mean and SEM of each group are indicated for the sera of day 20 and day 49 ( C ). The results for each group represent at least 12 mice out of 2–3 independent experiments with the exception from the results of group (HA) low , which based on a single experiment with 6 mice. (p<0.05): * vs. naïve; # vs. (F) hi ; + vs. (HA+F) low ; ∼ vs. (HA) low (1 way-ANOVA, Tukey post-test). 10.1371/journal.pone.0072217.g004 Figure 4 RSV-specific antibody response. Balb/c mice were immunized according to Table 1 . Sera were collected at days 20 and 49 and RSV-F-specific antibody responses were analyzed. To analyze antibodies binding RSV-F in its membrane bound conformation, the sera were incubated with RSV-F expressing 293T cells and bound antibodies were subsequently detected by FITC-labeled anti-mouse IgG antibodies. The mean fluorescence intensities (MFI) of each group (means+SEM) are shown for sera from day 20 and day 49 ( A ). The distribution of RSV-F-specific IgG1 and IgG2a were analyzed in an ELISA using RSV coated plates and HRP-conjugated anti-IgG1 and IgG2a antibodies. The means and SEM of the relative light units (RLU) are shown for sera from day 49 ( B ). The reciprocal value of the serum dilution which results in 50% inhibition of infection with rgRSV is given as neutralizing antibody titer (NT). The mean and SEM of each group are indicated for the sera of day 20 and day 49 ( C ). The results for each group represent at least 12 mice out of 2–3 independent experiments. (p<0.05): * vs. naïve; # vs. (HA) hi ; + vs. (HA+F) low ; ∼ vs. (F) low (1 way-ANOVA, Tukey post-test). Protective Efficacy Against RSV and IAV Infections Following confirmation of vaccine induced anti-RSV and anti-IAV immune responses, the protective efficacy was assessed by experimental infections via the intranasal route. In accordance with the high neutralizing antibody titers against IAV, all vaccinated animals were fully protected against disease progression indicated by constant weights after IAV challenge infection ( Fig. 5A ). In contrast, all animals of the two control groups, naïve and (F) hi , started to lose weight two days post infection and had to be sacrificed by day 6 due to loss of more than 25% of the initial body weight. To further analyze the protective efficacy achieved by vaccination, the viral loads were measured in the lung homogenates at day 6 post infection ( Fig. 5B ). There is no evidence of non-specific protection conferred by the application of the DNA electroporation as demonstrated by the animals of group (F) hi , which had comparable viral loads as the naïve control animals. The median viral load was reduced at least by 5 logs for all groups, which received the IAV-HA-expressing plasmid ( Fig. 5B ). The best protection was observed in the group (HA+F) hi , in which the viral loads of five out of six animals were below the detection limit of the qRT-PCR and therefore not distinguishable from non-infected controls. Nevertheless, there were neither statistical significant differences in the efficacy between the low and the high dose regimen nor between the single-component and the combinatory vaccines. 10.1371/journal.pone.0072217.g005 Figure 5 Protection from Influenza A Virus challenge. Balb/c mice were immunized according to Table 1 . Five weeks after the second immunization, the mice were challenged with 250 PFU IAV (PR/8/34). The weight loss of each animal was monitored daily and the means and SEM (n = 5–6) are indicated for each group up to day 6 post challenge ( A ). At day 6, viral loads in lung homogenates (vRNA copies/ml) were measured by qRT-PCR. Each animal is represented by one dot and the means of each group are marked by the line. The dotted line indicates the detection limit (428 copies/ml) ( B ). (p<0.05): * vs. naïve; # vs. (F) hi (1 way-ANOVA, Tukey post-test). In contrast to the IAV infection, the infection with the RSV is not lethal in mice and there is only a minor weight loss detectable in non-vaccinated mice. Although there are some variations in the weight loss from animal to animal, there were no significant differences between the groups at any time point ( Fig. 6A ). In contrast, the viral loads in the lung were statistically significant reduced in all animals which received the RSV-F expression plasmid compared to the naïve animals ( Fig. 6B ). Interestingly, the animals of group (HA) hi had also 3-fold lower copy numbers of viral RNA than the naïve animals. This is in line with the observation that in some animals of this group CD8 + T-cells reactive to the RSV-F specific peptides were detected ( Fig. 2B ). Again, there were no differences in the efficacy of the single-component, (F) hi and (F) low , and the combinatory vaccines, (HA+F) hi and (HA+F) low . The median viral load were reduced in the low dose groups by a factor of 83 for (F) low and 136 for (HA+F) low . Although it did not reach statistical significance in the multi-group comparison, the viral load could be further reduced by 1-log if the higher dose of the expression plasmid was used ( Fig. 6B ). 10.1371/journal.pone.0072217.g006 Figure 6 Protection from RSV challenge. Balb/c mice were immunized according to Table 1 . Five weeks after the second immunization, the mice were challenged with 10 6 PFU RSV (A/Long). The weight loss of each animal was monitored daily and the means and SEM (n = 5–6) are indicated for each group up to day 5 post challenge ( A ). At day 5, viral loads in lung homogenates (vRNA copies/ml) were measured by qRT-PCR. Each animal is represented by one dot and the means of each group are marked by the line. The detection limit of the qRT-PCR was 4280 copies/ml ( B ). (p<0.05): * vs. naïve; # vs. (HA) hi (1 way-ANOVA, Tukey post-test). We could demonstrate that the combinatory DNA vaccine protected at least as efficient as the single-component vaccine against infections of both viruses. Furthermore, the protection in both models strongly correlated with the neutralizing antibody titer (data not shown). Discussion In the present study, we thoroughly compared the immunogenicity and efficacy of a combinatory DNA vaccine against IAV and RSV to those of the single vaccines. Although Talaat et al . already reported on a protective combinatory DNA vaccine comprising of expression plasmids for HA (IAV), F (RSV) and gD (HSV) [11] , our study was different in regard to the application via electroporation and to codon-optimized sequences for both antigens, which dramatically increased the immunogenicity and efficacy of our DNA vaccines. Furthermore, we analyzed the cellular and humoral immune responses to both antigens in detail to detect any immunological interference between the two viral surface proteins. One of the most impressive advantages of gene-based vaccines is the induction of highly-efficient CTL responses in addition to the antibody response. Interestingly, co-administration of an RSV-F expressing plasmid reduces the CD8 + and CD4 + T-cell responses to IAV-HA, but the CD8 + T-cell response to RSV-F was not influenced by the addition of the IAV-HA expressing plasmid compared to the single plasmid vaccination. This is in contrast to the work of Patel et al. , in which the co-administration of an IAV-HA expressing plasmid of an H5 subtype led to reduced cellular responses to NA and NP, encoded by the second plasmid [13] . These observations suggest a kind of immunological hierarchy, which strongly depends on the chosen antigens and cannot be predicted for other combinations. This is in line with previously published reports on immunodominance during viral infections and that subdominant epitopes can become more immunogenic if the immunodominant was deleted [21] – [23] . An alternative explanation for the reduced T-cell responses against the IAV-HA might be a direct effect of immune-modulatory properties of the F protein. Since RSV-F was reported to be an TLR-4 agonist [24] and that TLR-4 signaling can trigger T H 2 responses [25] , [26] , it might have been possible that the HA-specific CD4 response was shifted from a T H 1 to a T H 2 response in the presence of RSV-F. But we could not detect a significant increase in IL-4, IL-5 or IL-10 production after HA-specific stimulation. In contrast, the IL-13 production was reduced in the same manner as it was observed for the IFN-γ production by HA-specific T-cells. This rather suggests a more general immunosuppression by RSV-F than a T H 2 bias. In this context, it was reported that the interaction with RSV-F could inhibit the proliferation of peripheral blood lymphocytes in vitro [27] , which might also result in lower T-cell priming in vivo . Since the HA used in this study is derived from the PR8/34 virus and is usually well expressed, the immunosuppressive effect of RSV-F might be even more prominent for HA variants with lower expression levels. This might be partially circumvented by using codon-optimized sequences, which could overcome instability issues of the RNA sequences [5] , but not in cases where the amino acid sequence itself influences the expression level. The high dose of the combinatory DNA vaccine induced substantial, polyfunctional CD8 + T-cell responses to both antigens, which should rapidly control viral replication once infection takes place. This might be of particular interest, since it was shown that CD4 + and CD8 + T-cell responses to influenza could partially confer protection from heterologous IAV infection [28] – [31] . It is further known that CTL responses play an important role in controlling RSV infection [32] – [34] . Interestingly, in some of the mice which received only the HA encoding plasmid, CD8 + T-cells produced IFN-γ after RSV-F-specific re-stimulation indicating some possible cross-reactivity which needs further evaluation. Consistently, the viral load in this group was 3-fold lower than in naïve animals which might be due to cytotoxic T-cells. Since there is no direct overlap in the RSV-F- and HA-specific peptides we used, it might be possible that some similarities in the amino acids at anchor positions might account for this cross-reactivity of the CD8 + T-cells as it was reported for HIV-specific T-cells [35] . Since the inactivated influenza virus vaccines approved for use in humans are known to be poor inducers of cellular immune responses, it is commonly accepted that protection from IAV infection is mainly based on antibody mediated mechanisms, like neutralization of the virus. We therefore analyzed carefully the humoral immune responses induced either by the combinatory vaccine or the single components. Although we found significant differences in the CD4 + T-cell responses to IAV-HA, the antibody response to IAV-HA seemed to be comparable for both groups. This was true in regard to the antibodies binding to membrane-anchored protein, to the neutralizing capacity and also to the IgG1/IgG2a distribution. Additionally our experiments demonstrate that two immunizations even with a low dose of 2 µg per plasmid were sufficient to provide protective antibody responses. This confirms the finding of Zhou et al. , who used a comparable vaccination protocol with either 5 µg or 30 µg of an H5 expressing plasmid and could show that the higher dose induced strong antibody responses already after the initial priming, whereas a second shot was needed for the lower dose [36] . In our previous work, we found the neutralizing antibody titers induced by this vaccination protocol to be comparable to those in humans after a single vaccination with a commercial H1N1(2009) vaccine (Pandemrix©) ( [5] and unpublished data). The humoral immune response to RSV-F seemed not to be influenced by the presence of the second antigen as well, which was also described in the previous study by Talaat et al . [11] . Nevertheless, the application via electroporation strikingly enhanced the antibody response compared to our previous study using conventional subcutaneous injections [3] and was nearly equal in strength to recombinant adenoviral vector immunizations [37] , underlining the great potential of DNA electroporation. Since it is still of debate that a dominant T H 2 response might aggravate the course of a subsequent RSV infection [38] , [39] , it is important to note, that our DNA vaccine induced a balanced T H 1/T H 2 immune response and that there was no sign of induction of an enhanced disease after challenge. We could demonstrate that the efficacy of the combinatory DNA vaccines was at least as high as the ones of the single-component vaccines. This was again in line with the previous report of Talaat et al. , in which the efficacy was not reduced by mixing IAV-HA, RSV-F and HSV-gD expressing plasmids. Nevertheless, we were able to reduce the viral load of RSV after infection by nearly 1000-fold, which was much more efficient than the 10-fold reduction described for the earlier combinatory DNA vaccine [11] and in the range of protection observed after the recombinant adenoviral vector immunization [37] . Furthermore, the vaccinated mice were fully protected against disease progression after an IAV infection and in half of them the viral load in the BALs and the lungs were even below the detection limit of the qRT-PCR, suggesting sterile immunity in these mice. In line with previous studies [3] , [40] , [41] , the protection efficacy correlates in both infection models with the neutralizing antibody titer, suggesting that the antibody-mediated inhibition of the initial virus infection might be the key factor for protection. In conclusion, we demonstrated the efficacy of a combinatory DNA vaccine comprised of two codon-optimized expression plasmids to protect against two severe viral respiratory tract infections. A comprehensive analysis revealed evidence of immunological interference for the cellular, but not for the humoral response. Taken together, this combinatory vaccine against RSV and IAV could have great implications on the rate of severe respiratory tract infections and could further reduce the number of necessary vaccinations. Supporting Information Figure S1 HA-specific CD4 + T-cell responses. Balb/c mice were immunized according to Table 1 . Since the low dose regimen did not result in substantial responses, only the groups which received a total of 40 µg of plasmid DNA were included. A) IAV-HA-specific CD4 + T-cell responses were analyzed one week after the second immunization by intracellular staining for the inflammatory cytokines IFN-γ, TNF and IL-2. The percentages of the different populations among the total CD4 + T-cells are shown. Mean values and standard errors of the means (SEM) represent 8 mice per vaccine group out of two independent experiments and 4 mice for the naïve group. (*** = p<0.001, ** = p<0.01, * = p<0.05; 1 way-ANOVA, Tukey post-test). B) Splenocytes were re-stimulated for 48 h in the presence of the HA-specific peptide and anti-CD28 antibody. Supernatants were analyzed in cytokine-specific ELISA for IL-4, IL-5, IL-10 and IL-13 (eBioscience, Frankfurt, Germany). Since no production of IL-5 and IL-10 could be detected, only the results for IL-4 and IL-13 are shown. Mean values and standard error of the means (SEM) represent 4 mice per vaccine group. The control group received 20 µg of empty pcDNA and 20 µg of empty pVAX. (*** = p<0.001, ** = p<0.01, * = p<0.05; 1 way-ANOVA, Tukey post-test). (PDF) Click here for additional data file.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4162053/

Biodegradable particles as vaccine antigen delivery systems for stimulating cellular immune responses

There is a need for both new and improved vaccination formulations for a range of diseases for which current vaccines are either inadequate or non-existent. Biodegradable polymer-based vaccines fulfill many of the desired properties in achieving effective long-term protection in a manner that is safe, economical, and potentially more practicable on a global scale. Here we discuss some of the work performed with micro/nanoparticles made from either synthetic (poly[lactic-co-glycolic acid] [PLGA] and polyanhydrides) or natural (chitosan) biodegradable polymers. Our attention is focused on, but not limited to, the generation of antitumor immunity where we stress the importance of particle size and co-delivery of antigen and adjuvant.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2998752/

Targeted Delivery of siRNA to Hepatocytes and Hepatic Stellate Cells by Bioconjugation

Previously, we successfully conjugated galactosylated poly(ethylene glycol) (Gal-PEG) to oligonucleotides (ODNs) via an acid labile ester linker (Zhu et al., Bioconjug Chem, 2008, 19 : 290-8). In this study, antisense strands of siRNA were conjugated to Gal-PEG and mannose 6-phosphate poly(ethylene glycol) (M6P-PEG) for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells (HSCs), respectively. These siRNA conjugates were purified by ion exchange chromatography and verified by gel retardation assay. To evaluate their RNAi functions, the validated siRNA duplexes targeting firefly luciferase and transforming growth factor beta 1 (TGF-β1) mRNA were conjugated to Gal-PEG and M6P-PEG, and their gene silencing efficiencies were determined after transfection into HepG2 and HSC-T6 cells. Disulfide bond between PEG and siRNA was cleaved by dithiothreitol, leading to the release of intact siRNA. Both Gal-PEG-siRNA and M6P-PEG-siRNA conjugates could silence luciferase gene expression for about 40% without any transfection reagents, while the gene silencing effects reached more than 98% with the help of cationic liposomes at the same dose. Conjugation of TGF-β1 siRNA with Gal-PEG and M6P-PEG could silence endogenous TGF-β1 gene expression as well. In conclusion, these siRNA conjugates have the potential for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells for efficient gene silencing in vivo .

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192967/

Calibration of an Upconverting Phosphor-Based Quantitative Immunochromatographic Assay for Detecting Yersinia pestis, Brucella spp., and Bacillus anthracis Spores

Yersinia pestis, Brucella spp., and Bacillus anthracis are pathogens that can cause infectious zoonotic diseases with high mortality rates. An upconverting phosphor-based quantitative immunochromatographic (UPT-LF) assay, a point-of-care testing method suitable for resource-limited areas, was calibrated to quantitatively detect pathogenic bacteria. The bacterial purity or activity were ensured via staining methods and growth curves, respectively. Growth assays showed that the classic plate-counting method underestimated bacterial numbers compared with the bacterial counting method recommended by the reference material of the National Institutes for Food and Drug Control, China. The detection results of the UPT-LF assay differed significantly between the bacterial cultures in liquid and solid media and between different strains. Accelerated stability assessments and freeze-thaw experiments showed that the stability of the corresponding antigens played an important role in calibrating the UPT-LF assay. In this study, a new calibration system was developed for quantitative immunochromatography for detecting pathogenic bacteria. The results demonstrated the necessity of calibration for standardizing point-of-care testing methods. Introduction Despite having lower sensitivities than sequencing and PCR methods, immunochromatographic assays (ICAs) are extensively employed for point-of-care testing in many developing countries and offer the first-line of defense against acute serious pandemic zoonotic diseases with high mortality rates. Such diseases include the plague, brucellosis, and anthrax, whose etiologic agents are Yersinia pestis, Brucella spp., and Bacillus anthracis , respectively. These pathogens have caused countless deaths during historical pandemics and are distributed worldwide in natural foci, each having natural reservoirs (such as rodents and fleas for Y. pestis ). In addition, these pathogenic bacteria are easily acquired and reproduced for use as bioterrorism agents and pose a great potential threat to public health. Timely diagnosis and therapy is crucial for saving patients' lives owing to limited therapeutic strategies and poor prognoses. Etiological diagnoses are especially critical because of the remarkable resemblance to clinical syndromes of diseases caused by bioterrorism agents and other factors. ICAs are rapid and portable and are widely used to detect pathogenic bacteria on-site for surveillance in natural foci and handling of public health emergencies, impeding the transmission of these diseases to the greatest possible extent. Correctly estimating the accuracy, detection limits, and comparability of ICAs is difficult because analyzing qualitative results based on naked-eye observations via the present colloidal gold ICA is unsuitable for refined calibration research. Upconverting phosphor-based technology based on the lateral flow assay (UPT-LF), a newly developed ICA method for quantitative detection, enables the establishment of a reliable calibration method for ICAs. UPT-LF assays for the detection of pathogenic bacteria have been developed and evaluated in our laboratory, and include assays to detect Y. pestis (Yan et al., 2006 ), B. anthracis spores (Li et al., 2006 ), Brucella spp. (Qu et al., 2009 ), Vibrio cholerae (Hao et al., 2017 ), Burkholderia pseudomallei (Hua et al., 2015a ; Liang et al., 2017 ), Francisella tularensis (Hua et al., 2015b ), Escherichia coli O157:H7 (Wang et al., 2007 ), Vibrio parahaemolyticus , and seven Salmonella spp. (Zhao et al., 2016 ). Our UPT-LF assays showed robust performance using many clinical and environmental samples such as biochemical reagents and various types of powders and viscera samples (Zhang P. et al., 2014 ; Hua et al., 2015a , b ), as well as field water samples (Hao et al., 2017 ). After the samples are loaded onto the strip, upconverting phosphor (UCP) particles that have first been combined with monoclonal antibodies (mAbs) against bacteria can capture the corresponding bacteria and are then captured by the other antibodies against bacteria fixed onto the test band (T band), while the remaining UCP-mAbs complexes are captured by goat anti-mouse IgG on the control band (C band). The visible light emitted by the UCP particles under the excitation of infrared light can be transformed into electric current signals. The ratios of the T- and C-band signals, or the T/C ratio, are used as the detection results in practical applications and are proportional to the bacterial concentration (Yang, 2019 ). Considering the principle of UPT-LF for quantitative bacterial detection, calibration of the assay should rely on the standard curve, using the detection value and target bacterial concentrations as parameters. However, the plate-counting method, the current international gold standard for counting bacteria, only counts live cells (Breed and Dotterrer, 1916 ) and thus may be unsuitable for evaluating the actual abilities of the ICA because antigens remain on the surfaces of dead cells, thus making calibration of the ICA difficult (Zhang X. et al., 2014 ; Gorsuch et al., 2019 ). Counting via microscopy or optical density measures are more exact methods for all cells. According to the Beer–Lambert law, absorbance measured via spectrophotometry can be used to quantify substances (Klumpp et al., 2009 ; Meyers et al., 2018 ), while the bacterial concentrations are proportional to the optical density at 600 nm (Dalgaard et al., 1994 ; Stevenson et al., 2016 ). The National Institutes for Food and Drug Control (NIFDC, China) developed a spectrophotometric method to determine the concentrations of Y. pestis (Wei et al., 2009 ), Brucella spp. (Li et al., 2011 ), and B. anthracis (Wei et al., 2010 ) according to the bacteria enumerated directly via direct microscopic counts. Reference Materials for the Bacterial Content of Plague Vaccines, Brucellosis Vaccines and Anthrax Vaccines (Approval No. National Biological Reference [2013] 0049, issued by the China Food and Drug Administration), were further developed, while the optical densities for three tubes containing glass fibers correspond to three concentrations for the three bacteria, and the standard curves of the two parameters can be used to quantify the bacteria in samples ( Table 1 ). In addition, the culture methods, including the various media and culture conditions (such as fluid medium or a solid plate) may significantly influence bacterial growth because the cellular components may differ under different conditions such as growth rate (Klumpp et al., 2009 ; Scott et al., 2010 ; Klumpp and Hwa, 2014 ) or temperature (Labrie et al., 2005 ), while heterogeneous populations may result from mutations (Schaechter and View From Here Group, 2001 ). In this study, a UPT-LF assay for detecting Y. pestis, Brucella spp., and B. anthracis was calibrated using the NIFDC bacterial reference material, and the influences of culture conditions and various strains were studied to form a solid foundation for calibrating and standardizing immunochromatographic assays. Table 1 Congruent relationship between the optical density at 600 nm of the NIFDC reference material and bacterial concentrations. Reference Material (RM) Corresponding concentration of Y. pestis (× 10 8 cell mL −1 ) Corresponding concentration of Brucella spp. (× 10 8 cell mL −1 ) Corresponding concentration of B. anthracis (× 10 8 cell mL −1 ) RM with low optical density 4.3 18 0.4 RM with Medium optical density 6.2 25 0.55 RM with high optical density 8.4 34 0.72 Materials and Methods Materials The strains used to calibrate the detection were Y. pestis 91001, Brucella abortus S19, B. anthracis Sterne, and other strains of Y. pestis, B. anthracis, Bacillus atrophaeus, Bacillus subtilis , and Bacillus cereus . All strains were preserved in our laboratory. The culture media, lysogeny broth (LB, including tryptone and yeast extract), Hiss agar, and brain heart infusion (BHI) were purchased from Oxoid, Ltd., Beijing Land Bridge Technology Co., Ltd. (Beijing, China), and Becton Dickinson and Company (New Jersey, USA), respectively. Gram stain kits were purchased from Beijing Solarbio Sciences & Technology Co., Ltd. (Beijing, China). Reference Materials for the Bacterial Content of Plague Vaccine, Brucellosis Vaccine, and Anthrax Vaccine were obtained from the NIFDC, China. The SpectraMax M2 Microplate Reader from Molecular Devices Co., Ltd. was used to measure optical density. UPT-LF strips for detecting Y. pestis, B. abortus , and B. anthracis were developed by our laboratory, and the UPT-LF detection kits, including strips with the corresponding sample-treatment buffer, were produced in workshops with air cleanliness up to the one million level of good manufacturing practices at Beijing Hotgen Biotechnology Co., Ltd. Radiofrequency identification devices (RFID) in the UPT-LF strips recorded the strip parameters, including the detection target, cutoff, and information from the standard curves (A and B value), which were automatically recognized by a UPT-3A biosensor. The UPT-3A biosensor reads the signals and exports the detection results of the UPT-LF assay ( Figures 1B–D ). The monoclonal antibodies for Y. pestis on the UPT-LF strip were prepared by injecting purified F1 antigen into BALB/c mice. The monoclonal antibodies for B. abortus and the polyclonal antibodies for B. anthracis on the strip were selected based on injecting whole inactivated bacterial cells into mice or goats; therefore, the information on their corresponding antigens was unclear. The standard curves of the UPT-LF strips in the commercial detection kit for quantifying Y. pestis, Brucella spp., and B. anthracis were based on the detection results for Y. pestis 91001, B. abortus S19, and B. anthracis Sterne cultured in liquid medium, as well as the numbers determined via the classic plate-counting method. Figure 1 The criteria program and an illustration of the UPT-LF assay. (A) Program for the UPT-LF assay criteria. (B) The photograph of UPT biosensor scanning of a strip. (C) The detection result shows the concentration of bacteria. The signal peak on the left is derived from the control band, and that on the right is derived from the test band. (D) Information stored by the UPT-LF biosensor including time, sample ID, detection target, concentration of the target, the value of the signal for the test band and control band, T/C ratio, as well as the parameters for standard curves (y = Ax + B, the logarithm of T/C-cutoff as x and the logarithm of concentration as y) for quantitation of the concentration of target bacteria, including the cut-off, A and B. Some information, with the exception of the detection result, was recorded in radiofrequency identification devices (RFID) for each commercial strip, and this can be revised through a new record after calibration. Criteria for Culturing and Identifying the Bacteria The program used to determine the UPT-LF assay criteria is summarized in Figure 1A . B. anthracis, B. atrophaeus, B. subtilis , and B. cereus spores were prepared and identified. For preincubation, 0.2 mL of bacteria were incubated in 5 mL of LB broth at −80°C, then cultured at 37°C with shaking at 200 rpm for 15.5 h. The bacteria were collected via centrifugation at 8,000 rpm for 5 min, then inoculated onto nutrient agar plates from Roche bottles (~30 × 15 cm) after being suspended in 2 mL of LB broth. The bacteria were cultured for 7–8 days, then suspended in normal saline to prepare the spores. To determine the spore quality, the bacteria were scraped into 0.2 mL of normal saline and stained with malachite green and crystal violet-iodine on slides treated with 3% hydrochloric acid. For the malachite green staining, a mixture of 0.1 mL of bacteria and 0.1 mL of malachite green were boiled at 100°C for 25 min, then 75% ethyl alcohol was used as a wash buffer after heat-fixing and staining. Using a Gram stain kit, the bacteria were stained with crystal violet for 1 min, washed, then soaked in iodine solution for 1 min, and washed in 20 mL of water. Y. pestis 91001 and B. abortus S19 were used for plotting growth curves. For preincubation, 0.2 mL of bacterial solution containing 30% glycerol preserved at −80°C was inoculated into tubes containing 5 mL of medium, then cultured at 37°C with shaking at 200 rpm for different times. The optical densities at 600 nm (OD 600 ) were detected three times each, using LB medium as the blank for Y. pestis 91001 and BHI medium as the blank for B. abortus S19. The preincubation time that resulted in an optical density of ~1.0 was considered the optimal preincubation time for the culture. For incubation, 1 mL of preincubated culture was inoculated into three Erlenmeyer flasks containing 19 mL of LB medium, and the OD 600 of the culture for all three flasks was measured via spectrophotometry for 1 or 2 h. The optical density of the bacteria was measured both in normal saline and culture medium. Three parallel Erlenmeyer flasks containing Y. pestis 91001 were preincubated for 16 h (to an OD 600 of ~1.0) and then cultured. At 2, 4, 6, 8, and 12 h, 0.4 mL of the bacterial culture was extracted and divided into two samples. One sample of the bacterial culture was used to directly measure the optical density with LB as a blank, and the other sample was centrifuged at 8,000 rpm at 4°C for 8 min to harvest the bacteria. The supernatant was then removed and the pellet was suspended in saline solution to the same volume as that used to measure the optical density. The optical density was then measured using saline solution as a blank. Similarly, three flasks of B. abortus S19 were preincubated for 17.5 h and cultured for 7.3, 8.3, and 9.5 h in BHI medium or normal saline, then the optical densities of the bacteria from the three flasks were measured using BHI medium and normal saline as the blanks, respectively. Comparing Counting Methods for Y. pestis 91001 and B. abortus S19 The standard curves for counting Y. pestis, Brucella spp., and B. anthracis were plotted using the OD 600 for each NIFDC reference material for different bacterial contents (including low, medium, and high concentrations) and measured three times using a spectrophotometer. The optical densities were determined for Y. pestis 91001 after a 16 h preincubation and 6 h incubation, B. abortus S19 after a 17.5 h preincubation and 7.3 h incubation, and B. anthracis stern spores after being collected and suspended in normal saline. After measuring their optical densities, their concentrations were calculated from the standard curve of the NIFDC reference material. For the plate-counting method, the bacterial cultures mentioned above for plotting the standard curves of NIFDC reference material were serially diluted 10-fold according to pre-estimated concentrations based on experience. The dilutions containing the final three low concentrations were inoculated onto Hiss agar plates containing 5% goat blood for Y. pestis , BHI plates containing 5% goat blood for B. abortus S19, and LB plates containing 5% goat blood for B. anthracis stern spores. The concentrations of each of the three flasks were measured for repetition. Plates containing 30–300 colony-forming units were used to calculate the bacterial concentrations in the original flask cultures based on the fold dilutions. UPT-LF Assay for Detecting Y. pestis 91001 and B. abortus S19 Cultured in Liquid and Solid Media First, different solid media were used to evaluate the bacterial growth states, then 0.2 mL of Y. pestis 91001 after a 16 h preincubation was smeared and cultured on LB and Hiss agar plates, while B. abortus S19 after a 17.5 h preincubation was smeared on BHI, BHI containing 5% goat blood, LB, and LB containing 5% goat blood. All bacteria were then cultured at 37°C. After 24 h, the bacteria on the plates were scraped into tubes containing 0.8 mL of normal saline. The bacteria in the liquid medium were then prepared according to the above-mentioned method for plotting the standard curves of NIFDC reference material. After harvesting from liquid or solid media and suspending in normal saline, the optical densities of the bacteria were measured, then their concentrations were determined from the standard curves of the NIFDC reference material. After dilution with sample-treatment buffer, 0.1 mL of Y. pestis 91001 at 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 cells ml −1 , B. abortus S19 at 1 × 10 6 , 1 × 10 7 , 1 × 10 8 , and 1 × 10 9 cells mL −1 , and B. anthracis Sterne at 1 × 10 5 , 1 × 10 6 , and 1 × 10 7 cells mL −1 were directly applied to the UPT-LF strips. To read the signals on the strips, the detection results, T/C ratios, and bacterial concentrations were retrieved directly using the UPT-3A biosensor. Inclusivity of the UPT-LF Assay After plate culturing and calibration of the concentrations using the NIFDC reference material, the Y. pestis strains 91001, EV, 0614F, Otten, Tjiusidej(R), MII, and M23 at concentrations of 1 × 10 4 , 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 cells mL −1 , Brucella suis S2, Brucella melitensis M5, and B. abortus S19 and 104M at 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 cells mL −1 , and B. anthracis Sterne, A16R, and CTN-1 at 1 × 10 5 , 1 × 10 6 , and 1 × 10 7 cells mL −1 were diluted with sample-treatment buffer and detected via the UPT-LF assay. All Y. pestis and Brucella spp. strains were also detected via a colloidal gold immunochromatographic assay (CG-ICA). For Y. pestis detection, 1, 10, and 100 ng mL −1 of F1 antigen were extracted and purified from the bacteria for use as a positive control and were tested via the UPT-LF assay. Antigen Stability Y. pestis and Brucella spp. (1 × 10 8 cells mL −1 ) were prepared and divided into three samples: the first was used as a control, the second was stored at 37°C for 5 d, and the third was freeze-thawed three times. After treatment, 1 × 10 5 , 1 × 10 6 , and 1 × 10 7 cells mL −1 of Y. pestis 91001, EV, 0614F, Otten, and Tjiusidej(R), and 1 × 10 7 and 1 × 10 8 cells mL −1 of B. abortus 104M, B. suis S2, and B. melitensis M5, were diluted with sample-treatment buffer and detected via the UPT-LF assay. Statistical Analysis All experiments were repeated three times. The statistical data were compared via paired-sample t -tests with a 95% confidence interval using Origin 8.0 software from OriginLab Corporation (MA, USA). Materials The strains used to calibrate the detection were Y. pestis 91001, Brucella abortus S19, B. anthracis Sterne, and other strains of Y. pestis, B. anthracis, Bacillus atrophaeus, Bacillus subtilis , and Bacillus cereus . All strains were preserved in our laboratory. The culture media, lysogeny broth (LB, including tryptone and yeast extract), Hiss agar, and brain heart infusion (BHI) were purchased from Oxoid, Ltd., Beijing Land Bridge Technology Co., Ltd. (Beijing, China), and Becton Dickinson and Company (New Jersey, USA), respectively. Gram stain kits were purchased from Beijing Solarbio Sciences & Technology Co., Ltd. (Beijing, China). Reference Materials for the Bacterial Content of Plague Vaccine, Brucellosis Vaccine, and Anthrax Vaccine were obtained from the NIFDC, China. The SpectraMax M2 Microplate Reader from Molecular Devices Co., Ltd. was used to measure optical density. UPT-LF strips for detecting Y. pestis, B. abortus , and B. anthracis were developed by our laboratory, and the UPT-LF detection kits, including strips with the corresponding sample-treatment buffer, were produced in workshops with air cleanliness up to the one million level of good manufacturing practices at Beijing Hotgen Biotechnology Co., Ltd. Radiofrequency identification devices (RFID) in the UPT-LF strips recorded the strip parameters, including the detection target, cutoff, and information from the standard curves (A and B value), which were automatically recognized by a UPT-3A biosensor. The UPT-3A biosensor reads the signals and exports the detection results of the UPT-LF assay ( Figures 1B–D ). The monoclonal antibodies for Y. pestis on the UPT-LF strip were prepared by injecting purified F1 antigen into BALB/c mice. The monoclonal antibodies for B. abortus and the polyclonal antibodies for B. anthracis on the strip were selected based on injecting whole inactivated bacterial cells into mice or goats; therefore, the information on their corresponding antigens was unclear. The standard curves of the UPT-LF strips in the commercial detection kit for quantifying Y. pestis, Brucella spp., and B. anthracis were based on the detection results for Y. pestis 91001, B. abortus S19, and B. anthracis Sterne cultured in liquid medium, as well as the numbers determined via the classic plate-counting method. Figure 1 The criteria program and an illustration of the UPT-LF assay. (A) Program for the UPT-LF assay criteria. (B) The photograph of UPT biosensor scanning of a strip. (C) The detection result shows the concentration of bacteria. The signal peak on the left is derived from the control band, and that on the right is derived from the test band. (D) Information stored by the UPT-LF biosensor including time, sample ID, detection target, concentration of the target, the value of the signal for the test band and control band, T/C ratio, as well as the parameters for standard curves (y = Ax + B, the logarithm of T/C-cutoff as x and the logarithm of concentration as y) for quantitation of the concentration of target bacteria, including the cut-off, A and B. Some information, with the exception of the detection result, was recorded in radiofrequency identification devices (RFID) for each commercial strip, and this can be revised through a new record after calibration. Criteria for Culturing and Identifying the Bacteria The program used to determine the UPT-LF assay criteria is summarized in Figure 1A . B. anthracis, B. atrophaeus, B. subtilis , and B. cereus spores were prepared and identified. For preincubation, 0.2 mL of bacteria were incubated in 5 mL of LB broth at −80°C, then cultured at 37°C with shaking at 200 rpm for 15.5 h. The bacteria were collected via centrifugation at 8,000 rpm for 5 min, then inoculated onto nutrient agar plates from Roche bottles (~30 × 15 cm) after being suspended in 2 mL of LB broth. The bacteria were cultured for 7–8 days, then suspended in normal saline to prepare the spores. To determine the spore quality, the bacteria were scraped into 0.2 mL of normal saline and stained with malachite green and crystal violet-iodine on slides treated with 3% hydrochloric acid. For the malachite green staining, a mixture of 0.1 mL of bacteria and 0.1 mL of malachite green were boiled at 100°C for 25 min, then 75% ethyl alcohol was used as a wash buffer after heat-fixing and staining. Using a Gram stain kit, the bacteria were stained with crystal violet for 1 min, washed, then soaked in iodine solution for 1 min, and washed in 20 mL of water. Y. pestis 91001 and B. abortus S19 were used for plotting growth curves. For preincubation, 0.2 mL of bacterial solution containing 30% glycerol preserved at −80°C was inoculated into tubes containing 5 mL of medium, then cultured at 37°C with shaking at 200 rpm for different times. The optical densities at 600 nm (OD 600 ) were detected three times each, using LB medium as the blank for Y. pestis 91001 and BHI medium as the blank for B. abortus S19. The preincubation time that resulted in an optical density of ~1.0 was considered the optimal preincubation time for the culture. For incubation, 1 mL of preincubated culture was inoculated into three Erlenmeyer flasks containing 19 mL of LB medium, and the OD 600 of the culture for all three flasks was measured via spectrophotometry for 1 or 2 h. The optical density of the bacteria was measured both in normal saline and culture medium. Three parallel Erlenmeyer flasks containing Y. pestis 91001 were preincubated for 16 h (to an OD 600 of ~1.0) and then cultured. At 2, 4, 6, 8, and 12 h, 0.4 mL of the bacterial culture was extracted and divided into two samples. One sample of the bacterial culture was used to directly measure the optical density with LB as a blank, and the other sample was centrifuged at 8,000 rpm at 4°C for 8 min to harvest the bacteria. The supernatant was then removed and the pellet was suspended in saline solution to the same volume as that used to measure the optical density. The optical density was then measured using saline solution as a blank. Similarly, three flasks of B. abortus S19 were preincubated for 17.5 h and cultured for 7.3, 8.3, and 9.5 h in BHI medium or normal saline, then the optical densities of the bacteria from the three flasks were measured using BHI medium and normal saline as the blanks, respectively. Comparing Counting Methods for Y. pestis 91001 and B. abortus S19 The standard curves for counting Y. pestis, Brucella spp., and B. anthracis were plotted using the OD 600 for each NIFDC reference material for different bacterial contents (including low, medium, and high concentrations) and measured three times using a spectrophotometer. The optical densities were determined for Y. pestis 91001 after a 16 h preincubation and 6 h incubation, B. abortus S19 after a 17.5 h preincubation and 7.3 h incubation, and B. anthracis stern spores after being collected and suspended in normal saline. After measuring their optical densities, their concentrations were calculated from the standard curve of the NIFDC reference material. For the plate-counting method, the bacterial cultures mentioned above for plotting the standard curves of NIFDC reference material were serially diluted 10-fold according to pre-estimated concentrations based on experience. The dilutions containing the final three low concentrations were inoculated onto Hiss agar plates containing 5% goat blood for Y. pestis , BHI plates containing 5% goat blood for B. abortus S19, and LB plates containing 5% goat blood for B. anthracis stern spores. The concentrations of each of the three flasks were measured for repetition. Plates containing 30–300 colony-forming units were used to calculate the bacterial concentrations in the original flask cultures based on the fold dilutions. UPT-LF Assay for Detecting Y. pestis 91001 and B. abortus S19 Cultured in Liquid and Solid Media First, different solid media were used to evaluate the bacterial growth states, then 0.2 mL of Y. pestis 91001 after a 16 h preincubation was smeared and cultured on LB and Hiss agar plates, while B. abortus S19 after a 17.5 h preincubation was smeared on BHI, BHI containing 5% goat blood, LB, and LB containing 5% goat blood. All bacteria were then cultured at 37°C. After 24 h, the bacteria on the plates were scraped into tubes containing 0.8 mL of normal saline. The bacteria in the liquid medium were then prepared according to the above-mentioned method for plotting the standard curves of NIFDC reference material. After harvesting from liquid or solid media and suspending in normal saline, the optical densities of the bacteria were measured, then their concentrations were determined from the standard curves of the NIFDC reference material. After dilution with sample-treatment buffer, 0.1 mL of Y. pestis 91001 at 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 cells ml −1 , B. abortus S19 at 1 × 10 6 , 1 × 10 7 , 1 × 10 8 , and 1 × 10 9 cells mL −1 , and B. anthracis Sterne at 1 × 10 5 , 1 × 10 6 , and 1 × 10 7 cells mL −1 were directly applied to the UPT-LF strips. To read the signals on the strips, the detection results, T/C ratios, and bacterial concentrations were retrieved directly using the UPT-3A biosensor. Inclusivity of the UPT-LF Assay After plate culturing and calibration of the concentrations using the NIFDC reference material, the Y. pestis strains 91001, EV, 0614F, Otten, Tjiusidej(R), MII, and M23 at concentrations of 1 × 10 4 , 1 × 10 5 , 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 cells mL −1 , Brucella suis S2, Brucella melitensis M5, and B. abortus S19 and 104M at 1 × 10 6 , 1 × 10 7 , and 1 × 10 8 cells mL −1 , and B. anthracis Sterne, A16R, and CTN-1 at 1 × 10 5 , 1 × 10 6 , and 1 × 10 7 cells mL −1 were diluted with sample-treatment buffer and detected via the UPT-LF assay. All Y. pestis and Brucella spp. strains were also detected via a colloidal gold immunochromatographic assay (CG-ICA). For Y. pestis detection, 1, 10, and 100 ng mL −1 of F1 antigen were extracted and purified from the bacteria for use as a positive control and were tested via the UPT-LF assay. Antigen Stability Y. pestis and Brucella spp. (1 × 10 8 cells mL −1 ) were prepared and divided into three samples: the first was used as a control, the second was stored at 37°C for 5 d, and the third was freeze-thawed three times. After treatment, 1 × 10 5 , 1 × 10 6 , and 1 × 10 7 cells mL −1 of Y. pestis 91001, EV, 0614F, Otten, and Tjiusidej(R), and 1 × 10 7 and 1 × 10 8 cells mL −1 of B. abortus 104M, B. suis S2, and B. melitensis M5, were diluted with sample-treatment buffer and detected via the UPT-LF assay. Statistical Analysis All experiments were repeated three times. The statistical data were compared via paired-sample t -tests with a 95% confidence interval using Origin 8.0 software from OriginLab Corporation (MA, USA). Results Criteria for Culturing and Identifying Bacteria Y. pestis 91001, B. abortus S19, and B. anthracis Sterne were used for the calibration. The bacterial purity and activity, including the cellular integrity and surface constituents, were critical for evaluating the accuracy of the immunoassay. This study primarily focused on the bacterial quality for calibration because the specificity of the UPT-LF assay for detecting Y. pestis, B. abortus S19, and B. anthracis had been demonstrated in a previous study (Zhang P. et al., 2014 ). For B. anthracis, B. atrophaeus, B. subtilis , and B. cereus , the bacterial spores can be dyed green by malachite green stain, whereas for the Gram-positive bacteria, their vegetative forms were recognized via crystal violet-iodine using a Gram stain. Both staining methods were used for identification to ensure the purity of spores after preparation ( Figure 2 ). The spore spheres were viewed by microscopy ( Figures 2A,B ). The spore culturing time should be extended if vegetative forms of the bacteria are visible, which appear as long rods after Gram staining ( Figure 2C ). Both the vegetative form and spores of B. subtilis could be recognized after culturing and Gram staining ( Figures 2D,E ). The B. anthracis spores with the highest purity were used as the foundation for calibrating the UPT-LF assay. Figure 2 Staining of the spores and vegetative forms of B. anthracis . B. anthracis Sterne (A) and B. cereus 41 (B) spores stained with malachite green. (C) Vegetative form of B. anthracis Sterne among spores stained with crystal violet-iodine. (D) Vegetative form of B. subtilis stained with crystal violet-iodine. (E) B. subtilis spores stained with crystal violet-iodine. After determining the optimal preincubation times by measuring the optical density at 600 nm, growth curves were plotted for Y. pestis 91001 in LB medium and B. abortus S19 in BHI medium at 37°C to ensure the bacteria were in an active state. The optimal preincubation times for Y. pestis 91001 and B. abortus S19 were 16 and 17.5 h, respectively, and the logarithmic phases lasted 3–10 and 7–15 h, respectively ( Figure 3 ). Figure 3 Preincubation times and growth curves for Y. pestis 91001 (A,C) and B. abortus S19 (B,D) in the LB and BHI media. Comparison of the Optical Densities in Normal Saline and Culture Media The optical densities of the bacteria in normal saline were measured because normal saline is the blank control used for the NIFDC reference material. After 16 and 17.5 h of preincubation, the optical densities of the Y. pestis 91001 and B. abortus S19 cultures incubated for different times were determined, using LB and BHI as blanks. The same cultures were also resuspended in normal saline after centrifugation and the optical densities were measured using normal saline as a blank. Paired-sample t -tests at the 0.05 level showed minimal differences between the two groups in terms of optical density for Y. pestis 91001, whereas the optical densities differed significantly for B. abortus S19 ( Figure 4 ). Interestingly, the optical density of Y. pestis 91001 in medium was higher than that in normal saline and vice versa for B. abortus S19, emphasizing that the optical densities differ for different bacteria and media. Figure 4 Optical densities of Y. pestis 91001 (A) and B. abortus S19 (B) in LB or BHI medium and normal saline. Asterisks denote significant differences in OD value ( t -test; p < 0.05; n = 3). Comparison of Bacterial Counting Methods The bacteria were counted via two methods, namely, plate counts and counting based on the NIFDC reference material ( Figure 5A ). Standard curves were constructed showing the optical densities at 600 nm for each reference material and the concentrations of Y. pestis, Brucella spp., and B. anthracis ( Figures 5B–D ), and the concentrations of the bacterial solutions, were confirmed when their optical densities were measured. The concentrations determined by the two counting methods were compared ( Figure 5E ). Although the method using the reference material yielded slightly higher numbers than the plate-counting method for B. abortus , the differences between the two methods for B. abortus S19, and between the two plate types for B. anthracis Sterne were not significant. Paired-sample t -tests revealed that Y. pestis 91001 and B. anthracis Sterne spores differed significantly in number, and the numbers obtained with the reference material method were 11- and 5-fold higher than those of the plate-count method. The differences between the two methods for counting Y. pestis may have partly resulted from the culture temperature being 37°C for the production of the F1 antigen, whereas 28°C is the best temperature for preparing the reference material. B. anthracis Sterne spores are considered more stable than those of other bacteria; however, we suspended and stored the spores in normal saline before counting, which resulted in low concentrations being determined via the plate-counting method because the NaCl in normal saline significantly influenced the number of viable cells (Wei et al., 2012 ). Thus, the number of dead cells in a culture may significantly influence the detection results of a quantitative immunoassay. Figure 5 Comparison of different bacterial counting methods. (A) Reference Material for Bacterial Content of the Plague Vaccine, Brucellosis Vaccine, and Anthrax Vaccine from NIFDC. Standard curves of the reference material for counting Y. pestis (B) , Brucella spp. (C) , and B. anthracis (D) were plotted based on optical density. (E) Comparison of the bacterial concentrations determined by the reference material method and the plate-counting method (Hiss agar plate containing 5% goat blood for Y. pestis 91001, BHI plate containing 5% goat blood for B. abortus S19, and LB plate and LB plate containing 5% goat blood for B. anthracis Stern spores). Asterisks denote significant differences in bacterial concentrations determined by the counting methods ( t -test; p < 0.05; n = 3). Differences in the Detection Results of the UPT-LF Assay After Culturing in Liquid or Solid Media First, Y. pestis 91001 and B. abortus S19 were cultured on different types of solid media. The growth rates of Y. pestis 91001 on LB plates were higher than those on LB inclined-plane medium, BHI inclined-plane medium, and Hiss agar plates ( Figure 6A ), while the growth rates of B. abortus S19 on BHI containing blood were higher than those on BHI and both LB plates ( Figure 6B ). However, bacteria cultured on plates containing goat blood were not used for the UPT-LF assay because goat blood cells may affect the results of the assay. Y. pestis 91001 and B. abortus S19 cultured on LB and BHI plates, as well as in the corresponding liquid media, were used for the UPT-LF assays after their concentrations had been determined from the NIFDC reference material ( Figures 6C,D ). Most of the T/C ratios for Y. pestis 91001 and B. abortus S19 grown in the liquid medium were significantly lower than those grown on solid medium, demonstrating that the amounts of certain antigens on the bacteria differed under different culture conditions. This phenomenon was consistent with previous reports (Klumpp et al., 2009 ; Klumpp and Hwa, 2014 ), inferring that culture methods should be screened for immunoassay calibration. After calibration, the standard curves for quantifying Y. pestis 91001, B. abortus S19, and B. anthracis Sterne spores ( Figures 6E–G ) were plotted to calculate the concentrations. The quantification results showed approximately 1.5- and 0.5-fold differences between the results with or without calibration for most Y. pestis 91001 and B. abortus S19 concentrations (data not shown). Figure 6 Differences in the UPT-LF detection results for Y. pestis 91001, B. abortus S19, and B. anthracis Sterne cultured in liquid and solid media. The growth conditions for Y. pestis (A) and B. abortus (B) differed on different solid media. UPT-LF detection results for Y. pestis differed significantly between liquid LB broth and LB plates (C) and for B. abortus S19 between the liquid BHI medium and BHI plates (D) . All bacterial concentrations were determined using the NIFDC reference material, and the standard curves for the UPT-LF assay for quantifying Y. pestis 91001 (E) , B. abortus S19 (F) , and B. anthracis Sterne spores (G) were plotted after calibration. Asterisks denote significant differences in UPT-LF results for bacterial culture of liquid and solid medium ( t -test; p < 0.05; n = 3). Inclusivity Evaluation for the UPT-LF Assay After culturing on plates, the concentrations of bacteria were determined according to the reference material, and seven Y. pestis , four Brucella spp., and three B. anthracis strains of different concentrations were detected via the UPT-LF assay ( Figure 7 ). The sensitivities of the assay for Y. pestis strains 91001, EV, Otten, 0614F, and Tjiusidej(R) were 10 5 cells mL −1 , while MII and M23 yielded negative results. Y. pestis M23 is a F1-negative strain that lacks the virulence determinant of the F1 envelope antigen (Burrows and Bacon, 1958 ; Seguin et al., 1987 ). Although its F1 genes ( caf1, caf1A, caf1M , and caf1R ) are 100% identical to those of Y. pestis 91001, Y. pestis MII is an attenuated strain with fewer surface proteins than those of other Y. pestis vaccine strains (Lei et al., 1997 ). B. suis S2, B. abortus 104M, and B. melitensis M5 can be detected with similar T/C ratios to that of B. abortus S19, which was the same for B. anthracis A16R and CTN-1 compared with B. anthracis Sterne. For most stains, the qualitative results were consistent between the CG-ICA assay and the UPT-LF assay ( Figure 7 ). Considering the inclusivity of the immunoassay, the quantitative performance of the bacterial concentrations using immunoassays should be defined more precisely for calibration. Using standard curves for each bacterial strain is impractical for an on-site detection method. The standard curves of the UPT-LF assay for quantifying Y. pestis were determined for Y. pestis 91001 in our laboratory. In conclusion, for calibration, the standard curves for quantifying concentrations were based on certain bacteria, while the true concentration for a specific bacterium hinges on the amount of F1 antigen on its surface. Figure 7 Detection results for different Y. pestis, Brucella spp., and B. anthracis strains. (A) Detection results for Y. pestis stains and the F1 antigen via a UPT-LF assay and CG-ICA. (B) Detection results for Brucella spp. strains via UPT-LF and CG-ICA methods. (C) Detection results for B. anthracis strains via a UPT-LF assay. Antigen Stability on the Bacterial Surface Corresponded With the Results of the UPT-LF Assay The stability of the antigen to which an immunoassay is targeted is a pivotal factor that influences the accuracy of the detection results. Because degradant levels are more than 100-fold greater under higher temperatures than under lower temperatures according to the Arrhenius equation (Waterman, 2011 ), we evaluated the stability of antigens stored at 37°C for 5 days, as well as after three freeze-thaw cycles to assess the accelerated stability ( Figure 8 ). The recovery rates, or the proportionality between the T/C ratios of the bacteria after treatment and those of the control, were all higher than 97% ( Table 2 ). In conclusion, the treatment had little influence on the results of the UPT-LF assay for Y. pestis strains 91001, EV, 0614F, Otten, and Tjiusidej(R), or Brucella spp., at various concentrations. Figure 8 Detection results for the UPT-LF assay for Y. pestis (A) and Brucella spp. strains (B) in accelerated stability assessments. Table 2 Recovery rate * for each strain under various parameters in accelerated stability assessments. Bacteria Parameter 37 ° C, 5 d Freeze-thaw 10 5 cell mL −1 10 6 cell mL −1 10 7 cell mL −1 10 5 cell mL −1 10 6 cell mL −1 10 7 cell mL −1 Y. pestis 91001 98.8% 98.9% 99.2% 100.7% 99.2% 99.0% Y. pestis EV 101.4% 100.2% 101.1% 98.8% 100.5% 99.8% Y. pestis 0614F 99.5% 99.5% 99.6% 98.3% 99.6% 99.8% Y. pestis Otten 100.8% 100.2% 99.9% 99.8% 100.6% 99.6% Y. pestis Tjiusidej(R) 100.9% 99.5% 99.4% - 99.9% 99.8% 10 7 cell mL −1 10 8 cell mL −1 10 7 cell mL −1 10 8 cell mL −1 B. abortus 104M 96.2% 98.2% 97.4% 98.3% B. suis S2 97.7% 98.5% 97.3% 98.1% B. melitensis M5 104.2% 101.2% 103.1% 100.1% * Recovery rate = (T/C ratio under various parameters)/(T/C ratio of the control) . Criteria for Culturing and Identifying Bacteria Y. pestis 91001, B. abortus S19, and B. anthracis Sterne were used for the calibration. The bacterial purity and activity, including the cellular integrity and surface constituents, were critical for evaluating the accuracy of the immunoassay. This study primarily focused on the bacterial quality for calibration because the specificity of the UPT-LF assay for detecting Y. pestis, B. abortus S19, and B. anthracis had been demonstrated in a previous study (Zhang P. et al., 2014 ). For B. anthracis, B. atrophaeus, B. subtilis , and B. cereus , the bacterial spores can be dyed green by malachite green stain, whereas for the Gram-positive bacteria, their vegetative forms were recognized via crystal violet-iodine using a Gram stain. Both staining methods were used for identification to ensure the purity of spores after preparation ( Figure 2 ). The spore spheres were viewed by microscopy ( Figures 2A,B ). The spore culturing time should be extended if vegetative forms of the bacteria are visible, which appear as long rods after Gram staining ( Figure 2C ). Both the vegetative form and spores of B. subtilis could be recognized after culturing and Gram staining ( Figures 2D,E ). The B. anthracis spores with the highest purity were used as the foundation for calibrating the UPT-LF assay. Figure 2 Staining of the spores and vegetative forms of B. anthracis . B. anthracis Sterne (A) and B. cereus 41 (B) spores stained with malachite green. (C) Vegetative form of B. anthracis Sterne among spores stained with crystal violet-iodine. (D) Vegetative form of B. subtilis stained with crystal violet-iodine. (E) B. subtilis spores stained with crystal violet-iodine. After determining the optimal preincubation times by measuring the optical density at 600 nm, growth curves were plotted for Y. pestis 91001 in LB medium and B. abortus S19 in BHI medium at 37°C to ensure the bacteria were in an active state. The optimal preincubation times for Y. pestis 91001 and B. abortus S19 were 16 and 17.5 h, respectively, and the logarithmic phases lasted 3–10 and 7–15 h, respectively ( Figure 3 ). Figure 3 Preincubation times and growth curves for Y. pestis 91001 (A,C) and B. abortus S19 (B,D) in the LB and BHI media. Comparison of the Optical Densities in Normal Saline and Culture Media The optical densities of the bacteria in normal saline were measured because normal saline is the blank control used for the NIFDC reference material. After 16 and 17.5 h of preincubation, the optical densities of the Y. pestis 91001 and B. abortus S19 cultures incubated for different times were determined, using LB and BHI as blanks. The same cultures were also resuspended in normal saline after centrifugation and the optical densities were measured using normal saline as a blank. Paired-sample t -tests at the 0.05 level showed minimal differences between the two groups in terms of optical density for Y. pestis 91001, whereas the optical densities differed significantly for B. abortus S19 ( Figure 4 ). Interestingly, the optical density of Y. pestis 91001 in medium was higher than that in normal saline and vice versa for B. abortus S19, emphasizing that the optical densities differ for different bacteria and media. Figure 4 Optical densities of Y. pestis 91001 (A) and B. abortus S19 (B) in LB or BHI medium and normal saline. Asterisks denote significant differences in OD value ( t -test; p < 0.05; n = 3). Comparison of Bacterial Counting Methods The bacteria were counted via two methods, namely, plate counts and counting based on the NIFDC reference material ( Figure 5A ). Standard curves were constructed showing the optical densities at 600 nm for each reference material and the concentrations of Y. pestis, Brucella spp., and B. anthracis ( Figures 5B–D ), and the concentrations of the bacterial solutions, were confirmed when their optical densities were measured. The concentrations determined by the two counting methods were compared ( Figure 5E ). Although the method using the reference material yielded slightly higher numbers than the plate-counting method for B. abortus , the differences between the two methods for B. abortus S19, and between the two plate types for B. anthracis Sterne were not significant. Paired-sample t -tests revealed that Y. pestis 91001 and B. anthracis Sterne spores differed significantly in number, and the numbers obtained with the reference material method were 11- and 5-fold higher than those of the plate-count method. The differences between the two methods for counting Y. pestis may have partly resulted from the culture temperature being 37°C for the production of the F1 antigen, whereas 28°C is the best temperature for preparing the reference material. B. anthracis Sterne spores are considered more stable than those of other bacteria; however, we suspended and stored the spores in normal saline before counting, which resulted in low concentrations being determined via the plate-counting method because the NaCl in normal saline significantly influenced the number of viable cells (Wei et al., 2012 ). Thus, the number of dead cells in a culture may significantly influence the detection results of a quantitative immunoassay. Figure 5 Comparison of different bacterial counting methods. (A) Reference Material for Bacterial Content of the Plague Vaccine, Brucellosis Vaccine, and Anthrax Vaccine from NIFDC. Standard curves of the reference material for counting Y. pestis (B) , Brucella spp. (C) , and B. anthracis (D) were plotted based on optical density. (E) Comparison of the bacterial concentrations determined by the reference material method and the plate-counting method (Hiss agar plate containing 5% goat blood for Y. pestis 91001, BHI plate containing 5% goat blood for B. abortus S19, and LB plate and LB plate containing 5% goat blood for B. anthracis Stern spores). Asterisks denote significant differences in bacterial concentrations determined by the counting methods ( t -test; p < 0.05; n = 3). Differences in the Detection Results of the UPT-LF Assay After Culturing in Liquid or Solid Media First, Y. pestis 91001 and B. abortus S19 were cultured on different types of solid media. The growth rates of Y. pestis 91001 on LB plates were higher than those on LB inclined-plane medium, BHI inclined-plane medium, and Hiss agar plates ( Figure 6A ), while the growth rates of B. abortus S19 on BHI containing blood were higher than those on BHI and both LB plates ( Figure 6B ). However, bacteria cultured on plates containing goat blood were not used for the UPT-LF assay because goat blood cells may affect the results of the assay. Y. pestis 91001 and B. abortus S19 cultured on LB and BHI plates, as well as in the corresponding liquid media, were used for the UPT-LF assays after their concentrations had been determined from the NIFDC reference material ( Figures 6C,D ). Most of the T/C ratios for Y. pestis 91001 and B. abortus S19 grown in the liquid medium were significantly lower than those grown on solid medium, demonstrating that the amounts of certain antigens on the bacteria differed under different culture conditions. This phenomenon was consistent with previous reports (Klumpp et al., 2009 ; Klumpp and Hwa, 2014 ), inferring that culture methods should be screened for immunoassay calibration. After calibration, the standard curves for quantifying Y. pestis 91001, B. abortus S19, and B. anthracis Sterne spores ( Figures 6E–G ) were plotted to calculate the concentrations. The quantification results showed approximately 1.5- and 0.5-fold differences between the results with or without calibration for most Y. pestis 91001 and B. abortus S19 concentrations (data not shown). Figure 6 Differences in the UPT-LF detection results for Y. pestis 91001, B. abortus S19, and B. anthracis Sterne cultured in liquid and solid media. The growth conditions for Y. pestis (A) and B. abortus (B) differed on different solid media. UPT-LF detection results for Y. pestis differed significantly between liquid LB broth and LB plates (C) and for B. abortus S19 between the liquid BHI medium and BHI plates (D) . All bacterial concentrations were determined using the NIFDC reference material, and the standard curves for the UPT-LF assay for quantifying Y. pestis 91001 (E) , B. abortus S19 (F) , and B. anthracis Sterne spores (G) were plotted after calibration. Asterisks denote significant differences in UPT-LF results for bacterial culture of liquid and solid medium ( t -test; p < 0.05; n = 3). Inclusivity Evaluation for the UPT-LF Assay After culturing on plates, the concentrations of bacteria were determined according to the reference material, and seven Y. pestis , four Brucella spp., and three B. anthracis strains of different concentrations were detected via the UPT-LF assay ( Figure 7 ). The sensitivities of the assay for Y. pestis strains 91001, EV, Otten, 0614F, and Tjiusidej(R) were 10 5 cells mL −1 , while MII and M23 yielded negative results. Y. pestis M23 is a F1-negative strain that lacks the virulence determinant of the F1 envelope antigen (Burrows and Bacon, 1958 ; Seguin et al., 1987 ). Although its F1 genes ( caf1, caf1A, caf1M , and caf1R ) are 100% identical to those of Y. pestis 91001, Y. pestis MII is an attenuated strain with fewer surface proteins than those of other Y. pestis vaccine strains (Lei et al., 1997 ). B. suis S2, B. abortus 104M, and B. melitensis M5 can be detected with similar T/C ratios to that of B. abortus S19, which was the same for B. anthracis A16R and CTN-1 compared with B. anthracis Sterne. For most stains, the qualitative results were consistent between the CG-ICA assay and the UPT-LF assay ( Figure 7 ). Considering the inclusivity of the immunoassay, the quantitative performance of the bacterial concentrations using immunoassays should be defined more precisely for calibration. Using standard curves for each bacterial strain is impractical for an on-site detection method. The standard curves of the UPT-LF assay for quantifying Y. pestis were determined for Y. pestis 91001 in our laboratory. In conclusion, for calibration, the standard curves for quantifying concentrations were based on certain bacteria, while the true concentration for a specific bacterium hinges on the amount of F1 antigen on its surface. Figure 7 Detection results for different Y. pestis, Brucella spp., and B. anthracis strains. (A) Detection results for Y. pestis stains and the F1 antigen via a UPT-LF assay and CG-ICA. (B) Detection results for Brucella spp. strains via UPT-LF and CG-ICA methods. (C) Detection results for B. anthracis strains via a UPT-LF assay. Antigen Stability on the Bacterial Surface Corresponded With the Results of the UPT-LF Assay The stability of the antigen to which an immunoassay is targeted is a pivotal factor that influences the accuracy of the detection results. Because degradant levels are more than 100-fold greater under higher temperatures than under lower temperatures according to the Arrhenius equation (Waterman, 2011 ), we evaluated the stability of antigens stored at 37°C for 5 days, as well as after three freeze-thaw cycles to assess the accelerated stability ( Figure 8 ). The recovery rates, or the proportionality between the T/C ratios of the bacteria after treatment and those of the control, were all higher than 97% ( Table 2 ). In conclusion, the treatment had little influence on the results of the UPT-LF assay for Y. pestis strains 91001, EV, 0614F, Otten, and Tjiusidej(R), or Brucella spp., at various concentrations. Figure 8 Detection results for the UPT-LF assay for Y. pestis (A) and Brucella spp. strains (B) in accelerated stability assessments. Table 2 Recovery rate * for each strain under various parameters in accelerated stability assessments. Bacteria Parameter 37 ° C, 5 d Freeze-thaw 10 5 cell mL −1 10 6 cell mL −1 10 7 cell mL −1 10 5 cell mL −1 10 6 cell mL −1 10 7 cell mL −1 Y. pestis 91001 98.8% 98.9% 99.2% 100.7% 99.2% 99.0% Y. pestis EV 101.4% 100.2% 101.1% 98.8% 100.5% 99.8% Y. pestis 0614F 99.5% 99.5% 99.6% 98.3% 99.6% 99.8% Y. pestis Otten 100.8% 100.2% 99.9% 99.8% 100.6% 99.6% Y. pestis Tjiusidej(R) 100.9% 99.5% 99.4% - 99.9% 99.8% 10 7 cell mL −1 10 8 cell mL −1 10 7 cell mL −1 10 8 cell mL −1 B. abortus 104M 96.2% 98.2% 97.4% 98.3% B. suis S2 97.7% 98.5% 97.3% 98.1% B. melitensis M5 104.2% 101.2% 103.1% 100.1% * Recovery rate = (T/C ratio under various parameters)/(T/C ratio of the control) . Discussion In this study, we developed a comprehensive immunochromatographic calibration method for quantitatively detecting bacteria. Our findings confirmed that: (1) bacterial purity and activity could be guaranteed via staining and growth curves, respectively. (2) Bacterial numbers should be confirmed using the NIFDC reference material, not plate-counting methods. (3) Antigen amounts differed significantly between cultures in liquid and solid media; therefore, the culture method should be fixed for calibration. (4) The amounts of specific antigen differed between each Y. pestis, Brucella spp., and B. anthracis strain, therefore the stains should be specified for calibration. (5) The stability of the corresponding antigens indicated the stability of the immunoassay, with surface antigens greatly influencing the assay results. After calibration, the UPT-LF assay yielded more exact bacterial quantification results, and all of the factors that influenced the detection results in this calibration method should be considered when performing other immunochromatographic assays. Because pathogenic bacteria responsible for serious infectious diseases can induce infections in several cell types and lead to death or disease breakouts, qualitative detection results are often sufficient. However, calibration of a quantification immunoassay may still have practical significance for diagnosing bacteria. First, it can help standardize pathogen detection and precisely evaluate the detection limits and inclusivity of an immunoassay. Second, the bacterial concentrations should be exactly determined for vaccine production, which can be achieved by a rapid quantification immunochromatographic method. Third, such an assay could be applied to detect foodborne bacteria, whose detection limits differ among various foods. In this study, the sensitivities of the UPT-LF assay for Y. pestis, Brucella spp., and B. anthracis were about 1 × 10 4 -10 5 cells mL −1 , 1 × 10 4 -1 × 10 5 cells mL −1 , 1 × 10 3 -1 × 10 4 cells mL −1 , respectively, which were at least 10-fold lower than that of the UPT-LF assay developed in our laboratory (Li et al., 2006 ; Yan et al., 2006 ; Qu et al., 2009 ). This may be because commercial, mass-produced UPT-LF strips were used for the calibration method for practicality reasons, but these strips may be of inferior quality, thereby resulting in decreased sensitivities. Nevertheless, UPT-LF detection in which the sample is directly added to the strip without any need for pre-treatment is especially useful as a field assay in resource-limited areas of developing countries, and meaningful calibration results can still be obtained. For example, the UPT-LF assay is practical for the surveillance of infected animals in natural foci for Y. pestis, Brucella spp., and B. anthracis , when the samples being tested (such as dead animals) contain a large number of bacteria. Furthermore, the sample volume for the UPT-LF assay is 100 μL, in other words, only 10 3 bacterial cells are applied to the strip when the concentration of the bacterial sample is 10 4 cells mL −1 . For comparison, the sensitivities of real-time or isothermal recombinase PCR are about 10 copies per reaction for Y. pestis (Qu et al., 2010 ), B. anthracis (Antwerpen et al., 2008 ; Bentahir et al., 2018 ), and Brucella spp. (Zeybek et al., 2019 ), which is equivalent to 2 × 10 3 -1 × 10 4 cells mL −1 in a 1–5 μL sample volume of template for each PCR, thereby enrichment of the target bacteria by culturing or extraction of nucleic acid is often required (Kane et al., 2019 ). Enrichment of the target bacteria, for example, by centrifugation, could be applied during sample preparation for the UPT-LF assay. Although low concentrations of bacteria could not be tested in this study, all of the factors addressed in the calibration method are relevant to other immunochromatographic assays, including the determination of bacterial purity and activity, the bacterial counting method, culturing conditions, and the different amounts of antigen between strains. Antibody fabrication limits the inclusivity of immunoassays for bacterial detection. For example, the F1 antigen is one of four virulence determinants for Y. pestis determined by the World Health Organization in 1970, but some low-virulence vaccine Y. pestis strains, such as M23 and MII, which lack or contain minimal amounts of the F1 antigen, were determined to be negative via the UPT-LF assay fabricated with antibodies against F1. Owing to the great differences in antigens on the surfaces of the various strains for one bacterial type, false-negative results are unavoidable for an immunoassay based on one or a few antibodies. Multiple detection chips based on various monoclonal antibodies against different antigens on the surfaces of some bacteria, as well as new bacterial biomarkers, could be used to promote the development of more accurate immunoassays. The relative limitations of a detection reagent should be illustrated in the instruction manual. Conclusion This study is the first to establish a calibration method for an upconverting phosphor-based quantitative immunochromatographic assay to detect pathogenic bacteria, which included guaranteeing the bacterial purity and activity, accurately counting the bacteria, and determining the antigen amounts under different culture conditions. It also ensured the inclusivity of the assay and the stability of the corresponding antigens. The results indicated that the bacterial preincubation and incubation times, the liquid and solid culturing methods, the bacterial counting method, quantification of different strains of a species, and antigen stability, should all be considered in calibrating immunochromatographic assays. In particular, bacterial strains, culturing and counting methods should be standardized for immunochromatographic assays. Data Availability Statement All datasets generated for this study are included in the article/supplementary material. Ethics Statement BALB/c mice (8 weeks old) and goats were used for the production of antibodies. The Committee of Welfare and Ethics of Laboratory Animals, Beijing Institute of Microbiology and Epidemiology (Beijing, China) reviewed and approved the animal care and experimental protocols concerning the preparation of antibodies (Permit No. IACUC-DWZX-2018- 006). All animal experiments were in compliance with the Guidelines for the Welfare and Ethics of Laboratory Animals of China. Author Contributions RY, DW, and PZ designed the experiments. PZ, YZhang, YZhao, YS, CN, ZS, and JW performed the experiments. PZ, RY, and DW analyzed the data and wrote the manuscript. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7908961/

The Methodological Basis of Defining Research Trends and Fronts

The methodological and technical aspects of identifying research fronts and trends in the development of science are considered. Based on the literature data, a comparison of scientometric methods for finding research fronts was carried out: analysis of publication activity, direct citation analysis, co-citation analysis, bibliographic coupling, and content analysis. The advantages of the combined application of various approaches are shown, the role of expert assessment and verification of the results of scientometric analysis is emphasized. We revealed topical problems associated with the detection of scientific fronts by scientometric methods and showed promising directions in their solution. INTRODUCTION The search for scientific trends and research fronts, that is, topical or promising research, is one of the most significant problems in science policy, scientometrics, and the history and philosophy of science and is of decisive importance at the stages of planning scientific activities. The topic of scientific trends and fronts is obvious if it is dictated by socio-political, environmental, and economic factors or threats to national health [ 1 ]. These can be natural disasters, terrorist attacks [ 2 ], economic crises, or the appearance of dangerous diseases in the human population, such as the outbreak of influenza A/H1N1 pandemic in 2009 [ 3 ] or SARS Cov2 in 2019–2020. In these cases, the scientific community, states, research and funding organizations are actively and consistently involved in the search for solutions to emerging problems. The fronts of science are much less obvious in the absence of such events; they then themselves become an object of study, requiring the development and use of methodological foundations and appropriate tools to identify them. Scientific trends and fronts, as a rule, are the object of research of science itself, and their identification is an attempt to search for new growth points, as represented by the most promising ideas and developments that are important for the further development of science and technology. In other words, a search is carried out for changing objects of research in their relation to existing knowledge and to each other [ 4 ]. When identifying research trends and fronts it is predominantly scientometric methods that are used. In a continuation of previous studies in the field of scientific trends in various fields of knowledge [ 5 – 7 ] and in the absence of reviews on the topic of detecting research fronts, we further consider the concepts of research trends and fronts, classify approaches, and describe the tools for their detection, as well as study the current issues that are pending their decision. When reviewing the literature, the Scopus and RJ "Informatika" databases of VINITI were used without restrictions on time and types of documents. The request included the following keywords: "research front", "research trend", and "research focus". Additionally, sources from lists of references based on search results were used. A METHODOLOGY FOR IDENTIFICATION OF RESEARCH TRENDS AND FRONTS In general, a research front is understood as the situation where the interests and needs of society coincide with the current scientific results [ 8 ]. The key object of analysis in identifying research fronts is the groups of scientific publications and their interrelationships. According to the classical definition of D. Price, a research front is a densely cited network of recently published papers [ 9 ]. In a more detailed definition, a research front is understood as a group of recently published articles with a common topic, which are strictly connected by a network of citations among themselves and weakly connected with publications outside the group [ 10 ]. At the same time, strong links between citations within a group are characteristic of a research front at the initial stage of its development, while at later stages, with an increase in the number of citations, including from other scientific areas, this connection weakens. The strength of citation links between publications of clusters is determined by predetermined threshold values that are unique for each scientific field. The sizes of research fronts also depend on the discipline, which usually ranges from a few publications to several dozen. As an example, in the latest report on research fronts from Clarivate Analytics the spread is from 2 to 50 articles [ 11 ]; sometimes a minimum threshold is set, for example, 10 publications [ 12 ]. The concept of a research trend is close in meaning to a research front. A research trend is the collective action of a group of researchers, each of which begins to pay considerable attention to a specific scientific topic: read scientific publications on this topic, refer to them, and publish the results of their own research [ 4 ]. At times the concepts of the research front and research trend are used synonymously [ 13 ]. The main types of research fronts according to the common classification of G. Small [ 8 ] are shown in Fig. 1 . The method for identifying the stage of a research front involves comparing clusters of publications for two or more equal consecutive periods of time. Fig. 1. Types of research fronts. The Clarivate Analytics together with the Chinese Academy of Sciences, in its periodic reports distinguishes only two types of research fronts: key ( key hot fronts ) and incipient ( emerging fronts ) [ 11 ]. Research fronts are also revealed by the Elsevier company based on SciVal data, where the most promising topics are determined by the Prominence indicator. Under the influence of various factors, the research fronts of the extensive phase can turn into an intensive one, for example, when new promising research methods appear, with increased funding for the field, when there is an urgent need to develop a topic under the influence of external factors, etc. [ 1 , 12 ]. As a result of the development of a research front, according to G. Small, it can either develop into a new discipline, or be absorbed by a broader field, which adapts the achievements of a research front to a wide group of studies [ 8 ]. In the first case, this indicates the growth of a scientific front, in the second, it indicates its influence on science. As a rule, scientific fronts of interdisciplinary research develop in separate directions, while the absorbed research fronts have little to do with interdisciplinarity, but are gaining citations faster. Study on research fronts is significant from both fundamental and applied points of view. At the theoretical level, they determine the vector of development of scientific progress and allow tracing the origin and evolution of one field or another, the division and merging of areas of knowledge, contribute to the spread of knowledge between scientific disciplines [ 14 ], and allow adjusting organizational processes when new knowledge meets traditional paradigms that dictate research topics, standards and regulations [ 15 ]. The identification of research fronts is of practical interest for a wide range of stakeholders involved in the definition of priority areas of scientific research and their funding. To date, three main scientometric approaches are widely used to identify research trends and fronts: analysis of the dynamics of changes in scientific production, citation analysis with its varieties, and content analysis, as well as their various combinations. Analysis of Publication Activity to Identify Research Trends Analysis of publication activity is usually used to identify research trends, while citation analysis is used to identify research fronts [ 4 , 16 ]. When analyzing scientific production, expressed by the number of publications, one resorts to models of the growth of scientific knowledge: (1) in the first model, the growth of knowledge is considered as the cumulative development of new ideas based on previous recent scientific achievements; (2) the second model assumes that the development of new ideas is based on the entire body of human knowledge, and not only on recent achievements. According to this model, there is a selective choice of grounds for a new idea from all of human scientific experience; (3) the third model is based on the theory of scientific revolutions by T. Kuhn [ 17 ] and presupposes an intensive growth of knowledge interrupted by periods of calm. There is no consensus about which of the proposed models most closely corresponds to reality, especially since each of them, to one degree or another, explains the ongoing scientific events in various disciplines. Each of these paradigms can correspond to some mathematical model of the growth of scientific literature, for example, linear or exponential [ 18 ]. In natural science disciplines, exponential growth often prevails; when identifying scientific trends researchers therefore turn to D. Price on the exponential growth and obsolescence of scientific literature [ 19 , 20 ]. The scattering law is used to identify a scientific information trend according to S. Bradford [ 21 ], which allows identification of the core of scientific journals of a given subject. An example of a study using this method is the work to identify research trends in the field of tourism [ 22 ]. A circle of authors and organizations that form a research trend on this topic was determined according to zones of concentration and dispersion of Bradford's scientific information, as well as the analysis of the scientific productivity and authoritativeness of publications. The analysis of research trends in the field of borehole geophysics was carried out by the authors of this work: the leading positions of this field in the field of earth sciences were identified, the most productive authors were detected and the redistribution of leading positions between countries over the past 20 years was shown [ 7 ]. Further identification of research trends and fronts in the field of geophysics is extremely important, since it is associated with the search for new research areas, primarily for the creation of innovative technologies. In the field of borehole geophysics, "cheap" logging technologies will be the most demanded by both large and small service companies in the near future, which is due to the end of time of "expensive" oil. Citation Analysis to Identify Research Fronts The main method in identifying research fronts is citation analysis, which makes it possible to trace the growth of interest and relevance of a particular topic by the dynamics of changes in the number of citations of publications of a particular field. Citation analysis is considered more objective in comparison with expert assessment, since it takes the opinion of the entire scientific world community of scientists expressed in references [ 23 ]. The approach is based on the observation that recent scientific publications are the most cited. Thus, the identification of thematic clusters of the most cited publications allows us to identify the research front of the corresponding discipline [ 9 ]. The response time to published papers varies across disciplines, but on average is 2–5 years, during which half of simultaneously published publications are cited [ 24 ]. Within the framework of citation analysis, where both cited and citing publications are clustered, a research front is understood as: (a) a group of the most cited publications identified by direct citation analysis [ 4 , 9 ]; (b) a group of co-cited publications identified by co-citation analysis, positions 6 and 7 in Fig. 2b [ 25 – 27 ]. The cluster of a research front, in addition to co-cited publications, may include citing publications, positions 1, 6 and 7 in Fig. 2b [ 28 ]. This definition of research fronts was used by E. Garfield [ 29 ]; this approach is still implemented by the Clarivate Analytics in periodic reports on research fronts using Web of Science databases [ 11 ]. There is also a third approach, where a research front refers to publications that cited a cluster of co-cited publications, position 1 in Fig. 2b [ 30 ]; Fig. 2. The principles of clustering publications used in identifying research fronts. A, direct citation analysis; B, co-citation analysis; B, bibliographic coupling. The top row usually represents recently published publications, the bottom row represents publications of the last 2–5 years. Citation analysis can cover out-of-sample publications. (c) a group of publications with similar references, identified by the bibliographic coupling method, positions 3 and 4 in Fig. 2b . According to this approach, the articles of a research front themselves may not have citations [ 2 , 12 , 31 – 33 ]; (d) with the joint application of the indicated approaches, a research front is understood, for example, as a group of co-cited publications plus a group of publications with similar references [ 30 , 34 – 37 ], a group of co-cited publications plus publications citing this group [ 38 ], or several groups of publications based on the results of all three approaches [ 28 , 39 , 40 ]. As a rule, when used together, each method is used separately, after which the results are compared or combined. However, it is possible to build complex combined approaches: for example, clustering by bibliographic coupling of those publications in which clusters of co-cited publications are cited; this is then clustering of the first and second levels [ 30 ]. The formal similarity with the clusters of publications of research fronts is demonstrated by artificially created groups of articles united by chief editors, for example, within the framework of special issues of journals, where articles of each issue abundantly cite each other. When analyzing research fronts, groups of publications united by similar publication models are usually excluded from the analysis [ 8 ]. When describing research fronts, the concept of an intellectual base ( knowledge base , knowledge foundation , intellectual base , or intellectual structure ) is used, which means literature cited by publications of a research front [ 2 , 4 , 41 ]. Many studies demonstrate the thematic proximity of an intellectual base and research fronts [ 13 , 31 , 36 , 42 ]. When analyzing co-citation, sometimes confusion of these concepts occurs; while some researchers understand co-cited publications as a research front, others consider them as an intellectual base, and the citing publications as a front (see Figure 2B ). In general, the scientometric task is to identify the points of intellectual displacement (research fronts) in the relatively stable scientific literature (intellectual base). Co-citation analysis was simultaneously proposed by I.V. Marshakova and G. Small [ 43 , 44 ]: two documents are considered co-cited and thematically related if they both appear in the reference list of a third document (with which the two cited documents also have a thematic relationship) and the citation rate is defined as the frequency with which two documents are cited together. Researchers usually choose a small group of publications that are highly cited within a given period of time as a basis for clustering. This could be 1 or 10% of the highly cited articles, the top 10, top 20 articles, etc. This approach to the search for scientific fronts has a drawback associated with the nature of citation [ 45 ]. Accordingly, the ability to take new publications into account, which are often of the greatest interest in the search for scientific fronts, is limited [ 46 ]. In other words, co-citation is suitable for identifying a research front at a relatively late stage, and not at the very moment of its emergence [ 8 ]. According to one of the developers of the method of G. Small, the analysis of socializing does not cover the entirety of publications on a scientific front, but rather informs about the emergence of such a front; it is designed to do a quick screening of the scientific landscape rather than a definitive delineation of some specific area [ 8 ]. The approach does not depend on the vocabulary and language of publications. The bibliographic coupling method proposed by M. Kessler [ 47 , 48 ] presupposes that two works have a meaningful relationship to each other and are thematically related if they have one or more similar references. Thus, a research front consists of publications that jointly cite other publications. Since references to the analyzed papers are not important and only their reference lists are investigated, the method is free from lag (especially if it is applied not to journal publications, but to preprints) and allows one to analyze newly published papers. The main idea of the method is as follows: (1) a separate bibliographic reference used in two publications is called one unit of coupling between these publications; (2) several publications form a linked group G if each member of the group has at least one coupling unit with the test paper P 0 ; and (3) the coupling strength between P 0 and any member of G is measured by the number of coupling units (n) between them. Like co-citation analysis, the bibliographic coupling method is independent of the vocabulary and language of publications and can be automated. In comparison with the analysis of co-citation analysis, the method of bibliographic coupling is used less often to search for scientific fronts [ 28 , 32 ]. One essential criterion for the study of research fronts is the choice of the citation window. The problem of choosing a citation window received full coverage in [ 32 ]: the model of a traditional static 5-year citation window was compared with a sliding overlapping citation window, as well as with the half-life of highly cited articles. Research with a static citation window was found to be the least labor-intensive; however, the most labor-intensive method with a sliding citation window helped to find more research fronts. At the same time, some of the emerging research fronts identified by the two methods did not intersect, which is why the joint use of static and sliding citation windows was recognized as the most effective. Since the main scientometric approaches to identifying research fronts involve a procedure for clustering bibliographic data, the results of the analysis can be influenced by clustering methods and the choice of threshold values for the measure of similarity between the grouped elements [ 30 , 31 ]. The object of citation analysis can be both the publications themselves and the authors of these publications, journals and, less often, subject categories [ 49 ]. Co-citation analysis is used to search for scientific fronts in various fields of knowledge: HIV/AIDS [ 15 ], scientific collaboration [ 13 ], library and information science [ 27 ]. The method of bibliographic coupling was used to study the historical development of research fronts in the field of anthrax research [ 12 ]. The joint use of methods of co-citation analysis and bibliographic coupling was carried out to search for scientific fronts in the library and information science [ 36 ] and in the field of battery research [ 37 ]. Author's citations and content analysis of links were used to identify research fronts in the field of bacterial infections [ 23 ]. The experience of identifying research fronts not for a discipline as a whole, but for an individual organization is remarkable: in [ 49 ], the intellectual base was studied by co-citation analysis; the corpus of publications cited by the organization, on the basis of which a research fronts of the organization itself were further identified. Similar studies of the publication activity and citations of a particular organization were carried out by the authors of this work for more effective information support of scientific projects [ 50 , 51 ], while the developed methods were also applicable for identifying research trends and fronts. The search for scientific fronts can also be carried out for a separate journal: for example, the Journal of the American Society for Information Science. Using the methods of bibliographic coupling and citation analysis, research fronts were identified and a significant closeness of the intellectual base with them was shown [ 31 ]. Content Analysis to Identify Research Fronts Methods for semantic analysis of metadata and full texts of scientific publications, including neural network technologies [ 52 , 53 ] and algorithms for detecting rapidly spreading, so-called burst terms, which express new phenomena, are widely used in identifying research fronts [ 2 , 14 , 42 , 54 ]. Content analysis investigates the frequency of the use of words in metadata and full texts and, separately, keywords, as well as their joint occurrence in publications. Analysis of the frequency and co-occurrence of keywords is carried out: (a) on the metadata of publications; in this case, author's or additional keywords assigned in systems are investigated (for example, KeyWords Plus [ 55 , 56 ] extracted from lists of cited literature) and words from various subject thesauri and authoritative dictionaries (for example, MeSH ), as well as automatically extracted keywords from titles and annotations; (b) on full texts, where keywords and terms are also extracted and semantically analyzed using software tools. Some researchers refer to the results of keyword co-occurrence analysis as a research focus, while the research front is considered to be the result of co-citation analysis [ 57 ]. To search for scientific fronts in the field of informatics and accounting, the content analysis method identified topics with growing and dying interest, as well as those that have lost their relevance [ 14 ]. To extract keywords, entity linking method was used that takes the context of the keyword into account. An approach based on the combined use of searching by association rules, keyword analysis and rapidly spreading terms is presented based on the example of anticancer developments in nanomedicine [ 54 ]. Using linguistic methods for searching for the semantic similarity of texts, the identification of research fronts was described in [ 46 ]: a method of comparing phrases and fragments of identical content, not necessarily expressed by the same keywords, was presented. Cluster analysis of author's keywords was carried out to search for scientific fronts in the field of social sciences: the result of a study in five countries was a map of national science, indicating promising areas [ 1 ]. Content analysis is often combined with citation analysis methods to identify scientific fronts. Thus, research fronts in the field of artificial intelligence were identified through the combined use of methods of bibliographic coupling and content analysis of keywords [ 58 ]. Methods of bibliographic coupling (by co-authors and documents) and content analysis were used to search for scientific fronts in the field of business [ 41 ]. A co-occurrence analysis method combined with co-citation analysis has been used to find research fronts in library and information science in Spain [ 42 ]. The same two methods were used to analyze co-citation fronts in astrophysical research [ 59 ]. A more sophisticated analysis of a research fronts of the interdisciplinary direction is presented using the example of magnetic nanoparticles, where co-citation and co-word networds were studied based on a sample of the 500 most-cited publications [ 60 ]. THE EFFICIENCY OF DIFFERENT TYPES OF SCIENTOMETRIC ANALYSIS IN REVEALING RESEARCH FRONTS A researcher's choice of a particular scientometric method is arbitrary in most cases, while in some situations it is necessary to correlate the method with the goals of the study and take the complexity of the calculations into account [ 28 , 39 ]. Different methods are more or less applicable to one type of research front or another. Thus, the emerging research fronts are better identified by the method of bibliographic coupling, which does not have a time delay. If topological clustering is preferable for research, then citation analysis turns out to be more applicable [ 39 ]. If it is necessary to cluster based on the textual similarity of publications, content analysis has proven itself better, in which the frequency analysis of words from metadata or full texts gives better results in comparison with the frequency analysis of an author's keywords. The choice of the approach has a significant impact on the results, as shown by the example of publications on environmental protection: the intersection of the results obtained in the co-citation analysis and the method of bibliographic coupling was only 33–41%, which in fact indicated different research fronts [ 30 ]. Comparison of methods of co-citation analysis and bibliographing coupling was carried out by M. Huang et al., who studies the methodological foundations of the search for scientific fronts [ 32 – 34 ]. In a series of publications, the advantages of the bibliographic coupling were shown: with its use, a greater number of fronts were identified, and several fronts were found at an earlier date [ 34 ]. The advantages of bibliographic coupling were disclosed in [ 39 ], although it was indicated that in certain narrow areas the method of direct citation analysis may be preferable, since significant publications may have few thematic links in their field but gain a large number of citations from related fields. A comparison of direct citation analysis, co-citation analysis, and bibliographic coupling was carried out in [ 61 ] using the example of research fronts in the field of carbon nanotubes, gallium nitride, and complex network: the direct citation method showed the best results for identifying the early stages of the formation of new topics and contributed to the identification of a larger number research fronts. The next most effective methods were the method of bibliographic coupling and co-citation analysis. Another example of comparing all three methods of citation analysis is the study of scientific fronts in biomedicine, where they were additionally compared with textual analysis [ 28 ]. To test the best approach, information on grants was analyzed: since publications on a grant are thematically similar by default, a search was made for the highest concentration of publications on specific grants in each of the clusters. Weighted Approaches to Improve the Accuracy in Identifying Research Fronts Over time, increasingly sophisticated approaches to defining research fronts are being developed, with the goal of increasing the accuracy of clustering. One of the trends in this field is the construction of weighted citation networks. With the assignment of weight to the publications of the cluster forming scientific fronts, a series of studies was carried out by K. Fujita et al., proving the benefits of weighted citation networks [ 39 , 40 , 53 ]. The weight of the publication, automatically determined using neural network training technologies, takes the year of publication, the number of citations of the publication, the field of knowledge, and the strength of the links between the reference list of publications and keywords into account [ 39 , 53 ]. A significant advantage of the research of this group is that various bibliometric methods are widely combined here. The analysis of collective dynamics of knowledge networks represented by weighted citation and keyword networks, which takes both incoming and outgoing connections between network elements into account, was presented in [ 4 ], which shows the advantages of this method over the analysis of direct citation networks, since it more closely approaches identifying research trends in small areas of knowledge. For more accurate clustering, the PageRank algorithm is used to assign different weights to publications of different significance levels: not only are the most cited publications recognized as the most significant in a cluster, but also publications cited by other equally significant publications of the cluster [ 35 ]. An analysis of links that establishes the relationship between the cited publications, taking their importance and position in the citation network into account, was carried out to search for research fronts in the field of shareholder activism: during the analyzed period, the development of this field was reconstructed by means of research fronts, including seven stages, from the theoretical origin of the concept to its practical implementation [ 62 ]. A weighted approach was used in the search for scientific fronts in chemical technology: 29 clusters were identified containing an average of 5.3 publications; for each cluster, the Price index was calculated, which quantitatively characterizes the degree of novelty of the field [ 38 , 63 ]. Together with the fundamental applicability of each of the approaches in identifying research trends and fronts, the results of most studies show that the least-accurate results are obtained by the direct citation analysis, although in certain situations it shows advantages over other approaches [ 39 , 61 ]. In the accuracy of its results the combination of the co-citation analysis and the bibliographic coupling is significantly superior to direct citation analysis, which does not take thematic links between publications into account [ 34 , 39 ]. The most accurate results in most cases are yielded by the method of bibliographic coupling; co-citation analysis lags slightly behind. The best results are achieved with the combined use of different approaches (and, if possible, different data sets), which should take the variability of publication activity and citation models in different disciplines into account, but such approaches are more laborious and time consuming [ 28 ]. Many researchers, for example [ 1 , 2 , 64 ], noted the importance of involving subject experts in the qualitative assessment of the results of scientometric analysis. Software for Revealing Research Fronts Significant attention is paid to the study of research fronts by software developers for visualization and mapping of science [ 65 , 66 ]. The visualization of bibliographic information is especially valuable for experts because it allows real-time detection of unexpected trends, gaps in scientific knowledge, statistical biases, and other important characteristics of research fronts [ 67 ]. VOSviewer [ 22 , 41 , 57 , 68 , 69 ] and CiteSpace [ 2 , 13 , 26 , 42 , 60 ] are most often used; however, ready-made programs are often seen as having limitations, since their functionality is standardized and often does not support innovative approaches [ 35 ]. Therefore, sometimes less common software products are used, for example, Cytoscape [ 15 ] or BibTechMon [ 37 ], including programs written for a specific study [ 12 ]. One of the most functional software for identifying research fronts is CiteSpace [ 2 ]. The capabilities of the program are presented by its developer using examples of the fields of "mass extinction" and "terrorism." Research fronts are understood as emerging transitional clusters of ideas, expressed by small groups (several dozen positions) of co-cited publications. At the same time, the study solved the problem of identifying new fields on the basis of linguistic analysis of terms from the metadata of publications (although some researchers insist on involving experts in the designation of new fields [ 12 , 23 ]). Experience in using VOSviewer was presented by the scientific library of Kent State University (United States): the methods of bibliographic coupling, citation analysis and content analysis were used to identify research fronts in the field of the Internet of things [ 69 ]. Dynamic keyword analysis in VOSviewer allowed them show changes in research fronts in this area over time. The Problem of the Reliability of the Results of Scientometric Analysis in Identifying Research Fronts Since the definition of research fronts is based on an array of scientific publications, the question of the legitimacy of such an approach often arises. In addition to the general criticism of bibliometric approaches, there are somewhat fair statements about the devaluation of the institute of scientific publications associated with an increase in the number of duplicate works, plagiarism, and "predatory" journals, as well as the frequent absence of descriptions of research methods in publications, which prevents their reproducibility. Another critisism concerns the role of publications in rewarding a scientist for his/her work instead of spread of knowledge and a shift of the central channels of scientific communication towards "invisible colleges". Taken together, this leads to the main question of how much one can rely on bibliometric research of publications to identify research trends and fronts. Other problems of identifying research fronts are associated with journal articles and, more broadly, with the market for periodicals and its internal standards. As an example, reputable international journals are more willing to publish research results on popular and global topics. Accordingly, in such a limited array of publications, research fronts that are important at the regional or national levels may not be found. The cautious attitude of reviewers and editorial boards to advanced ideas and developments, often at odds with the scientific tradition, remains an unresolved issue [ 70 ]. Modern publishing standards often imply a comprehensive coverage of a scientific problem and a description of a ready-made set of its solutions [ 71 ]. However, precisely in relation to research fronts, at the initial stages of developing a new problem, these requirements are the least feasible and force authors to bypass key issues, whose discussion is most important for understanding the essence of the problem and its causal mechanisms [ 64 , 71 ]. At times, the overestimated requirements of the editors of journals for breakthrough work lead to the rejection of publications that are significant for science and society. One illustrative example is the article by A.K. Geim and K.S. Novoselov on a new material, graphene, that was rejected by Nature 1 (it was later published by Science ). Another problem of using journal publications as a basis for searching scientific fronts includes the time lag from the submission of the manuscript to the editorial office to its publication. This adds to the subsequent delay in distributing the journal to libraries or indexing it in bibliographic databases. On average, the delay due to the technological publishing processes is estimated at 1 year [ 24 ]. Even if we compare this period with the total time from the birth of a scientific idea to its publication, which, for example, is 4 years in medicine [ 59 ], the publication delay appear to be significant. The databases for the selection of publications themselves have a significant impact on the identification of research fronts [ 27 ]. Most of research is based on publications indexed in Web of Science , and less often, Scopus . In addition to the delay in indexing, such systems have limitations in terms of regional and linguistic coverage of sources; the accuracy of bibliographic metadata is not always high [ 72 ]. Despite the annually expanding indexing of conference proceedings, where advanced scientific ideas are discussed much earlier than in print, international databases still tend to predominantly cover journal articles. The need for verification of automatically processed data was already noted in early works, caused by many discrepancies in the spelling of author's names, variations in the abbreviation of the names of journals, etc. [ 31 ]. (For more detail on the problems of identifying bibliographic objects, see [ 73 , 74 ].) Some questions remain open, others are eventually answered. Thus, in recent years, reviewers have paid more attention to the transparency of the methodological part of the articles; more and more often initial data are provided in the form of appendices to publications, which significantly increase the reliability and reproducibility of the results. Ethics committees are working to improve the research and publication culture of authors, preventing unfair approaches to the publication of scientific results [ 75 ]. At the philosophical level, the role of publications in the system of scientific information and the degree of their applicability to identifying research fronts are analyzed. The transformation of the main properties of a research front into the form of bibliometric indicators has been substantiated, including such front characteristics as novelty, relevance, interdisciplinarity, risk factors, and a combination of fundamental and applied significance [ 64 ]. The central place of publications in scientific research fronts is proved, since in addition to the main function of information delivery, they stabilize unstable networks of various scientific practices and elements [ 76 ]. The role of scientific publications is also demonstrated in the reconstruction of the evolutionary development of science: based on the example of research fronts in scientometrics and the historical processes of the intellectual organization of knowledge in this area, their codification and structuring with a simultaneous decrease in entropy have been shown [ 77 ]. Based on the example of one area of biomedical sciences, the methodology for constructing a time scale, which allows one to visualize the development of a research front and predict the emergence of new fronts, was presented [ 12 ]. On the basis of the theory of the aging of scientific literature, the speed of dissemination of scientific ideas is investigated and the depth of research fronts was revealed [ 24 ]. The problem of publishing breakthrough articles, whose material, methodology and results differ significantly from the scientific tradition, finds its solution in the widespread dissemination of open science, the publication of preprints, the development of repositories and models of open peer review. Publication of preprints solves the lag problem. This issue is partially resolved by the development of the system of "articles in print" that are published before the formation of printed issues, as well as early indexing of such publications in bibliographic databases. One possible solution to the problem of publication lag may include the analysis of network publications, whose rate of appearance is significantly higher, as shown by the example of the search for scientific fronts in the field of XML research [ 78 ]. In this case, unlike journal databases, special systems are used, for example, CiteSeer . It is proposed to solve the problem of publication delay of journal articles by analyzing information about the dates of the publication process (the time of receipt of the manuscript, its approval, and publication); clustering of publications taking these dates into account gives more accurate results in identifying research fronts [ 59 ]. CONCLUSIONS Over a relatively short period of studying research trends and fronts, a significant complication of the methodology is noticeable: combined approaches, neural networks, a wide range of bibliographic and network databases, and special software is increasingly used. Scientometric methods show their promise due to their rapid adaptation to the changing conditions of the functioning of science and new publication models for the dissemination of scientific information. The review of research carried out in this article shows that scientometric tools for identifying research fronts have proven themselves well as a source of reliable and objective information for subsequent expert assessment in various fields of knowledge. A wide methodological arsenal of various types of citation analysis and content analysis has been developed. The improvement of the approaches goes in the direction of specifying citation windows, objects of analysis, and identifying the advantages and disadvantages of each of the approaches, taking the types of scientific fronts and research goals into account. We see the immediate tasks on identifying research fronts and trends as follows. The problem of the initial distrust of the scientific community in breakthrough developments, whose results or methods do not agree well with scientific tradition, awaits a solution. A scientometric solution to this problem is outlined in a broader analysis of network publications. The second task is to increase the speed of identifying new fronts, if possible at the stage of publishing preliminary data on new fields. This requires a further search for methods to neutralize the effect of publication lag.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4274239/

Combination of Two Candidate Subunit Vaccine Antigens Elicits Protective Immunity to Ricin and Anthrax Toxin in Mice

In an effort to develop combination vaccines for biodefense, we evaluated a ricin subunit antigen, RiVax, given in conjunction with an anthrax protective antigen, DNI. The combination led to high endpoint titer antibody response, neutralizing antibodies, and protective immunity against ricin and anthrax lethal toxin. This is a natural combination vaccine, since both antigens are recombinant subunit proteins that would be given to the same target population.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750716/

A coupled human-natural systems framework of community resilience

This article compares and contrasts resilience frameworks to identify commonalities and gaps. It proposes use of a coupled human-natural systems framework (CHNS) to analyze community resilience to disasters. CHNS builds on the human ecosystem model that analyzes how institutions and social order shape fluxes and flows of resources between and within social and environmental systems. It expands on the model by including anthropological concepts of culture, agency, power, and discourse. The framework covers environmental and social legacies, pre-disaster trends and conditions, resilience measures, and system changes provoked by a disaster. The article proposes eleven categories of variables that affect resilience and discusses research steps for putting the framework into action. The CHNS framework can be used to predict system changes and identify resilience measures that allow communities to articulate and achieve their resilience goals.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10046508/

Immunosensors for Assay of Toxic Biological Warfare Agents

An immunosensor for the assay of toxic biological warfare agents is a biosensor suitable for detecting hazardous substances such as aflatoxin, botulinum toxin, ricin, Shiga toxin, and others. The application of immunosensors is used in outdoor assays, point-of-care tests, as a spare method for more expensive devices, and even in the laboratory as a standard analytical method. Some immunosensors, such as automated flow-through analyzers or lateral flow tests, have been successfully commercialized as tools for toxins assay, but the research is ongoing. New devices are being developed, and the use of advanced materials and assay techniques make immunosensors highly competitive analytical devices in the field of toxic biological warfare agents assay. This review summarizes facts about current applications and new trends of immunosensors regarding recent papers in this area. 1. Introduction Testing hazardous toxic materials is an important task in current analytical chemistry. Accurate and timely proof of hazardous materials in the environment or an organism is necessary for choosing the correct countermeasures or therapy. Various instrumental devices are available for the purpose, and accurate and sensitive assays of hazardous toxic materials are possible. Mass spectrometry, chromatography, electrophoresis, and immunochemical methods, such as enzyme-linked immunosorbent assay, can be standard analytical chemistry for toxins [ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ]. Although standard methods are available and fully applicable, they have disadvantages, such as the price of the device, cost per assay, and demands on staff and other laboratory equipment. Alternative methods are being sought, to serve in situations where standard methods are unsuitable. Simple devices usable in the field, small mobile laboratories, or by a sole investigator in terrain, or devices for point-of-care tests, could provide identification of toxins in sites where other methods are not convenient. Biosensors, chemosensors, aptasensors, and similar portable and low-cost analytical devices are generally suitable for use outside of laboratories. The concept of biosensors and biosensor-like devices brings an alternative to standard methods because the application of new materials and measurement procedures makes them sensitive up to the level of these standard methods [ 11 , 12 ]. They maintain the concept of simple portable devices that can even be integrated as wearable electronics in the future. Toxins with military relevance represent a group of harmful substances with serious pathological impacts on the human organism. The test of such toxins with small portable devices is highly desired. It can protect endangered persons, help choose proper therapy, and diagnose the true causative agent of poisoning. Biosensors with bound antibodies, immunosensors, are reviewed here. Recent discoveries are introduced, and the significance of immunosensors is discussed. 2. Toxins as a Part of Biological Warfare Agents Nuclear, radiological, chemical, and biological weapons of mass destruction exist. They are a threat when used by a state in war, by an organization, or by an individual perpetrator in a terrorist attack [ 13 , 14 ]. All types of mass destruction weapons are regulated by international treaties, and there is an effort to ban or at least restrict their possession. The Treaty on the Non-Proliferation of Nuclear Weapons from 1968, effective as of 1970, is the main international regulation for the first group of weapons of mass destruction. Most countries declared for abandoning nuclear weapons, except for Great Power states. Chemical and biological warfare agents are partially regulated worldwide per the so-called Geneva protocols of 1925. The Protocol for the Prohibition of the Use in War of Asphyxiating, Poisonous or other Gases, and of Bacteriological Methods of Warfare , however, was minimally effective. It did not force the signatories to stop arming themselves with these weapons; therefore, further treaties followed in the next decades. The Convention on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on their Destruction is the treaty regulating biological warfare. It was signed by most countries in the world in 1972 and entered into force in 1975. Chemical warfare agents have become fully banned internationally, the last of the mass destruction weapons. The Convention on the Prohibition of the Development, Production, Stockpiling and Use of Chemical Weapons and on their Destruction was signed in 1993 and entered into force in 1997. Despite extensive regulation of mass destruction weapon manufacturing, stockpiling, and use, their relevance and threat are still significant. The proliferation of such means of combat or terror can occur under certain circumstances, and active countermeasures still exist to protect against such threats [ 15 , 16 , 17 , 18 , 19 ]. Toxins are poisons of natural origin. They can be simple organic compounds and/or highly structurally arranged macromolecules. Anatoxin-a, with a molecular mass of 165 Da, and botulinum toxin, with a molecular mass of 150 kDa, can be mentioned as two toxic biological warfare agents of completely different sizes. Several toxins are considered biological warfare agents, and often the toxin itself and the producing microorganism are seen as a biological threat [ 20 ]. Functional subunits derived from the sizable toxins also have the status of biological warfare agents. Many toxic substances could be considered biological warfare agents; however, only a limited number have this status in practice. For instance, The Australia Group (Australia Group Secretariat, RG Casey Building, John McEwen Crescent, Barton Act 0221) coordinating 42 countries, plus the European Union, has a list of human and animal pathogens and toxins for export control. A total of 18 structurally close groups of toxins are on the list. Abrin (a protein toxalbumin from the plant Abrus pulchellus ) [ 21 ], aflatoxins (a low molecular weight mycotoxins from molds Aspergillus species) [ 22 ], botulinum toxins (all variants, protein toxins from the bacterium Clostridium botulinum ) [ 23 ], cholera toxin (a protein toxin from the bacterium Vibrio cholerae ) [ 24 ], Clostridium perfringens toxins (protein α, β1, β2, ε, ι toxins from bacterium Clostridium perfringens ) [ 25 ], conotoxins (a group of toxic peptides from marine cone snail, genus Conus ) [ 26 ], diacetoxyscirpenol (a low molecular weight mycotoxin from a group of trichothecenes and produced by the Fusarium fungi) [ 27 ], HT-2 toxin (a trichothecene mycotoxin produced by various fungi mainly of Fusarium species) [ 28 ], microcystins (cyanotoxins, a group of organic compounds produced by cyanobacteria) [ 29 ], modeccin (a glycoprotein from plant Adenia digitata ), ricin (a carbohydrate binding protein from plant Ricinus communis ) [ 30 ], saxitoxin (a cyanotoxin from various cyanobacteria, organic compound) [ 31 ], Shiga toxins (including Shiga-like toxins, verotoxins and verocytotoxins, a group of protein toxins from Shigella dysenteriae and some serotypes of Escherichia coli ) [ 32 ], Staphylococcus aureus enterotoxins (including hemolysin α-toxin and toxic shock syndrome toxin, a group of protein toxins from bacterium Staphylococcus aureus ), T-2 toxin (a trichothecene mycotoxin produced by various fungi mainly of Fusarium species) [ 33 ], tetrodotoxin (a neurotoxin organic substance produced by bacteria like Pseudoalteromonas , Pseudomonas , and Vibrio , it can be transmitted to other water organisms) [ 34 ], viscumin (viscumin albumin lectin 1, toxic lectins from mistletoe plant Viscum album ) [ 35 ], and volkensin (a toxic glycoprotein from Adenia volkensii plant) [ 36 ] are regulated substances according to the Australia Group. The mentioned toxins are given in Table 1 . The Center for Disease Control and Prevention (1600 Clifton Road, Atlanta, GA 30329-4027 USA) distinguishes three basic types of biological warfare agents labeled A, B, and C [ 37 , 38 ]. Group A contains the most dangerous biological warfare agents. Groups B and C are less important as the agents are less dangerous. Serious pathogens, such as Bacillus anthracis , Francisella tularensis , Yersinia pestis, and Variola major belong to group A. Clostridium botulinum toxin (Botulinum toxin) also belongs to the upper-priority group A as a representative of toxic substances. 3. Biosensors for the Toxic Biological Warfare Agents Assay Biosensors are analytical devices that combine a physicochemical transducer and a biorecognition element. While the physicochemical transducers work as a physical sensor, the biorecognition element is responsible for specificity, but it can also initiate chemical or physical processes detectable by the physico-chemical transducer. Biosensor analytical devices have progressed from simple detectors containing crude enzymes, such as glucose oxidase, to complex systems where purposely prepared biological origin molecules, nanomaterials, and other advanced techniques are used [ 39 , 40 , 41 , 42 ]. Immunosensors are a variant of a biosensor where an antibody plays the role of a biorecognition element, and an antigen is an analyte [ 43 , 44 , 45 , 46 , 47 ]. Conception in which an immunosensor containing an antibody is detected by an antigen is possible as well [ 48 ]. Toxins assay by an immunosensor can work on a direct interaction between immobilized antibodies specific to the toxin and the toxin itself presented in the sample. More complicated assay formats also exist, and sandwich immunocomplexes, competitive immunoassays, formation of complexes with nanoparticles, and other arrangements are known, as described in the chapter devoted to the specific examples. A general principle of an immunosensor for toxin assay is shown in Figure 1 . Biological warfare agents, including toxins, can be analyzed by a wide number of biosensors, as seen in the examples in the following text. The use of antibodies as a biorecognition element is quite common for a biological warfare agent assay. An electrochemical paper immunosensor for a B. anthracis assay is an example [ 49 ]. Antibodies react with a target molecule, called an antigen, and specifically recognize a site on the antigen called a paratope. The use of antibodies in various analyses has a long tradition, and specific antibodies can be attained in the market. On the other hand, antibodies are sizable molecules, and their production requires the use of animals (polyclonal antibodies) or biotechnology (monoclonal and recombinant antibodies). This means that the production of antibodies is not easily reproducible and can also require a high initial investment in material and work. Aptamers are another recognition element representing an artificial molecule based on polynucleotides, polydeoxynucleotides, or peptide-binding [ 50 , 51 ]. The use of aptamers for analyses became quite common, and the term aptasensor can be found in the current literature. Using an aptasensor for biological warfare agents is possible, and the application of bacillus anthracis is an example [ 52 , 53 ]. Aptamers exert affinity to the target molecule as antibodies do. Because aptamers are artificial biomolecules, they can be produced by typical chemical technologies, and thus the product can be more attractive to some manufacturers. On the other hand, aptamers can have problems with specificity and affinity concerning the target structures, though their production technology is proven, and some aptamers have good specifications. Molecularly imprinted polymers are another affinity material manufactured via chemical processes. Molecularly imprinted polymers can serve in the same way as a biorecognition element and gain specificity to the sensor device [ 54 , 55 , 56 ]. Molecularly imprinted polymers could be mass-produced by the chemical industry, and any structure can be imprinted in theory. There are, however, some shortcomings that should be taken into account. The specificity of the imprints can be limited. The affinity of the surface to the target molecule is based on the shape and molecular interactions, which are not guaranteed when a homogenous membrane is used, and testing small molecules with defined physical and chemical specifications is an easier task for sensors with molecularly imprinted polymers. Experiences with molecularly imprinted polymers for the preparation of sensors include the Helicobacter pylori virulence factor assay [ 57 ], specific extraction of aflatoxins by molecularly imprinted polymers [ 58 ], and the human immunodeficiency virus drug assay by Tenofovir [ 59 ]. The use of molecularly imprinted polymer will gain more applications when new materials are developed as a platform for in situ membrane manufacturing. Biological warfare agents can be analyzed by recognizing specific genes or sequences of their genetic information and using genetic probes, specific sequences of genetic information, etc. These devices proved their functionality in more applications, such as the Ebola virus assay [ 60 , 61 ], F. tularensis [ 62 ], F. tularensis , Y. pestis , B. anthracis , variola virus, Rift Valley fever virus Ebola virus, Sudan virus, and Marburg virus [ 63 ], variola major [ 64 ], B. anthracis [ 65 , 66 ], and Shiga toxin-producing E. coli [ 67 ]. Identifying biological warfare agents by detecting their genetic sequences is typically a sensitive and selective approach. These assays also have some disadvantages. First, genetic information is enclosed within cells or viral particles, and only rarely can the genetic information be attained directly. This may cause complications and will probably make it necessary to pretreat samples. Another disadvantage is that toxins cannot be assayed directly by genetic tests. Only a microorganism that produces the toxins can be analyzed. Biosensors, including immunosensors, are a group of portable analytical devices. Some highly complex biosensors are not suitable for use outside of laboratories, but most of the newly developed biosensors are miniaturized instruments suitable for field tests. The immunosensor for toxic biological warfare agents plays a role in fast identification in order to choose proper countermeasures. The immunosensor should be a single step or based on a limited number of steps, with a minimal requirement of sample pretreatment and personnel operating the device. It is not expected that the immunosensor will replace standard laboratory analytical methods, such as chromatography or mass spectrometry. The standard methods should verify results from the immunosensor when the situation allows. The role of an immunosensor in the toxic biological warfare agent assay can be compared with point-of-care tests based on lateral flow immunochromatography that were found to be useful during the Coronavirus 2019 pandmic, and that were taken as a less expensive, accessible, but less accurate diagnostic alternative to the standard polymerase chain reaction assays [ 68 , 69 , 70 , 71 , 72 , 73 ]. Biosensors also have shortcomings that should be considered when a new analytical device containing a biosensor is constructed. Compared to the universal standard analytical devices, biosensors are suitable for assaying a specific analyte or a group of defined analytes. The specificity depends on the type of biorecognition element or manufactured molecule. The recognition antibody has to be replaced in the case of an immunosensor for a toxic biological warfare agents assay. This replacement is quite elaborate and cannot be done by a user. Therefore, immunosensors are not universal devices but analytical tools for specific tasks. 4. Commercial Immunosensors for Toxic Biological Warfare Agents The research on immunosensors for toxic biological warfare agents is ongoing, and many interesting applications have already been commercialized. The already commercialized devices are outcomes of older research, and they have an actual use for safety purposes. On the other hand, the actual research outcomes are not involved in their construction. Both expensive automatically working analytical devices and cheap disposable detectors can be mentioned as successful adaptations of an immunosensor for the assay of biological warfare agents, including toxins. The analyzer Raptor by Research International (Monroe, WA, USA) is an automatic, portable fluorometric assay system for monitoring up to four toxins, viruses, bacteria, spores, fungi, and other diverse targets, and it can be designated an immunosensor. It is a battery-powered portable device of 28.0 × 17.3 × 20.5 cm and 6.45 kg and is suitable for indoor and outdoor applications. It works on the principle of fluorescence immunoassay, which takes place in four independent channels, meaning that up to four biological warfare agents can be analyzed simultaneously. One assay takes 15 min to complete. All steps are automated, and flow forced by a peristaltic pump is responsible for the delivery of samples and the solutions of monoclonal antibodies with bound fluorophore labels to a chamber where another antibody has already been immobilized. Optical fibers excite the fluorophore, and an optical waveguide detects fluorescence when an immunocomplex with the analyte is formed in the flow-through cell. The principle of the Raptor function is depicted in Figure 2 . The Raptor device can analyze a wide group of biological warfare agents. The exact type of agent depends on the regencies used. Toxic biological warfare agents can be proven with quite low detection limits: up to 0.1 ng/mL for staphylococcal enterotoxin B, 5 ng/mL for ricin, and up to 1 ng/mL for botulinum toxin [ 74 , 75 , 76 , 77 , 78 , 79 , 80 ]. Fluorescence measurement also uses another immunosensor for a biological warfare agents assay: Biosensor 220R by MSA (Pittsburgh, PA, USA). This immunosensor works automatically and uses magnetic microspheres with specific antibodies and a fluorescent tag with specific antibodies [ 81 ]. A complex is formed when an analyte is presented in a sample, the complex is held in a flow through a magnetic cell and washed, and its fluorescence is measured. The manufacturer does not disclose detailed information about the magnetic particles and antibodies. The whole device is suitable for indoor and outdoor performance. It is battery-powered, 27 × 25 × 14 cm in size, and weighs 2.7 kg. The manufacturer claims a sensitivity for ricin and staphylococcal enterotoxin B < 1 ng for an assay lasting 5 min. Lateral flow tests, also known as lateral flow immunoassays, are an analytical tool for the semiquantitative analysis of various chemicals, drugs, semiquantitative substances, biochemical and immunochemical markers, and microorganisms [ 82 , 83 , 84 , 85 , 86 , 87 , 88 ]. Toxic biological warfare agents can also be analyzed by lateral flow tests, and some manufacturers offer these immunoassay devices specific to toxins of security interest. Manufacturer Advnt Biotechnologies (Phoenix, AZ, USA) produces lateral flow tests for various biological warfare agents. There are tests for a single agent or for up to five agents analyzed in one assay. The tests for a single agent are named BADD (Biowarfare Agent Detection Devices); the tests for five simultaneous agents are called the Pro Strips Rapid Screening System. Other analytical specifications are the same for both tests. The detection limit for ricin and staphylococcal enterotoxin B is 10 ng/mL, the botulinum toxin variant A has a detection limit of 33 ng/mL, and the botulinum toxin variant B has a detection limit of 500 ng/mL for an assay that requires a sample size of 0.2 mL and a time of 3 min. The practical use of these strips was described in the papers cited for the assay of ricin [ 89 ] and the A variant of botulinum toxin [ 90 ]. Alexeter Technologies manufactures (Wheeling, IL, USA) similar lateral flow tests under the trade name BioDetect (test of a single biological warfare agent), RAID 5 (up to five contemporary assayed biological warfare agents), RAID 8 (up to eight contemporary assayed biological warfare agents), and RAID 10 (up to 10 contemporary assayed biological warfare agents). Ricin, staphylococcal enterotoxin B, and botulinum toxin are covered by these tests, but the manufacturer does not offer an assay for other toxic biological warfare agents. The assay takes 15 min to complete, though other analytical specifications are not disclosed by the manufacturer. Practical testing for ricin was described by Slotved et al. [ 89 ]. Lateral flow tests are also produced by other manufacturers. ANP Technologies (Newark, DE, USA) produce lateral flow tests for biological warfare agents and infectious microorganisms. Botulinum toxin A, ricin, and staphylococcal enterotoxin B tests are offered as tools for toxin assay. The manufacturer provides the tests for single target and multiplex assays suitable for the contemporary detection of two, four, five, and ten biological warfare agents. An example of a multiplexed lateral flow test is depicted in Figure 3 . The assay by lateral flow test can be further improved by using a digital reader to measure the coloration of lines, and automatizes manipulation with samples. The device BioHawk LF by Research International is an example. It can even collect samples from aerosols via an external wetted wall cyclone, and perform automated detection of biological warfare agents and their identification in a total elapsed time of 10–25 min. The device is suitable for outdoor use, and its small size of 47.0 × 24.8 × 36.5 cm with a weight of 13 kg makes it single-person portable. The commercially available immunosensors for the assay of toxic biological warfare agents are summarized in Table 2 . 5. Progress on Immunosensors for Toxic Biological Warfare Agents Assay Research on a new immunosensor for the assay of toxic biological warfare agents brings improvements, making devices more competitive to standard methods. New materials typically improve expected specifications, such as decreasing limits of detection and sample volume on one side and making the assay simple on the other. Miniaturization additionally leads to savings on raw materials and production costs. An immunosensor that works on the principle of the Raman scattering-lateral flow immunoassay was developed by Jia et al. [ 91 ]. Composite gold—silicon oxide nanoparticles were chosen for the assay as fluorescent labels. Variants of the immunosensor for the ricin, botulinum toxin, and staphylococcal enterotoxin B assay were developed. The toxins were analyzed with a detection limit of 0.1 ng/mL for ricin and botulinum toxin A, of 0.05 ng/mL for staphylococcal enterotoxin B, and the time per single measurement was 15 min. A voltametric immunosensor was developed to detect vacuolating cytotoxin A from Helicobacter pylori [ 92 ]. Although this toxin is not listed among biological warfare agents, the assay provides promising results and can be easily adapted for other bacterial toxins. The authors prepared a graphitic carbon nitride/zinc oxide nanocomposite electrochemically deposited on gold electrodes, further immobilized antibodies via carbodiimide and N-hydroxysuccinimide, and vacuolating cytotoxin A was detected by voltammetry. The detection limit for the assay was equal to 0.1 ng/mL for vacuolating cytotoxin A with a linear range of calibration between 0.1 and 12.8 ng/mL and a time per test of 10–15 min. An electrochemiluminescence immunosensor for a ricin assay was developed on a platform of screen-printed electrodes [ 93 ]. The immunosensor contained magnetic beads with antibodies specific for ricin immobilized through streptavidin-biotin. A sandwich was formed in the presence of ricin with CdSe/ZnS quantum dots, the immunocomplex formed on the magnetic beads was magnetically separated, and electrochemiluminescence was measured. The immunosensor had a detection limit of 5.5 pg/mL and a linear assay range of 0.01–100 ng/mL. Magnetic beads were also used in the work by Atanasova and colleagues concerning the detection of aflatoxin M1 [ 94 ]. The magnetic nanoparticle-based fluorescent immunoassay provided a limit of detection for aflatoxin M1 2.9 pg/mL and a linear calibration range of 3.0 to 100 pg/mL. An immunosensor for aflatoxins was also developed in the work of Peltomaa et al. [ 95 ]. They developed a non-competitive immunoassay in which a primary anti-aflatoxin antibody was bound via streptavidin to magnetic beads, and an immunocomplex was formed in the presence of aflatoxin B1 with a secondary Eu-labeled antibody. Fluorescence was measured after the magnetic separation. The assay had a detection limit of 70 pg/mL for an assay lasting 15 min. Botulinum toxin A was measured by an immunosensor, in which specific antibodies were attached to gold nanoparticles, a sandwich immunocomplex was formed with botulinum toxin and antibodies on fluorescent probe particles, and diffusivity was measured [ 96 ]. The assay had a detection limit of 10 pg/mL for a measurement time of 2 min, and botulinum toxin A was measured in a calibration range of 0.01–500 ng/mL. In another work, the simultaneous detection of botulinum toxins A and E was performed by a voltametric assay [ 97 ]. The immunosensor comprised magnetic core/metal-organic framework nanoparticles covered with antibodies specific to botulinum toxins and monoclonal antibodies labeled with polystyrene@polydopamine/cadmium and silver. The assay had a dynamic range of 0.1–1000 pg/mL and a limit of detection of 0.04 pg/mL for botulinum toxin A, and a dynamic range of 0.5–1000 pg/mL and a limit of detection of 0.16 pg/mL for botulinum toxin E. The botulinum toxin assay was also developed in the work of Kumar et al. [ 98 ]. They chose the toxoid form of botulinum toxin types C and D for their analysis, and the porous silicon Fabry-Perot interferometer as a platform for a competitive immunoassay. It was covered with a gelatin membrane and botulinum toxoid. Primary antibodies specific for toxoid and secondary antibodies labeled with horse radish peroxidase were used, and peroxidase-catalyzed oxidation of 4-chloro-1-naphthol using hydrogen peroxide created insoluble products. The botulinum toxin in a sample was completed with the immobilized toxoid for the antibodies applied. Reflectivity spectra were collected, and calibration was performed. The assay had a linear response of 10 pg/mL to 10 ng/mL and a limit of detection of 4.8 pg/mL for an assay occurring in nearly real-time. The immunosensor for toxins can also use complex and more expensive platforms to achieve outstanding specifications. Shiga toxins were, for instance, analyzed with surface plasmon resonance imaging [ 99 ]. This immunosensor contained immobilized immunoglobulin G on 50 nm gold film and proved Shiga toxoid Stx1 in a label-free mode with a detection limit of 50 ng/mL in an assay lasting 20 min. The signal can be further improved by applying gold nanoparticles covered with anti-Shiga toxin antibodies. The sensitivity of the assay improves when the immunosandwich forms, and the limit of detection is around 1 pg/mL. Surface plasmon resonance was used to detect ricin and abrin in another article [ 100 ]. A sandwich immunocomplex comprised of a protein G, a magnetic bead with an antibody, analyte, and secondary antibody, was formed and placed at the site of the proper sensor chip. The assay contained a magnetic separation step that enriched the analyte and improved sensitivity. The limit of detection for abrin and ricin assay was equal to 0.6 ng/mL. Immunocomplex formation on the surface plasmon resonance chip was also used in work by Stern et al. [ 101 ]. The authors co-immobilized antibodies against ricin, and agglutinins were assayed in the first step. Adding an antibody specific for ricin formed a sandwich immunocomplex, and the level of ricin could be differentiated from the level of agglutinin. The detection limit was equal to 3 ng/mL for ricin and 6 ng/mL for agglutinin in an assay providing the assay results in real-time. The total analysis time, including sample processing, was less than 30 min. The newly developed immunosensors for the toxic biological warfare agent assay are summarized in Table 3 . Introducing new immunosensors into practice is not an easy task. It requires not only assembling the particular parts but also using original nanomaterials and antibodies, and their production is a condition for getting an immunosensor into the market. Generally, producing biosensors and immunosensors has a great practical perspective, and their use by various consumers is expected [ 102 , 103 ]. Immunosensors for toxic biological warfare agents assays are devices designed for the military, police, or other organizations. The introduction of immunosensors to these consumers will highly depend on governmental support or acquisitions. The fact that one immunosensor typically detects only one type of toxic biological warfare agent is a disadvantage. Militaries tend to require a single analyzer for a wide number of analytes, and that the analyses are performed by trained staff for whom education in analytical chemistry, bioanalytical chemistry, or similar disciplines is necessary. There can also be problems with the manufacturing processes in which new materials are used, and shortcomings in quality or reproducibility can occur. The limitations mentioned here should be considered when the introduction of an immunosensor is planned. On the other hand, the benefits of small, portable, and cheap analytical devices for security practices are undeniable. The practical spread of the immunosensor for toxic biological warfare agents will depend on the verification of their potential by military specialists. If the first of the new types of immunosensors are at least partially successful, further propagation of them for toxic biological warfare agents can be expected. 6. Conclusions Toxins represent a substantial risk to human health; they can be present in the environment, food, and drugs or accompany infectious diseases. They are also a threat that can be misused for military or terrorist activities. Early detection is a necessity for helping to decide what countermeasures or therapies should be chosen. Although current analytical techniques are accurate and reliable, early test detection for outdoor measurement or point-of-care diagnosis is extremely helpful. Immunosensors can provide a highly sensitive assay and the possibility to perform testing outside of standard laboratories. Recent discoveries and the implementation of new materials make immunosensors highly sensitive and capable of detecting toxins in very low concentrations. At the same time, these devices are typically inexpensive, small, are readily integrated into portable or even wearable electronics, and perform point-of-care tests. The currently commercialized immunosensors are fully applicable. The newly developed ones will further improve possibilities for toxin assay.

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808437/

Lipid larceny: channelizing host lipids for establishing successful pathogenesis by bacteria

ABSTRACT Lipids are complex organic compounds made up of carbon, oxygen, and hydrogen. These play a diverse and intricate role in cellular processes like membrane trafficking, protein sorting, signal transduction, and bacterial infections. Both Gram-positive bacteria ( Staphylococcus sp., Listeria monocytogenes, etc .) and Gram-negative bacteria ( Chlamydia sp., Salmonella sp., E. coli , etc.) can hijack the various host-lipids and utilize them structurally as well as functionally to mount a successful infection. The pathogens can deploy with various arsenals to exploit host membrane lipids and lipid-associated receptors as an attachment for toxins' landing or facilitate their entry into the host cellular niche. Bacterial species like Mycobacterium sp. can also modulate the host lipid metabolism to fetch its carbon source from the host. The sequential conversion of host membrane lipids into arachidonic acid and prostaglandin E2 due to increased activity of cPLA-2 and COX-2 upon bacterial infection creates immunosuppressive conditions and facilitates the intracellular growth and proliferation of bacteria. However, lipids' more debatable role is that they can also be a blessing in disguise. Certain host-lipids, especially sphingolipids, have been shown to play a crucial antibacterial role and help the host in combating the infections. This review shed light on the detailed role of host lipids in bacterial infections and the current understanding of the lipid in therapeutics. We have also discussed potential prospects and the need of the hour to help us cope in this race against deadly pathogens and their rapidly evolving stealthy virulence strategies. Introduction Lipids are a class of complex organic compounds usually made up of carbon, oxygen, and hydrogen (nitrogen and phosphorous are present as well). These are soluble in an organic solvent, but they are either insoluble or partially soluble in water due to their hydrophobic or amphipathic nature. Lipids comprise fatty acids, glycerophospholipids, sphingolipids, glycolipids, prenol lipids, sterols, polyketides, and ether-linked glycerophospholipids. They have diverse cellular functions such as cell-membrane components, energy storage, signaling, and cellular trafficking. Moreover, it is well known that lipid homeostasis is lost during various cardiovascular and other metabolic diseases, indicating that they are crucial in maintaining a healthy state [ 1 ]. In correlation to host lipid interactions and hijacking, the pathogen's invasion into the host cells to maintain the intracellular niches has been well documented. The cell membrane harbors distinct lipid microdomains, composed of cholesterols and sphingolipids and membrane proteins known as Lipid Rafts. On the membrane, these rafts guide protein-protein and lipid-protein interactions, based on their properties to determine interacting proteins and the ability to knit together to generate a broader domain. Physiologically, they are reported to play a role in diverse cellular processes such as membrane trafficking, protein sorting, and signal transduction events. Thus, it also interplays with apoptosis, proliferation, adhesion, and migration [ 2 ]. A substantial amount of literature is available on lipid rafts' role in viral infection [ 3 , 4 ]. There has been an increase in reports relating to bacterial, fungal, and protozoan infections as well. Successful adhesion of bacteria to the host cell membrane leads to colonization of host tissue, followed by invasion, intracellular multiplication, and ultimately dissemination [ 5 , 6 ]. The adhesion and uptake of bacteria by the host cell membrane depends upon the structural modification of membrane lipids. Most of the gram-negative pathogenic bacteria synthesize and transport numerous virulent factors, effector proteins into the cytoplasm of the host cells by T3SS. These effectors change the host cell membrane's morphology by modulating actin filaments, etc [ 5 , 6 ]. The extracellular bacteria use cholesterols or GM1 receptors present on the eukaryotic membrane to bind and form pores on the host cell membrane. One of the notable examples includes AB toxins that use GM1 ganglioside receptors for attachment. Lipid microdomains also mediate bacterial entry into the host cells. Bacteria being a prokaryote, do not possess cellular tools to synthesize cholesterols and sphingolipids but have evolved to acquire the same from the eukaryotic host. These host lipids impart various roles in prokaryotic systems such as escaping host defense mechanisms, maintaining cell permeability, membrane fluidity, energy or nutrient sources, and signaling processes. Intracellular bacteria such as Chlamydiaceae sp., Salmonella sp., Shigella sp., Mycobacterium sp., Listeria sp ., and Enteropathogenic E. coli acquire host lipids to avoid the deleterious defense mechanism of the host and thus helps to maintain intracellular niches [ 7–13 ]. Host lipids are not only hijacked, but their metabolism is also altered during infection. One of the classic examples remains Mycobacterium spp ., which modulates host lipid metabolism and manages to use it as their sole carbon source during chronic infections [ 14 ]. Extracellular bacteria such as Mycoplasma sp., H. pylori , on the other hand, have also been reported to incorporate host lipids, primarily cholesterols, and convert them to glycol-cholesterol complex [ 15–17 ]. Lipids also have major functions as first and second messenger molecules in host defense signaling. Thus, bacteria usually modulate these to escape host immune signaling and thereby promoting their survival inside the host. One of the most important lipid mediators of the cell, PGE 2, elicits an immune response during chronic and acute infections [ 18 ]. It also promotes host protection against mitochondrial inner membrane disruption and limits bacterial spreading because of necrosis [ 19 ]. PGE 2 plays a role in the clearance of bacteria from the system by increasing the apoptosis of infected host cells and protecting against inner mitochondrial damage [ 20 ]. Another example of lipid in host protection remains the redistribution of glycerophospholipids in the membrane to mediate the Mycobacterium infected cells' efferocytosis and limit the growth [ 21 ]. Various host sphingolipids enhance bacteria's maturation containing phagosome into acidified lysosome upon its phosphorylation [ 22 ]. Active metabolites of vitamin D also help prevent bacteria's clearance by inducing paracrine signaling of macrophage and epithelial cells to enhance the release of antimicrobial peptides [ 22 ]. Hence, it can be concluded that host lipids are among the major targets of bacterial invasion among other host molecules. Macrophages, the professional phagocyte, and antigen-presenting cells are the host immune system's front-line defense and a link between innate and adaptive immunities. They are well known to be polarized either into inflammatory or anti-inflammatory macrophages. Increasingly, it is evident that lipids play an equitably important role in macrophages' function during polarization, thus influencing the outcome [ 23 ]. At different stages of infection, the inflammatory and anti-inflammatory responses are strictly regulated and pathogen-specific [ 24–26 ]. Perturbance of this equilibrium usually results in either excessive inflammation or failure to activate an immune response. Hence, various intracellular bacteria perturb this to thrive inside these otherwise bactericidal cells successfully. Lately, a major therapeutic strategy domain has been explored using lipids as a delivery agent. The amphipathic nature of lipids has been harnessed to encapsulate drugs inside the lipid layer called liposomes. These liposomes show better drug retention and excellent circulation of encapsulated drugs in the body. As a substitute for conventional lipid carriers like liposomes and emulsions, solid lipid nanoparticles (SLNs) have been developed recently. They have several advantages that confer them better stability [ 27 ]. Apart from SLN, there is another set of lipid carriers with a more structured framework called nanostructured lipid carriers (NLC), enhancing the drug's oral bioavailability [ 28 ]. Besides being an excellent carrier, lipids of bacterial origin and host origin (hijacked by bacteria) can be effective targets for antibiotics. So, therapeutic ventures have been made to tweak the in-vivo scenario, preventing pathogens [ 29 ]. One of the lipids playing inhibitory roles is sphingosine-1-phosphate, which has been extensively studied for potential therapeutic aspects [ 30 ]. There is also a marked difference between virus-infected individuals' lipid profile than that of bacteria-infected ones; these have been used as markers for quick probing and detection of the diseases and further analysis [ 31 ]. This review will be dissecting various roles of lipids, both during facultative and obligate intracellular bacterial infection. It is interesting to know that lipids exploitation by these two groups of bacteria is quite diverse; intracellular bacteria use host lipids to avoid phagosome maturation while extracellular bacteria use host lipids for their membranes' integrity. However, bacterial species' host lipid acquisition, especially cholesterol, is increasingly important in bacterial pathogenesis in recent literature. Bacterial and host lipids Lipids are the most common yet one of the differentiating biomolecules across all life domains; Eukarya, Archaea, and Bacteria. They are essentially and broadly two kinds: storage lipids (triglycerides-TAG) and membrane lipids (glycerophospholipids). Just a handful of bacteria can only synthesize storage lipids and, thus, are mostly limited to eukaryotes. On the contrary, it has been shown by several reports that those handfuls of bacteria such as Mycobacterium, Nocardia, Rhodococcus, Micromonospora, Dietzia , and Gordonia are often associated with harboring even neutral lipid bodies (LD), mainly comprising of TAG and wax esters (WE) [ 32 , 33 ]. On the other hand, glycerophospholipids or membrane lipids are part of every life domain [ 34 ]. However, there are striking differences between the membrane lipids of these three domains. In Eukarya and Bacteria, the fatty acyl side chains are linked to the sn -glycerol-3-phosphate backbone via an ester bond. In Archaea, there are ether linkages between isoprenoid hydrocarbon side chains and sn -glycerol-1-phosphate backbone [ 35 ]. In bacterial cell membranes, there are three major classes of phospholipids; phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipins (CL). Zwitterionic PE accounts for about 75% of the membrane lipids, anionic PG about 20%, and the rest 5% is CL [ 36 ]. However, these percentage contributions often vary, depending on which growth stage bacteria are in; for example, during the stationary phase in E. coli , the CL percentage is higher [ 37 ]. Apart from glycerophospholipids, various bacteria are capable of synthesizing diacylglycerols (DAG)-derived membrane lipids; these lack phosphorus in their structure, typical examples of these are glycolipids (GL), sulfolipids (sulfoquinovosyl diacylglycerol -SQDG), and diacyl-glyceryl trimethyl-homoserine (DGTS) lipids [ 38 ]. There are also membrane lipids which completely lack glycerol backbones, such as isoprenoids and hopanoids; these serve a specialized function in bacterial membranes and are a structural surrogate for sterols. Necessarily suggesting that bacteria have immense lipid diversity and all kinds of lipids exist in various bacterial species. It is widely known that apart from proteinaceous virulence factors, bacteria also harbor a set of lipids accounting for their virulence. These lipids are generally expressed on their surface, capable of modulating the host lipid metabolism and triggering an immune response. A major component of the Gram-negative bacterial cell wall is lipopolysaccharide (LPS), which comprises lipids A, core oligosaccharide region, and variable carbohydrate [ 39 ]. LPS has been extensively studied concerning orchestrating host lipid biosynthesis dynamics upon infection, discussed in detail later [ 40 ]. Also, it has been reported that LPS is often self-modulated by various pathogenic bacteria as a part of escaping strategies from the host immune system [ 41 ]. Apart from LPS, another bacterial lipid capable of host immune and metabolism modulating function is Trehalose-6,6ʹ- di-mycolate (TDM). TDM is expressed copiously on the cell wall of bacteria containing mycolic acid such as Mycobacterium, Nocardia, Corneybacterium species. TDM harbors trehalose as sugar esterified to two residues of mycolic acid, and the length of mycolic acid residue varies from 20–80, based on the bacterial species. It has been reported in several studies done in Mycobacterium that TMD is responsible for upregulating multiple cytokines (TNF-α, IL-1β, IL-1 m, IL-10, and MCSF-1) and chemokines (CCL3, CCL4, CCL7, CCL12, CCR12, CXCL1, CXCL2, and CXCL10). TMD also elicits the foamy cell formation and trigger activated foreign body and hypersensitivity-type granulomas in mice. There are vast literature and many detailed reviews available on the synthesis, structures, and functions of these lipids [ 37 , 38 , 42 ] Like bacterial lipids, host or eukaryotes also have lipids such as PE, PG, and CL, along with phosphatidylserine (PS). However, PG and CL are synthesized and confined to the mitochondrial membrane, primarily due to its prokaryotic origin. Apart from these, eukaryotic membranes also harbor phosphatidylcholine (PC) and phosphatidyl-inositol (PI) as glycerophospholipids. The cellular distribution of these membrane lipids is also based on individual lipid species' curvature and the given sub-cellular organelle requirement. Besides this, the endoplasmic reticulum (ER) serves as the main site for the synthesis of lipids, producing a lot of structural phospholipids and cholesterols, along with a decent amount of TAG and cholesteryl esters (having non-structural roles) [ 43 , 44 ]. ER also make ceramide (Cer), the precursor of sphingolipids, and GalCer is only produced in the epithelial and myelinating cell's ER. Apart from all of these lipids, ER also harbors various minor lipids playing a pivotal role in intermediates and end products of certain pathways (this includes: DAG, lysophospholipids, and dolichol) [ 45 ]. The other class of eukaryotic lipids, called sphingolipids, has an amino-alcohol backbone. Previously, these were only associated with membrane microdomains or rafts; however, more recent scientific development in the last decade shows that these are also involved in regulating several cellular functions such as autophagy and apoptosis. Sphingolipids are synthesized in the Golgi complex, producing sphingomyelin (SM) GlcCer and lactosylceramide (LacCer) and other higher-order glycerol-sphingolipids (GSL) [ 46 , 47 ]. The plasma membrane majorly comprises higher density glycerophospholipids, sphingolipids, sterols, and PI for various cell signaling cascades to sustain mechanical stress [ 48 ]. Besides this, there is a lot of dynamic fission and fusion of eukaryotic membranes in the endocytic pathways; those are mostly regulated by a dedicated system of phosphoinositide specific kinases and phosphatases. Essentially this manages the pool of phosphatidylinositol phosphates such as phosphatidylinositol 3-phosphate (PI3P), phosphatidylinositol 4-phosphate (PI4P), and phosphatidylinositol (4,5)- bisphosphate [PI(4,5)P2] on the target and vesicular membranes. Examples are PI4P in the Golgi, phosphatidylinositol 3-phosphate (PI3P) in endosomes, and [PI(4,5)P2] in plasma membranes [ 48 , 49 ]. Host lipids and bacterial toxins Toxins is a soluble protein known to have receptor-specific interactions present on the putative rafts [ 2 ]. Bacterial exotoxins are broad of two classes; (1) extracellularly acting and (2) intracellularly acting. Further, the extracellularly acting bacterial exotoxin are divided into two broad categories: a) non-membrane damaging and b) membrane damaging; there is a further division into different sub-categories based on the enzymatic activity possessed by the toxin. Since membrane damaging and intracellular acting toxins are involved in the interaction with host lipids, this section mainly focuses on them. The membrane damaging bacterial toxins, also known as pore-forming toxins (PFT), has three classes a) hemolysins, b) leukocidins, and c) phospholipases. PFTs account for 20–30% of all bacterial exotoxins, predominantly function to perforate the plasma membrane but can also act on intracellular organelle membranes [ 50 , 51 ]. The preliminary step for any toxin to act on the host cell is to identify and interact with its cell's either membrane-spanning or GPI-anchored-proteins or lipids microdomains. It has been well demonstrated that bacterial toxins have exploited the lipid microdomain of host cell membranes as binding and concentrating devices for toxins (Refer to Table 1) [ 52 , 53 ]. Lipid rafts help in inducing toxin oligomerization to form pores in the host cell membrane. Aerolysin from Aeromonas hydrophila is a water-dwelling, gram-negative rod-shaped bacterium, majorly causing diarrheal illness and sometimes necrotizing skin and soft tissue infections in individuals with immunocompromised conditions [ 54 ]. Upon binding to lipid rafts, Aerolysin undergoes the heptamerization process followed by pore formation [ 55 , 56 ]. Similar mechanisms of toxin oligomerization on lipid rafts of the host cell membrane have also been found in Staphylococcus sp. leukotoxin (octameric pore) and E. coli . ClyA (13-meric pore) [ 57 ]. The alpha-toxin produced by Clostridium sp., most potent of them is produced by Clostridium perfringens , harbors phospholipase C (PLC) activity [ 58 ], and the N-terminal domain retains only lecithinase activity and has a lower potency than the holoenzyme [ 59 ]. Cholera toxin (CT) is an AB toxin, which possesses a single A subunit and five B subunits in a pentameric ring fashion [ 60 ]. CT has been shown to interact with these lipid microdomains cluster of GM1 ganglioside receptors for attachment like Velcro mechanisms [ 61 ]. The A subunit's catalytic activity solely depends upon internalization into the host cell, resulting in the efflux of ions and water from the cells [ 62 , 63 ]. H. pylori classified as a group 1 carcinogen and one of the handfuls of bacteria that are directly linked to cancer [ 64 ]. VacA, from H. pylori , has been known to affect host cells by inducing cytoplasmic vacuoles, which support the survival of bacteria by increasing permeability of the host cell [ 65 ]. It has also been reported that the host sphingomyelin and depletion of cholesterol might have a role in toxin functionality, thus proposing strong pieces of evidence for the role of host lipid rafts in the activity of VacA [ 66 ]. Consequently, the selection of receptors harbored in rafts is critical in bacterial toxin-induced hyper-inflammatory responses, host defense, or endotoxin tolerance [ 67 ]. The diphtheria toxin is reported to enter the cell via clathrin-coated pits (COP-I), classically known to function independently of lipid rafts [ 68–70 ]. On the contrary, it has been shown that the depletion of sphingolipid facilitates the entry of Diphtheria toxins [ 71 ]. Anthrax, another toxin, also invades the cell via the clathrin-dependent process. This is mediated by a receptor called tumor endothelial marker 8 (TEM8, ANTXR1) and capillary morphogenesis gene 2 (CMG2, ANTXR2) but, the receptor-toxin cluster in the lipid raft and disruption of the same leads to a subsequent defect in toxin internalization [ 72 , 73 ]. Shiga toxin (Stx1/2), another AB5 toxin, interacts with amphipathic glycosphingolipid (GSL), which are globotriaosylceramide, and globotetraosylceramide. These are embedded in membrane lipid microdomains or lipid rafts [ 53 , 74 ]. Other sets of toxins have also been seen to utilize the lipid microdomains for attachment and entry into the cell; these are given as a tabulated form (Refer table 1). Few toxins help in survival inside the intracellular niches. Upon entry into the host cell, bacteria end up having two options: either to escape the vacuole or to deviate phagolysosome maturation, thereby securing an intracellular replicative niche. One classic example is employed by L. monocytogenes . To escape the vacuole and come into the cytoplasm, L. monocytogenes secretes pore-forming cytolysin, called listeriolysin O. It has been shown to interact with lipid rafts, and it belongs to the cholesterol-dependent cytolysin(CDC) family [ 75 ]. Besides this, Listeriolysin O also heightens the release of proinflammatory cytokines via the NF-KB pathway in macrophages [ 76 ]. This helps recruit a larger number of prospective host cells to the infection site and further helps the pathogen disseminate. Targeting the toxins' binding to the host cell by inhibiting the host lipid-binding sites serves as a potent therapeutic strategy against the toxins known to cause life-threatening conditions in the host. Recent literature in this field is vast, and many excellent reviews focus on each toxin's detailed mechanism [ 77–79 ]. This review has limited our focus to a more holistic aspect of the host-lipids race during bacterial infection. Host lipids and bacterial endocytosis Plasma membrane domains (enriched in cholesterol/sphingosine) function to sort the protein (based on the apical and basolateral side) of epithelial cells that mediate selective bacterial entry into the cell [ 80 ]. CD55, located in the apical membrane, acts as a receptor for various bacteria and viruses, causing mucosal infections [ 81–84 ]. Bacteria exclusively enter through the raft domain even when the entry receptors are available throughout the host cell membrane. Mañes et al. proposed that raft-mediated entry has two advantages: firstly, it activates cellular modulation for cytoskeleton remodeling along with membrane ruffling, and secondly, to circumvent intracellular degradative pathway [ 85 ]. Acquiring host lipid rafts to form phagosome also helps survive various intracellular bacteria, notably obligate intracellular such as Chlamydiaceae , and facultative intracellular such as Salmonella sp., Shigella sp., Mycobacterium sp. inside phagocytic cells [ 61 ]. E. coli K1 mediated infection causes meningitis. The route of access of E. coli K1 into the cells depends upon the integrity and cholesterol content of lipid rafts [ 86 ]. The Chlamydia trachomatis is the most common cause of all sexually transmitted diseases (causing urogenital trachoma and conjunctivitis infection) and has been reported to have lipid-mediated entry into the cell [ 85 , 87 , 88 ]. Lipid rafts mediate attachments, followed by the entry in the host cells of C. trachomatis . Some reports show that C. trachomatis serovars E and F enter through a membrane area rich in GM1, a lipid raft marker, along with caveolin1 and caveolin2 (Refer Figure 1 )[ 89–92 ]. A study done using chelator of cellular cholesterol has shown that the elementary body (infecting stage) of C. trachomatis serovars L2, D, E, and K attaches to the cholesterol-rich site in the membrane of epithelial cells [ 93 ]. On the contrary, C. trachomatis serovars A and C enter the cell through clathrin-mediated endocytosis and are uninhibited by cholesterol depletion [ 91 ]. Apart from this, Chlamydia spp. also traffic to the Golgi apparatus to hijack sphingolipids rich vesicles, thereby preventing lysosomal fusion (Refer Figure 1 )[ 90 ]. However, recent reports suggest that Chlamydia sp. utilizes heparan sulfate proteoglycans (HSPGs) on the host cell membrane for initial attachment followed by high-affinity binding to a battery of host cell receptors and thereby mediating entry into the host cells [ 94–96 ]. Figure 1. Host lipid raft in bacterial entry and phagosomal trafficking : Mycobacterial spp . incorporated raft-associated TACO (tryptophan-aspartate-containing coat protein) of the host cell and thus inhibit lysosomal fusion. Chlamydia trachomatis serovars E and F enter the host cell through lipid rafts associated with GM1 ganglioside receptor, caveolin 1/2 heteromeric complex. Upon entry, it recruits ceramide transfer to facilitate the transfer of ceramide to the Golgi bodies. Further upon conversion of ceramide to sphingolipid and cholesterol, it is recruited onto bacteria containing phagosome and thus escape fusion with Lysosome. Other species of Chlamydia, Brucella spp., E. coli, Salmonella sp., and Legionella sp . enters through lipid raft region rich in cholesterol. Chlamydia trachomatis 2 serovars A and C enters the cell through clathrin-dependent endocytosis which functions exclusive of lipid rafts. Pseudomonas aeruginosa enters and mediates an uptake by raft remodeling via CFTR, CD40, and CD95 dependent pathway Salmonella sp. and Shigella sp. are two Enterobacteriaceae that utilize type 3 secretion system (T3SS), a molecular syringe to inject effector protein into the membrane and cytosol the host cell that facilitates the entry in non-phagocytic cells and survival. T3SS has two effector proteins, which are part of these apparatus both in Shigella (IpaB and IpaC, invasion plasmid antigens) and in Salmonella (SipB and SipC) that are encoded by Salmonella pathogenicity islands (SPI). They are preassembled, but the T3SS does not get activated to translocate the effectors until the bacterium is in contact with the host lipid raft. IpaB and SipB are cholesterol-binding proteins, and it has been shown that membrane insertion of IpaB relies on lipid raft domains(Refer Figure 1 )[ 97 , 98 ]. However, experiments that were done using cells lacking the ability to form lipid rafts have shown that invasion and intracellular survival of Salmonella enterica serovar Typhimurium and C. trachomatis (certain serovars) is not dependent on cholesterol, unlike Coxiella burnetii [ 99 ]. A bacterial invasion is a complex event, and thus, not only lipids but also cholesterol/sphingosine associated membrane proteins play equally important roles. Similarly, in Mycobacterium spp. it has been reported that to hide from adaptive immune responses, they have incorporated or modified host lipids and proteins from rafts in their phagosome, thereby escaping antigen processing and presentation apparatus-phagosome containing Mycobacterium spp. The host cell actively binds TACO (tryptophan-aspartate-containing coat protein), which prevents the fusion of bacteria-containing phagosome to the lysosome [ 100 ]. This interaction of TACO with phagosome is cholesterol-dependent(Refer Figure 1 )[ 101 ]. Brucella abortus , which is Gram-negative, intracellular, zoonotic infection-causing bacteria, harbors Vir-B that requires macrophages' plasma membrane cholesterol for mediating internalization(Refer Figure 1 )[ 102 ]. Class A scavenger receptor, cholesterol, and ganglioside GM1 facilitated entry in macrophages is reported for B. ovis and B. canis (Refer Figure 1 )[ 103 ]. Another intracellular, zoonotic, Gram-negative, and facultative pathogen, Francisella tularensis , has been reported to have inhibited entry upon removal of GPI-anchored protein due to the destabilization of cholesterol/sphingosine [ 104 ]. Pseudomonas aeruginosa , a Gram-negative, opportunistic pathogen, causing nosocomial infection apart from causing chronic pulmonary infection in immunocompromised or cystic fibrosis patients [ 105–108 ]. Studies on primary human nasal and murine tracheal epithelial cells showed that it triggers lipid microdomain transformation into large platforms of insoluble membranes to stimulate its internalization [ 109 ], the stimulation of CD95 and CD40 trigger membrane reorganization subjecting acid sphingomyelinase (ASM) toward the extracellular leaflet. Thus, leading to the breakdown of sphingomyelin to ceramide [ 110 , 111 ]. This is further required to reorganize these microdomains into larger signaling platforms [ 112 ]. A T3SS mediated upregulation of CD95 is reported in Pseudomonas infection, which helps to internalize bacteria, induce apoptosis, and regulate cytokines release; and mice that are deficient in CD95 show higher susceptibility to Pseudomonas infection [ 109 ]. The other role of sphingosine and derivatives in infection with Mycobacterium sp. has been discussed later. Asialylated ganglioside receptor, namely asialoGM1, another sphingolipid, expressed on the surface of cystic fibrosis patients' respiratory epithelial cells enhances binding and invasion of Pseudomonas aeruginosa [ 113 ]. It can also be concluded that cholesterol/sphingosine and overall membrane integrity is vital for host defense mechanisms. Lipid rafts also cluster a chlorine channel CFTR (nonfunctional in Cystic Fibrosis), which colocalizes with GM1 marker [ 114 ]; a study using human corneal epithelial cells shows that P. aeruginosa enters via CFTR [ 7 ]. CFTR-mediated Pseudomonas infection activates the NF-KB pathway followed by cytokine release; however, methyl-β-cyclodextrin administration inhibits this NF-KB activation (Refer Figure 2 )[ 114 ]. Macrophage-mediated bacterial endocytosis by CFTR dependent lipid rafts is inhibited in stress conditions, thus challenging one of the immune system's potent arsenals [ 115 ]. Figure 2. Host lipid raft mediated Signaling modulation : Pseudomonas aeruginosa upon binding to cystic fibrosis transmembrane conductance regulator (CFTR) and triggers activity of acid sphingomyelinase (ASM). ASM then leads to the production of ceramide in a CD40 and CD95 mediated pathway, which remains knitted with lipid rafts clustering and activation of downstream signaling to inhibit IL-1β expression and thus again inducing CD95 dependent apoptosis Host lipids and intracellular bacteria Host lipid remodeling on to phagosome of intracellular bacteria helps to maintain growth and viability. A considerable amount of investigation regarding the same has been done. The fusion of pathogen containing vacuole with lipid droplets which are enriched with neutral lipids like TAG, sterol esters (SE) along with proteins like perilipin family proteins, adipose differentiation-related proteins (ADRP), tail interacting protein (TIP47), etc and surrounded by phospholipid hemimembrane facilitates the nourishment of bacterial pathogens such as Chlamydia trachomatis, M. tuberculosis, M. leprae , etc by supplying adequate amount of nutrients [ 116 ]. In the case of Chlamydiaece , upon entry, the elementary body, which is bound to the inclusion bodies derived from the host cell membrane, differentiates into reticulate bodies and establishes a replicative niche [ 117 ]. During this phase, the inclusion bodies avoid lysosomal fusion and are transported to the peri-Golgi region, where it interacts with various cellular components and trafficking routes to gain access to nutrients. Even though C. trachomatis can synthesize their membrane lipids; they have been found to harbor host-derived lipids such as sphingomyelin and cholesterol on their membranes. Chlamydia inclusion hijacks sphingomyelin and cholesterol-containing exocytic vesicles by a vesicular trafficking pathway which is Brefeldin A(inhibits activation of Arf1- crucial in vesicle formation) sensitive [ 8 , 118 ]. Ceramide is a precursor of sphingomyelin. Conversion from ceramide to sphingomyelin takes place in the Golgi. This further accumulates within the inclusion membrane at the early time points of infection [ 118 , 119 ]. Nonetheless, not all lipids are acquired from Golgi dependent pathway, and it is limited to only particular lipids. Rab-GTPase and SNARE proteins, which are key players of vesicle fusion in physiological conditions, could be involved in the acquisition of lipid from the host [ 95 , 120–124 ]. Apart from these mechanisms, Chlamydia also recruits neutral lipids from either de novo synthesis or lipid droplets through IncA (effector protein) rich site [ 8 , 125 , 126 ]. Chlamydia inclusion protein IncD facilitates the recruitment of ceramide-transfer protein (CERT) and channelizes ceramide trafficking from the endoplasmic reticulum into the inclusion membrane [ 127–130 ]. The CERT also executes the transfer of ceramide to the Golgi. Inhibiting the CERT leads to lesser recruitment of sphingomyelin to the inclusion membrane [ 130 ]. Disruption of host cellular organelles (such as Golgi fragmentation) can heighten lipids' uptake into the Chlamydia inclusion, resulting in increased C. trachomatis replication. This process is also governed by both host (Golgin-84) and Chlamydia proteases (chlamydial protease-like activity factor-CPFA) [ 131 ]. Abundant intracellular cholesterol levels trigger the esterification and packaging of Cholesterol ester into lipid droplets (LD). Chlamydia translocates various host proteins such as perilipin, Rab18, and ATGI, present on LD's membrane to the inclusion membrane. Conversely, many bacterial proteins on the inclusion membrane are trafficked to adjacent LD membranes [ 125 ]. This results in organelle mimicry and thus deceiving host machinery of lysosomal fusion. Mycobacterium spp. which are facultative intracellular pathogens, efficiently thrive in macrophages, also require cholesterol for their survival and uptake [ 101 ]. High polarity lipid derived from M. avium has been shown to interact with lipid rafts present on murine macrophages [ 132 ]. The ability to induce lipid droplet formation inside host cells is an important virulence factor for pathogenic Mycobacterium sp [ 133–135 ]. M. bovis BCG or M. tuberculosis derived LAM's cell wall component can induce lipid droplet formation in time and the dose-dependent manner by mimicking the host. Salmonella sp. upon entry mediated by T3SS encoded by SPI-1 in the non-phagocytic cell, maintain its intracellular replicative niche inside a vacuole called Salmonella containing vacuole (SCV) with the help of Salmonella pathogenicity island-1 and 2 (SPI-1 & 2) encoded T3SS and effector molecules both in non-phagocytic and phagocytic cells [ 136 , 137 ]. Remodeling of the outer membrane (OM) during Gram-negative bacterial infection helps intracellular survival and replications in tissue [ 10 , 138 ]. S. enterica serovar Typhi and S. enterica serovar Typhimurium both survive in acidified phagosomes facilitated by PhoPQ, ArcAB, mgtCB , and other regulons, a two-component regulator [ 139 ]. The genes are responsible for modifying OM in terms of increasing hydrophobicity and decreasing negative charge, resulting in the prevention of cationic antimicrobial peptide (CAMP) binding [ 9 ]. One recent report has shown that PbgA, under the control of the PhoPQ two-component system, traffics the cardiolipin from the inner membrane to the OM of Salmonella Typhimurium by binding to cardiolipin itself [ 140 ]. SseJ, another effector of SPI-2 having a glycerophospholipid cholesterol acyltransferase activity, localizes to the cytoplasmic face of SCV and esterifies the cholesterols. SseJ activity, along with localization, depends on the host protein, RhoA (GTPase) [ 141 ]. Cholesterol accumulation and CD55 protein recruitment on SCV suggests an important role for cholesterol in intracellular survival [ 11 ]. Another molecular complex of host CD44, the hyaluronan receptors, and IpaB (bacterial invasion) in the case of enteroinvasive Shigella infection partitions in the lipid rafts. Invasion of bacteria is reduced in the cells deficient for sphingolipids as it is utterly predictive of the possible roles lipid rafts play in shigellosis [ 142 ]. In L. monocytogenes , cholesterol is not required for entry into the cells. However, cholesterol plays a vital role in the event downstream to invasion, such as F-actin polymerization [ 12 ]. One of the reports demonstrates that, during enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) infection, cytoskeleton rearrangement was inhibited upon cholesterol depletion. Also, cholesterol depletion hampered adherence to epithelial cells in EPEC infection but not EHEC infection [ 13 ]. Coxiella burnetii genome has been reported to encode enzymes for the synthesis of fatty acids and phospholipids. Still, it lacks enzymes for de-novo synthesis of cholesterol [ 143 ]. However, it also encodes sterol reductase homolog, reducing double bonds of sterol to cholesterol in the final step. One of the studies showed that this enzyme was active in the yeast model system. Thus, it was proposed that C. burnetii could hijack and act on host sterols for their intracellular growth [ 144 ]. Contrary to the host lipid's role in the survival of intracellular pathogens trapped inside the vacuole, it also restricts bacterial proliferation. The attachment of ER-resident immune-related GTPase Irgm3 (47 kDa) with ADRP helps the dendritic cells cross-present phagocytosed exogenous antigen exogenous CD8 + T cells [ 145 ]. High-resolution single-cell Raman microscopy has revealed the association of arachidonate enriched lipid bodies with phagosomes in neutrophils, which finally activates NADPH phagocytic oxidase [ 146 ]. The derivatives of glycerophospholipid, phosphatidylinositol (PI) of host cells play a divergent role in the maturation of phagolysosome during bacterial infection [ 147 ]. Salmonella enterica uses SopB protein to dephosphorylate PI (3,5) P2, PI (4,5) P2, and PI (3,4,5) P3 of the host cell to prevent the maturation of phagolysosome [ 148 , 149 ]. The engagement of phosphatidylinositol-3-phosphate or PI (3) P on the phagosomal membrane recruits p40 phox that further activates NADPH phagocytic oxidase during FcɣIIA receptor-mediated phagocytosis of IgG coated RBCs in COS7 cell line [ 150 ]. M. tuberculosis can restrict the acidification of the phagosome by secreting SapM phosphatase that dephosphorylates PI (3) P of host cells [ 151–153 ]. The intracellular proliferation of Mycobacterium strain lacking PIP phosphatase MptpB that dephosphorylates PI (3) P, PI (4) P, PI (5) P, and PI (3,5) P2 is significantly attenuated in guinea pig macrophage [ 154 , 155 ]. Host cell lipids such as arachidonic acid, ceramide, sphingosine, sphingomyelin, and phosphatidylinositol-4,5-bisphosphate can restrict the intracellular proliferation of Mycobacterium tuberculosis and Mycobacterium avium by triggering the actin assembly, phagosome maturation, phagolysosome fusion, and acidification in J774A.1 macrophage cell [ 156 ]. Host lipids and extracellular bacteria Extracellular bacteria have also been reported to incorporate host lipids. Specifically, Mycoplasma spp. has been known to acquire free cholesterol from serum lipoprotein and modify it to glycolipids, cholesteryl ester to be specific [ 15–17 ]. Although lipid auxotrophy is not a surprising concept in biology, the spirochete also depends on external cholesterol reservoirs for replication and multiplication because it cannot synthesize cholesterols. However, they show hallmark characteristics of eukaryotic lipid rafts (containing phosphatidylcholine, phosphatidylglycerols, and lipoproteins) on their cell membrane [ 157–160 ]. Besides these, it also has free cholesterols and two cholesterol glycolipids, acylated cholesteryl galactoside (ACGal) and cholesteryl-galactoside (CGal), along with non-cholesterol glycolipid and mono-galactosyl diacylglycerol(MGalD) [ 161–164 ]. Sterols that help in lipid raft formation and many detergent resistance membranes are vital for B. burgdorferi membrane's integrity [ 157 ]. Although the mechanism of cholesterol acquisition from the host cell to spirochetes is not very clear, it is proposed to be mediated by lipid rafts-lipid rafts interactions [ 165 ]. Extracellular bacterial species H. pylori also incorporates cholesterols and converts it into the glucosyl-cholesterol complex. Glycolipids identified in cholesteryl glucosides are cholesteryl-α-D-glucopyranoside, cholesteryl-6-O-tetra-decanoyl-α-D-glucopyranoside, and plausibly cholesteryl-6-O-phosphatidyl-a-D-glucopyranoside. It has been known for a long time that H. pylori have a preference specific to cholesterols [ 166 ]. One study found that bacteria grown in the absence of cholesterol were more susceptible to antimicrobial peptides and antibiotics [ 167 ]. The presence of cholesterol on the H. pylori membrane helps modify the membrane to facilitate the colonization of bacteria in gastric mucosa without hampering the Lewis antigen's expression [ 168 ]. Host lipid serving as nutrients for bacteria Bacterial pathogens can use host lipid as a carbon source, and for energy as well, they efficiently hijack the host lipid metabolism to develop convenient reservoirs that help the pathogen to survive in the host during infection. Mycobacterium sp. is well known to establish infection via hijacking host lipids out of all intracellular pathogens. It is well reported that Mycobacterium sp. uses host lipid as a primary source of carbon in- vivo [ 14 , 169 ]. Based on their lipid composition and metabolism, M. tuberculosis and other genus members are one of a kind. They harbor a variety of lipids such as glycolipids, mycolic acids, and polyketides in the cell wall [ 170 ], the genes for biosynthesis is encoded in their genome. They possess approximately 250 enzymes, which are linked to fatty acid metabolism [ 171 ]. In different mouse models, it has been shown that the enzyme that governs the direction of degraded fatty acid product to a pathway also dictates the phenotype of the bacterium [ 172–174 ]. Interestingly M. tuberculosis does not depend on a single lipid source, shows plasticity in lipid utilization, and can co-catabolize different lipids as carbon sources [ 175 ]. M. tuberculosis makes use of host fatty acids as 1) substrates for β-oxidation, 2) acyl-primers for polyketide synthesis, and 3) incorporation into phospholipids and triacylglycerol (TAG) [ 176 , 177 ]. M. tuberculosis has a dedicated cholesterol import system that allows the bacterium to use lipid as a sole source of carbon and energy during persistent chronic infection inside activated macrophages. M. tuberculosis gene cluster mce4 encodes an importer of cholesterol, which serves as the sole carbon source for the pathogen inside the host cell [ 178 ]. The cholesterol usage comes with an added drawback for the bacterium, the terminal product of cholesterol catabolism being propionyl-CoA [ 179 ], a toxic compound that requires further metabolism. To ameliorate this toxic effect, M. tuberculosis has acquired various strategies to metabolize propionyl-CoA as an energy source and as building blocks for cell wall synthesis. They convert propionyl CoA into pyruvate and succinate via methyl citrate cycle and subsequently incorporate these into the tricarboxylic acid (TCA) cycle to produce energy [ 180 ]. It is perhaps due to the presence of Methyl-isocitrate lyase enzymatic activity. Another pathway utilized by Mycobacterium sp . is the conversion of propionyl- CoA into methyl-malonyl-CoA by the action of propionyl-CoA carboxylase, and further assimilated into the vitamin B12 mediated methyl-malonyl pathway that results in the production of succinyl CoA, that is used in the TCA cycle as well [ 181 ]. Finally, they have polyketide synthase, which can convert propionyl CoA into methyl-malonyl CoA and incorporate it into the bacterium's cell wall [ 182 ]. M. tuberculosis encodes for four phospholipase C proteins among them plcA, plcB and plcC are in an operon, and plcD is placed in a distant genomic region. These enzymes hydrolyze phospholipids, resulting in free fatty acids [ 171 ], and are essential for the pathogens' virulence [ 183 ]. They metabolize fatty acids via two pathways: β -oxidation or glyoxylate shunts. Even though the β -oxidation pathway involves only five enzymes, M. tuberculosis is known to have approximately 100 genes encoded for enzymes having a redundant function in this cycle [ 184 ]. The glyoxylate shunt has been long known as an anabolic pathway essential for carbon synthesis among prokaryotes. It has been reported in a proteomic and genomic-based study on Mycobacterium sp . that one of the key enzymes isocitrate lyase (ICLs) of glyoxylate shunt is upregulated during infection in macrophages [ 185 , 186 ]. There are two homologous genes for ICL, namely icl1 and icl2 ; studies on mutant lacking icl1 have demonstrated that it affects bacterium survival inside the activated macrophages. The double mutant of the icl mice model has also shown rapid clearance from the lungs of infected mice, thus, providing evidence for the essentiality of the icl gene and glyoxylate pathway in M. tuberculosis pathogenesis [ 187 ]. It is well known that M. tuberculosis can persist as a dormant infection within mononuclear cells with a characteristic and pathological hallmark. Granulomas are generally an assemblage of immune cells, comprising of different types of macrophages such as multinucleated enlarge cells, epithelioid cells, and foamy cells, enclosed by a border of lymphocytes. Foamy cells are one of the granulomas hallmarks and can be defined as macrophages enriched with lipid droplets [ 134 , 172 ]. To maintain the latency in host M. tuberculosis derives host triacylglycerol from these foamy cells and utilizes it as a carbon source [ 176 ]. In foamy cells, bacteria are responsible for diverting the glycolytic pathway toward ketone body synthesis [ 188 ], and various mycobacterial molecules are involved in accumulating lipids in foamy cells [ 189 ]. Lipid- a blessing in disguise Besides their roles in the augmentation of bacterial infection in host eukaryotic cells, which majorly include adhesion, invasion, and survival of bacterial pathogens, many times host lipids such as sphingolipids orchestrate significant inhibitory role during bacterial infection. Tuberculosis caused by Mycobacterium tuberculosis has been challenging humankind with its wrath since time immemorial. After several decades of decline, at present, tuberculosis cases are on the rise again. One of the reasons is its co-infection with HIV, and another reason is the emergence of MDR-strains. Also, TB treatments are prolonged (for six months), complex and expensive. At present, there is a dire need for new therapies to treat TB. M tuberculosis's success as an intracellular pathogen is attributed to its property to resist killing by fusion of lysosome with phagosome containing it. It has been found to employ multiple mechanisms for this purpose, including inhibition of phagosomal localization of vacuolar H + /ATPase that acidifies the phagolysosome, modulations of the vesicular soluble N-ethylmaleimide attachment protein cellubrevin, and alterations of the regulatory GTPases of the Rab family and its early endosomal marker Ag1. Sphingosine-1-phosphate, which is produced from ceramide by the consecutive catalytic activity of ceramidase and sphingosine kinase, has been reported to exert an inhibitory effect on the intracellular proliferation of nonpathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis H37Rv strains in human monocyte-derived macrophages (hMDM) and THP-1 cells [ 30 , 190 , 191 ]. The translocation of sphingosine kinase to the phagosomal membrane is inhibited by Mycobacterium tuberculosis , further diminishing the intracellular level of sphingosine-1-phosphate and ultimately resulting in the reduction of intracellular Ca2 + level required for the acidification of phagolysosome [ 192 , 193 ]. Sphingosine-1-phosphate can express iNOS in macrophages, differentiate the latter toward M1 phenotype, secretion of IFN-ɣ during infection, and enhance pulmonary infiltration CD11b + macrophages. Also, treatment with Sphingosine-1-phosphate enhances the expression of key signaling proteins of inflammatory response like phosphor-MAPK(pp38), phospho-NF-ĸβ, phospho-STAT3 in the lungs of 6–8 week old C57BL6 mice infected with Mtb [ 194 ]. Overexpression of Sphingosine Kinase (SphK-1) conferred resistance against M. smegmatis infection due to the enhanced generation of NO, iNOS species, pp38 LAMP-2 [ 195 ]. Burkholderia pseudomallei , the causative agent of melioidosis, improves its intracellular proliferation in murine macrophage by avoiding fusion with lysosome with the help of sphingosine-1-phosphate lyase, which is secreted into the host cytosol upon infection to degrade cellular pool of Sphingosine 1 Phosphate [ 196 ]. Shigella flexneri can evade the NFKB dependent proinflammatory immune response in adult mice's gut by reducing the level of sphingosine-1-phosphate with heightened activity S1P lyase and S1P phosphatase [ 197 ]. A recent study has illustrated that hydrolysis of sphingomyelin into ceramide and phosphorylcholine activates NADPH dependent phagocytic oxidase to generate ROS in macrophage and helps in the clearance of Salmonella Typhimurium and Pseudomonas aeruginosa [ 198 , 199 ]. Despite their bactericidal functions, a wide array of pro-bacterial roles of sphingolipids have also been extensively studied. The mice's enhanced resistance against the infection caused by Pseudomonas aeruginosa upon deleting sphingosine kinase 2, the enzyme required to synthesize sphingosine-1-phosphate, clearly indicates the contribution of sphingosine to the establishment of bacterial pathogenesis [ 200 ]. Legionella pneumophila lacking LegS2 gene coding for a novel sphingosine-1-phosphate lyase becomes hyper-proliferative in bone marrow-derived macrophages and hMDMs by downregulating the expression of proinflammatory cytokines [ 201 ]. Apart from sphingolipids, cholesterol has also been reported to possess antibacterial activity. As discussed earlier, cholesterol has a substantial role in supporting the survival of intracellular pathogens. On the contrary to this, it has been demonstrated that a high level of cholesterol in the parasitophorous vacuole (PV) either by supplementation or by treatment of U18666A, a drug that traps cholesterol in the parasitophorous vacuole, enhances the acidity of PV and successfully inhibits intracellular bacterial growth in both mouse embryonic fibroblast and THP1 macrophage cell lines [ 202 ]. A recent study has proved that oxysterol 25 hydroxycholesterol (25HC) can rapidly internalize the accessible fraction of cholesterol from the plasma membrane by activating acyl-CoA: cholesterol acyltransferase (ACAT) enzyme and builds up protection against invading bacterial pathogens such as Shigella flexneri, Listeria monocytogenes , etc [ 203 ]. As already discussed, ceramides are a group of sphingolipids that have been shown to have an antibacterial effect. Macrophages are front line soldiers associated with host defenses against foreign pathogens. They are one of the professional phagocytes and antigen-presenting cells of the host immune system and harbor various pathogen recognition and signaling receptors on their outer membrane's surface. They can efficiently uptake pathogens, digest them, and present them to T cells. Macrophages are generally classified into proinflammatory macrophages (classically activated, M1 macrophages) or anti-inflammatory macrophages (alternatively activated, M2 macrophages) based on their physiological features and state of polarization. They undergo polarization upon encountering foreign bodies/invading bacteria [ 204 ]. Proinflammatory cytokines such as IFN-ɣ and TNF-α are responsible for the induction of the M1 phenotype, further characterized by higher antigen presentation and heightened expression of IL-12, IL-23, reactive nitrogen species, and reactive oxygen species, etc. On the other hand, macrophages' M2 phenotype is augmented by anti-inflammatory cytokines like IL-4, IL-10, and IL-13 [ 205 ]. An increasing amount of research is going on, which demonstrates dynamic lipid remodeling during host defense mechanisms. M1 and M2 macrophages show two distinct metabolic pathways for energy generation; the former uses aerobic glycolysis, whereas the latter is skewed toward fatty acid oxidation. There is also a plethora of genes associated with the metabolism of fatty acids, which are upregulated and downregulated in each of these polarized states. For M1 macrophages, the upregulation of COX-2 and downregulation of COX-1, leukotriene A4 hydrolase, thromboxane A synthase 1, and arachidonate 5-lipoxygenase (5-LO) have been reported (Refer Figure 3 ). Contrary to this, in the M2 state, there is an upregulation of COX-1 and arachidonate 15 lipoxygenases (15-LO) [ 39 ]. Generally, it has been observed that the effects of eicosanoids in the intimate relation of host-pathogen can be categorized into two parts: proinflammatory and anti-inflammatory. Eicosanoids such as PGE2 and lipoxin A4 (LXA4) are the products from the oxidation reaction of arachidonic acid and other polyunsaturated fatty acids and extremely active lipid mediators of the immune response during bacterial infection [ 206 , 207 ]. These lipid mediators help the host by different mechanisms. During the infection of avirulent M. tuberculosis , PGE2 has been found to modulate host survival strategies against the perturbation of the inner mitochondrial membrane via EP2 receptors, which further restrict the pathogen's spread [ 19 ]. Figure 3 Role of host membrane lipid in immunomodulation during bacterial infection . After invading the host cell, bacteria (both gram positive and gram negative) synthesize and secrete effector proteins. Bacterial effector proteins/virulent factors aggravate the catalytic activity of cytosolic calcium-dependent phospholipase A2 (cPLA-2). As a result of which Arachidonic Acid is released from membrane glycerophospholipids into the cytoplasm. Subsequently, host cellular Cyclooxygenase-2 (COX-2) present in the cytoplasm 5 converts this AA into Prostaglandin H2 (PGH2) which represent a precursor of PGE2. Ultimately, PGH2 is transformed into PGE2 by the catalytic activity of the enzyme PGE2 Synthase. PGE2 performs a pivotal role in cytokine secretion & activation of several innate immune cells (like- macrophage, neutrophil, etc.). The production of PGE2 can elicit an immune response via any one of the four eicosanoid receptors termed as EP1, EP2, EP3, EP4. Interestingly, EP2 and EP4 represent the key players in developing the immunosuppressive response of PGE2, thus helping in survival and proliferation of bacteria inside immune cells. Downstream signaling by both EP2 and EP4, activate phosphatidylinositol 3- Kinase (PI3K) pathway. PI3K, in turn, activates ERK-1/2 to initiate immunosuppression Interestingly it has also been reported that PGE2 activates synaptotagmin 7, a calcium-sensing protein that plays a pivotal role in lysosome-mediated membrane repair to reseal the host plasma membrane [ 208 ]. This re-joining process is vital to prevent necrosis and promote apoptosis, leading to pathogen's clearance, thus helping the host. Strikingly the opposite role of PGE2, which potentiates the survival of intracellular pathogens in the host, has also been documented in great detail. PGE2 has been reported to acquire the ability to inhibit macrophage maturation and reduce the activation of NADPH oxidase and thus facilitates mycobacterial survival [ 209 , 210 ]. With the help of SPI-2 encoded virulent factor SpiC Salmonella Typhimurium can upregulate the expression of COX-2, which produces PGE2 in macrophages. PGE2 can enhance the production of IL-10 in a protein kinase A-dependent manner, which further accelerates Salmonella's intracellular survival [ 210 ]. During infection, the virulent strain of Mycobacterium sp . abrogates the expression of PGE2 and aggravates the expression of lipoxin A4 (LXA4), which is anti-inflammatory and produced by the catalytic activities of 5-LO and 15-LO [ 207 ]. The enhanced expression of lipoxin A4 promotes necrosis and mediates the pathogen's rapid spread [ 211 ]. Lipid and therapeutics Since the 20th century, the liposome or the phospholipid bilayer capsule has received quite some attention due to its potential to carry bio-active molecules into the living system. Liposomes are formed by allowing phospholipids to swell in an aqueous solution, whereby they form concentric layers of lipids. A liposome can entrap an aqueous phase inside it, and if a solute remains dissolved in the aqueous phase, it can also entrap it. The initial experimentation had started with the administration of enzymes using liposomes, but later the work was extended by using liposomes as carriers for drugs and other therapeutic materials [ 212 ], including actinomycin D [ 213 ] and methotrexate [ 3 ]. When these drugs were administered via liposome in rats, they were retained more in the body than when injected in free solution. Also, a larger fraction of the drugs reached the liver and spleen. Solid Lipid nanoparticles (SLNs) are colloidal carriers developed in the past decade as an alternative to traditional lipid carriers like emulsions and liposomes. They constitute a new generation of submicron-sized emulsions where the solid lipid has replaced the liquid lipid (oil). The use of SLNs is advantageous because they offer better stability and upgradability to the production scale than liposomes [ 27 ]. The use of SLNs to administer antitubercular drugs like rifampicin, isoniazid, pyrazinamide reduced their dosing frequency and increased patient compliance [ 214 ]. SLNs have also been used to transfer mRNA to the cells, a promising strategy for vaccine development. Entrapping RNA within SLNs acts to protect it from degradation by RNases [ 215 ]. However, few disadvantages regarding SLNs include poor drug loading capacity, drug expulsion after polymeric transition during storage periods, etc. The lipid matrix also governs the drug loading capacity of SLNs. If the lipid matrix is made of similar lipids, a perfect crystal structure with few imperfections is formed. Drugs are incorporated in the crystal imperfections, between lipid layers, and between fatty acid chains. Due to the less amount of crystal imperfections, drug loading capacity decreases. To address these problems, nanostructured lipid carriers (NLC) were developed. The idea was to mix different lipids with varying fatty acid chains, such that there is quite some distance between the fatty acid chains, which leads to the formation of imperfect crystals and can accommodate more drug molecules, thereby improving the drug loading scenario. In recent years, research is being conducted to characterize NLCs. NLCs demonstrated the faster release of the entrapped drug clotrimazole than low drug-loading SLNs, although there was no significant difference at high-drug loading [ 216 ]. NLCs were more stable than SLNs at 25°C. Also, the drug release profile of NLCs did not change after three months of storage, whereas there was a decline for SLNs [ 217 ]. NLCs have been tested to enhance the oral bioavailability of drugs like clotrimazole, apomorphine, ketoprofen, and lovastatin [ 28 ]. NLCs can be used for the targeted delivery of anti-tubercular drugs by the pulmonary route [ 218 ]. In addition to the oral route and pulmonary route, drug administration using NLCs is possible using the topical and intravenous routes. Administration through each route has its own set of advantages and shortcomings. Although very few NLCs have been used in clinical practice, they are promising candidates for lipid-nanocarrier mediated drug delivery [ 219 ]. Till now, we have dealt with how lipids can be used as carriers for drugs. From now, we will deal with how lipids can be used as targets for drugs. Lipid synthesis has been a significant target for antibiotic classes like lantibiotics, mannopeptimycins, and ramoplanins (Refer table 2) [ 220 ]. Lipid II has an extensively characterized role in cell wall synthesis. The cell wall of bacteria is composed of alternating units of n-acetylglucosamine (GlcNAc) and n-acetylmuramic acid (MurNAc), with these glycan chains cross-linked with a pentapeptide sequence [ 221 ]. The pentapeptide crosslinking confers strength and rigidity to the cell wall. The formation of a cell wall occurs in an extensive process involving the cytoplasmic face and the plasma membrane's periplasmic face. The assembly of the cell wall subunits starts from the plasma membrane's cytoplasmic side, where the assembly of UDP- MurNAc-pentapeptide is coupled to bactoprenol phosphate, yielding Lipid I. Next, the coupling of GluNAc occurs by peripherally membrane-attached protein MurG to yield Lipid II, the precursor of the cell wall, which is then translocated to the plasma membrane's exterior by an unknown mechanism. Different types of antibiotics form different types of interactions with lipid II [ 220 ]. Their roles have been summarized in (Table 2). Two new peptide antibiotics, katanosins, and plusbacins have been isolated from different bacterial strains Cytophaga and Pseudomonas , and they are found to have potent antibacterial activity against MRSA and Vancomycin-resistant Enterococci . Although their function has not been extensively characterized, they function Lipid-II mediated [ 222 , 223 ]. However, almost all these antibiotics have developed resistance against them, which raises the need to identify novel drug targets, and most importantly, identify novel therapeutic molecules. Lipid A is present mainly in Gram-negative bacteria where it is present as a hydrophobic anchor to lipopolysaccharide present exterior to the outer membrane. Inhibition of lipid A in LPS-deficient mutants led to growth defects, which indicate that lipid A is essential for the bacterium. Picomolar lipid A levels are seen to trigger TLR4/MD activation leading to acute inflammation in the mammalian immune system, thereby proving its potency as an endotoxin [ 224 , 225 ]. The biosynthetic pathway of Lipid A can be used to develop drugs. One such drug target is LpxC, the second enzyme in the biosynthetic pathway [ 226 ]. Several hydroxamates containing candidates are being developed, and clinical trials are on. This pathway can be further used for therapeutic purposes. As seen in the Kdo2-lipid molecule from E.coli , there are two phosphate groups, which are important for TLR4/MD2 activation [ 227 ]. Mono-phospholipid A can be prepared and used as an adjuvant due to its capability to partially activate TLR4/MD2 [ 228 ]. Some species of bacteria, like Francisella tularensis , produce amino-phospholipid A due to phosphatases like LpxE and LpxF, which remove the phosphates of Lipid A. The LpxE gene was chemically engineered into a Salmonella strain that acts as a potent oral vaccine candidate [ 229 ]. Lipid A itself cannot have any therapeutic application, although its modified form, like Lipid IV-A, can have therapeutic applications [ 230 ]. Lipid IV-A is an antagonist of TLR4 in humans and agonists in murine cells. Lipid IV-A could not be utilized due to its instability upon storage, leading to synthetic analogs like E5531 and E5564 [ 231 ]. Although E5564 was not able to reduce the mortality of septic patients, it protected mice from lethal influenza infection, showing that TLR4 can be one possible drug target in viral infection [ 232 ]. Numerous sphingolipids extracted and purified from natural sources have been known to have bactericidal activity against various microorganisms, including Gram-positive bacteria, Gram-negative bacteria, fungi, or microalgae. Ceramides, as discussed earlier too, are a group of sphingolipids that are structurally heterogeneous and complex, containing derivatives of fatty acids in amide linkage with a variety of fatty acids. Short-chain C6- ceramides and a functionalized ω-azido C6-ceramide were found to have antimicrobial properties against Neisseria meningitidis and Neisseria gonorrhoeae . Further studies have shown that these molecules were incorporated into the bacterial cell membrane and its functionality by dissipating its membrane potential [ 233 ]. As we know, Cystic fibrosis patients are predisposed to infection by Pseudomonas aeruginosa . Inhalation of mice with sphingosine or acid ceramidase helped in the recovery of this condition. This treatment successfully inhibited other pathogens that affected cystic fibrosis patients, including Acinetobacter baumannii, Moraxella catarrhalis, Hemophilus influenzae, Burkholderia cepacia opening a new horizon for treatment of lung infection by inhalation of sphingosine or ceramidase [ 234 ]. Administration of myriocin in intestinal epithelial SW480 cells, an inhibitor of de novo biosynthesis of sphingolipids, facilitates the intracellular proliferation of wildtype Salmonella Typhimurium by inhibiting autophagy and HBD-2 dependent response [ 235 ]. Also, ceramide analogs with modified 1-position, e.g., 1-O-methyl modification, have been found to have potent inhibition on Chlamydia trachomatis L2 strain with IC50 being in the micromolar or sub-micromolar range. The exact mechanism is yet to be elucidated, but it is speculated that these compounds may target a host molecule that the bacteria is using for its sphingosine acquisition [ 236 ]. The coating of hydroxyapatite surfaces with sphingosine and its derivatives decreased the adherence and biofilm-forming potential of Streptococcus mutans [ 237 ]. The supplementation of susceptible cystic fibrosis mice with C18-sphingosines by inhalation provided them a survival advantage against the infection caused by pulmonary Staphylococcus aureus [ 238 ]. As we already know, sphingosine-1 phosphate plays a critical role in inhibiting Mtb pathogenesis. Enhanced levels of Sphingosine-1-phosphate can be used as a novel therapeutic strategy to combat mycobacterial infections by boosting overall host immunity. Another common application of lipids is in sanitizers used for fresh poultry animal carcass sanitization to decrease human pathogens' bacterial burden, including Salmonella enterica. Cetyl pyridinium chloride (CPC) is a quaternary sanitizer that produces loss of membrane integrity in Salmonella , leading to loss of electrostatic repulsion between cells cytoplasmic content leakage and emulsification of the cytoplasmic contents by membrane lipids [ 239 ]. Lipids as emerging as a potential biomarker to differentiate between bacterial and viral infections. Detecting these infections molecular and culture-based techniques is available, but they take 24–48 hours to give results and are associated with many false-positive and false-negative cases. Also, antibiotics are administered as a precautionary measure, leading to the rampant misuse of these drugs. Hence, a more reliable and faster diagnostic test is the necessity of the moment. Comparing the lipidomic profile of two groups of febrile children with confirmed bacterial and viral infection revealed some glycerophosphoinositol species, sphingomyelin, lysophosphatidylcholine, and cholesterol sulfate that were higher in the virus-infected group. In contrast, some species of glycerophosphocholine, fatty acids, lactosylceramide, and bilirubin were higher in the confirmed bacterial group. The increase in cholesterol sulfate during viral infection might reflect its necessity in cellular lipid biogenesis and T cell signaling during viral infection. The elevated levels of lysophosphatidylcholine might be attributed to its role inducing membrane curvature needed for virus budding. Lactosylceramide LacCer (d18:1/24:1) and LacCer (d18:1/16:0) were higher in a bacteria-infected group than in a virally infected group. This might be due to lactosyl-ceramides' property to act as Pathogen Recognition Receptors (PRRs) to Pathogen Associated Molecular Patterns (PAMPs). Lactosyl-ceramide with long fatty acid chains, i.e., LacCer(d18:1/24:1), increased as it is essential for the formation of LacCer-Lyn complexes on neutrophils, which happen to be responsible for αMβ2- mediated phagocytosis. Sulfatides were higher in the group with bacterial infection as they are known for immune system regulation during infection. Five species of glycerophosphocholine, i.e., PC(16:0/18:2), PC(18:0/18:1), PC(18:0/18:2), PC (16:0/16:0), PC(16:0/18:1), and bilirubin were higher in case of bacterial infection, although their role in infection is still unclear. This study had been confined to children within the age of 1 month to 9 years, and as metabolism changes with age, further studies need to be done in adult groups [ 31 ]. Recent studies have also revealed that infection and inflammation can induce metabolic changes and changes in lipidomics. Infection and inflammation can induce the Acute-Phase Protein Response (APR) that can alter lipid and lipoprotein metabolism to neutralize invading microorganisms and participate in the local immune response. A notable increase in the plasma triglyceride levels can result from increased VLDL secretion due to adipose tissue lipolysis, suppression of fatty acid oxidation, and increased de novo fatty acid synthesis. In animals, this hyper-triglyceridemic effect is brought about by LPS and Lipoteichoic acid (LTA, also a component of the cell wall in Gram-positive bacteria) and is influenced by multiple cytokines. LPS and cytokines' hyper-triglyceridemic effect is rapid, start within 2 hours of administration, and continue till 24 hours. Also, it is seen that more severe infection leads to decreased VLDL clearance following decreased lipoprotein lipase and apolipoprotein E in VLDL. In rodents, increased hepatic cholesterol synthesis and decreased LDL clearance, conversion of cholesterol to bile acids, and cholesterol secretion to bile attribute to hypercholesterolemia. There are marked alterations in proteins important for HDL metabolism, leading to decreased reverse cholesterol transport and cholesterol delivery to immune cells increase. There is an increase in the oxidation of LDL and VLDL. HDL becomes a proinflammatory molecule. Lipoproteins become enriched in ceramide, glucosylceramide, and sphingomyelin, enhancing uptake by macrophages. Thus, it can be understood that molecular mechanisms that decrease the synthesis of many proteins during APR decrease several nuclear hormone receptors like liver X receptor and farnesoid X receptor. Thus, it can be understood that APR acts through lipid metabolism to protect the host cell from bacteria and viruses. Animals showed improved survival when infusions of synthetic chylomicrons or triglyceride-proteins were administered 30 mins after exposure to LPS, indicating that lipoprotein may have a therapeutic role during endotoxemia. Hypolipidemic mice were more sensitive to LPS-induced lethality, which was reversed by increasing serum lipid concentration to the physiological range [ 240 ]. Once fully characterized, these studies hold immense potential to be applied in laboratories as one important and reliant diagnostic test. Conclusion and future perspective Lipid plays many crucial roles in maintaining the cellular structure and function homeostasis, including cell membrane fluidity, endocytosis, signaling, energy reserve, and membrane trafficking. A vast amount of literature suggests distinct microdomains (enriched with cholesterols and sphingosine) that maintain proper receptor interaction, subsequently inducing signaling cascade inside the cell, as we know that these are time and again exploited by various pathogens. Various bacterial toxins utilize these plasma membrane microdomains to aid in its entry into the host cells. Pathogens that survive intracellularly employ various host-derived lipids for their entry into the cell. Also, Mycobacteria sp. is foremost to exploit host lipids because they often feed off host lipids as their sole carbon source. Few small lipid molecules play a role in immune signaling to escape the immune surveillance of bacteria such as Mycobacteria sp., Salmonella enterica, Enteropathogenic E. coli, Streptococcus pneumoniae, and Pseudomonas aeruginosa have developed stealthy strategies which bypasses the signaling via EP receptor-mediated pathway and hence mediate immunosuppression which in turn increases the bacterial proliferation. However, lipids such as sphingosine-1-phosphate have a much more debatable role in limiting or flaring up bacterial infections and can act in both ways. So, in cases where limiting the infection can be used as a therapeutic enhancement of the S1P function. There has been an increase in research to deduce the exact molecular mechanism employed by bacteria to execute these above-stated functions. This would not only help us to understand molecular pathogenesis but would also have implications on therapeutic target designing. As the antibiotic resistance era is a current emerging concern, targeting these host lipids hijacked by bacteria can be an excellent antibacterial molecule [ 241 , 242 ]. However, these will pose a greater overall risk to the host because targeting any host's intrinsic homeostatic biomolecules might significantly enhance side effects. Therefore, research around host lipid- bacterial infection dynamics is all the more important and demands more attention to the open-ended questions in this field. Hence, this review will be of benefit and interest to this field of research. Supplementary Material Supplemental Material Click here for additional data file. Disclosure statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Supplementary material Supplemental data for this article can be accessed here .

PMC

Anthrax

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2843228/

Anti-inflammatory Compounds Parthenolide and Bay 11-7082 Are Direct Inhibitors of the Inflammasome *

Activation of the inflammasome generates the pro-inflammatory cytokines interleukin-1β and -18, which are important mediators of inflammation. Abnormal activation of the inflammasome leads to many inflammatory diseases, including gout, silicosis, neurodegeneration, and genetically inherited periodic fever syndromes. Therefore, identification of small molecule inhibitors that target the inflammasome is an important step toward developing effective therapeutics for the treatment of inflammation. Here, we show that the herbal NF-κB inhibitory compound parthenolide inhibits the activity of multiple inflammasomes in macrophages by directly inhibiting the protease activity of caspase-1. Additional investigations of other NF-κB inhibitors revealed that the synthetic IκB kinase-β inhibitor Bay 11-7082 and structurally related vinyl sulfone compounds selectively inhibit NLRP3 inflammasome activity in macrophages independent of their inhibitory effect on NF-κB activity. In vitro assays of the effect of parthenolide and Bay 11-7082 on the ATPase activity of NLRP3 demonstrated that both compounds inhibit the ATPase activity of NLRP3, suggesting that the inhibitory effect of these compounds on inflammasome activity could be mediated in part through their effect on the ATPase activity of NLRP3. Our results thus elucidate the molecular mechanism for the therapeutic anti-inflammatory activity of parthenolide and identify vinyl sulfones as a new class of potential therapeutics that target the NLRP3 inflammasome.

PMC