Analysis of 16S rRNA databases showed the preferences of Binatota to terrestrial and freshwater ecosystems, hydrocarbon-rich habitats, and sponges, supporting their potential role in mitigating methanol and methane emissions, breakdown of alkanes, and their association with sponges. Our results expand the lists of methylotrophic, aerobic alkane-degrading, and pigment-producing lineages. We also highlight the consistent encountering of incomplete biosynthetic pathways in microbial genomes, a phenomenon necessitating careful assessment when assigning putative functions based on a set-threshold of pathway completion.The recent leveraging of genome-resolved metagenomics has generated an enormous number of genomes from novel uncultured microbial lineages yet left many clades undescribed. Here, we present a global analysis of genomes belonging to Binatota (UBP10), a globally distributed, yet-uncharacterized bacterial phylum. All orders in Binatota encoded the capacity for aerobic methylotrophy using methanol, methylamine, sulfomethanes, and chloromethanes as the substrates. Methylotrophy in Binatota was characterized by order-specific substrate degradation preferences, as well as extensive metabolic versatility, i.e., the utilization of diverse sets of genes, pathways, and combinations to achieve a specific metabolic goal. The genomes also encoded multiple alkane hydroxylases and monooxygenases, potentially enabling growth on a wide range of alkanes and fatty acids. Pigmentation is inferred from a complete pathway for carotenoids  production. Further, the majority of genes involved in bacteriochlorophyll  Distinct strategies are employed for the analysis of the deluge of obtained genomes. Site- or habitat-specific studies focus on spatiotemporal sampling of a single site or habitat of interest. Function-based studies focus on genomes from single or multiple habitats to identify and characterize organisms involved in a specific process, e.g., cellulose degradation  or sulfa efforts , 10. The efforts , as well efforts , 13. As Candidate phylum UBP10 has originally been described as one of the novel lineages recovered from a massive binning effort that reconstructed thousands of genomes from publicly available metagenomic data sets . UBP10 hn = 2), Binatales (n = 48), HRBin30 (n = 7), UBA1149 (n = 9), UBA9968 (n = 34), UBA12105 (n = 1), and UTPRO1 (n = 7), encompassing 12 families and 24 genera  and 22 high-quality  genomes, as defined by MIMAG standards . Binatot4 genera . 16S rRN classes . RDP II-bacteria .10.1128/mBio.00985-21.7TABLE S1Table S1, XLSX file, 0.02 MB.Binatota genomes used in this study number, their GTDB classification, and the corresponding classification in Silva and RDP databases, the source from which they were obtained, and the calculated Binatota abundances in metagenomes with available contig coverage data. Download Copyright © 2021 Murphy et al.2021Murphy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00985-21.8TABLE S2Table S2, XLSX file, 0.02 MB.Sequencing statistics for the genomic bins used in this study. Download Copyright © 2021 Murphy et al.2021Murphy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00985-21.9TABLE S3Table S3, XLSX file, 0.02 MB.General genomic features of the studied genomes. Download Copyright © 2021 Murphy et al.2021Murphy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mno), typically associated with Gram-positive methylotrophic bacteria (Actinobacteria and Bacillus methanolicus)  , was thecyclales , was enccyclales . All latcyclales .mau) was more common, with mauA and mauB enzyme subunits encoded in Binatales, HRBin30, UBA1149, UBA12105, and UTPRO1 (mauC) is the most probable electron acceptor for methylamine dehydrogenase (mau cluster) carried the full complement of genes for methylamine oxidation via the indirect glutamate pathway  involved in the degradation of dimethyl sulfone to MSA with the concomitant release of formaldehyde. Three of these nine genomes also encoded alkane sulfonic acid monooxygenase (ssuD), which will further degrade the MSA to formaldehyde and sulfite. Degradation of DMS via DMS monooxygenase (dmoA) to formaldehyde and sulfide was encountered in 13 genomes . Further, one Binatales genome encoded the dso system (enzyme class [EC]: 1.14.13.245) for DMS oxidation to dimethyl sulfone, which could be further degraded to MSA as explained above , and dimethyl sulfide (DMS). Nine genomes  encoded dimethyl sulfone monooxygenase (S-transferase (dcmA) capable of converting dichloromethane to formaldehyde.One Bin18 genome encoded the specific dehalogenase/glutathione Proteobacteria, Verrucomicrobia, and “Candidatus Methylomirabilis” (NC10)  methanotrophs (bmoA sequences (putative butane monooxygenase gene A) from Actinobacteria  and SAR324 (“Candidatus Lambdaproteobacteria”) (Nocardioides sp. strain CF8 demonstrated its capacity to oxidize short-chain (C2 to C4) hydrocarbons, but not methane, via its CuMMO, and its genome lacked methanol dehydrogenase homologues  3D model (Protein Data Bank ID: 3RGB) revealed a heterotrimeric structure (α3β3γ3) with the 7, 2, and 5 alpha helices of the PmoA, PmoB, and PmoC subunits, respectively, as well as the beta sheets characteristic of PmoA and PmoB subunits , thought to coordinate the Cu cofactor, were identified in all TUSC-affiliated and SAR324/Actinobacteria-affiliated Binatota CuMMO sequences (Actinobacteria/SAR324-type Binatota genomes using Methylococcus capsulatus (Bath) PmoB subunit (Protein Data Bank [PDB] ID: 3RGB) predicted the binding pockets for Cu in Binatota sequences (Genes encoding copper membrane monooxygenases (CuMMOs), a family of enzymes that includes particulate methane monooxygenase (pMMO), were identified in orders Bin18 (2/2 genomes) and Binatales (9/48 genomes) (” (NC10) –23  –. In addiive site , 25. Phynotrophs  (2 Binatcteria”) , 28  . Memberscteria”) . Previoucteria”) . Binatotmologues . Such dasubunits . Recentlsubunits . There hsubunits –33. Regaequences . Modelinequences .10.1128/mBio.00985-21.2FIG S1Methylococcus capsulatus (PDB ID: 3RGB). The alignment is showing conserved residues (red highlight). Of particular importance are the three conserved histidine residues His33, His137, and His139 (shown with a blue rectangle), thought to coordinate the Cu cofactor. Numbering follows the Methylococcus capsulatus strain Bath PmoB subunit (PDB: 3RGB). Alignment was created using the ENDscript webserver (http://espript.ibcp.fr/ESPript/ESPript/). (B and C) Predicted Cu methane monooxygenase (PmoABC) 3D structure (grey) from a cluster 2 TUSC-affiliated Binatota genome  and an Actinobacteria/SAR324-affiliated Binatota genome , both superimposed on CuMMO from the model methanotroph Methylococcus capsulatus strain Bath (PDB: 3RGB) (green) with a global model quality estimate of 0.7 and 0.62, respectively, and a quaternary structure quality score of 0.57 and 0.55, respectively. Download FIG S1, PDF file, 1.4 MB.(A) Alignment of the PmoB subunit of the 11 copper membrane monooxygenases predicted in Binatota genomes to PmoB from Copyright © 2021 Murphy et al.2021Murphy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 1 oxidation to formaldehyde, formaldehyde oxidation to CO2, and formaldehyde assimilation. Formaldehyde generated by C1 substrates oxidation is subsequently oxidized to formate and eventually CO2. Multiple pathways for formaldehyde oxidation to formate were identified in all Binatota orders , the H4F-linked pathway , the glutathione-independent formaldehyde dehydrogenase (fdhA), and the glutathione-dependent formaldehyde . Also shown is the distribution of the NAD-dependent formate dehydrogenase (EC: 1.17.1.9) (fdh) for formate oxidation. (B) Overview of the pathways for formaldehyde assimilation via the serine cycle (left) and glyoxylate regeneration via the ethylmalonyl-CoA pathway and the glyoxylate shunt (GS) (right). Names of enzymes are shown in red and their distribution in the Binatota genomes from different orders is shown in the heatmap in panel C. glyA, glycine hydroxymethyltransferase [EC: 2.1.2.1]; sgaA, serine-glyoxylate transaminase [EC: 2.6.1.45]; hprA, glycerate dehydrogenase [EC: 1.1.1.29]; gck, glycerate 2-kinase [EC: 2.7.1.165]; ppc, phosphoenolpyruvate carboxylase [EC: 4.1.1.31]; pckA, phosphoenolpyruvate carboxykinase; mdh, malate dehydrogenase [EC: 1.1.1.37]; mtkA/B, malate-CoA ligase [EC: 6.2.1.9]; mcl, malyl-CoA/(S)-citramalyl-CoA lyase [EC: 4.1.3.24 4.1.3.25]; aceA, isocitrate lyase [EC: 4.1.3.1]; aceB, malate synthase [EC: 2.3.3.9]; phbB, acetoacetyl-CoA reductase [EC: 1.1.1.36]; croR, 3-hydroxybutyryl-CoA dehydratase [EC: 4.2.1.55]; ccr, crotonyl-CoA carboxylase/reductase [EC: 1.3.1.85]; epi, methylmalonyl-CoA/ethylmalonyl-CoA epimerase [EC: 5.1.99.1]; ecm, ethylmalonyl-CoA mutase [EC: 5.4.99.63]; mcd, (2S)-methylsuccinyl-CoA dehydrogenase [EC: 1.3.8.12]; mch, 2-methylfumaryl-CoA hydratase [EC: 4.2.1.148]; mut, methylmalonyl-CoA mutase [EC: 5.4.99.2]; mcmA1/A2, methylmalonyl-CoA mutase [EC: 5.4.99.2]. Abbreviations: PEP, phosphoenol pyruvate; OAA, oxaloacetate. Download FIG S2, PDF file, 0.2 MB.Formaldehyde oxidation and assimilation capabilities encoded by Binatota genomes. (A) Heatmap of the distribution of formaldehyde oxidation genes in Binatota genomes from different orders. The heatmap colors (as explained in the key) correspond to the percentage of genomes in each order encoding a homologue of the gene in the column header. Shown are the different routes of formaldehyde oxidation, including the (myco)thiol-dependent formaldehyde dehydrogenase Copyright © 2021 Murphy et al.2021Murphy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Actinobacteria/SAR324-affiliated CuMMO to oxidize C1 to C5 alkanes and C1 to C4 alkenes as described above, some Binatota genomes encoded propane-2-monoxygenase (prmABC), an enzyme mediating propane hydroxylation in the 2-position yielding isopropanol. Several genomes also encoded medium-chain-specific alkane hydroxylases, e.g., homologues of the nonheme iron alkB (ladA homologues (enzyme class [EC]: 1.14.14.28)  known to have a broad substrate specificity for medium-chain-length (C3 to C10) mono- and dihaloalkanes, resulting in the production of their corresponding primary alcohol and haloalcohols, respectively    [EC: 1.1.1.80]; acmA, acetone monooxygenase (methyl acetate-forming) [EC: 1.14.13.226]; acmB, methyl acetate hydrolase [EC: 3.1.1.114]; aldehyde dehydrogenase (NAD+) [EC: 1.2.1.3]; acetaldehyde dehydrogenase (acetylating) [EC: 1.2.1.10]; acdAB, acetate-CoA ligase (ADP-forming) [EC: 6.2.1.13]; acs, acetyl-CoA synthase [EC: 2.3.1.169]; atoAD, acetate-CoA/acetoacetate CoA-transferase [EC: 2.8.3.8 2.8.3.9]; medium-chain acyl-CoA synthetase [EC: 6.2.1.2]; fadD, long-chain acyl-CoA synthetase [EC: 6.2.1.3]; pccA, propionyl-CoA carboxylase alpha chain [EC: 6.4.1.3]; epi, methylmalonyl-CoA/ethylmalonyl-CoA epimerase [EC: 5.1.99.1]; mut, methylmalonyl-CoA mutase [EC: 5.4.99.2]; mcl, malyl-CoA/(S)-citramalyl-CoA lyase [EC: 4.1.3.24 4.1.3.25]; mch, 2-methylfumaryl-CoA hydratase [EC: 4.2.1.148]; mct, 2-methylfumaryl-CoA isomerase [EC: 5.4.1.3]; meh, 3-methylfumaryl-CoA hydratase [EC: 4.2.1.153]; smtAB, succinyl-CoA:(S)-malate-CoA-transferase subunit A [EC: 2.8.3.22]; prpB, methylisocitrate lyase [EC: 4.1.3.30]; prpC, 2-methylcitrate synthase [EC: 2.3.3.5]; prpD, 2-methylcitrate dehydratase [EC: 4.2.1.79]; bcd, butyryl-CoA dehydrogenase [EC: 1.3.8.1]; acd, acyl-CoA dehydrogenase [EC: 1.3.8.7]; paaF, enoyl-CoA hydratase [EC: 4.2.1.17]; crt, enoyl-CoA hydratase [EC: 4.2.1.17]; paaH, 3-hydroxybutyryl-CoA dehydrogenase [EC: 1.1.1.157]; phbB, acetoacetyl-CoA reductase [EC: 1.1.1.36]; atoB, acetyl-CoA C-acetyltransferase [EC: 2.3.1.9]; fadJ, 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase/3-hydroxybutyryl-CoA epimerase [EC: 1.1.1.35 4.2.1.17 5.1.2.3]; fadA, acetyl-CoA acyltransferase [EC: 2.3.1.16]; dehH, 2-haloacid dehalogenase [EC: 3.8.1.2]; haloacetate dehalogenase [EC: 3.8.1.3]; glcDEF, glycolate oxidase [EC: 1.1.3.15]; (S)-2-hydroxy-acid oxidase [EC: 1.1.3.15]. Download FIG S3, PDF file, 0.3 MB.(A) Heatmap of the distribution of various chain-length fatty acid and haloacid degradation genes in Binatota genomes. The heatmap colors (as explained in the key) correspond to the percentage of genomes in each order encoding a homologue of the gene in the column header. (B) Propionyl-CoA degradation pathways encoded by the Binatota genomes. The methylmalonyl-CoA (MMCoA) pathway is shown in blue, while the 2-methylcitrate pathway is shown in green. In some genomes, the MMCoA pathway seems to be functional but with a slight modification (shown in purple) that includes glyoxylate assimilation and regeneration. Copyright © 2021 Murphy et al.2021Murphy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the actABCDEFG), and complex IV, as well as an F-type H+-translocating ATP synthase , 1f (22 sequences), 1i (1 sequence), and 1h (4 sequences) (2-tolerant hydrogenases) and methane (via pMMO) has been shown to occur in methanotrophic Verrucomicrobia to maximize proton-motive force generation and subsequent ATP production  . Simultaoduction . As well by pMMO  10.1128/mBio.00985-21.5FIG S4cytb/cyt1) and/or alternate cytochrome III , while genes encoding cytochrome c oxidase activities (complex IV) belonged to different families, including family A , family C , and/or cytochrome bd (cydAB). Possible electron transfer proteins between complex III  and complex IV belonging to different cytochrome c families are shown. Also shown in panel A is the distribution of the three subunits of the type I respiratory O2-tolerant H2-uptake [NiFe] hydrogenase (hyaABC) in Binatota genomes. (B) Maximum-likelihood phylogenetic tree showing the classification of the hyaA genes carried by the Binatota genomes (magenta) in relation to other [NiFe] hydrogenases. The [Fe-Fe] hydrogenase of Methanobacterium formicicum was used as the outgroup. Bootstrap support (from 100 bootstraps) is shown for branches with >50% support. Download FIG S4, PDF file, 0.3 MB.Electron transport chain in the Binatota. (A) Heatmap of the distribution of electron transport chain components in the Binatota genomes and electrons entry points from various substrates. The heatmap colors (as explained in the key) correspond to the percentage of genomes in each order carrying a homologue of the gene in the column header. All subunits of complexes I (NADH-quinone oxidoreductase [EC: 7.1.1.2]) and II (succinate dehydrogenase/fumarate reductase [EC: 1.3.5.1 1.3.5.4]) were encoded in all genomes but are shown here as single components for ease of visualization. Genes encoding quinone-cytochrome C reductase activities belonged to complex III  and oxygenated (xanthophyll) carotenoid biosynthesis capabilities. Carotenoids biosynthetic machinery in the Binatota included crtB for 15-cis-phyotene synthesis from geranylgeranyl pyrophosphate (PP), crtI, crtP, crtQ, and crtH for neurosporene and all-trans lycopene formation from 15-cis-phytone, crtY or crtL for gamma- and beta-carotene formation from all-trans lycopene, and a wide range of genes encoding enzymes for the conversion of neurosporene to spheroidene and 7,8-dihydro β-carotene, as well as the conversion of all-trans lycopene to spirilloxanthin, gamma-carotene to hydroxy-chlorobactene glucoside ester and hydroxy-γ-carotene glucoside ester, and beta-carotene to isorenieratene and zeaxanthins , third bchE (magnesium-protoporphyrin IX monomethyl ester cyclase [EC: 1.21.98.3]), and fourth bchLNB  steps were identified in the Binatota genomes (bchM (magnesium-protoporphyrin O-methyltransferase [EC: 2.1.1.11]) and the fifth bciA or bicB  or bchXYZ (chlorophyllide a reductase [EC 1.3.7.15]) steps were absent (a (BChl a) formation from chlorophyllide a (bchXYZ (chlorophyllide a reductase [EC 1.3.7.15]) and bchF (chlorophyllide a 31-hydratase [EC 4.2.1.165]) were not identified, while genes encoding bchC (bacteriochlorophyllide a dehydrogenase [EC 1.1.1.396]), bchG (bacteriochlorophyll a synthase [EC: EC: 2.5.1.133]), and bchP (geranylgeranyl-bacteriochlorophyllide a reductase [EC 1.3.1.111]) were present in most genomes (c (BChl c) and d (BChl d) formation from chlorophyllide a (bciC (chlorophyllide a hydrolase [EC: 3.1.1.100]) and bchF (chlorophyllide a 31-hydratase [EC: 4.2.1.165]) or bchV (3-vinyl bacteriochlorophyllide hydratase [EC: 4.2.1.169]) were not identified, while genes for bchR [bacteriochlorophyllide d C-12(1)-methyltransferase (EC: 2.1.1.331)], bchQ [bacteriochlorophyllide d C-8(2)-methyltransferase (EC: 2.1.1.332)], bchU (bacteriochlorophyllide d C-20 methyltransferase [EC: 2.1.1.333]), and bchK (bacteriochlorophyll c synthase [EC: 2.5.1.-]) were identified  and 1,213 (IMG/M) 16S rRNA genes affiliated with the Binatota orders were identified  are shown on the y axis (B). Further subclassifications for each environment are shown for (C) terrestrial, (D) freshwater, (E) marine, (F) host-associated, and (G) engineered environments. Details, including GenBank accession number of hit sequences, are shown in Extended Data 3. Download FIG S5, PDF file, 0.4 MB.(A) Maximum-likelihood phylogenetic tree based on the 16S rRNA gene representatives from six Binatota orders with representative hit sequences (number of sequences in parentheses following the order name) from the IMG and NCBI-nt databases identified by Blastn. Orders are color coded following the color scheme in Copyright © 2021 Murphy et al.2021Murphy et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In addition to the 16S rRNA-based analysis, we queried the data sets from which a Binatota MAG was binned using the sequence of their ribosomal protein S3 and estimating the Binatota relative abundance as the number of reads mapped to contigs with a Binatota ribosomal protein S3 as a percentage of the number of reads mapped to all contigs encoding a ribosomal protein S3 gene. Results showed relative abundances ranging between 0.1 and 10.21% (average 3.84 ± 3.21%) .Aplysina aerophoba. Analysis of the 16S rRNA data set suggests a notable association between Bin18 and sponges, with relatively high host-associated sequences , suggesting its widespread distribution beyond a single sponge species. The absolute majority of order Binatales sequences  were of a terrestrial origin  in Bin18 and Binatales , Firmicutes  (Verrucomicrobia (Candidatus Methylomirabilis” (NC10)  is especially notable, given the global magnitude of methane emissions and the relatively narrower range of organisms   , Verrucomicrobia , and “Ca” (NC10) . Further” (NC10) , 44, for” (NC10) , and met” (NC10) , in the  [NC10])  capable chanisms . All 11 1 oxidation to formaldehyde, formaldehyde oxidation to CO2, and formaldehyde assimilation. Within the world of methylotrophs, a wide array of functionally redundant enzymes and pathways have been characterized that mediate various reactions and transformations in such modules. In addition, multiple combinations of different modules have been observed in methylotrophs, with significant variations existing even in phylogenetically related organisms. Our analysis demonstrates that such metabolic versatility indeed occurs within the methylotrophic modules of Binatota. While few phylum-wide characteristics emerged, e.g., utilization of serine pathway for formaldehyde assimilation, absence of H4MPT-linked formaldehyde oxidation, and potential utilization of PEP carboxykinase (pckA) rather than PEP carboxylase (ppc) for CO2 entry to the serine cycle, multiple order-specific differences were observed, e.g., XoxF-type methanol dehydrogenase encoded by Bin18 and Binatales genomes, MDH2-type methanol dehydrogenase encoded by UBA1149 genomes, absence of methanol dehydrogenase homologues in HRBin30 genomes, absence of methylamine oxidation in order UBA9968, and potential utilization of the ethylmalonyl-CoA pathway for glyoxylate regeneration by the majority of the orders versus the glyoxylate shunt by UBA9968.As previously noted , methyloprmABC, propane monooxygenase), medium- , and long-chain alkanes (ladA) identified  for their activation and conversion to central metabolites  as well as order-specific  habitat preferences ; Fig. S5osystems , and we Within the phylum Binatota, it appears that orders HRBin30 and UBA1149 are abundant in thermal vents, thermal springs, and thermal soils, suggesting a specialization to high-temperature habitats . The preThe recovery of Binatota genomes from certain lakes could be a reflection of the high gaseous load in such lakes. Multiple genomes and a large number of Binatota-affiliated 16S rRNA sequences were binned and identified from Lake Kivu, a meromictic lake characterized by unusually high concentrations of methane . Biotica57–62–Finally, the occurrence and apparent wide distribution of members of the Binatota in sponges, particularly order Bin18, are notable and could possibly be viewed in terms of the wider symbiotic relationship between sponges and their microbiome. Presence of hydrocarbon degraders , 67, incAlphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria (including methano- and methylotrophs) and Bacteroidetes, Deinococcus, Thermus, Deltaproteobacteria, Firmicutes, Actinobacteria, Planctomycetes, and Archaea, e.g., Halobacteriaceae and Sulfolobus. Here, carotenoids could serve as antioxidants  are not pigmented. Indeed, root-associated facultative methylotrophs of the genus Methylobacterium have traditionally been referred to as “pink-pigmented facultative methylotrophs” and are seen as an integral part of root ecosystems . While such a pattern is tempting to propose phototrophic capacities in the Binatota, the consistent absence of critical genes , coupled with our inability to detect reaction center-encoding genes, prevents such a proclamation. Identification of a single or few gene shrapnel from the chlorophyll biosynthesis pathway in microbial genomes is not unique. Indeed, searching the functionally annotated bacterial tree of life AnnoTree  could possibly be encoded by general methyltransferases (EC: 2.1.1.-), the missing bciC (EC: 3.1.1.100) could possibly be encoded by general hydrolases (EC: 3.1.1.-), and the missing bchF (EC: 4.2.1.165) or bchV (EC: 4.2.1.169) could possibly be encoded by general hydratases (EC: 4.2.1.-).Accordingly, we put forward three scenarios to explain the proposed relationship between Binatota and phototrophy. The most plausible scenario, in our opinion, is that members of the Binatota are pigmented nonphotosynthetic organisms capable of carotenoid production but incapable of chlorophyll production and lack a photosynthetic reaction center. The second scenario posits that members of the Binatota are indeed phototrophs, possessing a complete pathway for chlorophyll biosynthesis and a novel type of reaction center that is bioinformatically unrecognizable. A minimal photosynthetic electron transport chain, similar to that of antiacus , with thantiacus , it has  species . A third12. Such auxotrophies are common in the microbial world and could be alleviated by nutrient uptake from the outside environment  were downloaded as assemblies from NCBI. In addition, 128 metagenome-assembled genomes with the classification “Bacteria;UBP10” were downloaded from the IMG/M database (April 2020). These genomes were recently assembled from public metagenomes as part of a wider effort to generate a genomic catalogue of Earth’s microbiome (n = 108) were retained for further analysis (All genomes classified as belonging to the Binatota in the Genome Taxonomy Database (GTDB) database , with the arbitrary cutoffs 56% and 68% for family and genus, respectively.Taxonomic classifications followed the Genome Taxonomy Database (GTDB) release r89 , 87 and 1, alkanes, and fatty acids metabolism, C1 assimilation, [NiFe] hydrogenases, electron transport chain complexes, and carotenoid and chlorophyll biosynthesis. To build the HMM profiles, Uniprot reference sequences for all genes with an assigned KO number were downloaded and aligned using Clustal-omega  were obtained from the pfam database . Additionally, HMM profiles were built for PscABCD (Chlorobia-specific), PshA/B (Heliobacteria-specific)  and reaction center type 2  , and theoflexota . The HMMoflexota , 99 was oflexota  to checkoflexota , 102, weoflexota . All ide2-tolerant H2-uptake [NiFe] hydrogenase large subunit (HyaA) were classified using the HydDB web tool (All sequences identified as belonging to the respiratory Oweb tool .Methylococcus capsulatus strain Bath (PDB: 3RGB) and to predict tertiary structure models. Predicted models were superimposed on the template enzyme in PyMol . Modeling of the active site was conducted similarly. The dicopper-binding site proposed for Methylococcus capsulatus strain Bath pMMO (3RGB) was used. Alignment of Binatota PmoB sequences with reference Methylococcus capsulatus strain Bath PmoB was performed with Clustal-omega . Two databases were searched: (i) GenBank nucleotide (nt) database (accessed in July 2020) using a minimum identity threshold of 90%, ≥80% subject length alignment for near full-length query sequences or ≥80% query length for non-full-length query sequences, and a minimum alignment length of 100 bp and (ii) The IMG/M 16S rRNA public assembled metagenomes using a cutoff E value of 1e−10, percentage similarity of ≥90%, and either ≥80% subject length for full-length query sequences or ≥80% query length for non-full-length query sequences. Hits satisfying the above criteria were further trimmed after alignment to the reference sequences from each order using Clustal-omega and inserted into maximum-likelihood phylogenetic trees in FastTree . The ecological distribution for each of the Binatota orders was then deduced from the environmental sources of its hits. All environmental sources were classified according to the GOLD ecosystem classification scheme . We alsohttps://github.com/ChelseaMurphy/Binatota. Maximum-likelihood trees (https://itol.embl.de/shared/1WgxEjrQfEYWk. Maximum-likelihood trees for chlorophyll biosynthesis genes are available at https://itol.embl.de/shared/34y3BUHcQd7Lh.Genomic bins, predicted proteins, and extended data for od trees  can be a


OBJECTIVES/GOALS: Using multi-state discharge data, to identify predictors of frequent emergency department (ED) use among the homeless patients seen in emergent care, and to compare frequent versus less frequent homeless ED users for their risk of serious health services utilization outcomes. METHODS/STUDY POPULATION: Based on the State Emergency Department Database and the State Inpatient Database, homeless individuals  who made at least one ED visit in four states  in 2014. In this retrospective cross-sectional analysis, patient-level demographic and clinical factors were assessed as predictors for increased ED use. Risks of opioid overdose, opioid-related hospital admission/ED visit, in-hospital mortality, mechanical ventilation, and number of hospitalizations were compared between individuals with 4 or more vs. 2-3 vs. 1 ED visit(s), adjusting for potential confounders including hospital fixed effects . RESULTS/ANTICIPATED RESULTS: Higher rates of ED use were associated with Medicare coverage <65; primary diagnosis of alcohol abuse, asthma, or abdominal pain; and co-morbidity of alcohol abuse, psychoses, or chronic pulmonary disease. Individuals with ≥4 visits had significantly higher adjusted risk of opioid overdose (3.7% vs. 1.2% vs. 1.0%), opioid-related hospitalizations/ED visits (17.9% vs. 8.5% vs. 6.6%), mechanical ventilation (9.8% vs. 7.0% vs. 4.7%), and greater # of hospitalizations (3.2 vs. 1.3 vs. 0.8) compared to individuals with 2-3 or 1 ED visit. Individuals with ≥4 and 2-3 ED visits had similar but increased risks of in-hospital mortality compared to individuals with 1 ED visit (2.8% vs. 2.8% vs. 2.3%). DISCUSSION/SIGNIFICANCE OF IMPACT: Homeless patients who were high ED users were more likely to be hospitalized and have other adverse outcomes. These findings encourage targeted interventions (i.e. housing) for the high-utilizer homeless population to reduce the burden of serious outcomes and costs for the patient and society.


BCOR and BCORL1. We report a distinct co-mutational pattern that suggests a role in disease progression rather than initiation, especially affecting mechanisms of DNA-methylation. Further, we found loss-of-function mutations of BCOR to be independent markers of poor outcomes in multivariable analysis. Therefore, loss-of-function mutations of BCOR need to be considered for AML management, as they may influence risk stratification and subsequent treatment allocation.Acute myeloid leukemia (AML) is a genetically heterogeneous disease. Clinical phenotypes of frequent mutations and their impact on patient outcome are well established. However, the role of rare mutations often remains elusive. We retrospectively analyzed 1529 newly diagnosed and intensively treated AML patients for mutations of BCL6 corepressor (BCOR) and its homolog, the BCL6 corepressor-like 1 (BCORL1), have been reported to be rare but recurrent mutations in AML. Previously, smaller studies have reported conflicting results regarding impacts on outcomes. Here, we retrospectively analyzed a large cohort of 1529 patients with newly diagnosed and intensively treated AML. BCOR and BCORL1 mutations were found in 71 (4.6%) and 53 patients (3.5%), respectively. Frequently co-mutated genes were DNTM3A, TET2 and RUNX1. Mutated BCORL1 and loss-of-function mutations of BCOR were significantly more common in the ELN2017 intermediate-risk group. Patients harboring loss-of-function mutations of BCOR had a significantly reduced median event-free survival : 1.005–2.134), p = 0.047), relapse-free survival (HR = 1.904 (95%-CI: 1.163–3.117), p = 0.01), and trend for reduced overall survival (HR = 1.495 (95%-CI: 0.990–2.258), p = 0.056) in multivariable analysis. Our study establishes a novel role for loss-of-function mutations of BCOR regarding risk stratification in AML, which may influence treatment allocation.Acute myeloid leukemia (AML) is characterized by recurrent genetic events. The  Acute myeloid leukemia (AML) is a genetically heterogeneous disease . In the BCOR) gene, and its homolog, the BCL6 corepressor-like 1 (BCORL1) gene, are located on chromosomes Xp11.4 and Xq26.1, respectively [The BCL6 corepressor  syndrome in heterozygous females, and prenatal death in hemizygous males [BCOR (mBCOR) and BCORL1 (mBCORL1) both occur in 4–6% of patients, and an association with poor outcomes has been suggested [Since BCOR is vital in ectodermal and mesenchymal differentiation, germline loss-of-function mutations in us males . Somaticus males ,16,17,18us males . It has us males ,20, chrous males , clonal us males , myelodyus males ,23,24,25us males ,26,27,28We retrospectively analyzed a multi-center cohort of 1529 AML patients. Eligibility criteria were newly diagnosed AML according to WHO definitions , AML200330). Remission and survival criteria were defined according to ELN2017 recommendations [AML was defined as de novo when neither previous malignancy nor previous treatment with chemo- and/or radiotherapy was reported. When myeloid neoplasms were documented prior to AML diagnosis, AML was defined as secondary (sAML). Prior exposure to chemo- and/or radiotherapy before the initial diagnosis defined therapy-associated AML (tMN). Early death was defined as death by any cause within 30 days of the initial diagnosis  using a TruSight Myeloid Sequencing Panel  was performed on pre-treatment bone marrow or peripheral blood, targeting 54 genes  associatn detail ,35. A DNWe compared categorical variables between groups using the chi-squared test, while continuous variables were compared using the Kruskal–Wallis test. The Kaplan–Meier method was used to estimate survival probabilities. The logrank test was used to compare survival time distributions between groups. Cox regression was used to estimate univariate and adjusted hazard ratios. For the binary endpoint of complete remission, logistic regression models were fitted to estimate univariate and adjusted odds ratios. A significance level of 0.05 was used to determine statistical significance. Calculations were performed in R 4.0.3.n = 1529), we found mBCOR in 71 (4.6%) and mBCORL1 in 53 (3.5%) patients. Twelve patients (0.8%) concomitantly harbored both mBCOR and mBCORL1. The median age for the entire cohort was 55 years (Interquartile range (IQR): 44–64). Median variant allele frequency (VAF) for mBCOR and mBCORL1 was 48% (range: 7–100%) and 47% (range: 5–100%), respectively (BCOR (87%) and mBCORL1 (83%) carried two or more additional mutations in other driver genes, while only one patient in each group had no other co-mutations detected by the panel , RUNX1 (30%), TET2 (23%), NRAS (20%), BCORL1 (17%), and STAG2 (17%). Low mutation frequencies were detected for other genes frequently mutated in AML, such as FLT3 , NPM1 (11%), and TP53 (4%). Similar to mBCOR, in mBCORL1 AML the majority of mutations had a loss-of-function effect , RUNX1 (34%), FLT3-ITD (25%), BCOR (23%), and TET2 (23%).However, due to the unknown effect of protein function, these mutations were classified as UFs (unknown functions) for sub-analysis. The co-mutational landscape of mBCOR AML F was chaect 60%, D and werect 60%, E were DNBCORL1 were more prevalent in females than in males , while for mBCOR no such association was observed. For both mBCOR and mBCORL1 no statistically significant associations were detected for age at diagnosis, the presence of a complex karyotype, hemoglobin levels, or platelet counts. With respect to disease status, the rate of sAML was significantly higher amongst patients harboring mBCOR than amongst wtBCOR patients , while no specific association with mutation type (LOF or UF) or mBCORL1 was found regarding AML type. Outcomes did not differ for patients with sAML and mBCOR or mBCORL1 compared to patients with de novo AML. In the ELN2017 intermediate-risk group we found a higher proportion of LOF of mBCOR compared to UF mBCOR and wtBCOR , as well as mBCORL1 compared to wtBCORL1 . Median white blood cell (WBC) counts in LOF mBCOR AML were significantly lower compared to UF mBCOR and wtBCOR (p < 0.001), while mBCORL1 showed no significant association with WBC. BCOR (LOF and UF) and mBCORL1, respectively.Mutations of BCOR and mBCORL1 were not associated with the rate of complete remission (CR) (mBCOR: OR = 0.781 (95%-CI: 0.469–1.301), p = 0.342 and mBCORL1: OR = 0.795 (95%-CI: 0.442–1.432), p = 0.445) or with ED30 (mBCOR: OR = 0.679 (95%-CI: 0.209–2.199), p = 0.518 and mBCORL1: OR = 1.288 (95%-CI: 0.454–3.649), p = 0.634). In contrast, mBCOR was associated with lower median measures of event-free survival (EFS) (2.8 months (95%-CI: 1.7–8.5) vs. 7.6 months (95%-CI: 6.8–8.5), HR = 1.485 (95%-CI: 1.147–1.922), p = 0.003; p = 0.026; p = 0.095; BCOR in general was not independently associated with EFS (HR = 1.243 (95%-CI: 0.89–1.736), p = 0.202), RFS (HR = 1.407 (95%-CI: 0.93–2.129), p = 0.106), and OS (HR = 1.216 (95%-CI: 0.846–1.748), p = 0.292).With respect to clinical outcomes, mBCOR, compared to UF mBCOR and wtBCOR, we found in both univariate and multivariable analysis significantly reduced median EFS (LOF mBCOR: 1.9 months (95%-CI: 1.4–8.0) vs. UF variant of mBCOR: 4.9 months (95%-CI: 1.676–n.a.) vs. wild-type BCOR: 7.5 months (95%-CI: 6.8–8.5), multivariable HR of LOF compared to wtBCOR = 1.464 (95%-CI: 1.005–2.134), p = 0.047, BCOR: 8.8 months (95%-CI: 7.3–24.0) vs. UF variant of mBCOR: 17.7 months (95%-CI: 8.317–n.a.) vs. wild-type BCOR: 18.7 months (95%-CI: 16.3–23.3), multivariable HR of LOF compared to wtBCOR = 1.904 (95%-CI: 1.163–3.117), p = 0.01, BCOR variants in univariate analysis (LOF mBCOR: 11.6 months (95%-CI: 8.5–33.2) vs. UF variant of mBCOR: 14.2 months (95%-CI: 11.045–n.a.) vs. wild-type BCOR: 18.4 months (95%-CI: 16.7–21.4), HR of LOF compared to wtBCOR = 1.409 (95%-CI: 1.010–1.965), p = 0.044), and a strong trend for reduced median OS in a multivariable analysis (HR = 1.495 (95%-CI: 0.990–2.258), p = 0.056, DNMT3A, TET2, and RUNX1 did not significantly affect HR. However, in order to precisely account for multiple interactions, an even larger cohort is needed, due to the rarity of LOF BCOR.For patients harboring LOF mBCORL1 in general compared to wtBCORL1, median EFS (3.6 months (95%-CI: 1.9–9.8) vs. 7.5 months (95%-CI: 6.7–8.3), HR = 1.204 (95%-CI: 0.885–1.639), p = 0.236; p = 0.263; p = 0.654; BCORL1 showed significantly reduced EFS (LOF mBCORL1: 3.5 months (95%-CI: 1.1–10.1) vs. UF mBCORL1: 6.2 months (95%-CI: 1.9–not reached) vs. wtBCORL1: 7.5 months (95%-CI: 6.7–8.3), HR = 1.521 (95%-CI: 1.045–2.213), p = 0.028); however, in a multivariable analysis adjusting for age, AML type, and ELN2017 risk, this was not statistically significant.In AML with mBCOR and mBCORL1 regarding outcomes in different ELN2017 risk groups. Since few patients with mBCOR and mBCORL1 were in the ELN2017 favorable  or ELN2017 adverse-risk groups , we focused on the ELN2017 intermediate-risk group . In ELN2017 intermediate-risk AML with mBCOR, median EFS and OS did not differ compared to wtBCOR, while there was a trend of lower median RFS in a univariate model (11.9 months (95%-CI: 7.0–24.0) vs. 14.8 months (95%-CI: 12.5–20.3), HR = 1.446 (95%-CI: 0.977–2.139), p = 0.065), as well as in a multivariable model adjusted for age and AML type (HR = 1.637 (95%-CI: 0.995–2.693), p = 0.052). In ELN2017 intermediate-risk AML with mBCORL1, median EFS and RFS did not differ, but there was a trend of lower median OS in a univariate model (14.9 months (95%-CI: 7.3–24.4) vs. 17.0 months (95%-CI: 14.0–20.6), HR = 1.401 (95%-CI: 0.954–2.058), p = 0.086), while in a multivariable model adjusted for age and AML type this difference was not statistically significant. There was no significant association between LOF of mBCOR or mBCORL1 and ELN2017 risk groups regarding outcomes.We did not find statistically significant differences in mBCOR and BCORL1. The respective proportions of mBCOR and mBCORL1 in the cohort were comparable to those reported in recent studies [BCOR with LOF and mBCORL1 to be more prevalent in patients in the ELN2017 intermediate-risk group [BCOR was significantly higher than in their wild-type counterparts, confirming previous reports [BCORL1 were significantly more prevalent in females than in males, as has been previously suggested [BCOR with LOF had significantly lower WBC, and a trend for lower peripheral blood blast counts. Mutations of BCOR frequently co-occurred with mutations of DNMT3A, RUNX1, TET2, NRAS, and BCORL1. Since both BCOR and DNMT3A function as epigenetic modifiers [BCOR and mutations of TET2, another epigenetic modifier [BCOR in our cohort, has been reported to induce MDS [BCOR and other essential regulators of normal myeloid development appears to be a factor that may promote leukemogenesis. Recent studies suggest, however, that perturbations of BCOR function alone do not suffice to induce malignant transformation [BCOR following an oncogenic event, e.g., MDS driver mutation triggers disease progression towards AML, as suggested by the higher frequency of sAML among mBCOR patients. Accordingly, in our cohort of patients with mBCOR AML, the majority had at least two co-mutations, while only one patient harbored no other co-mutations targeted by our panel (BCOR, co-mutations of mBCORL1 were RUNX1, DNMT3A, and TET2, as well as BCOR and FLT3-ITD, which were only rarely co-mutations of mBCOR. Again, the majority of patients harboring mBCORL1 had at least two other co-mutations, and only one patient had no other co-mutations revealed by our panel (BCOR, this suggests a potential interplay of impaired BCORL1 function with other dysfunctional mechanisms of DNA methylation, cell differentiation, and signal transduction in leukemogenesis.We analyzed a large cohort of newly diagnosed and intensively treated AML patients according to their mutational status of  studies ,21,26,27sk group . The pro reports . Mutatioodifiers , a synerodifiers ,40. In mmodifier  frequentduce MDS ,42. Thisduce MDS ,43. Therormation ,21,44, aur panel B. Similaur panel B. As forBCOR and mBCORL1 regarding CR rate and ED30, compared to wild-type patients. Interestingly, while we observed lower median EFS, RFS, and OS for both mBCOR and mBCORL1 in general, only LOF mutations of mBCOR were associated with significantly reduced EFS and RFS and a trend of reduced OS in multivariable testing adjusted for age, AML type, and ELN2017 risk. In MDS, Abuhadra et al. [BCOR, while general mutation status did not affect OS. Previous studies of mBCOR in AML have reported poorer outcomes to often be associated with distinct co-mutations; however, a significant association of LOF mutations of BCOR as independent markers of poor outcomes has not yet been reported in AML. Terada et al. [FLT3, and had intermediate-risk cytogenetics. Grossmann et al. [BCOR and a trend of reduced OS in normal-karyotype AML with no mutations in NPM1, FLT3-ITD, CEBPA, or MLL-PTD. However, in a validation cohort no association between mBCOR and OS was observed. Nevertheless, in both cohorts reduced EFS was detected [BCOR or SETBP1. Our findings underline the complexity of the mutational landscape of AML, where even mutational variants of rare mutations have to be considered in order to determine their clinical and prognostic effects. Future work needs to focus on the implementation of LOF mutations of BCOR in risk stratification tools for AML management.Regarding outcomes, we found no statistically significant differences between patients with ma et al.  recentlya et al.  reportedn et al.  reporteddetected . Recentldetected  reportedBCOR and mBCORL1 are rare but recurrent mutations in AML. While previous studies suggested poor outcomes for mBCOR in AML, especially in the context of co-occurring mutations, we found loss-of-function mutations of mBCOR to be independent markers of poor outcomes in AML, while mBCORL1 was not significantly associated with outcomes in multivariable testing.In conclusion, both m


Trimethoprim-sulfamethoxazole (TMP-SMX) is a high-bioavailability antibiotic associated with potentially serious adverse drug events (ADE). The objective of this study was to evaluate the safety of intravenous (IV) and oral (PO) high-dose TMP-SMX.≥ 18 years old and > 72 hours of renally adjusted high-dose TMP-SMX defined as ≥ 5 mg/kg/day of TMP. Exclusion: prophylaxis. Endpoints during treatment: hyponatremia with sodium < 135 mmol/L, hyperkalemia with potassium > 5 mmol/L, serum creatinine increase of ≥ 0.3 mg/dL or 1.5-1.9 times from baseline, and fluid overload on physical exam. Descriptive and bivariate statistics were performed.IRB-approved retrospective cohort of hospitalized patients from January 2016 to November 2020. Inclusion: Stenotrophomonas maltophilia (52% IV and 18% PO) and Pneumocystis jiroveci (16.3% IV and 62% PO). Median (IQR) days of inpatient therapy: 6 (5-7.5) PO vs. 7.5 (6-11.3) IV. Median (IQR) days of total duration: 9 (6-21.5) PO vs. 12 (7.8-14) IV (p=0.93). IV group: 88% of patients received >1 liter of D5W daily. Median (IQR) liters of D5W daily was 1 (1-1.5). 56% had a diuretic added, and 38% had a diuretic dose increase. Majority of patients (78%) on IV were taking other oral medications. 100% patients experienced any adverse event with IV vs. 70% with PO . Most common ADE in both groups: hyponatremia, hyperkalemia, and elevated creatinine. Hyponatremia: 92% with IV and 32% with PO . Edema on physical exam, an ADE specific to IV TMP-SMX, was the third most common side effect in the IV group. Relative changes from baseline in sodium, potassium, and creatinine from those who experienced hyponatremia, hyperkalemia and elevated creatinine were listed in Table 2.Each group included 50 patients (Table 1). Intensive care unit patients comprised 82% IV TMP-SMX compared to 32% PO. Most common infection: respiratory tract 86% IV and 68.1% PO. Most common organisms were Table 1. Baseline and Clinical CharacteristicsTable 2. Adverse EffectsPatients on IV TMP-SMX therapy were more likely to experience an ADE compared to PO, likely driven by the high volume of free water. Most patients on IV TMP-SMX were on other PO medications, suggesting a missed stewardship opportunity for IV to PO conversion to reduce patient harm.Susan L. Davis, PharmD, Nothing to disclose Michael P. Veve, Pharm.D., Cumberland (Grant/Research Support)Paratek Pharmaceuticals (Research Grant or Support) Rachel Kenney, PharmD, Medtronic, Inc.


Microbacterium foliorum NRRL B-24224. The 41,958-bp double-stranded DNA genome has 71 predicted protein coding genes and 1 tRNA. The lytic actinobacteriophage was extracted from soil samples collected in Stephenville, TX, and is related to cluster EB bacteriophages Didgeridoo and Lahqtemish.Microbacteriophage IndyLu was isolated from  Microbacterium foliorum NRRL B-24224 is a rod-shaped Gram-positive aerobic bacterium from the order Actinomycetales that contains no intact prophages or apparent antibacteriophage restriction-modification or CRISPR systems  (MH045566) and Lahqtemish (GenBank accession number MT889392). Auto-annotation using GLIMMER v3.02 (http://phagesdb.org/DNAMaster/), and PECAAN. Microbacteriophage IndyLu is predicted to contain 70 protein-coding genes and 1 tRNA coding for glutamine, identified using ARAGORN v1.2.38   showed gER v3.02 , 11 was ER v3.02 , DNA Mas v1.2.38  and tRNA v1.2.38 . Putativ v1.2.38 , 16 and OK318958. The raw reads are available in the SRA under accession number SRX12683423.The actinobacteriophage IndyLu genome sequence is available in GenBank under accession number


Theshort answer is to administer alpha-2 agonists slowly from admission orendotracheal intubation up to stabilized cooperative sedation. The “take home”message is as follows: a) alpha-2 agonists are jointly sympathetic deactivatorsand sedative agents; b) sympathetic deactivation implies maintaining the strokevolume and iterative assessment of volemia. Evidence-based medicine shoulddocument our propositions.Cardiac, ventilatory and kidney management in the critical care setting has beenoptimized over the past decades. Cognition and sedation represent one of thelast remaning challenges. As conventional sedation is suboptimal and as thesedation evoked by alpha-2 adrenergic agonists  represents a valuable alternative,this manuscript covers three practical topics for which evidence-based medicineis lacking: a) Switching from conventional to cooperative sedation(“switching”): the short answer is the abrupt withdrawal of conventionalsedation, immediate implementation of alpha-2 agonist infusion and the use of“rescue sedation” (midazolam bolus[es]) or “breakthrough sedation”  to stabilize cooperative sedation. b) Switching from conventional tocooperative sedation in unstable patients (e.g., refractory  Given the circulatory drawbacks in hypovolemic patients, only nicheindications are to be considered , which contradicts the“one size fits all” approach. Circulation is a major concern. In the setting ofsystolic15,16)( or diastolic17)( failure or cardiogenic pulmonary edema and a lowleft ventricular (LV) ejection fraction,18)( the sympathetic deactivation of capacitance(veins) and resistance vessels  is beneficial. Venous return is reduced, and ejectionimproves. In the hypovolemia scenario, alpha-2 agonists further reduce venous return and stroke volume (SV) and worsencirculatory distress .Alpha-2 adrenergic agonists  evoke “cooperative”, rousable sedation6,22-25)( or sleep26)( anti-inflammation31-35)$( and a reduced CCU stay.36), problems arise: a) how to switch from conventionalsedation to alpha-2 agonists (“switching”), e.g., in agitated or unstable patients,refractory delirium tremens (DT), circulatory/ventilatory distress,etc.; and b) how can alpha-2 agonists be prescribed as first-line sedatives de novoupon admission? This manuscript addresses the parasympathetic vs. sympatheticsystems, circulation, and ventilation.As alpha-2 agonists interfere with the autonomic system 46-48). Our clinical practice spanning the period of 1980 -2020 in several countries  is summarized . Physiol49)emergence delirium is encountered following deep sedation. However,is this delirium related to the pathology itself, the CCUenvironment, or conventional sedation? Moreover, b) deep sedation, bordering GA (1),is used in clinical practice for ARDS or increased intracranialpressure49)( without evidence.50), paralysis and proning.51,52)( is missing.50)the absence of a control group under adequate spontaneous breathing50)( and b) the tendency toshorten57,58)( without strong evidence50), traumatic brain injury,As most groups use cooperative sedation after conventional sedation, i.e., only whenthe patient is recovering and ready for tracheal extubation (“extubation”),switching from conventional to cooperative sedation is examined first.- Hypovolemia: See below.&), sick sinus syndrome,atrioventricular block II or III without a pacemaker.- Bradycardia (- Liver failure (Child-Pugh C): Clonidine and dexmedetomidine areexcreted through the kidney and liver, respectively. Moreover,clonidine and dexmedetomidine are useful in the scenarios of liverand kidney failure, respectively. Nevertheless, a) clonidine can beadministered in the setting of acute renal failure if renalreplacement therapy (RRT) is used, and b) dexmedetomidine can beused in the setting of liver cirrhosis.Dexmedetomidine and clonidine are sympathetic inhibitors in healthy restingsupine volunteers. In the CCU, given the increased sympathetic activity, theynormalize sympathetic hyperactivity back toward baseline, i.e., sympatheticdeactivators, with the following contraindications: no clinical relevance but is only an invitro finding.63). In contrast, clonidine p.o.allows for convenient oral administration (nonintubated patient with DT),transitioning alpha-2 agonists from i.v. dexmedetomidine to p.o. clonidine toavoid alpha-2 agonist withdrawal, etc. Sedation is achieved within 30 - 60minutes in healthy volunteers after clonidine 300µg p.o.5,12) < +1 occursimmediately before initiation of dexmedetomidine infusion23). Rescue sedation is used to achieve-2 <RASS < +123)(using either a) fentanyl infusion, followed by a propofol bolus (25 -50mg)22)( or b) midazolam  and fentanyl.23).A “ceiling” effect is reported with dexmedetomidine>1.5µg.kg-1.h-1.7) or propofol (25mg) to berepeated if necessary.65)( thesympathetic inhibition evoked by propofol68), blood pressure (BP) and cardiac output(CO).69) or a bolus of clonidine/dexmedetomidine. Consequently,severe bradycardia and hypotension may occur. To avoid such sideeffects, we used abrupt withdrawal. Abrupt withdrawal is performedduring the day shift only, starting in the earlymorning£. The prescription specifies the target (-2 <RASS < 0), the range of dose of dexmedetomidine (≤1.5µg.kg-1.h-1), the rescue versusbreakthrough sedation ,and the supplementation (neuroleptics: see refractory DT).Progressive switching and circulation: Simultaneous administration ofconventional sedation and cooperative sedation combines thesympathetic deactivation evoked by alpha-2 agonists,69)( withoutsequelae.71) led to no changein mortality (SPICE III).72), while ≈75% of dexmedetomidine patients receivedpropofol, midazolam or both.72,73)( isuseless.73)( to reassess the outcome and makethis large series72)( or CCU72).Intraoperative opioid-free anesthesia was administered:13)(4µg.kg-1/15 minutes during the induction of anesthesia for livertransplant28) is usually sufficient to cover the firstpostoperative day, with provision for opioid-free analgo-sedation andnicardipine (0.5mg to be repeated if needed). Technology addresses volume  orperfusion . If, after volume adjustment,the perfusion pressure is a concern, adjuncts  areused to counteract sympathetic deactivation. An additive effect between theincoming alpha-2 agonist and the opioid69)( should be eliminated. ii) Ifconventional GA has been administered, low-dose alpha-2 agonists will beintroduced slowly  toeffect.Intraoperative conventional GA has been administered: given premedication with analpha-2 agonist23). Second, if needed afterthis interval, neuroleptics are administered. This avoids thecognitive side effects of benzodiazepines (below: refractory DT).Midazolam is used only as rescue sedation during the switch, e.g.,to facilitate nursing.We do not use-2 < RASS < +1 as others do:82) or low-dose neuroleptics added to high-dosealpha-2 agonists .Elderly patients appear less sensitive to the sedation evoked byalpha-2 agonists than young, muscular, combative, andaddictedThe required RASS (-2 < RASS < 0) deserves comments: 7)or Behavioral Pain Scale (BPS) score, opioids22,23)( (fentanyl 0.5 - 1µg.kg-1 every 15minutes) or nonopioid analgesics can be selected. However, a) alpha-2 agonistsevoke analgognosia8)( and preserve respiratory genesis.9-11), low-dose ketamine (50mg.d-1) and tramadol(“weak” opioid: 400mg.d-1). These doses are reduced by 50 - 75%after 24 - 72 hours. This may be a consequence of accumulation orthe indifference to pain evoked by the alpha-2 agonist followingsteady-state cooperative sedation.or lidocaine 0.5mg.kg-1.h-1 infusion (loading dose: 1mg.kg-1.h-1) orketamine (0.25mgkg-1.h-1) infusion or gabapentin  or pregabalin or carbamazepine or amitriptyline. Low-dose opioids are employed as rescue analgesics, ifneeded.Once steady-state cooperative sedation is achieved, antinociception isconsidered. Patients presenting with medical conditions require littleantinociception84,85) leads to hypertension and low HR (60 -70 beats per min in a 2-year-old child) without sequelae upon reduceddexmedetomidine administration.86)..In the setting of ambulatory cardiology, very high-dose alpha-2 agonists lead toresistant hypertension (clonidine 5400 - 6000µg.d-1).90-92)( low-dose dexmedetomidine(0.7µg.kg-1.h-1) was successfully supplemented with haloperidol innonintubated patients . Dexmedetomidine achieves ≈93% satisfactory sedationlevels  and halves the CCU stay.93). Thus, a combination of drugsmanages a complex neurochemical pattern.DT involves hyperactivity or hypoactivity of several central pathways. Thesignal-to-noise ratio95)(-2 ≤ RASS ≤ 0) but“fairly alert”5)( or cognitivelyimproved24)( and thetemperature100)( and oxygen consumption(VO2)101-103) are insufficient to achieve-2 ≤ RASS ≤ 0(stringent absence of restlessness) without tremor, neuroleptics are employed assecond-line agents. When hallucinations were prominent, haloperidol  is administered. In contrast, when agitation wasprominent, loxapine (100mg x 4 p.o. or via the nasogastric tube) is selected.Neuroleptics, then alpha-2 agonists, are tapered as soon as the absence ofrestlessness without tremor is ascertained for 24 hours.104)( or low-dose midazolam (0.5mg.h-1) may beconsidered.Refractory DT patients with Gayet-Wernicke disease required clonidine 4µg.kg-1.h-1+loxapine 400mgx4 to achieve-2 ≤ RASS ≤ 0 andthe absence of tremor. To supplement a combination of high-dose alpha-2 agonist+ neuroleptic  and to avoid theadministration of higher doses of alpha-2 agonists + neuroleptics, baclofen (50- 150mg according to kidney function)93) and achieve quietness within 30 - 60 minutes. A similar regimenmay be used to transition from i.v. dexmedetomidine to oral clonidine,105)( up todiscontinuation.105) may be considered to initiate oral therapy or to transitionfrom i.v. dexmedetomidine to an oral alpha-2 agonist.Refractory DT in nonintubated patients106,107)( orrefractory111), the administration of alpha-2 agonistsis inadvisable in the setting of uncontrolled hemorrhage, septic or cardiogenicshock, etc. Indeed, for a short period of time, sympathetic activation is alifesaver in regards to control of the pathology, and exogenous vasopressorsand/or inotropes are required to maintain left ventricular perfusion pressureand/or contractility, in addition to endogenous sympathetic nervous activation.In contrast, AFTER controls acute cardioventilatory distress, and alpha-2agonists deactivate the prolonged sympathetic hyperactivity observed in the CCU.After circulatory optimization, normalized sympathetic hyperactivity towardbaseline may benefit metabolic syndrome, immunoparalysis, etc., e.g., in thefollowing settings: circulatory failure following cardiacsurgery108-110,112-114). Increasedpressor responsiveness to noradrenaline toward baseline follows.113,114)21,117)( Increased LV compliance17)( and vasomotorsympathetic deactivation in resistance vessels (arteries)15,16,118): PLR separates thevolume-responsive versus nonresponsive patients: thevolume-responsive patients are not necessarily in ahypovolemic state and do not necessarily need additionalvolume. Volume is minimized to prevent increased lungwater.122,123)( This may evokehypotension within the first 3 hours ofadministration125) or each incrementof alpha-2 agonist, absence of or minimal collapsibilityof the vena cava28,116,126)(O2 arteriovenousdifference127)( or superiorvena cava oxygen saturation (SsvcO2), carbondioxide (CO2) gap.- Second, the adequacy of CO and microcirculation areaddressed: diuresis, capillary refill, mottling,lactate,No hypovolemia: Following alpha-2 agonist administration, SVmaintenance is required:“start slow, goslow”, borrowed from the administration of beta-blockers in heartfailure128)( (124)( (21). We propose this overtlycautious approach and termed it ure(128) . No alph) ((124) .(21)Drugs combining sedation and sympathetic deactivation modify the circulation andrequire the following: In summary, bolus alpha-2 agonist administration with simultaneous conventionalsedation administration or without the iterative assessment of volemia leads tosevere bradycardia and hypotension.51,52)( presents shortcomings.50)( are in their infancy.Established practice129) imposed onhypovolemia worsens circulatory distress.Switching from conventional to cooperative sedation in the setting of hypovolemiaand vasopressor administration addresses only one circulatory issue. In thesetting of ventilatory distress, circulatory distress is intermingled withventilatory distress:130): fevercontrol,131-133)( systemic acidosis136-138),10,61)( and uprightpositioning.139)( in 55).140,141). Rescue sedation isadministered if needed. Muscle relaxants are withdrawn immediately beforesteady-state cooperative sedation is established, with reassurance. Spontaneousbreathing is established as soon as84,148)(versus deep sedation).In summary, the management becomes analytical: administration of an alpha-2agonist allows one to separate the physiological versus controlled mandatory ventilation150), and switchingfrom conventional to cooperative sedation (see above).Switching in a patient presenting with acute cardioventilatory distress underconventional sedation in the CCU involves prioritizing between simultaneousissues beyond the scope of this manuscript: stabilized circulation (see above),stabilized ventilatory distress , PaO2/FiO2(P/F), HR, and systolic BP normalize. The patient is discharged withoutintubation.151)( These reports need replication.Dexmedetomidine (infusion: 0.7µg.kg-1.h-1) addresses agitation in patientstreated with NIV presenting with postoperative ventilatoryfailure.153-155)( or status asthmaticus (dexmedetomidine: 0.2- 0.7µg.kg-1.h-1).156)( Clonidine (4µg.kg-1 p.o.) achieves thesame effect.157) andtitrated to effect: stringent ataraxia is requested when psychogenic stimuli arepresented.A similar positive outcome was observed in the setting of severe bronchospasm(dexmedetomidine: 0.25 - 0.8µg.kg-1.h-1)158)(or early diffuse ARDS150)( are beyond the scope of this section.SARS-CoV-2-ARDS (COVID-ARDS) is an inflammatory disease leading to a highrespiratory drive and an inflammatory vascular disease of the pulmonarycapillaries. Low or medium PEEP is required, with tight control of temperature,agitation and inflammationSeptic shock143,159-161) or very highO2 flow allows one to buy time, expeditepreoxygenation162) prevents the circulatory collapse observed immediatelyafter intubation + positive pressure ventilation + PEEP in hypovolemicpatients.163). Second, iterative rescue sedation allowsone to stabilize incremental cooperative sedation. Finally, P-SILI and hypoxemiaare addressed (Table 155).The dose of dexmedetomidine is a function of the circulation . As an extended CCU stayis likely, immediate stable cooperative sedation is not warranted. First,stabilization of the circulation should be achieved (volume vs. vasopressorswhen the diastolic pressure is low148) achieves-2 < RASS< 0.Tolerance to the sedative effects of alpha-2 agonists develops overweeks165), titration of alpha-2agonists to effect, reassurance and additional neuroleptics.Muscle relaxation suppresses P-SILI and patient-to-ventilatordyssynchronyTwo issues deserve comment: 13) toisolated ventilatory distress and then to acute cardioventilatory distress. A “startslow-go slow” approach is suggested. Neuroleptics supplement alpha-2 agonists, ifneeded, without benzodiazepines or propofol. Opioid-free analgesia is recommended.To avoid switching from conventional to cooperative sedation, alpha-2 agonistsshould be used as first-line sedatives.46), nociception, circulation . Evidencegathered from a randomized trial using a clear-cut design73,74)( may extend the preliminary outcomedata37-45)( and implement the present suggestions.In the critical care unit, alpha-2 agonists present intrinsicallyintertwined


Obsessive-Compulsive Disorder (OCD) is an anxiety disorder, which is the sixth largest contributor to non-fatal health loss globally. Coronavirus disease (COVID-19) pandemic, aside from its impact on physical health, has also had its effects on mental health. This study aimed to explore the frequency of new onset OCD symptomology in medical students amidst COVID-19 pandemic and its association with potential sociodemographic parameters.This cross-sectional study was conducted among medical students studying in Pakistani medical colleges. Data were collected after ethical approval from 1st January 2020 to 20th January 2020 during the second COVID-19 wave. Participants with a history of diagnosed psychiatric illness such as OCD, bipolar disorder, depression, anxiety and those taking relevant medications were excluded from the study. The online questionnaire included Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) and Revised Padua Inventory-Contamination Subscale (PI-CS), which were used to assess OCD symptoms and aversion for contamination respectively. Participants filled Y-BOCS twice, once for pre-pandemic score , and a second time during 2nd wave of COVID-19. Data were analysed using IBM SPSS v23.0 .The study included 711 participants  from over 46 medical colleges and over 44 cities of Pakistan. The mean pre-pandemic and mid-2nd wave Y-BOCS scores were 11.86 ± 6.02 and 15.61 ± 7.41 respectively. The mean PI-CS score was 17.27 ± 9.17. Twenty five percent (n = 176) of students developed new onset OCD symptomology during pandemic, while seventy percent (n = 497) suffered from worsened Y-BOCS score during pandemic. New onset OCD symptomology was associated with age less than 20 years (p = 0.02), higher PI-CS score (p = 0.001) and studying in preclinical years (p = 0.002). Worsening of YBOCS score had significant association with female gender (p = 0.02), attending pandemic related awareness seminar (p = 0.027), studying in preclinical years (p < 0.001) and age less than 20 years (p < 0.001). High Padua scorers (16 and above) showed significant association with increase in YBOCS score (p < 0.000), age less than 20 years (p = 0.005), preclinical years (p = 0.001), frequency of engagements in pandemic related discussions (p = 0.001) and change in YBOCS score (p < 0.001).Our findings indicate that the prevalence of OCD symptomology increased during the COVID-19 pandemic as demonstrated by increased Y-BOCS scores.Femal medical students and students in preclinical years are more likely to suffer from psycological impact of COVID-19 pandemic and heightened concerns and fear for contamination.


Lenacapavir (LEN) is a first-in-class HIV-1 capsid (CA) inhibitor in clinical development for treatment and prevention of HIV-1 infection. CALIBRATE is an ongoing, phase 2 clinical study evaluating subcutaneous (SC) or oral LEN, in combination with other antiretrovirals, in treatment-naïve people with HIV-1. High rates of virologic success (HIV-1 RNA < 50 copies/mL) were achieved with LEN-based regimens by FDA Snapshot analysis at Week 28. Here, we present interim resistance analyses through Week 28. Participants were randomized (2:2:2:1) to treatment groups (TG) : SC LEN + oral daily emtricitabine/tenofovir alafenamide (F/TAF); at Week 28, participants switch F/TAF to oral TAF (TG-A) or bictegravir  (TG-B); oral daily LEN + F/TAF (TG-C), or oral daily B/F/TAF (TG-D). Genotypic analyses (population sequencing) of HIV-1 reverse transcriptase and integrase, and genotypic (deep sequencing)/phenotypic analyses for CA were performed at screening; genotypic and phenotypic analyses were conducted at confirmed virologic failure. CALIBRATE Study Design182 participants were randomized and dosed in TG-A to D . Most participants had subtype B HIV-1 (92.9%). Sequence analysis of baseline samples found 65% of amino acid residues were conserved with < 1% variation across CA overall, and 55% of residues were fully conserved. No mutations were detected at 6 positions in CA associated with reduced susceptibility to LEN in vitro; residues were fully conserved at 5 positions , and < 2% variation was observed at 1 position (T107). Three participants met the criteria for resistance analysis: 2 participants resuppressed to < 50 copies/mL while continuing treatment. One participant on SC LEN + F/TAF developed emergent resistance to LEN (Q67H+K70R) and emtricitabine (M184M/I), followed by resuppression after starting dolutegravir, zidovudine + lamivudine, tenofovir disoproxil fumarate. Emergent resistance to LEN was uncommon in treatment-naïve participants receiving SC or oral LEN . These interim resistance findings support the ongoing evaluation of LEN for treatment and prevention of HIV.Laurie VanderVeen, PhD, Gilead Sciences  Nicolas Margot, MA, Gilead Sciences  Vidula Naik, MSc, Gilead Sciences  Silvia Chang, Masters, Gilead Sciences, Inc  Ross Martin, PhD, Gilead Sciences, Inc  Hadas Dvory-Sobol, PhD, Gilead Sciences  Martin Rhee, MD, Gilead Sciences  Christian Callebaut, PhD, Gilead Sciences


The vaccine is also licensed in ages 12 months and older in EU and certain other countries. We evaluated the safety and immunogenicity of MenACYW-TT compared to a licensed quadrivalent conjugate meningococcal vaccine (MenACWY-DT [Menactra®]) in Japanese children, adolescents and adults (2-55 years of age).MenACYW-TT [MenQuadfiA phase III modified double-blind, randomized study (NCT04368429) to evaluate the immunogenicity and safety of a single dose of MenACYW-TT versus MenACWY-DT was conducted in 360 participants (ratio 1:1) between ages 2 and 55 years in Japan. Serum bactericidal assays with human complement (hSBA) were used to measure antibodies against vaccine serogroups at baseline (Day 0) and 30 days post-vaccination (D30). Safety data were collected up to 30 days post-vaccination.Non-inferiority of immune responses for all four serogroups, based on percentages of participants achieving hSBA vaccine seroresponse as primary endpoint, was demonstrated for MenACYW-TT compared to MenACWY-DT at Day 30 in comparison to baseline: 85.6% vs 65.4% for serogroup A, 96.6% vs 62.6% for serogroup C, 87.4% vs 49.2% for serogroup W, and 97.7% vs 63.5% for serogroup Y. The proportions of individuals with hSBA titers ≥ 1:8 following MenACYW-TT administration were higher than those after MenACWY-DT administration for serogroups C (98.9% vs 81.0%), W (99.4% vs 91.1%) and Y (100 % vs 89.4%) and comparable for serogroup A (96.6% vs 92.7%). The hSBA GMTs were higher following administration of MenACYW-TT for all four serogroups. Immunogenicity results in participants 10 to 17 years of age and ≥ 18 years of age were comparable to those in the whole population (2-55 years). The safety profiles of MenACYW-TT and MenACWY-DT were comparable. There were no immediate adverse events (AEs), no AEs leading to study discontinuation, and no vaccine-related serious adverse events reported in the study.MenACYW-TT vaccine was well tolerated and demonstrated a non-inferior immune response compared to that for the licensed MenACWY-DT vaccine when administered as a single dose to meningococcal vaccine-naïve children, adolescents, and adults in Japan.Osamu Matsuoka, MD, Sanofi Pasteur  Mugen Ujiie, MD, Sanofi Pasteur  Hitoshi Kikuchi, MD, Sanofi Pasteur (Scientific Research Study Investigator)Sanofi Pasteur (Research Grant or Support) Danaya Chansinghakul, MD, Sanofi Pasteur (Employee) Takahiro Inoue, MSc, Sanofi Pasteur (Employee) Kucku Varghese, PhD, Sanofi Pasteur (Employee) Nuchra Sirisuphmitr, MSc, Sanofi Pasteur (Employee) Tomoyuki Hashiguchi, MSc, Sanofi Pasteur (Employee) Betzana Zambrano, MD, Sanofi Pasteur (Employee) Takahiro Nakama, MD, Sanofi Pasteur (Employee) Carina Frago, MD, Sanofi Pasteur  Emilia Jordanov, MD, Sanofi Pasteur  Mandeep S. Dhingra, MD, Sanofi Pasteur


GRF gene family has been performed in cotton, and their functions in flowering are largely unknown.In plants, 14-3-3 proteins, also called GENERAL REGULATORY FACTORs (GRFs), encoded by a large multigene family, are involved in protein–protein interactions and play crucial roles in various physiological processes. No genome-wide analysis of the GRF genes were identified in Gossypium herbaceum, G. arboreum, G. hirsutum, and G. raimondii, respectively, by genome-wide analyses and were designated as GheGRFs, GaGRFs, GhGRFs, and GrGRFs, respectively. A phylogenetic analysis revealed that these proteins were divided into ε and non-ε groups. Gene structural, motif composition, synteny, and duplicated gene analyses of the identified GRF genes provided insights into the evolution of this family in cotton. GhGRF genes exhibited diverse expression patterns in different tissues. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that the GhGRFs interacted with the cotton FLOWERING LOCUS T homologue GhFT in the cytoplasm and nucleus, while they interacted with the basic leucine zipper transcription factor GhFD only in the nucleus. Virus-induced gene silencing in G. hirsutum and transgenic studies in Arabidopsis demonstrated that GhGRF3/6/9/15 repressed flowering and that GhGRF14 promoted flowering.In this study, 17, 17, 31, and 17 GRF genes were identified in cotton, and their gene and protein features, classification, evolution, and expression patterns were comprehensively and systematically investigated. The GhGRF3/6/9/15 interacted with GhFT and GhFD to form florigen activation complexs that inhibited flowering. However, GhGRF14 interacted with GhFT and GhFD to form florigen activation complex that promoted flowering. The results provide a foundation for further studies on the regulatory mechanisms of flowering.Here, 82 The online version contains supplementary material available at 10.1186/s12870-021-02923-9. The R package “pheatmap” was used to generate the expression patterns of the GhGRF genes in these tissues.To obtain the expression profiles of A490626) . The expGhGRF genes were independently cloned into the pMD19-T vector, and positive plasmids containing each gene, as identified by sequences analyses, were then introduced into the plant binary vector pCAMBIA2300-35S-OCS [35S:GhGRF3, 35S:GhGRF6, 35S:GhGRF9, 35S:GhGRF14, and 35S:GhGRF15. All of the constructs were introduced independently into Agrobacterium tumefaciens GV3101 and transformed into A. thaliana Col-0 using the floral dip method [The coding sequences of the five -35S-OCS  to generp method . FloweriBD-GhFT vector used in this study was previously published [GhFD and 17 GhGRFs were amplified by PCR using gene-specific primers . The ublished . The codeviously .GhFD and five GhGRFs were amplified using gene-specific primers Additional file 4: Fig. S1. Chromosomal distributions of the Gossypium spp. GRF genes. (a) G. herbaceum. (b) G. arboreum. (c) G. hirsutum. (d) G. raimondii. Chromosomal locations were shown from top to bottom on corresponding chromosomes according to cotton genome annotation.Additional file 5: Fig. S2. Multiple amino acid sequence alignment of cotton GRF proteins. Amino acid sequence alignment of 82 GRF proteins from G. herbaceum, G. arboreum, G. hirsutum, and G. raimondii. Nine α-helices were marked as α1-α9.Additional file 6: Table S4. Seven conserved amino acid motifs and annotation of cotton GRF proteins.Additional file 7: Table S5. Syntenic gene pairs between G. hirsutum and other three cotton species.Additional file 8: Table S6. The duplicated GRF gene pairs from G. herbaceum, G. arboreum, G. hirsutum, and G. raimondii, respectively.Additional file 9: Fig. S3. Distributions of Ka/Ks values of cotton GRF gene pairs. (a) Ka/Ks ratios for paralogous genes in four cotton species. (b) Boxplot showing the Ka/Ks ratios for orthologous genes among cotton genomes. The center line in each box indicates the median, and the box limits indicate the upper and lower quartiles of divergence.Additional file 10: Fig. S4. The percentage of various responses about cis-acting elements on 2.0-kb promoter of the GRF genes in (a) G. herbaceum, (b) G. arboreum, (c) G. hirsutum, and (d) G. raimondii.Additional file 11: Fig. S5. Information of type, quantity, and location of various response elements in cotton.Additional file 12: Table S7. The FPKM value of GhGRFs in different tissues.Additional file 13: Fig. S6. Interaction of GhFT and each GhGRF protein. Plasmids transformed into AH109 yeast strains were screened on the -LT and -LTHA medium.Additional file 14: Fig. S7. Flowering times and the expression levels of the floral-meristem identity genes in the control and GhGRF silencing plants. (a–e) Statistics of flowering times in the control and GhGRF silencing plants. qRT-PCR analysis of the expression levels of GhAP1 (Gh_D13G0878) (f–j) and GhSOC1 (Gh_A11G0755) (k–o). A cotton Ubiquitin7  gene was used as an internal reference gene. Values are means ± SD (n = 3). Asterisks indicate significant differences between control TRV:00 and TRV:GhGRFs silencing lines .Additional file 15: Fig. S8. qRT-PCR expression analysis of GhGRF, AtSOC1, and AtAP1. ACT2 (At3g18780) was used as an internal reference transcript. Values are means ± SD (n = 3). Asterisks indicate significant differences between Col-0 and the 35S:GhGRFs transgenic lines .Additional file 16: Table S8. PCR primers used in this study.


Scrophulariae Radix (SR) has an important role as a medicinal plant, the roots of which are recorded used to cure fever, swelling, constipation, pharyngitis, laryngitis, neuritis, sore throat, rheumatism, and arthritis in Asia for more than two thousand years. In this paper, the studies published on Scrophularia buergeriana (SB) and Scrophularia ningpoensis (SN) in the latest 20 years were reviewed, and the biological activities of SB and SN were evaluated based on in vitro and in vivo studies. SB presented anti-inflammatory activities, immune-enhancing effects, bone disorder prevention activity, neuroprotective effect, anti-amnesic effect, and anti-allergic effect; SN showed a neuroprotective effect, anti-apoptotic effect, anti-amnesic effect, and anti-depressant effect; and SR exhibited an immune-enhancing effect and cardioprotective effects through in vitro and in vivo experiments. SB and SN are both known to exert neuroprotective and anti-amensice effects. This review investigated their applicability in the nutraceutical, functional foods, and pharmaceutical industries. Further studies, such as toxicological studies and clinical trials, on the efficacy and safety of SR, including SB and SN, need to be conducted. Scrophularia consists of more than 300 different herbs, and Scrophularia buergeriana (SB) and Scrophularia ningpoensis (SN) are representative plants plants of this genus. SB is called “Hyun-sam” in Korea; it is a perennial plant with a strong fragrance that grows up to 1.8 m [Scrophulariae Radix (SR), specifically, SB and SN. The physiological properties discussed in this review have been verified using in vitro and in vivo studies, and these results are the scientific basis for the development of health foods or therapeutics.The genus to 1.8 m . It is nto 1.8 m ,5. SN isto 1.8 m . In thisLee et al.  and JeonMost of the compounds isolated from SN were found in the roots and isolated by solvent fractionation. Iridoids and iridoid glycosides ,27,28,29p < 0.01), IL-6 (SBE: p < 0.01), and MMP-9  mRNA expression levels in LPS-induced Raw 264.7 cells. In addition, SBE significantly suppressed the expression of MMP-9  and p65 phosphorylation . The mRNA expression levels of IL-6 and TNF-α in LPS-induced Raw 264.7 cells were reduced by SBE treatment with Bay11-7085 (NF-κB inhibitor).Shin et al.  reportedp < 0.05), IL-2 , and IL-4  in MOLT-4 cells. Moreover, IgG (p < 0.05) production in the SNU 265 human B cell line increased with 1 mg/mL SB treatment. The cells treated with SB and SB plus IFN-γ showed increased IL-12  production and induced NO  level and iNOS  expression in mouse peritoneal macrophages.Kim et al.  demonstrp < 0.01) treatment increased the cell viability in lymphocytes. In addition, ERS markedly decreased cAMP/cGMP (1 mg/mL: p < 0.05), IFN-γ/IL-10 (1 mg/mL: p < 0.01), and MDA content (1 mg/mL: p < 0.01) and increased the SOD content (1 mg/mL: p < 0.01) in lymphocytes isolated from ICR mice spleen.Gong et al.  demonstrp < 0.001) suppressed TRAP-positive cell formation at 200 μg/mL concentration and had a resorption inhibition effect on mature osteoclasts. The resorption area (p < 0.001) was also observed to decrease after SB treatment.Nam et al.  reportedp < 0.01) in SH-SY5Y cells with glutamate-induced cell toxicity. Acetylcholinesterase activity  was decreased, and total glutathione content (SBE: p < 0.01) was increased in a dose-dependent manner. Glutamate-induced cell morphology changes and DNA fragmentation were measured using DAPI staining and TUNEL assays, and SBE was observed to reduce glutamate-induced fragmentation (SBE: p < 0.01).Lee et al.  demonstrp < 0.01), SOD-2 (SBE: p < 0.05), and GPx-1 (SBE: p < 0.01) expression levels, but it decreased Bax (SBE: p < 0.01) protein, cleaved caspase-3 , and cleaved PARP (SBE: p < 0.01) levels. SBE treatment also reduced the phosphorylation of p38 . In contrast, 500 μg/mL of SBE significantly (p < 0.01) increased B-cell lymphoma-2 (Bcl-2) expression levels.Increased antioxidant enzyme activity is known to protect nerve cells by reducing oxidative neuronal damage. SOD-1, SOD-2, and GPx-1 are antioxidant enzymes, and when reduced, they promoted the expression of apoptosis factors such as Bcl-2-associated X (Bax), cleaved caspase-3, and cleaved poly (adenosine diphosphate (ADP)-ribose) polymerase (PARP). SBE treatment markedly increased SOD-1  demonstrated the neuroprotection effects in PC12 cells. RSAE differently increased according to pretreatment hour. Pretreatment RSAE for 4 h did not affect the cell viability and pretreatment RSAE for 8 h (12.5 μg/mL: p < 0.0.05), 16 h (12.5 μg/mL: p < 0.05), and 24 h (6.25–25 μg/mL: p < 0.00001) statistically increased the cell viability. LDH (RSAE 12.5 μg/mL: p < 0.0001), MDA (RSAE 12.5 μg/mL: p < 0.001), and NO (RSAE 12.5 μg/mL: p < 0.01) levels are decreased and SOD (RSAE 12.5 μg/mL: p < 0.05), CAT (RSAE 12.5 μg/mL: p < 0.05), and GSH-Px (RSAE 12.5 μg/mL: p < 0.0001) activities are recovered with RSAE in oxygen-glucose deprived and reperfusion (OGD/R)-induced PC12 cells. JC-1 red fluorescence for mitochondrial membrane potential change detection was significantly increased by treating with 12.5 μg/mL RSAE in PC12 cells. OGD/R treatment induced MMP destruction and significantly suppressed the red/green fluorescence ratio, but it significantly (RSAE 12.5 μg/mL: p < 0.0001) enhanced the red/green fluorescence ratio.Meng et al.  reportedScrophularia ningpoensis water extract (RSN) presented the anti-apoptotic activity in HaCaT cells. RSN showed the IC50 value at 0.032 mg/mL concentration. TNF-a stimulation activates the NF-κB pathway and induces inflammation. In addition, ERK upstream of NF-κB affects cell proliferation and induces apoptosis. Pretreatment with 0.032 mg/mL RSN inhibited NF-κB translocation induced by TNF-α, which was observed using immunofluorescence staining of HaCaT cells. RSN decreased ERK phosphorylation, and ERK increased gradually with dose and time. RSN did not affect the cell cycle phase in G1/G0, S, and G2/M (at 0.016–0.064 mg/mL for 6–96 h) in HaCaT cells, and a tumor-preferred effect was not detected in Colo 38, SK-Mel-28, and MRI-221 cells. These results suggest that RSN regulates ERK and NF-κB signaling.Shen et al.  demonstrp < 0.05, SBE 1000 μg/mL: p < 0.001) and histamine  in RBL-2H3 cells. The release of pro-inflammatory cytokines, TNF-α (p < 0.01) and IL-4 (p < 0.001), after antigen induction in RBL-2H3 cells was decreased after 1000 μg/mL SBE treatment. Furthermore, the cells treated with 100 and 1000 μg/mL SBE showed inhibition of ERK  and p38 phosphorylation .Kim et al.  reportedp < 0.01), macrophages (SBE: p < 0.01), neutrophils , lymphocytes number , and total cells (SBE: p < 0.01) in bronchoalveolar lavage fluid (BALF) and also decreased airway hyper-responsiveness (SBE 20 mg/kg with methylcholine (MC) 30 mg/mL: p < 0.05, SBE 40 mg/kg with MC 20 mg/mL: p < 0.05, SBE 40 mg/kg with MC 30 mg/mL: p < 0.01).Shin et al.  reportedp < 0.01), IL-13 , and IL-17  in BALF and total IgE  and OVA-specific IgE  levels in the serum decreased after SBE treatment. SBE treatment significantly decreased the inflammatory index and mucus production index  in the lung tissue. In the asthma model induced by OVA, SBE administration decreased MMP-9 (SBE: p < 0.01) expression and p65 phosphorylation (SBE: p < 0.01).The levels of pro-inflammatory cytokines, including IL-5 , as well as treatment with 3–200 mg/kg KD-501  for 15 days significantly increased the step-through latency in the passive avoidance test. Acute and prolonged (for 15 days) treatment of 100 mg/kg KD-501 improved spatial memory ability by reducing escape latency in the Morris water maze test conducted for 4 days. The acetylcholinesterase activity of the cortex (KD-501: p < 0.05) and hippocampal  tissue was significantly increased by the acute administration of KD-501.Jeong et al.  reportedp < 0.001 in hippocampus and increasing glutathione reductase  and SOD  activities in the cortex and hippocampal tissue of amnesic mice. The prolonged (for 15 days) oral treatment with 100 mg/kg KD-501 showed antioxidant activity by decreasing GSSG/total GSH (KD-501: p < 0.001) and increasing glutathione reductase (KD-501: p < 0.001) and SOD (KD-501: p < 0.001) activities in the cortex and hippocampus.Acute treatment with 100 mg/kg KD-501 resulted in antioxidant activity by decreasing GSSG/total GSH (KD-501: These results showed that KD-501 may be used for the prevention and therapeutics of Alzheimer’s disease.p < 0.05) in the passive avoidance test that decreased after scopolamine injection was significantly increased by treatment with 30 and 100 mg/kg SBE, and escape latency and swim distance were decreased in the Morris water maze test. Moreover, in the probe trial conducted on day 28, administration of SBE (30 and 100 mg/kg) significantly increased the crossing number (SBE: p < 0.01). The decreased acetylcholine  level was increased, while increased acetylcholinesterase (SBE 100 mg/kg: p < 0.01) activity was decreased in the hippocampus after SBE (30 and 100 mg/kg) administration.Lee et al. ,69 demonp < 0.01) expression level, and 30 and 100 mg/kg SBE administration significantly increased CREB (SBE: p < 0.01) phosphorylation. Furthermore, SOD-1 (SBE: p < 0.01) and SOD-2 (SBE 100 mg/kg: p < 0.05) expression levels were increased and IL-1β (SBE 100 mg/kg: p < 0.01), IL-6 , and TNF-α (SBE 100 mg/kg: p < 0.01) gene expression levels were decreased in mice with SBE. The group injected with scopolamine showed increased expression levels of Bax, cleaved caspase-9, and cleaved PARP and decreased expression level of Bcl-2. The SBE treatment decreased Bax (SBE: p < 0.01), cleaved caspase-9 (SBE 100 mg/kg: p < 0.01), and cleaved PARP (SBE: p < 0.01) expression levels and increased Bcl-2 (SBE 100 mg/kg: p < 0.01) expression level.Scopolamine injection reduced the BDNF expression levels and CREB phosphorylation. However, 100 mg/kg SBE treatment markedly increased the BDNF  and decreased escape latency and swim distance in the Morris water maze test. The crossing number  was also increased after SBE treatment. Glutathione reductase activity (SBE: p < 0.01) and SOD-1 (SBE 100 mg/kg: p < 0.01), SOD-2 (SBE 100 mg/kg: p < 0.01), and GPx-1 (SBE 100 mg/kg: p < 0.01) expression levels were decreased by β-amyloid injection and increased in the mice administered SBE. Furthermore, Bax (SBE: p < 0.01), cleaved caspase-9 (SBE: p < 0.01), and cleaved PARP (SBE: p < 0.01) levels were markedly decreased, and Bcl-2 (SBE 100 mg/kg: p < 0.01) expression level was increased after SBE treatment. The β-amyloid (SBE: p < 0.01) and phosphorylation of Tau (SBE: p < 0.01) were significantly decreased in the mice treated with SBE. According to the in vitro and in vivo studies, SBE improved spatial memory and cognitive ability by inhibiting cell apoptosis.The mice injected with β-amyloid showed decreased step-through latency in the passive avoidance test and increased escape latency and swim distance. SBE treatment (30 and 100 mg/kg) in these mice increased the step-through latency  and MDA (RSAE 2.4 g/kg: p < 0.01) and NO (RSAE 2.4 g/kg: p < 0.01) levels in the ischemic hemisphere. 2,3,5-Triphenyltetrazolium chloride (TTC) staining presented that the corrected infarct volume increased by MCAO/R operation was significantly decreased by RSAE (2.4 g/kg: p < 0.0001) administration. In the cortex and CA1 region, significantly increased neuronal damage due to MCAO/R was observed compared to the sham group. RSAE treatment significantly decreased neuronal damage by increasing neuron density in the ischemic cortex (RSAE 2.4 g/kg: p < 0.01) and hippocampus CA1 region (RSAE 2.4 g/kg: p < 0.01).Meng et al.  reportedp < 0.05) expression and increased Bcl-2 (RSAE 2.4 g/kg: p < 0.0001) expression in focal cerebral ischemia and markedly decreased the phosphorylation of ERK1/2 (RSAE 2.4 g/kg: p < 0.05), p38 MAPK (RSAE 2.4 g/kg: p < 0.01), and JNK1/2 (RSAE 2.4 g/kg: NS). According to the in vitro and in vivo studies, RSAE inhibits apoptosis and exhibits neuroprotective effects by regulating the MAPK pathway.The MCAO/R group showed increased cell apoptosis by an increase in Bax-positive cells and a decrease in Bcl-2-positive cells. However, RSAE significantly reduced Bax  remarkably decreased the number of escape failures and had an anti-depressive effect on the animal model.Xu et al.  demonstrThese results suggest that II could be used for the treatment of depression.Kim et al.  reportedp < 0.05, SBE 100 μg at 48 h: p < 0.01, SBE 100 μg at 72 h: p < 0.001, 1000 μg at 48, 72 h: p < 0.001) and weight  and decreased TNF-α  and IL-4 (SBE 1000 μg: p < 0.01) levels in ear homogenates in a dose-dependent manner.SBE application reduced ear thickness  has a ventricular remodeling effect. Left ventricular remodeling was induced in rats orally administered with 60, 120, and 240 mg/kg EERS for 14 weeks. The ventricular remodeling group showed decreased systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) when compared with the sham-operated control. The ERRS-treated rats showed increased SBP (CAL plus 120 mg/kg EERS: p < 0.05), DBP , and MAP , and their increased heart rate  was significantly reduced. The hemodynamic parameter left ventricular systolic pressure  was remarkably increased by ERRS. Increased by left coronary artery ligation (CAL), cardiac weight indexes such as the left ventricular weight index (LVWI)  and heart weight index (HWI)  and cardiocyte cross-section area  were decreased in the ERRS-treated group.Huang et al.  demonstrp < 0.01) and I/III collagen  in the interstitial space were remarkably decreased. The interstitial collagen volume fraction (ICVF)  and perivascular collagen volume fraction were also significantly reduced with ERRS treatment. The serum angiotensin II (Ang II)  concentration was decreased in the ERRS-treated rats. Endothelin (ET)-1 , atrial natriuretic peptide (ANP) , hydroxyproline (Hyp) , MMP-2 , and TNF-α  concentrations were decreased by ERRS administration. The rats with ventricular remodeling that were administered ERRS showed a significant decrease in angiotensin-converting enzyme (ACE) , ET-1 , and ANP  mRNA expression levels.The ERRS-treated group showed that types I and III collagen volume  possesses ventricular remodeling inhibition effects. Spontaneously hypertensive (SHR) male rats were used for the experiment and orally treated with 70, 140, and 280 mg/kg ACRS for 21 weeks. The cardiac mass indexes LVWI , HWI , and ICVF , perivascular collagen area (PVCA)  ratio, collagen I  and III  volumes, I/III ratio , serum norepinephrine , myocardium Ang II , and serum TNF-α levels were significantly decreased by ACRS treatment in the SHR rats.Zhang et al.  showed tp < 0.01), TGF-β1 , and ACE  mRNA expression levels and decreased phosphorylation of p44/42 MAPK , SANP/JNK , and p38 MAPK . These results show that ACRS inhibited ventricular remodeling through MAPK pathway regulation, and it may be used to prevent and treat heart failure.The ACRS-treated SHR rats showed reduced collagen I , HWI , myocardium Ang II , and Hyp (ERS-L and H: p < 0.01) decreased with the ERS treatment. To analyze the cardiocyte cross-section area (ERS-L and H: p < 0.01) by H&E staining, ERS administration was reduced similarly to that in the captopril group used as the positive control. In addition, PVCA (ERS-L and H: p < 0.01) and CVF (ERS-L and H: p < 0.01), which are increased by ventricular remodeling, were significantly decreased by the ERS treatment.Gu et al.  showed tp < 0.01) and III collagen contents  were significantly decreased and AT1R (ERS-L and H: p < 0.01), TNF-α (ERS-L and H: p < 0.01), and TGF-β1 (ERS-L and H: p < 0.01) mRNA expression levels were reduced in the myocardium by the ERS treatment.Subtypes I  was observed to significantly increase, and body temperature (ERS: p < 0.05), heart rate (ERS: p < 0.05), average speed (ERS: p < 0.01), and upright time (ERS: p < 0.01) were found to markedly decrease. Measurement of serum MDA and SOD levels and cAMP/cGMP suggested that ERS treatment decreased the MDA (ERS: p < 0.05) level and cAMP/cGMP (ERS: p < 0.05) and increased SOD (ERS: p < 0.05) level. The ERS-treated mice showed a statistical decrease in IL-6 (ERS: p < 0.01) expression level in the serum and Na+-K+ATP enzyme content (ERS: p < 0.01) in the liver homogenates. According to the in vitro and in vivo studies, ERS showed its immune regulation and antioxidant activities, and it can be used as dietary supplement for better health.Gong et al.  reportedIn this review, we summarized the biological effects of SB, SN, and SR based on the previous in vitro and in vivo studies. SB was administered orally at a minimum dose of 20 mg/kg to a maximum of 200 mg/kg (mice), SN was administered orally at a minimum dose of 5 mg/kg to a maximum of 2.4 g/kg (mice), and SR was orally administered at 2 g/kg (rat). SB showed anti-inflammation activity, immune-enhancing effects, bone disorder prevention activity, neuroprotective effect, anti-amnesic effect, and anti-allergic effect. SN exhibited neuroprotective effect, anti-apoptotic effect, anti-amnesic effect, cardioprotective effect, and anti-depressant effect; and SR showed immune-enhancing activity and cardioprotective effects. However, clinical trials need to be conducted to investigate the efficacy and safety of SR, including SB and SN.As per studies published on SB, SN, and SR in the last 20 years, these plants have been processed using water or ethanol as a solvent. This is thought to reduce side effects when administered orally, and will help increase their applicability to various formulations when developing health functional foods or therapeutics in the future.Taken together, we suggested the possibility of utilizing SB, SN, and SR for developing health functional foods or therapeutics for various applications on the basis of previously reported literature.


Scientific Reportshttps://doi.org/10.1038/s41598-018-29965-8, published online 02 August 2018Correction to: The original version of this Article contained errors in the Reference list. The authors omitted the below paper, which is listed as Reference 22.Kirshenbaum AS, Akin C, Wu Y, Rottem M, Goff JP, Beaven MA, Rao VK, Metcalfe DD. Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia; activation following aggregation of FcepsilonRI or FcgammaRI. Leuk Res. 2003 Aug;27(8):677–82.As a result, in the Materials and Methods section,“The LAD2 human mast cell line was kindly provided by Dr. D. Metcalfe, (NIH Washington).”now reads:22.”“The LAD2 human mast cell line was kindly provided by Dr. D. Metcalfe, (NIH Washington)Consequently, References 23–26 were incorrectly listed as References 22–25.The original Article has been corrected.