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+{"text": "Protegrin-1 (PG-1) is known as a potent antibiotic peptide; it prevents infection via an attack on the membrane surface of invading microorganisms. In the membrane, the peptide forms a pore/channel through oligomerization of multiple subunits. Recent experimental and computational studies have increasingly unraveled the molecular-level mechanisms underlying the interactions of the PG-1 \u03b2-sheet motifs with the membrane. The PG-1 dimer is important for the formation of oligomers, ordered aggregates, and for membrane damaging effects. Yet, experimentally, different dimeric behavior has been observed depending on the environment: antiparallel in the micelle environment, and parallel in the POPC bilayer. The experimental structure of the PG-1 dimer is currently unavailable.Although the \u03b2-sheet structures of the PG-1 dimer are less stable in the bulk water environment, the dimer interface is retained by two intermolecular hydrogen bonds. The formation of the dimer in the water environment implies that the pathway of the dimer invasion into the membrane can originate from the bulk region. In the initial contact with the membrane, both the antiparallel and parallel \u03b2-sheet conformations of the PG-1 dimer are well preserved at the amphipathic interface of the lipid bilayer. These \u03b2-sheet structures illustrate the conformations of PG-1 dimer in the early stage of the membrane attack. Here we observed that the activity of PG-1 \u03b2-sheets on the bilayer surface is strongly correlated with the dimer conformation. Our long-term goal is to provide a detailed mechanism of the membrane-disrupting effects by PG-1 \u03b2-sheets which are able to attack the membrane and eventually assemble into the ordered aggregates.In order to understand the dimeric effects leading to membrane damage, extensive molecular dynamics (MD) simulations were performed for the \u03b2-sheets of the PG-1 dimer in explicit water, salt, and lipid bilayers composed of POPC lipids. Here, we studied PG-1 dimers when organized into a \u03b2-sheet motif with antiparallel and parallel \u03b2-sheet arrangements in an NCCN packing mode. We focus on the conformations of PG-1 dimers in the lipid bilayer, and on the correlation between the conformations and the membrane disruption effects by PG-1 dimers. We investigate equilibrium structures of the PG-1 dimers in different environments in the early stage of the dimer invasion. The dimer interface of the antiparallel \u03b2-sheets is more stable than the parallel \u03b2-sheets, similar to the experimental observation in micelle environments. However, we only observe membrane disruption effects by the parallel \u03b2-sheets of the PG-1 dimer. This indicates that the parallel \u03b2-sheets interact with the lipids with the \u03b2-sheet plane lying obliquely to the bilayer surface, increasing the surface pressure in the initial insertion into the lipid bilayer. Recent experimental observation verified that parallel PG-1 dimer is biologically more active to insert into the POPC lipid bilayer. Protegrin-1 (PG-1) is composed of 18 amino-acids (RGGRL-CYCRR-RFCVC-VGR) with a high content of cysteine (Cys) and positively charged arginine (Arg) residues. The peptide displays an antimicrobial activity . A nucleThe major role of PG-1 in the microorganism is the formation of an oligomeric structure in the membrane that creates a pore/channel. It has been suggested that the self-association of PG-1 into a dimeric \u03b2-sheet can take place in two ways: an antiparallel \u03b2-sheet with a turn-next-to-tail association or a parallel \u03b2-sheet with a turn-next-to-turn association . These m19F spin diffusion showed that PG-1 dimers are populated in the POPC bilayer, while the dimer fraction is reduced as the peptide concentration decreases [et al. for the PG-1 aggregation from 2D solid-state NMR experiments on PG-1 aggregates in phosphate buffer saline (PBS) solution suggested that a parallel \u03b2-sheet in an NCCN  packing mode is the sole repeat motif in the ordered aggregates [Oligomerization or aggregation of PG-1 dimers into ordered aggregates is responsible for the membrane disrupting effects. It has been reported that PG-1 is immobilized in palmitoyl-oleyl-phosphatidylcholine (POPC) lipid bilayers, suggesting aggregation in the lipid bilayer . The resecreases . The modgregates . The recgregates .The formation of the PG-1 dimer is important for pore formation in the lipid bilayer, since the dimer can be regarded as the primary unit for assembly into the ordered aggregates. Experimental observations for other cytolytic peptides have verified that dimer seeding accelerates the formation of ordered aggregates, such as fibrils made by \u03b2-amyloids and prions . HoweverPG-1 dimers in a \u03b2-sheet motif were used in the all-atom simulations. In our simulations, PG-1 dimers in two different \u03b2-sheet arrangements, antiparallel (turn-next-to-tail) and parallel (turn-next-to-turn) \u03b2-sheets in an NCCN packing mode, were considered . Fig. 1 QH-bond, for the antiparallel (A1 & A2) and parallel (P1 & P2) \u03b2-sheets of PG-1 dimer at the amphipathic interface of the lipid bilayer. We calculated the fraction using QH-bond = NH-bond/\t\t\t\tNH-bond is the number of the intermolecular and intramolecular backbone H-bonds that are monitored during the simulations, and Figure arallel A & A2 andQH-bond lines are observed. To compare the dimeric behavior between the water and on the lipid, the \u03b2-sheets used in the water simulation are the same (A1 and P1) as those used in the lipid simulation. Although the PG-1 dimers in water exhibit a partially ordered or slightly collapsed \u03b2-sheet conformation compared to the \u03b2-sheet conformations on the lipid bilayer, two intermolecular backbone H-bonds between two cysteine residues at the C-terminal strands from each monomer retain strongly the dimer interface. The formation of PG-1 dimer in water implies that the PG-1 dimer can exist in many different environments and acts as a seed in the formation of ordered aggregates in a proper environment.In the bulk water environment, the \u03b2-sheets structures of PG-1 dimer are less stable, since in Fig. The detailed secondary structures for the \u03b2-sheet conformations are investigated. Fig. The orientation of the PG-1 \u03b2-sheet on the lipid bilayer is monitored by calculating the angle between the backbone carbonyl bond, C=O, and the normal to the bilayer surface. Only the backbone carbonyl bonds located in residues that form the \u03b2-strands (residues 4 to 8 and 13 to 17 of each \u03b2-hairpin) are considered in the calculation. The probability distribution for the angle of the C=O vector relative to the membrane normal is shown in Fig. For an \u03b1-helical peptide, the peptide orientation in the lipid bilayer can be measured experimentally by polarized ATR-FTIR spectroscopy ,24, and ij is the angle between two vectors of the backbone carbonyl bonds, C=O, in the i and j residues, and N is the total number of the vector pairs. In the calculation, only the C=O bonds located in \u03b2-strands (residues 4 to 8 and 13 to 17 of each \u03b2-hairpin) are considered. Fig. SC=O, over the 20 ns trajectories. For the A1 and P1 \u03b2-sheets, where the initial \u03b2-hairpin monomer structure is from NMR [SC=O are 0.75 and 0.77, respectively. These values are much smaller for the A2 and P2 \u03b2-sheets, where the initial \u03b2-hairpin monomer structure is from our previous simulation [SC=O reveal that for the antiparallel \u03b2-sheets (A1 & A2), the distribution curves are located around SC=O = 0.3, while the curves are found around SC=O = 0.2 for the parallel \u03b2-sheets (P1 & P2). Both parallel \u03b2-sheets have smaller values of the peptide order parameter, indicating that the values of the peptide order parameter strongly depend on the \u03b2-sheet topology.where \u03b8mulation , which aps and averaged over the 20 ns simulations for each monomer separately. For the antiparallel \u03b2-sheets (A1 & A2), the strength of the interaction energy of each monomer with the lipid is similar. However, for the parallel \u03b2-sheets (P1 & P2), at least one monomer interacts with the lipid more strongly than the other. The strong interaction reflects the strong electrostatic interaction of the Arg residues with the lipid headgroups. For the P1 \u03b2-sheet, the terminal Arg residues of the monomer whose apolar surface faces the bilayer surface closely contact the lipid, while for the P2 \u03b2-sheet the loop Arg residues of the monomer whose apolar surface faces the bulk region interact strongly with lipid. These strong Arg-lipid interactions observed in the parallel PG-1 \u03b2-sheet are closely related to the bending or tilting of the \u03b2-sheet, ultimately causing the membrane thinning and disruption.The interaction energy between the PG-1 \u03b2-sheets and lipids is calculated in order to understand the dominant forces that stabilize the \u03b2-sheet structure. Fig. ps and averaged over the 20 ns simulations for each monomer separately. The figure also presents the averaged lipid accessible surface areas for the Arg residues (dark gray bars). As expected, the lipid accessible surface area is strongly correlated with the interaction energy of the PG-1 \u03b2-sheets as seen in Fig. Fig. SPOP, which calculates the angle between the positional vector connecting two adjacent phosphate atoms and the plane of the bilayer surface,In our previous study of the PG-1 monomer on the lipid bilayers , we haverij is the distance between two phosphate atoms, and ith and jth residue, respectively. The notation <i,j> means that the sum is restricted to adjacent pairs of phosphate atoms. The plane order parameter SPOP can measure the flatness or roughness of the bilayer surface, indicating that for SPOP < 1 the bilayer surface is a perfect smooth plane, while for SPOP < 1 the bilayer surface is bent or contains many troughs. Fig. SPOP >, at the top leaflet (solid bars) and at the bottom leaflet (gray bars) of the lipid bilayer during the simulations. The PG-1 \u03b2-sheet is located at the top leaflet of the lipid bilayer. As expected, the bilayer surface at the top leaflet is rougher than the bottom leaflet of the lipid bilayer, since the top leaflet contains the \u03b2-sheet. Note however that the bilayer surface at the top leaflet containing the parallel \u03b2-sheets (P1 & P2) is rougher than that at the same leaflet containing the antiparallel \u03b2-sheets (A1 & A2). This suggests that the parallel \u03b2-sheets are very active and more strongly interact with the lipid bilayer, with the activity closely related to the bilayer disruption.where P) for five different component groups of POPC, water, and salts as a function of the distance from the POPC lipid bilayer center. The POPC headgroup is divided into four subunits, choline , phosphate , glycerol , and carbonyl . The tail group involves two fatty acids with terminal methyl . The PG-1 \u03b2-sheet is located at the top leaflet of the lipid bilayer (positive z area), while the bottom leaflet of the lipid bilayer (negative z area) contains lipids only. For the antiparallel \u03b2-sheets (A1 & A2), the symmetric distributions of the lipid headgroups at both sides of the bilayer indicate that there are no disturbances in the lipid arrangement induced by the \u03b2-sheets. However, for the parallel \u03b2-sheets (P1 & P2), the asymmetric distributions of the lipid headgroups indicate that there are great disturbances in the lipid arrangement, especially at the top leaflet of the lipid bilayer. This is consistent with the result presented in Fig. The disruption of the lipid bilayer reflects disordering of lipid molecules around the PG-1 \u03b2-sheets. The average positions of lipid groups may illustrate how lipids are distributed in the bilayer in response to the dimer invasion. Fig. SCD, was calculated usingTo investigate the average structure in the interior of the bilayer, the deuterium order parameter, where \u03b8 is the angle between the C-H bond vector and the membrane normal, and the angular brackets indicate averaging over time and over lipids. Fig. We simulated the Protegrin-1 (PG-1) dimers with different \u03b2-sheet arrangements in an aqueous solution and in a lipid bilayer composed of POPC. To create the PG-1 dimer, the two \u03b2-hairpins were initially assembled into \u03b2-sheets with both antiparallel (turn-next-to-tail) and parallel (turn-next-to-turn) \u03b2-sheet motifs in an NCCN packing mode . The monIn this work we wish to compare between different dimer conformations in different environments and to find the connection between the dimer conformation and the activity of the PG-1 dimer in the early stage of the membrane disruption. In this early stage, the PG-1 dimer is in contact with the bilayer surface, preparing the penetration into the hydrophobic core region of the lipid bilayer. Thus, our lipid simulations start with a PG-1 dimer that is initially located at the top of the lipid bilayer without giving any stress to the lipid bilayer. The dimer quickly moves to the amphipathic interface immediately following the start of the simulation. This indicates that our simulation results are independent of the initial dimer location with respect to the interface, since this kind of relaxation takes a very short time. If the PG-1 monomer or dimer is fully embedded in the lipid bilayer, the membrane disruption effects are enhanced. In this case, the initial position and orientation of the peptides in the lipid bilayer are crucial, since the peptides cause hydrophobic mismatch between the lipid bilayer and the PG-1, inducing a highly curved bilayer surface. In this paper, we do not target the dimer insertion into the lipid bilayer, since the timescale for such spontaneous dimer relocation may range from few hundreds of nanoseconds to a microsecond, which is far beyond the timescale of our simulations. In our simulation, the antiparallel PG-1 \u03b2-sheets bind within the top leaflet of a lipid bilayer with its apolar surface of the \u03b2-sheet containing the four disulfide bonds facing the bilayer surface, closely following the experimental suggestion . Unlike The simulations of the PG-1 \u03b2-sheets with different monomer \u03b2-hairpin conformations have shown that the behavior of the \u03b2-sheets on the bilayer surface strongly depends on the topology. For example, parallel \u03b2-sheets have smaller values of the peptide order parameter, stronger lipid interaction, and they induce a bilayer disruption effect. In the turn-next-to-turn association of the parallel \u03b2-sheet, charge distributions due to the positively charged arginine side chains are separated into two regions, \u03b2-turns and both termini. As compared to the flexible motions in the arginine residues in order to avoid electrostatic repulsion at both termini, the dynamic motions of the arginine side chains at the \u03b2-turns are stiffened due to the restricted backbone motions, clustering positively charges at the one side of the \u03b2-sheet. These confined repulsive forces by the positive charges at the \u03b2-turns induce distortion of the \u03b2-sheet plane, lying obliquely to the bilayer. As a result, one monomer of the parallel \u03b2-sheets interacts with the lipid more strongly than the other and has a larger lipid accessible surface area than the other as seen in Fig. In our previous PG-1 monomer simulations , PG-1 exThe model of PG-1 dimerization in the POPC lipid bilayer  has suggHere, we present the PG-1 dimer structures in different environments. In the bulk water environment, PG-1 dimers are partially folded \u03b2-sheets, while both the antiparallel and parallel \u03b2-sheet conformations of the PG-1 dimer are well preserved at the amphipathic interface of the lipid bilayer. Our simulation results provide important guidelines for how the environment supports the \u03b2-sheets of the PG-1 dimer and how the dimer activity depends on the \u03b2-sheet arrangements. We conducted four simulations starting at different initial configurations, two for antiparallel dimers and two for parallel dimers (page 4). As expected, these simulations do not converge to the same point, since molecules should have conformational and energetic distributions in nature, especially when embedded in the complex membrane environment. Similarly, a recent PG-1 monomer simulation work observed a \"kick-shaped\" conformation only in one of the two simulations . In the Cl-) were inserted to electrically neutralize the lipid bilayer system, since there are six positively charged amino acid residues in each monomer. To obtain a physiological salt concentration near 100 mM, additional 20 Na+ and 20 Cl- were added.A unit cell containing two layers of lipids with almost 65,000 atoms is constructed. The \u03b2-sheet of the PG-1 dimer is initially located at the amphipathic region in the top leaflet of the lipid bilayer. Since our simulation method closely follows the previous method of the PG-1 monomer simulation, in this paper we only briefly describe key parameters used for the dimer simulations. The details of the simulation method are described elsewhere . A lipidOur simulation employed the NPAT  ensemble, an effective (time-averaged) surface tension, with a constant normal pressure applied in the direction perpendicular to the membrane. An alternative protocol would involve using variable surface area controlled by constant surface tension with the NP\u03b3T  ensemble, where \u03b3 is the applied surface tension. When \u03b3 = 0, NP\u03b3T is equivalent to NPT. Although experimentally measured macroscopic property of \u03b3 should be close to or zero for the non-stressed lipid bilayers , a nonzeK. The entire pre-equilibration cycle took 5 ns to yield the starting point. For the production runs of 20 ns, any constraint applied to the peptides was removed, and the simulations were performed with the same parameter sets as used in the pre-equilibrium simulations. The system reached to the equilibration after initial 3\u20134 ns. The NAMD code [ns duration simulations can reveal details of the interactions of lipid molecules with inner and outer membrane proteins [The CHARMM program  and CharAMD code  on a Bioproteins ,39.HJ carried out the computational simulations, theoretical calculations, and analysis of the results and drafted the manuscript. BM and RN conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "Retrotransposons are key players in the evolution of eukaryotic genomes. Moreover, it is now known that some retrotransposon classes, like the abundant and plant-specific Sireviruses, have intriguingly distinctive host preferences. Yet, it is largely unknown if this bias is supported by different genome structures.copia retrotransposons. We discovered that Sireviruses are unique among Pseudoviridae in that they constitute an ancient genus characterized by vastly divergent members, which however contain highly conserved motifs in key non-coding regions: multiple polypurine tract (PPT) copies cluster upstream of the 3' long terminal repeat (3'LTR), of which the terminal PPT tethers to a distinctive attachment site and is flanked by a precisely positioned inverted repeat. Their LTRs possess a novel type of repeated motif (RM) defined by its exceptionally high copy number, symmetry and core CGG-CCG signature. These RM boxes form CpG islands and lie a short distance upstream of a conserved promoter region thus hinting towards regulatory functions. Intriguingly, in the envelope-containing Sireviruses additional boxes cluster at the 5' vicinity of the envelope. The 5'LTR/internal domain junction and a polyC-rich integrase signal are also highly conserved domains of the Sirevirus genome.We performed sensitive comparative analysis of the genomes of a large set of Ty1/in silico methods highlighted the unique genome organization of Sireviruses. Their structure may dictate a life cycle with different regulation and transmission strategy compared to other Pseudoviridae, which may contribute towards their pattern of distribution within and across plants.Our comparative analysis of retrotransposon genomes using advanced  ENV) gene that allows them to enter the extracellular space and infect other individuals. Retrotransposons lack the ENV gene and cannot escape the cell, however they are free to reinfect their host genome. Long Terminal Repeat (LTR) retrotransposons form the most abundant transposable element type in plants, largely accounting for the vast differences in genome sizes [Arabidopsis (121 Mbp) and rice (389 Mbp) are sparsely populated by LTR retrotransposons, 5.6% [Retrotransposons and retroviruses (collectively referred as retroelements) can replicate their genomes via an RNA intermediate and insert the copies into new chromosomal locations of the host organism ,2. This me sizes . Small gns, 5.6%  and 17% nd rice 39 Mbp arens, 5.6%  and 70% ns, 5.6% .copia (Pseudoviridae) and Ty3/gypsy (Metaviridae) [gag gene and the pol gene region. gag encodes a capsid protein that forms the virus-like particle (VLP), which houses one or two RNA genomes and the enzymes for the cytoplasmic step of reverse transcription. pol encodes the enzymatic proteins required for the production of the DNA copy from the RNA template and the insertion of the new copy in the host genome: an aspartic protease (AP), integrase (INT), reverse transcriptase (RT) and RNaseH (RH) [cis-acting transcriptional regulators, the promoter and termination transcription points. The cis-acting boxes are often recognition sites of stress-related DNA binding factors (DBFs) and may be organized as arrays of two or three repeated motifs (RM) in tandem [gag, while the linker domain connects pol and the 3'LTR. At the junctions of the 5' and 3'LTR with the internal retrotransposon genome reside the primer binding site (PBS) and the polypurine tract (PPT), respectively, that prime cDNA synthesis during reverse transcription [The two main superfamilies of LTR retrotransposons are the Ty1/viridae) , which dseH (RH) . LTRs fln tandem ,15. A 5'cription .Pseudoviridae family [ENV-like gene in their linker domain [copia genera . Sireviruses have successfully proliferated within plant genomes, comprising a large proportion of the available Ty1/copia populations [RT domain revealed that Sireviruses are less diverse than classic Ty1/copia retrotransposons, indicating a possible recent evolutionary origin and colonization of their host genomes [The International Committee on the Taxonomy of Viruses (ICTV) has classified Sireviruses into the e family  togethere family . Sirevirr domain , which dulations . Exampleulations , and Osrulations . Phylogegag and pol as a single open reading frame (ORF), Sireviruses exhibit variation in respect to their organization. Besides the consensus, many elements have pol in two unusual +1 frameshifts relative to gag, of which the one suggests novel ways of translational recoding for pol expression, involving internal ribosomal entry or a bypass strategy [gag/pol boundary may provide a favorable secondary structure at the RNA level. Sireviruses also have a much larger gag gene compared to classic retrotransposons, which is characterized by a central RNA-binding CCHC zinc knuckle and a downstream predicted coiled-coil domain [Sireviruses differ in the sequence and genomic organization compared to other elements. Whereas the vast majority of plant retrotransposons encode strategy . For thel domain ,18. The Pseudoviridae genomes enabled the identification of several short but highly conserved sequence motifs in the otherwise diverse Sirevirus genome. The motifs are found in key regions of the non-coding genome, regions which are necessary for the activation, reverse transcription and integration of the element, or even its virulence capacity through the expression of the ENV gene. Interestingly, one of the motifs is semi-conserved in classic retrotransposons, implying a global fundamental role of its function. Our results suggest that Sireviruses are an ancient retrotransposon lineage that maintains core domains with absolute or high similarity. These domains appear to attribute novel features to the Sirevirus life cycle and may partially explain their distribution within and across plants.In this study, the in depth comparative analysis of Pseudoviridae, 21 Sireviruses were collected from various monocot and eudicot species including  that are re-classified as Sireviruses during this work. The classic Ty1/copia retrotransposon population consisted of 30 elements (25 from plants and five from other organisms), representing the other two genera that make up the Pseudoviridae family: 26 Pseudoviruses and four Hemiviruses , whilst the Sirevirus linker domain and LTRs are extremely diverse  the genome of classic retrotransposons Figure . In factENV-gene  reside after the ENV-gene , which is found inverted within the 3'LTR (right IR arm). The IR is present in all but three elements  and shares high nucleotide identity even between Sireviruses that colonize evolutionary distant monocot and eudicot plant species Figure . The maiPseudoviridae. In all cases the left arm extends downstream till the beginning of the predicted PPT, which occupies the 5' border of the loop sequence  cluster within the upper half of each LTR, as they start 200-400 bp after the PPT/3'LTR junction and extend to the following 300-500 bp. As much as 32 units can occur in one element, while all Sireviruses contain at least eight copies of their RM. The overall nucleotide conservation within most elements is remarkable, commonly displaying more than 85% identity  does a predicted island occur in a region other than the LTRs or the ENV. Among the RM-containing Sireviruses, the RM clusters of HOPIE, OPIE-2, PREM-2, Vitis, Barbara, Inga and Usier are the only CpG islands in their genome, whilst one or two additional islands are present in Osr10, Osr9, Citrus and Osr8 . Only seven have LTR-derived islands, whilst nine lack such a domain and 14 have irregular conformations where CpG islands are present in varied non-LTR locations.CpG islands are known to be strongly associated with genes, as they typically occur within or close to their 5' terminus near the transcription start site and have been found both in vertebrates  and planAnother feature that differentiates Sireviruses from classic retrotransposon genomes is the presence of a conserved TATA box motif in the Sirevirus LTRs. The promoter element 5'-cTATAA/TAT/AAg-3', where lower case letters represent less critical bases , residesmet tRNA, which is therefore the first primer during reverse transcription. The sharp decrease in sequence conservation after the ninemer implies that Sireviruses paradoxically use only a short segment of the tRNA molecule to prime cDNA synthesis. The intervening sequence is unusually long (5'-ATTGG-3') and in six elements a G replaces the T at the second position. Consequently, the Sirevirus upstream attachment site is the heptamer 5'-CA/ATTGG-3', whilst over the whole length of the 16 bp region the genus maintains 92.7% identity. In contrast, classic elements contain a more variable 5'LTR/PBS junction, although and due to the common met tRNA-derived PBS in many LTR retrotransposons [The second most popular sequence motif of the Sirevirus genome according to TEIRESIAS is the 5'LTR/internal domain junction that accommodates the PBS. The highly conserved upstream junction is divided in three subdomains that in the 5' to 3' direction consists of the universal CA end of the 5'LTR, the intervening sequence and the PBS Figure . The Sirnsposons , TEIRESIINT and the attachment sites of the element [INT to the viral DNA is achieved through additional interactions that extend at least 25 bp inwards at the 3' end of the LTR in a domain that is called the 'integrase signal' [The successful integration of a new retrotransposon copy in the host genome is highly dependent on the interactions between  element . However signal' ,33. TEIRcopia retrotransposons (Table copia elements (5.6 kb). The difference derives from the large LTRs (1.28 kb compared to 0.56 kb), linker domain (1.5 kb to 78 bp), expanded gag (660 to 330 amino acids) and ENV (1.8 kb when present). The sum of the extended regions equals to 5.5 kb and hence matches the size difference.A comparative analysis of the size between Sireviruses and classic elements revealed that Sireviruses share an exceptionally large genome that clearly differentiates them from other Ty1/ns Table . So far in silico analysis of the Sirevirus genome and the comparison to a group of representative classic elements provided what we believe are major insights into the structure of LTR retrotransposons. Our data show that Sireviruses form an ancient lineage, at least as old as those of other classic Ty1/copia elements. The main characteristic of their genome is the high sequence divergence of their large non-coding domains, which are however full of highly conserved microdomains in key regions  the junctions contain highly conserved PBS, PPT and attachment sites, whilst an IR accurately surrounds the terminal PPT, iii) multiple identical PPTs cluster next to the 3'LTR and iv) the integrase signal occurs at the predicted domain at the 3' end of the LTRs. These features may be among the underlying factors that contribute towards the Sirevirus distribution in plants.The novel s Figure  that indINT recognizes and cleaves the intervening nucleotides from the 3' ends of the linear cDNA, thereby preparing the new copy for nucleophilic attack [The PPT and the pairing of the tRNA molecule to the PBS are used as primers during reverse transcription to initiate plus and minus strand cDNA synthesis, respectively. Both steps are followed by template switching events (or jumps) that transfer the partial DNA fragments to the other side of the template, so as to resume and complete cDNA synthesis . During c attack .INT with the Sirevirus LTRs. In contrast, the respective regions of classic elements show extensive sequence variation, although a large proportion maintains a common PPT structure that relates to the Sirevirus PPT octamer. We propose that the primer for plus strand cDNA synthesis in the majority of Pseudoviridae consists of eight bases, which may tolerate some variability in classic elements, but is strictly conserved in Sireviruses.According to the TEIRESIAS algorithm the two most conserved motifs of the Sirevirus genome are the PPT/3'LTR and 5'LTR/PBS junctions. The extreme sequence conservation of the two domains, which appear to be very important for reverse transcription and integration , reflectENV-containing Ty3-gypsy elements Athila4 and Calypso [RH degradation of the tRNA after it is copied in the natural PPT-primed cDNA may prohibit strand transfer from internally initiated cDNAs [The presence of multiple PPTs within retroelements is not unprecedented, however their role in plus strand cDNA synthesis is unclear. One extra PPT is present in the linkers of the  Calypso , whilst  Calypso . PPT-pri Calypso . Yet, th Calypso . In Sireed cDNAs .INT processing, the cDNA intermediate will be unable to insert in the new genomic location. On the other hand, the strict nucleotide integrity of the upstream PPTs, their high copy number and preferential clustering next to the 3'LTR point towards selection. They may facilitate genetic recombination between the two RNA genomes in the VLP [ENV genes contain near their 3'LTR repeats that show similarity to PPTs, Havecker et al. (2004) proposed their involvement in reverse transcription.The upstream PPTs also lack the correct attachment site and the IR motif, which further obscures their functionality. The transposition ability of the retroelement is dependant on the attachment site . If the  the VLP , or offe the VLP . In any RH processing. Additional recognition signals in the form of secondary structures near the PPT domain may be needed to affect the overall conformation of the region and attract the RH for PPT formation and subsequent DNA elongation [RH for precise cleavage, which will automatically generate the highly conserved attachment site. Many classic Ty1/copia elements also contain the IR, which suggests that this motif may have a universal role in retrotransposon life cycle.Research in Ty1 has shown that the PPT sequence by itself is not sufficient to promote correct ongation . A highlENV-gene does not seem random and they also form CpG islands that are by default found at the 5' side of genes regulating their expression [ENV are particularly CG-rich and generate CpG islands (data not shown). Despite these unique characteristics, we could not identify a common binding site targeted by a single DBF. It appears that minor variation, notably the presence of one intervening base or a substitution in the symmetrical motif, may confer different binding properties that may enable interactions with different nuclear factors. Future promoter analysis experiments could elucidate the role, if any, of the RMs in the Sirevirus activation network.Sireviruses share highly similar versions of a novel RM, which in conjunction to the exceptional high copy number and flanking sequence divergence, make the conservation remarkable. Symmetry is a frequent feature of regulatory sequences, including box I of Tnt1 , an actipression . InteresENV-gene in plant retroelements has not been experimentally proven and due to the plant cell wall its role remains highly controversial [ENV-containing plant retroelements express their ENV either through a spliced ENV-like mRNA, for example splice acceptor sites have been predicted for the Ty3-gypsy BAGY2 and Athila elements [pol and ENV are separated only by a stop codon. The sequence of the Sirevirus ENV-gene is highly variable [ENV may be expressed from an internal promoter, although they were not able to identify promoters or transcription factors-binding sites upstream of the ENV. Our work indicates that specific motifs, which are also the epicentre of CpG islands, may have a regulatory role in the 5' vicinity of ENV, which offers support to the internal promoter hypothesis for ENV expression, although we were not able to conclusively identify one. Intriguingly, CpG islands are predicted in the same domain for six ENV-containing Sireviruses that lack the RM boxes, even in the pol/ENV junction of SIRE1-1. Collectively, we believe our observations to be the first set of evidence concerning the Sirevirus ENV expression and their potential capacity to become virulent during their life cycle.The function of the oversial . So far elements ,43, or telements . Howevervariable . PetersoPseudoviridae genus to contain an ENV gene, which is hypothesized as being funtional. Has horizontal transmission, which is a sporadic incident, aided their proliferation? Possibly as plant viruses do, Sireviruses accumulate in the cytoplasm within their envelope and wait for a feeding invertebrate to ingest and transfer them to another plant [gag capsid may act as a retrovirus movement protein and enable VLP transport outside the cell to infect different tissues of the same host, including meristem or germ cells, thereby enhancing the vertical transmission rate [Sireviruses are abundant and widely dispersed in plants . Moreoveer plant . If horiion rate ,46.pol and particularly gag. These contradict the sequence differentiation that has been reported for the Tnt1 superfamily and to the best of our knowledge this is the first time such a pattern of sequence conservation is shown. The Tnt1 superfamily consists of retrotransposons with surprisingly homologous genomes that colonize species of the Solanaceae family [cis-acting motifs [The conservation of the motifs is exemplified in the closely related OPIE-2 and PREM-2 maize elements. The alignment of their LTRs provides striking results, as the microdomains are the only highly similar segments amidst diverse sequences  [Aided by the novel computational analysis which was orchestrated by the TEIRESIAS pattern discovery algorithm, we identified a plethora of highly conserved motifs in specific non-coding regions of the Sirevirus genome Figure  and we h/dicots) .cis-regulatory elements to host genomes [As vast sequence data become available from whole-genome sequencing projects, the findings of this work should enable the detailed analysis of how Sireviruses interact with their plant hosts and shape their genomes, towards which the fact that Sireviruses inhabit only plants may have important implications. While our knowledge on the evolutionary depth, distribution and genomic organization of Sireviruses will expand, more informed functional analysis of the herein introduced motifs will elucidate how and why the Sirevirus life cycle differs from other retrotransposons. In light of such knowledge, in the modern era of large-scale transcriptome analysis, and in possession of new evidence for the major influence of retrotransposon acticity on the cell transcriptional output  ,55, we s genomes , SirevirPseudoviridae genus is provided  of our Sirevirus database is available at http://bat.ina.certh.gr/downloads/Sirevirus_sequence_data.doc.The Sirevirus and classic retrotransposon datasets were constructed by retrieving sequences from various sources. The information for each element, including the source, host species and http://www.ebi.ac.uk/. MEGA4 [RT-RH peptide alignment used for the analysis is provided (Additional file http://tandem.bu.edu/tools.html. The RMs were compared against known plant cis-acting regulatory sequences in the PLACE database [Multiple alignments were carried out using ClustalW at default parameters  and the /. MEGA4  was used/. MEGA4 . Bootstr/. MEGA4 . The RT-database ,65, in odatabase  from theDiscovery of sequence motifs was performed with the use of the TEIRESIAS algorithm . ImportaPPT: polypurine tract; LTR: long terminal repeat; ENV: envelope; VLP: virus-like particle; AP: aspartic protease; INT: integrase; RT: reverse transcriptase; RH: RNaseH; DBF: DNA binding factor; RM: repeated motif; UTR: untranslated region; PBS: primer binding site; ICTV: International Committee on the Taxonomy of Viruses; ORF: open reading frame; IR: inverted repeat.AB conceived and designed the study, carried out the data collection, analysis, and interpretation, and drafted the manuscript. ND carried out pattern discovery with the TEIRESIAS algorithm, assisted in data interpretation and critically revised the manuscript. AT assisted in data interpretation and critically revised the manuscript. SP conceived and designed the study, and critically revised the manuscript. All authors read and approved the final manuscript.Pseudoviridae dataset used in this analysis. The This file contains supplementary Table S1 showing the source, accession number, host species and Pseudoviridae genus for each retrotransposon.Click here for filecopia retrotransposons. Phylogenetic and structural domain analysis of the Sirevirus and classic Ty1/This file contains supplementary Figure S1 showing the phylogenetic analysis of the Ty1/copia retrotransposons based on the RT/RH domains, supplementary Figures S2 and S3 with alignments of the IR arms of each Sirevirus and classic retrotransposon respectively, supplementary Figure S4 showing the distribution of the novel Sirevirus RM boxes, and supplementary Figure S5 with the 5'LTR alignment of the OPIE-2 and PREM-2 Sireviruses.Click here for fileThe stress-related DNA binding factors that target core sites within the RMs. This file contains supplementary Table S2 with information related to the binding site and its orientation within each RM and the stress nature of the DBFs.Click here for fileRT/RH peptide sequences that were used for the construction of the Ty1/copia phylogenetic tree.List of the Click here for file"}
+{"text": "A steady state numerical groundwater flow model has been calibrated to characterize the spatial distribution of a key hydraulic parameter in a crystalline aquifer in southwestern Ghana. This was to provide an initial basis for characterizing the hydrogeology of the terrain with a view to assisting in the large scale development of groundwater resources for various uses. The results suggest that the structural entities that control groundwater occurrence in the area are quite heterogeneous in their nature and orientation, ascribing hydraulic conductivity values in the range of 4.5\u2009m/d to over 70\u2009m/d to the simulated aquifer. Aquifer heterogeneities, coupled possibly with topographical trends, have led to the development of five prominent groundwater flowpaths in the area. Estimated groundwater recharge at calibration ranges between 0.25% and 9.13% of the total annual rainfall and appears to hold significant promise for large-scale groundwater development to support irrigation schemes. However, the model suggests that with reduced recharge by up to 30% of the current rates, the system can only sustain increased groundwater abstraction by up to 150% of the current abstraction rates. Prudent management of the resource will require a much more detailed hydrogeological study that identifies all the aquifers in the basin for the assessment of sustainable basin yield. Groundwater resources constitute arguably the most reliable buffer against the unremitting effects of climate change/variability and the concomitant ramifications on sustainable agriculture especially in the developing world. This is so because groundwater is mostly protected from high surface temperatures and the corresponding high evapotranspiration rates that affect surface flows and impoundments and thus render them ineffective sources of irrigation water supply to sustain large-scale irrigation activities. In the light of this, regional hydrogeological studies that lead to the development of aquifers for sustainable abstraction and management of groundwater resources is crucial.There are various approaches available for regional hydrogeological investigations and for providing the necessary information required for optimal aquifer and basin yield management . They inIn Ghana, erratic rainfall patterns have been noted in recent times. This has affected rain-fed agricultural activities in most areas. Efforts are being made at phasing groundwater resources at reasonable depths to meet growing domestic water needs whilst exploring the possibilities of developing the same resources for large-scale irrigation activities to augment agricultural activities. This would enhance food security and ultimately contribute to poverty reduction in the rural farming communities. Towards achieving these objectives, detailed hydrogeological investigations have been proposed to effectively characterize aquifers for proper development of the resource. This study forms one of the initial stages of this effort and utilizes a steady state numerical groundwater flow model to characterize the spatial variations in the hydraulic conductivities of a crystalline basement aquifer in southwestern Ghana. In addition to providing a conceptual framework of the groundwater flow system in the local environment, the methodology is of international importance as the utility of groundwater models in such endeavours can be applied in other terrains.The study area  is chara2/d and 119\u2009m2/d, with an average of 7.4\u2009m2/d  \u201321 19\u201321This equation has been used in diverse forms to model groundwater flow depending on the prevailing conditions. For instance, where it cannot be safely assumed that density and viscosity are largely constant,  is modifIn the current study, steady state conditions were assumed. In this respect, the time variable nature of the hydraulic head on the right hand side of  was regaLOW-2000 , was choLOW-2000 , which cThe hydrogeological data for this study were obtained from the offices of the Community Water and Sanitation Agency, CWSA, the SAL Consult , the Water Resources Commission, and the Geological Survey Department of Ghana. Data from borehole logs and the historical account of the geology and hydrogeology assisted in the vertical conceptualization of the terrain. The conceptualization was based on logs from 22 boreholes. The base map  was imported and registered in the GMS using the Map tools. Spatial discretization for the aquifer hydraulic parameters was facilitated by the local geology.Q, Cd, and dh are, respectively, the flow across the boundary, the conductance of the material, and the hydraulic head difference across the boundary.On the basis of the available data, the terrain was conceptualized as a single layer model. The lower limits of the aquifer system were conceptualized as a confining layer to coincide with the impervious material beneath. The upper limit was modelled as a convertible layer to coincide with semiconfining conditions prevailing in the area. In the absence of physical boundaries to limit flow on all sides, the vertical walls were all conceptualized as General Head Boundaries, GHB . This woAn observation coverage was created to accommodate hydraulic data from 12 boreholes for the purpose of calibration. Coverages were similarly created for the hydraulic conductivity and recharge fields as well as the GHB condition. Data of the upper and lower limits of the aquifer were imported as text files as part of the conceptualization process. A grid system was then automatically developed to overlay the domain. A uniform rectangular grid system was used for the entire domain as the intended purpose was to general flow without focus on any particular location.The conceptual model was converted into a numerical MODFLOW model to begin the simulation. The thickness of the layer was defined by mapping the top and bottom elevations obtained from the borehole logs into MODFLOW. The appropriate solver and flow packages were then selected to begin the simulation of flow. An elaborate account of documentation of the MODFLOW code is contained in the US Geological Survey reports . HoweverThe Preconditioned Conjugate Gradient (PCG) solver  uses botThe only observation data used to calibrate the model were hydraulic head data as there are no known springs and other drains in the area. The calibration objective was therefore to ensure that the model computed hydraulic head data closely matched the observed data within a margin of error established during the conceptualization process. Manual calibration was first performed by altering the values of the hydraulic conductivity and recharge and running the model each time. When the model stabilized, the calibration was switched over to the automatic calibration mode through the Parameter Estimation, PEST , 27, 28.Sensitivity analysis is recommended after calibration of every model. The objective is to test the stability of the model in the face of subtle variations in some of the key aquifer parameters. A highly sensitive model to any of the parameters is regarded as unstable and therefore not reliable for use in predicting scenarios. In this project, sensitivity analysis was performed automatically through the PEST. In this way, histograms would be generated at the end of the calibration to indicate parameter sensitivities.Since the model was conceptualized and calibrated under steady state conditions, it is not appropriate for modelling fluctuations in groundwater storage. However, it can be used in a limited fashion to evaluate recharge and abstraction scenarios. For each of the observation wells used in this study, the yields estimated during pumping tests were applied as the initial abstraction rates. In the scenario analyses, abstraction rates were increased by 10%, 20%, 50%, 100%, and 150% whilst maintaining recharge at the calibrated rates. In the next scenario, the recharge was deliberately reduced by 10%, 20%, and 30% whilst abstraction rates were increased at 10%, 20%, 50%, 100%, and 150%. This scenario was expected to simulate possible reduction in groundwater recharge rates due to decreasing rainfall amounts at the recharge areas.The model was deemed calibrated when the computed heads for all the wells were well within 2.5\u2009m of the observed heads. Five prominent flow paths have been defined in the study area , throughYidana et al.  predicteA smooth map of the horizontal hydraulic conductivity field in the domain has been established through the pilot point method \u201329 in th3/day of the total groundwater input into the system. Contributions of subsurface flows are largely responsible for the observed groundwater flow geometry in the study area. This much of recharge holds significant promise for groundwater resources development in the area. The estimated rates are compatible with the estimates of Yidana et al. [Estimated vertical groundwater recharge in the area ranges between 3\u2009mm and 109.5\u2009mm per annum, equivalent to 0.25% and 9.13% of the average annual precipitation in the basin. Apparently, a significant proportion of the total groundwater input in the terrain results from subsurface flows through the GHB condition, contributing to over 35,000\u2009ma et al. , 18, 26.Freeze and Cherry  defined This research proves that model calibration is one of the best approaches towards constraining the spatial distribution of aquifer hydraulic parameters in large-scale regional hydrogeological assessments. Therefore, in regional hydrogeological studies, the use of numerical groundwater models is very much recommended for achieving the goals of aquifer characterization. In this study, a hydraulic conductivity field has been developed for a crystalline basement aquifer in Southern Ghana through model calibration. It suggests that aquifer hydraulic conductivity in the area ranges between 4.50\u2009m/d and over 70\u2009m/d. The heterogeneity in the predicted dataset appears to be dictated by the heterogeneities in the structural entities that govern the hydrogeological properties of the aquifers in the area. Local flow systems appear to be predominant due to the observed heterogeneities in the aquifer properties in the area. Five prominent flow lines have been identified in the study area where groundwater recharge rates range between 0.25% and 9.13% of the total annual precipitation in the area. A substantial proportion of this recharge appears to accrue from subsurface flows. This much recharge holds promise for large-scale development of groundwater resources for irrigation in the area as suggested by the results of the various scenarios of groundwater abstraction simulated in the terrain. Even with a reduction in recharge by up to 30% of the current rates, the domain can sustain an increase in groundwater abstraction by up to 150% of the current abstraction rates. Evaluation of the effects of climate change/variability on groundwater resources sustainability and livelihood will require the use of groundwater flow models especially when such models are calibrated for transient conditions."}
+{"text": "To determine the underlying genetic cause of Duane retraction syndrome (DRS) in a non-consanguineous Chinese Han family.sal-like 4 (SALL4) gene were amplified with polymerase chain reaction and analyzed with direct sequencing in all the recruited family members and 200 unrelated control subjects.Detailed ophthalmic and physical examinations were performed on all members from a pedigree with DRS. All exons and their adjacent splicing junctions of the SALL4 identified a novel heterozygous duplication mutation, c.1919dupT, which was completely cosegregated with the disease in the family and absent in controls. This mutation was predicted to cause a frameshift, introducing a premature stop codon, when translated, resulting in a truncated SALL4 protein, i.e., p.Met640IlefsX25. Bioinformatics analysis showed that the affected region of SALL4 shared a highly conserved sequence across different species. Diversified clinical manifestations were observed in the c.1919dupT carriers of the family.Clinical examination revealed a broad spectrum of phenotypes in the DRS family. Mutation analysis of SALL4 gene that leads to diversified clinical features of DRS in a Chinese family. This mutation is predicted to result in a truncated SALL4 protein affecting two functional domains and cause disease development due to haploinsufficiency through nonsense-mediated mRNA decay.We identified a novel truncating mutation in the  Duane retraction syndrome (DRS) is a congenital disorder of ocular motility, which is pan-ethnic and accounts for approximately 1% of the total cases of strabismus . Occurresal-like 4  gene on chromosome 20 have been linked to DRS or DRS associated with radial forearm malformations, also known as Okihiro syndrome [SALL4-related disorders include Duane-radial ray syndrome  and acro-renal-ocular syndrome (AROS). Okihiro syndrome, characterized by radial malformation associated with Duane congenital abnormalities, is used interchangeably with DRRS. Another SALL-4 related syndrome is known as acro-renal-ocular syndrome (AROS), characterized by radial ray malformation, Duane abnormality, renal malformation , and coloboma [SALL4 have been reported in association with these disorders. The SALL4 gene is located in chromosome 20q13\u201320q13.2, with a length of 18.14 kb and 4 exons, encoding for a 1053 amino-acid-residue protein. SALL4, a new member of the SAL family of proposed C2H2 zinc finger transcription factors, plays important transcription roles during human embryogenesis [SALL4 is actually the first identified disease-causing gene of DRS and likely plays a crucial role in the development of the abducens motoneuron. Although several mutations have been reported in SALL4, disease phenotypes vary greatly among different SALL4 mutations, and the phenotype\u2013genotype correlation remains elusive. In this paper, we report for the first time a Chinese family with members presenting with isolated DRS or DRS associated syndromes in a dominant trait.Linkage analyses of DRS have successfully mapped its associated loci to chromosomes 2q13, 4q27, 8q13, 22q11, and 20q13 -13. Recesyndrome -17. SALLcoloboma . More thogenesis ,18. SALLThis study was approved by the Research Ethics Committees of the Chinese University of Hong Kong and conducted in accordance with the tenets of the Declaration of Helsinki. Informed consent was obtained from each participant after detailed explanation of the nature of the study.A three-generation Chinese Han family with DRS was recruited at the Department of Ophthalmology, Prince of Wales Hospital, Hong Kong . AffecteAlso included in this study was a group of 200 unrelated Chinese control subjects (age >40 years) who had undergone complete ocular examination and were confirmed to be free of major ocular and systemic disorders. Peripheral blood samples were collected from each control subject after informed consent was received.SALL4 were amplified with PCR and analyzed with direct sequencing in all study subjects. In total, seven pairs of primers, designed by using Primer3  referring to the sequence of human SALL4 gene from the Ensembl database , were used  according to the supplier's instructions. All exons and intron-exon boundaries of ere used . TargeteNCBI Reference Sequence database . Multiple alignments of SALL4 orthologs from different vertebrate species were conducted using a Web-based program, T-Coffee .Sequences of SALL4 orthologs in other vertebrate species were retrieved from the As illustrated in The proband (III:1) was a 4-year-old boy, who was referred to us by his pediatrician for suspected convergent squint. He was born with an uncomplicated normal delivery and enjoyed normal development and visual acuity. Ophthalmic examination showed that he had right eye abduction weakness and noticeable narrowing of palpebral fissure on attempted abduction gaze. He also presented with right esotropia at the primary position of gaze . Other oThe proband\u2019s younger sister (III:2), a 6-month-old girl, was referred at the same time to our clinic for convergent squint. She was born full term, with normal prenatal and postnatal checkups. Ophthalmic examination showed that she had large angle convergent squint at the primary position of gaze. Narrowing of bilateral palpebral fissure was noticed on both sides in attempted abduction. MRI was performed to rule out any lesion occupying intracranial space causing bilateral sixth nerve palsy. MRI of the brain was normal, except the right abducens nerve was absent . The braSALL4 gene. A heterozygous duplication c.1919dupT (Direct sequencing was performed to cover all exons and intron-exon boundaries of the 1919dupT  in exon To evaluate the functional significance of this mutation, multiple alignments of SALL4 sequences were applied to compare the sequences of human, chicken, zebrafish, rhesus monkey, cattle, African clawed frog. The results showed that the affected region of SALL4 was highly conserved across species and revealed the strong identity of the deleted region of protein at SALL4 zinc finger domains .SALL4 mutation in a Chinese family with DRS presenting with variable phenotypes. The mutation showed complete disease cosegregation and is highly likely to be disease-causing, although further functional characterization is needed to confirm the mutation\u2019s role in disease etiology. Autosomal dominant DRS has a high penetrance, although considerable variability within a pedigree also exists. This may explain the wide-spectrum phenotypes observed in the family evaluated. Until now, several sporadic cases or pedigrees and mutations implicated in DRS have been reported in Caucasians. To the best of our knowledge, this is the first genetic study, and the first pedigree report, on DRS in the Chinese population.In this study, we identified a novel SALL4 gene have been shown to cause DRS and DRS-associated disorders, namely, Okihiro syndrome, acro-renal-ocular syndrome, Holt-Oram syndrome, or suspected thalidomide embryopathy [SALL4 gene consists of four coding exons and encodes a protein with three highly conserved C2H2 double zinc finger domains, the second of which has a single C2H2 zinc finger attached at its C-terminal end, as well as an N-terminal C2HC zinc finger motif [SALL4 mutations have been described in Duane-related syndromes, especially Okihiro syndrome [Mutations in the ryopathy -22. Seveer motif ,18,23,24syndrome -17,21,23SALL4 associated with Duane-related syndromes are likely to be disease-causing via haploinsufficiency [SALL4 mutations affect the disease phenotype through nonsense-mediated mRNA decay.The c.1919dupT mutation identified in this study was a duplication located in exon 2. This mutation is predicted to generate a frameshift resulting in a truncated protein denoted as p.Met640IlefsX25. Thus, when translated, the premature termination codon would truncate the SALL4 polypeptide by 414 residues, i.e., appropriately 40% of the total length. The codon would truncate the second C2H2 double zinc finger domain and lead to a complete deletion of the third domain due to premature termination. The region containing these three functional domains was conserved; our sequence analysis also showed the deleted residues in the second domain of SALL4 to be highly conserved across different vertebrate species. This heterozygous c.1919dupT mutation plays a role in the disease pathogenesis via haploinsufficiency through a pathway known as nonsense-mediated mRNA decay, an mRNA surveillance mechanism found in all eukaryotic organisms that leads to a degradation of the transcripts with introns in the 3\u2032 untranslated region. The mutation subsequently prevents the synthesis of truncated proteins that may have toxic effects such as dominant negative interactions . In factficiency . This prficiency . The c.1SALL4 mutations with the severity of the phenotypes of DRS and DRS-associated syndromes. Although the mutation p.R865X has been reported to cause either a mild or severe phenotype [To date, there is insufficient information to correlate henotype , c.1919dSALL4 mRNA expression has been detected in the embryo and well described during different developmental periods; the expression in the progress zone of the limb buds fitted well with the radial ray anomalies in patients with Duane-related syndromes [SALL4 could be detected in isolated DRS cases, suggesting other genetic influences in addition to SALL4 [CHN1 have been found in association with isolated families with DRS [Kohlhase et al. demonstrated that the expression of SALL4 in human could be detected only in the testis and ovary . In animyndromes . In addiyndromes . Two indto SALL4 ,29. Recewith DRS .SALL4 mutation cosegregating with DRS in a Chinese pedigree. This mutation is predicted to result in a truncated SALL4 protein affecting two functional domains, and haploinsufficiency due to nonsense-mediated mRNA decay may be responsible for the disease pathogenesis. The results of this study provide further evidence for understanding the genetic causes of DRS and extend its phenotypic and mutational spectra.In summary, we have identified a novel heterozygous"}
+{"text": "We set out to evaluate the novel compound, [125I]WYE-230949, as a potential radionuclide imaging agent for the histamine H3 receptor in brain. [125I]WYE-230949 had a high in vitro affinity for the rat histamine H3 receptor (Kd of 6.9 nM). The regional distribution of [125I]WYE-230949 binding sites in rat brain, demonstrated by in vitro autoradiography, was consistent with the known distribution of the histamine H3 receptor. Rat brain uptake of intravenously injected [125I]WYE-230949 was low (0.11 %ID/g) and the ratio of specific: non-specific binding was less than 1.4, as determined by ex vivo autoradiography. In plasma, metabolism of [125I]WYE-230949 into a less lipophilic species occurred, such that less than 38% of the parent compound remained 30 minutes after injection. Brain uptake and metabolism of [125I]WYE-230949 were increased and specific binding was reduced in anaesthetised compared to conscious rats. [125I]WYE230949 is not a potential radiotracer for imaging rat histamine H3 receptors in vivo due to low brain uptake, in vivo metabolism of the parent compound and low specific binding.Histamine H WYE-230949  NaOH was purchased from Perkin Elmer Life and Analytical Sciences, Boston, MA (specific activity 81.76GBq/\u00b5M); iodophenpropit dihydrobromide was purchased from Tocris Bioscience, Bristol, UK. Other reagents and chemicals were purchased from Riedel de Ha\u00ebn, Seelze, Germany and Sigma-Aldrich, Gillingham, UK, and were used without further purification. WYE230949 and the tributylstannyl precursor of WYE-230949 were obtained from Wyeth Research, Princeton, NJ.Sodium  made up to 50 \u00b5l with 0.05 M NaOH was added 20 \u00b5l of 1 M HCl, 0.3 mg of the tributylstannyl precursor of WYE-230949 in 100 \u00b5l ethanol, and 50 \u00b5l chloramine-T solution (1 mg/ml). The reaction was mixed via vortex and incubated at room temperature for 5 min, after which it was quenched by addition of 200 \u00b5l of mobile phase. The reaction mixture was analysed by analytical HPLC before purification by preparative HPLC. The fraction containing the desired product was collected and the solvent was removed by rotary evaporation. The product was reconstituted with 0.9% saline, and finally passed through a 0.22 \u00b5m filter. The radiochemical purity was determined by analytical HPLC and the specific activity of the final product was calculated using a concentration-response curve obtained using the corresponding cold standard.The radiolabelling of WYE-230949 has previously been reported d determination. Unlabelled WYE-230949 was added prior to purification by preparative HPLC in order to allow the accurate measurement of specific activity from the preparative HPLC trace. For in vivo studies of [125I]WYE-230949, 6 mg ascorbic acid was added to during the formulation and the radioligand prepared as described above stored in a refrigerator and protected from light until use.For in vitro studies, carrier WYE-230949 was added in order to obtain the higher concentration of WYE-230949 required for K125I]WYE-230949 was determined in octanol/water and at physiological pH using a modification of the shake-flask method 125I]WYE-230949 was mixed with 1 ml of octanol and 1 ml of water (for logP) or with 1 ml of octanol and phosphate buffer . The radioactivity incorporated in both phases was determined using a gamma counter . The assay was performed in triplicate on 3 separate occasions. LogP was calculated to be log [counts in octanol/counts in water] and logD was calculated to be log [counts in octanol/counts in pH 7.4 buffer].The lipophilicity of [125I]WYE230949 binding assays were carried out in a final incubation volume of 500 \u00b5l containing Tris-HCl buffer , brain homogenates (1.47 to 2.27 mg/ml of protein) and [125I]WYE230949 (0.04\u2013102 nM). Non-specific binding was defined in the presence of 10 \u00b5M iodophenpropit. Samples were incubated at 30\u00b0C for 30 min, then filtered rapidly through Whatman GF/B filters  using a Brandel M24R cell harvester and the filters washed with 3\u00d74 ml ice-cold Tris buffer. Radioactivity on the filters was determined by liquid scintillation counting a minimum of 48 h after filtration. All assays were performed in triplicate. Specific binding was determined by subtracting non-specific binding from total binding at each concentration; Kd and Bmax values derived using GraphPad Prism (version 4.03), and expressed as mean \u00b1SEM of the triplicate assays.Animals  were killed by cervical dislocation. Whole brains were immediately excised and placed into ice-cold Tris-HCl buffer , homogenised and centrifuged at approx 40,000 g for 10 min at 4\u00b0C (Beckman J2-21M/E). The pellet was resuspended in 25 ml of buffer and centrifuged as previously, then the final pellet was resuspended in 10 ml of ice cold Tris buffer and stored at \u221250\u00b0C until required. Protein content was analysed using Bio-Rad reagent; absorbance was read on a spectrophotometer at 562 nm 2O/O2) for the duration of the experiment. Anaesthetised rats had their respiration and temperature monitored throughout. [125I]WYE-230949 was administered via a femoral vein cannula to anaesthetised rats or via the lateral tail vein to conscious rats. The injected dose for each rat was calculated by measuring the syringe in a dose calibrator before and after [125I]WYE-230949 administration. The radioactive and radiochemical amounts administered to individual rats ranged from 12.6 to 24.5 MBq and 0.32 to 0.70 \u00b5g/kg respectively in all in vivo studies.Animal experiments were conducted under the UK Animals (Scientific Procedures) Act 1986. This work was approved by the ethical review committee at the University of Glasgow (PPL: 60/3436) prior to the start of study. Na\u00efve male Sprague-Dawley rats , weighing 217 to 352 g were either conscious or anaesthetised . Rats were anaesthetised for the duration of scan (2\u20133% isoflurane in 70/30% N2O/O2) and physiological parameters were maintained as described previously 3 bindings sites including cortex, striatum, hippocampus and thalamus was captured; the first slice being 14.4 mm caudal to the eyes, approximately at the level of the caudate nucleus. Scanning was commenced 12 min prior to intravenous injection of [125I]WYE-230949 via the tail vein. Ten sequential coronal slices (400 \u00b5m/slice) were collected over a total scanning distance of 4 mm with a scanning time of 12 min per repetition. This protocol was repeated over the same 4 mm slice for a total of 17 repetitions. SPECT images were co-registered with corresponding T2-weighted MRI images obtained from a strain- and weight-matched rat using anatomical landmarks from a previously registered whole brain scan. MRI was carried out on a Bruker Biospec 7T using a T2-weighted sequence with an isotropic resolution of 300 \u00b5m. The MR image set was manually aligned to the SPECT images set using AMIDE  freeware. Each post injection SPECT stack was compiled to produce a 170 slice stack of the entire scan over time using Image J freeware . In order to determine whole brain uptake a region of interest (ROI) encompassing cortical, striatal, hippocampal and thalamic structures was defined using anatomical information on the MRI data set. The mean intensity of the ROI in each slice of the SPECT images was measured by plotting a z-axis profile. These were averaged over 12 minute time bins to produce a mean intensity for the entire 4 mm slice. These were converted to emissions per second per mm3 using the scan scaling factor and then to disintegrations per second per ml or Bq/ml. Finally these values were expressed as % of injected dose per ml of tissue and plotted against time.MicroSPECT imaging was performed in order to image the uptake of [125I]WYE-230949 uptake. At 30 or 120 min following [125I]WYE-230949 administration rats were killed by cervical dislocation. These time points were chosen based on the biological half life of [125I]WYE-230949 determined from microSPECT imaging studies. Serial arterial blood sampling via a femoral artery cannula was performed in anaesthetised rats only. Blood was collected into heparin-coated tubes for analysis of plasma and into EDTA coated tubes for analysis of whole blood. At either 30 or 120 min following [125I]WYE-230949 administration rats were killed by cervical dislocation and the brain, heart, lungs, kidneys and liver dissected. Samples were counted on a Gamma Scintillation Counter  and counts per minute (cpm) converted to kBq using a standard curve. Radioactivity was corrected for decay of 125I and expressed per sample weight and as a percentage of injected dose (%ID/g tissue).Ex vivo biodistribution studies were performed in both conscious and anaesthetised rats in order to investigate the effect of anaesthesia on [125I]WYE-230949 uptake. At either 30 or 120 min following [125I]WYE-230949 administration rats were killed by cervical dislocation. The brains were removed, frozen in isopentane at \u221242\u00b0C and stored at \u221220\u00b0C before sectioning (20 \u00b5m) in a cryostat (Bright Instrument Company Ltd). Three sections were taken every 400 \u00b5m for the entire length of the rat brain and thaw mounted onto poly-L-lysine coated slides. Sections were dried at room temperature and apposed to X-ray film (Kodak Biomax MR-1) for approximately 2 weeks. The resultant autoradiograms were analysed using computer-based densitometry . Relative optical density (ROD) measurements were obtained from 14 ROIs defined with reference to a rat brain atlas Regional brain uptake was determined by ex vivo autoradiography, studies were performed in both conscious and anaesthetised rats in order to investigate the effect of anaesthesia on [125I]WYE-230949 for 90 min at room temperature. Non-specific binding was defined in adjacent sections in the presence of 5 \u00b5M iodophenpropit dihydrobromide. Sections were washed (2\u00d730 min in Tris buffer) and briefly (15 s) rinsed in distilled water before drying and apposition to Kodak Biomax for 24 h. These autoradiograms were generated only for visual comparison with those obtained ex vivo.Sections (20 \u00b5m) cut from a frozen rat brain were pre-incubated in Tris-HCl buffer  for 15 min then incubated in 5 nM [125I]WYE-230949 blood samples were obtained by terminal cardiac puncture from anaesthetised rats or via the lateral tail vein from conscious rats at either 30 or 120 min. Rats were then immediately killed by cervical dislocation and the brain removed. Plasma was separated from whole blood by centrifugation and the protein precipitated by combining 400 \u00b5l of plasma with 400 \u00b5l of ice-cold acetonitrile. A sample (200 \u00b5l) of the supernatant was made up to 1 ml with distilled water and injected onto the HPLC column. The brain was homogenised in 2 ml saline, protein precipitated by addition of 2 ml of ice-cold acetonitrile. Samples were then centrifuged  and the supernatant added to 1 ml acetonitrile before being centrifuged again . The acetonitrile was evaporated under a stream of argon gas and the remaining 1 ml injected onto the HPLC column. Analysis of the brain and plasma metabolite samples was performed using the analytical HPLC methodology described. The % parent compound present was calculated.After intravenous injection of [All data are presented as mean \u00b1SEM. Data obtained at 30 and 120 min in conscious and anaesthetised groups were compared using two-way ANOVA with time and anaesthesia as variables with a single rat as the unit of analysis.125I]WYE-230949 was synthesized with an isolated radiochemical yield of 50.7\u00b11.7% (n\u200a=\u200a22). For in vivo and ex vivo experiments, [125I]WYE-230949 was synthesised with specific activity 82.9\u00b17.4 GBq/\u00b5mol (n\u200a=\u200a14). For in vitro experiments, [125I]WYE-230949 was synthesised with a lower specific activity of 8.5\u00b11.1 GBq/\u00b5mol (n\u200a=\u200a5) due to the addition of carrier needed to obtain the WYE-230949 concentration required for Kd determination. HPLC analysis showed that [125I]WYE-230949 had>99% radiochemical purity.[7.4 values for [125I]WYE-230949 were 1.59\u00b10.04 (n\u200a=\u200a3) and 1.64\u00b10.04 (n\u200a=\u200a3) respectively.The experimentally determined logP and logD125I]WYE-230949 to rat brain homogenates was saturable (d of 6.9\u00b11.3 nM and Bmax of 508.6\u00b134.9 fmol/mg protein (n\u200a=\u200a8). Non-specific binding was linear and represented 39\u00b13% of total binding at 6.6\u00b10.1 nM (n\u200a=\u200a3).Binding of [aturable  and disp125I]WYE-230949 and the dynamics of [125I]WYE-230949 washout in vivo. Cerebral uptake of [125I]WYE-230949 was relatively low (125I]WYE-230949 was 0.14\u00b10.01% ID/ml in the first 12 min following injection of [125I]WYE-230949 (n\u200a=\u200a5). The half-life of [125I]WYE-230949 in rat brain was 45.4 min.Serial microSPECT scanning was performed in order to provide information on the brain uptake of [vely low . The max125I]WYE-230949 detected by SPECT imaging could have been due to anaesthesia. Brain uptake was low in both conscious and anaesthetised rats, the maximal amount measured being 0.11%ID/g of tissue at 30 min in anaesthetised rats. The brain uptake in anaesthetised rats was significantly higher than in conscious rats at both time points, 0.11\u00b10.01%ID/g vs. 0.06\u00b10.01%ID/g at 30 min and 0.06\u00b10.01 vs. 0.03\u00b10.003%ID/g at 120 min  and in whole blood was 3.14\u00b10.26%ID/g of tissue at 0.50\u00b10.03 min (n\u200a=\u200a6). The radioactive concentration in plasma and whole blood reached a plateau of approximately 0.3%ID/g of tissue after 2 min , substantia nigra (p\u200a=\u200a0.002), core (p\u200a=\u200a0.0002) and shell of the nucleus accumbens (p\u200a=\u200a0.006), posterior cortex (p\u200a=\u200a0.02) and amgydala (p\u200a=\u200a0.003) (125I]WYE-230949 the pattern of [125I]WYE-230949 distribution was more homogeneous compared with the heterogeneous pattern of radioligand binding obtained from in vitro autoradiography (In areas with high histamine H=\u200a0.003) . However=\u200a0.003) . In addiiography .125I]WYE-230949 in vivo. At both time points and in conscious and anaesthetised rats two metabolites were detected in plasma, one polar metabolite  and one metabolite of intermediate lipophilicity . Both of the metabolites were less lipophilic than [125I]WYE-230949   . The amo.89 min)  and the 125I]WYE-230949. There was more parent compound present in the brains of conscious compared to anaesthetised rats at both time points (p\u200a=\u200a0.035). The amount of parent compound remaining in the brain was similar at 30 min and 120 min after administration of [125I]WYE-230949 in anaesthetised and conscious rats , radiochemical yield (average 50%) and purity (>99%).The aim of these studies was to evaluate [125I]WYE-230949 had promise as a potential radionuclide imaging agent for the histamine H3 receptor. Using standard methodology the experimentally determined logP and logD values for [125I]WYE-230949 were 1.59 and 1.64 respectively, which are comparable with other radiotracers that readily enter the brain such as [11C]raclopride (LogP 1.2) and [11C]GSK189254 (LogD of 1.7) 125I]WYE-230949 has characteristics that are predictive of good BBB penetration Initial in vitro characterisation suggested [125I]WYE-230949 has high affinity (Kd of 6.9 nM) for the rat histamine H3 receptor in whole rat brain homogenates. A Bmax value of 509 fmol/mg protein in whole rat brain is higher than previously reported values of rat histamine H3 receptor densities. For example, the Bmax determined using [123I]iodoproxyfan in rat striatum was 78 fmol/mg of protein max of [125I]iodophenpropit in rat cortex was 268 fmol/mg of protein 3H]GSK189254 in rat cortex was 283 fmol/mg of protein max in whole brain would be lower that the Bmax in striatum due to the presence of low density regions in the whole brain homogenate. It is not clear why our values are higher. Our data suggests that a secondary binding site is unlikely as the data were best fit with a one site binding model and the Scatchard plot was linear except for an expected increased variability at very low bound values  mediated transport and plasma protein binding, BBB penetration is a complex process making prediction of in vivo brain uptake from in vitro assays challenging. Recent studies have highlighted that measurement of lipophilicity should not be relied upon as a predictor of BBB penetration 125I]WYE-230949 administration in the caudate, amygdala, core and shell of the nucleus accumbens, substantia nigra and posterior cortex, all regions previously shown to have high histamine H3 receptor densities. The increase in specific binding over time indicates that some of the injected [125I]WYE-230949 was binding to histamine H3 receptors in these regions It should be noted that higher specific binding was present at 120 min compared with 30 min after [125I]WYE-230949. Brain uptake was lower in conscious rats -citalopram to serotonin transporters and decreases [125I]PE2I binding to dopamine transporters Radioligand brain uptake, distribution and metabolite studies were performed in conscious as well as anaesthetised rats to rule out the possibility that anaesthesia may have confounded the images obtained from microSPECT imaging of [125I]WYE-230949 is not a useful radiotracer for imaging rat histamine H3 receptors in vivo due to low brain uptake, in vivo metabolism of the parent compound and low specific binding.In conclusion, [S1 Fig125I]WYE-230949 injection.MicroSPECT/MRI images showing low brain and high glandular uptake following [ High uptake was observed in the thyroid, Hardarian and exorbital lacrimal glands suggestive of radio-deiodination. The glands are indicated on the microSPECT images by the white arrows; HG, Hardarian gland; ELG, exorbital gland; Th, thyroid gland.(TIF)Click here for additional data file."}
+{"text": "Studies have shown that structural lesions may be present in patients with non-radiographic axial spondyloarthritis (nr-axSpA). However, the relevance of structural lesions in these patients is unclear, particularly without signs of inflammation on magnetic resonance imaging (MRI). We assessed the presence of structural lesions at baseline on MRI in the sacroiliac joints (SIJ) of patients with nr-axSpA with and without SIJ inflammation on MRI.Bone marrow edema (BME) was assessed on short tau inversion recovery (STIR) scans from 185 patients with nr-axSpA, by two independent readers at baseline using the Spondyloarthritis Research Consortium of Canada (SPARCC) score. Structural lesions were evaluated on T1 weighted spin echo scans, with readers blinded to STIR scans, using the SPARCC MRI SIJ structural score. Disease characteristics and structural lesions were compared in patients with SIJ BME (score \u22652) and without SIJ BME (score <2).P\u2009<\u20090.001), backfill , fat metaplasia , and ankylosis . Significantly more patients with both SIJ BME and structural lesions were male and/or HLA-B27 positive than patients with only SIJ BME. Mean (SD) spinal scores  were significantly higher in patients with SIJ structural lesions than without: 6.5 (11.5) vs 3.3 (5.1), respectively, P\u2009=\u20090.01.Both SIJ BME and structural lesions scores were available for 183 patients; 128/183 (69.9%) patients had SIJ BME scores \u22652 and 55/183 (30.1%) had scores <2. Frequencies of MRI structural lesions in patients with vs without SIJ BME were: erosions  contains supplementary material, which is available to authorized users. Treatment of axial spondyloarthritis (axSpA) is increasingly aimed at intervention early in the disease course before radiographic sacroiliitis has appeared and when response to treatment is greatest. Thus, magnetic resonance imaging (MRI) is an important tool for the diagnosis of SpA because it can identify inflammation prior to the detection of radiographic changes \u20135.The Assessment of SpondyloArthritis international Society (ASAS) classification system for axSpA is based on whether patients meet clinical criteria or imaging criteria \u20138. PatieRecent studies have described several structural lesions on MRI in the sacroiliac joints (SIJ) \u201311. ErosAlthough limited studies have demonstrated that structural lesions may already be evident in non-radiographic SpA defined according to physician expert opinion , 2, the Details of the EMBARK study , a phase IIIb, 104-week clinical study, have been published previously , 15. BriThe study was conducted in accordance with the International Conference on Harmonisation guidelines for Good Clinical Practice and the ethical principles of the Declaration of Helsinki. Written informed consent was obtained from all patients prior to enrollment. The institutional review board or independent ethics committee at each participating center reviewed and approved all consent forms and the study protocol (see \u201cAcknowledgments\u201d for details).This was a post-hoc cross-sectional analysis of patients with baseline lesion scores. The primary analysis was assessment of the presence of structural lesions on MRI in patients with SIJ BME (score \u22652) versus without SIJ BME (score <2). Sensitivity analysis was performed by evaluating the presence of structural lesions in patients who were ASAS MRI positive versus negative.Two independent central readers, blinded to treatment and patient characteristics, assessed STIR scans of the SIJ and spine for BME using the Spondyloarthritis Research Consortium of Canada (SPARCC) scores \u201318. For Two different readers (who developed the SPARCC scoring method), scored structural lesions using the SPARCC MRI SIJ structural score (SSS), which assesses fat metaplasia, erosion, backfill, and ankylosis on T1WSE MRI . The reaThe two readers scored the presence of lesions dichotomously (yes/no) in SIJ quadrants for erosion and fat metaplasia and in SIJ halves for backfill and ankylosis using a data entry system based on a diagram of the SIJ . Five coErosion: on T1WSE is the full-thickness loss of the dark appearance of iliac or sacral cortical bone at its anticipated location as well as loss of the normal bright appearance of adjacent bone marrow.Backfill: on T1WSE is the complete loss of iliac or sacral cortical bone at its anticipated location as well as increased signal clearly demarcated from adjacent normal marrow by dark signal with irregular contour reflecting sclerosis at the border of the eroded bone.Fat metaplasia: increased signal on T1WSE sequences. To be scored in the SPARCC SSS method, there must be homogeneous signal across the lesion, and it must extend >1\u00a0cm in depth from the joint surface.Ankylosis: bone marrow signal on T1WSE between the sacral and iliac bone marrow with a full-thickness loss of the dark appearance of the iliac and sacral cortical bone.This study used standardized definitions of structural lesions of the SIJ on MRI, which were developed by the Canada-Denmark MRI Working Group and then extended in a subsequent report to include backfill , 10, 12.Proportions of patients with lesions scored >0 (i.e. with any lesions or specific lesions) were determined according to the presence or absence of SIJ BME (primary strata) and according to ASAS MRI positive and negative status (secondary strata); differences in proportions were tested using Cochran-Mantel-Haenszel (CMH) tests. The presence of SIJ BME and structural lesions was evaluated according to the radiographic grade of sacroiliitis using CMH tests. Differences in demographics and baseline disease characteristics, including 23-DVU spinal score, were analyzed by presence or absence of any SSS lesions (>0 or =0) overall and separately by presence or absence of SIJ BME (\u22652 or <2). Each type of structural lesion was evaluated in this manner as well. These analyses were performed using analysis of variance, the chi-square test, or the Wilcoxon-Mann-Whitney test.Cumulative probability plots were derived to compare the SSS lesion scores of specific structural lesions according to the presence or absence of SIJ BME. These data were also analyzed using simple descriptive statistics with one-way analysis of variance and the Wilcoxon-Mann-Whitney test to test for differences in SIJ BME subgroups. Inter-observer intra-class correlation coefficients (ICC) were determined for structural lesion scores to evaluate scoring consistency between the two MRI readers. Additionally, the proportion of patients with individual lesion scores >0 was compared between two different definitions: scores from both readers were >0, and the score from at least one reader was >0. Spearman correlation was tested to analyze the relationship between continuous spinal inflammation, i.e. the 23-DVU spinal score, and continuous patient characteristics, i.e. age and symptom duration, and between continuous spinal inflammation and continuous structural lesions. Multivariate stepwise regression analysis was used to determine whether age, gender, symptom duration, presence/absence of SIJ BME, or presence of specific structural lesions was significantly associated with the 23-DVU spinal score.MRI scans at baseline were available for 185 patients. The mean (standard deviation (SD)) age was 32.0 (7.8) years; 60.5% were male, and the mean (SD) duration of disease symptoms was 2.4 (1.8) years. A total of 133/182 (71.9%) patients were HLA-B27 positive and 152/185 (82.2%) met the ASAS MRI imaging criteria.P\u2009<\u20090.001). Relative frequencies of MRI structural lesions in patients with versus without SIJ BME were: erosion , backfill , fat metaplasia , and ankylosis )  patients had \u22651 structural lesion on MRI comprising erosion , backfill , fat metaplasia , and ankylosis . SIJ BME data were available for 183 patients; 128/183 (69.9%) patients had SIJ BME and 55/183 (30.1%) did not. Of the patients with SIJ BME, 69/128 (53.9%) had a structural lesion on MRI, compared with 7/55 (12.7%) patients without SIJ BME (P\u2009=\u20090.03). For ASAS MRI positive versus negative patients, relative frequencies of structural lesions were: erosion , backfill , fat metaplasia , and ankylosis   had a structural lesion on MRI, compared with 8/33 (24.2%) who were ASAS MRI negative (P\u2009<\u20090.001). Additionally, age was significantly lower in the group with both SIJ BME and structural lesions (30.3\u00a0years) compared to the group with only SIJ BME . Symptom duration was similar across the groups. More patients were HLA-B27-positive who had both SIJ BME and structural lesions (87.0%) than those with only SIJ BME . Mean BASDAI and mean BASFI were significantly higher in the group with SIJ BME and no structural lesions  than in the group with both SIJ BME and structural lesions . For the patients without SIJ BME, the small sample size of only seven patients in the SSS >0 category precluded interpretation of this subgroup.A comparison of the demographics and baseline disease characteristics between patients with the presence (SSS >0) or absence (SSS\u2009=\u20090) of any structural lesion according to those with and without SIJ BME is provided in Table\u00a0P\u2009=\u20090.01. Similar differences in demographic and baseline disease characteristics were also observed when comparisons were made according to the presence or absence of individual structural lesions, with more male patients, higher B27 positivity, and greater spinal inflammation, especially in patients with erosion or backfill  23-DVU spinal scores were higher overall in those patients with structural lesions in the SIJ: 6.5 (11.5) vs 3.3 (5.1), respectively, The cumulative probability plots demonstrate visually that the structural lesions of erosion, backfill, and fat metaplasia occurred to a greater extent in patients with than without SIJ BME Fig.\u00a0, which iAn analysis of the presence of SIJ BME and structural lesions according to radiographic grade did not identify a linear relationship between these measurements. However, the frequency of MRI structural lesions was greatest in those patients with radiographic unilateral grade 1 and contralateral grade 2 changes in the SIJ Table\u00a0.Table 3MP\u2009=\u20090.02; backfill: 4.4 (1.7), P\u2009=\u20090.01.A univariate analysis of the relationship between 23-DVU spinal score and select dichotomous patient characteristics, including structural lesions, determined that male patients and patients with SIJ BME had significantly higher 23-DVU spinal scores Additional file 2:Lesions seen on T1 weighted spin echo MRI. (PDF 128\u00a0kb)"}
+{"text": "Treatment options for metastatic urothelial carcinoma (mUC) remained relative unchanged over the last 30\u00a0years with combination chemotherapy as the mainstay of treatment. Within the last year the landscape for mUC has seismically shifted following the approval of five therapies targeting the programmed cell death protein (PD-1)/programmed cell death ligand 1 (PD-L1) axis. Notably, the anti-PD-1 antibody pembrolizumab demonstrated improved OS relative to chemotherapy in a randomized phase III study for second line treatment of mUC; this level 1 evidence led to approval from the U.S. Food and Drug Administration (FDA). The PD-1 antibody nivolumab also demonstrated an overall survival benefit, in this case in comparison to historical controls. Similarly, antibodies targeting PD-L1 including atezolizumab, durvalumab, and avelumab have now received accelerated approval from the FDA as second line treatments for mUC, with durable response lasting more than 1 year in some patients. Some of these agents are approved in the first line setting as well - based on single-arm phase II studies atezolizumab and pembrolizumab received accelerated approval for first-line treatment of cisplatin ineligible patients. Despite these multiple approvals, the development of clinically useful biomarkers to determine the optimal treatment for patients remains somewhat elusive. In this review, we examine key clinical trial results with anti-PD1/PD-L1 antibodies and discuss progress towards developing novel biomarkers beyond PD-L1 expression. Approximately 79,000 new cases of bladder cancer are estimated in the United States in 2017, resulting in 16,870 deaths \u20135. WithiThe five immunotherapy agents that are FDA approved for the treatment of metastatic urothelial carcinoma all have similar objective response rates (ORR) - between 15 and 23% in unselected patients in the second line setting  noted with atezolizumab at the prespecified dose level in cohort I and cohort II were fatigue, diarrhea, and pruritis with rare instances of autoimmune phenomena commonly associated with PD-1 therapy including elevation in liver enzyme tests (3%), pneumonitis (2%) and hypothyroidism (7%). Recently, the randomized phase III IMvigor 211 study evaluating atezolizumab in comparison to chemotherapy as second line treatment was announced to have failed to reach its primary endpoint of improved overall survival, independent of PD-L1 expression status. More extensive data on that trial were not available at the time of writing, but that unexpected outcome highlights the need for improved patient stratification beyond PD-L1 testing to select appropriate mUC patients for immunotherapy.Nivolumab, a fully-human IgG4 anti-PD1 antibody hinge-modified to improve half-life, received accelerated approval from the FDA for second line therapy in previously platinum treated mUC. This approval was based on data from the checkmate 275 trial, a single-arm phase II study that enrolled 270 patients to receive nivolumab at 3\u00a0mg/kg every 2\u00a0weeks . The PD-P\u00a0=\u00a00.002). Progression free survival was not improved versus chemotherapy; this has been observed in other phase III studies of PD-1 blocking agents [P\u00a0=\u00a00.001). Consistent with what was observed in cohort I of IMvigor 210 with atezolizumab and Checkmate-275 with nivolumab, overall response rate was similar between groups with low and high PD-L1 expression as measured by tumor cell (TC) and immune cell (IC) PD-L1 expression using the Dako assay and the 22C3 antibody. I.E. the ORR was 21.1% in the total population versus 21.6% in the group with PD-L1 score of >10%. The lack of correlation between response rates and PD-L1 combined score again demonstrates an unmet need for biomarkers for treatment selection. The median overall survival of the PD-L1 high composite score group (>10%) was 8.0\u00a0months (CI 5.0-12.3) with pembrolizumab in comparison to 5.2\u00a0months (CI 4.0-7.4) in the chemotherapy group. While pembrolizumab clearly provides a survival benefit relative to chemotherapy, higher PD-L1 expression was not associated with increased survival relative to the entire pembrolizumab treatment group. Grade 3 or 4 adverse events were less frequent in the pembrolizumab group (15% with pembrolizumab vs 49.4% in the chemotherapy arm). Similar to nivolumab the most frequently reported side effects were pruritus (19.5%), fatigue (15.0%), nausea (11.3%), and diarrhea (10.1%).Pembrolizumab is a hinge-stabilized, humanized IgG4 anti-PD1 antibody that, like nivolumab, disrupts the engagement of PD-1 with its ligands PD-L1 and PD-L2. Of the FDA-approved antibodies blocking the PD-1/PD-L1 interaction, pembrolizumab is the only agent approved based on data from a randomized, phase III study . Approvag agents , 15. Then\u00a0=\u00a042) [Pembrolizumab was also approved for use as first-line therapy in cisplatin ineligible patients in mUC based on early data from the phase II Keynote-052 study , 16. Oven\u00a0=\u00a042) . In an en\u00a0=\u00a042) . This con\u00a0=\u00a042)  and showeither tumor cells (TC) or immune cells (IC) demonstrated \u226525% staining by IHC [Durvalumab, an FcR binding deficient anti-PD-L1 antibody, was approved in May 2017 based on a single-arm phase I/II study evaluating 61 patients with platinum-treated advanced UC . Patientg by IHC . The FDAThe single-arm JAVELIN phase I study evaluated avelumab, an IgG1 anti-PD-L1 antibody that blocks the interaction between PD-1 and PD-L1 but not PD-1 and PD-L2. In an initial phase Ib study of unselected patients with platinum refractory mUC the ORR was 18.2% with a reported median OS of 13.7\u00a0months . All patExtrapolating from studies in melanoma  and NSCLA number of on-going trials are evaluating novel targets in combination with PD-1 therapy, including traditional chemotherapy , intra-vWith the continued success of PD-1 targeted therapies in the metastatic setting a number of studies are evaluating immune checkpoint blockade in BCG-refractory non-muscle invasive bladder cancer. Early phase clinical trials evaluating BCG in combination with both pembrolizumab   to rvalumab ), and Chrvalumab ).One potential reason for these discrepancies is the use of 4 distinct assays for PD-L1 IHC scoring. For instance, pembrolizumab and nivolumab clinical trials use the Dako assay with the 22C3 and 28-8 antibody clones respectively. In contrast, durvalumab and atezolizumab use the Ventana assay and SP26 and SP142 antibody clones respectively . In the n\u00a0=\u00a073) or basal (n\u00a0=\u00a0122) subtypes. Enrichment in PD-L1 immune cell expression was noted in the basal subtype (60% vs 23%), while tumor cell PD-L1 expression was noted almost exclusively in basal subtypes (39% vs 4%). Responses to atezolizumab were documented in all subtypes with a statistically higher response rate noted in luminal cluster II subtype  relative to luminal cluster I, basal cluster I, and basal cluster II . A similar trend was noted in Cohort I of IMvigor with atezolizumab with the highest percentage of responses noted in the luminal cluster II group . The TCGA subtypes were also an exploratory endpoint in the phase II Checkmate-275 study of nivolumab; by contrast here basal I subtype tumors represented the highest proportion of responders . Luminal cluster II tumors treated with nivolumab had an overall response rate of ~25%. The reasons for these discrepancies in the mUC subtype most likely to respond might be related to tissue source. Both cohorts of IMVigor210 and Checkmate-275 allowed biopsy specimens from primary tumor, lymph nodes, or metastatic lesions for TCGA subtyping which may lead to inappropriate tumor classification. Second, the criteria for molecular subtyping differs in each study, highlighting a challenge in standardizing TCGA classification. Taken together these results are consistent with the notion that TCGA subtype is not likely to prove a strong predictive biomarker, especially across agents.Exploratory analyses in several trials retrospectively correlated The Cancer Genome Atlas (TCGA) urothelial cancer subtype with response to PD-1 / PD-L1 directed immunotherpy . In cohoP\u00a0<\u00a00.0001) [s showing a similar RR and the top 1/3rd deriving a progression free survival benefit. In both melanoma  and NSCL\u00a00.0001) . Smoking\u00a00.0001) . There w\u00a00.0001) , 50. An \u00a00.0001) . Patientnegative predictive value of TMB, however, is unclear, as responses were observed in some patients with low mutation burden.Other studies used retrospective data to evaluate the relationship between the number of non-synonomous mutations and immunotherapy responses. Data in NSCLC using targeted exome sequencing of cancer specific genes identified an association between high mutation burden and durable overall response . A compoSignificant challenges confront the use of TMB as a predictive biomarker for immunotherapy. First is the challenge of unifying and standardizing the definition of mutation burden. For instance, some assays standardize for the size of the genome covered by targeted sequencing on a per megabase level. Others report based on absolute mutational burden which may fail to represent the true tumor mutation burden relative to the depth of sequencing performed. Second, gene fusions, truncations, and translocations may not be adequately covered by targeted sequencing panels and the value of these genetic events relative to single nucleotide variants in predicting response to immunotherapy remains to be determined. Third, germline variants may not be silenced by informatics techniques that filter common germline single nucleotide polymorphisms. As a consequence, uncommon germline variants may artificially increase the calculated tumor mutation burden, which highlights the need to improve standardization between tumor mutation burden assays. The somatic mutation burden is also likely to change dependent on other variables through the treatment course such as prior chemotherapy treatment and a biopsy at a single time point may not adequately reflect the relative antigenicity of the tumor. Despite these limitations there is now strong evidence that TMB correlates with durable responses to PD-1 blockade in multiple tumor types and with further standardization TMB will likely be a reliable surrogate to predict immunotherapy response.Other surrogate measures of mutation burden such as chronic carcinogen exposure , defects in DNA repair mechanisms such as microsatellite instability/mismatch repair defects, and POLE mutations have emerged as potentially useful clinical biomarkers , 54. BasComposite variables integrating PD-L1 expression, TCR sequencing/TCR clonotypes, epigenetic analysis, and tumor mutation burden may delineate characteristics that predict responses to immunotherapy due to inherent advantages and disadvantages of each biomarker as a stand alone test Fig.\u00a0. These iThere are now numerous examples across solid tumor types including head and neck squamous cell cancer, NSCLC, melanoma, and urothelial cancer exploring correlation between composite markers and response to anti-PD1 , 63. In Ongoing efforts evaluating combined measurements of mutational load with gene expression signatures show promise. Gene expression profiling performed in longitudinal tumor biopsies showed dynamic changes in multiple genes after the initiation of PD-1 therapy . As thesAn inherent difficulty in using PD-L1 status as a predictive biomarker is that subjective scoring of IHC sections provides information regarding only a single factor in the tumor microenvironment, and doesn\u2019t take into account other features that might more accurately segregate \u201chot\u201d from \u201ccold\u201d tumors , 67. In p\u00a0=\u00a00.0003, CR or PR in 20/59 patients with high IFN-\u03b3 signature relative to CR or PR in 19/118 patients with medium or low IFN-\u03b3 signature). Similar gene expression analysis performed with a chemokine panel showed enrichment in responses from individuals with high expression of CXCL9 and CXCL10 demonstrating the potential to use gene expression profiling as a biomarker. Similar to TMB measurements, the negative predictive value of this gene panel remains problematic as some responses were noted in some patients with a non-inflamed cytokine signature.In the Checkmate 275 study with nivolumab in mUC, a 25-gene interferon-gamma (IFN-\u03b3) signature derived from crude extract  was used to assess 177 tumor samples from pretreatment biopsies. Higher values in the IFN-\u03b3 gene signature were correlated with response to nivolumab relative to low value IFN-\u03b3 expression score  uses 6-color bar codes to identify specific RNA sequencing without gene amplification as is required with traditional RNA sequencing or qPCR technologies. Using a small subset of 19 melanoma patients from Keynote-001, 680 different genes were profiled by Nanostring. A subset of 18 specific genes including interferon-gamma signaling (IDO1 and STAT1), antigen presentation , NK T cell signaling , and additional immunomodulatory proteins Fig.  were tesThe FDA approvals of atezolizumab, nivolumab, pembrolizumab, avelumab, and durvalumab represent a major paradigm shift in treating mUC. The recent results of the Phase III IMvigor 211 study, however, suggest the possibility that not all PD-1/PD-L1 reagents will have comparable efficacy. Standardized, reproducible biomarkers  are needed to accurately guide treatment decisions as no single test has as of yet demonstrated reproducibility to predict responders to immunotherapy. This is of particular importance as there are potentially subgroups of patients with low mutation burden who may respond more favorably to chemotherapy as was noted in Checkmate 026. Although composite biomarkers are of interest, the next generation of predictive biomarkers for immunotherapy might involve either an assessment of tumor mutational burden (TMB) or a targeted gene expression profile with particular attention to T cell gene signatures; these ongoing studies are of critical importance in optimizing precision immunotherapy for patients with metastatic urothelial cancer."}
+{"text": "Monoclonal antibodies that block immune regulatory proteins such as programmed death-1 (PD-1) have demonstrated remarkable efficacy in controlling the growth of multiple tumor types. Unresectable or metastatic basal cell carcinoma, however, has largely gone untested. Because PD-Ligand-1 (PD-L1) expression in other tumor types has been associated with response to anti-PD-1, we investigated the expression of PD-L1 and its association with PD-1 expression in the basal cell carcinoma tumor microenvironment. Among 40 basal cell carcinoma specimens, 9/40 (22%) demonstrated PD-L1 expression on tumor cells, and 33/40 (82%) demonstrated PD-L1 expression on tumor-infiltrating lymphocytes and associated macrophages. PD-L1 was observed in close geographic association to PD-1+ tumor infiltrating lymphocytes. Additionally, we present, here, the first report of an objective anti-tumor response to pembrolizumab (anti-PD-1) in a patient with metastatic PD-L1 (+) basal cell carcinoma, whose disease had previously progressed through hedgehog pathway-directed therapy. The patient remains in a partial response 14\u00a0months after initiation of therapy. Taken together, our findings provide a rationale for testing anti-PD-1 therapy in patients with advanced basal cell carcinoma, either as initial treatment or after acquired resistance to hedgehog pathway inhibition. Basal cell carcinoma (BCC) is the most common human cancer, though most tumors are eradicated using locally-directed therapies . In the Over the past several years, monoclonal antibodies that block immune checkpoint proteins , PD-Ligand-1 (PD-L1)), have demonstrated remarkable efficacy in controlling the growth of multiple tumor types . In seveIn the current study, we characterized PD-L1 and PD-1 expression patterns in the tumor microenvironment of 40 archived BCC specimens. Additionally, we studied a pre-treatment tumor specimen and clinical and radiographic response characteristics from one patient who experienced a durable, objective anti-tumor response to pembrolizumab (anti-PD-1) monotherapy.Following Institutional Review Board approval, 40 surgical pathology specimens from 40 unique patients with BCC were identified from the Johns Hopkins Hospital surgical pathology archives. Slides were reviewed by a board-certified dermatopathologist (JMT) to confirm the diagnosis, and one representative paraffin block was chosen for PD-L1 and PD-1 immunohistochemistry (IHC). PD-L1 expression was assessed using the murine anti-human PD-L1 monoclonal antibody 5H1  at a concentration of 0.1\u00a0\u03bcg/mL, as previously described . PD-L1 eForty specimens from 40 unique patients were evaluable. All were primary lesions that were locally aggressive or recurrent. The tumors ranged in size from 2 to15\u00a0cm in greatest dimension. Nine of forty (22%) demonstrated PD-L1 expression on tumor cells, and 33/40 (82%) demonstrated PD-L1 expression on tumor-infiltrating lymphocytes (TIL) and associated macrophages. All  of cases had varying degrees of either true TIL present or lymphocytes present in the immediate extratumoral stroma. PD-1 expression was seen on lymphocytes in the tumor microenvironment of each case, including the 16/40 (40%) cases that had only rare lymphocytes present. All (33/33) cases with PD-L1 expression on tumor or macrophages showed an association with PD-1+ TIL . Although radiographically her best overall response per RECIST criteria was stable disease, clinically, her symptoms related to tumor growth improved dramatically for several months, then worsened about 12\u00a0months after having initiated therapy.She then pursued treatment on a phase 1 clinical trial of AZD6244 (MEK inhibitor) and IMC-A12 ; clinicaltrials.gov ID# NCT01061749). Her disease progressed within 6\u00a0months, requiring palliative radiotherapy.In late 2014 her neck pain increased and CT scans demonstrated multiple lung metastases. Re-initiation of Hh inhibition was considered but, based on previous clinical experience, the likelihood of anti-tumor activity was felt to be low . The patAlthough we had not yet assessed PD-L1 expression in this patient\u2019s tumor, support for the use of PD-1 pathway-directed therapy came from pre-clinical studies demonstrating that BCC typically caries a high genetic mutation burden, a characteristic associated with response to PD-1 blockade in other tumor types , 14. AddPembrolizumab (anti-PD-1) was administered at 2\u00a0mg/kg IV every 3\u00a0weeks beginning in December 2015. The following month, the patient reported that her pain medication requirement had decreased substantially. CT scans performed 4\u00a0months into therapy demonstrated a partial response (PR) to therapy per RECIST 1.1 criteria , and will require a large cohort of patients.In conclusion, our laboratory and clinical findings suggest that PD-1 blockade should be considered as salvage therapy in patients with advanced BCC whose disease has progressed after standard Hh pathway inhibition. Our results also provide a rationale for ongoing clinical trials of PD-1 pathway blockade agents in patients with advanced BCC, either as first-line therapy or in combination with or after treatment with Hh inhibitors ."}
+{"text": "Gastrointestinal (GI) malignancies are the most prevalent tumors worldwide, with increasing incidence and mortality. Although surgical resection, chemotherapy, radiotherapy, and molecular targeted therapy have led to significant advances in the treatment of GI cancer patients, overall survival is still low. Therefore, alternative strategies must be identified to improve patient outcomes. In the tumor microenvironment, tumor cells can escape the host immune response through the interaction of PD-1 and PD-L, which inhibits the function of T cells and tumor-infiltrating lymphocytes while increasing the function of immunosuppressive T regulatory cells. The use of an anti-PD-1/PD-L blockade enables reprogramming of the immune system to efficiently identify and kill tumor cells. In recent years, the efficacy of PD-1/PD-L blockade has been demonstrated in many tumors, and this treatment is expected to be a pan-immunotherapy for tumors. Here, we review the signaling pathway underlying the dysregulation of PD-1/PD-L in tumors, summarize the current clinical data for PD-1/PD-L inhibitors in GI malignancies, and discuss road toward precision immunotherapy in relation to PD-1/PD-L blockade. The preliminary data for PD-1/PD-L inhibitors are encouraging, and the precision immunotherapy of PD-1/PD-L inhibitors will be a viable and pivotal clinical strategy for GI cancer therapy. Gastrointestinal (GI) cancers are the most common human tumor worldwide, and the incidence and mortality are increasing every year , 2. SeveBecause the antigens of tumor cells are \u201cself\u201d antigens, the immune system is unable to recognize cancers. Thus, tumors are able to escape the host immune response through a variety of mechanisms at the level of the tumor microenvironment . These mRecently, the effectiveness of immunotherapy targeting immune checkpoints in the treatment of numerous forms of cancers has been studied. PD-1, an immune checkpoint, plays a major role in tumor immune escape , 10. TheThe myriad of genetic and epigenetic variations and alterations that are features of all cancers supply a varied set of antigens that are utilized by the immune system to distinguish tumor cells from their normal counterparts. Regarding T cells, the ultimate extent and quality of the response is regulated by a balance between co-stimulatory and inhibitory signals, which are initiated through antigen recognition by the T cell receptor (TCR) . Co-stimT cells have become the core of cancer immunotherapy efforts owing to their capacities to selectively recognize peptides derived from the cytolysis tumor cells, directly recognize and kill antigen-expressing cells, and integrate adaptive and innate effector mechanisms to orchestrate diverse immune responses such as helper and regulator T cells . TherefoPD-1, also known as CD279, is a cell surface co-inhibitory receptor that induces immune inhibition and promotes tumor immune escape from the cytotoxic T cell immune response during carcinogenesis . PD-1 isSeveral studies have been devoted to the discovery of molecules that interact with PD-1. Programmed cell death ligand-1 (PD-L1), also called B7 homolog 1 (B7-H1) or CD274, was previously identified as an inhibitor of the human T cell response in vitro. PD-L1 was later determined to be a binding and functional partner of PD-1 . AnotherPD-1 is expressed on a large proportion of tumor-infiltrating lymphocytes (TILs) from many different cancer types. PD-L1 is expressed in 20\u201350% of human tumors and can provide immune evasion in many cancers by its overexpression (PD-L1 or PD-L2) and an augmented tumor immune response by its (PD-1) abrogated ligand interaction . Based oSince tumors can escape the T cell immune response by expressing inhibitory molecules such as PD-1 or PD-L1, blocking the PD-1/PD-L pathway by interfering with binding between PD-1 and its ligands may become a therapy for the treatment of cancer.Ranked as the sixth leading cause of cancer-related morbidity worldwide, esophageal cancer is one of the least studied but most lethal medical conditions . Compare+ advanced esophageal carcinoma [+ advanced esophageal carcinoma. Similarly, Kojima et al. conducted a phase II study of nivolumab, a fully humanized IgG4 mAb PD-1 inhibitor, in patients with advanced esophageal cancer [+ patients, whereas nivolumab was used for unselected patients. PD-1/PD-L blockade alone or combined with radiotherapy and chemotherapy will be a future research direction in the treatment of advanced esophageal cancer (Table\u00a0Pembrolizumab is a PD-1 inhibitor that blocks the interaction between PD-1 and PD-L1 . Doi et arcinoma . PD-L1 el cancer . Sixty-fThe Cancer Genome Atlas network divides gastric cancer (GC) into four molecular subtypes: (1) Epstein-Barr virus (EBV)-positive tumors, (2) microsatellite instable tumors (MSI), (3) genomically stable (GS) tumors, and (4) tumors with chromosomal instability (CIN) . PD-L1 e+; a total of 39 patients enrolled in the trial and 36 patients were evaluable for a response. ORR was 33% by investigator review. These results indicated that pembrolizumab exhibited antitumor activity in PD-L1+ advanced gastric cancer. Most recently, a clinical phase III trial was conducted to assess the efficacy and safety of nivolumab in patients with unresectable advanced GC/GEC [.) 4.14\u00a0months among patients with placebo, and the OS rates at 6 and 12\u00a0months were 46.4 vs. 34.7% and 26.6 vs. 10.9%, respectively. The ORR was 11.2% with nivolumab vs. 0% with placebo. The median PFS was 1.61\u00a0months with nivolumab vs. 1.45\u00a0months with placebo . Pembrolizumab was evaluated as monotherapy and in combination with cisplatin + 5-fluorouracil in participants with recurrent or metastatic GC/GEC (NCT02335411). Durvalumab monotherapy, durvalumab in combination with tremelimumab, or tremelimumab monotherapy are currently being assessed for the treatment of metastatic or recurrent GC/GEC (NCT02340975).Hepatocellular carcinoma (HCC) is the most common primary liver malignancy . The oven\u2009=\u200948), and a phase II dose-expansion study was initiated in four cohorts (n\u2009=\u2009214): sorafenib intolerant/na\u00efve, sorafenib progressors, HBV infected and hepatitis C infected. During dose escalation, no maximum tolerated dose was reached. In the dose expansion phase, the ORR was 20% and the 9-month OS rate was 74%. The median duration of response (DOR) was 9.9\u00a0months, and the disease control rate (DCR) was 64%. ORRs of 21 and 23% were observed in the uninfected sorafenib-treated and intolerant/naive patients, respectively  tumor tissue and was found to be associated with poor survival, suggesting that PD-1/-L1 inhibitors may serve as adjuvant therapy , 54. In Despite a deep understanding of the genetic mechanisms underlying pancreatic cancer (PC), current therapies for this malignancy are still limited . The immThe majority of colorectal cancers (CRCs) develop through a CIN pathway, and approximately 15% show defective mismatch repair (dMMR), which can be measured by either the presence of MSI9 or by the lack of DNA mismatch repair proteins , 63. dMMThe clinical activity of immune checkpoint blockade with pembrolizuma was evaluated in a phase II study conducted by Le and colleagues . PembrolAn important phase II study evaluating the clinical activity of nivolumab in patients with dMMR/MSI-H mCRC was reported at the 2017 Gastrointestinal Cancers Symposium of the American Society of Clinical Oncology (ASCO) . SeventyIn these trials, PD-1 inhibitor demonstrated clear efficacy in patients with MSI-H CRC; however, MSS CRC patients still had a low response to PD-1 inhibitor. Fortunately, preclinical studies performed in mice have shown that MEK inhibitors lead to the upregulation of MHC I on tumor cells, inducing T cell infiltration and enhancing PD-L1 activity . TherefoAnal cancer accounts for 2\u20133% of GI cancers, including squamous cell carcinomas (SCCs), adenocarcinomas, basal cell carcinomas, melanomas and gastrointestinal stromal tumors (GIST) . As the NCT02314169 explored the use of the anti-PD-1 antibody nivolumab for the treatment of metastatic SCC of anal cancer . AccordiPrecision medicine is broadly defined as \u201can emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person\u201d . In the Patients with increased tumor cell and TIL expression of PD-L1 have demonstrated trends toward increased rates of a response to anti-PD1/PD-L1 inhibitors across various clinical trials . HoweverThe tumor mutation burden (TMB) is measured by the overall number of somatic protein encoding mutations in the tumor . Tumor cThe MSI/MMR status can be determined by polymerase chain reaction (PCR) or IHC at specific microsatellite foci , 90. We Neoantigens generally established by either somatic mutation genes or viral genes and presented by MHC on the surface of tumor cells have the potential to induce specific anti-tumoral immunity . Next-geAlternative biomarkers, such as tumor etiology, the presence or absence of TILs, composition of TIL effectors, circulating cytokine levels, neutrophil-to-lymphocyte ratio, and baseline and on-treatment immune effector composition, appear to correlate with antitumor activity and represent desirable predictors of responses to immunotherapy , 98. ChaImmunotherapy can provide patients with a better clinical effect, and we also note that unselected patients who receive anti-PD-1 and anti-PD-L1 immunotherapy have a response rate of only approximately 20%, necessitating other treatment strategies to allow the remaining 80% non-responders to be converted to responders. Radiation therapy has the advantage of interfering with the primary tumor site and potentially restoring some of the established immunosuppressive barriers present in the tumor microenvironment, ideally restoring the primary tumor as an effective immunogenic center. Local radiation also triggers a systemic effect that can be used in combination with immunotherapy to elicit a response external to the radiation field . Two triAlthough anti-PD-1/L monotherapy can lead to profound and sustained tumor responses in some cases, a small subset of patients treated with anti-PD-1/L inhibitor appear to exhibit hyperprogression of disease (HPD) . CompareAlthough combination therapy is becoming more prevalent, few studies are designed to optimize clinical efficacy based on the timing of administration. In fact, timing is another critical factor for determining the outcome of immunotherapy, and the optimal timing varies . RadiatiImmunotherapy can result in a unique spectrum of immune-related adverse effects (irAEs) . HoweverThere are several criteria for assessing tumors, including the World Health Organization (WHO), modified WHO, RECIST 1.0, RECIST 1.1, and modified RECIST criteria. RECIST and mWHO criteria are used in clinical trials to assess responses to cytotoxic chemotherapy , 135. UnDespite the compelling anti-tumor efficacy of antibodies targeting the PD-1/PD-L immune checkpoint in a variety of cancers, many patients do not respond to therapy, and more concerning, the initial response of some patients to immunotherapy showing encouraging results eventually leads to drug resistance. A recent study showed that of 78 patients with melanoma treated with a PD-1 inhibitor, 42 had an objective response and 15 subsequently developed disease progression . The resImproved survival and tumor reduction after RECIST-defined progression was observed in a subset of patients . Immunot. 7%), and no significant differences in the likelihood of obtaining PR were found according to sex, age, tumor histology, type of salvage chemotherapy regimen and number of prior chemotherapy regimens, indicating that patients with advanced NSCLC who have progressed following treatment with a PD-1/PD-L1 checkpoint inhibitor have a 30% better chance of achieving at least PR with salvage chemotherapy compared with those who have received prior chemotherapy but not a PD-1/PD-L1 checkpoint inhibitor. Immunotherapy can alter the natural history and microenvironment of the tumor, making it more sensitive to chemotherapy. These preliminary findings may facilitate the development of a new approach to drug resistance to immunotherapy.Immune checkpoint inhibitors are active for advanced cancer patients who have progressed following chemotherapy . A retro. everolimus  is beyond the willingness-to-pay (WTP) threshold of $100,000/QALY [Despite advances across various tumors, it is recommended that the high cost of PD-1/PD-L1 inhibitors be carefully evaluated to ensure their economic sustainability for the health care industry and benefit to all cancer patients . In this000/QALY . The cha000/QALY . However000/QALY . The ICE000/QALY . A cost 000/QALY . If the 000/QALY . In addi000/QALY . However000/QALY . AlthougThe clinical data from GI tumor trials has demonstrated that immunotherapy targeting immunocheckpoints have produced exciting clinical benefits. However, the response rate is not as high as expected, and therefore treatment with PD-1/PD-L inhibitors must be subjected to precision immunotherapy to improve efficiency. Ongoing and future research should explore the genetic and molecular mechanisms involved in the response and resistance to PD-1/PD-L inhibitors and develop a correct criterion for evaluating the efficacy of PD-1/PD-L blockade. It will also be important to identify predictable and reliable combined biomarkers that will help to select patients who may benefit from PD-1/PD-L inhibitors while minimizing toxicities and maximizing cost-effectiveness. After integrating these approaches, individualized and precise immunotherapies will hopefully lead to a more effective treatment, perhaps even conquest, of GI tumors."}
+{"text": "Immune checkpoint inhibitors have led to a breakthrough in solid tumor immunotherapy, but related studies on musculoskeletal tumors are few, especially for PD-L2.We examined expression of three molecular effectors of the PD-1 axis in 234 patients with musculoskeletal tumors, including osteosarcoma, chondrosarcoma, synovial sarcoma, and giant cell tumor. Survival analyses and potential mechanisms were investigated in osteosarcoma per the Gene Expression Omnibus (GEO) and immunohistochemistry analyses. In vivo, humanized mice were used to evaluate the effect of nivolumab on osteosarcoma.PD-L1, PD-L2, and PD-1 expression levels were significantly different between the histologic types of the musculoskeletal tumors. For osteosarcoma, PD-L1 was negatively correlated with prognosis, while PD-1 had a negative correlation tendency with overall survival (OS). Meanwhile, PD-L2 had a positive correlation trend with OS. Nivolumab inhibited osteosarcoma metastasis in humanized mice by increasing CD4+ and CD8+ lymphocytes and the cytolytic activity of CD8 lymphocytes in the lung but did not affect primary osteosarcoma growth.We systematically detected the expression patterns of PD-L1, PD-L2, and PD-1 in musculoskeletal tumors for the first time and demonstrated the prognostic roles and underlying mechanisms of PD-1 axis in osteosarcoma. Furthermore, PD-1 blockade could effectively control osteosarcoma pulmonary metastasis in vivo. Therefore, the PD-1 axis may be a potential immunotherapeutic target for metastatic osteosarcoma.The online version of this article (10.1186/s13045-018-0560-1) contains supplementary material, which is available to authorized users. Sarcomas, characterized by high heterogeneity, are the main types of malignant bone and soft-tissue tumors , and neoPD-L1 and PD-L2 are both ligands of PD-1, and these interactions transduce co-inhibitory signals for T cell activation, suppress T cell function, which is called T cell exhaustion, and ultimately promote tumor evasion of the immune system , 4. For In our study, we systematically investigated the expression patterns of PD-L1, PD-L2, and PD-1 in sarcomas including osteosarcoma, chondrosarcoma, synovial sarcoma, and giant cell tumors (GCTs) and further evaluated the association between PD-L1, PD-L2, and PD-1 expression and clinical prognosis of osteosarcoma to provide a therapeutic strategy guide. Then, we investigated the therapeutic effect of nivolumab on osteosarcoma and its underlying mechanism.Three tissue microarray (TMA) slides were used to evaluate the expression patterns of the PD-1 axis. One was constructed using samples acquired from the Musculoskeletal Tumor Center, Peking University People\u2019s Hospital , and the relevant tumor tissues, including osteosarcoma (62 cases) and dedifferentiated chondrosarcoma (4 cases), were acquired at the time of definitive surgery and disease recurrence  with several paired samples included on the array. Core tissue (3\u00a0mm in diameter) was obtained from each donor block and placed in the recipient tissue array block. TMA sections (5-\u03bcm thickness) were sliced and preserved properly at room temperature for subsequent experiments. Informed consent was obtained from each patient, and the study was approved by the ethics committee of Peking University People\u2019s Hospital. Clinical and histopathologic data were collated through a retrospective review of patient records. The other two TMAs (OS803 and SS1501) were purchased from US Biomax, Inc. Among them, the SS1501 TMA included chronic synovitis (9 cases), giant cell tumor (14 cases), and synovial sarcoma (127 cases); the OS803 TMA included 27 cases of chondrosarcoma. Some core tissues were removed from the slide during staining for immunohistochemistry (IHC); thus, the presented results only included the samples that remained on the slide and could be graded.2.HOS, KHOS, 143B, MNNG, U2OS, SAOS-2, MG63, and NIH3T3 cells were obtained from American Type Culture Collection (ATCC). The KHOS cell line used for in vivo experiments was recently authenticated in Beijing Microread Genetics Co., Ltd. by STR analysis and was passaged for less than 3\u00a0months after resuscitation. HOS, KHOS, and U2OS cells were cultured in RPMI 1640 medium (HyClone). 143B, MNNG, SAOS-2, MG63, and NIH3T3 cells were maintained in DMEM (HyClone). Cell culture media were supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Invitrogen). All cell lines were cultured at 37\u00a0\u00b0C with 5% COWestern blotting was performed as previously described . BrieflyAll osteosarcoma cell lines were analyzed for PD-L1 and PD-L2 expression by flow cytometry. The cells were prepared and incubated with the primary antibody for 30\u00a0min at 4\u00a0\u00b0C and then washed with phosphate-buffered saline (PBS) according to the manufacturer\u2019s instructions. After washing, cells were assayed using an Accuri C6 flow cytometer . Fluorescent antibodies, including PE-PDL1 (12-5983), APC-PDL2 (17-5888) and the corresponding isotype controls (17-4714 and 12-4714), were purchased from eBioscience. The single-cell suspensions isolated from the mouse tumors were similarly examined for human lymphocyte infiltration by flow cytometry, and the fluorescent antibodies included APC-mouse CD45 , PE-human CD45, PerCP-human CD3, FITC-human CD4, and PE-human CD8a .Paraffin sections were incubated with the corresponding antibodies and stained with nonimmune serum in PBS instead of the primary antibody as the negative control. Based on the average percentage of positive cells calculated from at least 10 representative fields (\u00d7\u2009400 magnification), positive staining was defined as a positive cell percentage \u2265\u200910%. Staining intensity was classified as follows: 0, no staining or staining in <\u200910% of tumor cells; 1+, weak to moderate staining in 10 to 20% of tumor cells; 2+, strong staining in 10 to 20% of tumor cells or weak staining in 20 to 50% of tumor cells; 3+, moderate to strong staining in 20 to 50% of tumor cells or staining in 50% of tumor cells. More than 10 representative areas (\u00d7\u2009400 magnification) were calculated for the tumor-infiltrating lymphocyte analysis. The immunostaining assessment was conducted by two independent pathologists without any previous knowledge of the clinical characteristics and outcomes. Antibodies for IHC against PD-L1 (M442) and PD-1 (M569) were purchased from Spring Bioscience. Anti-PD-L2 (82723) was purchased from Cell Signaling Technology, and anti-CD4 (19068-1-AP), anti-CD8a (17335-1-AP), anti-granzyme B (13588-1-AP), and anti-interferon gamma (15365-1-AP) were purchased from Proteintech Group Inc.For immunofluorescence assay of colocalization of PD-L1 and PD-1 or PD-L2 and PD-1, paraffin sections were incubated with anti-PD-L1 and anti-PD-1 or anti-PD-L2 and anti-PD-1 antibody overnight at 4\u00a0\u00b0C, then washed three times with PBS and incubated with Alexa Flour 594-conjugated goat anti-mouse IgG and Alexa Flour 488-conjugated goat anti-rabbit IgG for 1\u00a0h at room temperature. The sections were viewed using confocal microscopy .Total RNA was isolated using Trizol (Invitrogen), and cDNAs were synthesized with purified RNA and OligdT primers using SuperScript III First-Strand Synthesis SuperMix (Invitrogen). Real-time quantitative PCR was performed using the SYBR-Green PCR Master Mix  on Bio-Rad CFX96 . Relative transcript expression was normalized to GAPDH. All protocols were conducted as per the manufacturer\u2019s instructions.The primer sequences were as follows: PD-1 forward 5\u2032-AAGCTTATGTGGGTCCGGC-3 and PD-1 reverse 5\u2032-GGATCCTCAAAGAGGCC-3\u2032; PD-L1 forward 5\u2032-ACGCATTTACTGTCACGGTTCC-3\u2032 and PD-L1 reverse 5\u2032-CGATGGGGTTCCGGCTTCAG-3\u2032; PD-L2 forward 5\u2032-AAAGAGCCACTTTGCTGGAG-3\u2032 and PD-L2 reverse 5\u2032-GAGGACGTAGTAACGAAAGT-3\u2032; GAPDH forward 5\u2032-GCACCGTCAAGGCTGAGAAC-3\u2032 and GAPDH reverse 5\u2032-ATGGTGGTGAAGACGCCAGT-3\u2032.The osteosarcoma dataset from the Gene Expression Omnibus (GEO)  (accessiscidIl2rgnull) were purchased from Beijing Vitalstar Biotech. Co. Ltd. The two- to threefold diluted blood samples were subjected to centrifugation on a lymphocyte separation medium  at a density of 1.077\u00a0g/ml, and the nucleated cell layer between the plasma and separation medium was gathered. After two washes with RPMI 1640 medium, the PBMC pellets were suspended in RPMI 1640 medium at a density of 5\u2009\u00d7\u2009107 cells/ml. Then, 1\u2009\u00d7\u2009107 cells were injected via tail vein for each NPG mouse. Mice were housed in an SPF facility and accessed food and water ad libitum. The PBMC-transplanted mice were bled retro-orbitally every week, and the human CD45-positive cell rate in the mouse peripheral blood was analyzed by flow cytometry. The mice with more than 25% human CD45-positive cells in their blood were considered successful human PBMC-engrafted mouse models  and injected intraperitoneally with sterile saline or nivolumab at a dose of 10\u00a0mg/kg every 5\u00a0days for a total of five injections. The volume of the xenograft was measured every 5\u00a0days (tumor volume = (length \u00d7 width2)/2). The mice were sacrificed after 30\u00a0days. At the termination of the study, the lungs were processed for routine hematoxylin-eosin (H&E) staining, and the number of metastatic nodules in the lung was determined. The tumors collected from the mice were chopped into small pieces and digested with 1\u00a0mg/ml collagenase type IV  solution for 30\u00a0min. The dissociated tissues were processed through a 70-\u03bcm strainer, and the single-cell suspensions were subjected to flow cytometric analysis for human CD4+ and CD8+ lymphocyte infiltration.To evaluate the effect of the nivolumab treatment on primary tumor growth and spontaneous metastasis, 5\u2009\u00d7\u200910t tests. Data are expressed as the mean\u2009\u00b1\u2009S.D. In all statistical analyses, a P value <\u20090.05 was considered statistically significant in the two-sided test.All statistical analyses were performed using the SPSS v.21.0 software  and the GraphPad Prism software. For survival analysis, overall survival (OS) was defined as the time interval between the confirmed diagnosis and death or last follow-up. Survival analysis was per the Kaplan-Meier method with the log-rank test. The association between the expression levels of the PD-1 axis effectors and the clinicopathological variables, along with the relationships among the expression levels of the PD-1 axis effectors, were assessed by using the chi-square analysis. Statistical evaluations were performed using Student\u2019s PD-L1, PD-L2, and PD-1 expression patterns were examined in a musculoskeletal tumor TMA (234 cases), including osteosarcoma (62 cases), chondrosarcoma (31 cases), synovial sarcoma (127 cases), and GCT (14 cases), using IHC. Representative positively and negatively stained images for each pathological type are shown in Fig.\u00a0As shown in Table\u00a0P\u2009=\u20090.036 for PD-L2 versus PD-1). In particular, similar results were observed in chondrosarcoma (P\u2009=\u20090.016 for PD-L2 versus PD-1), while no association was observed in synovial sarcoma and osteosarcoma. However, when PD-L1 and PD-L2 expression was taken together for analysis with PD-1 expression, the expression levels of the two PD-1 ligands were significantly correlated with PD-1 expression in the sarcomas (P\u2009=\u20090.000) and in osteosarcoma (P\u2009=\u20090.002), synovial sarcoma (P\u2009=\u20090.017) and chondrosarcoma (P\u2009=\u20090.002).As shown in Table\u00a0P\u2009=\u20090.000 for PD-L1; P\u2009=\u20090.004 for PD-L2; P\u2009=\u20090.000 for PD-1), while no significant difference regarding age and sex was evident for the sarcomas. Specifically, the PD-L1, PD-L2, and PD-1 expression levels were not significantly different among primary, recurrent, and metastatic osteosarcoma. In synovial sarcoma, PD-L1 expression was significantly different according to clinical stage (P\u2009=\u20090.011), while no difference was evident for PD-L2 (P\u2009=\u20090.912) and PD-1 (P\u2009=\u20090.103) , while PD-1 expression had no correlation with OS (P\u2009=\u20090.749). PD-L2 expression had a positive correlation trend with OS (P\u2009=\u20090.106)  and found that PD-L1 expression had a negative correlation tendency with OS (06) Fig.\u00a0.Fig. 2SuP\u2009=\u20090.017). Similarly, PD-1 expression was also negatively associated with OS (P\u2009=\u20090.016). Unlike PD-L1 and PD-1, the PD-L2-positive group had a longer OS time than the PD-L2-negative group, although the P value did not achieve statistical significance (P\u2009=\u20090.166), which meant that PD-L2 expression had a positive correlation trend with OS in osteosarcoma  in PD-L1-positive group, and the PD-L2-positive subgroup exhibited a borderline positive correlation trend with OS (P\u2009=\u20090.076) in the PD-L1-negative group. In contrast, PD-L1 expression indicated a negative correlation tendency with OS (P\u2009=\u20090.051) in the PD-L2-positive group. Similarly, in the PD-L2-negative group, PD-L1 expression predicted a negative correlation with OS (P\u2009=\u20090.000).Based on the PD-L1 and PD-L2 expression levels, the osteosarcoma TMA patients were divided into four subgroups. As shown in Fig.\u00a0n\u2009=\u200910), (II) positivity for either PD-L1 or PD-1 (n\u2009=\u200919), and (III) negativity for both PD-L1 and PD-1 (n\u2009=\u200933). The doubly positive group had a distinctly worse OS than the doubly negative group (P\u2009=\u20090.001), whereas the statistical significance between the doubly negative group and the singly positive group was borderline (P\u2009=\u20090.085). The doubly positive group tended to have a worse OS than the singly positive group, though statistical significance was not achieved   positivity for both PD-L1 and PD-1 (15) Fig.\u00a0.P\u2009=\u20090.043). Similarly, in the PD-L2-negative group, PD-1 expression had a negative correlation with OS (P\u2009=\u20090.024). Conversely, in the PD-1-negative group, PD-L2 expression indicated a longer OS time (P\u2009=\u20090.084). The most obvious discrepancy in median survival was between the PD-L2(+) and PD-1(\u2212) subgroup and PD-L2(\u2212) and PD-1(+) subgroup (P\u2009=\u20090.014). The doubly positive subgroup was not significantly different from the doubly negative subgroup (P\u2009=\u20090.864).On the basis of the PD-L2 and PD-1 expression levels, four subgroups were formed for an OS analysis. As shown in Fig.\u00a0Taken together, these survival analyses of the IHC and GEO data reveal that PD-L1 was negatively correlated with prognosis, while PD-1 had a negative correlation tendency with OS. Meanwhile, PD-L2 had a positive correlation trend with OS.An overview of the expression patterns of immune checkpoint-related genes  and their associations with OS and HUVOS grade in the 53 osteosarcoma samples is shown in Fig.\u00a0The GSEA analysis results show that PD-1 is positively correlated with activation of immune response pathways, docetaxel resistance, and metastasis-related signatures Fig.\u00a0. PD-L1 iGene annotation network analyses were performed on the differentially expressed genes  or sterile saline every 5\u00a0days for a five-injection treatment course.No significant differences were observed in the primary tumor volume and growth rate between the nivolumab-treated group and control group Fig.\u00a0. This reOverall, nivolumab markedly suppressed the metastatic potential of osteosarcoma but not primary osteosarcoma in vivo.The flow cytometry results showed that the primary tumors in the humanized PBMC-NPG mice were infiltrated with human CD4+ and CD8+ lymphocytes, but both groups displayed similar proportions of these T cells regardless of treatment Fig.\u00a0.The IHC results indicated that CD4+ and CD8+ lymphocytes were more frequently observed in the lungs of the nivolumab-treated group than in the lungs of the control group, while CD4+ and CD8+ lymphocytes showed no statistically significant differences in the primary tumors from both groups, which was consistent with the flow cytometry results. PD-L1 and PD-1 were also detected by IHC in tumors and lung metastases, and no differences were observed between the two groups Fig.\u00a0. AdditioThese data reveal that nivolumab enhances tumor lymphocyte infiltration and the cytolytic activity of CD8 lymphocytes in lung metastases, which may be the mechanism by which nivolumab inhibits lung metastasis.Known as the B7 family members, PD-L1 and PD-L2 both provide negative costimulatory signals during antigen-specific T cell activation by binding to the PD-1 receptor. Therefore, these three effectors play important roles in forming the tumor immunosuppressive microenvironment. Several studies have shown that inhibition of the PD-1 axis restores and enhances the immune response in vitro and in vivo , 36. MeaFew studies have analyzed the clinical significance of the PD-1 axis, especially PD-L2, in sarcomas. In our study, we examined the expression levels of PD-L1, PD-L2, and PD-1 in multiple sarcomas, as indicated in the \u201cBecause the PD-1 axis effectors are differentially expressed in various sarcomas, the immunotherapeutic efficiency may vary widely due to the pathological type of the sarcoma, so further investigation of each sarcoma is urgently needed. Based on our findings, osteosarcoma, which is the most common primary malignant bone tumor with a high mortality and disability rate, exhibits relatively high expression levels of PD-1 axis effectors. Furthermore, the IHC experiments and data mining both indicate that PD-L1 was negatively correlated with prognosis, while PD-1 had a negative correlation tendency with OS. Meanwhile, PD-L2 had a positive correlation trend with OS.To investigate the potential mechanisms that underlie the PD-1, PD-L1, and PD-L2 associations with prognosis, mRNA expression of 10 immune checkpoint-related genes in the osteosarcoma samples were clustered and visualized through datamining and bioinformatic analyses. The expression patterns, GSEA, and gene annotation network analysis of the genes associated with PD-1, PD-L1, or PD-L2 have also been presented. In addition to immune suppression, our results indicate that PD-1 may be correlated with docetaxel resistance and activation of MAPK and metastasis-related pathways. The PD-L1-associated poor prognosis may due to immune suppression, cisplatin resistance, and metastasis-related pathway activation, whereas PD-L2 may slow osteosarcoma progression by repressing DNA repair, stem cell-related pathways, and doxorubicin resistance.Based on the negative prognostic role of PD-1/PD-L1 and the immune response associated with the PD-1 axis in the datamining analysis, we investigated whether blockade of the PD-1 axis could generate an antitumor effect in osteosarcoma. In our study, we revealed that treatment with nivolumab resulted in effective control of pulmonary metastasis in an osteosarcoma model in humanized mice, while no obvious effect was evident on localized osteosarcoma. Moreover, the tumor-infiltrating lymphocyte (TIL) investigation indicated that nivolumab increased CD4+ and CD8+ lymphocytes in the lung but not in the primary lesion. Furthermore, nivolumab enhances the cytolytic activity of CD8 lymphocytes in the lung. The limitation of this animal model was that the mature human lymphocytes gradually initiated severe graft-versus-host disease in the mouse due to the PBMC injection, which resulted in a relatively short survival time. Therefore, we could not determine whether nivolumab could control tumor growth over the long term, even though the tumor growth rate of the nivolumab-treated group had begun to slow down in our study.As we know, ICI inhibits tumor development by restoring the functions of T cells to kill the tumor cells, and the quantity of TILs plays an important role in the immunotherapy effect. In our study, nivolumab inhibited osteosarcoma metastasis by increasing the number of lymphocytes in the lung but have been ineffective towards primary osteosarcoma. Interestingly, in recent studies , 39, patIn summary, this study is the first to systematically investigate the expression patterns of PD-1/PD-L1/PD-L2 in osteosarcoma, chondrosarcoma, synovial sarcoma, and GCT. The diversity of the expression levels of PD-1, PD-L1, and PD-L2 may indicate the underlying basis for the different immunotherapy outcomes. The bioinformatic analysis and our TMA results revealed that PD-L1 was negatively correlated with prognosis, while PD-1 had a negative correlation tendency with OS. Meanwhile, PD-L2 had a positive correlation trend with OS in osteosarcoma. The PD-1- and PD-L1-associated poor prognosis in osteosarcoma may due to immune suppression, chemotherapy resistance, and metastasis-related pathways. Our in vivo experiments demonstrated that nivolumab inhibited lung metastasis of osteosarcoma rather than primary tumor growth by increasing the numbers of CD4+ and CD8+ lymphocytes as well as cytolytic activity of CD8 lymphocytes in the lung. Further experiments are needed to confirm the mechanism involved and whether the PD-1 axis is a potential and promising immunotherapeutic target for other sarcomas.Additional file 1: Table S1.The summary of examination for human CD45 positive cells in humanized mice by flow cytometry. (DOCX 13\u00a0kb)Additional file 2: Figure S1.Representative images of the assessment of the human CD45 positivity cell rate in humanized mice and immunofluorescence assay for PD-L1/PD-1 and PD-L2/PD-1 in osteosarcoma. (DOCX 1962\u00a0kb)Additional file 3: Figure S2.PD-L1, PD-L2 and PD-1 expressions in osteosarcoma. (DOCX 1426\u00a0kb)"}
+{"text": "Older adults are known to have lesser cognitive control capability and greater susceptibility to distraction than young adults. Previous studies have reported age-related problems in selective attention and inhibitory control, yielding mixed results depending on modality and context in which stimuli and tasks were presented. The purpose of the study was to empirically demonstrate a modality-specific loss of inhibitory control in processing audio-visual information with ageing. A group of 30 young adults  = 1.86) and 22 older adults  performed the audio-visual contour identification task (AV-CIT). We compared performance of visual/auditory identification  with that of visual/auditory identification in the presence of distraction in counterpart modality . The findings showed a modality-specific effect on inhibitory control. Uni-V performance was significantly better than Multi-V, indicating that auditory distraction significantly hampered visual target identification. However, Multi-A performance was significantly enhanced compared to Uni-A, indicating that auditory target performance was significantly enhanced by visual distraction. Additional analysis showed an age-specific effect on enhancement between Uni-A and Multi-A depending on the level of visual inhibition. Together, our findings indicated that the loss of visual inhibitory control was beneficial for the auditory target identification presented in a multimodal context in older adults. A likely multisensory information processing strategy in the older adults was further discussed in relation to aged cognition. Ageing is a process that leads to a weakening of perceptual and cognitive capacity. With a decrease in the accuracy of sensory perception, older adults show declines in various type of cognitive functions  , which fSuch age-related changes need to be considered in relation to multisensory processing since our experience is multimodal in nature. Multisensory integration refers to a process by which inputs from two or more senses are combined to form a product that is distinct from, and thus cannot be easily broken down to, each component . At an eOur brain is highly plastic and reversible, however, and develops a compensatory strategy when necessary . Older aIndeed, research on multisensory integration with ageing has been made in the audio-visual paradigm  using static visual and auditory stimuli. A series of studies examined the modality-specific effect in audio-visual information processing in aged cognition. They found that visual target detection against the auditory non-target was intact, but filtering visual distraction against auditory target was hampered ,29,30. IRecent evidence also suggests that older adults exhibit impaired suppression of visual distraction at the cortical level ,36. SincAnother series of studies, conversely, reported a salient effect of auditory interference with ageing. Lutfi-Proctor et al. re-designed the conventional Stroop test for an audio-visual version and found that auditory interference  heavily deteriorated the visual task performance . InteresThe purpose of the study was, thus, to empirically demonstrate the modality-specific effect of audio-visual information processing with a selective attention and an inhibitory control framework with ageing. For this, we noted that both visual and auditory information can form perceptual contours, and developed a nonverbal, audio-visual contour identification task (AV-CIT). Using the AV-CIT, we also aimed to determine whether the characteristics of aged cognition  would play a modality-specific role in processing audio-visual information. p < 0.05). All participants were given instructions regarding the experiment and a consent form prior to the study. This study was approved by the Clinical Research Ethics Committee of Hanyang University Hospital (HYUH 2013-08-017-002).Thirty younger adults and thirty older adults participated in this study. The younger adults were voluntarily recruited from the course titled \u201cIntroduction to Psychology\u201d at Hanyang University, Seoul, South Korea. The older adults, aged 40\u201360 years, were recruited through online advertisements. Eight older adults were excluded because they did not complete the whole experimental session. Thus, the data from 30 younger adults (Mean (M) = 25.23 years, Standard Deviation (SD) = 1.86, 15 women) and 22 older adults  were used in this analysis. All participants had no medical history of sensory defects and reported normal vision and hearing capacities. The younger group had significantly fewer years of formal education  than the older adult group  (t(50) = \u22122.67, All sounds were generated by a musical instrument digital interface (MIDI) synthesizer  with a digital audio workstation and MIDI sequencer . The auditory stimuli were four different types of melodic contours see , adoptedThe visual stimuli with dynamic balls were designed to be equivalent to the melodic contour see : (1) ascOur experimental task, AV-CIT, consisted of four successive contour identification subtasks: (1) Unimodal Auditory Contour Identification Task (Uni-A), (2) Unimodal Visual Contour Identification Task (Uni-V), (3) Multimodal Auditory Contour Identification Task with Visual Distraction (Multi-A), and (4) Multimodal Visual Contour Identification Task with Auditory Distraction (Multi-V). Each CIT was performed 24 times, with a two-minute break between the four CITs. The four types of contour directions see  were ranThe experiment was performed in a sound-proof and light-controlled room to minimize other distractions and ensure that full attention was committed to the test. The participants were individually tested. They were asked to hold the tablet with their non-dominant hand and conduct the experiment with their dominant hand. All the participants put on headphones. The experimental setting is shown in For the data analysis, the response time and the accuracy of each test were collected. The response times (milliseconds) and accuracy (percentage of correct answers) were analyzed by two-way within-subject analysis of variance (ANOVA). Missing values as a result of technical problems were handled by using the linearly predicated value . As the  = 11.96, p < 0.05, \u03b7p2 = 0.19), indicating that younger adults performed better than the older age group in all tasks (p < 0.01). The main effect of target modality was significant  = 229.61, p < 0.001, \u03b7p2 = 0.82), indicating that both groups required significantly shorter time in the Uni-V than in the Uni-A (p < 0.001), and in the Multi-V than in the Multi-A (p < 0.001). There was also a significant main effect of task type  = 9.37, p < 0.01, \u03b7p2 = 0.16), indicating that response time was significantly shorter in the unimodal task type (p < 0.01). Next, we performed a three-way mixed ANOVA using the target modality , task type (uni vs. multi), and Group (younger vs. older) . As depi = 5.90, p < 0.05, \u03b7p2 = 0.11) and between the target modality and task type  = 84.48, p < 0.001, \u03b7p2 = 0.63)  = 4.07, p < 0.05, \u03b7p2 = 0.08). The interaction between task type and group was not significant (p > 0.05). Post hoc analysis using Bonferroni correction revealed that both groups performed better in unimodal than multimodal tasks in visual modality . The reverse trend, however, was found in auditory modality. That is, both age groups spent less time in Multi-A than Uni-A (p < 0.001), but significance was observed only in the findings for the older group (p < 0.001). There was a significant two-way interaction between the target modality and group (F = 0.63) . There w = 81.49, p < 0.001, \u03b7p2 = 0.62) and target modality  = 162.99, p < 0.001, \u03b7p2 = 0.77). The findings indicated that younger adults performed significantly better (p < 0.001) and both groups performed better in the visual  than in the auditory modality . Moreover, there was a significant two-way interaction effect between the target modality and group  = 71.94, p < 0.001, \u03b7p2 = 0.59). Post hoc analysis revealed that the performance difference between groups was much larger in auditory  than in visual modality . Unlike the findings for response time, a three-way interaction was not significant (p > 0.05). With regards to performance accuracy, as shown in Collectively, our behavioral data analysis indicated the dominance effect of visual modality and the effect of aging on cognitive function. Interestingly, we found a modality-specific effect on multisensory task performance only in the older adult group. That is, auditory target identification (Multi-A) was enhanced by visual non-target information, while visual target identification (Multi-V) was hampered by auditory non-target information only in the older adults. How this happens is further analyzed in the next section. Our behavioral findings indicated that older adults exploited a modality-specific effect in multisensory information. To determine if unattended stimuli play a certain role of \u2018reference\u2019 or \u2018distraction\u2019, we analyzed the influence of visual information processing on enhancement between Uni-A and Multi-A, because no significant enhancement between Uni-V and Multi-V was found see a. Note tFor this assessment, we recoded a new variable by subtracting the response time of Multi-A from that of Uni-A . In our hypothesis, the Multi-A condition reveals the occurrence of auditory selective attention and visual inhibition at the same time. Therefore, those who performed better for the visual non-target stimuli against the auditory target stimuli would be better at visual inhibition. This indicates that these people have a special visual inhibition capability, so they can exploit this capability as a negative reference information to detect the auditory target. Conversely, those who do not have this special visual inhibition capability might suffer more from the distracting visual non-target information. In order to test this hypothesis, we grouped all the participants into three subgroups using the response time in the Multi-A condition: \u2018high\u2019 for those who scored in the highest quartile, \u2018low\u2019 for those who scored in the lowest quartile, and \u2018middle\u2019 for the rest of them. The grouping criterion followed the criterion frequently used in the education research . t-tests between younger and older adult groups in each subgroup. The high and middle performance subgroups in the Multi-A were not significantly different between the younger and older adult groups; however, the older adults in the low performing subgroup showed significant enhancement with the visual non-target (p < 0.05). In This finding may indicate that an ability to deal with distracting visual information in a multimodal context plays an important role in the Multi-A enhancement, specifically in the older adults group. This hints that though the older adults showed an obvious decline in general inhibitory control as compared to the younger adults, the loss of inhibition control can be partially compensated for by employing visual stimuli as a \u2018negative\u2019 reference. The present study examined the effect of ageing in multisensory information processing. The younger and older adult groups showed a shared but distinctive tendency of performance. First, both groups outperformed with visual contour identification presented in a unimodal context. Second, the auditory stimuli alone  were not sufficiently salient for identifying the contour direction. However, when visual non-target stimuli were given with auditory target identification, the response time was shortened. This trend was more obvious in the older than in the younger adult group, indicating that the older adults were more supported by the visual distracting information, which showed their special modality-specific multisensory information processing. Our findings suggest that the loss of inhibitory control with ageing can benefit from a special modality of distraction. This interpretation may be a result of the nature of our experimental apparatus . The comparison between our experimental data with scores from a computerized version of Stroop Word Color Test  were notVision is dominant, so auditory distraction is less prominent in visual target identification. Our findings again confirmed that both younger and older groups required significantly less time when the target was presented in the visual modality and this trend was the same between the younger and older adult groups. In effect, it seemed that visual stimuli play a leading role in object recognition ,47,48,49In addition, the finding that auditory distraction did not severely deteriorate visual selective attention was possibly due to the distinct filtering mechanisms within the visual and auditory modalities, which might be differentially affected by ageing. Specifically, auditory distractors appear to be filtered out at both central ,53 and pThis account is also confirmed by Stothart and Kazanina . They coBoth age groups spent less time in Multi-A than Uni-A, while they spent more time in Multi-V than Uni-V. The findings indicated that auditory distraction seemed to hamper the participants\u2019 performances, while visual distraction played a different role in that case. The current findings importantly suggested that when presented with visual distraction, auditory target detection was enhanced, while visual target detection was hindered by auditory distraction. This asymmetric interaction is inconsistent with the previous findings reporting that older adults were more susceptible to task-irrelevant auditory suppression during visual attention tasks than task-irrelevant visual suppression during auditory attention tasks ,58. One plausible explanation can be the role of visual stimuli as a negative reference. Note that in our study, the Multi-A condition used the auditory contour stimuli as a target and the visual contour as a non-target, by which the participants seemed to use visual information as a negative feedback reference item to quickly identify the target auditory contour direction. The more interesting finding is that this observation was noted in the older adult group. Amer et al.  also notIn brain studies, the principle of inverse effectiveness well represents the fact that a decrease in sensory perception increases the magnitude of multisensory enhancements . Neuro-iThis study suggests that the younger and older adult groups showed a shared but distinctive tendency of performance in the multisensory information processing. Although performance of visual target identification was impeded by auditory distraction, performance of auditory target identification was enhanced by visual distraction. This trend was prominent in the older adult group, implying that the loss of visual inhibitory control supported the older adults\u2019 modality-specific multisensory information processing. There are some limitations that limit the generalizability of the findings. First, we recruited younger and older adults whose mean ages were in the mid-20s and mid-50s, respectively. More confirmation with data from patients aged over 65 years is urgently needed. A relatively small sample size would be problematic, so a scaled-up experiment is indeed planned soon. A similar study could also be performed in a simulated real-world situation employing various modalities at the same time . Another type of future study might be the use of AV-CIT in the patients with cognitive impairment ."}
+{"text": "Ordered mesoporous metal oxides have attracted more and more attention due to their large surface areas and pore volumes, unblocked pore structure, and good thermal stabilities. Compared with non-porous metal oxides, the most prominent feature is their ability to interact with molecules not only on their outer surface but also on the large internal surfaces of the material, providing more accessible active sites for the reactants. This review carefully describes the characteristics, classification and synthesis of ordered mesoporous metal oxides in detail. Besides, it also summarizes the catalytic application of ordered mesoporous metal oxides in the field of carbon dioxide conversion and resource utilization, which provides prospective viewpoints to reduce the emission of greenhouse gas and the inhibition of global warming. Although the scope of current review is mainly limited to the ordered mesoporous metal oxides and their application in the field of CO The porous materials have been widely investigated and applied in many fields owing to their outstanding structural properties. According to the definition of International Union of Pure and Applied Chemistry (IUPAC), porous materials can be categorized into three types: microporous materials (pore size < 2 nm), mesoporous materials 2\u201350 nm) and macroporous materials (pore size > 50 nm) . The mos0 nm and 3O4 and iron-nitrogen co-doped ordered mesoporous carbon-silicon nanocomposite (Si-Fe/NOMC) [3/ZnO (OM-WO3/ZnO) using the soft template method. Compared with ordered mesoporous non-metal oxide materials, the ordered mesoporous metal oxides are widely investigated in the field of energy conversion and storage, catalysis, sensing, adsorption and separation due to high specific surface area and ordered pore structure. Therefore, the design and fabrication of the ordered mesoporous metal oxides have been the research focus. The development of mesoporous materials in nanometer range is beneficial to industrial process. Meanwhile, the uniform and adjustable mesopores provide enough monodisperse pore space for macromolecules, breaking through the size limitation of traditional microporous materials, and have advantages in catalysis, adsorption, separation, and drug and Deoxyribose Nucleic Acid (DNA) transfer ,9,10. InFe/NOMC) ,15,16. XFe/NOMC)  synthesiFe/NOMC)  synthesi2 capture [Climate change is considered to be one of the greatest environmental threats of our current globe . Continu capture .2 is attractive in the manufacture of commodity chemicals, fuels, and materials since CO2 is an abundant, non-toxic, nonflammable, typically renewable, and easily available synthon in organic synthesis. In recent years, various processes of thermochemistry, electrochemistry, photocatalysis and biology directly convert CO2 into value-added chemicals such as methanol, methane, carbon monoxide and formic acid. From both the economic and environmental point of view, one of the key points to achieve the efficient conversion of CO2 is the effective activation of CO2 through the highly active catalytic system [2 because of their excellent textural properties and outstanding performances of CO2 activation [As a typical renewable compound, COc system . It is nc system . In recetivation . Compare2 hydrogenation to methanol, CO2 reforming of methane, CO2 methanation, synthesis of dimethyl ether, and reverse water\u2013gas shift. It can be used to highlight the potential trend for future development in this area. In this review, the development progresses of the ordered mesoporous metal oxides and their applications in the field of CO2 catalytic conversion in recent years are carefully summarized and the future development trends are also prospected.In this review, we mainly analyzed the synthesis and properties of ordered mesoporous metal oxides, providing an effective basis for their stronger stability and higher catalytic activity. The role of ordered mesoporous metal oxides in the catalytic conversion of carbon dioxide is also reviewed to reduce carbon dioxide emission into the atmosphere through various chemical processes, such as COMesoporous material refers to the porous material with pore size between micropore and macropore, typically between 2 and 50 nm. According to IUPAC classifications, mesoporous materials can be ordered or disordered in nature. The pores of the ordered mesoporous material are arranged uniformly . GeneralThe ordered mesoporous non-metal oxides can be classified into silica-based mesoporous materials and carbon-based mesoporous materials according to the texture of the skeleton. The following is a brief introduction to these two types of mesoporous materials.Silica is the most abundant type of mesoporous material, both in structure and morphology. In 1992, the M41S (MCM for Mobil Composition of Matter) series was synthesized by Mobil Corporation using a cationic surfactant as a template, which including a two-dimensional hexagonal phase MCM-41 with a space group of p6mm and cubic MCM-48 with a space group la3d . Zhao et3O4-OMC) using a one-pot hydrothermal method using F127 as a template. Ryoo et al. [The porous carbon materials have many excellent properties such as good electrical conductivity, strong skeleton rigidity, and a large specific surface area. These characteristics make the researches on porous carbon materials enduring. The synthesis of porous carbon materials can be roughly divided into hard template method and soft template method ,29. Manyo et al.  used meso et al.  successf3O4 [2 [3 [2O3 [2 [2 [2O3 [2O3 [2O3 [2 [Ordered mesoporous metal oxides have promising applications in many fields due to their structural regularity, adjustable pore size, and high specific surface area . Up to n3O4 ,36,37, T3O4 [2 ,39, WO3 O4 [2 [3 ,41, Al2O [3 [2O3 ,43,44, Z [2O3 [2 ,46,47, CO3 [2 [2 ,49,50, NO3 [2 [2 ,52, Cr2O [2 [2O3 ,54, Sm2O2O3 [2O3 , In2O3 [2O3 [2O3 , and UO2 [2O3 [2  have bee2 can effectively enhance its functions in photocatalysis and photoelectric conversion, showing a broad application prospect in the treatment of sewage, air purification, solar cell materials, nano-material microreactors, and biological materials [2 hollow microspheres with highly crystalline thin shells. 2 prepared after calcination.Antonelli et al.  first syaterials . Zhang eaterials  found thaterials  used EISaterials  synthesiaterials  synthesi2 synthesized by a surfactant template route with high specific surface area has attracted great interests of the scientists in catalytic field [4)2 as the zirconium source and long-chain quaternary ammonium salt surfactant as template to synthesize hexagonal or layered mesoporous zirconia according to the electrostatic mechanism of \u2018S+X\u2212I+\u2019. They found that the nature of the surfactant, crystallization temperature and crystallization time are the main factors affecting the synthesis of mesoporous ZrO2. Knowles et al. [2 in an alkaline medium (PH = 11.4\u201311.7) to obtain a mesoporous ZrO2 powder. Its interplanar spacing d is independent of the length of the hydrocarbon chain of the surfactant, and the surface spacing d of the calcined sample is approximately linear with the length of the hydrocarbon chain of the surfactant. Zelcer et al. [2 films by evaporation induced self-assembly. 2 prepared with different surfactants after calcination at 623 K. Large ordered mesoporous domains were observed in all polymer template samples [Sulfuric acid zirconia (SZ) has potential application value in the fields of hydrocracking and hydroisomerization as highly effective solid acid catalyst, but the specific surface area of traditional of SZ is low so that its catalytic efficiency is not high. Therefore, mesoporous ZrOic field ,64. Reddic field  used Zr has been widely used in the fields of catalysis, electrochromism, energy storage of electrode materials and microwave materials [3. It was found that potential of hydrogen (PH) value control is the most important factor affecting the mesoporous structure of WO3. The optimum PH value for synthesizing the hexagonal phase structure is about 4 to 8, and when the PH value is greater than 9, the synthesized mesoporous WO3 has two sets of layered coexisting structures. Zhu et al. [3 with a pore structure by surface modification using mesoporous silica (SBA-15) as a template, but eventually only WO3 nanowires were obtained. Subsequently, many literatures reported the use of phosphotungstic acid as a tungsten source and mesoporous silica (KIT-6) as a hard template. The ordered mesoporous tungsten trioxide with a specific surface area and a large pore size is prepared by using tungsten trioxide to crystallize at a high temperature in the mesopores of KIT-6 and then removing the template with Hydrogen Fluoride (HF) [3 film by employing a template-assisted peroxopolytungstic acid sol\u2013gel method. The TEM images of WO3 films are presented in As a functional material, tungsten trioxide  ,72,73. Fide (HF)  prepared2/g using an electrically neutral polyoxyethylene ether nonionic surfactant as a template and aluminum alkoxide as an aluminum source for the first time. Compared to other metal oxides, the alumina surface contains a large number of Lewis acid sites, so that the surface acidity can be regulated. The synthesis of materials is related to the pH value of the mixed gels. With different template agents, the specific surface area and average pore size of the synthesized mesoporous alumina vary greatly, which was summarized in Alumina is a well promising catalytic material widely used in petroleum, chemical and other industries. The traditional microporous alumina catalysts have the disadvantages of causing clogging of the pores due to coking in the practical application processes, thereby rapidly deactivating. Therefore, synthesizing mesoporous alumina with large pore diameter is of great significance for reducing coking on the catalyst surface, blocking pores, and prolonging catalyst life. Bagshaw et al.  synthesi2O3. TEM images of \u03b3-Al2O3 are displayed in parts a and b of 2O3 decreases, which will affect its industrial application [2O3. Therefore, how to suppress its transformation and to improve the thermal stability of mesoporous alumina has been a problem that people are keen to solve. The introduction of other metals oxides is one of the most effective methods for improving the thermal stability of alumina.The activity of alumina with different crystal forms is different, with \u03b3-type alumina having the highest activity. It is beneficial for applications such as catalysis, adsorption and separation . Yuan etlication . The strThe core of synthetic ordered mesoporous metal oxide is how to \u201cmake holes\u201d in order at nano-scale . The pre3, Nb2O5, Fe2O3 using the soft template method (cationic surfactants and anionic surfactants) [Soft template is constructed by the polymerization of surfactant molecules. During the process of studying the synthesis of mesoporous materials by the soft template method, many scientists proposed a variety of synthesis mechanisms for preparing ordered mesoporous materials using soft template methods . As showactants) . CompareThe degree of polycondensation of inorganic species to form a stable intermediate product could be increased after going through hydrothermal, aging and other processes. After washing, filtration, and drying, an organic\u2013inorganic composite precursor is obtained. Further calcining or solvent extraction to remove the surfactant can give mesoporous materials . During the synthesis of ordered mesoporous materials, it is critical to choose suitable template agents. The type and nature of template agents have a great influence on the formation of ordered mesoporous structures. It can even change the synthetic route of the reaction system. The surfactants used in the synthesis of ordered mesoporous materials can be either cationic, anionic, or nonionic .\u2212S+, I+S\u2212, I+X\u2212S+, I\u2212M+S\u2212  [2/g. The pore size distribution is within 2 nm and the orderliness is relatively good [4. Zhao et al. [2 materials via a sol-gel route using sodium dodecyl benzene sulfonate (SDBS) surfactants as soft templates. A mesoporous material is formed by charge matching between the ionic surfactant and the inorganic source. Ionic surfactants can be used for the synthesis of aqueous systems. The main synthesis mechanisms are I cation) . Cationi cation)  prepared cation)  synthesi cation) . The dis cation)  studied  cation)  discusseely good . Holloanely good  used sodely good  synthesio et al.  prepared20PO70EO20, EO106PO70EO106, EO75BO45 to prepare a series of mesoporous metal oxides such as Ta2O5, WO3 and TiO2. Zhao et al. [2 with crystal walls by soft membrane method. Nonionic surfactants are structurally oriented, self-assembled, and highly adaptable. They are widely used in the synthesis of ordered mesoporous materials with ordered frameworks and pore structures. Nonionic surfactants synthesize mesoporous materials through hydrogen bonding between organic templates and inorganic sources. Non-ionic surfactants mainly consist of polyepoxides such as polyethylene oxide (PEO), polypropylene oxide (PPO). Block copolymer surfactants are mainly composed of epoxide blocks of different chain lengths. Yang et al.  used a bo et al.  showed to et al. . Bagshawo et al.  used PPOo et al. . Compareo et al.  used trio et al.  successfThe hard template method, firstly reported by Ryoo et al with the synthesis of ordered mesoporous carbon (CMK-1) , is cons2O3 [3O4 [2O3 [2 [3 [2O3 [3O4 [2 [Up to date, several mesoporous silica  have been used as hard templates for the synthesis of mesoporous crystals ,103. The2O3 , Co3O4 [2O3 [3O4 ,105, In23O4 [2O3 ,107, NiO3O4 [2O3 ,52,108,  [2O3 [2 ,109, WO3O3 [2 [3 ,110, Fe2 [3 [2O3 , Fe3O4 [2O3 [3O4 , and MnO [3O4 [2 ,114.2O4 by nanocasting method using mesoporous silica SBA-15 as hard-template. For the same purpose, Tian et al. [3O4, In2O3, Cr2O3, Mn3O4, CuO, NiO, and CeO2. The mesoporous silica treated with microwave method not only removes the surfactant but also leaves a rich hydroxyl group in the mesopores. The hydrophilic hydroxyl groups in the pores facilitate the entry of hydrophilic inorganic salt precursors. Therefore, the synthesized material has good continuity [The silica template can be easily removed with the etching of HF or concentrated NaOH solution. The former is generally carried out at room temperature and can be completely removed by one treatment. The latter is safer but generally requires repeated treatment at higher temperatures (353 K) to remove most of the silica. It mainly depends on the chemical stability of the target product in both solutions. The most typical example of the synthesis of mesoporous materials using mesoporous silica as a template is the synthesis of mesoporous carbon. Ryong Ryoo et al.  first usn et al.  used micntinuity . In geneUsing mesoporous carbon as a hard template to the synthesis of mesoporous materials is also an effective method . The harThe carbon template is mainly removed by calcining in air. For target substances that are easily oxidized by air at high temperatures, carbon templates can be removed at high temperatures by oxygen . Mesopor3OH, CO, CH4, and dimethyl ether (DME), is considered as an attractive CO2 recovery method to control its emission into the atmosphere [The cumulation of carbon dioxide in the atmosphere is widely recognized as the main cause of global warming, which may pose a huge threat to human living environment and human beings. Climate change experts recommend that it should be developed and utilized as soon as possible so as to effectively manage carbon dioxide within the limits of the atmosphere. The chemical conversion of carbon dioxide into useful products and fuels, such as CHmosphere ,130.2. In recent years, many scholars have achieved a series of promising and excellent results in this field. Here we briefly review the application of mesoporous metal oxides as catalysts in the catalytic conversion of CO2 via heterogeneous catalysis process.Due to the large specific surface area, developed pore structure and wide pore size, ordered mesoporous metal oxides have been considered as promising catalyst candidates for the catalytic conversion of CO2 has been used as a substitute for CO in methanol production and CO2 hydrogenation to methanol has been widely recognized as an effective CO2 utilization method [2 is considered to be the most economical way to alleviate the greenhouse effect caused by the significant increase in CO2 concentration. It is mainly because that methanol is not only an important chemical intermediate to produce some chemicals such as formaldehyde and acetic acid, but also an excellent fuel due to its cleaner emissions in comparison with other fossil fuels [In the past two decades, COn method . Under cil fuels .2 hydrogenation process, the main reaction is the formation of methanol and the reverse water\u2013gas-shift reaction is a side reaction [In the COreaction :Formation of methanol:Reverse water\u2013gas-shift reaction:2, an increase in the reaction temperature (>513 K) favors the activation of CO2, which in turn forms methanol. The reverse water gas shift reaction results in additional hydrogen consumption and a reduction in methanol production. The formation of a large amount of water has an inhibitory effect on the active metal, resulting in deactivation of the catalyst. Therefore, the hydrogenation of CO2 to methanol requires a more selective catalyst to avoid the production of unwanted by-products [The methanol production is an exothermic reaction, in which the number of reactive molecules is reduced. Therefore, the decrease in temperature and the increase in pressure favor the reaction of thermodynamic analysis. However, considering the reaction rate and the chemical inertness of COproducts . 2 molecule is inherently very stable and inert. However, the simple, less cumbersome and cost-effective method of CO2 conversion based on photo-catalytic reduction has become quite attractive. In photo-catalytic CO2 reduction, the electron-hole pairs generated on the surface of a semiconducting photo-catalyst mediates photo-oxidation and photo-reduction reactions that result in the desired end product [Most methods of conversion require huge amounts of energy and lengthy procedures and complex instrumentation, owing to the fact that the CO product .2O3) by nanocasting technique, in which highly ordered mesoporous silica (SBA-15) was used as structural matrix. The results showed that the introduction of mesoporosity in indium oxide, and the consequent enhancement of positive attributes required for a photo-catalyst, transformed photo-catalytically weak indium oxide into an effective photocatalyst for the conversion of CO2 into methanol. Richardson et al. [2 conversion to methanol. Compared to commercial catalysts, the band gap of Mn and Cu doped TiO2 is less than 3 eV. Due to the rapid transport of excited state electrons to the metal dopant, the coupling between Mn and Cu is achieved, which enhances the ability of CO2 photocatalytic reduction to methanol. Gondal et al.  synthesin et al.  prepared4 and CO2 are rich in natural resources. Therefore, it is important to convert these two molecules into high-value added compounds [2 reforming of CH4 to produce syngas  can be used in chemical energy transfer systems as well as in the production of liquid fuels [2/CO ratio.CHompounds . The reaid fuels . In the 2O3 [2O3 [2O3 [2O3 [2O3 [2O3 (OMA) further confirmed the existence of ordered mesoporous channels. The images are shown in x-Al2O3 (NMA) catalyst by modified EISA method and evaluated its catalytic performance. Since MgO has a strong Lewis basicity and promotes CO2 activation, the M-NMA catalyst is more resistant to carbon formation than the non-promoted catalyst. Compared with the Ni/Al2O3 catalyst (NA), the M-NMA catalyst has a larger surface area and a narrower pore size distribution [2 and finally participate in the process of CRM reaction and carbon elimination via their redox properties so that it could be considered as a series of promising catalyst carriers for CRM. This typical process was expressed in For the CRM reaction catalysts, most of the VIII family metal catalysts were investigated, including non-precious metal Ni and Co based catalysts and noble metal catalysts . The Ni-2O3 , CoO-MO 2O3 [2O3 , xCoyNi-2O3 [2O3 , Ni/-Cex2O3 [2O3 , Ni/CaO-2O3 [2O3  and NiO-2O3 [2O3  by EISA ribution . The catribution . Besides2 methanation is more thermodynamic favorable because of its strong exothermic properties [Natural gas is considered as a potential source of energy due to its clean nature. Therefore, the conversion of carbon dioxide to methane can not only reduce greenhouse gas emissions but also develop carbon dioxide. Compared with the production of hydrocarbons and alcohols, COoperties .CO2(g) 2 molecules. Therefore, a material having a large surface area, a large pore volume, and a clear channel such as a mesoporous metal oxide can be used as a carrier or catalyst for the CO2 methanation catalyst [Catalytic supports have a significant effect on the high dispersion of metal active sites, which greatly promotes the activation and dissociation of Hcatalyst ,150. Comcatalyst . The ordcatalyst ,152.2 methanation catalysts have focused on the loading of Ru, Rh, Ni and Pd on the catalyst. However, their high prices and high hydrogenation temperature (>573 K) limit the application of precious metal catalysts in catalytic hydrogenation of CO2 [2 methanation has become the challenge and research focus in this field.Previous studies on COn of CO2 ,154. Then of CO2 . The disxCoyNi [xCa [xMg [2 methanation reaction. The conversion of CO2 on the OMA-10Ni and OMA-10Ni5Mg catalysts is also much higher than the 10Ni/Al2O3 catalyst, especially in the low temperature. Owing to the excellent textural properties of the ordered mesoporous catalysts, the gaseous reactants are easily to diffuse toward the accessible metallic Ni active site. The effects of reaction temperature on the catalytic activity and selectivity of OMA-10Ni, OMA-10Ni5Mg and 10Ni/Al2O3 were investigated in 2O3 (NCOMA) catalysts for CO2 methanation. The ordered mesoporous 10N3COMA catalyst has high activity with the maximum CO2 conversion rate of 78% and the CH4 selectivity of 99% under specific conditions . The results showed that the Co species could significantly increase the H2 uptake and the catalyst showed high stability and superior anti-sintering property due to the confinement effect of the ordered mesostructure. The CO2 photoreduction is also considered as an effective route to produce CH4. Wang et al. [2 with crystalline walls. Ordered mesoporous TiO2 has higher CH4 production efficiency and better CO2 photoreduction stability than the disordered mesoporous counterpart. The superior performance of ordered mesoporous TiO2 for CO2 photoreduction may be due to the limited spatial effects of ordered mesoporous structures. In the ordered mesoporous structure, the mass transfer of gas molecules is more stable than that of disordered mesoporous structures.Xu et al. successfully synthesized NiO-OMA , OMA-xCoxCoyNi , OMA-10NyNi [xCa , OMA-10NxCa [xMg , and OMAxCa [xMg , which w2 or syngas can produce DME. Generally, this reaction can be divided into two steps. The first step is the synthesis of methanol from carbon dioxide or synthesis gas under the action of a catalyst. In the second step, methanol is dehydrated on the catalyst to produce DME. The following are the main reactions that occur during the synthesis of DME from CO2/H2 gas mixtures.DME is considered as a kind of future fuel, especially as a diesel alternative. DME has higher thermal efficiency compared to conventional fuels. Besides, it also does not emit sulfur oxides or soot. In addition, DME can be used as a raw material for a range of chemicals such as oxygenates, olefins, and hydrocarbon fuels . Therefore, DME will become a very important clean fuel in the view of sustainable development, which will help the effective management of the future energy . The hydMethanol synthesis reaction:Methanol dehydration reaction:2/H2 involves methanol synthesis and methanol dehydration [It can be seen from the above reactions (5) and (6) that the synthesis of DME by COydration ,163,164.2 and acid sites for the continuous dehydration of alcohols to ethers, especially in the reaction of syngas directly synthesizing DME. Both the active site of the solid acid and the active site of the metallic copper are related to the catalytic activity [2O3. In the direct synthesis of DME from syngas, the catalytic activity and stability was improved because the amount of copper crystals accumulated was small. The copper nanoparticles form a strong interaction with the acidic sites on the surface of ordered mesoporous Al2O3 through the formation of CuAl2O4 interface which provides some effective acidic sites for DME methanol dehydration. The highly dispersed Cu nanoparticles have a strong interaction at the rich acidic sites of the ordered mesoporous Al2O3, which greatly improves the stability of the catalyst and the DME selectivity. The TEM image of Cu/mesoAl is displayed in the 2 to dimethyl ether. Luan et al. [2O3, which was used for dehydration of dimethyl ether and helped convert CO2 to dimethyl ether.Thus, many bifunctional (or mixed) catalytic systems contain metal sites for the hydrogenation of COactivity ,166. Hamactivity  uses then et al.  successf2 to CO, which can be further converted into hydrocarbons by the Ficher\u2013Tropsch synthesis process. The RWGS reaction is another key reaction in the field of catalytic hydrogenation of CO2, which has been considered as a promising candidate for large-scale conversion of CO2 and renewable H2 [The reverse water gas shift reaction (RWGS) converts COwable H2 ,170. ThePt, Co, and Ni based catalysts have been widely investigated in the RWGS reaction ,172,173.2 is a typical rare earth metal oxide with a cubic fluorite structure. Under a reducing atmosphere, the surface oxygen of CeO2 can be reduced and oxygen vacancies will be generated subsequently. Oxygen vacancies play a key role in the catalytic reaction, especially in the catalytic reduction of RWGS [2 by hard template to carry out the RWGS reaction and compared it with non-porous CeO2. According to the TEM results in 2 (b), (c) catalysts have low porosity and small specific surface area, which is disadvantageous for absorption and activation of reactant molecules. The ordered mesoporous CeO2 (a) catalyst has a good ordered mesoporous structure to facilitate the adsorption and activation of CO2 and H2 molecules. Therefore, ordered mesoporous CeO2 (a) catalyst has a good catalytic activity in CO2 RWGS reaction [CeO of RWGS . Dai et  of RWGS  synthesireaction .2 lattice to form a solid solution, generating oxygen vacancies [2 catalysts with different Co contents by colloidal solution combustion method and carried out the CO2 RWGS reaction. In the Co-CeO2-M catalyst, the Co3O4 particles embedded in the pore walls were separated by fine CeO2 particles and strongly interacted with CeO2. The results showed that the 5% Co-CeO2-M catalyst was provided with the best catalytic performance for RWGS reaction. Dai et al. [2  catalysts by hard template method and carried out CO2 RWGS reaction. The CO2 RWGS reaction performance has a great relationship with the d orbital holes of the transition metal. It can be seen from 2 catalysts, the conversion rate of CO2 increases as the reaction temperature increases.The addition of other metal elements to the catalyst also can increase the catalytic activity of the RWGS and the selectivity of the catalytic reaction . The traacancies . Wang etacancies  preparedi et al.  preparedThere are many strategies for the design and preparation of ordered mesoporous catalysts. Ordered mesoporous materials have attracted wide attention due to their rich unique properties, functions and potential application prospects. In recent years, many achievements have been made in its synthesis and structural characterization. The classification of ordered mesoporous materials, the synthesis of ordered mesoporous metal oxides and their applications in catalytic conversion of carbon dioxide are reviewed. It is believed that relevant scientists can obtain information on the synthesis, properties and potential applications of these materials to facilitate their research. Ordered mesoporous metal oxide materials are important components of catalytic materials and have the unique physicochemical properties of metal oxides.In recent years, a variety of ordered mesoporous metal oxides have been synthesized by soft and hard template methods and their properties and applications investigated. The methods and mechanisms for the synthesis of ordered mesoporous metal oxides by soft and hard template methods have matured, but both methods have advantages and disadvantages. The advantages of the soft template method are that the template cost is relatively low, the synthesis method is simple and the conditions are mild. The main disadvantages are that the metal ions in the hydrolysis and polymerization processes are relatively sensitive to moisture, and the products are often amorphous and have poor thermal stability. The advantages of the hard template method are that it is universal and the mesoporous structure of the target material can be controlled by selecting hard templates with different structures. The disadvantage of the hard template method is that when the template is removed with sodium hydroxide or hydrofluoric acid, the mesoporous metal oxide synthesized must be resistant to strong acids and bases. The preparation process is cumbersome and complex. 2 conversion is the activation of either CO2 or co-reactant at different conditions, especially at low temperature. In this way, catalytic conversion of CO2 has been carried out by different methodology, including CO2 reforming of methane to syngas production over catalysis, CO2 hydrogenation for methanol synthesis by mesoporous catalyst, CO2 methanation over a Ni based ordered mesoporous catalyst, synthesis of DME from CO2/H2 gas mixture, and CO2 reverse water\u2013gas shift. In the future research, not only should the catalytic conversion of CO2 be further improved, but also the hydrothermal stability, chemical stability and environmental compatibility of ordered mesoporous metal oxide need to be greatly optimized. Meanwhile, for the wide application of materials in industry, the cost of materials should be minimized and it is required to create new ordered mesoporous metal oxide materials that are easy to be synthesized in large scale with low cost.Ordered mesoporous metal oxide can catalyze the conversion of carbon dioxide to value-added chemicals and fuels. As a rich natural raw material, carbon dioxide has attracted great interests in recent years. The key point to CO"}
+{"text": "PBPK) similar to those implemented in physiologically based pharmacokinetic (PBPK) models (AS0.75\u2009+\u2009MFPBPK). A PBPK-based simulation workflow was used, including hypothetical drugs with a wide range of properties and metabolized by different isoenzymes. Adult clearance values were scaled to seven typical children between one day and four years. Prediction errors of \u00b1\u00a050% were considered reasonably accurate. Isoenzyme maturation was found to be an important driver of changes in hepatic metabolic clearance in children younger than five years, which prevents the systematic accuracy of ADE scaling. AS0.75\u2009+\u2009MFPBPK, when accounting for maturation of isoenzymes and microsomal protein per gram of liver (MPPGL), can reasonably accurately scale hepatic metabolic clearance for all low and intermediate extraction ratio drugs except for drugs binding to alpha-1-acid glycoprotein in neonates. As differences in the impact of changes in system-specific parameters on drugs with different properties yield differences in clearance ontogeny, it is unlikely that for the remaining drugs, scaling methods that do not take drug properties into account will be systematically accurate.Previous research showed that scaling drug clearance from adults to children based on body weight alone is not accurate for all hepatically cleared drugs in very young children. This study systematically assesses the accuracy of scaling methods that, in addition to body weight, also take age-based variables into account for drugs undergoing hepatic metabolism in children younger than five years, namely scaling with (1) a body weight-based function using an age-dependent exponent (ADE) and (2) a body weight-based function with fixed exponent of 0.75 (AS0.75) combined with isoenzyme maturation functions (MFThe online version of this article (10.1208/s12248-019-0295-0) contains supplementary material, which is available to authorized users. Accurate scaling of drug plasma clearance (CLp) from adults to children is important for the definition of first in child doses and hence robust study design involving younger children. To date, physiologically based pharmacokinetic (PBPK) models represent the most mechanistic method to scale CLp across the paediatric age range. PBPK models quantify the interactions between drug-specific and system-specific parameters and predict paediatric CLp by accounting for developmental changes in the system-specific parameters and how they impact drugs with specific properties. Application of these models is considered best practice in pharmaceutical industry, but obtaining PBPK ontogeny functions for a given drug is time-consuming and complex due to the requirement of a wide range of drug-specific and system-specific information. Moreover, all information may not always be available for each drug or each population. This leads to a need for simplified scaling functions which are more convenient for defining paediatric CLp in pharmacometrics. As multiple system-specific parameters may change in the paediatric population and as the impact of each of these changes on paediatric CLp may be different for each given drug with different characteristics, the challenge in developing simplified scaling functions is to aggregate all relevant information in functions with a limited number of scaling variables. Various simplified clearance scaling methods for the paediatric population have been proposed. Allometric scaling using a fixed exponent of 0.75 (AS0.75) is one of the simplest scaling methods, as it only uses body weight as scaling variable. However, AS0.75 has been shown to lead to large over-predictions of hepatic metabolic CLp in children younger than 5\u00a0years, especially when isoenzymes are immature ,2.et al. have proposed the age-dependent exponent method (ADE) that was found to outperform AS0.75 in young children   . In addime drugs .PBPK represent potentially viable options to accurately scale clearance in children under five years of age  or alpha-1 acid glycoprotein (AAG). The unbound drug fraction in plasma (fu) in adults ranged from 1 to 100%, with 8 equidistant intermediate values. Equations by Rodgers and Rowland ( Rowland . The affBlood-to-plasma partition coefficient (Kp). Kp values of 0.35 and 0.8 and values from 1 to 40 with 38 intermediate equidistant values were selected, reflecting different extents of drug diffusion into the red blood cells . Total CLint,mic ranged between 0.56\u00b710\u22126 and 0.209\u00b710\u22123\u00a0mL\u00a0min\u22121\u00a0\u03bcg\u22121 microsomal protein in adults  was implemented as a near continuous variable. To do so, first, a realistic range of isoenzyme maturation values was defined for each age by taking the maximum and minimum isoenzyme maturation values reported for 15 isoenzymes. A minimum limit of 5% isoenzyme maturation was set. For all isoenzymes but SULT1A1, isoenzyme maturation values were taken from the Simcyp\u00ae library. For SULT1A1, maturity was taken to have been reached at birth ,,PBPK scaPBPK corresponds to the different percentages of isoenzyme maturation defined for each age, as also used in the PBPK model for the generation of \u2018true\u2019 relative paediatric CLps \u20133\u00a0months, >\u20093\u00a0months\u20132\u00a0years, and >\u20092\u20135\u00a0years, respectively (PBPK in use and both were investigated in this work. MFPBPK was either expressed as percentage of adult unbound intrinsic clearance per gram of liver (MFPBPK-liver), which accounts for maturation in both isoenzyme activity and microsomal protein per gram of liver (MPPGL). Alternatively, MFPBPK was expressed as percentage of adult unbound intrinsic clearance per microgram of microsomes (MFPBPK-microsomes), which only accounts for maturation of isoenzyme activity. Therefore, for MFPBPK-liver, maturation in MPPGL as implemented in the PBPK model for the generation of \u2018true\u2019 relative paediatric CLps was also used.In literature, there are two different interpretations of MFFor comparative purposes, Eq.  was usedPBPK-based CLp scaling was numerically assessed using the prediction error (PE). PE was computed for each \u2018true\u2019 paediatric hepatic metabolic CLp generated in step 1 and its corresponding scaled value in step 2 using Eq. ;drugs influenced neither by plasma protein maturation (fu\u2009=\u20091) nor by haematocrit maturation (Kp\u2009=\u20091) ;all hypothetical drugs binding to HSA, including drugs with fu\u2009=\u20091 .all hypothetical drugs binding to AAG, including drugs with fu\u2009=\u20091 , intermediate , or high  ER in adults.These categories were, then, further subcategorized based on the extraction ratio in adults (ER) as having either a low  which reflects both MFPBPK_microsomes and maturation in MPPGL, while the x-axis in Fig.\u00a0PBPK_microsomes).Figures\u00a0Figure PBPK-liver does not generally lead to over-prediction of hepatic metabolic CLp in the studied age range, but under-predictions may occur, especially when isoenzyme maturation is low. When enzyme maturation in this approach is expressed relative to adult intrinsic activity per microgram of microsomes (AS0.75\u2009+\u2009MFPBPK-microsomes), both over- and under-predictions of paediatric CLp for different drugs are observed in all ages , this method leads to PE of all hypothetical drugs between \u00b1\u00a030 and \u00b1\u00a050% for drugs with a low or intermediate ER in adults, respectively, except for drugs bound to AAG in term neonates of 1\u00a0day. This is due to the decreasing variability in relative paediatric CLp with decreasing ER values, because isoenzyme maturation is the main driver of relative paediatric CLp for drugs that have a low or intermediate ER in adults. The lack of accuracy in one day term neonates for drugs binding to AAG is due to the steep increase in AAG concentration in the first days of life, leading to a wide variation in relative paediatric CLp for different hypothetical drugs binding to this plasma protein to varying extents . Expressing isoenzyme maturation as percentage of adult intrinsic clearance per microgram of microsomes (MFPBPK-microsomes) leads to inaccurate CLp predictions regardless of drug properties in children under five years of age. Until 2008, MPPGL maturation had not been characterized and, therefore, isoenzyme maturation was expressed as percentage of adult intrinsic clearance per gram of liver (MFPBPK-liver)  . AfterwaK-liver)  and becaPBPK scaled predictions. The explanation may be that these estimated maturation functions also aggregate the impact of drug properties on clearance maturation that are not properly accounted for. This is in line with previous finding from Strougo et al. ESM 2(DOCX 34\u00a0kb)ESM 3(DOCX 1071\u00a0kb)ESM 4(DOCX 1229\u00a0kb)ESM 5(DOCX 1164\u00a0kb)ESM 6(DOCX 1223\u00a0kb)"}
+{"text": "Sulfolobales, a taxonomic group within the Crenarchaeota whose cellular features have been suggested to have a close relationship to the last archaea-eukaryote common ancestor. In this study, we genetically dissect how the two previously characterized S-layer genes as well as a newly identified S-layer-associated protein-encoding gene contribute to the S-layer architecture in Sulfolobus. We provide genetic evidence for the first time showing that the slaA gene is a key cell morphology determinant and may play a role in Sulfolobus cell division or/and cell fusion.The S-layer is considered to be the sole component of the cell wall in  Archaea but whose in vivo function is unknown. Here, we investigate the architecture and cellular roles of the S-layer in the hyperthermophilic crenarchaeon Sulfolobus islandicus. Electron micrographs of mutant cells lacking slaA or both slaA and slaB confirm the absence of the outermost layer (SlaA), whereas cells with intact or partially or completely detached SlaA are observed for the \u0394slaB mutant. We experimentally identify a novel S-layer-associated protein, M164_1049, which does not functionally replace its homolog SlaB but likely assists SlaB to stabilize SlaA. Mutants deficient in the SlaA outer layer form large cell aggregates, and individual cell size varies, increasing significantly up to six times the diameter of wild-type cells. We show that the \u0394slaA mutant cells exhibit more sensitivity to hyperosmotic stress but are not reduced to wild-type cell size. The \u0394slaA mutant contains aberrant chromosome copy numbers not seen in wild-type cells, in which the cell cycle is tightly regulated. Together, these data suggest that the lack of SlaA results in either cell fusion or irregularities in cell division. Our studies show the key physiological and cellular functions of the S-layer in this archaeal cell.Rediscovery of the ancient evolutionary relationship between archaea and eukaryotes has revitalized interest in archaeal cell biology. Key to the understanding of archaeal cells is the surface layer (S-layer), which is commonly found in  Bacteria, it has been shown that the S-layer is associated with either the peptidoglycan or the outer membrane  crystalline matrix coating the outside of the cell, called the surface layer (S-layer). Despite the broad distribution of the S-layer in cells from two domains, its generalized function has not been identified. One unifying concept suggests that the S-layer functions as an exoskeleton that interacts with other proteins in the cell membrane to coordinate diverse internal and external cell functions . In BactD crystaloenzymes , maintaioenzymes , resistioenzymes , regulatoenzymes , as welloenzymes \u20138. In coenzymes \u2013. So far,enzymes \u2013, 10 sincears ago .in vivo functions have not been studied extensively, but it has been proposed that the S-layer plays a role in osmotic stress  and SlaB (\u223c45\u2009kDa) in olobales \u201320. The \u2009kDa and ole cell . Compensole cell . The S-lole cell . Instabill shape  and buddll shape , 25. It ll shape . Thus faS. islandicus M.16.4 cell survival under standard lab conditions  in S. islandicus M.16.4. Reverse transcription-PCR (RT-PCR) analysis showed that slaA and slaB are cotranscribed , in agreement with a previous study in a related Sulfolobus species, Sulfolobus solfataricus P2 , slaB (\u0394slaB), or both genes (\u0394slaAB) (As in other ricus P2 . To diss(\u0394slaAB)  (see MatSulfolobus species in SEM and thin-section transmission electron microscopy (TEM) images  and 0.2 to 6.0\u2009\u03bcm (n\u2009=\u2009667), respectively, compared to that of wild-type cells . Around 21% of cells in the \u0394slaA mutant and 17% of cells in the \u0394slaAB mutant were larger than the wild-type cell size range and very few (\u223c0.9% for the \u0394slaA mutant and \u223c0.8% for the \u0394slaAB mutant) were below this range. The \u0394slaB mutant cells varied in size between 0.1 and 1.6\u2009\u03bcm (n\u2009=\u2009646), with approximately 99% of cells in the wild-type cell size range.) images , 28. In nt cells . For theartially . These dartially  as well tion TEM . The celhttps://doi.org/10.6084/m9.figshare.8285423]) but not in the \u0394slaA  or \u0394slaAB  mutant cells. A partial or discontinuous outermost layer was frequently observed in the \u0394slaB mutant cells , demonstrating that SlaA was present on the outside of the cell but was fragile and unstable and, thus, was easily detached from the cytoplasmic membrane following TEM sample preparation without the support provided by the SlaB stalk. These observations are in agreement with those obtained recently from our thin-section TEM experiments   strategy . The growth profiles of \u0394M164_1049 and \u0394slaB \u0394M164_1049 mutants have no significant differences from the parental RJW004 and \u0394slaB strains, respectively, in that they exhibited comparable growth rates and could reach approximately the same terminal optical densities at 600 nm (OD600) .Electron microscopy  analyses, presented both here and in a previous study , revealeFig. S4) . We nextstrategy , which rM164_1049 strain has a cell morphology very similar to that of its parental strain RJW004 (https://doi.org/10.6084/m9.figshare.8285423]). In contrast to \u0394slaB mutant cells, a partial coat of SlaA is not observed in the \u0394M164_1049 mutant cells. The \u0394slaB \u0394M164_1049 mutant appears to lack an S-layer  in the \u0394slaB \u0394M164_1049 mutant are less than 0.5\u2009\u03bcm in diameter whereas only 0.5% of cells (n\u2009=\u2009778) in that range are found in the wild type. The conventional whole-cell TEM analysis revealed that an intact and smooth outermost layer, i.e., SlaA, was consistently observed to encompass the whole cell of the \u0394M164_1049 mutant , which was further validated by thin-section TEM . In agreement with the SEM analysis, the outermost layer was not observed in the mutant strain lacking both slaB and M164_1049 genes in TEM images . These results suggest that M164_1049 may assist SlaB to firmly anchor the SlaA to the cytoplasmic membrane but is not redundant in its functioning in S-layer stabilization.SEM images showed that the \u0394 S-layer  to g, sislaA mutant cultures, which generally contain hundreds or thousands of cells in a single clump or aggregate . About 67% of cells in the \u0394slaA mutant have a diameter greater than 2\u2009\u03bcm whereas in the wild type only 5% of cells are found in this range  . We used live-dead fluorescence microscopy to see whether the aggregated cells in large clumps are alive or dead, and it revealed that most of the aggregated cells in the \u0394slaA mutant are not permeable by propidium iodide (PI)  . This incultures . The pheslaB gene caused small aggregates in the cell cultures . We found that around 16% of cells in the \u0394slaB mutant have a diameter greater than 2\u2009\u03bcm (Table S1). No obvious cellular aggregation was seen in the single \u0394M164_1049 mutant cells .Deletion of the single cultures , and thent cells , similarnt cells . Interese mutant  comparedslaA mutant and wild-type cultures have comparable growth rates . Large aggregates would result in more light passing through the liquid culture when the optical density is measured, causing a lower OD600 value, as observed for the \u0394slaA mutant. Aggregated cells may grow as one CFU in solid medium, consistent with the observation that there is a more than 10-fold reduction in the CFU count of the \u0394slaA mutant in spotting assays . We cannot exclude the possibility that the lower CFU count may be caused or contributed by other factors such as an increased sensitivity to cell processing procedures.The formation of aggregates in liquid culture is supported by the fact that although the \u0394th rates , we obseslaA mutant cells in liquid medium supplemented with increasing amounts of sucrose. The wild-type strain was able to grow in the presence of 2% (wt/vol) sucrose with a growth rate that was still comparable to that of cells that grew in sucrose medium with concentrations ranging from 0% to 1%; however, cell growth was severely impaired when cells were cultivated in 5% sucrose medium (slaA mutant was significantly delayed (n\u2009=\u2009788) and 26% (n\u2009=\u2009740) when cells were cultured in 2% and 5% sucrose medium, respectively . For the \u0394slaA mutant, the estimated proportion of permeable cells increased by more than 50% and 90% in the presence of 2% and 5% sucrose, respectively; in particular, the membranes of relatively enlarged cells were more prone to be permeable at high concentrations of sucrose . Even under high-osmotic-stress conditions, impermeable cells in the \u0394slaA mutant were significantly larger than wild-type cells , indicating that large cells observed in the \u0394slaA mutant are not expanding due to turgor pressure alone. These results collectively showed that S. islandicus cells deficient in S-layer are more sensitive to hyperosmotic stress than wild-type cells.In agreement with a theoretical study , we hypoe medium . In cont delayed . We furtSulfolobus cells with a cell division defect often have an enlarged-cell-size phenotype  and four copies of chromosome (4C) as well as a long trailing tail became distinct, suggesting the existence of multiple chromosomes in the cells . We note that in contrast to the wild-type cells, \u0394slaA cells contain a greatly reduced population in the process of replicating between the 1C (1 copy of chromosome) and 2C (2 copies of chromosome) peak . The cell size distribution of \u0394slaA and \u0394slaB mutants was further evaluated by fluorescence-activated cell sorting (FACS) analysis. We found that a substantial proportion of enlarged cells existed in the \u0394slaA mutant; however, the size distribution profiles between the \u0394slaB and wild type were hardly distinguishable .henotype , 31. To  species , 33. Howhe cells . It shouS. islandicus S-layer gene deletion mutants  present in both the N and C termini of M164_1049 suggests that M164_1049 has a membrane topological organization, implying that there is a potential connection between M164_1049 and the outermost layer (SlaA). Interestingly, the M164_1049 homolog in a related Sulfolobus species, S. solfataricus P2  has been shown to be an N-glycosylated protein  or by the physical absence of SlaA as in the \u0394slaB \u0394M164_1049 mutant cells, form large aggregates composed of cells with increased diameters. The \u0394slaB mutant also exhibits a high level of cellular aggregation , but the sizes of aggregates along with the cell diameters are smaller than those of the \u0394slaA mutant. We proposed that the high level of cellular aggregation but of relatively small-size aggregates in the \u0394slaB mutant may be due to a large proportion of cells that retained a partial outermost layer (SlaA), as revealed by electron microscopy analysis of the \u0394slaB mutant. This viewpoint was further supported by the FACS analysis in which the cell size distribution of the \u0394slaB mutant exhibits a pattern as similar to that of the wild type. Taken together, these data suggest that SlaA, not SlaB, is the key determinant of cellular aggregation and cell morphology in S. islandicus though the exact contribution of SlaB to these phenotypes remains to be investigated.slaA mutant: cell fusion and a cell division defect. In the cell fusion model, the SlaA-deficient cells aggregate, possibly because of interactions between outer membrane proteins or attraction between hydrophobic membranes themselves when cells are not protected by the S-layer outer shell. Within aggregates, cells fuse, resulting in an increase in cell size. As the cells continue to grow over time, a wide range of cell diameters would be created, as observed in the \u0394slaA mutant. We suspect that this type of hypothesis alone would result in mutants with a greater increase in even-number chromosome copy numbers due to the higher chance of contact between the cells that are in an extensive G2 stage during the Sulfolobus cell cycle -III-1 , a paralog of ESCRT-III that was shown to play an auxiliary role in cell division , suggesting the possibility that cells were no longer dividing to 1C but replicating before the division was completed.We suggest two models to explain the combination of large cell sizes within aggregates and surprisingly variable odd numbers of chromosomes observed in the \u0394ll cycle . The dist)-III-1 ; howeverdivision . Though ivision 35, 38, 39egulated . In addiSulfolobus and archaeal cell biology as a whole as well as of interactions with fundamental processes such as chromosome partitioning.The cell fusion and cell division hypotheses are clearly not mutually exclusive, and both cell fusion and irregularities in the cell cycle may be linked to S-layer function. Addressing these questions will allow for a more complete understanding of the S-layer function in https://doi.org/10.6084/m9.figshare.8285423]). The E. coli Top10 strain  was used to maintain and propagate plasmid DNA and grown in Luria-Bertani (LB) liquid medium at 37\u00b0C. When required, 100\u2009\u03bcg/ml ampicillin was supplemented in LB agar plates to select ampicillin-resistant clones. S. islandicus RJW004, the genetic host used in this study, was originated from wild-type isolate S. islandicus M.16.4 , as described previously  with a CO8000 cell density meter .The strains and plasmids used in this study are listed in Table S2 . Genomic DNA and total RNA were used as templates for positive and negative controls in PCR amplification, respectively. The resulting PCR products were separated by electrophoresis with a 1.2% (wt/vol) Tris-acetate EDTA (TAE) agarose gel.Ten milliliters of cell cultures taken from exponentially growing strains was harvested and lysed with 1\u2009ml of TRIzol reagent . Total RNA was extracted and isolated with a PureLink RNA Minikit  and then cleaned and concentrated with an RNA Clean & Concentrator-25 kit . To exclude potential DNA contamination, the total RNA (\u223c2\u2009\u03bcg) was treated with 2\u2009\u03bcl of amplification-grade DNase I . Approximately 500\u2009ng of DNase I-treated total RNA was reverse transcribed using 2 \u03bcM gene-specific reverse primers and 1 U of SuperScript IV reverse transcriptase  according to the manufacturer\u2019s guidelines. Two microliters of synthesized cDNA was used as a template in a standard PCR amplification reaction performed in a thermocycler , using Phusion High-Fidelity DNA polymerase  and the gene-specific primers binding the regions inside the S. islandicus cells was extracted using a DNeasy Blood & Tissue kit  according to the manufacturer\u2019s instructions. Sequencing libraries of genomic DNA from wild-type RJW004 and the \u0394slaA, \u0394slaB, and \u0394slaAB mutants were constructed with a Nextera XT DNA Library Preparation kit  individually. Libraries were sequenced using HiSeq MMD (250-bp paired-end). The raw sequencing reads were processed with a quality filtering and then mapped to the reference genome of S. islandicus M.16.4 (the ancestor strain of RJW004) with breseq  harbored a 363-bp deletion in M164_1772 annotated as a hypothetical protein. Although the function of M164_1772 is unclear, it should not contribute to the phenotypes as observed in the \u0394slaAB mutant because characterizations of another \u0394slaAB isolate without any M164_1772 mutation (\u0394slaAB-2) demonstrated the same phenotype as that of the \u0394slaAB-6 mutant (this study) under electron microscopes (data not shown).Genomic DNA of h breseq  using deM164_1049 in the genetic background of RJW004 and RJW012  was accomplished by replacing it with an arginine decarboxylase expression cassette (StoargD) (the argD gene from Sulfolobus tokodaii), using a recently developed MMGI approach (+) transformants were selected on dextrin-tryptone plates containing 20\u2009\u03bcg/ml of uracil but lacking agmatine. The M164_1049 deletion mutants were verified by a colony PCR procedure described previously  uranyl acetate for 15 to 30\u2009s. Samples used for thin-section TEM analyses were essentially prepared as described by Bautista et al.  were placed on 20-\u03bcl droplets of a et al.  with mina et al. . The griS. islandicus strains were grown to the mid-log phase. Five microliters of cell cultures was spotted on a cleaned microscope slide using a blunted 20-\u03bcl pipette tip in order to avoid potential disruption of the large aggregates formed in the S-layer gene deletion mutants. A coverslip was added immediately, and then the slide was observed on a Zeiss Axiovert 200M microscope with a 63\u00d7/1.40 oil objective. Images were captured using a Zeiss Axiocam 506 microscope camera and visualized with Zeiss Zen imaging software.S. islandicus cells was fixed with 700\u2009\u03bcl of ice-cold absolute ethanol, and the mixture was then briefly vortexed. Fixed samples were stored at 4\u00b0C for at least 12\u2009h. Afterwards, the fixed cells were centrifuged at 13,000\u2009rpm for 5\u2009min and resuspended in 1\u2009ml of cold Tris-MgCl2 buffer . Then the samples were precipitated and resuspended again in 300\u2009\u03bcl of Tris-MgCl2 buffer containing 2\u2009\u03bcg/ml of Sytox Green nucleic acid stain  and 100\u2009\u03bcg/ml of RNase A . After at least a 30-min incubation on ice in the dark, samples were analyzed using an LSR II  flow cytometer, with a 488-nm (50 mW) blue laser as excitation light and a 530/30-nm band pass filter. A total of 100,000 cell counts for each sample were collected. Flow cytometry data were processed and analyzed with De Novo FCS Express 6 software. The cell population was gated with three steps as described in the Fig. S10  to obtain a final DNA content histogram.Three hundred microliters of slaA mutant strain were grown to mid-log phase. For the wild-type RJW004, 1\u2009ml of cultures was transferred to a 1.5-ml microcentrifuge tube and then centrifuged at 10,000\u2009rpm for 5\u2009min to pellet. The pellet was resuspended with 1\u2009ml of dextrin-tryptone (1\u00d7) medium to wash. Centrifugation was repeated once, with the resulting cell pellet then resuspended in 500\u2009\u03bcl of dye solution. For the \u0394slaA mutant cells, to preserve the aggregate phenotype, a total of 5 microcentrifuge tubes containing 1\u2009ml of cultures were allowed to settle for 15\u2009min to collect cells at the bottom of the tubes. Cells of all 5 microcentrifuge tubes were then combined, with supernatant removed. The remaining cells were resuspended in 1\u2009ml of fresh dextrin-tryptone liquid medium. Cells were allowed to settle for an additional 10\u2009min. Supernatant was removed, and the cells were gently resuspended in 500\u2009\u03bcl of the dye solution, as per protocol  . Cells were covered and incubated at room temperature in darkness for at least 15\u2009min before viewing. Five microliters of stained cells was then spotted directly on a microscope slide using a blunted 20-\u03bcl pipette tip, immediately covered with a coverslip, and observed on a Zeiss LSM 710 confocal microscope with a 63\u00d7/1.40 oil M27 objective at excitation wavelengths of 561\u2009nm (for PI detection) and 488\u2009nm (for Syto 9 detection) using Zeiss Zen software.RJW004 and the \u0394S. islandicus cells were transferred into the medium to make an initial OD600 of 0.02. Three biological replicates were established for each treatment. Cells were grown at 78\u00b0C without shaking, and their growth was monitored for 7\u2009days by measuring the OD600 usually every 12 or 24\u2009h. Cell cultures after a 48-h incubation were examined with a Zeiss LSM 710 confocal microscope.For the osmotic stress assay, a 50% sucrose solution was added to 45\u2009ml of dextrin-tryptone liquid medium, making the final concentrations (wt/vol) of sucrose 0%, 0.05%, 0.10%, 0.2%, 0.5%, 1.0%, 2.0% and 5.0%. Proper volumes of S. islandicus M.16.4 were previously published (GenBank accession no. CP001402.1). The raw sequencing data of the parental strain S. islandicus RJW004 and its derived S-layer deletion mutants are available upon request.The genome sequence data of the reference strain"}
+{"text": "Large-scale, defect-free, micro- and nano-circuits with controlled inter-connections represent the nexus between electronic and photonic components. However, their fabrication over large scales often requires demanding procedures that are hardly scalable. Here we synthesize arrays of parallel ultra-long (up to 0.75 mm), monocrystalline, silicon-based nano-wires and complex, connected circuits exploiting low-resolution etching and annealing of thin silicon films on insulator. Phase field simulations reveal that crystal faceting and stabilization of the wires against breaking is due to surface energy anisotropy. Wires splitting, inter-connections and direction are independently managed by engineering the dewetting fronts and exploiting the spontaneous formation of kinks. Finally, we fabricate field-effect transistors with state-of-the-art trans-conductance and electron mobility. Beyond the first experimental evidence of controlled dewetting of patches featuring a record aspect ratio of  Fabricating defect-free micro- and nano-circuits over large scales with controlled interconnections remains a challenge. Here, Bollani et al. show a dewetting strategy for engineering arrays of parallel Si-based nanowires up to 0.75 mm and complex interconnected circuits of monocrystalline Si on a chip. Their disruptive potential has been demonstrated in photonics3 (e.g. for lasers4 or quantum optics5), electronics6, thermoelectricity7, energy storage with batteries8, gas9 and mechanical10 sensing, topological quantum states11, and much more. For these reasons their growth has been tackled with a plethora of techniques aiming at the production of controlled, ultra-long structures matching the needs of high yield, scalability (e.g. integration of a large number of devices on the same monolithic nano-wire) and material quality (e.g. smooth interfaces).Semiconductor nanowires exhibit superior and configurable electronic and optical properties1, from direct top-down design, as, e.g., three-dimensional mesoscale lithography12, superlattice nanowire pattern transfer13 up to the exploitation of natural phenomena such as the renowned Plateau-Rayleigh instability14. Several bottom-up, self-assembly methods can be employed to obtain high-quality parallel wires15. Nevertheless, a full control over their morphology, size, position, direction, inter-connection, and electrical isolation remains a challenge as current techniques are not versatile and are limited to a few micrometers length. On the other side, lithographic top-down methods can be used to precisely prepare a substrate for further engineering, as for instance shown for the cases of controlled wetting16 or nano-imprint lithography17. Hybrid top-down/bottom-up approaches21 can marry the crystalline quality of epitaxial self-assembly with the ultimate control of nano-structures position and size and create quantum-grade materials, such as complex assemblies of connected wires and membranes. However, their exploitation in scalable devices is often hindered by the complexity of their implementation requiring too many, cumbersome nano-fabrication steps. In fact, these approaches often need high-resolution etching  and provide out-of-plane objects eventually requiring further processing before their implementation in a device22. These methods are hardly scalable as they exploit complex epitiaxial growth steps, often involving a metallic catalyst and are limited to structures extending only over a few micrometers.As such, the range of available approaches to grow elongated crystalline structures steadily increasesHere we fabricate arrays of in-plane, ultra-long nano-wires (up to 0.75\u2009mm) and complex inter-connected circuits of monocrystalline silicon using a catalyst- and epitaxy-free, hybrid top-down/bottom-up approach based on the natural morphological evolution of thin solid films. We control the metamorphosis of a commercial (001)-oriented ultra-thin silicon film on insulator (UT-SOI) relying only on low-resolution etching and annealing, directly transforming it in monocrystalline nano-wires. The final structures have a lateral size up to five times smaller than the etched patch width, are obtained with size- and position-control and are electrically-isolated from the substrate. Phase field simulations of surface-diffusion-limited kinetics elucidate the key role of the surface-energy anisotropy in stabilizing the dewetting outcome against breaking. They quantitatively reproduce the main features of the morphological evolution of the patches.23 which hinders the onset of the typical Rayleigh-like instability along the wire axis24 we extend these results to arbitrary in-plane crystallographic directions, building complex circuits of wires featuring splitting, changes in their directions and inter-connections. Finally, with a simple spin-on-dopant post-fabrication method, we render the wires conductive, demonstrating the possibility to use them as field-effect transistor6 with trans-conductance and electron mobility similar to state-of-the-art nanowires devices.Exploiting the orientation-dependent edge faceting promoted by surface energy minimization26. It leads to a complete metamorphosis of the flat layer in three-dimensional structures through hole nucleation, rims formation (where mass accumulates while receding), followed by finger-like structures and finally, in isolated islands. This natural shape evolution can, however, be controlled by engineering the dewetting fronts by patterning the UT-SOI prior to annealing28.Dewetting of monocrystalline thin silicon films is a spontaneous phenomenon where capillary forces drive mass transport via surface-diffusion-limited kinetics30, the need of a precise and controlled dewetting front in order to form regular nano-architectures requires mono-crystalline wafers where the dewetting process only affects the shape of the layer which always remains a mono-crystal33. The potential of templated dewetting was showcased for a mono-crystalline 12\u2009nm thick UT-SOI where, in analogy with metals34, arrays of complex nano-architectures of islands and wires  were reported28. The key tool used to enhance the stability of the dewetting outcome against breaking leading to reproducible patterns was the creation of ad hoc dewetting fronts triggering the formation of opposite rims that move one towards the other. So far, this approach was limited to patches extending over a few 28.Although silicon dewetting is also possible starting from an amorphous UT-SOI28.Following this concept, we tested the stability against annealing of a 12 nm thick UT-SOI  we observe the formation of extremely long wires, with a length limited only by the patch design  even when considering a film in contact with a substrate40: the dewetting process for the larger pitches , is not concluded at the end of the annealing step. It is worth noting that, although the process for In a similar sample, entclass1pt{minima14.In all investigated cases, the fluctuations around the average full width at half maximum (FWHM) and heights of the wires were only a few nanometers Fig.\u00a0f\u2013g accou42, including surface-energy anisotropy44 solved by a finite element approach46 For simulations reproducing mentclass2pt{minim(ii)The simulations for entclass1pt{minima(iii)49. For Surface faceting is found to play a central role in determining a quantitative outcome of the process(iv)50 at the cost of a significant increase in the computational budget without however, delivering relevant, additional information than those discussed so far.For the largest patch, the experimental rims are smaller than the prediction by phase field simulations and the valleys next to the rims are not visible. This feature is attributed to an effective larger stiffness/anisotropy of the real structures with respect to those considered in these simulations. This could be readily accounted for by phase field simulationsThe relevant features emerging from this analysis are summarized as follows:In a different sample, similar patch arrangements are etched with a slight mis-cut of 2\u00b0 circa, with respect to a stable dewetting front. We now consider patches size of 800\u2009nm in width  may improve the quality of the final outcome.This demonstrates that it is possible to control the continuity, connectivity and curvature of the wires up to several degrees of misalignment with respect to the stable dewetting front without any optimization. A more appropriate choice of etching design and experimental conditions .In order to rule out the presence of crystalline defects in the dewetted structures we performed atomic-resolution scanning transmission electron microscopy (STEM) imaging on a wire Fig.\u00a0. In line54. All metal contacts  are 130\u2009nm thick gold and were placed by e-beam deposition on 15 parallel nano-wires. S and D partially wet the silicon wires whereas G is separated from the wires by a 100\u2009nm thick silicon dioxide (deposited via e-beam evaporation) covering the wires for about 30\u2009Through the templated dewetting process we demonstrated crystalline silicon wires formed directly on an insulating substrate. To show the potential of these structures for electronic circuits a doping procedure involving phosphorus spin-on-dopant (SOD) deposition and rapid thermal annealing treatment has been carried out on 70\u200955. From the I\u2013V curves obtained on 15 parallel nanowires for different G tension, we estimate a trans-conductance . This transistor works in enhancement mode, where the saturation and the S-D voltage are characteristic of a n-channel FET56 and neglecting spurious effects57, we can estimate the electron mobility with the formula Adopting the common approximations for nanowire-based transistorsThere are several differences and advantages of our wires with respect to previous demonstrations of similar structures implemented via dewetting and other fabrication techniques, particularly regarding the simplicity and versatility of implementation, improved comprehension of the dewetting mechanism and improved quality of the structures enhancing the electrical properties of the implemented devices. In what follows we discuss all these features with respect to the existing state-of-the-art.28 to 0.75 mm . This is a record aspect ratio of a In this work, we extend the coherent control of dewetting by more than 2 order of magnitude, going from patches of a few 28 is the control of patch evolution for patterns oriented along unstable fronts. The formation of kinks during dewetting  allows to adjust the macroscopic directions of the final structures without breaking. This feature has never been reported so far in semiconductor dewetting and it allows to curve, split and connect the wires ad libitum in order to form complex circuits.An important difference with respect to previous reports of Si dewettingdd28: above 3\u200928). These spurious effects could be attributed to extrinsic disorder locally perturbing the rim evolution and affecting in a more marked way larger patches with respect to smaller ones (e.g. native oxide not properly removed or other impurities present in the ultra-high vacuum used for silicon dewetting). Furthermore, small tips found at the rim/BOX interface can perturb the dewetting dynamics28. Thus, although these defects allow the formation of kinks along the wires and thus to curve them with respect to the preferential axes orientations, they may be a limit for a coherent control of larger patches . In the present case of 12\u2009nm thick UT-SOI, a coherent control of the patch evolution  is limited to <3\u2009The limit of this method is evident when considering patches larger than a few entclass1pt{minimaentclass1pt{minima28. This was in stark contrast with the real systems featuring a much smaller value of 58 and strong50 anisotropy. However, in all these cases the patch aspect ratio was at most 1/60 which is pretty far from the actual experimental conditions used for metal and semiconductor dewetting. Here we used a phase field model taking into account surface diffusion and surface-energy anisotropy for a 1/1 scale case (aspect ratio up to 1/330).In the present work, we also managed to compare real systems with realistic models taking into account anisotropic surface diffusion. So far, simulations of templated dewetting of UT-SOI based on a phase field approach considered patches featuring an aspect ratio of, at most, 1/160Our novel theoretical understanding of the anisotropic dewetting problem allows therefore to correctly predict long-time evolution of the main features observed in experiments showing that the presence of facets (due to anisotropic surface energy) stabilizes the dewetting outcome against breaking. Isotropic models  fails in this task, at least for larger patches.21 (e.g. for III\u2013V-based structures).From a fabrication standpoint our approach offers several advantages with respect to other bottom-up methods. We implemented our wires on commercial UT-SOI wafers, available in a large set of device thickness, orientation, doping, composition (e.g. SiGe and Ge on insulator), BOX thickness, and up to 12 inches in size. These features are not matched by other methods implemented on, at most, a few inches and expensive epilayers59  and an easier implementation with respect to recent demonstrations were the wires were bound together with complex patterning and epitaxial nano-fabrication methods61 .Our structures are implemented only in two steps, etching and annealing. Thus, templated dewetting of wires offers a versatility in directions, splitting and connections not attainable with catalyst-based approaches for in-plane wires growth62, droplet epitaxy and droplet etching (only for III\u2013V)63, vapor-liquid-solid growth via gold catalyst59, or more advanced hybrid top-down/bottom-up approaches19. All these strategies can be exploited only when a precise epitaxial relation holds between the substrate and the deposited material. As such, they require a crystalline support. In contrast to this, we directly produce well-ordered, monocrystalline nano-architectures on amorphous SiO21 that becomes insulating only at very low temperature19. Another important difference between dewetting and recent reports of advanced epitaxial structures based on selective area growth, is the lack of strain and alloy disorder that is known to complicate the interpretation of the device behavior20.Other self-assembly methods are not suitable for the formation of crystalline structures on amorphous SiO21. In this respect, it is worth stressing that a high resolution for electron-beam lithography inevitably requires a reduced writing field, limiting the size of nanowires to rather short channel length (a few 64 and bottom-up21 approaches). In our case, a low resolution allows for mm scale structures providing an aspect ratio of the order of 1/10The width of our wires is 4\u20135 times smaller than the initial patch width, implying that a low-resolution etching  can in principle be exploited. This constitutes an advantage with respect to current hybrid top-down/bottom-up approaches, where the resolution of the lithography directly sets the final size of the structuresOur approach allows to tune the wires height on the same substrate in contrast with top-down approaches that lead to structures with a height fixed by the thickness of the UT-SOI in use or by the etching depth. We achieved this by setting the initial patch lateral width 65. Thus, trans-conductance and electron mobility are important figures of merits that account for the quality of the interfaces and the limits of a wire-based device.One of the most important features of nano-wires is their electrical properties. For instance, the changes in conduction of wire-based transistors can be efficiently exploited for sensing67 even when considering wires having size close to those shown here6.In our wires, the measured value of trans-conductance is 66. For top-down FET wires  typical values of 68. Larger values of 70. Thus, even without any kind of optimization of our devices, we can reach relatively high electron mobility. We interpret this feature as a possible consequence of the reduced surface roughness in our structures with respect to those obtained via vapor-liquid-solid growth and top-down methods.From the electric characterization, a rough estimation of electron mobility in our FET transistors provides 34, and semiconductors 1/40028) forming extremely elongated silicon mono-crystals.In conclusion, we showed that dewetting, a spontaneous shape instability common to many different thin films of organic and inorganic substances, can be efficiently controlled in order to form extremely long and connected circuits of monocrystalline silicon wires on SiOWe directly compare the experimental outcomes with 1/1 scale phase field simulations of surface diffusion that quantitatively reproduce their morphological evolution. We show a clear evidence of the key role played by faceting in stabilizing the dewetting outcome against breaking and thus for the reproducibility of the process and the stability of the final structures. Phosphorous-doped conductive Si wires are implemented showcasing the possibility to fabricate conducting nano-channels and transistors on an insulating substrate. Since the proposed approach is very general, it can be adapted to tune the Si wires aspect ratio by choosing suitable UT-SOI thickness and pattern periodicity combined with more complex, connected nano-architectures towards a full exploitation of their record length and atomically smooth facets.32 similar results can be extended to these materials rendering this method attractive for wires formation with different materials with the perspective of band-gap engineering and carrier mobility enhancement.Owing to a similar dewetting dynamic ruled by surface-diffusion-limited kinetics observed in SiGe alloysdA 12\u2009nm-thick UT-SOI on a 25\u2009nm thick buried oxide (BOX) was etched by electron-beam lithography and reactive etching with parallel trenches, from 0.75\u2009mm to 70\u2009Deposition of Spin on dopant  was performed by spin-coating at 4000\u2009rpm for 1\u2009min and baking on a hot plate (10\u2009min at 120\u2009Scanning transmission electron micrograph have been acquired in z-contrast with a Cs probe corrected JEOL ARM 200F operated at 60\u2009keV.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7"}
+{"text": "Escherichia coli to examine how organisms address translation at unassigned UAG codons, which obstruct propagation of UAG-containing viruses and plasmids. Using mass spectrometry, we show that recoded organisms resolve translation at unassigned UAG codons via near-cognate suppression, dramatic frameshifting from at least \u22123 to +19 nucleotides, and rescue by ssrA-encoded tmRNA, ArfA, and ArfB. We then demonstrate that deleting tmRNA restores expression of UAG-ending proteins and propagation of UAG-containing viruses and plasmids in the recoded strain, indicating that tmRNA rescue and nascent peptide degradation is the cause of impaired virus and plasmid propagation. The ubiquity of tmRNA homologs suggests that genomic recoding is a promising path for impairing horizontal gene transfer and conferring genetic isolation in diverse organisms.Organisms possessing genetic codes with unassigned codons raise the question of how cellular machinery resolves such codons and how this could impact horizontal gene transfer. Here, we use a genomically recoded  Usually, DNA passes from parent to offspring, vertically down the generations. But not always. In some cases, it can move directly from one organism to another by a process called horizontal gene transfer. In bacteria, this happens when DNA segments pass through a bacterium\u2019s cell wall, which can then be picked up by another bacterium. Because the vast majority of organisms share the same genetic code, the bacteria can read this DNA with ease, as it is in the same biological language.Horizontal gene transfer helps bacteria adapt and evolve to their surroundings, letting them swap and share genetic information that could be useful. The process also poses a threat to human health because the DNA that bacteria share can help spread antibiotic resistance. However, some organisms use an alternative genetic code, which obstructs horizontal gene transfer. They cannot read the DNA transmitted to them, because it is in a different \u2018biological language\u2019. The mechanism of how this language barrier works has been poorly understood until now.Escherichia coli bacteria with an artificially alternated genetic code. In this E. coli, one of the three-letter DNA \u2018words\u2019 in the sequence is a blank \u2013 it does not exist in the bacterium\u2019s biological language. This three-letter DNA word normally corresponds to a particular protein building block. Using a technique called mass spectrometry, Ma et al. analyzed the proteins this E. coli forms. The results showed that it has several strategies to deal with DNA transmitted horizontally into the bacterium. One method is destroying the proteins that are half-created from the DNA, using molecules called tmRNAs. These are part of a rescue system that intervenes when protein translation stalls on the blank word. The tmRNAs help to add a tag to half-formed proteins, marking them for destruction.Ma, Hemez, Barber et al. investigated this using This mechanism creates a \u2018genetic firewall\u2019 that prevents horizontal gene transfer. In organisms engineered to work from an altered genetic code, this helps to isolate them from outside interference. The findings could have applications in creating engineered bacteria that are safer for use in fields such as medicine and biofuel production. The standard genetic code allows faithful translation of proteins across nearly all living organisms and enables horizontally\u00a0transferred genetic elements (HTGEs), such as conjugative plasmids and viruses, to exploit a host\u2019s translational machinery . Since nEscherichia coli  to recognize codon(s) during translation. One possible alteration of the genetic code is the loss of a codon assignment through the deletion or modification of an aminoacyl-tRNA or release factor, removing the cell\u2019s ability to decode that codon . Such unhia coli . We recehia coli  and estaE. coli can resolve translation at unassigned UAG codons. We hypothesize that three mechanisms could resolve translation at prokaryotic ribosomes encountering these unassigned codons, each resulting in peptides with different C-terminal sequences  supequences . The tmRequences . tmRNA-Sequences . The ssribosomes . ArfA anibosomes . tmRNA, ibosomes . A possiibosomes , followeibosomes .Studies of ribosomal stalling arising at rare codons  or in coEscherichia coli strain in which all UAG codons were mutated to UAA, permitting the deletion of release factor 1 (RF1) and resulting in an organism that lacks a codon assignment of UAG. This genomically recoded organism (GRO)   exhibitesm (GRO)  attributsm (GRO) , enablinprfA gene (GRO.DD.prfA+) or in GRO cells lacking prfA and consequentially without UAG assignment (GRO.DD) (We assembled plasmids (pUAG-GFP and pUAA-GFP) encoding GFP genes with C-terminal 6x-His tags positioned immediately upstream of a UAG or UAA stop codon. We\u00a0then expressed GFP from pUAG-GFP and pUAA-GFP in GRO cells containing the RF1-encoding (GRO.DD) . To dist(GRO.DD)  to identssrA DD-tag appended directly to the C-terminus of\u00a0GFP (LEHHHHHHAANDENYALDD) appeared in both UAG- and UAA-ending transcripts in GRO.DD and GRO.DD.+prfA  , and we E. coli is estimated to be\u00a0~1 in 10,000 bases  in strains with wild-type ssrA sequence (GRO.AA) to determine whether protein production from UAG-ending transcripts in \u0394RF1 cells could be restored to levels seen in\u00a0+RF1 cells. Using recombineering  because the resulting phenotype is synthetic lethal (600)  as compared to the GRO.AA (54% increase in doubling time)  . HoweverssrA in the UAG-GFP-expressing GRO partially restored protein production to levels seen in its UAA-GFP-expressing counterpart with no knockouts (31 \u00b5g/ml for GRO.AA.\u2206ssrA [pUAG-GFP] versus 68 \u00b5g/ml for GRO.AA [pUAA-GFP]) and deletion of both ssrA and arfB fully restored protein production (70. \u00b5g/ml). These ssrA deletion strains likely demonstrate increased GFP expression and reduced growth rate , and the strain showed an increase in doubling time of only 6% compared to a 28% increase for GRO.AA (p<0.0001). We observed similar results for\u00a0GRO.AA.\u2206ssrA.\u2206arfB. However, single deletion of arfB halved RK2 conjugative efficiency  compared to GRO.AA (3.30 \u00d7 104 events), arfA deletion (3.41 \u00d7 104 events), and arfB deletion (3.47 \u00d7 104 events). GRO.AA.\u2206ssrA.\u2206arfB and GRO.AA.\u2206arfA.\u2206arfB exhibited 5.2- and 2.3-fold decrease in conjugative efficiency when compared to GRO.AA.\u2206ssrA and GRO.AA.\u2206arfA single deletion strains, respectively  or alongside deletion of arfB (p<0.0001)\u2014recovers \u03bb infection of the GRO to levels similar to wild-type, with about 108 plaque forming units per mL (PFU/mL)  . These r the GRO .ssrA deletion strains exhibit both significantly increased UAG-GFP yields  containing an unassigned UAG codon as a model to investigate the molecular mechanisms that obstruct the propagation of HTGEs in organisms with alternative genetic codes. We demonstrate that unassigned stop codons elicit near-cognate suppression, frameshifting, and the action of ribosomal rescue mechanisms . tmRNA-mP yields  and recoP yields , consistradation . Our GROGUGHis-tRNA anticodon)   and any ticodon)  exists uticodon) . From ouMass spectrometry analysis also revealed truncated mistranslation products that possibly represent loss of translational fidelity and termination by RF2 downstream of an initial mistranslation event at the UAG codon, known as post-peptidyl transfer quality control , a resulE. coli should tolerate unassigned codons as intermediates toward codon reassignments in genomic recoding, efforts for which are underway in numerous prokaryotic and eukaryotic species  in which mutS is replaced by a zeocin resistance cassette  to increase stability of tagged proteins was performed with MAGE as described previously  media as described previously .R instead of kanR. The complete nucleotide sequence for the plasmid is available in NCBI database, Accession L27758.1 and GI 508311. We obtained the F plasmid from the Yale CGSC  and added KanR from plasmid pZE21 for antibiotic selection.For viral relative titers, we used phage \u03bb cI857 obtained from Dr. John Wertz at the Yale Coli Genetic Stock Center (CGSC) because it is obligately lytic at 37\u00b0C, preventing possible confounding factors from lysogeny. We used the conjugative plasmid RK2 described in R)carrying a copy of the tet repressor gene (tetR) to prevent leaked gene expression. We then modified the stop codon of the eGFP construct to end in either a UAG or UAA stop codon.To create the UAG-GFP and UAA-GFP constructs for protein expression, we cloned an eGFP construct with a C-terminal 6xHis tag downstream of pLtetO into a modified pZE21 vector with kanamycin resistance (kan600 of 1.0 and induced protein expression with the addition of 30 ng/uL anhydrotetracycline (aTC). After incubation overnight, we pelleted cells and resuspended them in sterile phosphate buffer solution, then lysed cells via sonication. Cell debris was then pelleted by centrifugation and GFP purified from supernatant via a nickel resin affinity column. To concentrate protein and exchange buffer for subsequent trypsin digest, we then concentrated GFP via Millipore Amicon spin columns.To obtain GFP for analysis via mass spectrometry, we transformed UAG-GFP and UAA-GFP constructs into wild-type and GRO strains carrying the AA->DD modification in the ssrA tag to prolong the half-life of tagged peptides. Experiments in the absence of the AA->DD modification yielded no peptides with ssrA degradation tags (data not shown). We then grew 50 mL cultures of each strain at 33\u00b0C in LB Lennox with 30 \u03bcg/mL kanamycin to an OD600 of 0.15 in fresh media containing 30 \u03bcg/mL kanamycin and 30 ng/uL aTC for 20 hr. To quantify protein expression and compare across strains, we normalized the OD600 of all cultures to 2.5 and pelleted 1 mL of this culture, which we placed in the \u221280C for 2 hr. We then re-suspended cell pellets in lysis buffer described previously  17,500 resolution, 1 \u00d7 106 AGC target, top 10 mode, 1.6 m/z isolation window, 27 normalized collision energy, 90 s dynamic exclusion, unassigned and\u00a0+1 charge exclusion. Peptide identification from collected spectra was performed using MaxQuant v1.5.1.2 (E. coli proteome (EcoCyc K-12 MG1655 v17). The searches considered carbamidomethyl (Cys) as a fixed modification and the following variable modifications: acetyl , oxidation (Met), deamidation , and phosphorylation (Ser/Thr/Tyr). Discovered peptides had a minimum length of five amino acids and could contain up to three trypsin miscleavage events. A 1% false discovery rate was used. The mass spectrometry proteomics data and the custom search databases have been deposited to the ProteomeXchange Consortium  via the PRIDE partner repository . We then ran 10 \u00b5l of 1/150 diluted lysate per lane of the SDS-PAGE gel. We obtained primary mouse anti-GFP antibody from Invitrogen  and goat anti-mouse antibody from AbCam . Western blots were developed using Bio-Rad Clarity Western ECL Blotting Substrate and Imaged on a GE Amersham Imager 600. We performed quantification of western blot bands as described previously  cells in 3 mL of TK soft agar and poured onto TK solid agar plates. Starter cultures of cells were diluted to an OD600 of 0.5 into three separate culture tubes, and cells within each tube were infected with phage lambda in parallel . Each tube was plated on a separate TK solid agar plate. We incubated plates overnight at 37\u00b0C, and counted plaques the next day.To quantify relative titers, we mixed 100-fold dilutions of phage with 300 \u00b5L of mid-log  code that we generated ; Yitzhak Pilpel (Reviewer #2); Alexander Mankin (Reviewer #3).Thank you for submitting your article \"Organisms with alternative genetic codes resolve unassigned codons via mistranslation and ribosomal rescue\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.E. coli strain lacking UAG codons and RF1. This question is important because there are a number of examples in nature of codon reassignment, but it is not clear how organisms would make the transition from a canonical code if there is a large penalty for having an unassigned codon. It has also been suggested that reassignment might limit transfer of foreign DNA. The authors used mass spectrometry to identify the proteins produced from a reporter gene with a UAG codon and find that multiple mechanisms help cells deal with the unassigned codon, including suppression, frameshifting, and general mistranslation. The authors also tested the effect of inactivation of three ribosome rescue systems  on the reporter expression and on propagation of conjugative plasmids or phages that have genes terminating in UAG. These data suggested that tmRNA accounts for the resistance of their E. coli mutant strain to horizontal gene transfer.In this manuscript, Ma et al. investigate how bacteria contend with unassigned codons by expressing genes with a UAG stop codon in a genetically recoded This is an interesting study that expands our knowledge of how cells deal with difficult-to-translate codons. It is also useful for the future use of genetically recoded organisms.Essential revisions:ssrA is deleted, ArfA will release a complete polypeptide identical to what would be produced by RF1. If a sense codon were unassigned, neither trans-translation nor ArfA activity could produce active proteins. The authors should either provide an explanation for why the ribosome rescue pathways would have the same impact on unassigned codons within a gene, or they should clarify that their interpretation is restricted to unassigned stop codons.1) The manuscript consistently refers to unassigned codons, implying that the work addresses this general issue. For example, in the Introduction: \"Our work reveals mechanistic details into how cells rescue ribosomes stalled at unassigned codons\". However, the effects observed when ribosome rescue pathways are deleted are almost certainly unique to unassigned stop codons. Trans-translation activity on ribosomes stalled at unassigned stop codons will remove the protein, but when 2) For the experiment in Figure 2, the basis for some of the assignments are unclear. For example, how is it known that LEHHHHHHMVR results from a +19 skip instead of from loss of fidelity like LEHHHHHHYQR? The presence of two additional His residues in the His-tail of the GFP-His6 reporter may indeed indicate a -6 frameshift, as the authors propose, but may also indicate two consecutive -3 frameshifting events. Authors do not discuss the second possibility and suggest that they have detected \"the furthest frameshift backward\". Without strong evidence that they are indeed dealing with a -6 frameshift, instead of two -3 frameshifts, this seems to be an overstatement. Some of the peptides could also result from transcription errors rather than translation mistakes. A more quantitative summary of the types of peptides found in the mass spec, including the types of peptides found in the strain expressing the UAA-containing construct, would be very useful.ssrA-DD  or wild-type ssrA? This is critical for the interpretation. All strains and plasmids need to be described at a level of detail that would allow others to reproduce the work.3) Were the experiments in Figure 3 and Figure 4 done with strains containing ratio between the expressed and non-expressed constructs. It is hard to know whether the effects shown are due to effects of the mutations on the expressed or non-expressed construct. To resolve this issue, the authors should show the effects of the different mutations on the actual max OD600 and doubling time values of each strain separately (i.e. not just on the ratio).4) Figure 3A is completely not clear and perhaps even misleading since it shows the effect of the different mutations on the 600 ratio of the ssrA arfB double mutant \u2013 Figure 3A, the opposite trend in GFP values shown for the ssrA arfB compared to arfB and ssrA alone \u2013 Figure 3C etc.). There is no clear explanation to these peculiarities in the text. A more elaborate discussion in the context of epistatic interactions shown is needed, such a discussion could address the effects of double mutations compare to single ones. Likewise, the authors should try to comment on the observation that knockout of arfA increases GFP production , whereas ArfA is expected to facilitate termination of the GFP translation at the UAG codon.5) The results shown for some of the strains  are surprising and not expected  In Figure 4B, the authors interpreted the change in doubling time as an indication for the ability of RK2 plasmid to replicate \u2013 some explanation to this should be added to text.eLife. Your revised article has been favorably evaluated by Gisela Storz (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled \"Organisms with alternative genetic codes resolve unassigned codons via mistranslation and ribosomal rescue\" for further consideration at The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:1) For the experiments in Figure 3 and Figure 4, explain whether the replicates are biological replicates or technical replicates and at what step the replication occurred. From the source data, it appears that all of the growth data is from a single 96 well plate. Were replicate wells in the plate inoculated from independent overnight cultures or from the same culture? This distinction is important to determine if the small changes observed are likely to be physiologically relevant. It is confusing that there are 3 plates of data in the source file, but data in the figure all seems to come from a single plate. Likewise, for the conjugation experiments, do the data come from one mating that was plated three times, from three matings made with the same cultures, or from three matings performed with independently grown cultures?2) Figure 3C shows negative protein concentration, and the explanation is that the band was quantified, and the value was plugged into a formula from a standard curve, giving a negative result. Because it is physically impossible to have negative protein concentration and there is quite clearly a band on the blot, the explanation provided suggests that the standard curve was not accurate. From the source data, it appears that the standard curves are derived from two points at 1 ng and 100 ng fit to a line. Unfortunately, almost all of the samples are outside the 1-100 ng range. It appears the negative value is the result of a large negative number from blot 3 which also seems to have an anomalously low value for 100 ng in the standard curve. The differences in protein production from the different samples are clear, but the quantification is clearly inaccurate. This problem could potentially be addressed by running another replicate of the experiment or reporting the data normalized to one of the samples such as GRO.AA [pUAG-GFP]. We will not publish a negative concentration.ssrA tagging occur at unassigned codons\u201d: Spontaneous termination of translation could refer to untemplated termination by RF2 or to spontaneous (nonenzymatic) hydrolysis of the peptidyl-tRNA.Text clarifications:a) In subsection \u201cSuppression, ribosomal frameshifting, and ssrA and arfB mediate degradation of proteins containing unassigned UAG codons\u201d: Fitness is best used in relation to competitive growth experiments because it is possible for a strain to grow to a lower OD600 in monoculture but outcompete other strains and therefore have higher fitness. It would be clearer here to refer to growth rate or OD600 instead of fitness.b) In subsection \u201cssrA and arfB mediate degradation of proteins containing unassigned UAG codons\u201d: What is the knockout of arfB being compared to here? In Figure 3A, the induced/uninduced ratio for the arfB strain looks very similar to that for the isogenic +arfA strain, which would seem to suggest that ArfB does not play a role.c) In subsection \u201cssrA and arfB mediate degradation of proteins containing unassigned UAG codons\u201d is unclear: \"Interestingly, a single knockout of arfB significantly reduced production of protein from UAG-GFP to low levels similar to those mapped to quantified GFP standards.\"d) The sentence in the last paragraph of subsection \u201cssrA and arfB mediate degradation of proteins containing unassigned UAG codons\u201d, the comparison for \"fully restore protein expression from UAG-ending transcripts\" appears to be GRO.AA[pUAA-GFP] instead of ECNR2[pUAG-GFP], but \"restore\" suggests it should be the latter. The comparison and the meaning behind which strain is used for the comparison should be clarified.e) In subsection \u201cssrA restores conjugative plasmid propagation and viral infection in the GRO\u201d, it is not clear what comparison was used for the 2.4-fold increase in doubling time. The graph in Figure 4B shows a 38% increase for the arfA strain versus 28% for the isogenic wild type.f) In subsection \u201cDeletion of g) Discussion section: What is the evidence supporting the demonstration of ribosome stalling? It seems that ribosome stalling was assumed based on the addition of the SsrA tag. Experiments such as ribosome profiling could demonstrate ribosome stalling, but these were not done. I think stalling is part of the model here but has not been demonstrated.h) Discussion section: Similar to (g) above, the regulatory relationship between tmRNA and ArfA was used to explain the data, so it would be circular reasoning to then use this explanation to validate the regulatory relationship.i) Discussion section: \"extensive\" suggests there is a large amount of frameshifting, but the frameshifting events cannot be quantified using the techniques in this work. Perhaps something like \"a wide variety of frameshifting events\" would be more accurate.j) Figure 1: The cartoon shows the SsrA-tagged protein going into the protease N terminus first, but all the proteases recognize the tag and start at the C terminus . In the legend, the word \"hypothesized\" should be removed from the last sentence \u2013 the lack of modification has been observed.k) Figure 3 legend: For panels A and B, the legend does not match the labels. A is doubling time and B is max OD. In this manuscript, Ma et al. investigate how bacteria contend with unassigned codons by expressing genes with a UAG stop codon in a genetically recoded E. coli strain lacking UAG codons and RF1. This question is important because there are a number of examples in nature of codon reassignment, but it is not clear how organisms would make the transition from a canonical code if there is a large penalty for having an unassigned codon. It has also been suggested that reassignment might limit transfer of foreign DNA. The authors used mass spectrometry to identify the proteins produced from a reporter gene with a UAG codon and find that multiple mechanisms help cells deal with the unassigned codon, including suppression, frameshifting, and general mistranslation. The authors also tested the effect of inactivation of three ribosome rescue systems  on the reporter expression and on propagation of conjugative plasmids or phages that have genes terminating in UAG. These data suggested that tmRNA accounts for the resistance of their E. coli mutant strain to horizontal gene transfer.This is an interesting study that expands our knowledge of how cells deal with difficult-to-translate codons. It is also useful for the future use of genetically recoded organisms.We thank the reviewers for recognizing the significance of our work and relevance to both the translation of rare codons found in nature and to the translation of codons engineered into genomically recoded organisms.Essential revisions:1) The manuscript consistently refers to unassigned codons, implying that the work addresses this general issue. For example, in the Introduction: \"Our work reveals mechanistic details into how cells rescue ribosomes stalled at unassigned codons\". However, the effects observed when ribosome rescue pathways are deleted are almost certainly unique to unassigned stop codons. Trans-translation activity on ribosomes stalled at unassigned stop codons will remove the protein, but when ssrA is deleted, ArfA will release a complete polypeptide identical to what would be produced by RF1. If a sense codon were unassigned, neither trans-translation nor ArfA activity could produce active proteins. The authors should either provide an explanation for why the ribosome rescue pathways would have the same impact on unassigned codons within a gene, or they should clarify that their interpretation is restricted to unassigned stop codons.ssrA is deleted, fully-functional protein is made that is detectable both by western blot for GFP and phenotypic rescue for conjugative plasmids and viruses, which simplifies study of the molecular mechanism and (2) our model remains the only organism to date engineered to have an unassigned codon. Given that recoding of sense codons is actively underway , such GROs with unassigned sense codons would offer opportunities to conduct a similar study on sense codons to directly answer such questions.It is true that our work is limited to unassigned stop codons and we agree that ribosomal rescue through trans-translation or ArfA at an unassigned sense codon would likely produce a truncated, nonfunctional protein and think this greatly supports our argument made in the Discussion section that further modifying the genetic code in engineered organisms could lead to even greater barriers to horizontal gene transfer. However, we also recognize prior literature that reports that these ribosomal rescue mechanisms would not be restricted to unassigned stop codons. As seen previously, these ribosomal rescue mechanisms are active at rare sense codons  and during periods of amino acid starvation , as we note in the Discussion section, suggesting they would also rescue stalled ribosomes at unassigned sense codons. In this manuscript, we empirically examine this in the context of an unassigned stop codon for two reasons: (1) when 2) For the experiment in Figure 2, the basis for some of the assignments are unclear. For example, how is it known that LEHHHHHHMVR results from a +19 skip instead of from loss of fidelity like LEHHHHHHYQR? The presence of two additional His residues in the His-tail of the GFP-His6 reporter may indeed indicate a -6 frameshift, as the authors propose, but may also indicate two consecutive -3 frameshifting events. Authors do not discuss the second possibility and suggest that they have detected \"the furthest frameshift backward\". Without strong evidence that they are indeed dealing with a -6 frameshift, instead of two -3 frameshifts, this seems to be an overstatement. Some of the peptides could also result from transcription errors rather than translation mistakes. A more quantitative summary of the types of peptides found in the mass spec, including the types of peptides found in the strain expressing the UAA-containing construct, would be very useful.ssrA tagging occur at unassigned codons\u201d is that LEHHHHHHMVR could be genetically encoded with a +19 skip, while the other peptides could not be encoded unless multiple mistranslation events occurred. With regard to LEHHHHHHHH resulting from a -6 frameshift or two -3 frameshifts, it would be impossible to distinguish between these two scenarios using mass spectrometry data. As we have not found anything in the literature to denote which scenario is more likely, we have modified both the Abstract and text in subsection \u201cSuppression, ribosomal frameshifting, and ssrA tagging occur at unassigned codons\u201d, to read: \u201cWe detected ribosomal frameshifting of up to -3 (LEHHHHHHH) and +19 nucleotides (LEHHHHHHMVR), as determined by presence of fragments from all three reading frames appended immediately following the C-terminal peptide of LEHHHHHH. Additionally, the LEHHHHHHHH peptide may indicate a -6 frameshift, although it is not possible to determine whether this peptide arises from a single -6 frameshift or two -3 frameshifts between histidine incorporation.\u201dIt would be difficult to determine using mass spectrometry whether LEHHHHHHMVR results from a + 19 skip or a loss of fidelity, but given that the skips of up to +50 bases have been validated in vivo  and loss of fidelity has only been observed in vitro prior to this manuscript, we hypothesize that this peptide arises from skipping. In addition, a key difference between LEHHHHHHMVR and the 5 tripeptides described in subsection \u201cSuppression, ribosomal frameshifting, and ssrA tagging occur at unassigned codons\u201d, which reads: \u201cit is unlikely these fragments arose from routine errors in mRNA transcription because this would require \u22652 transcriptional errors in a 30-nucleotide span. The transcription error rate in E. coli is estimated to be ~1 in 10,000 bases  and our strains have no known mutations that would lead to greater error rates in transcription.\u201dAlthough some of the tripeptides we identified could be the result of transcription mistakes, the number of transcriptional mistakes required to produce the degenerate tripeptides we identified would be between 2 and 6 transcriptional errors in a 30-nucleotide span. Given that the transcriptional error rate is 1 in 10,000 bases  and our strains have no mutations that would lead to greater error rates in transcription, we find this hypothesis to be less likely than loss of translational fidelity. We have revised the text to clarify this in subsection \u201cSuppression, ribosomal frameshifting, and ssrA tagging occur at unassigned codons\u201d, the only detectable GFP C-terminal peptide from UAA-ending constructs (and from strains containing RF1) were LEHHHHHH and LEHHHHHHAANDENYALDD. Mass spectrometry datasets that include ion intensity scores are available in Supplementary file 1 and Supplementary file 2. We agree that the quantification of the relative abundances of the various observed translational products would be very useful. Unfortunately, the amino acid sequence of each unique tryptic peptide confers different ionization properties, meaning that some peptides are much more amenable to observation by mass spectrometry than others. By the same token, the intensity of the peptides observed by mass spectrometry cannot be used to directly quantify difference in abundance between different peptides and mistranslated proteins. We therefore view our experiments as a \u201cscouting\u201d technique to identify the possible translational outcomes in response to the unassigned UAG codon, but further experimentation using a targeted collection of isotopically labeled peptides would be necessary for absolute peptide quantification, which is beyond the scope of the current work. However, even rigorously quantitative measures pose challenges because protein turnover rate is difficult to assess, and quickly-degraded proteins such as those with the tmRNA tag may be undervalued by simply comparing peptide abundances. A detailed description of mass spectrometry and proteomic analysis was added to the Materials and methods section of the manuscript.A summary of the C-terminal peptides identified from our mass spectrometry data is available on Supplementary file 1 and Supplementary file 2, and this has been modified to include the source strain for each peptide identified. As noted in the manuscript in subsection \u201cSuppression, ribosomal frameshifting, and 3) Were the experiments in Figure 3 and Figure 4 done with strains containing ssrA-DD  or wild-type ssrA? This is critical for the interpretation. All strains and plasmids need to be described at a level of detail that would allow others to reproduce the work.ssrA variants used in Figure 3 and Figure 4, and for pointing out the need to describe the strains and plasmids used in this study in greater detail. Strains used for the protein yield  and conjugation/infection  experiments contain wild-type ssrA (ssrA-AA). We added the following sentence to the manuscript to clarify this (subsection \u201cssrA and arfB mediate degradation of proteins containing unassigned UAG codons\u201d): \u201cSince mass spectrometry data indicated that a combination of mechanisms could resolve stalled translation at the unassigned UAG codon, we constructed targeted deletions of the ribosomal rescue systems  in strains with wild-type ssrA sequence (GRO.AA) to determine whether protein production from UAG-ending transcripts in \u0394RF1 cells could be restored to levels seen in +RF1 cells.\u201dWe thank the reviewers for their comment on the ssrA tag, as well as the figure attribution for each strain.We have also compiled a new table of strains (Table 1) and a list of plasmids (see Key Resources Table) used in this study. The strain table includes information on the status of the 4) Figure 3A is completely not clear and perhaps even misleading since it shows the effect of the different mutations on the ratio 600 and doubling time values of each strain separately (i.e. not just on the ratio).between the expressed and non-expressed constructs. It is hard to know whether the effects shown are due to effects of the mutations on the expressed or non-expressed construct. To resolve this issue, the authors should show the effects of the different mutations on the actual max OD600with and without GFP expression . The quantitative values that comprise these figures are also available on Supplementary file 3.We thank the reviewers for this comment and agree with their assessment. We have thus modified Figure 3 to consist of bar graphs demonstrating the doubling times with and without GFP expression  and max OD5) The results shown for some of the strains  are surprising and not expected . There is no clear explanation to these peculiarities in the text. A more elaborate discussion in the context of epistatic interactions shown is needed, such a discussion could address the effects of double mutations compare to single ones. Likewise, the authors should try to comment on the observation that knockout of arfA increases GFP production , whereas ArfA is expected to facilitate termination of the GFP translation at the UAG codon.600) but not both simultaneously. In ssrA knockout strains, GFP expression increase at the expense of cellular fitness because GFP peptides stalled on ribosomes are freed by ArfA or ArfB and not tagged for degradation. We have clarified this point in the manuscript in subsection \u201cssrA and arfB mediate degradation of proteins containing unassigned UAG codons\u201d: \u201cThese ssrA knockout strains likely demonstrate increased GFP expression and reduced fitness  because translation of GFP transcripts sequester cellular resources at the expense of cellular replication, producing GFP peptides that are freed from stalled ribosomes via ArfA or ArfB without addition of a degradation tag.\u201dWe thank the reviewers for raising these points and would hypothesize that, given limited cellular resources, cells are capable of expressing high levels of protein or achieving high fitness . These ArfB deletion data, together with the fitness reduction observed in the GRO, suggest that ArfB is constitutively expressed and relieving low levels of ribosomal stalling in E. coli.\u201dWe have also addressed the unusual results in the arfA knockout strain is not statistically significant when compared to the GRO strain expressing the same UAG-GFP construct, we do not think emphasizing such nuanced differences is warranted and would require detailed follow-up in future work.Since the increased GFP expression observed in the 6) In Figure 4B, the authors interpreted the change in doubling time as an indication for the ability of RK2 plasmid to replicate \u2013 some explanation to this should be added to text.trfA gene on RK2, which initiates plasmid replication, reduces cell growth and increases doubling time in the GRO unless its UAG stop codon is recoded to UAA when grown in media selecting for plasmid maintenance . To clarify this point, we changed the sentence in which we discuss these results  to the following: \u201cPrevious research indicates that the UAG stop codon in the trfA gene on RK2 leads to impaired conjugation efficiency and replication in the GRO , likely because the trfA protein is required to initiate plasmid replication. Phenotypically, this manifests as reduced efficiency of plasmid transfer in conjugation experiments and increased doubling times for RK2+ strains in media selecting for plasmid maintenance due to loss of plasmid and concomitant antibiotic resistance genes.\u201dWe thank the reviewers for identifying the need to clearly articulate our interpretation of doubling time data. The basis for interpreting changes in doubling time as an indication of RK2\u2019s ability to replicate stems from a prior study we conducted that demonstrated the UAG-ending The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance, as outlined below:1) For the experiments in Figure 3 and Figure 4, explain whether the replicates are biological replicates or technical replicates and at what step the replication occurred. From the source data, it appears that all of the growth data is from a single 96 well plate. Were replicate wells in the plate inoculated from independent overnight cultures or from the same culture? This distinction is important to determine if the small changes observed are likely to be physiologically relevant. It is confusing that there are 3 plates of data in the source file, but data in the figure all seems to come from a single plate. Likewise, for the conjugation experiments, do the data come from one mating that was plated three times, from three matings made with the same cultures, or from three matings performed with independently grown cultures?Thank you for pointing out the need to distinguish between biological and technical replicates in our experiments. We used the definitions for biological and technical replication outlined in Blainey et al., (2014): Biological replicates are parallel measurements of different biological samples subjected to the same experiment, and technical replicates are parallel measurements of a single biological sample subjected to experimentation. The data represented in all figures except for Figure 4A and 4C are the result of biological replicates. Figure 4A and 4C represent technical replicates (see below for more details).600\u2019s of indicated strains. File contains doubling times and maximum OD600\u2019s for three separate experiments conducted on different plate reader machines. Each experiment tested each sample in biological triplicate. Only the biological triplicate data from Plate 3 is represented in Figure 3A and 3B.\u201dThe source data file for Figure 3A and 3B  contains data from three identical 96-well plate experiments conducted using different 96-well plate reader models. Each experiment tested each sample in biological triplicate. Although data from the first two experiments are consistent with data from the third experiment, variability among plate reader machines precludes us from averaging the data obtained in the different experiments to draw conclusions of biological relevance. As such, we only represent the data obtained from \u201cPlate 3\u201d in Figure 3A and 3B. We modified the legend for Figure 3\u2014source data 2 to clarify this: \u201cAnalysis of doubling times and maximum ODTo clarify that the data obtained from our conjugation experiments  are the result of technical replication, we have added the following sentence to the legend for Figure 4A and 4C: \u201cData are obtained from technical triplicates generated from a single biological sample.\u201d We also note that these data are the consequence of technical replication in the legends for the appropriate source data files .We have written a new paragraph within our Materials and methods section to outline our definitions of biological and technical replication, and have clarified the nature of replication for each experiment within relevant paragraphs of the Materials and methods section. We updated our Transparent Reporting Form to include the above-mentioned definitions of biological and technical replication.2) Figure 3C shows negative protein concentration, and the explanation is that the band was quantified, and the value was plugged into a formula from a standard curve, giving a negative result. Because it is physically impossible to have negative protein concentration and there is quite clearly a band on the blot, the explanation provided suggests that the standard curve was not accurate. From the source data, it appears that the standard curves are derived from two points at 1 ng and 100 ng fit to a line. Unfortunately, almost all of the samples are outside the 1-100 ng range. It appears the negative value is the result of a large negative number from blot 3 which also seems to have an anomalously low value for 100 ng in the standard curve. The differences in protein production from the different samples are clear, but the quantification is clearly inaccurate. This problem could potentially be addressed by running another replicate of the experiment or reporting the data normalized to one of the samples such as GRO.AA [pUAG-GFP]. We will not publish a negative concentration.We agree with the reviewers that reporting a negative GFP value does not make sense, and we have amended our data analysis to provide better quantitation of the amount of GFP present in each sample. Images of the blots were provided in our initial submission as Figure 3\u2014source data 3-5. The raw values for our reanalysis are provided in an updated version of Figure 3\u2014source data 6..\u2206arfB samples and the 1ng standard, suggested that the antibody signal is sublinear in the 0-10ng regime. To address this problem, we revised the data analysis as follows: we created new standard curves using only the 10ng, 50ng and 100ng standards for each replicate western blot , and fit data points with an intensity greater than that of the 10 ng standard to this curve. 20 of 24 samples fell within or slightly above this range, with the highest-intensity sample quantified as 136ng. We created a second set of standard curves using the 1ng and 10ng standards to quantify the protein abundances of three samples whose intensities fell within this range. The one remaining sample had a weaker intensity than that of the 1ng standard, and we quantified protein in this sample through a linear approximation between the 1ng sample intensity and a blank lane with an intensity of zero.For the western blots represented in Figure 3C, 1ng, 10ng, 50ng and 100ng GFP-6xHis standards were run on each blot next to the experimental samples. The majority of the samples fell within this range of GFP standards because the y-axis of Figure 3C, in \u00b5g/mL GFP, corresponds to a 6x correction factor based on the loading volume of lysate in each lane of the SDS-PAGE gels, to convert the ng of protein detected to \u00b5g/mL in the total lysate. Our previous data analysis, which yielded negative values for the GRO.AAThe results of this analysis are not substantively different than our previous analysis, but this method is clearly superior in calculating the GFP concentration from low-intensity samples and allows us to accurately report non-negative protein abundances. The standard curves from our reanalysis are provided in Figure 3\u2014figure supplement 1.We have revised the subsection \u201cWestern blot\u201d to reflect these changes and to describe our analysis procedure in greater detail. The subsection now reads: \u201cWestern blots were run as described previously using SDS-PAGE gels . [\u2026] We repeated three western blots in parallel for each strain induced in separate culture tubes .\u201dText clarifications:a) In subsection \u201cSuppression, ribosomal frameshifting, and ssrA tagging occur at unassigned codons\u201d: Spontaneous termination of translation could refer to untemplated termination by RF2 or to spontaneous (nonenzymatic) hydrolysis of the peptidyl-tRNA.ssrA tagging occur at unassigned codons\u201d. However, we recognize that our methods do not distinguish between these possible events and have thus modified subsection \u201cSuppression, ribosomal frameshifting, and tmRNA-mediated peptide ssrA tagging occur at unassigned codons\u201d to read: \u201cGiven this, we hypothesize that these five peptides result from loss of translational fidelity after stalling at the UAG codon that may lead to (1) spontaneous termination of translation due to the untemplated action of RF2 following mistranslation or (2) ArfA- or ArfB-mediated release predicated on 3\u2019 exonuclease degradation of the mRNA. The rare event of spontaneous hydrolysis of the peptide from the ribosome is also possible.\u201dWe thank the reviewers for pointing this out. The nonenzymatic hydrolysis of peptidyl-tRNA is posited to be a rare event , and we refer to this possibility in subsection \u201cSuppression, ribosomal frameshifting, and tmRNA-mediated peptide b) In subsection \u201cssrA and arfB mediate degradation of proteins containing unassigned UAG codons\u201d: Fitness is best used in relation to competitive growth experiments because it is possible for a strain to grow to a lower OD600 in monoculture but outcompete other strains and therefore have higher fitness. It would be clearer here to refer to growth rate or OD600 instead of fitness.ssrA tagging occur at unassigned codons\u201d to \u201cgrowth rate and cell density.\u201dWe thank the reviewers for making this distinction. We have replaced the term \u201cfitness\u201d in subsection \u201cSuppression, ribosomal frameshifting, and tmRNA-mediated peptide + strain, which would seem to suggest that ArfB does not play a role.c) In subsection \u201cssrA and arfB mediate degradation of proteins containing unassigned UAG codons\u201d: What is the knockout of arfB being compared to here? In Figure 3A, the induced/uninduced ratio for the arfB strain looks very similar to that for the isogenic arfAssrA knockout and a strain with both an ssrA and arfB knockout. We intend to point out that the knockout of both ssrA and arfB is needed to recover GFP production to levels seen in GRO cells that produce GFP from UAA-ending transcripts. We revised this sentence (and the sentence before it) to clarify the nature of our comparisons, and to make explicit the nature of the p-values we are reporting . The paragraph beginning in subsection \u201cssrA and arfB mediate degradation of proteins containing unassigned UAG codons\u201d now reads:In the sentence that the reviewers mention, we are comparing GFP production from UAG-ending transcripts between a strain with ssrA deletion strains likely demonstrate increased GFP expression and reduced growth rate and cell density  because translation of GFP transcripts sequesters cellular resources at the expense of cellular replication, producing GFP peptides that are freed from nonstop ribosomes via ArfA or ArfB without addition of a degradation tag.\"\u201cWe then investigated the impact of unassigned codons on protein production using western blot densitometry, and found that the GRO expressing UAG-GFP produced less than one-fourth of the protein amount than does ECNR2 expressing UAG-GFP . [\u2026] These arfB does not seem to affect the induced/uninduced doubling time ratio. Indeed, ArfB does not seem to play a substantial role in influencing the growth rate of the GRO when it is expressing protein from UAG-ending transcripts. However, our data on protein expression represented in Figure 3C suggests that ArfB has a significant role to play in protein production. We emphasize this point on in subsection \u201cSuppression, ribosomal frameshifting, and tmRNA-mediated peptide ssrA tagging occur at unassigned codons \u201c.The reviewers are also correct to point out that, in Figure 3A, knockout of d) The sentence in the last paragraph of subsection \u201cssrA and arfB mediate degradation of proteins containing unassigned UAG codons\u201d is unclear: \"Interestingly, a single knockout of arfB significantly reduced production of protein from UAG-GFP to low levels similar to those mapped to quantified GFP standards.\"arfB GRO strain leads to very low protein production levels , and that the resulting protein abundance from this strain is near the lower limit of detection of our assay. We have revised subsection \u201cSuppression, ribosomal frameshifting, and tmRNA-mediated peptide ssrA tagging occur at unassigned codons\u201d to read: \u201cA deletion of arfB leads to strikingly low protein abundances from UAG-GFP transcripts that approach the lower limit of detection of our assay, although this apparent reduction in protein production was not statistically significant in comparison to protein production by GRO.AA [pUAG-GFP].\u201dWe thank the reviewers for pointing this out. Our intention is to demonstrate that UAG-GFP expression in the \u2206e) In subsection \u201cssrA and arfB mediate degradation of proteins containing unassigned UAG codons\u201d, the comparison for \"fully restore protein expression from UAG-ending transcripts\" appears to be GRO.AA[pUAA-GFP] instead of ECNR2[pUAG-GFP], but \"restore\" suggests it should be the latter. The comparison and the meaning behind which strain is used for the comparison should be clarified.ssrA.\u2206arfB [pUAG-GFP] with GRO.AA [pUAA-GFP]. Because of the genetic differences between ECNR2 strains and GRO strains (Table 1), it would be inaccurate to compare protein production from GRO.AA.\u2206ssrA.\u2206arfB [pUAG-GFP] with that of ECNR2 [pUAG-GFP]. Our intention in making this comparison is to show that the knockout of both ssrA and arfB is needed to enable the GRO to translate peptides from UAG-ending transcripts at abundances similar to the translation of peptides from UAA-ending transcripts. We have revised subsection \u201cssrA and arfB mediate degradation of proteins containing unassigned UAG codons\u201d to clarify the nature of our comparison, and it now reads: \u201cThese data also suggest that while deletion of ssrA partially recovers protein production from UAG-ending transcripts in the GRO, deletion of both ssrA and arfB is necessary to fully recover protein expression from UAG-ending transcripts to levels seen from the translation of UAA-ending transcripts in the GRO.\u201dThe reviewer is correct, and we are indeed comparing protein production from GRO.AA.\u2206f) In subsection \u201cDeletion of ssrA restores conjugative plasmid propagation and viral infection in the GRO\u201d, it is not clear what comparison was used for the 2.4-fold increase in doubling time. The graph in Figure 4B shows a 38% increase for the arfA strain versus 28% for the isogenic wild type.ssrA restores conjugative plasmid propagation and viral infection in the GRO\u201d now reads: \u201cRK2 conjugation efficiency in GRO.AA.\u2206ssrA improved to 99% (compared to 87% in GRO.AA), and the strain showed an increase in doubling time of only 6% compared to a 28% increase for GRO.AA (p < 0.0001). We observed similar results for GRO.AA.\u2206ssrA.\u2206arfB. However, single deletion of arfB halved RK2 conjugative efficiency . This strain also exhibited a 38% increase in doubling time when bearing RK2, compared to the 28% increase in doubling time seen in the GRO with no ribosomal rescue gene deletions .\u201dWe thank the reviewers for pointing this out. We revised the sentence in question to accurately reflect the data shown in the figure. Subsection \u201cDeletion of g) Discussion section: What is the evidence supporting the demonstration of ribosome stalling? It seems that ribosome stalling was assumed based on the addition of the SsrA tag. Experiments such as ribosome profiling could demonstrate ribosome stalling, but these were not done. I think stalling is part of the model here but has not been demonstrated.We appreciate the reviewer\u2019s question and believe that clarifying the terminology used in the manuscript will resolve the issue. The term \u201cribosomal stalling\u201d refers to the situation in which a ribosome reaches the 3\u2019 end of an mRNA without encountering a stop codon, or when translation slows or pauses in the middle of an mRNA . Stalling at the 3\u2019 end of an mRNA results in ribosomal rescue . Slowed translation within an mRNA can arise when the ribosome encounters a rare codon or a codon with depleted or inefficient cognate decoding elements, and can result in near-cognate suppression, frameshifting, or mRNA cleavage followed by ribosomal rescue .i.e., ribosomal rescue, frameshifting, and near-cognate suppression) consistent with resolution mechanisms for stalled ribosomes.The reviewer is correct to point out that we do not present data to support the claim that unassigned codons result in ribosomal stalling as we have defined the term above. However, our mass spectrometry data does reveal that unassigned codons elicit translational responses .\u201d We conclude this paragraph with the additional sentence: \u201cThese mechanistic outcomes that occur as a consequence of ribosomal stalling could be further investigated via ribosomal profiling in future work.\u201dh) Discussion section: Similar to (g) above, the regulatory relationship between tmRNA and ArfA was used to explain the data, so it would be circular reasoning to then use this explanation to validate the regulatory relationship.We thank the reviewers for pointing this out, and we agree that our data does not explicitly validate previously observed regulatory relationships between tmRNA and ArfA . Our data does, however, reveal the physiological relevance of this relationship. In order to make this point more explicit, we have revised the sentence in the Discussion section to read: \u201cOur GRO model thus sheds light on the functional significance of previously-described regulatory relationships while elucidating the unique mechanistic contributions of different ribosomal rescue systems in resolving translation at unassigned stop codons.\u201di) Discussion section: \"extensive\" suggests there is a large amount of frameshifting, but the frameshifting events cannot be quantified using the techniques in this work. Perhaps something like \"a wide variety of frameshifting events\" would be more accurate.We agree with the reviewer on this point. We have changed \u201cextensive frameshifting\u201d to \u201ca wide variety of frameshifting events.\u201dj) Figure 1: The cartoon shows the SsrA-tagged protein going into the protease N terminus first, but all the proteases recognize the tag and start at the C terminus . In the legend, the word \"hypothesized\" should be removed from the last sentence \u2013 the lack of modification has been observed.We thank the reviewers for this observation. We have modified the Figure to show the protease hydrolyzing the C-terminal end of the peptide and have removed the word \u201chypothesized\u201d from the figure legend.k) Figure 3 legend: For panels A and B, the legend does not match the labels. A is doubling time and B is max OD.We thank the reviewers for pointing this out. We have altered the figure legend accordingly."}
+{"text": "These results demonstrate that hT-SerCs can functionally model elements of transport across the human BTB, potentially leading to identification of other transport pathways for xenobiotics, and will guide drug discovery efforts in developing effective BTB-permeable compounds.The blood-testis barrier (BTB) formed by adjacent Sertoli cells (SCs) limits the entry of many chemicals into seminiferous tubules. Differences in rodent and human substrate-transporter selectivity or kinetics can misrepresent conclusions drawn using rodent in vitro models. Therefore, human in vitro models are preferable when studying transporter dynamics at the BTB. This study describes a hTERT-immortalized human SC line (hT-SerC) with significantly increased replication capacity and minor phenotypic alterations compared to primary human SCs. Notably, hT-SerCs retained similar morphology and minimal changes to mRNA expression of several common SC genes, including AR and FSHR. The mRNA expression of most xenobiotic transporters was within the 2-fold difference threshold in RT-qPCR analysis with some exceptions . Functional analysis of the equilibrative nucleoside transporters (ENTs) revealed that primary human SCs and hT-SerCs predominantly express ENT1 with minimal ENT2 expression at the plasma membrane. ENT1-mediated uptake of [ Polarized Sertoli cells (SCs) are the fundamental epithelial cells that line the seminiferous tubules of the testes and are essential for supporting and protecting developing germ cells. These cells are commonly referred to as the \u201cnurse\u201d cells of the testes because they are responsible for regulating the growth and development of germ cells by providing nutrients or phagocytosing apoptotic cells ,2. SCs eAdjacent SCs express junctional proteins that congregate and form the physical blood-testis barrier (BTB), including the tight junction proteins claudin-11 (CLDN11), occludin (OCLN), and tight junction protein-1/zonula occludens-1 (TJP1/ZO-1) ,7. The B14C] TEA or [3H] carnitine into primary rat SCs. The uptake of [14C] TEA was attributed to two transporters (OCT1 and OCTN2), which were referred to as high-affinity transporters in rat SCs [14C] TEA revealed a low-affinity component attributed to OCT3 and OCTN1 [3H] carnitine uptake in primary rat SCs [3H] uridine transport with the selective ENT1-inhibitor, NBMPR, across polarized monolayers of rat SCs plated on Transwell inserts  Uridine (specific activity: 35.8 or 35.2 Ci/mmol) was purchased from Perkin-Elmer . Uridine was purchased from Sigma-Aldrich . The 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (NBMPR) was purchased from Tocris . Oligonucleotide primers were designed in house and synthesized by Integrated DNA Technologies . Additional reagents were purchased from ThermoFisher Scientific  unless otherwise noted. uridine (~25 nM) and 5 mM unlabeled uridine, 100 nM NBMPR, or 100 \u03bcM NBMPR. Cells were plated into standard 24-well TC plates or Nunc MicroWell 96-well optical bottom plates  and grown to confluence before each experiment. Cells cultured in 24-well plates were used to assess total [3H] uridine uptake after 10 min and cells cultured in 96-well plates were used to assess [3H] uridine uptake every minute for 10 min.Transport experiments were performed as previously described with minor modifications ,39,40. A3H] uridine (~25 nM) and 5 mM unlabeled uridine, 100 nM NBMPR, or 100 \u03bcM NBMPR. Transport was terminated after 10 min by washing the cells three times with WB and the cells were lysed with a lysis solution  for 20 min. The NaOH was neutralized using 1 N HCl and the extract was transferred to a 96-well plate. Approximately 200 \u03bcL of MicroScint-20 scintillation cocktail  was added to each well with extract before sealing the plate with microplate film and incubating at room temperature for at least 2 hrs before measuring total accumulated radioactivity measured using a Wallac 1450 MicroBeta TriLux liquid scintillation counter .Confluent cells plated in 24-well plates were washed once with room temperature WB before incubating with 300 \u03bcL WB transport buffer supplemented with 1 \u03bcCi/mL [3H] uridine (~25 nM) and 5 mM unlabeled uridine, 100 nM NBMPR, or 100 \u03bcM NBMPR every minute from 0 to 10 min. Transport was terminated after the first column of wells had been incubated for 10 min by washing the cells twice with WB using a Biotek 405 LS Microplate Washer . After washing, 200 \u03bcL of MicroScint-20 scintillation cocktail was added to each well and the steps described above were followed to quantify uptake.Confluent cells plated in 96-well plates were washed once with room temperature WB before incubating one column of wells with 50 \u03bcL WB transport buffer supplemented with 1 \u03bcCi/mL  uridine (~25 nM) and serially diluted NBMPR (0\u201310 nM) to the cells. Transport was terminated after 10 min by washing the cells twice with WB using the Biotek plate washer. After washing, total accumulated [3H] uridine was measured as described above. Non-transport mediated uptake of [3H] uridine was subtracted from each data point to separate transport-mediated uptake for data analysis. Data were normalized as a percentage of the uninhibited [3H] uridine uptake control.To determine the IC2 averages for each cell and the fold change was calculated relative to the primary human SCs. The microarray data were deposited in the NIH NCBI GEO database with the series accession number: GSE158400.Total RNA was isolated from primary human SCs at passages 4\u20136 and post-passage 20 hT-SerCs using a RNeasy Mini Kit  according to the manufacturer\u2019s protocol. RNA quality and concentration were measured using a Spectrophotometer NanoDrop ND-1000 . Approximately 100 ng of total RNA in 10 \u03bcL for both cells was submitted to the University of Arizona Cancer Center Genomics Shared Service Core to perform whole genome RNA expression analysis on an Affymetrix GeneChip human Clariom D microarray . Gene expression signals were collected as log50 values and statistics were completed using GraphPad Prism 8 . The IC50 values for NBMPR inhibition of [3H] uridine uptake were reported with 95% confidence intervals. Data are reported as the mean \u00b1 S.D. unless otherwise noted. Statistical analysis was not performed for the RT-qPCR data since fold-change values are not normally distributed.Each experiment was completed with cells cultured from at least three separate passages with multiple replicates. Western blots are representative of at least three independent experiments. Generation of ICPrimary human SCs obtained from MandalMed were previously isolated from a 36-year-old donor and characterized by their morphology, gene expression profiles, and function ,29. The TERT expression in hT-SerCs indicated a fold change in expression relative to primary human SCs of over 25,800 , Fas ligand (FASLG), inhibin subunit beta b (INHBB), sex hormone-binding globulin (SHBG), transferrin (TF), and Wilms\u2019 tumor protein (WT1) revealed minor changes to most SC markers in post-passage 20 hT-SerCs relative to primary human SCs at passages 4\u20136. A 2-fold difference threshold in expression was chosen to identify more robust changes in RT-qPCR gene expression as indicated by the dashed lines  was evaluated. Western blot analysis indicated a negligible difference in FSHR expression between both cells B, which BCRP expression by microarray analysis was close to the threshold but was not considered a robust change  and the SLC22 families, which have varying organ-specific expression levels. Therefore, the mRNA expression of several members of these two families was evaluated. hreshold B; howevend OCTN2 . In RT-qanalysis C. The mRcroarray .CNT1 and CNT2 mRNA was detectable in both cells; however, the expression change relative to primary human SCs was inconsequential ; however, unlabeled uridine and NBMPR continued to block uptake to the same extent. These data suggest that uridine uptake into both cells is primarily mediated by ENT1 with minimal contribution by ENT2.The expression of the ENTs in SCs has been well described and provides a route for endogenous nucleosides and nucleoside analog drugs to cross the BTB ,14. To vof NBMPR A. The tofmol/cm2 A Interes3H] uridine was measured over a period of 10 min with data points collected at each minute. The uptake of [3H] uridine was linear during this time and was also reduced by 85\u201390% in the presence of 5 mM unlabeled uridine and either 100 nM or 100 \u03bcM NBMPR   is the [3H] uridine concentration, and [S] is the NBMPR concentration. Results were normalized to percent of the control. The apparent IC50 value of NBMPR on ENT1-mediated [3H] uridine uptake is 1.35 \u00b1 0.37 nM . The R2 for the goodness of fit of Equation (1) was 0.86.A dose\u2013response curve generated by using serially diluted concentrations of NBMPR from 0 to 10 \u03bcM inhibited  uridine accumulation is measured based on transport across the plasma membrane. These differences in plasma membrane expression may be attributed to species differences, inter-individual variability, or tissue-specific expression of each transporter. More importantly, the uptake of [3H] uridine into primary human SCs and hT-SerCs was linear over 10 min. Although the maximum capacity for [3H] uridine uptake was 4- to 5-fold lower compared to HeLa S3 cells after 7 min, the linear uptake and response to NBMPR inhibition was consistent with previous studies  uridine into these cells remains unchanged even after hundreds of population doublings. These data suggest that hT-SerCs may be a robust and suitable model for studying transport of selected xenobiotics at the BTB. As a result, these cells may be a valuable tool for identifying the transport mechanisms of known testicular toxicants, antivirals, cancer chemotherapeutics, chemical contraceptives, and infertility treatments across the BTB. The identification of these pathways will be essential for the development of novel therapeutics that require access to the MGT.In conclusion, hT-SerCs have a significantly enhanced lifespan while retaining many phenotypic characteristics of the original primary human SCs from which they were derived. The phenotypic characteristics of these cells also appears to be stable, with no apparent morphological or growth changes being detected after 40 passages. Consequently, hT-SerCs may be suitable for other applications that involve SC biology, but further studies are necessary to explore their potential. The xenobiotic transporter expression in hT-SerCs appears to have minimal changes at the transcriptional level, with some exceptions. Importantly, the overall transport of ["}
+{"text": "Familial hypercholesterolaemia (FH) is a common inherited disorder, causing lifelong elevated low-density lipoprotein cholesterol (LDL-C). Most individuals with FH remain undiagnosed, precluding opportunities to prevent premature heart disease and death. Some machine-learning approaches improve detection of FH in electronic health records, though clinical impact is under-explored. We assessed performance of an array of machine-learning approaches for enhancing detection of FH, and their clinical utility, within a large primary care population. A retrospective cohort study was done using routine primary care clinical records of 4,027,775 individuals from the United Kingdom with total cholesterol measured from 1 January 1999 to 25 June 2019. Predictive accuracy of five common machine-learning algorithms  were assessed for detecting FH. Predictive accuracy was assessed by area under the receiver operating curves (AUC) and expected vs observed calibration slope; with clinical utility assessed by expected case-review workload and likelihood ratios. There were 7928 incident diagnoses of FH. In addition to known clinical features of FH , machine-learning (ML) algorithms identified features such as raised triglycerides which reduced the likelihood of FH. Apart from logistic regression , all four other ML approaches had similarly high predictive accuracy (AUC\u2009>\u20090.89). Calibration slope ranged from 0.997 for gradient boosting machines to 1.857 for logistic regression. Among those screened, high probability cases requiring clinical review varied from 0.73% using ensemble learning to 10.16% using deep learning, but with positive predictive values of 15.5% and 2.8% respectively. Ensemble learning exhibited a dominant positive likelihood ratio (45.5) compared to all other ML models (7.0\u201314.4). Machine-learning models show similar high accuracy in detecting FH, offering opportunities to increase diagnosis. However, the clinical case-finding workload required for yield of cases will differ substantially between models. FH affects ~1 in 200\u2013500 of the general population4. However, most individuals with FH and affected family members remain undiagnosed worldwide5. In patients with heterozygous FH, lipid-lowering therapy such as the use of moderate- to high-intensity statins, or newer PCSK9 inhibitors markedly improves prognosis6 \u2013 reducing risk of coronary heart disease and all-cause mortality by at least 44%8. Patients who remain unidentified will be untreated or be sub-optimally treated with low-intensity statins and assumed to have commoner multifactorial causes for raised cholesterol.Familial hypercholesterolaemia (FH) is a common inherited genetic disorder causing high cholesterol levels from birth2, Dutch Lipid Clinic Network criteria (DLCN)9, Make Early Diagnosis to Prevent Early Deaths (MEDPED)10, or Japanese Atherosclerosis Society (JAS) criteria11 13 used an electronic version of the DLCN criteria, while the FAMCAT tool in the UK16 and FindFH model in the US17 have been recently developed from prediction modelling. Using standard logistic regression, area under receiver operating curves (AUC) for FAMCAT were from 0.86 in its development database 18, to 0.83 and 0.84 in two separate external validation databases15 (QRESEARCH and RCGP Surveillance Network). Developed as a data-driven machine-learning (ML) algorithm from US administrative health data, the recent FindFH model resulted in an AUC of 0.89 using a random forest models approach17.Hence, there has been a drive to develop bespoke algorithms derived from large electronic health records (EHRs) to detect FH. The SEARCH study in the US19, with the potential of improving prediction, identifying latent variables which are unlikely to be observed but might be inferred from other variables. ML, therefore, offers an alternative approach to standard prediction modelling20. The aims of this study were firstly, to evaluate the performance of a range of different ML algorithms to identify patients with FH within a large UK general primary care population; secondly, we sought to determine potential differences in the clinical utility of using different ML algorithms.ML algorithms have diverse applications including disease modellingThere was a total of 4,157,705 individuals in CPRD study population with either a total cholesterol or LDL-cholesterol record during the study period. 129,930 (3.1%) individuals were excluded from the analysis for either having outlying cholesterol measurements, data entry errors, having a death or transfer out date before study start date or a diagnosis of FH before the study start date. The complete study cohort for analysis was made up of 4,027,775 individuals, with 7928 (0.2%) having a documented diagnosis of FH  was randomly sampled to become the training cohort and the remaining 25% of the cohort  assigned as the validation cohort. Table To develop the FH models, 75% of the complete cohort . The discrimination accuracy based on AUC, c-statistics, is presented in Fig. To predict the risk/probability of having FH for each individual, the algorithms were applied to the validation cohort , we determined the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of each machine-learning algorithm shown in Table We determined positive and negative likelihood ratios (LR) for each machine-learning algorithm Fig. . The posWe have assessed the ability of five different machine-learning (ML) algorithms to detect cases of familial hypercholesterolaemia (FH) in over 4 million patients\u2019 routine primary health care records.17; and a 3\u20136% improvement on the UK FAMCAT tool using standard logistic regression18.We found four ML models  all had similarly high predictive accuracy, with AUC\u2009>\u20090.89. This is highly consistent with that found for the FindFH model, using a random forest algorithm in US administrative health dataWe found substantial differences for clinical utility between ML algorithms. Despite their similar overall accuracy (apart from logistic regression), our analysis highlights a trade-off that will be necessary between specificity and sensitivity of these models for binary risk stratification. Specificity and negative predictive values were consistently high across all methods, due to the low prevalence of FH in the general population. However, numbers identified as high probability of FH, sensitivity and positive predictive values varied between approaches.For instance, we found a deep learning model would identify ~10% of the population as probable FH, generating a very high case load for clinicians to screen, review and test. This would have the highest sensitivity  but the detection rate would be poor . Conversely, ensemble learning would identify only 0.73% of the population as probable FH requiring clinical review. Although this has lower sensitivity, it would be more efficient in having a higher detection rate .n\u2009=\u20092640) of individuals would have had a registered cholesterol measured21. Using a deep learning model would identify 264 probable FH needing clinical assessment and testing. This strategy would yield the maximum absolute number of FH cases identified but would also be the most resource intensive. Conversely, ensemble learning would minimise the number of probable FH to <20 patients for clinical assessment. As the ensemble learning algorithm has the greatest positive likelihood ratio whilst maintaining a significant negative likelihood ratio, this may arguably be the most viable ML model to implement for FH case-finding in primary care practice, given workload and resource implications. Following a model-based approach to case-finding for potential FH, the patient would require a detailed clinical assessment and confirmatory diagnosis by genetic testing to identify a pathogenic mutation. Hence, the extent of false-positive results would have significant resource implications.For example, in an average sized UK primary care practice of 8800 patients, an estimated 30%  which is similar to the initial triage of primary care electronic health records recommended by English NICE guideline recommendations through identification of elevated cholesterols alone to systematically identify those with possible FHThis research offers a number of strengths. Our study evaluates a range of different ML models for detection of FH in primary care and has done so using not only conventional AUC but also the sensitivity, specificity, positive predictive value and negative predictive values. We employed a large sample size of over four million patients, embracing 6% of the entire UK population, enhancing generalisability of the findings. In particular, this work has also assessed the clinical value of these ML algorithms by exploring diagnostic test accuracy metrics, seldom reported for prediction models using machine-learning.17 confirm that ML approaches are viable to use in EHR systems and can significantly enhance detection FH. This offers major opportunities to increase diagnosis of FH and to prevent premature heart disease and early deaths. Moreover, while replication of ML methods can be questioned24, the use of different datasets in the UK and US, with consistency between their analysis by different study teams is now available, supporting the generalisability of these ML approaches. In this regard, we have also made fully available, in GitHub, the codes for our models to assist with replication, validation and implementation.The current UK study and recent study in the USHowever, we acknowledge several study limitations, in common with other research using large health care databases. These include lack of formal adjudication of diagnoses, information bias and potential bias due to missing data. Missing data could potentially introduce bias in the effect estimates of the prediction models as well as a reducing power. However, we used imputation methods for variables which were sufficiently missing-at-random and a very large sample size to mitigate these effects. The specific coding of FH recorded in UK general practice records will include patients identified with phenotypic FH, who may or may not have been confirmed by genetic testing. A recognised issue in EHRs is that some patients FH could potentially be misclassified, have not yet been identified, or might not have had cholesterol assessed.Future research should validate and replicate our ML models in other large clinical datasets in other populations. Secondly, further evaluation of the feasibility and acceptability of machine-learning applications in clinical practice is needed. The computational capacity of health care systems continues to evolve; and electronic health records are increasingly moving to cloud-based servers with data centralisation. This presents exciting opportunities to exploit machine-learning as a realistic option to detect uncommon conditions of major health importance, such as familial hypercholesterolaemia.25 and is representative of the UK general population26. Over 5.2 million of these patient records are currently of active research-quality, making CPRD one of the most widely used real-world data sources for healthcare research. Information routinely collected from primary care practices, as part of the database, include demographics, lifestyle, diagnoses, prescriptions, diagnostic tests, referrals to specialists and secondary care and death status. Secondary care activity is incorporated in the primary care records through hospital discharge letters from hospital or referral notes from specialists.The study cohort was obtained from the Clinical Practice Research Datalink (CPRD). CPRD contains anonymised electronic medical records from 836 general practices with over 11 million research-usable patients22. Patients with a FH diagnosis prior to study entry date (1 January 1999) or with a prior diagnosis of other inherited lipid disorders were excluded.A record of cholesterol level is essential to establish a diagnosis of FH hence, all patients included in the study had at least a single record of total cholesterol measurement between the baseline date of 1 January 1999 or the earliest date the CPRD primary care practice started contributing data to the database after 1 January 1999 and the end date of 25 June 2019 or the latest date the CPRD primary care practice finished contributing data prior to 25 June 2019. Where follow-up was not completed by a patient, the end date was specified as the date of death, transfer out of practice, or final practice visit. Patients with a diagnosis of FH, had the date of the diagnosis specified as their end date. Patients aged 16 years and younger were excluded as the cholesterol level thresholds for the diagnosis and treatment of FH vary when compared to adultsClinical features incorporated into all machine-learning models are documented in Box 3. Where a patients had both LDL and total cholesterol levels recorded, the LDL-cholesterol was prioritised, given its importance in recommended diagnostic criteria. For patients with multiple cholesterol levels recorded, the highest cholesterol value at any point between 1 January 1999 and 25 June 2019 was used. Each patient\u2019s recorded triglyceride record at the time of each cholesterol measurement was extracted \u2013 raised levels are a negative predictor of FH23. We assessed for outliers (cholesterol and triglyceride levels \u22640\u2009mmol/L or >5 positive standard deviations [SD] from the mean) and data entry errors. A prior history of CHD <60 years may also lead to a higher likelihood of being diagnosed with FH27.Identifying patients with possible FH using Simon-Broome criteria is based on the following variables: total cholesterol, LDL-cholesterol, family history of MI, and family history of raised cholesterol22. while not specifically included in previous criteria/guidelines, family history of FH was also examined. Family history variables were dichotomised to either having a family history or not. In the event that family history was not evaluated, we assumed that there was no family history. Additional information was not available to further categorise family history to identify the relative affected and age at diagnosis for the condition28.Family histories of MI and of raised cholesterol were included as likely diagnostic variables27, patients with elevated cholesterol levels may be receiving lipid-lowering therapy. Consequently, the prescribing and potency of lipid-lowering therapy were included as variables of interest. Cholesterol level was considered treated when the most recent prescription of lipid-lowering therapy ended within 30-days or overlapped with the date of the cholesterol measurement. A 30-day washout period was used to account for any residual effects of the lipid-lowering drugs when the drug treatment had been stopped29. Statin potency was classified using the most recent recommendations for statin intensity in the clinical guidance of the UK National Institute of Health and Care Excellence (NICE), which is based on a previous meta-analysis30.Although current diagnostic criteria use untreated cholesterol levels to evaluate probability of FH22. The following important secondary conditions were, therefore, included in our assessment: liver disease , diabetes mellitus (type I and type II), hypothyroidism , kidney disease  and nephrotic syndrome.Secondary causes of hypercholesterolaemia are currently recommended as negative predictors of FH in clinical guidelinesBox 1 Baseline predictor variables included in predicting familial hypercholesterolaemiaSex Tendon xanthomata Family history of Familial Hypercholesterolaemia Family history of coronary heart disease, excluding myocardial infarction Family history of myocardial infarction Family history of raised cholesterol Family history of all coronary heart disease DNA test for apoB-100 Any diagnosis of hypertension ever Any diagnosis of nephrotic syndrome ever Any diagnosis of coronary heart disease ever Any diagnosis of cerebrovascular accident ever Any diagnosis of peripheral vascular disease ever Any diagnosis of kidney disease ever Any diagnosis of hypothyroidism ever Any diagnosis of diabetes ever Any diagnosis of liver disease ever Most recent smoking status Most recent alcohol status Most recent alcohol consumption (units/week)Highest potency statin ever prescribed Highest total cholesterol level ever recorded (mmol/L)Age at time of highest total cholesterol record (years)Whether high total cholesterol was treated Treatment for high total cholesterol Triglyceride level at the time of highest total cholesterol record (mmol/L)Diastolic blood pressure closest to time of highest total cholesterol record (mmHg)Systolic blood pressure closest to time of highest total cholesterol record (mmHg)Hypertension control at the time of highest total cholesterol record Hypothyroidism control at the time of highest total cholesterol record Diabetes control at the time of highest total cholesterol record Liver damage at the time of highest total cholesterol record Kidney disease at the time of highest total cholesterol record Highest LDL-cholesterol level ever recorded (mmol/L)Age at time of LDL-cholesterol measurement (years)Whether high LDL-cholesterol was treated Treatment for high LDL-cholesterol Triglyceride level at the time of highest LDL-cholesterol record (mmol/L)Diastolic blood pressure closest to time of highest LDL-cholesterol recordSystolic blood pressure closest to time of highest LDL-cholesterol recordHypertension control at the time of highest LDL-cholesterol record Hypothyroidism control at the time of highest LDL-cholesterol record Diabetes control at the time of highest LDL-cholesterol record Liver damage at the time of highest LDL-cholesterol record Kidney disease at the time of highest LDL-cholesterol record The primary outcome was a documented incident diagnosis of FH in the patient records during the specified study period. FH is explicitly coded using the internationally recognised Read coding system in UK primary electronic health records (EHRs). This diagnostic code is entered into primary care electronic records after lipid specialist assessment, based on clinical phenotype, and/or by genetic test. To ensure temporality between predictors and the outcome, the diagnosis of FH must have occurred after the predictor variables.31, random forest32, gradient boosting machines33, deep-learning neural networks34 and ensemble learning35. Ensemble learning model was a combination of the four (4) other ML algorithms. Using the library package h2o (http://www.h2o.ai) in R Studio, the risk algorithms were developed in the training cohort and applied to the validation cohort. A grid search was used to determine the hyper parameters for each model and 10-fold cross-validations was done to determine the values for the best performance using the training cohort  in which the FH algorithms were derived and a \u2018validation\u2019 cohort (remaining 25% of the cohort) in which the algorithms were applied and tested. The data split was computer-generated using a uniform distribution to generate random numbers in STATA. The five commonly used algorithms were used \u2013 logistic regression36. The imputed datasets were pooled into a single dataset using Rubin\u2019s rule37.Descriptive characteristics for the study population are reported as numbers with percentages or mean with standard deviation (SD) for categorical and continuous variables, respectively. The level of missing values ranged between 2.4% for systolic blood pressure to 23.3% for body mass index (BMI) 4 to reflect the expected prevalence of FH in the general population. Stata 16 MP4 version was used for statistical analyses to assess model performance.Harrell\u2019s c-statistic, a measure of the total area under the receiver operating characteristic curve (AUC), was calculated using the validation cohorts to determine the predictive accuracy of the models developed in the training cohort. A jack-knife procedure was used to estimate the standard errors and 95% confidence intervals for the c-statistic estimatesEthical approval for this study was obtained from the Independent Scientific Advisory Committee (ISAC) \u2013 study protocol number 19_083R. De-identified (anonymised) patient data was obtained from CPRD hence this study was exempt from obtaining informed consent from patients.Further information on research design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationReporting Summary"}
+{"text": "This paper is an attempt to study the nonlinear vibration of a pre-stressed single-walled carbon nanotube (SWCNT) with water-filled and simply supported ends. A new analytical formula is obtained for the nonlinear model based on the simplified Donnell\u2019s shell theory. The effects of internal fluid on the coupling vibration of the SWCNT\u2013water system are discussed in detail. Furthermore, the influence of the different nanotube thicknesses and radiuses on the nonlinear vibration frequencies is investigated according to the shell theory. Numerical calculations are done to show the effectiveness of the proposed schemes. The results show that the nonlinear frequency grew with the increasing nonlinear parameters (radius and thickness of nanotube). In addition, it is shown that the influence of the nonlinear parameters is greater at the lower mode in comparison with the higher mode for the same nanotube thickness and radius. Carbon nanotubes have become one of the most important nanomaterials for nanotechnology; they have distinguished mechanical and electrical properties, and have had notable applications in nanodevices in recent years ,4,5,6,7.In this work, the nonlinear vibration of an initially stressed water-filled single-walled carbon nanotube is investigated using shell theory. Furthermore, the influence of the different nanotube thicknesses and radiuses on the nonlinear vibration frequencies is investigated according to Donnell\u2019s shell theory. Numerical calculations are done and shown graphically.u, v and w are the axial, circumferential and radial displacements, respectively.Assuming small strains and displacements, and considering the thin shell theory, h is the tube thickness, The nonlinear shallow-shell equations of motion based on Donnell\u2019s theory are given by Amabili :(1)D\u22074wtn and its first derivative with respect to the argument and For the water shell, n is the An approximate solution will be used to solve Equations (1) and (2). First, we choose a vibration mode for olutions :(10)wt=\u2211The particular solution of the function To solve Equation (1), we will substitute Equations (10)\u2013(12) into Equation (1). Galerkin\u2019s procedure provides a very powerful approximation method by employing any set of basic functions Galerkin\u2019s weighting function is obtained from the first derivative of Equation (10) with respect to time.After evaluating the integral in Equation (14), the ordinary differential system with unknown functions The non-linear ordinary differential Equations (16)\u2013(19) can be solved approximately by using the method of averaging . This meBy applying the assumptions that steady state vibrations and ThenBy substituting (26\u201328) into Equations (16\u201319), we getEquations (29) and (30) were used to evaluate the first and second modes of the nonlinear frequency of a SWCNT, which has been modeled by Donnell\u2019s nonlinear model. For simplicity, it is assumed that SWCNTs are geometrically and physically identical and the numerical calculation has been done for Equations (29) and (30) using the geometries of SWCNTs  that werIn this section, the effects of the nonlinear parameter h). The results show that the effects of the nanotube thicknesses are notable at low vibration frequency.R). The figure shows that the nonlinear frequency grew with the increasing nanotube radius (R). From this figure, it is also clear that the small change in the nanotube radius corresponds to a notable change in the vibration frequency.h). The results show that the nonlinear frequency grew slowly with the change in the nanotube thickness compared with the case of the first mode of vibration.R). From this figure, it is clear that the small change in the nanotube radius corresponds to a small increase in the vibration frequency compared with the same case of the first mode of vibrations.From In this paper, the nonlinear vibration of pre-stressed fluid-filled single-walled carbon nanotubes with simply supported ends is investigated based on von Karman\u2019s geometric nonlinearity and Donnell\u2019s simplified shell model and the effects of the different nanotube thicknesses and radiuses on the nonlinear vibration frequencies have been discussed in detail. Galerkin\u2019s procedure was used to discretize the governing partial differential equations into ordinary differential equations of motion. A nonlinear analytical formula was obtained for the model and the effects of internal fluid on the vibration of single-walled carbon nanotubes with the different nonlinear parameters have been discussed. As a case study, the mechanical and dimensional properties of the SWCNT were obtained from Gupta et al. . The res"}
+{"text": "Christof Daetwyler was born in Zollikerberg in Z\u00fcrich, Switzerland, he was the older of two brothers. A significant person we have worked with and greatly appreciated since very many years suddenly slipped out of our lives, leaving a great and irreplaceable loss. We received the unexpected message about Christof Daetwyler\u2019s sudden death with great sadness. He passed away in the morning of December 11https://www.gma-dach.org], as co-organiser of the Slice of Life community and in the AMEE, he was a friend, a colleague and an expert who regularly shared his new ideas at these conferences. Christof was well known and highly esteemed for his contributions to medical education and his innovative technological approaches, many of which were pathbreaking. For many members of the Association for Medical Education (GMA) ). At the same time, working part time at the AUM, he studied documentary movie making at the \u201cHigher school of fine Arts\u201d in Bern (1995-1996). Continuing to work full time at the AUM, he completed his MD thesis (1999) on the issue of technology enhanced medical education. During his time in the AUM, he contributed significantly to the development of computer assisted learning modules for medical education (e.g.: neurology/headache interactive). His work in this area was pioneering and he developed several price-winning interactive learning programs. In his own words, he described his time at the AUM as follows: He completed his basic education in Z\u00fcrich. His thirst for thorough knowledge in the fields that interested him, first led him to the \u201cHigher School of Fine Arts\u201d in Z\u00fcrich, where he completed classes in painting and drawing (1985-1986). After completion of the art studies, he studied medicine at the university of Z\u00fcrich (1986-1993). With his background in the arts, it was clear to him that he wanted to use his medical skills outside the clinical field. Already during his medical education, he worked part time as a data-base expert in two different companies. Then, from 1994 he started to actively combine his medical and media-technical skills at the \u201cDepartment for Instructional Media\u201d (AUM), at the \u201cInstitute of education and further medical training\u201d in Bern, Switzerland  computer applications for medical education at the Department of Educational Media (AUM). After one year, I concentrated my energy on my job at the AUM. As often in life, it\u2019s people who are the most important ingredient to success. In my case, I was extremely lucky to meet and collaborate with Marco Mumenthaler, a \u201cGrand Senior\u201d of Neurology \u2013 and a great teacher. Our work was very productive and successful, in terms of projects (they received many awards and were translated into different languages) as well as for personal reasons since we became friends. I stayed at the AUM for almost 8 years, until 2001 [http://webcampus.drexelmed.edu/interactive/cda/cv/index.html].In 2001, Christof set off to the United States to start a new chapter of his remarkable career. As a research assistant professor at the \u201cDepartment of Community and Family Medicine\u201d, at Dartmouth Medical School, he was invited by Joe Henderson to work as a lead designer and multimedia Developer at the interactive media laboratory (directed by Joe Henderson). After three creative years, he changed to the Drexel University College of Medicine in 2004 as a research assistant professor in the Department of Family, Community and Preventive Medicine (directed by Dennis Novack), where he started a successful and creative cooperation with Dennis Novack. Christof described his activities at Drexel as follows: In October 2004, I joined the Faculty of Drexel University College of Medicine in Philadelphia as an assistant research professor. My task was to continue on my way and to research, develop and integrate computer technology for the enhancement of medical education. My first large-scale project was \u201cdoc.com\u201d, a series of 41 media-rich on-line modules for the teaching, learning, and assessment of skills needed in healthcare communication. Another project I'm working on is \u201cWebOSCE\u201d, an on-line technology to allow students to practice new knowledge and skills on on-line standardized patient [http://webcampus.drexelmed.edu/interactive/cda/cv/index.html].https://webcampus.drexelmed.edu/doccom/user/] as well as the \u201cWeb-OSCE\u201d [http://www.iamse.org/websem/webosce-an-online-tool-for-remote-encounters-between-learners-and-standardized-patients-for-the-practice-assessment-and-remediation-of-clinical-skills/] projects represent typical achievements of Christof\u2019s work and add to the series of fruitful and stimulating cooperation with colleagues, this time with Dennis Novack. Today \u201cDoc.Com\u201d is successfully implemented as a learning resource for doctor patient communication in many prestigious universities in the US and abroad. An offspring of Christof\u2019s work is already well established in the German speaking countries in Europe. With the support of Dennis Novack, he initiated the generation of a European, German speaking, version of the \u201cDoc.Com\u201d platform, simply called \u201cDocCom.Deutsch\u201d [https://doccom.iml.unibe.ch/startseite/]. The first release was a team effort, with Christof, Wolf Langewitz , Kai Schnabel and Sissel Guttormsen (both IML), supported from the Novartis foundation for men and environment and by many experienced experts from the GMA community . As for the US-original, the German production was created based on the insight that successful doctor-patient communication can be trained, and that online media, when designed well, deliver important resources for basic communication training. This tool, today managed and developed further by the IML, somehow bridges Christof\u2019s early work at the IML with his later achievements. It was important for Christof to close this circle, and we are grateful for the enrichment this cooperation has become to us. It was also an important concern for him to support advancements in the education and particularly the employment of simulated patients (SPs). He promoted their education and enabled them to work from home, organising their encounters flexibly directly with the respective students  and in Germany . Since 2018 he also held a formal position at the University Basel, in the student dean\u2019s office for the development of a web-based video processing application supporting students in reflecting on their own performance. Indeed, the \u201cdoc.com\u201d [http://webcampus.drexelmed.edu/interactive/cda/publications/index.html]), as well as conference talks and workshops. As an engaged presenter, he was regularly invited as a speaker at national and international events. Throughout his career, he also continued to use his talent and expertise in arts e.g. as a photographer and as a painter. Christof strength was to inform and to bring out the message about the importance and relevance of high-quality media productions for the sake of effective medical education. His publications also include, book chapters and editorship for four books, a long list of about 70 communications (accessible online [In 2019 Christof decided to retire early from Drexel. He moved with his wife Magalene Daetwyler-Pina to Brasilia. There he continued to work as a freelancer, as a researcher and developer, he helped is step-son set up a brewery, and started organising music festivals. We will miss his enthusiasm, never ending energy for new ventures, creative ideas - and above all his heartly and generous nature; he was a remarkable person, self-assure and vulnerable at the same time. Christoph saw his success as a medical educator and media specialist in that he was trained in different disciplines, and thereby could combine his knowledge in the arts, video-documentation and medicine for the development of complex learning applications. He was an idealist tirelessly taking on a great workload and with a strong commitment to get his projects done. He managed successfully the art of promoting his ideas and to nourish recognition for his remarkable work. His enthusiasm was sometimes a curse and a blessing at the same time, particularly when the potential of his ideas could not be realised. We are left with gratitude for what he has created and a great loss for the new work that will no longer emerge. The authors declare that they have no competing interests."}
+{"text": "Sulfolobus islandicus type I-D Cas10d large subunit exhibits an unusual domain architecture consisting of a Cas3-like HD nuclease domain fused to a degenerate polymerase fold and a C-terminal domain structurally similar to Cas11. Crystal structures of Cas10d both in isolation and bound to S. islandicus rod-shaped virus 3 AcrID1 reveal that the anti-CRISPR protein sequesters the large subunit in a non-functional state unable to form a cleavage-competent effector complex. The architecture of Cas10d suggests that the type I-D effector complex is similar to those found in type III CRISPR\u2013Cas systems and that this feature is specifically exploited by phages for anti-CRISPR defence.A hallmark of type I CRISPR\u2013Cas systems is the presence of Cas3, which contains both the nuclease and helicase activities required for DNA cleavage during interference. In subtype I-D systems, however, the histidine-aspartate (HD) nuclease domain is encoded as part of a Cas10-like large effector complex subunit and the helicase activity in a separate Cas3\u2019 subunit, but the functional and mechanistic consequences of this organisation are not currently understood. Here we show that the  Sulfolobus islandicus type I-D large subunit Cas10d, containing a nuclease domain, reveals unusual architecture. The structure of Cas10d in complex with anti-CRISPR protein AcrID1 suggests that the latter sequesters Cas10d in a nonfunctional state.In type I-D CRISPR\u2013Cas systems, the nuclease and helicase activities are carried out by separate subunits. The crystal structure of  CRISPR\u2013Cas immunity is obtained through a three-step process11 consisting of adaptation , involving incorporation of fragments of invading DNA or RNA into genomic CRISPR arrays; expression, transcription and maturation of CRISPR-RNAs (crRNAs) carrying a sequence complementary to the invading nucleic acids and assembly of the Cas\u2013crRNA surveillance complex; and finally, interference, cleavage of foreign nucleic acids that have been encountered before by a Cas\u2013crRNA effector complex11. CRISPR\u2013Cas systems have been divided into two main classes depending on whether their effector complexes consist of multiple (Class 1) or a single  protein, six types (I\u2013VI) based on the configuration of the signature Cas proteins, and finally 33 subtypes on the basis of functional and mechanistic features12. Type I (Class 1) CRISPR\u2013Cas systems encode a large multi-subunit effector complex termed Cascade , which also includes a crRNA that recognises the invading DNA sequence during the interference stage. Formation of a DNA\u2013RNA heteroduplex (an R-loop) inside the effector complex then triggers recruitment of stand-alone helicase and nuclease activities, which unwind and cleave DNA, respectively. In most type I systems, both these functions are carried out by the signature protein Cas32, which contains an N-terminal metal-dependent histidine-aspartate (HD) nuclease domain, responsible for endo-nucleolytic cleavage of the target, fused to an ATP-dependent and single-stranded DNA (ssDNA)-stimulated superfamily-2 (SF2) helicase  and Cas3\u201d (nuclease). Type III CRISPR\u2013Cas systems also have multi-protein effector complexes for which the large subunits are members of the Cas10 family2. Cas10 proteins contain an N-terminal HD nuclease domain that is evolutionarily distinct from Cas3\u201d, two RNA recognition motif (RRM) domains homologous to those found in polymerases and cyclases, and a helical Thumb/C-terminal domain 18. Cas10d thus contains an N-terminal HD domain similar to those in type I Cas3 and Cas3\u201d proteins, which is distinguished by lacking a circular permutation found in type III large subunit HD nucleases that results in a conserved His residue shifting from the N to the C terminal end of the domain2. The Cas10d HD domain is fused to a predicted inactivated polymerase consisting of two RRM domains reminiscent of those found in type III Cas10 subunits19. Recently, it was shown that an archaeal type I-D system from Sulfolobus islandicus is capable of cleaving both double-stranded DNA (dsDNA) and, remarkably, also ssDNA, which could be targeting rudivirus (SIRV) replicative intermediates20. dsDNA cleavage is catalysed by the Cas10d HD domain and in the presence of the Cas3\u2019 helicase and ATP, multiple cleavage events take place on both DNA strands. ssDNA cleavage, on the other hand, appears to follow a 6-nucleotide (nt) ruler-like mechanism and is catalysed by the backbone subunit Csc2 (Cas7), in a way reminiscent of the protospacer RNA transcript cleavage observed in type III systems20. Here, we present the crystal structure of the Cas10d large effector complex subunit from the Sulfolobus islandicus I-D system, both in isolation and in complex with the Sulfolobus islandicus rod-shaped virus 3 (SIRV3) anti-CRISPR protein, AcrID1. The structures reveal an unusual domain architecture of the large subunit, which supports that the effector complex is similar to those found in type III CRISPR\u2013Cas systems. Moreover, we show that binding of the anti-CRISPR protein sequesters Cas10d in a non-functional state, in which it cannot engage in interference. Taken together, our data provide a framework for understanding essential mechanistic features that distinguish type I-D from other type I and type III CRISPR\u2013Cas subtypes.Type I-D CRISPR\u2013Cas systems, which are found both in bacteria and archaea, are exceptional in that they contain a hybrid large subunit, Cas10d, bearing resemblance to both the signature proteins found in type I (Cas3) and type III (Cas10)Sulfolobus islandicus LAL14/1 carries one subtype I-D CRISPR\u2013Cas locus containing spacers matching the genome of the lytic rod-shaped virus, Sulfolobus islandicus rod-shaped virus 2 (SIRV2), for which it is a laboratory host21. To counter the CRISPR\u2013Cas immunity, SIRV2 on its part expresses a 12.2\u2009kDa anti-CRISPR (Acr) protein, AcrID1, which is conserved in many archaeal viruses including Sulfolobus islandicus rod-shaped virus 3 (SIRV3)22. SIRV3 AcrID1 was shown to inhibit type I-D immunity in S. islandicus through direct binding to the large subunit of the effector complex, Cas10d23. To understand the molecular architecture of the unique type I-D system as well as mechanism of inhibition by AcrID1, we reconstituted the Cas10d\u2013AcrID1 complex in vitro and determined the crystal structure to 3.5\u2009\u00c5 resolution by single-wavelength anomalous dispersion (SAD) phasing using Se\u2013Met substitution , a Cyclase/RRM domain (187\u2013425), a zinc finger domain (426\u2013490), a Palm/RRM domain (491\u2013623), and a C-terminal helical region consisting of a polymerase-like Thumb domain (624\u2013719) and an extension with predicted homology to Cas11 (720\u2013847)  7) Figs.\u00a012. The s26, which is configured to function as an endo and/or exo-nuclease in CRISPR\u2013Cas interference29. HD nuclease domains are found in a wide range of nucleic acid enzymes and consist of a conserved core of 6\u20137 helices that organise the residues of the active site16. Outside this core motif, the structures vary considerably. The HD domain found in Cas10d appears to be a minimalised version of the core motif found in other type I systems, such as Thermus thermophilus Cas3 (Cas3Tth) and Thermobifida fusca Cas3 , but compensates with an N-terminal extension (residues 1-26), part of which forms a helix  that spatially overlaps with \u03b18 in Cas3Tth despite running in the opposite direction. Interestingly, this N-terminal topology is also found in type I-A Cas3\u201d proteins, such as Methanocaldococcus jannaschii Cas3  and Cas3 . Finally, at its C-terminal end (residues 158\u2013186), the Cas10d HD domain contains two additional helices (\u03b19\u2013\u03b110), which spatially overlap with a helix and a short beta-hairpin in full length Cas3Tth  for activity16. In the active site of the Cas3Tth HD domain, two His, two Asp, and a Ser residue coordinate a single divalent metal ion16. Other Cas3 HD domains, such as the one found in Cas3Tfu, have an extended set of residues that includes two additional histidines that come together to coordinate two ions  are not present, suggesting that this HD domain only binds a single  cation ons Fig.\u00a0, left14.31. The first of these RRM domains (residues 187\u2013425) maintains the interleaved \u03b2\u03b1\u03b2-\u03b2\u03b1\u03b2 structure characteristic of an RRM domain but has two very short \u03b2-strands and appears partially degenerate  is more canonical except for a small \u03b2-hairpin insertion surrounding the helix of the first \u03b2\u03b1\u03b2 RRM motif  and does not have the conserved \u03b2-hairpin motif (GGDD) nor the P-loop required for ATP binding, both of which are conserved in the Cmr2 and Csm1 type III subunits  and a helical bundle, which is structurally similar to the small effector complex subunit Csm2/Cmr5/Cas11 , Csc2 (Cas7) and crRNA was mixed with Cas10d co-purified with SS , Cas3\u2019, ATP, and increasing amounts of AcrID1 before addition of double-stranded target DNA radio-labelled on the non-target strand (dsNTS). Upon incubation of the effector complex components with the dsNTS, but in the absence of AcrID1, several cleavage products appeared, which have been shown to be the result of the activity of the Cas10d HD domain  or AcrE1 (anti-type I-E) reveal little similarity  and A. fulgidus Cmr (type III-B) complexes determined by cryo-EM41. Comparison of Cas10d with the conformation of the corresponding large subunit in the Csm complex, Csm1, and taking the observed flexibility of Cas10d into account suggests that the helical extension of the Cyclase/RRM domain likely folds back up upon interaction with the effector complex and target DNA  reveals a striking structural overlap of the Cas11-like domain of Cas10d with the Cas11 subunit of the type III-B system , suggesting that the C-terminal domain of Cas10d in fact plays the role of the small subunit inside the type I-D effector complex , which likely results from an alternative translation start position at the beginning of the Cas11 domain in the gene encoding the large subunit44. Comparison with the Cmr complex, which contains two copies of the Cmr5 (Cas11) small subunits further suggests that in the type I-D effector complex, these roles may be played by multiple copies of the C-terminal Cas11 domain of Cas10d generated by the alternative translation start. Structural studies of Cas10d in the context of the effector complex will hopefully shed light on this intriguing aspect of the type I-D effector complex41.Finally, super-positioning of Cas10d on the large subunit of the lex Fig.\u00a0. This is6 tags23. Separate 2\u2009L cultures of E. coli B834 (DE3) cells (Novagen) carrying plasmids expressing either Cas10d or AcrID1 were grown in lysogeny broth (LB) medium supplemented with 50\u2009\u00b5g/mL kanamycin at 37\u2009\u00b0C with shaking, until OD600 reached approximately 0.6\u20130.8. The cells were harvested by centrifugation (1500\u2009\u00d7\u2009g for 10\u2009min at 4\u2009\u00b0C) and used to inoculate 6\u2009L of M9 minimal medium (Molecular Dimensions), supplemented with 50\u2009\u00b5g/mL kanamycin, glucose, vitamins and amino acids with the exception of L-methionine. The cells were grown for 3\u2009h at 37\u2009\u00b0C after which 20\u2009mg/mL seleno\u2013methionine  was added and the cells were further incubated until OD600 reached 0.8. Protein expression was then induced with 0.5\u2009mM isopropyl-\u03b2-D-thio-galactopyranoside (IPTG) and expression continued overnight at 20\u2009\u00b0C. For protein purification, cells were resuspended in lysis buffer  supplemented with 1\u2009mM phenyl methyl sulfonyl fluoride (PMSF) and 10\u2009\u00b5g/mL DNase I. The cells were lysed by sonication and cell debris removed by centrifugation . The cell lysate was heated at 75\u2009\u00b0C for 20\u2009min to remove the bulk of E. coli proteins as a first step of purification, then centrifuged to clear the supernatant containing either Cas10d-His or AcrID1-His. The samples were then applied to a pre-packed 5\u2009mL HisTrap column (Cytiva), pre-equilibrated in lysis buffer and eluted using a stepwise gradient into lysis buffer with increasing concentrations (30\u2013300\u2009mM) of imidazole. The eluate containing the protein of interest was then applied on a 1\u2009mL Heparin column (Cytiva), pre-equilibrated in 50\u2009mM Bicine, pH\u2009=\u20098.5, 50\u2009mM NaCl, 5\u2009mM \u03b2-ME, and eluted with a linear gradient into the same buffer containing 1\u2009M NaCl. The sample was finally purified by size exclusion chromatography using a Superdex 200 increase 10/300 column (Cytiva), pre-equilibrated in 25\u2009mM Hepes-NaOH, pH\u2009=\u20097.5, 150\u2009mM NaCl, 5\u2009mM \u03b2-ME. Peak fractions of Cas10d were pooled and concentrated to 5\u201315\u2009mg/mL and used for crystallisation in isolation or to form the AcrID1-bound complex23. For complex formation, Cas10d and AcrID1 were mixed in a 1:2 (Cas10d:AcrID1) molar ratio and incubated at 75\u2009\u00b0C for 1\u2009min. The complex was then isolated using a Superdex 200 increase 10/300 column (Cytiva) pre-equilibrated in 30\u2009mM Hepes-NaOH, pH\u2009=\u20097.5, 150\u2009mM NaCl, 5\u2009mM \u03b2-ME and concentrated to 4\u2009mg/mL. For the isolated Cas10d structure, a sample of the protein was treated with bovine trypsin in a 1:500 (trypsin:Cas10d) molar ratio for 50\u2009min at room temperature. The proteolysis reaction was quenched with 1\u2009mM PMSF after which the sample was purified on a Superdex 200 increase 10/300 column (Cytiva), pre-equilibrated in 25\u2009mM Na-Hepes, pH\u2009=\u20097.5, 300\u2009mM NaCl, 5\u2009mM \u03b2-ME. The protein eluted in a similar position to the uncut protein, but sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of two bands corresponding to approximately 45 and 50\u2009kDa. The protein was concentrated to ~5\u2009mg/ml using a 30\u2009kDa MWCO VivaSpin sample concentrator (Sigma-Aldrich) before crystallisation screening. All protein concentrations were estimated using a NanoDrop measuring OD280 and theoretical extinction coefficients calculated from the known protein sequences.The plasmids pET30a-cas10d and pET30a-acrID1 were used for expression of recombinant Cas10d and AcrID1, both with C-terminal His\u00ae nanoliter protein crystallisation robot (TTP Labtech). Initial crystals appeared after two days at 19\u2009\u00b0C and further optimised for data collection. All crystals were cryo-cooled in the reservoir buffer supplemented with 30% (v/v) ethylene glycol. After extensive crystal screening and optimisation, a single-wavelength Se anomalous dataset extending to 3.5\u2009\u00c5 (CC1/2\u2009=\u20090.55 at 3.48\u2009\u00c5) was obtained at 100\u2009K and 0.9793\u2009\u00c5 X-ray wavelength, identified using a fluorescence energy scan for selenium using mxCUBE at the PETRA III beamline P14 in Hamburg, Germany  likely owing to the high level of flexibility of Cas10d  PEG 8000, 5% (v/v) ethylene glycol by sitting drop vapour diffusion prepared in Swissci MRC 2-drop crystallisation plates (Hampton Research) using a Mosquito45, and data quality was assessed using Phenix.xtriage47. The structure was determined using molecular replacement (Phenix.phaser)51 using Cas10d from the Cas10d\u2013AcrID1 complex as a search model. The crystals of isolated Cas10d contain two copies of the protein per crystallographic asymmetric unit, one of which appears to have a partial RRM/Cyclase domain, presumably due to the proteolytic treatment. In the other molecule, which is more intact, the RRM/Cyclase domain has shifted ~5\u2009\u00c5 compared to the complex structure. Inspection of the packing further revealed that parts of the intact structures would clash in the new crystal, suggesting this is the reason that proteolysis allowed crystallisation of the isolated protein. Both chains were rebuilt in Coot and iteratively refined using Phenix.refine. The final structure has R-work\u2009=\u200934.3% and R-free\u2009=\u200937.0% and a very high, average B factor (228\u2009\u00c52), presumably due to the additional flexibility induced by cleavage of the protein  PEG 8000, 8 % (v/v) ethylene glycol and further optimised by addition of 1% (w/v) PEG 3350. The crystals were cryo-cooled in the reservoir buffer supplemented with 25% (v/v) ethylene glycol before native, monochromatic x-ray data extending to 4.0\u2009\u00c5 were collected at the Diamond Light Source beamline I04 in Oxford, UK. The raw data were processed and scaled using XDS and XSCALE32P]-ATP (PerkinElmer) and T4 polynucleotide kinase (New England Biolabs) before double-stranded DNA (dsDNA) was generated by annealing the labelled non-target strand with 2-fold molar excess of unlabelled target strand.DNA oligonucleotides  essentially as described before20. Briefly, cells were grown in SCVY medium and the His-tagged complex purified using a His-trap column (Cytiva) followed by gel filtration using a Superdex 200 10/300 column (Cytiva) eluting in 20\u2009mM Hepes-OH, pH\u2009=\u20097.5, and 300\u2009mM NaCl. Recombinant Cas10d for the functional assays co-purified with small subunit (SS) and was purified from E. coli as described previously23. Recombinant MBP-Tev-Cas3\u2019-His was overexpressed from pMAL-TEV-Cas3\u2019 in E. coli BL21 (DE3) Rosetta overnight at 20\u2009\u00b0C and purified by HisTrap and gel filtration as described above, cleaved by Tev protease overnight at 4\u2009\u00b0C, and purified by HisTrap and gel filtration again, eluting in a final buffer containing 20\u2009mM Hepes-OH, pH\u2009=\u20097.5, and 300\u2009mM NaCl.A 2, 1\u2009mM DTT) at 65\u2009\u00b0C for 1\u2009h. Reactions were stopped by adding 2\u00d7 RNA loading dye (New England Biolabs). For electrophoresis, samples were heated for 3\u2009min at 95\u2009\u00b0C and analysed by 12% denaturing polyacrylamide gel electrophoresis (PAGE). For visualisation, a phospho-imager screen was exposed to the gel overnight and scanned with Typhoon FLA-7000. DNA cleavage gels  and was incubated at 65\u2009\u00b0C for 30\u2009min. An equal volume of 2\u00d7 native loading buffer  glycerol) was added and the samples were loaded on an 8% native PAGE running at 25\u2009\u00b0C in 40\u2009mM Tris, 20\u2009mM acetic acid, pH\u2009=\u20098.4. Migration on the gel was visualised by exposure to a phospho-imager screen overnight, which was subsequently scanned on a Typhoon FLA-7000.Each reaction mixture contained 0.02\u2009mg/mL backbone complex, 0.03\u2009mg/mL Cas10d, 20\u2009nM dsDNA substrate and the indicated amounts of AcrID1 in cleavage buffer (20\u2009mM MES-NaOH pH\u2009=\u20096.0, 10\u2009mM MgClFurther information on research design is available in the\u00a0Supplementary InformationPeer Review FileReporting Summary"}
+{"text": "To compare the effectiveness and safety of carbon dioxide , intraocular pressure (IOP), number of medications, surgical success rate, and complications were analyzed. P > 0.05). There were higher rates of delayed anterior chamber formation (21.3%) and thin-wall filtrating blebs (10.6%) in the Trab group. Meanwhile, the peripheral anterior synechiae were only observed in the CLASS group, and the ratio was 30%.  The mean follow-up periods were 27.89\u2009\u00b1\u20092.94 months and 26.11\u2009\u00b1\u20092.06 months in the CLASS and Trab groups, respectively. 30 eyes (24 patients) underwent CLASS and 47 eyes (38 patients) underwent Trab. The BCVA in the CLASS and Trab groups was recovered to baseline at postoperative 1 week and 1 month, respectively. At last follow-up visits, a remarkable reduction in the IOP and number of medications was observed in both groups, and no significant difference was found in those between the two groups. The complete success rates were 51.7% and 47.7% in postoperative 24 months in the CLASS and Trab groups, respectively ( CLASS is an effective and safe treatment modality for POAG, with fewer filtering bleb-related complications and quicker visual recovery in the early postoperative stage than trabeculectomy. The efficacy of lowering intraocular pressure was similar for both procedures. This surgery is mainly suitable to treat those with open-angle glaucoma [Trabeculectomy (Trab) is considered a classical surgical approach and the gold standard surgery for treating primary open-angle glaucoma (POAG) . Neverthglaucoma , 6. Thisglaucoma . Accordiglaucoma , 7. Howeglaucoma , few stuP > 0.05). All diagnoses of POAG followed the guidelines of the International Society of Geographical and Epidemiological Ophthalmology. In addition, all surgeries were performed by two experienced ophthalmologists.The inclusion criteria were patients aged \u226518 years with the following: POAG , medicalAll procedures were performed under topical anesthesia with proparacaine hydrochloride eye drops and subconjunctival anesthesia with 0.2\u2009mL of 2% lidocaine.2 with one-third scleral thickness was dissected 1.5 mm anterior to the corneal limbus for thorough exposure of the TDM. Under the conjunctiva and scleral flap, a piece of sponge soaked in 0.2\u20130.4\u2009mg/mL mitomycin was applied for 3-4\u2009min and then rinsed with 100\u2009mL of balanced salt solution. Subsequently, the CO2 laser system  with the desired scanning dimensions and shape (marked with red laser beam) that could be altered in the range of 4\u2009\u00d7\u20093\u2009mm was used to repeatedly ablate >90% scleral tissue until the suprachoroidal space was detected and the residual scarring issue was removed. Mitomycin was applied again to the scleral pool for 1\u2009min, which was washed with 100\u2009mL of balanced salt solution. Following that, CO2 laser was applied to ablate the TDM area, outer wall of Schlemm's canal, and trabecular meshwork until a slow and continuous percolation of aqueous humor was seen; the ablated area measured about 4\u2009\u00d7\u20091\u2009mm in width. The laser energy was set to and maintained at 21\u201318\u2009W for all cases, and the interval between the laser applications was 2-3\u2009s to detect continuous aqueous humor percolation of the ablated area. Then, the scleral flap was sutured with 10-0 nylon sutures. Finally, the conjunctiva was fixed with 10-0 nylon sutures.The fornix-based conjunctival flap was used. A scleral flap measuring 5\u2009\u00d7\u20095\u2009mmTrabeculectomy was conducted in the superior quadrant according to the surgical procedure described in a previous study . A sclerThe following parameters were examined and analyzed before surgery, at 1 week, and at 1, 3, 6, 12, and 24 months after the surgery. The best-corrected visual acuity (BCVA) was measured with the Snellen chart, and the results were described with the logarithmic minimum angle of resolution (logMAR). IOP was measured with a calibrated Goldman applanation tonometer. Gonioscopy (a single mirror Gonio diagnostic lens), number of medications, surgical success rate, and complications were recorded. All examinations were performed by experienced ophthalmologists and technicians.\u03bcm. The laser energy was adjusted according to the iris reaction that the iris was atrophied, but no bubbles were formed. The energy for laser peripheral iris boring was 4\u20138\u2009mJ, the exposure time was 11\u2009ns, and the laser hole was >200\u2009\u03bcm. Furthermore, seven eyes (23.33%) preoperatively underwent LPI combined with the YAG laser boring hole as close as possible to the peripheral iris, just corresponding to the surgical site. All manipulations were done by experienced ocular technicians.In the CLASS group, the patients underwent preoperative routine ultrasound biomicroscopy (UBM)   was performed if PAS still existed  , 17. Fort-test, except for the gender composition ratio that was compared using chi-square test (\u03c72). Repeated-measures analysis of variance (ANOVA) was used to analyze the changes in BCVA, IOP, and number of medications from preoperative baseline to 24 months between the two groups. Success rates between the two groups were compared with chi-square test (\u03c72). The postoperative complications were compared using Fisher's exact test. The cumulative probability of success was illustrated using Kaplan\u2013Meier survival curves, and the Log-rank test was performed for group comparisons. A P value less than 0.05 was considered statistically significant.SPSS 19.0  was used for all statistical analyses. Patients' information of both groups before surgery was compared and analyzed using unpaired After the surgery, patients were followed up for 27.89\u2009\u00b1\u20092.94 months in the CLASS group and 26.11\u2009\u00b1\u20092.06 months in the Trab group. One patient in the CLASS group experienced severe hematencephalon after 3 months and was lost to follow-up. In the Trab group, three patients were lost to follow-up after 6 months, 1 year, and 2 years, respectively.P=0.67). In 1 week and 1 month after surgery, the BCVA in the CLASS group was 0.28\u2009\u00b1\u20090.22 and 0.27\u2009\u00b1\u20090.24, while in the Trab group it was 0.27\u2009\u00b1\u20090.24 and 0.31\u2009\u00b1\u20090.31. Repeated-measures analysis showed the Mauchly sphericity test P < 0.01, and the Greenhouse\u2013Geisser estimation was conducted . Interestingly, the BCVA in CLASS group was significantly improved compared to Trab group at 1 week after the operation (P=0.02); no statistical difference was found 1 month after the operation (P=0.55). These data suggested that the BCVA was restored to the preoperative level in the CLASS group one week after surgery. In the Trab group, however, the BCVA was reduced 1 week after operation and gradually recovered to the preoperative level 1 month after surgery . No significant difference of group effect was observed  in the CLASS group and (37.87\u2009\u00b1\u20095.74\u2009mm Hg) in the Trab group preoperatively to (15.36\u2009\u00b1\u20093.74\u2009mm Hg) and (16.34\u2009\u00b1\u20093.25\u2009mm Hg) postoperatively at 24 months, respectively . However, no significant difference in the follow-up doses of medications was found between the two groups  in the CLASS group and (3.60\u2009\u00b1\u20090.99) in the Trab group preoperatively to (1.31\u2009\u00b1\u20091.54) and (1.30\u2009\u00b1\u20091.32) postoperatively at 24 months, respectively .As shown in During the following visits, four eyes in the CLASS group with uncontrolled IOP finally underwent gonioscopy-assisted transluminal trabeculectomy, and six eyes in the Trab group underwent reoperation, including two eyes that underwent trabeculectomy and four eyes that underwent trabeculectomy combined with cataract surgery.As listed in GPT and LPI were not performed in the Trab group. The formation of the anterior chamber was delayed in 10 (21.3%) eyes in the early stage after the surgery. Two eyes recovered after anterior chamber reformation, and the rest recovered after receiving medical conservative therapy during the follow-up. Anterior chamber hemorrhage occurred in four eyes, and the hemorrhage was absorbed 2 weeks after the surgery with no further intervention. Choroidal detachment took place in five eyes, of which four recovered after one-week conservative therapy and the other eye recovered after draining fluid from the suprachoroidal space. One eye suffered low-tension maculopathy and gradually recovered after injecting autologous blood into the filtration bleb. The intervention of bleb needling with MMC injection was performed on 13 eyes (28.9%), one year after surgery. During the follow-up visits, a functional thin-wall filtration bleb was found in five eyes with well-controlled IOP.2 laser (wavelength: 10600\u2009nm) is highly effective in ablating the deep scleral tissue and outer wall of Schlemm's canal. Under a scleral flap, the laser not only ablates the TDM but also keeps the inner wall of the Schlemm canal and trabecular meshwork intact. Subsequently, the scleral pool, intrascleral  space, and TDW are constituted, which help to reduce the aqueous humor outflow resistance and ensure continuous drainage of the aqueous humor from the anterior chamber to the scleral pool and suprachoroidal space, thereby decreasing the intraocular pressure (IOP). In addition, the coagulation ablation effect of CO2 laser not only plays a role in hemostasis and blood vessel blockade but also reduces the risk of scleral reservoir scarring after surgery [The far-infrared radiation of the CO surgery .P < 0.001), while the variation in IOP tended to stabilize with prolonged observation time, with no significant intergroup difference in IOP 24 months after the surgery (P > 0.05). The number of antiglaucoma medications markedly decreased in both groups up to 24 postoperative months (P < 0.001). Compared with the Trab group, there were no significant differences in the follow-up doses of medication in the CLASS group. This result suggests the same efficacy for the CLASS and Trab groups on lowering the IOP. Skaat et al. studied 15 eyes with open-angle glaucoma treated using CLASS. The results at one year after the surgery showed that CLASS could safely and effectively decrease IOP in patients with POAG and exfoliative glaucoma, with a complete success rate of 45.5% following surgery for 12 months (76.9% patients used MMC intraoperatively) [As shown in this study, the postoperative IOP significantly decreased as compared with the preoperative level in both groups (atively) . In a Chatively) . Geffen y P > 0.0. The numatively) , 20. Theatively) . This isatively) , 22.The present study indicated that BCVA in the CLASS group was restored to the preoperative level one week after surgery. However, in the Trab group, the BCVA was reduced and gradually restored to the baseline level, one month after surgery. The reasons for this might be as follows: (1) The anterior chamber was not penetrated during CLASS surgery, leading to the formation of a barrier by the intraocular tissue and a small fluctuation in the intraoperative and postoperative anterior chamber and IOP, which maintained the depth and stability of the anterior chamber . (2) Adjin situ, ensure moderate tightness, and prevent overfiltering, which is very important to impede the PAS. (4) Seider et al. and He et al. [We found that the early complications in the Trab group mainly included shallow anterior chamber 21.3%), choroid detachment (10%), and hyphema (8.5%); relative to the CLASS group, no complication was observed. Other investigations have presented a similar result (42%) for complications following trabeculectomy . As expe1.3%, choe et al. , 24 sugge et al. . In our This study had some limitations such as the single-center design, single ethnic population, small cohort of patients, and short-term follow-up. In the future, a more rigorous, large-scale, multicenter study encompassing patients of different ethnicities and with a longer follow-up should be performed to further verify and validate the effect of CLASS.As a modification of nonpenetrating deep sclerectomy, CLASS was believed to be remarkably less effective compared with trabeculectomy, although CLASS has a better safety profile. In the study, we confirmed that CLASS is an effective and safe treatment modality for POAG, with fewer filtering bleb-related complications and quicker visual recovery in the early postoperative stage than trabeculectomy. The efficacy of lowering intraocular pressure was similar for both procedures."}
+{"text": "Forming long-term memories is crucial for adaptive behavior and survival in changing environments. The molecular consolidation processes which underlie the formation of these long-term memories are dependent on protein synthesis in excitatory and SST-expressing neurons. A centrally important, parallel process to this involves the removal of the memory constraint quinone reductase 2 (QR2), which has been recently shown to enhance memory consolidation for novel experiences in the cortex and hippocampus, via redox modulation. However, it is unknown within which cell type in the cortex removal of QR2 occurs, nor how this affects neuronal function. Here, we use novel taste learning in the mouse anterior insular cortex (aIC) to show that similarly to mRNA translation, QR2 removal occurs in excitatory and SST-expressing neurons. Interestingly, both novel taste and QR2 inhibition reduce excitability specifically within SST, but not excitatory neurons. Furthermore, reducing QR2 expression in SST, but not in PV or excitatory neurons, is sufficient to enhance taste memory. Thus, QR2 mediated intrinsic property changes of SST interneurons in the aIC is a central removable factor to allow novel taste memory formation. This previously unknown involvement of QR2 and SST interneurons in resetting aIC activity hours following learning, describes a molecular mechanism to define cell circuits for novel information. Therefore, the QR2 pathway in SST interneurons provides a fresh new avenue by which to tackle age-related cognitive deficits, while shedding new light onto the functional machinations of long-term memory formation for novel information. Quinone reductase 2 (QR2) removal from the cortex and hippocampus demarcates novelty, and facilitates memory formation for the associated events to a novel experience. The newly described QR2 pathway, and the circuit that is responsible for late consolidation of novel internal representations, are both currently being investigated and are not yet wholly understood. Presently, a hitherto unknown phenomenon of reduced SST activity in the hours following novel taste learning via QR2 removal is shown, providing a molecular mechanism to define a brain state enacted by distinct neuronal populations. This provides both new molecular and cellular information regarding an important time period in memory formation, and also describes the key cell types involved in the underlying circuit, which is as yet unexplored.Learning new information about the environment is critical for behavioral adaptation and survival . Novel tThe aIC is a complex cortical structure that associates external events with their visceral consequences and acts across several forms of learning and behavior . It is cad libitum. Experimentation was approved by the University of Haifa Animal Care and Use Committee . Before experimentation, mice were allowed 7\u2009d of acclimatization. Animals were handled in accordance with University of Haifa practices and standards, in compliance with the National Institutes of Health guidelines for the ethical treatment of animals.Male and female mice, 8\u201316\u2009weeks old, weighing 20\u201330 g from the following strains were used: C57BL/6 (Envigo), Gad2-IRES-Cre, SST-IRES-Cre, B6 PV-Cre and VIP-IRES-Cre . The mice were kept in a temperature-controlled facility (22\u201324 \u00b0C), on a 12/12 h light/dark cycle (light phase 7 A.M. to 7 P.M.) at the University of Haifa, with water and food provided Mice were taught to drink water from pipettes (2 ml/pipette) once a day for 20\u2009min, over 3 d. They were then given pipettes containing a novel taste (0.5% saccharin). Following novel taste consumption, animals were killed 3 h later, at the time point when QR2 mRNA expression reduction is best measured . To assehttp://n2t.net/addgene:84882; RRID:Addgene_84882), using conventional cloning techniques. HEK293FT cells were seeded at 25\u201335% confluence to produce AAV vectors, and were transfected 24 h later. The plasmids encoding AAV rep, cap of AAV1 and AAV2, and a vector plasmid for the rAAV cassette, expressing the relevant shRNA, were applied using the PEI method (g), resuspended in lysis solution  and lysed via X3 freeze-thaw cycles. Treatment of the crude lysate with 250\u2009U benzonase (Sigma) per 1 ml of lysate at 37\u00b0C for 1.5 h was then done, to degrade genomic and unpackaged AAV DNA, followed by centrifugation at 3000\u2009\u00d7\u2009g for 15\u2009min to pellet cell debris. Heparin-agarose columns were used to purify the virus particles from the supernatant, which were then washed with PBS and concentrated with Amicon (Merck Millipore) columns. The resultant viral suspension was then aliquoted and stored at \u221280\u00b0C. Viral titer was determined by qPCR. AAV vectors used for injections had genomic titers ranging between 2 \u00d7 1010 and 5 \u00d71010 genome copies per milliliter (gc/ml). The AAV p315 (ssAAV-1/2-mCaMKII\u03b1-EGFP_2A_iCre-WPRE-SV40p(A), physical titer 4.6\u2009\u00d7\u20091012 vg/ml) was purchased from the University of Zurich Viral Vector Facility (https://vvf.ethz.ch/), to express Cre under the CamKII promoter.QR2 shRNA sequences (shNQO2 and scrambled control) were cloned into pAAV-Sico-Red plasmid, which was a gift from Eun Mi Hwang , given an analgesic  and 40\u2009min later were placed in a robot stereotaxic device (Neurostar). The scalp was opened, and bregma and \u03bb were marked as reference points for the drilling and injection site . Following syringe (Hamilton) insertion, 5\u2009min were given before 0.2- to 0.8-\u03bcl virus was injected, at 0.05\u2009\u03bcl/min using StereoDrive and InjectoMate software (Neurostar). The syringe was then left in the injection site for a further 10\u2009min to prevent capillary motion of the virus from the site. Upon completion, incisions were dressed with 5% synthomycine ointment (Rekah), sealed shut with Vetbond (3M), 0.5\u2009mg/kg enrofloxacin (Baytril) was given and mice were administered 0.25\u2009mg/kg atipamezole hydrochloride (Antisedan) and placed in a heated recovery cage for 1\u20132 h. A month-long period of mouse recovery and virus expression was then given, following which experimentation began.Mice were swiftly killed by cervical dislocation, brains were removed and instantly flash frozen with liquid nitrogen and stored in \u221280\u00b0C. Brain sections and tissue punches were conducted in a Leica CM 1950 cryostat. Samples for qPCR were dissected from 1-mm-thick coronal slices . Relative quantitation was done using the 2\u0394\u0394Ct method  was then conducted as previously described (https://imagej.net/Fiji), where regions of interest (ROIs) were manually drawn to denote aIC subregions, using the Allen Mouse Brain Atlas (http://mouse.brain-map.org/) for reference (2) to avoid counting any remaining debris or incomplete cell segments and other artifacts. The fluorescent QR2 signal was then measured within these cell-defined ROIs, using the FIJI Command Manager Measure function, and data were collated into relevant bins . QR2 signal within these cells was then averaged for each animal, and each animals mean QR2 expression per brain area and cell type was then used. In order to minimize signal variation because of technical factors existing between experiments , the average QR2 expression from every animal in each of the groups  was divided by the control (familiar water) group mean QR2 signal, in each experiment separately. This normalized QR2 expression from each separate experiment was then combined, and a comparison was done between experimental groups . When comparing relative contribution of QR2 signal from within excitatory or inhibitory cell types, cells were pooled from all animals in either familiar or novel taste groups and binned into either vGlut or GAD positive pools. The sum total QR2 signal from each of the pooled cell types was then divided by the total cell number within each experimental group (familiar or novel taste). Then, the reduction in QR2 signal was calculated by deducting the novel taste group QR2 signal, from each cell type, from that of the familiar taste group.Over two separate experiments, mice received either a familiar  or a novel taste (first exposure to saccharin), were killed 3 h later and fresh frozen brains were sliced into 20-\u03bcm sections in a Leica CM 1950 cryostat, and then mounted onto SuperFrost Ultra Plus Adhesion slides (Thermo Fisher Scientific). RNAscope (ACD) fluorescent escribed  by an exeference . Standarm sucrose, 60 mm NaCl, 3 mm KCl, 1.25 mm NaH2PO4, 28 mm NaHCO3, 0.5 mm CaCl2, 7 mm MgCl2, 5 mm D-glucose, and 0.6 mm ascorbate, Sigma-Aldrich). The slices were placed in artificial CSF  for a 30\u2009min recovery period at 37\u00b0C and then kept at room temperature for at least an additional 30\u2009min before electrophysiological recording. Throughout, ACSF was continually gassed, using carbogen .Mice were killed 3 h following water or saccharin consumption by decapitation, following anesthesia with isoflurane. Brain slices were cut at 300\u2009\u03bcm in coronal sections using a vibratome (Campden-1000) in ice chilled cutting solution . Recordings were done from soma of pyramidal neurons and SST interneurons expressing mCherry, from the aIC of C57BL/6 and SST Cre mice with 0.5 \u03bcm S29434  in 0.5% DMSO, or vehicle, in the patch pipette. No online correction was done for liquid junction potentials (10\u2009mV), and current clamp recordings were low-pass filtered at 10\u2009kHz and sampled at 50\u2009kHz. Only cells with resistance smaller than 20 M\u2126 were included, once compensation for pipette capacitance and series resistance was done.Intrinsic properties were measured as previously reported . Slices SS/\u0394V max) \u00d7 100%] of the voltage response to \u2013150 pA, as previously described . Injection of 500-ms, 50-pA current steps, from 50 to 400\u2009pA, was used to measure neuron firing rate . A hyperpolarizing current pulse (\u2212150 pA) elicited a voltage response which was used to calculate the input resistance (Rin). A sag ratio was calculated using [. Data obtained were tested for normality, using Shapiro\u2013Wilk normality test. Normally distributed data were then analyzed by unpaired Student\u2019s t test, one-way ANOVA or repeated measures two-way ANOVA, followed by Tukey\u2019s or Sidak\u2019s post hoc analysis. For non-parametric analysis, Mann\u2013Whitney tests or Kruskal\u2013Wallis followed by Dunn\u2019s multiple comparisons tests were conducted. All data are presented as means with SEM. All descriptive statistics, normality tests, parametric and non-parametric tests were conducted using GraphPad Prism 7 software.Male and female subjects were randomly allocated to experimental groups. Group size range estimation was based on previously published results using similar methods, as well as an online power calculator , water (highly familiar), or a novel taste  neuron cell count, and therefore a reduced ratio of excitatory-to-inhibitory neurons (\u223c3.6:1). However, despite this artificial reduction in total excitatory neuron number  by automn\u2009=\u20098; PV, novel 1.013\u2009\u00b1\u20090.054\u2009AU, n\u2009=\u20097; Student\u2019s t test, t\u2009=\u20090.2208 df\u2009=\u200913, p\u2009=\u20090.8287; VIP, familiar 1\u2009\u00b1\u20090.062\u2009AU, n\u2009=\u20098; VIP, novel 1.081\u2009\u00b1\u20090.175\u2009AU, n\u2009=\u20097; Student\u2019s t test, t\u2009=\u20090.4621 df\u2009=\u200913, p\u2009=\u20090.6517), the dgIC , the agIC , layers 2/3  or layers 5/6 . In contrast, a significant reduction in QR2 was measured in SST neurons within the agIC, with strong trends seen in other regions, most notably layers 5/6  injected with a Cre-dependent mCherry reporter, or wild-type (WT) mice, were given a familiar (water) or novel (saccharin 0.5%) taste to drink. The mice were then killed 3 h later to perform whole-cell patch-clamp recordings of the intrinsic properties of SST or pyramidal neurons of the aIC A. Interem S29434  within tm S29434 H but notm S29434 I neuronsm S29434 B\u2013E. Addim S29434 I, and QRm S29434 F,G. Othem S29434 A\u2013H. Howem S29434 A. Togetht, GAD Cre-QR2 shRNA 9.306\u2009\u00b1\u20090.032 \u0394Ct) showed both significantly reduced QR2 expression  showed no improvement in novel taste memory  were injected with Cre-dependent QR2 shRNA-expressing viral vectors. In PV Cre animals  a \u223c6%  an \u223c7%  a \u223c6% I and in ) an \u223c7% K non-sig results , QR2 shR results J, while  results H. CombinLearning is a process defined by time, in which animals first acquire the information and later consolidate it molecularly (hours) and systemically (days to months; An intriguing possibility lies in the concept of a second wave of proteostasis rearrangement, occurring \u22653 h following learning, proposed last century by Hansjurgen Matthies , and latThe QR2 pathway has an intercellular and intracellular affect. The former is by reducing the firing rate of interneurons, thus transiently altering E/I balance. The latter is redox modulation, which may have any number of molecular targets . We haveHere, as well as in recent findings, SST interneurons have emerged as an important focal point in which it is necessary to identify both molecular and circuit wide mechanisms underlying memory consolidation. The QR2 pathway is one such mechanism that must be further investigated in these cells, as it is the molecular determinant by which SST interneurons galvanize the distinct memory that is formed to differentiate novel from familiar."}
+{"text": "Living marine resources (LMRs) contribute considerably to marine economies. Oceans continue to respond to the effects of global change, with environmental factors anticipated to impact future seafood production and its associated economic performance. Here we document novel relationships between primary productivity and LMR-based economics for US regional marine ecosystems and 64 international large marine ecosystems (LMEs). Intermediate relationships between production, total biomass, fisheries landings, revenue, and LMR-based employment are also elucidated. We found that all these factors were dependent on the amount of basal production in a given system. In addition, factors including human population, exploitation history, and governance interventions significantly influenced these relationships. As system productivity plays a foundational role in determining fisheries-based economics throughout global LMEs, greater accounting for these relationships has significant implications for global seafood sustainability and food security. Quantifying the direct link between primary production and fisheries economic performance serves to better inform ecosystem overfishing thresholds and their economic consequences. Further recognition and understanding of these relationships is key to ensuring that these connections are accounted for more effectively in sustainable management practices. Total LMR-related revenue and employment have increased over the past decades, with US marine fisheries currently valued at 210 billion USD and contributing on average ~\u20092.5% of US ocean gross domestic product (GDP)4. Fisheries economic production is limited by factors including ecosystem-level constraints that have not been fully explored in past investigations9. As oceans continue to undergo global change, environmental and ecological factors are anticipated to become more limiting on seafood production and affect marine economies10. Addressing these future challenges will require broader management approaches that consider both marine ecosystem dynamics and human dimensions in concert13. Here we examine these socio-ecological relationships more closely for both US and all International Large Marine Ecosystems (LMEs)8.In both the United States (US) and globally, living marine resources (LMRs) and associated fisheries are important contributors to ocean economies14, but have not always examined limiting factors. Previous studies have determined that sustainable fisheries harvest is related to the level of primary production  available within a given ecosystem16, with this limitation being a primary consideration when accounting for ecosystem overfishing18. Investigations in estuarine and lacustrine environments have similarly quantified the influence of nutrient loading (which impacts primary production) on fish production, with some hinting at ultimately limiting the magnitude of fisheries economics20. Yet there have been no examinations of the relationship among fisheries economic performance and primary productivity for marine waters. Theoretical studies, marine ecosystem models, and empirical examinations have estimated transfers of primary and higher-order production to biomass throughout trophic levels, identified shifts and perturbations in trophic composition, and demonstrated how LMR production and associated fisheries landings are ultimately limited by ecosystem production21. These relationships have also been shown to differ among regions, and are influenced by multiple human-related and environmental factors22. In this study, we detail these connections between primary productivity and economic performance at the LME scale.Seminal works have addressed the value and sustainability of natural capital, including its connections to multiple marine ecosystems24, primary production accounts for a major, foundational component of this variability , reinforcing that primary productivity is also positively related to total landings , Northeast US, Gulf of Mexico, and US Pacific  regions. Fisheries landings in the North Pacific are several times higher than in other US regions, while total revenue ranges have been generally comparable within North Pacific, Northeast US, and Gulf of Mexico systems  contain greater pelagic biomass supported by intermittently productive waters32, these productivity-biomass trends still generally persist. Observed parabolic relationships between productivity and biomass  and low landings and fisheries value (associated with overfishing)41 are observed  as in others due to the limitations of primary production. Ultimately, some regions are inherently more productive than others, which could translate into higher economic benefits that might be sustained over greater periods of time if exploitation rates remain at or below system thresholds34. In general, these relationships hold, but there are also outliers, which reinforce the importance of monitoring, understanding, and knowing the particular characteristics of each system. While regional LMR-based economies are also dependent on other human  and environmental factors, their dependence on system-level primary production should be recognized more strongly, particularly in terms of limiting catch and LMR-based economics. Thus, currently productive systems in the US such as the North Pacific and Northeast US could see future climate-related changes to their basal productivities, which could extend to the sustainability of these major LMR-based economies38. The same would apply to other regions around the globe.If these relationships generally hold true throughout all LMEs, then significant implications for LMR-based economics and management follow. Chiefly, these results show that for any given marine ecosystem, its associated economic performance  is ultimately limited by inherent basal ecosystem productivity, as has been demonstrated for fisheries landings44. There is a foundational role that system productivity plays in determining ecosystem biomass, and both directly affect sustainable harvest rates and LMR-based economics throughout the majority of US and international LMEs. Specifically, greater accounting for the linkages between basal production, system-level biomass, landings, ecosystem overfishing, and LMR-based revenue and employment in management is integral for the successful future sustainability of marine fisheries and their dependent communities. This focus is especially warranted given that altered global primary production is predicted to occur as a result of ongoing climate change effects to the world\u2019s ocean38. While these effects may not be observed for total, global oceanic primary production, future changes in the seasonality, distribution, and composition of the phytoplankton community may alter the productivity available to higher trophic levels within LMEs, with large ramifications for fisheries-associated economies40. In addition, continued natural and anthropogenic impacts to LMEs may lead to large-scale regime shifts, such as those currently observed for multiple systems, including the Baltic Sea, Benguela Current, California Current, and North Sea47. Redirected trophic flow and altered ecosystem structure would have significant effects on the primary production and biomass required to sustain catch, thereby affecting future harvest potential and its associated economics.The global ramifications of these findings are not trivial, reinforcing studies that have highlighted concerns about the future sustainability of fisheries production and economics18. Efforts to prevent ecosystem overfishing, particularly in areas that have already exceeded thresholds 48 would wisely account for the relationships noted here under future management. These approaches also account for natural and human-related factors that influence system-level production, biomass, landings, and economics, whose characterizations are especially important in explaining trends and interrelationships in outlier ecosystems. As compounding natural and human-related stressors continue to affect marine ecosystems, and multiple fisheries stocks remain overfished or of unknown status, the urgency for these more holistic and adaptive approaches cannot be overstated.These relationships have particular application for ecosystem overfishing, where management of system-level landings, revenue, and LMR-based economics are considered relative to system productivity and biomass49. However, these trends are expected to change during the twenty-first century as a result of multiple factors, including climate-related production shifts, continued exploitation, and consequential increases in fishing-associated costs51. Global landings have effectively plateaued51, and together with their revenue are ultimately limited by primary production. While limited quantity and high demand may cause prices to increase by an order of magnitude for particular taxa in a given ecosystem, the associated maximum potential revenues and their variability are ultimately constrained by the amount of catch available, which itself is constrained by primary production. Accounting for these factors and their influences and greater commitment to monitoring global primary production, especially in under-resourced areas with high human population growth, is warranted. These relationships are especially worth monitoring for marine ecosystems that contain the most lucrative fisheries resources, and in those having greatest risks of ecosystem overexploitation; the estimation, validation, and assessment of primary production dynamics is a minor cost relative to the value of those fisheries.Global fisheries value and employment continue to increase and have remained high over the past decades52, with strengthened understanding of the socioecological connections among marine ecosystems, natural phenomena, and human dimensions will allow for more thorough implementation and benefits of ecosystem-level approaches. To be able to consider these multiple factors together, a rethinking of how we view and approach both fisheries harvest and management is necessary. Efforts to shift from a traditional single-species focus to a more comprehensive ecosystem-based approach have been underway for several decades, with concrete implementation plans for US fisheries developed over the last few years52. The benefits of establishing maximum biomass removal caps, such as those for groundfish in the North Pacific53, continue to be observed. Their utility as a management tool for preventing ecosystem overfishing and ensuring sustainable LMR-based economics is reinforced by the findings of our study. The needs and benefits of ecosystem-based fisheries management are gaining in awareness, acceptance and implementation; these systematic marine management approaches better allow for the accounting of interconnected environmental, ecological, economic, and system-level tradeoffs52. Ultimately, greater recognition that foundational primary production within an ecosystem indeed limits its sustainable harvest, and thus the associated economic performance and benefits derived from its marine resources, is key to ensuring that these connections are accounted for more effectively in management approaches.Continuing to invest in broader, ecosystem-based approaches3, from primary productivity, total surveyed fish and invertebrate biomass, fisheries landings, economic revenue, and living marine resource (LMR)-based employment for US regions  and international LMEs, we used the following methods and data sources. Primary productivity estimates for each region or identified LME were measured by spatially characterizing annual regional primary productivity (g\u00a0C\u00a0m\u22122\u00a0year\u22121) and mean annual chlorophyll concentrations from NASA Ocean Color Web Data SeaWiFS years 1998\u20132007 and MODIS-Aqua years 2008\u20132014 (4\u00a0km resolution)54, using the Behrenfield and Falkowski Vertically Generalized Production Model (VGPM) estimation method56. Primary productivity values were averaged over published Large Marine Ecosystem (LME) areas42. These were then converted from units of Carbon to wet weight using a common scalar6. Nearshore benthic production throughout vegetated habitats  is important in some of these systems, but data are less comprehensively available as the extents of many of these areas are not well-mapped57. Given these limitations, and that the scale of this study occurred throughout the entire US EEZ, we did not incorporate benthic primary productivity estimates. Although performed in other studies that examine satellite data58, no additional correction for chlorophyll or productivity values were made for optically shallow waters.To evaluate relationships between components of the pathway proposed by Link and Marshak60. Given surveying methodology constraints, total biomass was not estimable for the US Caribbean, US Western Pacific and US South Atlantic regions. Additionally, all productivity, landings, and economic data for the US Western Pacific region were limited to the Hawaiian Insular platform to correspond with the identified LME. Trends in total regional commercial and recreational landings  reported by NOAA Fisheries  were examined (years 1998\u20132014). For social and economic indicators, regional trends in total LMR employment , including their related percent contributions to the total oceanic workforce of a given region as defined and recorded by the National Ocean Economics Program61, were calculated per year over the past decades (years 2005\u20132014). Additionally, the total USD value of all commercially landed species as reported by NOAA Fisheries and regional FMCs was examined4 and normalized to the year 2017 to align with other variables  from that timeframe. We note that although more recent data are available, we stopped in 2014 to be consistent for data availability across all data sets  and to avoid any potential misinterpretation for current fishery management actions. Although landed highly migratory species are included in NOAA fisheries statistics for all regions, including the Western Pacific, the numbers and values are underestimated in light of the international jurisdictions of these species and records of capture beyond US waters throughout their range62. Thus, they are not used here and were considered to be low estimates.During the same time period (1998\u20132014), annual estimates of total surveyed  fish and invertebrate biomass were summed from NOAA Fisheries seasonal fishery independent surveys of most US regions2. Regressions were chosen to illustrate relationships among production, biomass, landings, and economic factors through easily understood, stepwise means that demonstrated the logic sequence of linked dependencies across those variables. Although generalized additive models (GAMs), mixed models (GAMMs), or multivariate approaches may allow for more detailed and integrated investigation of these relationships, they were not the primary means of analysis given our objective of establishing sequential relationships among the variables. And particularly with respect to possible multivariate statistical approaches the separate multiple regressions used to examine these relationships simultaneously accounted for all factors and covariates  to examine the direct effect of a given independent variable  on a specific dependent variable (n\u2009=\u20095), and its significance . In this analysis, the separate effects of landings on biomass, and of biomass on landings, in relation to relevant independent variables and covariates were examined. Annual total biomass, fisheries revenue, and LMR-based employment (including their percent contributions to a given ocean economy) for each region were examined as a function of primary productivity in all regions. Relationships were similarly examined between total biomass and fisheries landings, and for fisheries revenue and LMR-based employment as a function of landings. In addition, separate multiple regressions were employed to examine relationships among these dependent variables and ten other covariates from all US regions. Factors included human population density, continental shelf area for each US region, fishing effort, standardized landings per shelf area (km\u22122), fisheries stock status, total Fishery Management Plan interventions , and percentage of EEZ permanently protected from fishing as of 2017. Human population density values were derived from NOAA Digital Coast US Census decadal data available within coastal counties from the past three decades . Coastal counties are defined by NOAA and the US Census Bureau as those counties where at least 15% of a county\u2019s total land area is located within the US coastal watershed63.Relationships for these metrics throughout the US EEZ were quantified using multiple non-linear and linear regressions; the form of regression chosen was that which had the lowest Akaike Information Criterion (AIC) and higher R64. Fishing effort (kilowatt sea days) was obtained from Watson65. Each managed US fisheries stock was examined for its June 2017 overfishing, overfished, or unknown status as reported for NOAA\u2019s Fish Stock Sustainability Index (FSSI) and non-FSSI stocks66, and totals and proportions of stocks of a given status were summarized per region. All regional states marine fisheries commissions and federal FMPs and fishing regulations were examined for the total number of modifications it had undergone since its original release (as of 2017), and all values were summed per region. Finally, the total spatial extent of marine protected areas where commercial and/or recreational fishing is prohibited was tallied per region using NOAA\u2019s Marine Protected Area Inventory . Afterward, the percent coverage of areas where commercial and/or recreational fishing is permanently prohibited was calculated relative to the EEZ of a given region (as of 2017).Population values were summed for all coastal counties within a given region and divided by county area. Total shelf area per region was calculated using NOAA Office of Coast Survey US maritime boundaries and limits spatial shapefile data65 and examined for the years 1998\u20132014 for each LME as related to primary productivity. LME primary productivity values were obtained as noted above for US LMEs. Relationships among LME productivity, fisheries landings, and USD value were cumulatively examined at continental and global scales.Additionally, for international LMEs (n\u2009=\u200964), total fisheries landings and USD value (standardized to year 2017) were obtained from WatsonSupplementary Figures."}
+{"text": "A. variegatum (46.3%), Rh. decoloratus (20.1%), A. cohaerens (15.7%), A. gemma (11.9%), and Rh. pulchellus (6.04%), respectively. The prevalence of tick infestation between different risk factors such as sex, age, and body condition of cattle was statistically significant (p < 0.05). The overall male-to-female ratio of ticks was 2.29\u2009:\u20091. Also, it was reported that, in A. variegatum, A. cohaerens, and A. gemma, the number of male exceeded that of female, but female number exceeded male number in case of Rh. decoloratus. The result also reported difference in attachment site preference, for example, Amblyomma genus was attached mostly to the scrotum/udder and axial and Rh. pulchellus was specified on the ear and perianal area, while Rh. decoloratus was non site selective. In conclusion, findings of this study suggest that ticks were the most important problems of cattle of the study areas. Therefore, the increasing threat of ticks warrants urgent strategic control including application of acaricides and creation of awareness among livestock owners about the veterinary importance of ticks for the integrated tick control.A cross-sectional study was conducted from December 2017 to April 2018 to determine the prevalence and identify major species of ixodid ticks of cattle and tick burden of different sex, age, breed, and body condition of cattle. Standard physical and direct stereomicroscopy techniques were employed for identification of tick species. During the study period, a total of 353 cattle were examined for presence of ticks and around 447 ticks were collected. The study showed that 34.3% cattle were infested with one or more tick species. The study reported different species of ticks in the order of their prevalence:  Livestock mainly cattle in Ethiopia represent the pillar of the economy and plays vital roles in generating income to farmers, ensuring food security, contributing to asset, social, cultural, and environmental values , 2. DespAmong the ectoparasites, ticks are well known for substantial effects in livestock production. Ticks are very common and extensively distributed in all agroecological zones especially in tropical and subtropical areas including Ethiopia . They arAmblyomma, Rhipicephalus, Haemaphysalis, Rhipicephalus (Boophilus), and Hyalomma [Amblyomma variegatum and Rhipicephalus (Boophilus) decoloratus are considered to be the most prevalent and predominant tick species in higher rainfall season in Ethiopia [A. variegatum was the most common and widely distributed cattle tick in Ethiopia [Ticks that are most important to domestic animals' health in Africa include about seven genera and forty species . In EthiHyalomma . SeveralHyalomma , 59.6% pHyalomma , and 74.Hyalomma . The seaEthiopia . From maEthiopia . Dry envEthiopia .Rh. decoloratus (47.8%), A. variegatum (28.4%), A. gemma (12.5%), Rh. pulchellus (9.3%), and Rhipicephalus evertsi evertsi (2.02%) in Haramaya district. Another author reported 33.2% prevalence and three species of hard ticks, A. variegatum, A. cohaerens, Rh. evertsi evertsi, Rh. pulchellus, and Rh. decoloratus were recorded. This study also showed that A. variegatum was the most abundant of all tick species comprising 38.9% of the collected ticks in Haramaya district [Boophilus (51.0%), Amblyomma (58.3%), Hyalomma (48.2%), and Rhipicephalus (53.1%) [A study carried out six years before by Desalegn et al.  in the sdistrict . A very  (53.1%) . There iThe survey of identification of major ixodid ticks of cattle was conducted in Haramaya district, Ethiopia. Haramaya district is located 506\u2009km east of Addis Ababa capital of Ethiopia. According to Haramaya district agricultural statistics information, the district has about 115,989 cattle, 88,144 sheep, 133,520 goats, 33,466 donkeys, 578 camels, and 137,545 chickens. The production system of the district is mixed type. Topographically, it is situated at an altitude of 1600\u20132100\u2009m above sea level with the mean annual temperature and relative humidity of 18\u00b0C and 65%, respectively. The district receives an annual rainfall of approximately 870\u2009mm with a range of 560\u20131260\u2009mm and bimodal distribution pattern, picking in mid-April and mid-August. The vegetation that constitutes the available pasture lands in this area are predominantly native grasses and legumes interspersed with open acacia shrub land. Geographically, it is located at 9\u00b024\u2009N latitude and 42\u00b001\u2009E longitude, and ecologically, the area has 65% midland and 35% lowland zones [n\u2009=\u2009sample size, Pexp\u2009=\u2009expected prevalence (0.25), and d\u2009=\u2009desired precision (0.05).Study cattle were sampled using simple random sampling technique at feeding burn from Haramaya University beef farm, and cattle were taken to Haramaya town veterinary clinic. The required sample size was determined by Thrusfeild  formula According to Desalegn et al. , the preA cross-sectional study was conducted from December 2017 to April 2018 to determine the prevalence and identify major species of ixodid ticks of cattle and tick burden of different sex, age, breed, and body condition of cattle. In this study, two study areas selected, namely, Haramaya University farm and Haramaya veterinary clinic, because of the easy access to cattle from different kebeles. Animals were randomly selected during the visit to Haramaya University farm using the lottery method, while Haramaya veterinary clinic animals were randomly sampled from cattle visiting for different diseases except for the case of ectoparasites spray. Different color dyes were used to mark sampled cattle to avoid repeated sampling in the clinic, while ear tags were used in Haramaya University farm. Age was determined using owner's information and dentition parameters. Age of the animals were determined as young (<1 year), adult (1\u20133 years), and old (>3 years) . The bodSample collection was made after proper physical restraining of the animals; detectable ticks were carefully removed from the host for identification and count using hand. The whole body surface of the animals was examined for the presence or absence of ticks. Ticks were collected from different body parts of cattle such as head, ear, dewlap, scrotum, udder, perianal region, and the tail, which were kept separately in well-labeled bottle. During collection, ticks were removed manually from different attachment sites of the animal body by a rotating manner to retain their body parts for identification. Data collection format was used to register the data during tick collection, and proper labeling was made on universal bottles with a permanent marker. Code of animal, species, sex, age, body condition, and sites of attachment were included in the labeling. The collected ticks were placed into the universal bottle containing 70% ethanol for preservation and transported to Haramaya University Veterinary Parasitology Laboratory where ticks were counted and genus and species level was identified using a stereomicroscope, according to standard identification keys given by Walker et al. .\u03c72) test was used to point out the possible association of factors with the prevalence of tick infestations. Effects were reported as statistically significant in all cases if the value is less than 0.05 at 95% confidence interval (CI).All the data were entered on Microsoft Excel data base system, and they were analyzed by using Statistical Package for Social Sciences (SPSS) Version 20. Descriptive statistics was used to determine the prevalence of tick infestation in cattle. The overall prevalence of tick was determined by dividing the number of positive animals by total sample size and was expressed as percentage. The chi-square  than in female cattle 28.6%) with statistical significance difference p < 0.05 . This fi.6% with p \u2264 0.05). This finding was strengthened by the finding of Desalegn et al. [The proportion of tick infestation was higher in older than in adult and young age groups . Differen et al.  who repon et al. , 23, 31 p < 0.05    in tick  minimal , 34. In A. variegatum and the least one was Rh. pulchellus, as shown in A. variegatum (46.3%), Rh. decoloratus (20.1%), A. cohaerens (15.7%), A. gemma (11.9%), and Rh. pulchellus (6.04%), respectively. This finding agreed with Kassa and Yalew [A. variegatum was the most abundant (38.87%), Rh. decoloratus (31.54%) was the second, and Rh. pulchellus was the least (6.64%). Several authors also strengthened the report that A. variegatum is the most common and widely distributed cattle tick in Ethiopia and African countries [Amblyomma variegatum occurs in areas with a wide variety of climates ranging from highland Savannah to lowlands [A. variegatum has a great economic importance because it is an efficient vector of Cowdria ruminantium, the causative agent of cowdriosis or heartwater in cattle [Rickettsia, Coxiella burnetii, and Borrelia spp [A. cohaerens was found to be the most abundant tick species with a prevalence range of 36.4\u201350.5% in different parts of Ethiopia. This difference can be due to seasonal variation and agroecological difference.Overall, a total of 447 ticks were collected from 121 positive cattle. The most abundant species of tick was nd Yalew  who repooloratus .1%, A. countries , 31, 35.ohaerens .7%, A. gbundant 3.87%, Rh.lowlands . A. varin cattle , the gren cattle , 38, andelia spp . In contelia spp , Musa anelia spp , and Belelia spp  reportedRh. decoloratus; the finding coincides with reports of different researchers. In Ethiopia, Rh. (Boophilus) decoloratus is the widely distributed tick species in different agroecological conditions and seasons of the country [Rh. decoloratus is similar to A. variegatum accompanied by wetter highlands and subhighlands receiving more than 800\u2009mm rainfall [The second abundant tick was  country , 28, 41.rainfall .A. gemma and Rh. pulchellus reported in the study area is consistent with Pegram et al. [A. gemma and Rh. pulchellus are confined to semiarid areas and the lowland tick densities are usually greater than those in the highlands. It is also reported that A. gemma is an important tick of cattle and camels in eastern and southeastern parts of Ethiopia particularly in Afar, Somalia, and Harar and Eastern Tigray, Amhara, and SNNP regional states [A. gemma was obviously associated with dry types of vegetation or semiarid rangelands and in lowland areas [A. gemma is widely distributed in woodland, bushland, and grassland in arid and semiarid area between the altitude 500\u20131750 meter above sea level and receiving 350\u2013750\u2009mm annual rainfall [Rh. pulchellus has been reported as the most predominant tick species on camels in eastern Ethiopia, on small ruminants in eastern part of Ethiopia, and on cattle in Borena zone in Oromia region [R. pulchellus has been associated with a wide variety of pathogenic organisms affecting both animals and human, i.e., anaplasmosis, brucellosis, and anthrax [Theileria taurotragi which causes benign bovine theileriosis. It has been implicated as a probable vector of Nairobi sheep disease that exists in north of Somali [Rickettsia conorii, causing tick typhus, and transmission of the virus of Crimean-Congo haemorrhagic fever. It may occur on some hosts in sufficient numbers to cause direct parasitic harm [The lowest proportion of m et al.  who repol states . A. gemmnd areas , 43. A. rainfall . Rh. pula region , 45. R.  anthrax . This tif Somali . It can tic harm .A. variegatum, A. cohaerens, and A. gemma, the number of male exceeded that of female, but female number exceeded male number in case of Rh. decoloratus. Generally, the overall male-to-female ratio was 2.29\u2009:\u20091 (Rh. decoloratus outnumbered males in this study probably due to small size of male which may not be seen during collection [In 2.29\u2009:\u20091 . This fillection .A. variegatum, A. cohaerens, and A. gemma attach mostly to the scrotum/udder and axial but uncommon in other areas. Rh. decoloratus was distributed in all parts of cattle body except rare case on axial, scrotum, and udder, while Rh. pulchellus was frequently attached to perianal and inner parts of ear (Rh. decoloratus was found on the dewlap, udder, belly, head, neck, back, and scrotum, Rh. pulchellus showed high preference to the inside ear and anogenital region of the body. It was also reported earlier by Kassa and Yalew [With regard to predilection site for attachment, different tick species show different site preferences. s of ear . The finnd Yalew . Site prnd Yalew . Genera nd Yalew . Generalnd Yalew .A. variegatum was found to be the most prevalent tick species identified. In addition, Rh. decoloratus, A. cohaerens, A. gemma, and Rh. pulchellus were also reported. Animal-related factors such as age, sex, breed, and body condition showed significant difference in tick infestation. Therefore, ticks are economically very important ectoparasites, which cause direct and indirect substantial economy losses to livestock sectors. Taking into account the effects of tick on livestock productivity, it is important to minimize the impact through effective tick control program which should be formulated and implemented at national and regional level based on the distribution pattern of ticks and factors responsible for their distribution. Moreover, attention should be given in creating community awareness about the impact of ticks, health care services, and management practices of cattle so as to control ticks which interns control problems that affect livestock production as a result of tick infestation.The present study indicated 34.3% overall prevalence of tick in cattle in the study area."}
+{"text": "In Ethiopia, ixodid ticks and associated tick-borne pathogens (TBPs) are of great importance from both a veterinary and public health point of view. This review aimed at compiling available published data on the distribution of ixodid tick species and TBPs in the country.A standard review approach was employed using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. Published peer-reviewed articles and theses/dissertations reporting on ixodid ticks and TBPs in Ethiopia were searched using different keywords in many electronic databases including PubMed, Scopus, Web of Science, Google Scholar, African Journals OnLine, and institutional repositories. Articles were screened based on inclusion and exclusion criteria using the PRISMA flowchart. Data were retrieved from eligible articles and recorded in a preformed data record sheet. Descriptive statistics were employed to present data using graphs. QGIS (Quantum GIS) software version 3.4.5 was used to show the distribution of ixodid tick species and TBPs.Anaplasma, two Ehrlichia, two Rickettsia, five Theileria, two Babesia, and one Coxiella species are the major pathogens in both livestock and humans.Overall, 35 articles that met the inclusion criteria were included in this review. Of these, 24 articles report only on ixodid ticks of domestic animals, six articles report only on TBPs in livestock or ticks, and five articles report on both ticks and TBPs in either animals or ticks. Of these studies, 54% were in the Oromia region, while only 3% of studies were in the Benishangul-Gumuz region. The Gambela region lacked studies on ticks and TBPs. At least 19 ixodid tick species have been recorded from different domestic animals including cattle, small ruminants, donkeys, horses, camels, dogs, and cats. Morphological characterization appears to be the sole method of tick species identification in the country. The distribution and abundance of specific tick species depend on geographical locations and agroecological factors. Sixteen molecularly confirmed TBPs have been identified in animal and tick tissue using molecular methods from only four administrative regions, despite the wide distribution of ticks. Among TBPs, five Many ixodid ticks circulate in a wide geographical zone of Ethiopia. However, the limited reports on TBPs at the country level in general, and the absence of either tick or TBP reports around the border region with neighboring countries in particular, highlights the need for further study.The online version contains supplementary material available at 10.1186/s13071-022-05221-x. In Ethiopia, the contribution of domestic animals to socioeconomic well-being remains substantial. Cattle, sheep, and goats contribute to the national gross domestic product (GDP) by providing meat and milk for local and international markets . They arIxodid ticks are a group of ectoparasites whose life-cycle depends on the vertebrate host. The importance of ixodid ticks as ectoparasites is their habit of blood-feeding on domestic and wild animals and humans to complete their life-cycle. In the feeding process, the affected host may become anemic if the parasite burden is high  or may bRhipicephalus appendiculatus, presumed to be prevalent in East African countries including in the highland region of Kenya, does not thrive in the border area of two countries where the unrestricted movement of livestock takes place. Unfavorable natural climatic barriers might have prevented the introduction of this tick to Ethiopia from neighboring Kenya [Ticks are distributed worldwide, although the distribution of individual tick species varies according to climatic factors, such as temperature, humidity, altitude, and vegetation types. For instance, the tick ng Kenya . This suBabesia bovis, which causes bovine babesiosis in several regions, was wrongly reported from the southwestern part of Ethiopia, where neither of its vectors Rhipicephalus (Boophilus) microplus nor Rhipicephalus (Boophilus) annulatus was reported [B. bovis in the area using molecular techniques. These examples illustrate the possibility of predicting TBPs without requiring expensive laboratory techniques when their vector distribution is known.Information concerning the distribution and abundance of ticks is of paramount importance, as it helps to predict the occurrence of tick-borne infections in animals so that control measures against ticks and associated infections can be set. This information can be used as a criterion during the morphological identification of tick fauna by inexperienced researchers . Moreovereported . A lettereported . Furtherreported  confirmeIndigenous breeds of livestock are resilient to ticks and TBPs, but genetically improved breeds, which contribute to sustainable food security, are highly susceptible , 15. VarTherefore, this literature review is designed to present local secondary data on the distribution and abundance of ixodid tick species infesting various domestic animals in Ethiopia. It also demonstrates available evidence on TBP distribution in the country. This information is of paramount importance in designing control measures against ticks and TBPs in domestic animals.The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) checklist and flowchart for data extraction and inclusion of eligible studies, respectively, were used for this study .Literature databases including PubMed, Web of Science, Scopus, African Journals OnLine (AJOL), and Google Scholar were used to retrieve published articles on ixodid ticks and TBPs affecting domestic animals in Ethiopia. To maximize the search, the key terms \u201cixodid ticks of domestic animals,\u201d \u201cticks of livestock,\u201d \u201cectoparasite of domestic animals,\u201d \u201cectoparasite of livestock,\u201d ticks of ruminants,\u201d \u201cticks of equines,\u201d \u201cticks of cats and dogs,\u201d ticks of the camel,\u201d \u201ctick and tick-borne disease of livestock,\u201d AND \u201cEthiopia\u201d were used. All works published from 2001 to October 30, 2020, were included. In addition, PhD/MSc theses that had not yet been published in peer-reviewed journals were searched from different institutional repository databases. A systematic screening of available pieces of literature was conducted by assessing the title, abstract, and detailed aspects of manuscripts.All articles indexed in the aforementioned databases that report on ixodid ticks alone or ixodid ticks in combination with other ectoparasites and TBPs in ticks and/or in domestic animals, including ruminants, camels, equines, and pets (dogs and cats) in Ethiopia were included. To investigate differences in the geographical and host abundance of tick species, only those articles that revealed the proportion of individual tick species in infested animals and those articles reporting TBPs in ticks/animals were included. However, studies in which ticks were not identified to species level and tick species proportions were not clearly reported were excluded. Mendeley Desktop version 1.15.3 was used to catalog the initial literature search results and manage citations.Bereha), semi-arid (Kola), warm sub-humid (Woinadega), cool-humid (Dega), or cold-moist (Wurch) based on altitude, average annual rainfall, and temperature of the study districts [From eligible studies, the following data were extracted: the first author, year of publication, geographical location, administrative region of study, host species, proportion of tick species on animals, TBPs in ticks or animals, agroecology, and georeference of the study sites. The georeference data were obtained from the internet if it was not mentioned in the publication. The geographical locations of study sites were systematically classified into five categories, namely central, western, eastern, southern, and northern Ethiopia, based on the distance and direction of specific study districts from the capital city (Addis Ababa). Accordingly, studies conducted within a radius of 150\u00a0km from Addis Ababa were categorized as central. The designations southern, northern, eastern, and western were used for studies reported from areas more than 150\u00a0km from Addis Ababa in their respective directions. The agroecology of the study area was classified as arid  software version 3.4.5  was used to present the geographical distribution of individual tick species and TBPs on the map.Two volunteer individuals see  section n\u2009=\u200945) were subjected to detailed assessment based on the eligibility criteria. Finally, 10 studies that did not meet eligibility criteria were removed, leaving only 35 studies for review.The PRISMA flow diagram indicating the selection process for eligible studies is presented in Fig.\u00a0n\u2009=\u200918), Amhara , Southern Nations, Nationalities, and Peoples\u2019 Region (SNNPR) , Tigray , Somali , and Benishangul-Gumuz (B-G) , and one study in both Amhara and Oromia districts (3%) . They were from six administrative regions of the country, including Oromia  Tables , 3. Of t) Tables .Table 2LTicks were collected from different domestic animals, including cattle, donkeys, horses, sheep, goats, camels, dogs, and cats. TBPs were detected from either the blood of the host or tick tissue. Molecular techniques  were employed for the identification of TBPs in all reports but one . No studAmblyomma variegatum infests a wide range of domestic animals including cattle, donkeys, horses, camels, dogs, cats, sheep, and goats. However, the infestation burden was highest in donkeys, followed by cattle, goats, sheep, dogs, horses, camels, and cats  decoloratus affects all domestic animals included in this report except cats. Donkeys are the host most preferred by this tick, followed by goats, cattle, sheep, horses, camels, and dogs  decoloratus is like Rh. evertsi evertsi in that it covers wide geographical locations and is presumed to exist in almost all agroecologies. Nonetheless, its higher burden in warm sub-humid agroecology indicates its importance in this area. A similar finding was reported in the warm humid forest zone of Cameroon [Cameroon . Thus, sCameroon , and thiRh. sanguineus s.l. in Ethiopia is primarily associated with dogs but can infest other animals as well. The same observation was reported in Sudan, where Rh. sanguineus s.l. infests cattle [As in other parts of the world, s cattle . The ides cattle  is a goos cattle  thus mayRh. pravus in the lowland arid and semi-arid agroecologies of Ethiopia justifies its drought-resistant nature. This allows its survival during the dry season, thus becoming endemic throughout the year in the environment. Infestation of this tick on cattle and other domestic animals suggests that it is not host-specific. The high burden of this tick next to Rh. pulchellus on camels reared in the lowland arid agroecological zone of Kenya [A significant abundance of of Kenya  supportsRhipicephalus species identified in Ethiopia, Rh. lunulatus is seldom reported, found only in Oromia and southern Ethiopia. The absence of this tick in many surveyed regions and lack of data within the past 10\u201320\u00a0years remains a great concern. This might be linked with climate change that could have hampered the ecology and biology of the tick. In Nigeria, one study [Rh. lunulatus on cattle in sub-humid agroecology during the rainy season. However, the issue of seasonal impact on its abundance needs further investigation. Moreover, the pathogens they may transmit in livestock have never been identified. Hence, studies on the seasonal abundance and TBPs in this tick are highly encouraged.Unlike other ne study  identifiRh. muhsamae thrives in the lowland arid and semi-arid agroecology of Ethiopia. The presence of this tick has been demonstrated in almost all regions in Sudan [Rhipicephalus species, its limited distribution only in Ethiopia and Sudan raises questions as to the specific factors contributing to its distribution.As with other drought-resistant ticks, in Sudan . NonetheRhipicephalus praetextatus appears to coexist with many drought-resistant Rhipicephalus species in Ethiopia. Its geographical distribution in lowland semi-arid agroecology in Ethiopia accords with reports from neighboring countries, such as Kenya and Sudan [nd Sudan , 57.Anaplasma species, namely Anaplasma centrale, Anaplasma marginale, Anaplasma sp. \u2018Omatjenne,\u2019 Anaplasma phagocytophilum, and Anaplasma ovis, along with other unidentified species [Anaplasma pathogens, almost all are endemic in the western region of Ethiopia, as many suitable tick vectors are present in this region. However, some of them, such as A. centrale, A. marginale, and A. sp. \u2018Omatjenne,\u2019 are found to infect livestock in central and southern areas of the country, suggesting the existence of common tick vectors for these pathogens in western, central, and southern geographical locations. Moreover, reports of A. ovis and A. phagocytophilum infections of livestock in western and central regions confirm the occurrence of their respective tick vectors in the areas. Teshale et al. [Rh. evertsi evertsi and Rh. (B.) decoloratus are vectors of A. ovis. In Kenya, this pathogen was identified in Am. variegatum, Rh. pulchellus, and Am. gemma [A. ovis should be suspected in areas in which these ticks are prevalent. They are abundant in almost all geographical locations, including in western, southern, and central areas. Meanwhile, Am. cohaerens, which circulates in many areas including in western and central regions, was found to transmit A. marginale [A. phagocytophilum [Ixodes ricinus [A. centrale and A. sp. \u2018Omatjenne\u2019 could not be identified. From the perspective of controlling ticks and TBPs, the vectors for these pathogens should be determined.To date, five  species , have bee et al.  demonstrm. gemma , provingarginale  and A. ptophilum , which i ricinus , confirm, Ehrlichia ruminantium, and other unconfirmed species were detected in Rh. (B.) decoloratus in the southwestern region of Ethiopia. Bovine infection with this pathogen had been elucidated in the southwestern region of the country [Am. gemma, in the eastern Somali region [E. ruminantium. In neighboring Sudan, Am. variegatum and Am. lepidum have been implicated as the major vectors [Am. gemma, Am. variegatum, and Rh. evertsi evertsi were found to harbor E. ruminantium in Kenya [Ehrlichia species in Rh. (B.) decoloratus collected from the southwestern area could be E. minasensis, as infection of livestock with this agent was discovered in the same region [The causative agent of bovine ehrlichiosis country . Interesi region ; nonethe vectors , whereasin Kenya . This eve region .Rickettsia africae, an agent for African tick bite fever (ATBF), and unconfirmed species have been reported from western and eastern regions. Rhipicephalus (B.) decoloratus and Rh. evertsi evertsi are the major vectors for R. africae in western locations [Am. gemma was found to be a vector responsible for this pathogen in the eastern Somali region [Am. variegatum, Am. gemma, and Rh. evertsi evertsi are implicated as vectors of R. africae [ocations , while Ai region . In Keny africae , 77. Thi africae .Rickettsia aeschlimannii, an agent of spotted fever group (SFG) rickettsiosis, was confirmed in three tick species, including Hy. impeltatum, Hy. rufipes, and Hy. truncatum, in eastern Somali using a molecular technique [Hy. truncatum, Hy. rufipes, and Rh. pulchellus in Kenya [Hyalomma species are implicated as vectors for R. aeschlimannii in Egypt and Algeria, suggesting potential vector competence of Hyalomma species. Thus, SFG rickettsiosis should be considered when diagnosing patients with a history of tick bites in regions where Hyalomma species are abundant. Furthermore, Hyalomma species are found to carry Francisella-like endosymbiont [echnique , 79. Simin Kenya . In addisymbiont , which iTheileria species  decoloratus and Rh. evertsi evertsi appear to harbor a mild form of Theileria pathogens. However, a pathogenic strain, Theileria annulata, that causes tropical theileriosis is endemic in the southern region of the country [More than five molecularly confirmed  country .T. annulata in this region is interesting. Its vector is not yet identified in the country; however, the occurrence of this pathogen in southern areas reveals the existence of a specific tick, which is deemed uncommon in western regions. In Sudan, researchers were able to identify T. annulata from Hy. anatolicum [Hy. anatolicum might be a potential vector for T. annulata in Ethiopia. Therefore, surveillance of tropical theileriosis should be considered in the area in which Hy. anatolicum circulates.There have been many studies on TBPs in the western area, but the absence of atolicum , which iBabesia species in Ethiopia. Of the Babesia species, only B. bigemina seems to have been implicated as an agent of bovine babesiosis, as it was confirmed in cattle with a molecular technique [B. bovis in the same area by microscopic examination [Rh. (B.) decoloratus, Rh. (B.) annulatus, and Rh. (B.) microplus are thought to be responsible vectors for B. bigemina in many tropical countries, while only Rh. (B.) annulatus and Rh. (B.) microplus are major vectors for B. bovis in Latin America and other parts of the world [Rh. (B.) annulatus and/or Rh. (B.) microplus in the southwestern region of Ethiopia and low sensitivity of microscopic examination for pathogen detection might have contributed to misleading positive diagnosis in the previous study. Reporting from Sudan confirms the presence of B. bovis and B. bigemina in Rh. (B.) annulatus, and the absence of B. bovis in Rh. (B.) decoloratus supports the argument that Rh. (B.) decoloratus does not transmit the agent B. bovis. In addition, B. caballi has been isolated from the tick species [There have been controversial issues as regards the reporting of echnique . On the mination . The tiche world , 81. The species . HoweverCoxiella burnetii, a zoonotic pathogen that causes Q fever in humans, was detected from Ethiopia in hard ticks using molecular techniques [C. burnetii in the country. However, a significantly higher infection rate occurs in Am. gemma and Rh. pulchellus [. In addition, other Amblyomma species, including Am. variegatum and Am. cohaerens, have been found to carry the C. burnetii genotype. This highlights that Am. gemma, Am. variegatum, and Am. cohaerens seem to be the major vectors in southwestern Ethiopia given their high burden in this region. Moreover, the seropositivity of Q fever in humans and livestock in eastern Somali [chniques , 82. Then Somali  and the n Somali  reveals Rh. appendiculatus, a vector of East Coast fever, is prevalent in South Sudan, a country bordering Gambela, Ethiopia. TBPs have been reported from only a few administrative regions of Ethiopia, even though ticks are widely distributed in the country. Thus, the author encourages future studies to detect TBPs from ticks and livestock using molecular techniques to gain a clearer picture of TBDs and to aid in the design of control techniques. Individual tick distribution and abundance depend on the nature of agroecology and the presence of a suitable host. This helps identify the areas of lowest and highest risk for better management of genetically improved livestock breeds. Moreover, all ixodid ticks can infest a wide range of mammalian hosts, including humans; however, there has never been a report on ticks infesting wild animals in the country. Hence, the contribution of wild animals to the epidemiology of ixodid ticks and TBPs in the country should be established.Ixodid ticks and TBPs pose significant health threats to livestock in Ethiopia. To date, ticks have been reported from all administrative regions of the country except Gambela and Afar. This highlights the need for future study on ticks and TBPs in these regions in general and the Gambela region in particular, as Additional file 1: Fig. S1. Abundance of tick species on different domestic animals in Ethiopia.Additional file 2: Fig. S2. Abundance of tick species in various geographic locations of Ethiopia.Additional file 3: Fig. S3. Abundance of tick species in different agroecological zones of study areas of Ethiopia."}
+{"text": "Mentha spicata is one of the most popular species in the genus, and it is of great interest as a gastrointestinal and sedative agent in the folk medicine system. In this study, different M. spicata extracts, obtained by the use of four solvents  were chemically characterized using HPLC-ESI-MS n, which allowed for identification of 27 phenolic compounds. The extracts\u2019 antioxidant and enzyme inhibitory properties were investigated. In addition, neuroprotective effects were evaluated in hypothalamic HypoE22 cells, and the ability of the extracts to prevent the hydrogen peroxide-induced degradation of dopamine and serotonin was observed. The best antioxidant effect was achieved for all the extraction methods using acetone/water as a solvent. These extracts were the richest in acacetin, eriodictyol, hesperidin, sagerinic acid, naringenin, luteolin, chlorogenic acid, chrysoeriol and apigenin. The intrinsic antioxidant and enzyme inhibition properties of the acetone/water extract could also explain, albeit partially, its efficacy in preventing prostaglandin E2 overproduction and dopamine depletion (82.9% turnover reduction) in HypoE22 cells exposed to hydrogen peroxide. Thus, our observations can provide a scientific confirmation of the neuromodulatory and neuroprotective effects of M. spicata. Mentha is one of the most important genera of the Lamiaceae family, and its members are generally common in the temperate zone. The genus Mentha is represented worldwide with about 38 species \u2212 at m/z 717 and fragment ions at m/z 537 and 519. This fragmentation pattern was already reported for salvianolic acids B, E and L \u2013 at m/z 551 and fragment ions at m/z 519 and 359, was tentatively characterized as monomethyl lithospermate [The characterization of the phytochemicals  was carrmoieties . Compounand 135) . Compoun[m/z 271  after th E and L , but the spicata . Compoun spicata . Compounm/z 268) , so it wspermate . CompounM. spicata is rich in polar compounds. In all acetone/water extracts, 20 compounds were identified as common. Citric and caffeic acids were only detected in MAC\u2013acetone/water extract. In acetone/water extracts, chrysoeriol and luteolin were only found in HAE and MAC  were also found. For all extraction techniques, the use of acetone/water as an extractant was clearly more efficient than the use of water (see TIPC in O-rutinoside (compound 7) and luteolin-O-hexoside and luteolin-O-glucuronide (compounds 12 and 13). However, in the acetone/water extracts, the concentrations of sagerinic acid (compound 20) were similar to those of luteolin glycosides and approximately 50% of eriodictyol-O-rutinoside.The main components of the extracts were quantified by HPLC with UV detection. Commercial standards of caffeic acid , chlorogenic acid (320 nm), hesperidin  and apigenin  were used. Calibration graphs were constructed in the 0.5\u2013100 mg/L range and Relative Standard Deviations lower than 5% were observed in all cases. The results are shown in M. spicata extracts were investigated by cell-free chemical methods , while in MAC, no significant difference was observed between the chloroform (2.38 mmol TE/g) and acetone (2.54 mmol TE/g) extracts.The phosphomolybdenum (PBD) assay is based on the ability of anti-oxidant compounds to reduce Mo (VI) to Mo (V). Since all antioxidants (phenolics and non-phenolics) play a reducing role in the PBD assay, this test could be considered as one of the assays describing the total antioxidant capacity of the extracts. In contrast to free radical scavenger and reducing power assays, each solvent registers different results when combined with a different extraction method. For example, among the extracts obtained by the homogenizer-assisted method, the best ability in the PBD assay was found in hexane extract (3.23 mmol TE/g). The two highest effects for ultrasound assisted extracts and maceration were determined by acetone/water (3.48 mmol TE/g) and acetone (2.54 mmol TE/g), respectively. In addition, all extracts from HAE and UAE showed statistical differences in the phosphomolybdenum assay (p > 0.05).In the metal chelating assay, the ability of the extract to chelate transition metals, which is due to the inhibition of the production of hydroxyl radicals in the Fenton reaction, was measured. The hexane and acetone extracts by the homogenizer-assisted method showed the strongest metal chelating abilities, with values of 24.09 and 21.80 mg EDTAE/g, respectively. The acetone extract values were significantly higher than the other solvent when combined with both UAE and MAC methods . In HAE, the lowest values were provided by the chloroform (3.30 mg EDTAE/g) and acetone/water (7.98 mg EDTAE/g) extracts, and they exhibited similar abilities  on the extract composition [When the results of the antioxidant assays are evaluated together, the free radical scavenging and the reducing power appear in line with their total phenolic content. This fact was also confirmed by Pearson\u2019s correlation analysis, and the results are shown in position ,59,60. As for the quantitative analysis of chemical constituents in the tested extracts, acetone and acetone/water extracts contained more compounds than hexane and chloroform ones. For example, rutin and chlorogenic acid were only present in acetone/water extracts in the tested extraction methods. As shown in M. spicata extracts towards enzymes, known for playing master roles in diverse chronic and degenerative pathologies, were evaluated. Specifically, we measured the inhibition of the activity of \u03b1-amylase and \u03b1-glucosidase as targets of antidiabetic therapy, tyrosinase and cholinesterases (AChE and BChE), which play master roles in skin disorders and Alzheimer\u2019s disease (AD), respectively [In the present study, the inhibitory properties of ectively ,68,69,70When the results of enzyme inhibition assays were correlated to the total phenolic content of the extracts, only \u03b1-glucosidase inhibitory effect was moderately correlated with the flavonoid content (R > 0.5). The correlation value can be associated with the presence of some flavonoids such as eriodictyol, luteolin and apigenin, which had higher concentrations in the acetone/water extracts. These compounds were described by several researchers as potent inhibitors of \u03b1-glucosidase ,72. SohrIn addition to flavonoids, the phenolic acids, including caffeic, chlorogenic and sagerinic acids, could form the basis of the observed enzyme inhibitory properties. Several researchers reported that chlorogenic acid possesses a large spectrum of enzyme inhibitory properties ,74,75,76Mentha, including M. spicata [M. spicata are still unexplored.In the literature, several studies have examined the effects of the enzyme inhibitory properties of the members of the genus  spicata ,80,81,82M. spicata is considered a powerful source of natural antioxidants and enzyme inhibition agents, and the obtained results may open new avenues for future pharmaceutical applications.Considering the aforementioned biological properties of the extracts, 2O2 300 \u00b5M . The HAE acetone/water extract, in particular, showed the most relevant effects in protecting the cells from death induced by H2O2. This extract was then chosen for further analysis.All twelve extracts were administered to HypoE22 cells at 10, 100 and 1000 \u00b5g/mL, and cell viability was evaluated both in basal condition and when cells were exposed to an oxidative stress, represented by 3 h exposure to H2 300 \u00b5M A\u2013C. Half2O2 300 \u00b5M and treated with HAE acetone/water extract at 1\u2013100 \u00b5g/mL for 48 h, and the extracellular levels of 5-HT, 5HIIA, DA, DOPAC and L-dopa were evaluated in the supernatants. The hydrogen peroxide stimulus was able to increase the turnover of 5-HT and DA, represented by the ratios of 5HIIA/5-HT , in the 2020 summer season (August). The plants were confirmed by a botanist in Selcuk University, Science Faculty, Konya, Turkey, and one voucher specimen (N. GA-20-001) has been deposited in the Department of Biology, Selcuk University. The aerial parts of the plant samples were dried in shade conditions for 10 days at room temperature. The plant samples were powdered by using a laboratory mill and the powdered plant samples were stored in dark conditions at room temperature. In the present work, we used hexane, chloroform, acetone and acetone/water (70%) as solvents. Homogenizer-assisted extraction (HAE), ultrasound-assisted extraction (UAE) and maceration (MAC) were used as extraction methods. Briefly, plant materials (10 g) were homogenized with 200 mL of solvents by using Ultra-turrax at 6000 g for 5 min in HAE. In UAE, the plant materials (10 g) were sonicated with 200 mL of solvents at 30 \u00b0C for 20 min. In MAC, the plant materials (10 g) were macerated with 200 mL of solvents at room temperature for 24 h. Then, all extracts were filtered and the solvents were removed using a rotary evaporator. All extracts were stored at 4 \u00b0C until analysis.Total phenolic content (TPC) and total flavonoid content (TFC) were determined according to previously described methods ,95. TPC 18 analytical column  with a Polar C18 Security Guard cartridge (4 \u00d7 3.0 mm), both purchased from Phenomenex. Detailed chromatographic conditions are available in [Chromatographic analyses were performed with an Agilent Series 1100 HPLC system with a G1315B diode array detector  and an ion trap mass spectrometer  with an electrospray interface operating in negative ion mode. Separation was performed in a Luna Omega Polar Clable in .In the current work, the antioxidant effects of the tested extracts were detected by different assays . The assv/v) heat-inactivated fetal bovine serum and penicillin\u2013streptomycin (100 \u00b5g/mL) . The cells were grown at 37 \u00b0C in a humidified atmosphere of 5% CO2. When indicated, the cells were treated with H2O2 (300 \u00b5M) for 3 h and with different concentrations (10\u20131000 \u00b5g/mL) of the various extracts of M. spicata.The HypoE22 rat-hypothalamus cell line was purchased from Cedarlane Corporation  and cultured in Dulbecco\u2019s Modified Eagle Medium (DMEM) supplemented with 10%  growth assay , based on the capability of viable cells to reduce MTT to a colored formazan product. Details about the protocol were reported in our recent study .2O2 (300 \u00b5M) for 3 h and with different concentrations (1\u2013100 \u00b5g/mL) of the HAE acetone/water extract of M. spicata. At 48 h, the cell supernatants were harvested and the secretion of prostaglandin E2 (PGE2) in the culture media was evaluated by an ELISA kit , according to the manufacturer\u2019s instructions. The optical density values were obtained by measuring the absorbance at 405 nm using a Multiscan GO microplate spectrophotometer .The HypoE22 rat-hypothalamus cell line was treated with HDA, 5-HT, 5HIIA, L-dopa and DOPAC levels were analyzed through a HPLC apparatus consisting of a Jasco  PU-2080 chromatographic pump and an ESA  Coulochem III coulometric detector, equipped with a microdialysis cell (ESA-5014b) porous graphite working electrode and a solid-state palladium reference electrode. The detailed description of the chromatographic analysis is fully described in a previous study .Gene expression of COX-2 was conducted as previously reported . BrieflyPutative targets were identified according to the bioinformatics method recently described by Gu et al. . Brieflyp < 0.05 was considered statistically significant.The experimental data related to in vitro studies were analyzed through the analysis of variance (ANOVA) followed by Newman\u2013Keuls post hoc test. The GraphPad Prism software (9.0) was employed for statistical analysis. M. spicata extracts obtained by different extraction methods and solvents. In general, the acetone/water extracts had the highest content in total phenols and exhibited the strongest antioxidant effects, regardless of the extraction method used. In particular, the HAE extract was able to protect HypoE22 hypothalamic cells from the oxidative stress injury suffered after exposure to hydrogen peroxide. Indeed, the acetone/water extract prevented the hydrogen peroxide-induced dopamine depletion and reduced the production of pro-inflammatory PGE2 in vitro. Hence, neuroprotective effects are partly related to the prominent content in the extract of phytochemicals, namely acacetin, eriodictyol, hesperidin, sagerinic acid, naringenin, luteolin, chlorogenic acid, chrysoeriol and apigenin. Based on these findings, the current work provided new scientific bases for anti-inflammatory and neuromodulatory properties of M. spicata extract and its potential applications in the pharmaceutical industry. However, further studies are needed to improve our knowledge about the aforementioned effects by the extracts through the use of independent in vitro models, including astrocytes which have been described to support neuron function and response to xenobiotics [This study was intended to explore the phytochemical composition and bio-pharmacological effects of obiotics ."}
+{"text": "CSRNP) family has prognostic value for various cancers. However, the association between this proteins and prognosis of clear cell renal cell carcinoma (ccRCC) remains unclear. This study aimed to determine the prognostic value of the CSRNP family for patients with ccRCC. Therefore, the gene expression profiling interactive analysis database was used to analyze the mRNA expression of CSRNP family members (CSRNPs) in relation with survival. Combined and independent prognostic values of CSRNPs were evaluated using SurvExpress and multivariate Cox regression analyses, respectively. Potential signaling pathways impacted by CSRNPs were evaluated using Metascape. Associations between the CSRNP family and immunocyte infiltration were determined from single-sample gene set enrichment analysis. Both cBioPortal and MethSurv were used to explore whether genomic and epidemic alterations might influence prognosis. We found that when both CSRNP1 and CSRNP3 had a low expression, patients with ccRCC had a worse overall survival (OS). Therefore, a prognostic signature was constructed as follows: risk score = \u22120.224 \u00d7 expmRNA ofCSRNP1 + 0.820 \u00d7 expmRNA ofCSRNP2 \u2212 1.428 \u00d7 expmRNA ofCSRNP3. We found that OS was worse in patients from the high- than from the low-risk groups (AUC = 0.69). Moreover, this signature was an independent predictor after adjusting for clinical features. Functional enrichment analysis positively associated CSRNPs with the acute inflammatory response and humoral immune response pathways. This was validated by correlating each CSRNP with 28 types of immunocytes in tumor and normal tissues. A higher expression of CSRNP1 and CSRNP3 was associated with a better prognosis in both the high- and low-mutant burden groups. Cg19538674, cg07772537, and cg07811002 of CSRNP1, CSRNP2, and CSRNP3, respectively, were the predominant DNA methylation sites affecting OS. The CSRNP gene family signature may serve as a prognostic biomarker for predicting OS in patients with ccRCC. The association between CSRNPs and immune infiltration might offer future clinical treatment options.The cysteine-serine-rich nuclear protein ( Renal cell carcinoma (RCC) has multiple histological subtypes; together, they account for nearly 3% of all human malignant carcinomas . The incDrosophila to humans (The cysteine-serine-rich nuclear protein (CSRNP) family members, CSRNP1, CSRNP2, and CSRNP3, have been considered as nuclear proteins . Their co humans , play eso humans , and mouo humans .CSRNP1  in mouse T cells; it expresses a 1.7 kb transcript with five exons in some malignant cancers, such as kidney, liver, lung, and colon carcinomas  is a brain-specific gene , to investigate genomic functionality. We obtained the expression profile of TCGA-KIRC from UCSC Xena (https://xenabrowser.net/datapages/?cohort=GDC%20TCGA%20Kidney%20Clear%20Cell%20Carcinoma%20(KIRC)&removeHub=https%3A%2F%2Fxena.treehouse.gi.ucsc.edu%3A443) and the expression profile and clinical features of GSE29609 from the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/).We explored whether the expression of CSRNP family members, which are involved in different clinical stages and affect the prognosis of ccRCC, differed between ccRCC and normal tissues. We therefore analyzed mRNA expression, stage-specific expression, overall survival (OS), and CSRNPs matching normal and genotype-tissue expression (GTEx) data derived from The Cancer Genome Atlas (TCGA), using the Gene Expression Profiling Interactive Analysis (GEPIA) online tool  (http://http://bioinformatica.mty.itesm.mx:8080/Biomatec/SurvivaX.jsp) was utilized to construct and evaluate the prognostic value of the CSRNP family signature. Here, a risk score formula was obtained, and the risk score for each patient was automatically generated. Patients were assigned to high- or low-risk groups based on the median cutoff value of the risk scores. Moreover, the independent prognostic value of the CSRNP family signature was determined using multivariate Cox regression analysis incorporating age, gender, grade, stage, and the signature.We aimed to construct a comprehensive CSRNP family signature to better predict the OS of ccRCC patients. The SurvExpress online tool  (http://2FC| > 1 and P < 0.05) were detected using volcano plots. Then, samples were classified as belonging to the high- or low-risk groups based on the median cutoff of the risk score model; DEGs between these two groups (|log2FC| > 1 and P < 0.05) were also identified via volcano plots. Finally, DEGs that merged in Venn diagrams were considered as risk-related DEGs and selected for further analysis by Metascape (http://metascape.org/gp/index.html).We then investigated the correlations between potentially critical pathways and the risk score model. First, DEGs between normal or adjacent tissues and ccRCC  . SubsequSince the transcriptional gene expression profile could be affected by genetic and epigenetic changes , 17, we http://www.cbioportal.org/). Patients were assigned to groups with a high- or a low-mutant burden based on the median cutoff value, and their survival was analyzed using Kaplan-Meier (K-M) curves according to the genetic alterations found in each CSRNP family gene.First, genetic alterations, which mainly comprised missense and truncating mutations, amplification, and deep deletion, were analyzed using cBioPortal  . Moreover, the prognostic values of all methylation sites associated with CSRNP family members were assessed.Then, we assessed epigenetic changes in the MethSurv   and a 95% confidence interval (95% CI). Paired t-tests were conducted to compare tumor and adjacent normal tissues from patients from the TCGA-KIRC dataset. OS was evaluated using the K-M curves. CSRNP1, CSRNP2, and CSRNP3 were significantly less abundant in ccRCC (n = 532) than in normal (n = 72) tissue sample data from TCGA-KIRC database  and CSRNP3  were significantly correlated with favorable OS . Moreover, the area under the curve (AUC) of a time-dependent ROC increased to 0.69 during the follow-up period (We constructed a CSRNP family signature risk score model as follows: risk score = \u22120.224 \u00d7 expP = 0.0163).Results from a multivariate Cox regression analysis suggested that the CSRNP family signature was an independent predictor for the prognosis of patients with ccRCC , cg23618218 , and cg07811002  of CSRNP1, CSRNP2, and CSRNP3, respectively, were the most powerful and DNA methylation locational risk factors. Overall, nine, ten, and two CpGs of CSRNP1, CSRNP2, and CSRNP3, respectively, indicated aberrant prognosis (DNA methylation also plays a pivotal role in the regulation of gene expression and affects clinical outcomes. The DNA methylation sites of the CSRNP family genes in ccRCC using online bioinformatics tools.With the rapid development of bioinformatics tools for analyzing multiple databases with many clinical samples, outcomes, and different clinical features, prognoses can be predicted and specific cancers can be detected using biomarker molecules, especially some gene families. This study mainly explored the prognostic value and biology of CSRNP1 has been considered as an immediate early gene (in vivo indicated that the expression of CSRNP1 could be highly induced by IL-2 in mouse T lymphocytes (CSRNP1 expression is positively associated with the infiltration of type 2 T helper cells in both normal and ccRCC tissues, confirming the previous findings. Noteworthy, in Drosophila, upregulated CSRNP1 disturbs cell cycle progression by downregulating Cdk1 activity and promoting apoptosis in a JNK-dependent manner (CSRNP1 was associated with a poor prognosis in patients with ccRCC; whereas stage-specific expression profiles significantly differed. Moreover, in terms of potential genetic and epidemic alterations, CSRNP1 acts as a protective factor in patients with high- and low-mutant burdens. In addition, nine CpGs of CSRNP1 were correlated with a significantly aberrant prognosis.rly gene  that binphocytes . In thist manner . Besidest manner .\u00a0These fCSRNP2 showed significant hazard ratios, suggested that CSRNP2 might be a meaningful target gene for epigenetic therapy.CSRNP2 has been positively associated with many aberrant non-cancerous diseases, including obesity and type 2 diabetes mellitus . MoreoveCSRNP3 was found to encode a transcriptional factor for muscle development in growing pigs (CSRNP3, like that of CSRNP1, was lower in ccRCC, and was associated with a poor prognosis. Moreover, CSRNP3 may be a protective factor in patients with high- and low-mutant burdens. In addition, two CpGs of CSRNP3 positively correlated with significantly aberrant prognosis, which might help clarify detailed biological functions.ing pigs , and wasing pigs . HoweverCSRNP genes were consistent with the above details. We constructed a novel risk score model based on the expression of the CSRNP family to improve the prediction of OS. We also classified all the samples into high- and low-risk groups according to the median cutoff value of the risk score. The expression profiles of the CSRNP family members were different between these groups, especially those of CSRNP1 and CSRNP3. The low-risk group had a better OS. Importantly, the AUC of the time-dependent ROC curve reached 0.69 over time. Moreover, this signature was an independent predictor of prognosis among patients with ccRCC. Our model exhibited good diagnostic and predictive capacities, but further improvement is needed. The CSRNP family, particularly CSRNP1 and CSRNP3, was validated as a useful prognostic biomarker for patients with ccRCC.The prognostic values of via immune-related biological pathways. We found that immunocyte infiltration was higher in tumor than in paracancerous tissues. The immune infiltration profile of the CSRNP family genes in ccRCC tumor tissues was different from that in normal tissues; natural killer cells and plasmacytoid dendritic cells showed positive correlations. It is known that natural killer cells destroy various cancer cells (Further investigation on functional enrichment analysis implied that the CSRNP family might function er cells \u201329, incler cells . Plasmacer cells  and melaer cells . In addier cells  and cytoer cells . Consistin vitro or in vivo experiments, and clinical validation are urgently needed. Limitations are also imposed by the retrospective design of the study and the small sample size. Therefore, we plan to cooperate with several urological centers to conduct a prospective study and maximize the sample size. We will also continue to conduct in-depth investigations into the occurrence and development of CSRNP family genes in ccRCC to support our conclusion that the CSRNP family could serve as a useful prognostic biomarker.There were some limitations to this study. The most important was that we generated conclusions mostly based on online integrative bioinformatics analysis tools; therefore, data from In conclusion, we comprehensively explored the prognostic value of the CSRNP family using online integrative bioinformatics analysis tools. The CSRNP family signature may serve as a prognostic biomarker to predict the OS of patients with ccRCC. The risk score model based on the CSRNP showed good diagnostic and independent predictive capacity. The association between the CSRNP family and immune infiltration might offer another clinical treatment option.The original contributions presented in the study are included in the article/GY designed the study. HZ and XQ performed the bioinformatics analyses and wrote the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the Science and Technology Planning Project of Guangzhou City, Guangdong, China, under grant 201904010035, and the Natural Science Foundation of Guangdong Province, China, under grant 2018A030313905.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Drugs can treat different diseases but also bring side effects. Undetected and unaccepted side effects for approved drugs can greatly harm the human body and bring huge risks for pharmaceutical companies. Traditional experimental methods used to determine the side effects have several drawbacks, such as low efficiency and high cost. One alternative to achieve this purpose is to design computational methods. Previous studies modeled a binary classification problem by pairing drugs and side effects; however, their classifiers can only extract one feature from each type of drug association. The present work proposed a novel multiple-feature sampling scheme that can extract several features from one type of drug association. Thirteen classification algorithms were employed to construct classifiers with features yielded by such scheme. Their performance was greatly improved compared with that of the classifiers that use the features yielded by the original scheme. Best performance was observed for the classifier based on random forest with MCC of 0.8661, AUROC of 0.969, and AUPR of 0.977. Finally, one key parameter in the multiple-feature sampling scheme was analyzed. Drugs are important in treating various diseases; however, their therapeutic effects are accompanied by negative effects called side effects. In the pharmaceutical field, drug side effect is classified as an adverse drug reaction (ADR), the harmful or accidental reactions of qualified drugs that are irrelevant to the purpose of their use under normal usage and dosage. Some market-approved drugs may generate unaccepted side effects that can be harmful to the human body and bring high risks to pharmaceutical companies. For example, fluconazole and atorvastatin have potential hepatotoxicity and nephrotoxicity that can increase transaminase when used in specific patients such as those with liver disease. Side effects are one of the major obstacles in launching new drugs and delaying their development. Thus, determining all the side effects for a given drug is an important topic in drug development. Despite their efficiency in identifying side effects, solid clinical trials are time consuming and expensive and thus cannot meet the demand of large-scale tests. Thus, rapid and cheap methods for the identification of drug side effects must be developed.Many advanced computational algorithms have been proposed \u20135 to proIn this study, an efficient binary classifier was proposed for the identification of drug side effects. Drugs and side effects were also paired as samples \u201322. The http://sideeffects.embl.de/) [Data on 841 drugs and their side effects 824) \u201322 were  \u201322 wereDS1, DS2, DS3, DS4, and DS5.In addition to PDS, a negative dataset (NDS) was necessary in building an efficient binary classifier. A total of 57,058 drug\u2013side effect pairs were produced by randomly pairing one drug and one side effect , 21. HowTwo drugs with strong associations always share similar functions \u201329. Sided1 and d2 was denoted by Gf.Simplified molecular input line entry specification (SMILES) string  is a widhttps://www.genome.jp/tools/simcomp/) reported in the KEGG [d1 and d2 was denoted by Gs.In addition to SMILES string, another popular drug representation scheme is graph-based method. Here, each drug is represented by a graph with nodes depicting atoms and edges indicating bonds. The association of two drugs can be assessed by considering the similarity of two corresponding graphs. \u201cSIMCOMP\u201d .The ATC system is a widely accepted and used in drug classification. Each drug in such system is assigned five-level ATC codes that indicate its essential properties. For two drugs, their association can be measured according to their ATC codes. This study used the same method in  to evaluhttp://stitch4.embl.de/) [d1 and d2, their literature association was denoted by Gtm.Given the extensive literature on drugs, the association of two drugs can be measured from their cooccurrence in some literature and natural language processing methods. The well-known public database, STITCH  , providehttps://go.drugbank.com/) [d1 and d2 was denoted as Gt.Target protein is the basic property of drugs. Hence, the association of two drugs can be estimated by comparing their target proteins. In this study, the target proteins of drugs were retrieved from DrugBank (nk.com/) . Each drp = <d, s>, where d and s indicate one drug and one side effect, respectively, let S be a set consisting of drugs having side effect s that have been extracted from the training dataset. If d also has side effect s, then, it would not be included in S. For one type of drug association, all values between d and drugs in S are selected. Denoted by \u03a8k(p) , a candidate feature list for p is then constructed with the decreasing order of above values. The top value in this list has been previously chosen as exclusive feature [d and side effect s. On the basis of the different selection models, two strategies were proposed, namely, discrete and continuous strategies. Their procedures are shown in In  feature , 22. Selk(p) are selected to indicate the distribution of values in the list. In this way, these selected values can fully indicate the linkage between drug d and side effect s. This process can be achieved by selecting some discrete values in the list. For example, the value at the first place or that at the top q% place can be selected. These values comprise a set of features from one type of drug association.In this strategy, several values from the list \u03a8d and side effect s is highly indicated by some top values in the list, these values must be properly selected because they may fully contain the essential information. For an integer q between 1 and 100, the top q% values in the list \u03a8k(p) were selected as features.This strategy differs from the first one. Given that the linkage of drug A proper classification algorithm is important in building an efficient classifier. In this study, RF  was adopn samples, randomly select n samples with replacement from such dataset. The second procedure is to select features to split each node. The selected features should be much less than overall features. After the predefined number of decision trees has been constructed, RF integrates them by major voting. For a query sample, each decision tree gives its prediction. The majority prediction is the predicted result of RF. Although a decision tree is a relative weak classification algorithm, RF is extremely powerful and has always been an important candidate to build different classifiers.RF is an integrated classification algorithm containing several decision trees, each of which is constructed by two random selection procedures. The first procedure is to select samples. Given a dataset with In this study, \u201cRandomForest\u201d in Weka  was direK-nearest neighbor (KNN) [In addition to RF, the following classification algorithms were used to build corresponding classifiers: support vector machine (SVM)  , Adaboosor (KNN) , decisioor (KNN) , PART [5or (KNN) , logistior (KNN) , multilaor (KNN) , and Repor (KNN) . The goaTen-fold cross-validation \u201359 was aF1-measure. Their definitions are as follows:For a binary classification problem, four entries can be counted by comparing the predicted and true classes of each sample, that is, true positive (TP), false positive (FP), true negative (TN), and false negative (FN). The following measurements were based on these four entries: sensitivity (SN) , specificity (SP), prediction accuracy (ACC), Matthews correlation coefficient (MCC) , 60\u201363, F1-measure use all four entries and thus are more important than the other three measurements. Receiver operating characteristic (ROC) curve [x-axis and SN as the y-axis, and PR curve takes recall as x-axis and precision as y-axis. Areas under these two curves (AUROC and AUPR) are important measurements to evaluate the performance of classifiers. Among the abovementioned parameters, MCC was selected as the main measurement.ACC, MCC, and C) curve  and precA novel feature extraction method was proposed to extract essential features from drug\u2013side effect pairs. On the basis of these features, efficient classifiers to predict drug side effects were established. All procedures are illustrated in q% place in the list was also selected. In this study, q was set as 5, 10, 15, and 20. Values with high ranks in the candidate feature list are more important than those with low ranks, that is, the top value is the most important, followed by values at 5%, 10%, 15%, and 20%. Incremental feature selection was adopted to generate four feature subsets as listed in column 1 of The discrete strategy picks some discrete values in the candidate feature list. Given that the top value in such list is the most important and has been previously selected as the exclusive feature , 65, thiThe ROC and PR curves of these four RF classifiers were investigated, and the results are shown in q% values in the candidate feature list can be chosen as features. Here, some q values including 10, 20, 30, and 40 and four feature subsets were tested. A RF classifier was also built on each of the five datasets by using the feature subsets derived from the five types of drug associations. Each classifier was assessed by 10-fold cross-validation, and the average performance is listed in q = 20 (top 20%), the RF classifier yielded the highest MCC of 0.8661 and generated the ACC of 0.9312, F1-measure of 0.9278, SN of 0.8852, SP of 0.9771, and precision of 0.9747. Compared with the RF classifiers with discrete strategy, the best RF with continuous strategy had higher measurements, particularly for MCC (by 15%), ACC (by 7%), and F1-measure (by 7%). These results indicated that the features obtained by continuous strategy were more powerful in identifying drug side effects than those yielded by discrete strategy.Different from discrete strategy, continuous strategy selected values from the candidate feature list in a continuous way. As mentioned in The ROC and PR curves of RF classifiers with continuous strategy were plotted as shown in A multiple-feature sampling scheme was proposed to extract essential features from each drug\u2013side effect pair. Previous studies , 22 onlyF1-measure was 0.7988. Other three measurements  were 0.7948, 0.8049, and 0.8030, respectively. The best performing (highest MCC) RF classifiers with discrete and continuous strategies were selected for comparison and are also listed in The average performances of RF classifiers with single-feature sampling scheme are listed in The RF classifiers with features yielded by multiple sampling (discrete strategy) were superior to those with features yielded by single sampling, and the RF classifiers with continuous strategy were better than those with discrete strategy. However, the relevance of this result to the selection of classification algorithms must be explored. In this section, 12 classification algorithms mentioned in F1-measure as illustrated in The performances of classifiers with single sampling and the best performance of classifiers with multiple sampling are listed in q is a key factor that determines the number of selected features from the candidate feature list. Here, its influence on the performance of classifiers was investigated.For the continuous strategy, the parameter q = 20 (q = 20 always yields the best performance as shown in q = 20, occupying 76.92%. Meanwhile, two yielded the best performance when q = 30. This phenomenon was reasonable. When q is extremely small, some essential information of drug\u2013side effect pairs cannot be included. When q is large, several noises may be employed. Current investigation revealed that the values of q can be taken in an interval [For RF classifiers, the highest MCC of 0.8661 was achieved when q = 20 . For othThis study prevents a novel investigation on drug side effects. The contributions contained two aspects. One was the multiple-feature sampling scheme that can extract essential features from drug\u2013side effect pairs, and other one was novel computational methods for the identification of drug side effects based on the features yielded by the multiple sampling scheme. Classifiers were built on the basis of different classification algorithms. By comparison, the classifiers using features yielded by the multiple sampling scheme performed better than those using features yielded by the single sampling scheme. The proposed classifiers can be useful tools to identify drug side effects, and the novel feature extraction scheme can be applied to other similar biological or medical problems."}
+{"text": "Retrospective.To investigate the efficacy of cervical single open-door laminoplasty with and without local lateral mass screw fixation and fusion as treatments for cervical spinal cord injuries accompanied by multisegmental spinal canal stenosis.The Second Affiliated Hospital, School of Medicine, Zhejiang University.Of all enrolled patients, 42 formed a stable group who underwent cervical single open-door laminoplasty alone and 14 formed an unstable group who underwent the procedure combined with lateral mass screw fixation and fusion. Neurological function was evaluated before surgery, at discharge, and at final follow-up using the American Spinal Cord Injury Association (ASIA) impairment scale and the Japanese Orthopedic Association (JOA) score.P\u2009>\u20090.05). Final follow-up JOA scores reflected 49.2%\u2009\u00b1\u200931.7% improvement in the stable group and 47.1%\u2009\u00b1\u200939.2% improvement in the unstable group (P\u2009>\u20090.05).ASIA scores reflected improved neurological function in 52.5% of the stable group (15 with grade-D and 4 with grade-A injuries did not improve) and 45.5% of the unstable group (3 with grade-D and 3 with grade-A injuries did not improve). Postoperative JOA scores reflected 19.1%\u2009\u00b1\u200921.6% improvement in the stable group and 18.6%\u2009\u00b1\u200918.4% improvement in the unstable group (Laminoplasty combined with local fusion aided the treatment of unstable cervical spinal cord injuries and spinal stenosis. Such stenosis is the main pathological factor causing multiple spinal cord compressions in patients with cervical spinal cord injuries. Additional intervertebral disc injuries, anterior longitudinal ligament injuries, and vertebral body avulsion fractures, when present, were stabilized via local lateral mass screw fixation and fusion in addition to laminoplasty. The improvements in neurological symptoms afforded by these treatments, the effects of surgical timing, and the risk factors for local instability were also investigated.Many high-energy injuries are osseous in nature; however, many low-energy spinal cord injuries in patients with pre-existing cervical stenosis are not associated with any fracture or other cause of instability. The treatment of central cord syndrome without associated instability remains controversial. Classically, nonsurgical treatment was considered to yield results similar to those of surgery . This poBetween December 2014 and August 2017, 56 patients underwent surgery to treat traumatic cervical spinal cord injuries accompanied by multisegmental cervical spinal canal stenosis. The stenosis and cord injury were examined by magnetic resonance imaging (MRI). All patients evidenced greatly increased T2 signal changes in the spinal cord and cervical spinal canal stenosis [cervical sagittal diameter\u2009<\u200913\u2005mm on MR images ] at threAccording to MRI and CT findings, the patients were divided into stable and unstable groups. Stability and instability were defined as the absence and presence, respectively, of intervertebral disc or anterior longitudinal ligament rupture or teardrop-like fracture at the anterior edge of the vertebral body. Simple cervical single open-door laminoplasty was indicated for the stable group. The open-door side was that of the most severe symptom. When symptoms on both sides were similar, we chose the side on which stenosis was more obvious on imaging. On the open-door side, fixation was performed using small titanium-alloy plates  to support the lamina and prevent postoperative \u201cdoor closing.\u201d Single open-door laminoplasty with local lateral mass screw fixation was indicated for the unstable group. Single open-door laminoplasty was used to treat the stenotic segment, and laminoplasty plus fixation with lateral mass screws was used to treat the local unstable segments. The lateral mass screws were placed on the hinge side and/or the open-door side. The facet joints of fusion segments were decorticated using a high-speed drill. Next, bone fragments from the autogenous spinous process were implanted in the facet joints. Methylprednisolone (80\u2005mg QD) was prescribed for the first 2 postoperative days and was then reduced to 40\u2005mg QD for the next 2 days to prevent spinal cord edema. Cefuroxime was prescribed at 2\u2005g BID for 2 days postoperatively to prevent infection of the incision. The drainage tube was removed 48\u2005h after surgery, or within 8\u2005h when the drainage volume was <50\u2005ml. After tube removal, the patients were encouraged to leave the bed and walk. The patients were told to protect their necks with a cervical collar for 2 weeks after surgery, and then to commence exercises that rehabilitated neck muscle function. Imaging data from a typical case are provided in We recorded patient sex and age, cause of injury, time from injury to operation, duration of operation, amount of intraoperative blood loss, pathological type of cervical spinal stenosis, numbers and distributions of high-signal segments in the cervical spinal cord, and unstable segment pathological type and distribution. Patients' neurological status was evaluated before surgery, at discharge, and at the final follow-up using the ASIA impairment scale, and was classified as grades A\u2013E based on symptom severity. Improvement was defined as at least one increment of improvement in the ASIA score at the final follow-up assessment. Neurological function was also evaluated using the JOA score before surgery, at discharge, and at the final follow-up. The JOA recovery rates at discharge and final follow-up were calculated using the Hirabayashi method as: [(postoperative JOA score\u2014preoperative JOA score)/(17\u2014preoperative JOA score)]\u2009\u00d7\u2009100%.t test, Mann\u2013Whitney U test, and chi-squared test were used, as appropriate, for group comparisons. P values\u2009<\u20090.05 were considered to be statistically significant.All statistical analyses were performed using SPSS software . We present means\u2009\u00b1\u2009standard deviations. The unpaired independent-samples P\u2009>\u20090.05). Pre- and postoperative radiographic data from a typical case (from the unstable group) are provided in The stable group comprised 42 patients , including 1 patient who died and another who was lost to follow-up. The mean age was 59.1\u2009\u00b1\u20099.5  years, and the average time from injury to surgery was 10.1\u2009\u00b1\u20096.5  days. All patients in the stable group underwent cervical single open-door laminoplasty. Thirty-two patients required surgery from C3 to C7, eight patients required C3\u2013C6 laminoplasty, and two patients required C4\u2013C7 laminoplasty. The mean operation time was 128.6\u2009\u00b1\u200938.1\u2005min and the average intraoperative blood loss was 136.3\u2009\u00b1\u2009120.9\u2005ml. The unstable group consisted of 14 patients , including 3 who died during follow-up; thus, data from only 11 patients are provided in P\u2009=\u20090.230). In the stable group, 64.3% of cervical canal stenoses were developmental; 9.5% of these cases were combined with OPLL. OPLL alone was observed in 21.4% of cases and simple multilevel disc herniation was observed in 14.3% of cases in this group. In the unstable group, all cervical spinal canal stenoses were developmental, and 35.7% of them were combined with OPLL (P\u2009=\u20090.03). Intervertebral disc injuries and vertebral body avulsion fractures were located principally in the regions of stress concentration in patients with multisegmental OPLL or intervertebral joint stiffness; the proportion of such patients in the unstable group was 57.1%.High-energy injuries  accounted for 52.5% of injuries in the stable group and 72.7% of injuries in the unstable group (P\u2009>\u20090.05). JOA scores from the final follow-up assessments reflected 49.2%\u2009\u00b1\u200931.7% improvement in the stable group and 47.1%\u2009\u00b1\u200939.2% improvement in the unstable group. Improvements reflected by JOA scores obtained at discharge and final follow-up did not differ between groups. JOA scores improved in patients with grade-B\u2013D neurological function according to the ASIA impairment scale.Neurological function improvements are shown in We found that 73.2% of patients with cervical spinal cord injuries but no significant fracture or dislocation had developmental spinal canal stenosis . The relationship between such stenosis and cervical spinal cord injury has been of concern to researchers. In asymptomatic populations, the incidences of cervical spinal cord compression and signal change are related closely to developmental spinal canal stenosis . Morishin\u2009=\u200931) than among those who underwent nonoperative treatment (n\u2009=\u200929 cases). The degree of improvement in the JOA score at the final follow-up was about 50%. The JOA scores of all patients with grade-B\u2013D function improved, whereas those of patients with grade-A function did not; no patient evidenced neurological deterioration after surgery. A prospective study on this topic would be difficult to conduct because the conditions and pathogenic factors of patients with spinal cord injuries are complex. Further research is needed.The use of surgery to treat cervical spinal cord injuries with no significant fracture or dislocation remains controversial. Similar outcomes of nonoperative and operative treatments have been reported , 16. HowThe optimal timing of surgery remains controversial. Fehlings et al.  found novia the anterior approach, but avoids the posterior scar formation and risk of C5 nerve root palsy associated with laminectomy and internal fixation  affects the neurological outcomes."}
+{"text": "NOD was diagnosed in 26.48% of patients. The severity of the infection, hospital admission values for fasting plasma glucose, lactate dehydrogenase (LDH), PaO2/FiO2 ratio, the peak values for leucocytes, neutrophils, C-reactive protein, triglycerides, and the need for care in the intensive care unit were predictors for the occurrence of NOD in univariate analysis, while only LDH level remained a significant predictor in the multivariable analysis. In conclusion, the results of the study showed a high incidence of NOD in patients hospitalized with COVID-19 and identified LDH levels at hospital admission as a significant predictor of NOD during SARS-CoV-2 infection. However, the persistence of NOD after the COVID-19 infection is not known, therefore, the results must be interpreted with caution.The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is one of the world\u2019s most disruptive health crises. The presence of diabetes plays an important role in the severity of the infection, and a rise in newly diagnosed diabetes cases has been identified. The aim of this retrospective study was to determine the incidence of new-onset diabetes (NOD) and predictive factors with their cut-off values for patients hospitalized with COVID-19. All patients ( The coronavirus disease 19 (COVID-19) pandemic given by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is still a public health issue with global impact , the neeIt is established that a severe/critical form of COVID-19 increases the risk of a poor prognosis, including death of patients. A number of studies have identified that people who develop those forms of SARS-CoV-2 infection have underlying conditions, such as hypertension 49.7%), obesity 48.3%), chronic lung disease 34.6%), and cardiovascular diseases (27.8%) 9.7%, obe,6. In th%, and ca.3%, chroIt is well-known that DM plays a key role in global morbidity and mortality . WorldwiThus, a bidirectional link has emerged between DM and SARS-CoV-2 infection: having diabetes increases the risk of developing a severe form of the disease and the infection may increase the risk of NOD . A varieTo date, as far as we know, there are no previous data regarding the bio-humoral markers to predict the development of diabetes in SARS-CoV-2 infection. The aims of this study were to describe the incidence of NOD in patients with SARS-CoV-2 infection and to investigate these biological predictors of NOD and their cut-off values.This study was a retrospective study conducted on a consecutive series of patients admitted to the \u201cLeon-Daniello\u201d Pulmonology Hospital in Cluj-Napoca, Romania between 10 November 2020, and 31 January 2021 with a confirmed SARS-CoV-2 infection. Patients with all forms of COVID-19 severity were included in the research if their age was above 18 years and the COVID-19 was confirmed through real time-polymerase chain reaction (RT-PCR) according to World Health Organization (WHO) case definition applicable in 2020 . The excTo diagnose NOD during the hospitalization, the American Diabetes Association (ADA) criteria were used: 2 values of fasting plasma glucose (FPG) \u22657.0 mmol/L (\u2265126 mg/dL) or HbA1c \u2265 6.5% or random glucose level \u2265 11.1 mmol/L (\u2265200 mg/dL) along with symptoms of hyperglycemia in the absence of a medical history of hyperglycemia .For each participant, there were registered demographic data: age, gender, body mass index (BMI), medical history , symptomatology , and peripheral oxygen saturation (SpO2) at admission. Other data collected included the number of days since COVID-19 symptoms onset until hospitalization, the evolution of the respiratory status with progression to a critical form of COVID-19, with the need for an intensive care unit (ICU). The severity of COVID-19 was determined according to the Romanian Protocol for the therapy of patients with SARS-CoV-2 infection  as folloFor this study, there were collected parameters routinely assessed to detect the predictors of NOD in SARS-CoV-2 infection. Each of the biomarkers is accessible in any medical service: complete blood count, C-reactive protein (CRP), D-Dimer, ferritinemia, interleukin-6, procalcitonin, lactate dehydrogenase (LDH), FPG, triglycerides, creatinine, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), and arterial blood gas. All analyses were performed using standard clinical chemistry techniques in the clinical laboratory of the hospital where the study was performed. The samples were collected in the morning before breakfast and we extracted data from the first day of admission, during hospitalization (days 5\u20137), and upon discharge.The software used for statistical data processing were IBM SPSS Statistics V26.0  and MedCalc 20.026 . SPSS was used to find out the correlation between variables and for uni and multivariate logistic regression, while MedCalc was used to identify the cut-off values of continuous variables assessed as risk factors for NOD and ROC, AUC curves. p-value < 0.05) were included in the univariate logistic regression. Variables significantly associated with NOD in univariate logistic regression were further included in a multivariable regression analysis. For the variables associated with NOD in the multivariable logistic regression, receiver-operating characteristic (ROC) curve analysis, area under the curve (AUC), sensitivity, and specificity were used to test the predictive power and to determine cut-off values of risk factors for NOD. The optimal cut-off values of continuous variables assessed as risk factors for NOD were identified using the Youden index method [p-value < 0.05 was considered as statistically significant.Baseline characteristics  are presented as frequencies and percentages or as median and quartiles 1 and 3 . Mann and Whitney and Chi-square tests were used to compare variables between groups with and without NOD. All variables that were statistically significantly different between groups . Given the retrospective nature of the research, we took the information from the database of the hospital, and the signature of informed consent by participants was not required, as per local regulations.Of the 514 patients hospitalized for COVID-19 during the study period, 165 were excluded due to the short duration of hospitalization (<48 h), missing investigations, or readmission of the same patient. Additionally, 130 patients had a prior diagnosis of DM and were excluded from the current analysis. Of the 219 patients who fulfilled the inclusion criteria, had no exclusion criteria, and were included in this analysis, 58 had NOD diagnosed during the hospitalization, giving an incidence of NOD in the present population of 26.5%. p < 0.05). Moreover, patients with NOD more often developed a severe form of COVID-19 compared to those without NOD (p = 0.001) and had a more frequent need for ICU care (p = 0.043). The median length of hospital stay was also longer (12.5 vs. 9.0 days), with a p-value < 0.0001. No statistical significance was observed between the two groups regarding the use of corticoid therapy (p = 0.155), nor regarding the time from symptoms onset to hospital admission or confirmation of COVID-19.p = 0.043). Moreover, the frequency of NOD increased in parallel with the severity of COVID-19 .The study has also assessed the frequency of NOD according to the need for ICU care and the severity of COVID-19. There was found a higher frequency of NOD in patients who needed ICU care as compared to those without ICU care , LDH and FPG (p < 0.0001) at admission were significantly higher in NOD group. In terms of blood gas analysis, hypoxemia was more pronounced among patients with NOD (53.12 vs. 63)\u2014but statistically insignificant (p > 0.05), while the acute respiratory distress syndrome (p = 0.024) was statistically significant in NOD group than in those without NOD. The peak values of biochemical analysis showed that leukocytosis, neutrophilia, ferritin, CRP, LDH, FPG and triglycerides are significantly higher in NOD patients (p < 0.05).Ferritin . The association of NOD with COVID-19 severity was previously reported by Birabaharan et al. [Regarding the COVID-19 severity, the univariate logistic regression showed that patients with NOD had a 2.7 higher risk of a severe form . Thus, the presence of these comorbidities cannot be associated with the onset of diabetes.The main risk factors for diabetes are overweight and obesity, prediabetes, genetic component, and family history, age, and physical inactivity . Morevoep = 0.001 and p < 0.001). The phenomenon was described [In the present study, the NOD group more often had dyspnea with acute respiratory distress syndrome (ARDS), but with only slightly lower peripheral saturation compared with the control group. This agrees with other studies. Fadini et al.  and Faraescribed , as \u201csilescribed . On the escribed ,41, so tp < 0.001). Moreover, the authors found that patients with NOD had a more severe form of SARS-CoV-2 infection, with a higher ARDS and rate of mortality [In the present analysis, higher FPG levels at admission were observed in patients with NOD than in those without. The results are consistent with those of Fadini et al. , who fouortality . Similarortality ,43. p < 0.001 and 36.3 vs. 55.4, p < 0.009). It is known that patients with diabetes have an immune system imbalance [Hyperinflammation and cytokine storm have an important effect on insulin resistance, resulting in hyperglycemia . In the mbalance  and a himbalance . The infmbalance .p < 0.001 [p = 0.028) was an independent risk factor for disease severity. Although this study there were also found higher ferritin levels at admission and higher peak values in the NOD group than non-NOD patients (0.015 and <0.001), ferritin was not a predictor for NOD. An extreme form of inflammation is found in patients with a severe form of the coronavirus disease who go through the cytokine storm\u2014an uncontrollable hyperinflammation that can lead to cell damage in multiple locations . During  < 0.001 . A retro < 0.001  showed tNeutrophils are the main source of cytokines and chemokines. The paradoxical influence of hyperglycemia on neutrophil count is explained by its chemotaxis, reduced phagocytosis produced by neutrophils, macrophages, and monocytes, and lower apoptosis in neutrophils ,49. Bothp < 0.001). Another recent study showed that, in patients with COVID-19, triglycerides had higher values in the NOD group than in patients without NOD [Higher hypertriglyceridemia was seen in the NOD group vs. control group . In 453 patients admitted for SARS-CoV2 infection, NOD patients were more likely to need ICU and invasive mechanical ventilation than normal glucose patients, due to the highest prevalence of coronavirus-related complications and longer hospital stay [The current study showed that in patients with COVID-19, there is a higher percentage of people with NOD who needed ICU than patients without NOD (tal stay . Yang ettal stay  concludeThis investigation has several limitations. The first one is represented by the fact that the persistence of NOD after the COVID-19 infection is not known, therefore, the results must be interpreted with caution. Second, given that it is a retrospective study, certain methodological disadvantages may occur such as limited availability of certain biochemical deteriorations in all patients. Prospective studies are needed to determine the persistence of NOD and the true value of biomarkers analyzed here.The present study found a high incidence of NOD in patients hospitalized for SARS-CoV-2 infection and identified LDH levels at hospital admission as a significant predictor of NOD during the SARS-CoV2 infection. LDH could be of real use in closely monitoring the glycemic values with early established antihyperglycemic treatment in order to prevent long-term complications and provide a good quality of life. To the best of our knowledge, this is the first study that highlighted the factors that predict the occurrence of NOD in patients infected with SARS-CoV2. The challenge remains to find the optimal strategy for primary prophylaxis of diabetes and to correct risk factors, regardless of the circumstances in which diabetes may occur."}
+{"text": "The resulting iodine lignin-based carbon fibers had better tensile strength (89 MPa) than that of PAN carbon fibers produced by electrospinning technology.Bio-renewable carbon fibers are fabricated and employed as high-strength composite materials in many fields. In this work, a facile and low energy consumption method was developed to fabricate high-strength lignin-based carbon fibers. Using iodine treatment, the thermodynamic stability of the lignin-based precursor fibers increased significantly, and thus energy consumption during the preparation of the carbon fibers was reduced. The influence of the iodine treatment on fibers was analyzed by differential scanning calorimetry (DSC), scanning electron microscopy (SEM), Raman spectroscopy, tension testing,  via electrostatic spinning.A simple low-energy method to fabricate lignin-based carbon fibers with excellent mechanical properties  Currently, almost 90% of the commercially available carbon fibers are produced using polyacrylonitrile (PAN) as the precursor,8\u201312 which is a non-renewable material and contributes about 50% to the total manufacturing costs of carbon fibers.13\u201316 Therefore, finding an alternative precursor which is bio-renewable and inexpensive is a significant challenge for the carbon fiber industry.17\u201320Carbon fibers are an exciting kind of reinforced material, which consist of more than ninety-five percent carbon with a lamellar graphite structure and have been widely used in sporting goods, construction, national defense, and automotive industries. Their excellent physical and chemical properties, such as excellent tensile strength, low density, high creep resistance, and good resistance to chemicals, have attracted tremendous interest.21 coatings,22 engineering plastics,23 and hydrogels.24 Unique advantages of lignin including low cost, high carbon content, and an aromatic structure make it an attractive choice as an alternative precursor for the carbon fiber industry.25\u201327 Several strategies have been used to develop the lignin-based carbon fibers (CFs) with novel microstructures and excellent mechanical properties using chemical modification and/or physical blending.28\u201332 To produce the CFs, the section of PAN in lignin-based precursor fibers must undergo thermostabilization to obtain N-containing ladder-type polymers, and carbonization, which removes the other elements aside from carbon and rearranges the carbon bonds to obtain a lamellar graphite structure.13,14,33 It is worth noticing that the uniform diameters and lack of structural defects obtained by optimal thermostabilization are critical to increase the mechanical properties of carbon fibers. However, to achieve these good mechanical properties, precursor fibers have a relatively low heating rate and long preparation time during thermostabilization to prevent the melting and deformation of lignin-based stabilized fibers (SFs),34,35 which lead to higher energy consumption, compared with just PAN precursor carbon fibers.24,25 Therefore, increasing the heating rate and reducing the preparation time in thermostabilization has become a major challenge.Lignin, as the second most abundant natural polymer in the plant cell wall, only next to cellulose, has received increasing attention for use in renewable adhesives,28 However, there are no studies relating to iodine treatment to improve the mechanical properties, the influence of ordered graphitic crystallites, and the defects of interior carbon fibers.In this study, we dissolved lignin and PAN in a suitable solvent, and then the resulting solution was pulled into continuous precursor fibers by electrostatic forces, which was a simple and low-cost method for the preparation of carbon fibers. Subsequently, the precursor fibers were treated by iodine, and finally, the carbon fibers were obtained through thermostabilization and carbonization. The iodine treatment effectively enhanced the rigidity of the lignin chain, which was positive for the increase in morphology retention ability of the precursor fibers during thermostabilization. With a relatively high heating rate during the thermostabilization, the iodine treated lignin-based stabilized fibers (ISFs) have a uniform diameter and no structural defects, compared with the no-iodine SFs. The resulting carbon fibers which were treated with iodine (ICFs) have excellent mechanical properties, even beyond those of the just PAN precursor carbon fibers, which were prepared by the same electrospinning preparation technique. This is a promising method to prepare low-cost, bio-renewable, and high strength carbon fibers for the wider application of carbon fibers. The preparation mechanism for iodine treated lignin and high strength lignin-based carbon fibers is illustrated in Iodine assisted carbonization of biomaterials has been reported in several studies.2.2.1N,N-Dimethylformamide (DMF) and ethanol were purchased from Sinopharm Chemical Reagent Corporation. All chemicals were used as received.Corn residues after enzymatic hydrolysis were purchased from COFCO Corporation. Polyacrylonitrile  and iodine crystals were purchased from Sigma-Aldrich. 2.2Corn residues (200 g) and 50% (v/v) ethanol (2000 mL) were mixed in a 3 L three-neck boiling flask, and then the mixture was stirred at 60 \u00b0C for 4 h. After cooling, the resulting mixture was filtered, and the filtrate was poured into three volumes of water, the pH was adjusted to 1 and the lignin precipitate was formed. The resulting lignin was washed with deionized water several times, and was then dried in a vacuum.2.3\u22121, and an applied voltage power of 20 kV (+15 kV and \u22125 kV). The resulting precursor fibers were carefully removed from the collector and were dried in a vacuum at 60 \u00b0C.To obtain the electrospinning solutions of lignin\u2013PAN blends, lignin (5 g), PAN (5 g) and DMF (40 g) were mixed in a 100 mL boiling flask. The precursor fibers were manufactured by electrospinning with a working distance of 20 cm, a feed rate of 1.0 mL h2.4The precursor fibers were placed into a ceramic dish, and then the ceramic dish was put into a jar (4 L) with iodine (10 g). Subsequently, the jar was kept in a 70 \u00b0C oven for 12 h to vaporize the iodine. After being cooled, the iodine treated lignin-based precursor fibers (I-precursor fibers) were obtained.2.5\u22121 in a muffle furnace  and were kept there for 60 min. Carbonization was performed using a heating rate of 4.0 \u00b0C min\u22121 to 1400 \u00b0C and held for 120 min in a tube furnace .The precursor fibers were heated to 220 \u00b0C under air atmosphere with two heating rates of 0.2 or 2.0 \u00b0C min2.6\u22121. X-ray diffraction (XRD) was measured on a Shimadzu XRD-7000S diffractometer with Cu K\u03b1 radiation  and a scanning step of 0.02\u00b0. The carbon structure of the resulting fibers was analyzed by Raman spectroscopy, and the spectra were recorded on an InVia spectrometer  with back-scattered light from a 480 nm laser. Differential scanning calorimetry (DSC) experiments were carried out on a TA Discovery DSC250 at a heating rate of 10 \u00b0C min\u22121 under liquid nitrogen. Thermostabilization was assessed on an SDT Q500 analyzer  with a heating rate of 10 \u00b0C min\u22121 under a nitrogen atmosphere. Scanning electron microscopy (SEM) was performed on a JEOL JSM 7800F electron microscope with a primary electron energy of 15 kV and energy dispersive spectrometry (EDS) was performed on an Oxford X-Max 50 spectrometer. The mechanical properties of the carbon fibers were only assessed by uniaxial tensile strength testing using an Instron tension tester . Each sample was cut to a length of 4 cm and a width of 1 cm before testing and the strain rate was 2 mm min\u22121. The effective length for the tests was 3 cm.FT-IR spectra were recorded on a Perkin-Elmer Spectrum TWO FT-IR infrared spectrometer in the wavenumber range from 4000 to 400 cm3.3.1Iodine is widely used to form charge transfer complexes with electron-rich molecules, such as aromatic rings. One iodine atom can take a proton from aromatic rings to form a HI molecule at high temperatures, and the other iodine atom can be caught by aromatic rings to form the charge transfer complexes. As shown in \u22121, which was assigned to the O\u2013H stretching in lignin. The peaks at 2920 and 2849 cm\u22121 were assigned to methylene C\u2013H asymmetric stretching and symmetric stretching, respectively. The typical absorbance peaks at 1604, 1513, and 1462 cm\u22121 were assigned to the aromatic skeletal vibrations. The absorption for a benzene ring at 834 cm\u22121 weakened obviously after iodine treatment. This result suggested that the p\u2013\u03c0 conjugated structure of I-benzene was created during iodine treatment, and was consistent with the EDS findings.36The existence of iodine on I-precursor fibers was further confirmed by FT-IR spectra. As shown in 37Due to the strong electron donating ability of iodine, the charge transfer complexes with iodine had a full \u03c0 electron orbit, which was effective for enhancing \u03c0\u2013\u03c0 interaction in macromolecules and changed the thermodynamic properties of the macromolecules. As an important thermodynamic property of the macromolecules, the glass transition temperature was reliable and convenient to expound the phase behavior of the materials. As shown in \u22121 under air atmosphere) were observed by SEM. As shown in The morphologies of lignin and I-lignin  had many structural defects (\u22121 (CFs-2.0) collapsed thoroughly (\u22121 (ICFs-0.2 and ICFs-2.0) still kept the original morphologies and had no structural defects , and the units of stress (N Tex\u22121) were converted into the tensile stress (GPa) by multiplying the density of the test specimen, which was determined using a Sartorius Secura balance. The tensile stress and Young\u2019s moduli of the CFs-0.2, CFs-2.0, ICFs-0.2, and ICFs-2.0 are shown in \u22121. It is worth noting that the mechanical strength of the ICFs-2.0 (89 MPa) had exceeded the PAN-based carbon fibers (41 MPa) produced using electrospinning.40 The density of the CFs-0.2, CFs-2.0, ICFs-0.2, and ICFs-2.0 was between 1.62 and 1.69 g cm\u22123, which was determined using a Mettler Toledo balance and density kit. The corresponding properties of the carbon fibers are listed in The areal density was the weight of the sample (g) divided by its area  and (100) in the graphite structure, respectively. In addition, the broad peak at 2\u03b8 of 24\u00b0 indicated that the sizes of the graphite crystallites possibly were very small and the disordered carbonaceous structure probably was relatively high, probably due to the fact that 4\u20137% nitrogen, oxygen and other non-carbon impurity atoms still existed in the fibers after carbonization.41,42 According to the Bragg method, the average interplanar crystal spacings for (002) in the CF-0.2, CF-2.0, ICF-0.2, and ICF-2.0 samples were calculated to be 0.3711, 0.3630, 0.3573 and 0.3528 nm, respectively. It was found that the average interplanar spacing in the ICFs was lower than that in the CFs, which was due to the increase of the density of fibers.27 The difference in the porosity of the fiber mats also caused the density to change. Furthermore, it suggested that the pores were formed in untreated fibers according to 42,43XRD patterns of carbon fibers were produced to characterize the crystalline structures of carbonaceous materials, as presented in \u22121 in the spectrum were referred to as the D and G band, respectively. The D band was related to disordered carbonaceous structure while the G band was related to ordered graphitic structures. The D band was attributed to a hybridized vibrational mode related to graphene layer edges, indicating the number of defects in the graphitic structure, and the G band originated from an in-plane stretching mode of sp2 carbon bonds existing in the ideal graphitic lattice. The \u201cR-value\u201d of the \u201cD-band\u201d to \u201cG-band\u201d is often used to indicate the degree of structural disorder in carbonaceous materials. The R-values of CFs-0.2, CFs-2.0, ICFs-0.2, and ICFs-2.0 were 1.03, 0.94, 0.91, and 0.85, respectively. With the increase in the heating rate during thermostabilization (from 0.2 to 2.0 \u00b0C min\u22121), the R-value decreased. This suggested that some disordered carbonaceous structures were converted to ordered graphitic crystallites with the increased heating rate during thermostabilization. It is worth noting that the amount of ordered graphitic crystallites also increased after iodine treatment. As a result, iodine treatment on lignin based precursor fibers can improve the mechanical properties of fibers by increasing the content of ordered graphitic crystallites.44 This observation was consistent with the SEM results, mechanical properties, and XRD results.Raman spectroscopy was used to determine the structure of the carbon fibers . The two4.via electrostatic spinning. As a result, the study on the effects of iodine treatment on lignin-based carbon fibers suggests that the energy consumption during the preparation of high-strength carbon fibers reduces significantly with the increase of rigidity in the lignin chain. This finding is of great significance to the development of the green carbon fiber industry. These high-strength lignin-based carbon fibers are the more promising candidate instead of the PAN-based carbon fibers used in civilian areas, such as sporting goods and electrodes.We have developed a simple low-energy method to fabricate lignin-based carbon fibers with excellent mechanical properties There are no conflicts to declare.RA-008-C7RA10821D-s001"}
+{"text": "Background: The main objective of this study was to assess different aspects of family physicians  regarding their competencies, attitudes, and procedures towards their patients\u2019 sexuality. We also sought to develop a valid questionnaire to perform this task. Methods: A cross-sectional study was performed among family and community medicine physicians in southeast Spain. Results: A total of 259 family physicians participated. Overall, 69.9% were women, 80.7% were heterosexual, 80.7% had a partner, and 50.6% had not received specific sexology training. Homosexual physicians showed a slightly more positive attitude toward sexuality. Training in sexuality established differences in competencies and procedures, but no differences were found in the attitude regarding whether the physicians had a partner or their training. While younger ages were correlated with a more positive attitude, the global score was positively correlated with the age of the professionals. Conclusions: Competences, attitudes, and knowledge of procedures do not depend on whether the professional has a partner, but there may be slight differences regarding attitude when considering the sexual orientation of the physicians. The attitude toward sexuality may not depend on previous training. Albeit younger family physicians have a more positive attitude, all providers become more involved with sexuality as they gain professional experience. The World Health Organization (WHO) recognizes sexual health as an essential aspect of the life of human beings and strives to promote recognition and attention to sexual rights through policies, education, and integration in health systems . DespiteFamily physicians have an essential role in this regard, being the first to be consulted on these issues and being able to detect problems in the sexual sphere of their patients ,5,6. EleThe level of training in sexual health among professionals is usually related to a more excellent approach to patients\u2019 sexuality in their clinical practice, and understanding the development of this training is a priority to improve the skills of professionals ,12. The Family doctors tend not to ask about sexuality and focus on the biological rather than psychological aspects of their patients\u2019 sexuality ,11,17. HThere have been studies that present a nonstandardized method in their analyses , with a Therefore, the main objective of this study was to assess the family physicians\u2019 competence and attitude regarding sexuality quantitatively. To perform this, we assessed three spheres of family physicians\u2019 practice regarding patients\u2019 sexuality: their attitudes or mindset, their aptitudes or capacity to approach this issue, and their procedures or actual labor in this area. A questionnaire was developed to perform this task. We also sought to assess the validity of the questionnaire.A cross-sectional observational study was performed to evaluate physicians\u2019 performance toward patients\u2019 sexuality according to their attitudes, aptitudes, and procedures in clinical practice. The study was carried out among family and community medicine physicians in primary care of the Andalusian Health Service in Almer\u00eda, located in southeastern Spain. This location was selected for reasons of plausibility and methodological soundness. According to the Spanish National Statistics Institute, these professionals attended to a population of 731,792 inhabitants as of 1 January 2022 . The queThe sample size was calculated utilizing the Epi Info\u2122 app, from Atlanta CDC, with the following parameters: population size of 731,792 , 80% conThe eligible populations were family and community medicine physicians in the Andalusian Health Service primary care in Almer\u00eda. Population peculiarities were not contemplated, as the subject of the study were physicians and not their patients. The questionnaire was sent by email from the Almeria Primary Care Management of the Andalusian Health Service to the professionals potentially eligible to participate in the study. The inclusion criteria were: being family and community medicine physicians, working in the Andalusian Health Service in Almer\u00eda, working in primary care, and being able to speak and read Spanish fluently. The exclusion criteria were: not meeting any of the referred inclusion criteria and not wishing to participate in the study, despite meeting the criteria.Content validation was performed by an expert panel made up of nine family physicians and sexologists. The tool\u2019s reliability was measured using Cronbach\u2019s alpha . The splStatistical analyses and the exploratory factorial analysis were performed using SPSS version 28 . Univariant and bivariant analyses were conducted. The statistical software AMOS version 26.0.0  was used for confirmatory factor analysis. The study was conducted in accordance with the Declaration of Helsinki. Informed consent was shown at the beginning of the questionnaire. Personal data were not collected. The confidentiality of the participants was absolute as no personal data were collected or stored, and the researchers only could access completely anonymous questionnaires. Although the responses were anonymous and, therefore, participants could not be identified, the questionnaires were stored in encrypted servers of the Andalusian Health Service. This study was approved by the Research and Ethics Committee of Nursing, Physiotherapy, and Medicine Department of the University of Almeria (Spain), with approval number EFM 205/2022.Two hundred and fifty-nine family and community medicine physicians participated in the research. Their mean age was 37.3 years (range 24\u201365), with a standard deviation (SD) of 11.8. One hundred eighty-one (69.9%) were women, two hundred and nine (80.7%) were heterosexual, two hundred and nine (80.7%) had a partner, and one hundred thirty-one (50.6%) had not received any specific training related to sexology .p-value < 0.001. The split-half method did not detect significant differences in the domains or the global evaluation .When analyzing the scores of the participant regarding their sexual orientation , signifiThe analysis of the participants\u2019 scores regarding sexology training  showed tThe main objective of this study was to assess the family physicians\u2019 work regarding sexuality quantitatively through a questionnaire that evaluated their attitudes , their aptitudes (what they could do) and the procedures (what they currently did) of professionals of primary care regarding the sexuality of their patients. We also sought to assess the validity of the questionnaire to perform this specific task.Most of the physicians that participated in this research were women, which can be representative of the current composition of primary care physicians, as stated by other authors . This fiThe exploratory factor analysis excluded 38 items from the initial 50-item questionnaire. This process allowed us to define a much shorter validated questionnaire, also easier to fulfill, based on the 12 items that contributed most to the construct of the questionnaire. This figure may appear significant but aligns with other studies in other ambits ,35. MoreThe confirmatory factorial analysis was utilized to establish the questionnaire\u2019s underlying conceptual structure . Albeit Contrary to some authors who state that physicians\u2019 attitudes toward sexology could be improved ,39, the The analysis of the different domains of the questionnaire regarding different aspects of the family physicians, such as sex, sexual orientation, training in sexuality, or if they had a partner, showed some interesting findings. Similar to other research ,21, womep-value was slightly under the significance value. Even more, in the rest of the domains or the global score, no significant differences were found regarding the sexual orientation of the Family physicians. Therefore, we can conclude that the attitude toward sexuality was independent of the sexual orientation of the healthcare providers, excepting a possible worse attitude from heterosexual professionals. However, given the slight differences, this specific finding should be studied in more detail in future research.Some authors have described that heterosexual healthcare providers have a more positive attitude toward sexuality when they address heterosexual people . HoweverIt seems logical that the professionals with higher training in sexuality scored higher in the global score and the domains that defined the professional procedures on sexuality, family planning, and professional competence. However, contrary to several authors\u2019 findings ,40,41,42The last aspect analyzed, the age of the family physicians, shows some striking results not reflected in the literature. It seems logical that younger physicians score higher in the domain that defines the attitude toward sexuality, while older participants score higher in the domains that define the knowledge of professional procedures on sexuality and family training. However, there was a positive correlation between the age of the physicians and the global score of the questionnaire. A possible conclusion of these findings is that, independently of their age, family physicians seem to become more involved with their patients\u2019 sexuality as they gain professional experience. We believe that this is another critical finding that seems logical and promising.A significant interpretation of our results is the potential applicability for clinical practice and professional training programs\u2019 development. The validation of this questionnaire was intended to allow further application abroad, nationally or internationally, to comprehend family medicine physicians\u2019 situation towards sexuality. This potential future research may allow the development of strategies to improve the situation when required. The data obtained from its appliance could also raise awareness among professionals about the relevance of education in these matters. Future research could dig into themes such as practitioners\u2019 flaws and the resources they consider more helpful to acquiring sexuality concerns in their day-to-day work.This research has some limitations. The most important is the selection bias due to various factors. Our sample was obtained from the family physicians of a province of 731,792 inhabitants, but it could not be representative of other regions or countries. In addition, participation was voluntary, which contributes to the potential selection bias. We must also consider that the questionnaire and the study were in Spanish. Larger sample sizes could also help increase the confidence interval. These aspects must be considered when assessing the external validity of our conclusions, which should be interpreted with caution.This research also has some strengths. The most important one is that the questionnaire obtained excellent results in the exploratory and confirmatory factor analyses and the reliability studies. These aspects give almost complete validity to the final questionnaire and support the possibility of using it in future studies. Another important strength is that the study has been performed with current real healthcare professionals from primary care, and some of the aspects assessed are innovative. Thus, our findings can be helpful for current clinical practice and future research.This study could be the first to describe the percentage of heterosexual, homosexual, and bisexual family physicians or the percentage with a partner and correlate these aspects to their competencies, attitudes, and knowledge of procedures regarding sexology and family planning in their patients. While female family physicians may show a more positive attitude toward sexuality, males feel more confident globally. Competences, attitudes, and knowledge of procedures are the same, independent of whether the professional has a partner, and there may be slight differences regarding attitude when considering the sexual orientation of the physicians. One of the most important findings is that the attitude toward sexuality does not depend on their previous training in this topic. Even more, albeit younger family physicians have a more positive attitude toward sexuality, all providers seem to become more involved with their patients\u2019 sexuality as they gain professional experience. These are critical findings that can break some clich\u00e9s. Finally, the developed questionnaire is a valid tool to assess these aspects and could be translated and culturally adapted to other languages or countries for future research."}
+{"text": "Chlorella vulgaris is a green microalga with a high chlorophyll content, representing a valuable source of green pigments for food applications. As the application of whole biomass can promote an unpleasant fish-like flavor, the use of chlorophyll extract can overcome this drawback. However, chlorophylls tend to easily degrade when out of the chloroplasts, decreasing their potential as a food ingredient. Thus, to study the suitable conditions for isolated chlorophylls preservation, in this work, the influence of temperature (4 to 60 \u00b0C), light (dark or 24 h photoperiod), alkaline conditions (with or without aqueous NaOH addition), and modified atmosphere (air or argon atmosphere) on the stability of the color in ethanolic solutions obtained from C. vulgaris were studied. The loss of green color with temperature followed the first-order kinetics, with an activation energy of 74 kJ/mol. Below 28 \u00b0C and dark conditions were suitable to preserve isolated chlorophylls. The addition of NaOH and an inert argon-rich atmosphere did not exhibit a statistically positive effect on color preservation. In the case study, cooked cold rice was colored to be used in sushi. The color remained stable for up to 3 days at 4 \u00b0C. Therefore, this work showed that C. vulgaris chlorophylls could be preserved in ethanolic solutions at room or lower temperatures when protected from light, allowing them to obtain a suitable natural food ingredient to color foodstuffs. Chlorella is a great source of chlorophylls, namely chlorophylls a (Chl a) and b (Chl b), being one of the highest chlorophyll contents found in nature, reaching up to 45 mg per g of dry weight [Chlorella species, such as Chlorella vulgaris, can be added to processed foods as a green coloring agent. However, the addition of green microalgae to food formulations could potentially develop food products with a slight fish flavor that can be negatively perceived by consumers [C. vulgaris with organic solvents [C. vulgaris [Chlorophylls are green pigments that have been used in the food industry as a natural food ingredient in processed foods. Additionally, due to their strong green pigmentation and consumers\u2019 demand for natural and sustainable foods, following a clean-label market trend, chlorophylls are gaining major importance as food coloring ingredients . Indeed,y weight . Thus, Csolvents ,7. Ethanvulgaris . Chlorophylls are sensitive compounds since they can degrade easily by various mechanisms when exposed to organic solvents, pH variations, heat, oxygen, or light. Acidification and/or thermal processing result in a perceivable discoloration of chlorophylls from bright green to an olive-green or olive-yellow color, known as pheophytinization. During this reaction, occurs the substitution of the magnesium ion in the porphyrin ring by two hydrogen ions, transforming the natural chlorophylls to their corresponding pheophytins ,9. The s2+ and Zn2+ [Consequently, efforts in preserving the green chlorophylls mostly in foods ,14,15,16and Zn2+ ,15. Studand Zn2+ ,18. C. vulgaris pigments were extracted with ethanol, a food-grade solvent. The influence of combined storage conditions, namely temperature, light, atmosphere, and alkaline environment, on the stability of ethanol solutions of C. vulgaris chlorophylls/color was studied. These aim to set the most appropriate storage conditions for C. vulgaris ethanolic extract to stabilize the color to further allow their application as a suitable green coloring ingredient without affecting the organoleptic characteristics of the food products.This study aims to obtain a proper green extract to incorporate into food products. Thus, v/v) and acetone, were also used to evaluate the C. vulgaris chlorophyll\u2019s extraction yield  allowed us to obtain a total chlorophyll content of 7.9 mg/g, agreeing with the literature [C. vulgaris did not reveal a particularly interesting efficiency. The liposoluble chlorophylls are usually extracted with organic solvents such as chloroform, methanol, ethanol, and acetone or their mixtures . Althougon yield . The extterature , correspterature , dried Ca content was higher than Chl b content (v/v), which agrees with the higher content of Chl a in C. vulgaris biomass [C. vulgaris for further studies. Chl  content  when ext biomass . This alv/v/v) and n-hexane:acetone . The separation was carried out on the principle of affinity of substances to the stationary phase and solubility in the mobile phase according to their polarity as follows: the fastest mobility compound was carotene, pheophytin, Chl a, Chl b, and xanthophylls  was performed using two mobile phases with different polarities: petroleum ether:1-propanol:water . The evaluation of pigments/green color degradation over time was carried out with 16 experiments in total, measuring the absorbance.Since Chl ntensity , the evaC. vulgaris ethanolic extract was performed  for the 16 experimental storage conditions are represented in a degradation. The major absorbance decrease was observed for the first period measured, 48 h of storage. In the conditions that have in common the temperature of 4 \u00b0C in the dark, it was observed that with the addition of NaOH in the presence of argon, a lower absorbance decrease (\u22120.001 \u0394abs/h) was obtained when compared with the other ones (average of \u22120.003 \u0394abs/h) a. NevertC. vulgaris ethanolic extract was also measured using the CIELAB system, which complements the spectrophotometric method since color vision is a complex phenomenon and its measurement can be more complex than absorption at specific wavelengths [The color of elengths . Thus, telengths ,18. Figua degradation and to understand the way that those conditions interact between them, an unreplicated 24 full factorial design with two levels was performed for 48 h of storage. The two levels are coded (+1) and (\u22121) for the higher and lower limits of each one, respectively. The absorbance at 418 nm (Y1) and \u2212a* (Y2) for the full factorial design is represented in In order to better understand which conditions have more influence in Chl 2) and temperature (X1) exhibited a significant effect on pigment degradation. This is shown by the bars of the standardized effect that are beyond the vertical red line, representing the statical significance at a 95% confidence level, which does not happen when considering their interactions. Moreover, the Pareto chart evidenced that the linear light variable exerts the most preponderant effect. Accordingly, the Pareto chart in 2) and temperature (X1) showed a significant effect on the green color vanishing (\u2212a*) of C. vulgaris ethanolic extract, being the presence of light the most significant factor as well. According to both Pareto charts (3) and the alkaline environment (X4) on ethanolic solutions had no statistical impact on the color loss. Contrary to the Pareto chart of the response at Abs 418 nm (1X2). When an interaction is significant, it means that the effect of a term on the response is distinct at different levels of another independent variable [The Pareto chart a represeo charts , under ts 418 nm a, the Pas 418 nm b showed variable . a, Chl b, and lutein) of pistachio kernels since no differences were observed during storage, irrespective of the use of oxygen scavengers and high gas barrier plastic films [When chlorophyll in organic solvents such as ethanol, acetone, or benzene is exposed to light in the presence of oxygen, it is irreversibly bleached by photo-oxidation, resulting in colorless derivates . The preic films . a in ethanolic solutions increases with temperature (tested from 20 to 50 \u00b0C). Moreover, the stability of Chl a or green color with temperature in broccoli and green beans treated from 40 up to 96 \u00b0C [C. vulgaris ethanolic extract, the solution changed immediately from bright green to olive brown (results not shown). However, the addition of NaOH did not significantly influence the chlorophyll degradation in the present study. Alkaline conditions have been reported to induce oxidation of the isocyclic ring and de-esterification of phytol in chlorophylls. These reactions do not significantly affect the color of the product since these compounds retain intact the basic structure of the chromophore group with Mg2+ linked to the porphyrin ring [Temperature is also a well-established main factor influencing the stability of chlorophylls  since hito 96 \u00b0C  and spinto 96 \u00b0C  was alsorin ring . Consequrin ring  or even rin ring .C. vulgaris ethanolic extract in different storage conditions, another set of experiments (set 2) was performed, starting with a higher-concentrated ethanolic pigment solution. Since the presence of NaOH seemed to prevent the degradation of long-term chlorophyll at 4 \u00b0C in the dark under air atmosphere  and green color (\u2212a*), particularly in the presence of light, with higher slopes observed (The samples with higher chlorophyll concentration (set 2) had a higher degradation rate of Chl observed . In dark1 (abs 418 nm) and Y2 (\u2212a*) are represented in 2) on the decreasing absorbance (418 nm) at 9.5 h of storage, as shown in the Pareto chart (1) and alkaline environment . For the same time of storage (9.5 h) of C. vulgaris ethanolic extract, similar behavior was observed in the stability of the \u2212a* parameter (1) also exhibited a lower preponderant influence on the decrease of absorbance without statistically affecting the decrease of \u2212a*. The evaluation of the CIELAB parameter \u2212a* reflects the vanishing of green color, which is a crucial visual parameter to take into account for further application of these colorants in the food industry. The full factorial analysis was employed at 9.5 h and at 65.5 h, and the responses Yto chart a, and tharameter b, where 2) and temperature (X1) were not observed, contrarily to the experiments performed at 60 \u00b0C, allowing us to infer that the temperature of 28 \u00b0C did not promote a significant extent of chlorophyll degradation as observed at 60 \u00b0C. It was reported that both refrigerator and room temperatures are suitable to store Chl a dissolved in acetone, which was kept for 84 days in the dark [a of pistachio kernels was only slightly different at 10 and at 25 \u00b0C [Significant two 2-way interactions between the terms light  was plotted vs. time , from which the rate constants (k) were calculated. C. vulgaris ethanolic extract preserved in the dark at different temperatures, namely 4, 15, 28, 45, and 60 \u00b0C. The correlation coefficient was >0.84 in all cases, confirming that the fading of the visual green color followed first-order kinetics at all temperatures. These results are in line with the first-order reaction found for Chl a degradation in solutions with different percentages of ethanol (1\u201360%) [The CIELAB \u2212a*/\u2212aperature . Using l (1\u201360%)  and in v (1\u201360%) , broccol (1\u201360%) , and grek vs. the inverse of temperature. The Arrhenius plot for color loss of C. vulgaris pigments in ethanol revealed that the main color loss occurred between 28 and 60 \u00b0C under the studied conditions, emphasizing the importance of the higher temperatures in the green color degradation. When plotting the three higher temperatures, it was obtained an activation energy of 74 kJ/mol  was added to cooked cold white rice. The visual color stability was observed over time at 4 \u00b0C and is shown in C. vulgaris ethanolic extract, it does not have an influence when applied to the rice. The application of the green extract in cooked cold rice proved the potential of C. vulgaris as a source of a food green color additive, mainly for the immediate addition and consumption of processed food, meeting the need of consumers for natural colorants. As a request from the food industry to have green-colored rice used in sushi dishes, the green ct 1 and  mL was aC. vulgaris microalgae dry biomass. Thus, 10 mL of ethanol 96%, acetone, or chloroform:methanol  were added to 100 mg of biomass, and the respective suspensions were homogenized for 10 min at room temperature. The suspensions of all extractions were centrifuged for 3 min (4000\u00d7 g rpm), and the supernatant was collected. Two other extractions with 5 mL were performed, and all supernatants were combined.Different solvents and extraction methodologies were tested to obtain the pigments (green color) from C. vulgaris at one of the maximum absorption wavelengths that slightly changed according to the organic solvent used. The absorbance was measured at 649 and 664 nm to determine chlorophyll a (Chl a) and b (Chl b) contents when ethanol was used [v/v) extract, the absorbance was measured at 648 and 666 nm [a and Chl b and expressed as mg/g biomass dry weight [Absorption measurements of chlorophyll were made on an Eon Microplate Spectrophotometer . The equations proposed by Wellburn  and Lichwas used . For acewas used . For chld 666 nm . The toty weight .C. vulgaris extracted with ethanol 96%, a thin layer chromatography (TLC) was performed. Thus, C. vulgaris ethanolic extract was applied to 4 cm \u00d7 10 cm silica plates. Two different eluents were used, namely petroleum ether:1-propanol:water  and n-hexane:acetone . To identify the pigments of C. vulgaris pigments were extracted with ethanol to evaluate their storage stability under different conditions, namely temperature, light, atmosphere, and alkaline environment. In the data set, 1.4 g of C. vulgaris biomass was mixed with 300 mL of ethanol 96% and stirred for 15 min to extract pigments. Thereafter, the ethanolic solution was centrifuged and filtered with glass fiber filters under a vacuum. More ethanol 96% was added until the absorbance at 418 nm reached 0.6 (addition of about 800 mL ethanol). NaOH 1 M (100 \u03bcL) was added to 300 mL of the solution. Since the solutions were not aqueous, the pH value that could be measured by a pH meter would not be reliable. For that reason, the variation in the concentration of NaOH in the ethanolic solutions was taken as a measure of the concentration of variation of H+ in the samples.The ethanolic extracts were distributed in glass bottles (25 mL). Eight bottles (four with and four without NaOH addition) were placed at 4 \u00b0C (refrigerator temperature) and the other eight bottles at 60 \u00b0C (a representative extremely high temperature) in a bath with paraffin (to avoid long-term evaporation). At each temperature, four bottles were protected from the light (two with and two without NaOH addition), and four bottles were subjected to light with a photoperiod of 24 h (about 3500 lx). In each temperature, half of the bottles were under an air atmosphere, and the other half were under an argon atmosphere, according to the scheme represented in 4 full factorial design with two levels was used to evaluate, after 48 h of storage, the effect of temperature , light , modified atmosphere , and alkaline environment . The two levels are coded (+1) and (\u22121) for the higher and lower limits of each one, respectively. In a two-level full factorial design, 2k runs are required, where k represents the number of factors to be analyzed, which results in 16 runs performed. The experimental data were statistically analyzed using Minitab v17 software. The Pareto charts were developed for the linear terms and for their interactions that showed statistical relevance to simplify the model. The Pareto chart with the linear and all the 2-way interaction terms are presented in To statistically analyze the data, an unreplicated 23 full factorial design with two levels to evaluate the effect of temperature , light , and alkaline environment  for 9.5 and 65.5 h, using a more concentrated C. vulgaris ethanolic solution (8 g of C. vulgaris biomass extracted with 600 mL of 96% of ethanol), resulting in 8 runs performed. The preparation of the bottles with the ethanolic extract was performed as described for data set 1, except for the use of a water bath to monitor the temperature at 28 \u00b0C instead of a bath with paraffin. The absorbance and color stability were evaluated over 14 days  was performed with an unreplicated 2C. vulgaris ethanolic extract, the increase of parameter \u201ca*\u201d values from a more negative value towards zero (\u2212a*) was considered a visual parameter to describe the green color degradation at different temperatures [k) was calculated using the following equation:t) and The bottles with ethanolic extract (data set 2) were placed at five different temperatures: 4, 15, 28, 45, and 60 \u00b0C , protecteratures . The firaE is the activation energy (kJ mol\u22121), k is the first-order reaction rate constant (s\u22121), k0 is the pre-exponential factor, R is the universal gas constant (8.3145 J mol\u22121 K\u22121), and T is the absolute temperature (K).Temperature dependence of green color degradation was determined by the Arrhenius equation:C. vulgaris was obtained using 1 g of biomass and 10 mL of ethanol 96%. This extract (1 mL) was added to two containers with 18 g of cold-cooked rice, the minimum volume necessary to confer an appropriate coloration to the rice. Moreover, 2 mL was also added to two containers with the same amount of rice to evaluate the effect of the chlorophyll concentration in the applied food product. A fifth container was used as a control, only containing the cooked rice. All containers were placed at 4 \u00b0C, two in the presence of light in a 24 h photoperiod and two in the absence of light; the control was maintained at 4 \u00b0C in the presence of light  had no statistical effect on the green color preservation. The loss of color in the ethanol solution with temperature followed the first-order kinetic, being more significant between 28 and 60 \u00b0C, with an activation energy of 74 kJ/mol. These results showed that C. vulgaris chlorophylls could be preserved in a food-grade solvent at room or lower temperatures when kept in the dark, obtaining an easy-to-handle extract that can be used as a natural food ingredient without conferring an unpleasant flavor. Moreover, the green residue left is also a non-odorant food ingredient rich in protein, starch, and cell wall polysaccharides, thus with high potential to be further valued. This work allowed us to obtain a food-grade and stable extract to be applied as a green colorant ingredient, pursuing the market tendency for clean-label products. The assessment of the color stability of a"}
+{"text": "Hyperglycemia has various adverse health effects, some of which are due to chronic oxidative and inflammatory impairment of bone marrow (BM), hematopoietic stem cells (HSCs), and mesenchymal stem cells (MSCs). Astaxanthin (ASTX) has been shown to ameliorate hyperglycemia-associated systemic complications and acute mortality, and this effect is partially associated with restoration of normal hematopoiesis. Here, the effects of ASTX on diabetes-induced complications in BM and BM stem cells were investigated, and the underlying molecular mechanisms were elucidated. Ten-week-old C57BL/6 mice received a single intraperitoneal injection of streptozotocin  in combination with oral gavage of ASTX (12.5 mg/kg) for 30 or 60 consecutive days. Supplemental ASTX ameliorated acute mortality and restored the STZ-impaired bone mass accrual and BM microenvironment in STZ-injected mice. Oral gavage of ASTX suppressed osteoclast formation in the BM of STZ-injected mice. Specifically, supplementation with ASTX inhibited oxidative stress and senescence induction of BM HSCs and MSCs and ameliorated hematopoietic disorders in STZ-injected mice. These effects of ASTX were associated with BM restoration of angiopoietin 1, stromal cell-derived factor 1, \u03b2-catenin, and Nrf2. Long-term ASTX gavage also recovered the STZ-induced dysfunction in migration, colony formation, and mineralization of BM-derived stromal cells. Further, a direct addition of ASTX exhibited direct and dose-dependent inhibition of osteoclastic activation without cytotoxic effects. Collectively, these results indicate that ASTX protects against diabetes-induced damage in the BM microenvironment in BM, HSCs, and MSCs and restores normal hematopoiesis and bone accrual in STZ-injected mice. Diabetes mellitus (DM) is a metabolic syndrome characterized by impaired insulin secretion, insulin sensitivity, or both . DM caus-Sca-1+c-Kit+ (LSK) cells in the BM [+ cells, along with reduced mobilization of hematopoietic progenitor cells (HPCs) in diabetic patients, were found to be associated with DM-mediated hematopoietic complications [Numerous studies have shown that long-term diabetic disorders are orchestrated by impairments in the bone marrow (BM) microenvironment and hematopoietic development ,10,11,12n the BM ,14. In aications . STZ-indications . Togetheications .Interaction of HSCs with osteoblastic niche cells is important for their retention and development in BM. Osteoblastic cells secrete various cytokines and chemokines that support the survival and differentiation of BM HSCs . In addiHyperglycemia-related local and systemic disorders have been attributed to prolonged oxidative and inflammatory damage to cells and tissues. Oxidative stress and inflammatory conditions in the BM are also hallmarks of impaired BM retention and senescence induction of hematopoietic and mesenchymal stem cells in the bone ,10,11,12In this study, how supplemental ASTX affects diabetes-triggered complications in BM and BM stem cells was explored using a STZ-induced diabetic mouse model. To understand the cellular mechanisms by which supplemental ASTX protected against diabetic complications, the expression of various chemokines and signaling molecules involved in the regulation of homeostatic hematopoiesis and bone mass accrual was evaluated. The ex vivo effects of supplemental ASTX on migration, colony formation, and mineralization were also investigated using bone marrow stromal cells (BMSCs) derived from experimental mice. Additionally, the in vitro effects of ASTX on osteoclast formation, DNA damage, and viability were examined using primary cultured bone marrow monocytes (BMMs). Collectively, the current findings not only support a protective role for supplemental ASTX on diabetes-associated impairments in the hematopoietic system and BM microenvironment, but also provide further evidence of its clinical usefulness.ASTX (CAS No. 472-61-7) and STZ (CAS No. 18883-66-4) were purchased from Sigma-Aldrich Co. LLC , while fetal bovine serum (FBS) was obtained from HyClone Laboratories, Inc. . Antibodies specific to Nrf2 (BS1258) and receptor activator of nuclear factor (NF)-\u03baB ligand  were purchased from Bioworld Technology, Inc.  and Enzo Life Sciences, Inc. , respectively. Antibodies specific to interleukin-1\u03b2 , interferon-\u03b3 , tumor necrosis factor \u03b1 , and \u03b2-actin (sc-47778) were obtained from Santa Cruz Biotechnology . 2\u2032,7\u2032-Dichlorodihydrofluorescein-diacetate (DCF-DA), and anti-osterix (ab209484), anti-osteopontin , anti-heme oxygenase 1 , anti-bone morphogenetic protein 2 , and anti-\u03b3-H2AX (ab26350) antibodies were purchased from Abcam . Unless otherwise specified, other reagents were purchased from Sigma-Aldrich Co. LLC, while laboratory consumables were from Falcon Labware .n = 5/cage) were housed at 22 \u00b1 1 \u00b0C and 55 \u00b1 5% humidity on a 12 h light/dark auto-cycling with free access to food and water and monitored 12 h/day during the experimental period. At 2 weeks post-acclimatization, STZ and STZ+ASTX groups received STZ, as described previously [\u00ae Advantage, Roche Diagnostic, Mannheim, Germany); mice with a glucose level greater than 300 mg/dL were used as hyperglycemic diabetes mice in subsequent experiments. The STZ+ASTX group received 12.5 mg/kg ASTX via oral gavage in 100 \u00b5L olive oil (vehicle) daily for 30 or 60 consecutive days. The vehicle and STZ groups received 100 \u00b5L olive oil only. ASTX group received 12.5 mg/kg ASTX in 100 \u00b5L olive oil daily for 60 consecutive days without STZ injection. Blood glucose levels and body weight changes were monitored in all groups every 10 days after hyperglycemia induction. Consumption of water, food intake, and the survival rate of mice were observed daily. Experimental samples, including skeletal long bones, peripheral or whole blood, pancreas, and whole BM cells and BM supernatants were harvested 12 h after the final supplementation with ASTX.Eight-week-old C57BL/6 male mice  were supplied by Orient Bio  and were randomly divided into five groups: non-diabetic control, vehicle, STZ, STZ+ASTX, and ASTX groups. Mice (eviously . Briefly3), bone volume percentage , bone mineral density , trabecular number , and trabecular thickness  in trabecular and cortical regions of femoral bones were evaluated from the reconstructed 3D images, as described previously [Femoral bones were scanned using a desktop scanner  60 days post hyperglycemia induction. Maximum voltage was 100 kV and maximum current was 100 \u00b5A using a 1-mm filter with a tomographic rotation of 360\u00b0 (rotation step of 0.6\u00b0). Images were obtained at 18 \u00b5pixels, and data were analyzed using the SkyScan NRecon reconstruction package . Image slices were also reconstructed using cone-beam reconstruction software based on the Feldkamp algorithm . Values of bone-specific parameters, namely bone volume  and counterstained with 0.25% Eosin Y stain . Finally, stained sections were observed under a light microscope .+ osteoclasts formed in trabecular or cortical bone surface (mm2).Osteoclasts formed in trabecular and cortical zones of femoral bones were quantified by staining tissue sections for TRAP using a leukocyte acid phosphatase kit . The TRAP-stained sections were counterstained with hematoxylin and photographed using a light microscope (EL-Einsatz 451888). Osteoclastic activation in the bones was determined by counting the number of TRAPLevels of HO-1 and Nrf2 in the pancreas were evaluated by IHC. Briefly, tissue sections were stained with primary antibody (1:200 dilution) specific to HO-1 or Nrf2. Expression patterns were determined using a mouse-anti-Vectasta in ABC DAB-HRP kit , followed by observation under a light microscope (EL-Einsatz 451888). The area (%) of cells positively stained with HO-1 or Nrf2 was determined using ImageJ software . Unless otherwise specified, five mice per group were used for the staining assays, such as H&E, TRAP, and IHC, in which at least 5 tissue sections per mouse were analyzed in each assay.Gapdh) was used as the endogenous control.Total RNA was extracted from whole BM cells isolated from mice at 60 days post-hyperglycemia induction using TRIzol reagent . RNA samples (1 \u03bcg/sample) were subjected to cDNA synthesis using an AmpiGene cDNA synthesis kit , following the manufacturer\u2019s instruction. qRT-PCR was performed using Power SYBR Green PCR Master Mix  and an ABI StepOnePlus Real-Time PCR system (Applied Biosystems). Thermocycling conditions were 95 \u00b0C for 10 min for pre-denaturation, followed by denaturation at 95 \u00b0C for 15 s, annealing at 60 \u00b0C for 30 s, and extension at 72 \u00b0C for 30 s for 40 cycles. Oligonucleotide primers specific to SDF-1, Ang1, \u03b2-catenin, CXCR4, and DKK1 were designed . Glycerag) for 10 min and processed for the removal of red blood cells (RBCs). After washing with PBS, cells were analyzed by multicolor flow cytometry , and phenotypical identification of cell populations was performed using FlowJo software  at the Center for University-Wide Research Facilities of Jeonbuk National University, as described previously [\u2212Sca-1\u2212c-Kit+ cells , and CD150+CD48\u2212LSK cells (HSCs) were phenotypically identified after staining with lineage markers following phycoerythrin (PE)-Cy7-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD45R, anti-CD11b, anti-Gr-1, and anti-TER-119. In addition, cells were stained with PE- or fluorescein isothiocyanate (FITC)-conjugated anti-stem cell antigen 1 (Sca-1), an allophycocyanin (APC)-conjugated anti-c-kit, PerCP/Cy5.5-conjuated anti-CD150 , and APC-Cy7-conjugated anti-CD48. MSCs (CD29+CD105+LSK) were characterized using a PE-Cy7-conjugated lineage cocktail, APC-Cy7-conjugated anti-Sca-1, PE-conjugated anti-CD29, and APC-conjugated anti-CD105 antibodies. Flow cytometric analysis was also carried out to assess oxidative stress, DNA damage, or senescence-related phenotypes in BM HSCs and MSCs. Briefly, mitochondrial superoxide anion level and senescence-associated \u03b2-galactosidase  activity was assessed with kits containing MitoSox Red  or C12FDG  according to the manufacturers\u2019 instructions. Levels of cell cycle regulatory factor p16INK4a (Santa Cruz Biotechnology) and the DNA damage marker \u03b3-H2AX  in hematopoietic and mesenchymal stem/stromal cells were determined using Alexa Fluor 488-conjugated and PE-conjugated antibodies, respectively, after fixation and permeabilization. Alternatively, intracellular expression levels of Nrf2 in BM cells were measured by flow cytometry. To this end, whole BM cells isolated from femur and tibial bones were fixed, permeabilized, and stained with PE-conjugated Nrf2 antibody , followed by flow cytometric analysis. Here, cell fixation and permeabilization for intracellular staining were performed using BD Cytofix/CytopermTM , according to the manufacturer\u2019s instructions. Unless specified otherwise, other antibodies used in flow cytometric assay were purchased from BD Biosciences.Hematopoietic and mesenchymal stem/stromal cells in the BM were harvested from long bones of control, STZ, and STZ+ASTX mice at 30 or 60 days post-hyperglycemia induction. In brief, the ends of femoral and tibial bones were cut and flushed with phosphate-buffered saline (PBS) using a 5-mL syringe without crushing bones or treating with collagenase. BM cells were collected by centrifugation (1000\u00d7 eviously . In this2EDTA-treated tubes (BD Biosciences), respectively, 30 and 60 days after hyperglycemia induction. Levels of circulating granulocytes, lymphocytes, RBC, and white blood cells (WBC) were analyzed using an automated blood cell counter .Peripheral and whole blood samples were collected from mouse groups via tail vein cutting and cardiac puncture into Kg in serum separator tubes. Serum level of RANKL was measured using a mouse-anti-RANKL ELISA Kit  using a microplate reader , following the manufacturers\u2019 instructions.Serum was obtained from whole blood samples at 30 or 60 days after hyperglycemia induction by centrifuging whole blood samples at 1500\u00d7 g for 3 min. Pellets were resuspended, seeded onto 60-mm tissue culture plates, and incubated in growth medium . After a 24-h incubation, non-adherent suspended cells were carefully removed, and adherent cells were collected by centrifugation at 2000\u00d7 g for 5 min. Here, the adherent cells were used as BMSCs for ex vivo experiments. BMMs were also obtained from the long bones of 10-week-old B6 mice that did not receive STZ or ASTX treatment. Briefly, RBC-free BM cells were incubated for 24 h in growth medium and, then, non-adherent and suspended cells were harvested and collected to use as BMMs.Whole BM cells were isolated from long bones of mouse groups 60 days post-hyperglycemia induction. BM cells were collected by centrifugation for 10 min before the removal of RBCs. After washing with PBS, BM cells were resuspended in \u03b1-minimum essential media  and centrifuged at 2000\u00d7 6 cells/well). When these cells reached approximate 70% confluence, the bottoms of the plates were scraped to create a 1-mm-wide wound area. Twenty-four hours after the wound area was created, photographs were taken using an optical microscope (EL-Einsatz 451888), and wound areas (relative area (%) of migrated cells to that of control) were calculated using ImageJ software. BMSCs were also spread onto 6-well culture plates (106 cells/well) in growth medium. After 14 days of incubation, adherent cells were fixed in 10% formalin for 10 min and stained with 0.5% crystal violet dissolved in 100% methanol. BMSC-derived colonies (CFU-F) containing more than 50 cells per colony were counted. In addition, BMSCs isolated from mouse groups were incubated in osteogenic medium supplemented with 5% FBS, 100 nM dexamethasone, 50 \u00b5M of ascorbic acid, and 10 mM \u03b2-glycerophosphate (DAG). Culture media was changed every 3 days throughout the incubation period. After 21 days of incubation, the degree of mineralization was determined by staining cells with Alizarin Red S after fixation for 30 min in 4% paraformaldehyde. Cells were stained with the red dye and photographed under a light microscope (EL-Einsatz 451888). The stained cells were also treated with 10% acetylpyridinum chloride, and the amount of red dye was quantified by measuring the dye-specific absorbance at 405 nm using a microplate reader (SPECTROstar\u00ae Nano).The effect of supplemental ASTX on the migration of BMSCs into wound areas was initially evaluated on culture plates. In brief, control-, STZ-, and STZ+ASTX-derived BMSCs were spread onto 12-well culture plates (106 cells/well) in growth medium. After 24 h of incubation, culture medium was changed to DAG-supplemented medium followed by an additional incubation for 5 days. At the end of the incubation, whole protein lysates were extracted from BMSC cultures, and extracts (20 \u03bcg/sample) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12% gels. After electroblotting onto polyvinylidene difluoride membranes, blots were washed with buffer (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.05% Tween-20) and treated with 5% skim milk for 1 h followed by incubation with primary OPN, osterix, BMP2, or \u03b2-actin antibodies (1:1000 or 2000 dilution). Blots were exposed to horseradish peroxidase-conjugated secondary antibodies, and immunoreactive bands were visualized by enhanced chemiluminescence , followed by X-ray film exposure .BMSCs were spread onto 6-well culture plates (2 \u00d7 106 cells/well) in \u03b1-MEM supplemented with 5% FBS, 30 ng/mL monocyte-colony stimulating factor (M-CSF) and 50 ng/mL RANKL in the presence or absence of ASTX (30 or 60 \u03bcM). During the incubation, medium was replaced with fresh medium every 3 days. After 7 days of incubation, BMMs were stained with TRAP, and TRAP-positive multinucleated cells (MNCs) were counted as osteoclasts. BMMs cultured for 7 days were also permeabilized with Triton\u2122 X-100 and stained with FITC-conjugated phalloidin (green) to visualize the formation of F-actin rings. In addition, BMMs were incubated in osteoclastic medium with and without ASTX (30 or 60 \u03bcM). After 2 days of incubation, expression levels of osteoclastogenic molecules such as nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), TRAP, and cathepsin K (CTSK) were evaluated on qRT-PCR. All procedures for PCR followed the same methods described above. Primer sequences of the osteoclastic molecules are shown in BMMs were spread onto 12-well culture plates (2 \u00d7 106 cells/well) or 96-multiwell culture plates (2 \u00d7 103 cells/well) in growth medium supplemented with 30 or 60 \u03bcM ASTX. After 48 h of incubation, the level of \u03b3-H2AX, a well-known marker of DNA double-strand breaks, was detected by flow cytometry using PE-conjugated antibodies. Viability of BMMs incubated in the same medium for 2 days was determined using a Cell Counting Kit-8 , according to the manufacturer\u2019s instructions.To evaluate the direct effects of ASTX on DNA and BMM viability, BMMs were seeded onto 6-well . Scheffe\u2019s multiple range test was used for multiple comparisons among groups. The significance of differences between two groups was analyzed by unpaired Student\u2019s A schematic diagram of the experimental design, including mouse groups, treatments, and schedules for sample collection, is shown in It was examined whether hyperglycemic conditions actually impaired the BM microenvironment and bone mass accrual. Two-dimensional (2D) images A and rec+ regions in trabecular bone than did control, vehicle, STZ+ASTX, or ASTX group. Significantly higher numbers of TRAP+ osteoclasts were found in the trabecular region of the STZ group compared with that of control (p < 0.001), vehicle (p < 0.001), STZ+ASTX (p < 0.01), or ASTX group (p < 0.001) (+ osteoclasts compared with that of other groups (data not shown). In addition, the STZ group exhibited higher concentrations of RANKL, a ligand for the receptor RANK, in serum than did the control, vehicle, STZ+ASTX, or ASTX groups both 30 and 60 days post-hyperglycemia induction  B. Corticnduction C. There p < 0.05) or STZ group (p < 0.01)  B. In add < 0.01) C,D.+ HSCs (p < 0.01 or p < 0.001) or STZ+ASTX group (p < 0.05 or p < 0.01). The BM level of senescent HSCs was also evaluated in mice by determining the percentages of p16INK4a+ and C12FDG+ HSCs 60 days after hyperglycemia induction. The STZ group exhibited approximately 2.1- and 1.7-fold higher levels of p16INK4a+ HSCs than the control and STZ+ASTX groups, respectively (12FDG+ HSCs than did the control (p < 0.01) or STZ+ASTX group (p < 0.05)  G. These p < 0.01 or p < 0.001) than that in the control or STZ+ASTX group both 30 and 60 days after hyperglycemia induction  or STZ+ASTX group (p < 0.05) (INK4a+ (12FDG+ MSCs (p < 0.05 or p < 0.01) diminished by long-term oral gavage of ASTX  A,B. The   augmented by up to more than two-fold in control- and STZ+ASTX-derived BMSCs (p < 0.001) or STZ+ASTX (p < 0.01) group  group D. In par1) group E,F. Toge+ MNCs from BMMs in a dose-dependent manner (NFATc1, TRAP, and CTSK (+ BMMs (+ BMMs (It was previously found that the BMMs derived from STZ group showed greater osteoclast forming activity than did the cells from control or STZ+ASTX group . To furtt manner A,B. Parat manner C,D. The and CTSK E\u2013G. Howe (+ BMMs H or CCK- (+ BMMs I. These ASTX exists naturally in seafood with a red-orange xanthophyll carotenoid . FDA appROS accumulation and subsequent oxidative damage are thought to be the main causes of diabetes-related complications; investigators have therefore focused their efforts on developing diabetes-specific antioxidant therapies. Ohshima et al. reported that antioxidants attenuate diabetes-induced disorders, such as cellular ROS accumulation and dysregulated BM retention of stem cells . In addiThe maintenance, self-renewal, and peripheral migration of BM cells are tightly regulated by several signaling pathways. Wnt signaling plays crucial roles in embryogenesis, tissue development, homeostasis , and theWhile long-term hyperglycemia causes chronic oxidative and inflammatory complications in various organs, diabetes-mediated death is correlated with the impaired retention, function, and peripheral circulation of BM HSCs . The curThis study shows that long-term oral gavage of ASTX protects against STZ-induced damage to the BM microenvironment and the functions of BM HSCs and MSCs. This study also suggests that supplemental ASTX ameliorates STZ-induced hematopoietic development and bone mass accrual in mice, and that this is closely associated with its ability to inhibit oxidative damage and maintain the Nrf2-related antioxidant defense system. Overall, the current findings highlight the clinical potential of ASTX in ameliorating diabetes-induced oxidative complications in various organs and preventing excessive induction of stem cell senescence."}
+{"text": "Sowohl nach COVID-Infektion als auch nach ein oder mehreren COVID-Impfungen k\u00f6nnen rheumatische Beschwerden beginnen. In beiden F\u00e4llen scheint der Mechanismus \u00e4hnlich zu sein und mit dem Coronavirus oder seinen spezifischen Folgen zusammenzuh\u00e4ngen. Zumeist wird von einer reaktiven Arthritis gesprochen, wenngleich die Bezeichnung COVID-19-assoziierte Arthritis f\u00fcr das Beschwerdebild eher zutreffen d\u00fcrfte. In Relation zur Zahl der COVID-Infizierten und der COVID-geimpften ist die Zahl der F\u00e4lle, in denen es zu COVID-assoziierten Beschwerden kommt, au\u00dferordentlich gering und die Prognose scheint eher gut zu sein. Seit Beginn der Pandemie Ende 2019 mehren sich Berichte, dass nach COVID-Infektion chronische entz\u00fcndlich-rheumatische Erkrankungen beginnen k\u00f6nnen. Rheumatische Beschwerden im Zusammenhang mit viralen oder bakteriellen Infekten sind nicht ungew\u00f6hnlich, k\u00f6nnen auch einige Tage \u00fcber die Infektion anhalten und sind \u00fcblicherweise selbstlimitierend. Demgegen\u00fcber stehen Beschwerden, die Tage bis Wochen nach einem Infekt beginnen, \u00fcber Wochen bis Monate anhalten und sich mitunter zur chronischen entz\u00fcndlich-rheumatischen Erkrankung entwickeln.https://www.sozialministerium.at/Corona/allgemeine-informationen/long-covid.html). Sch\u00e4tzungen zufolge leiden etwa 10\u202f% der COVID-19-\u00dcberlebenden an Long COVID [Nach Abklingen einer akuten Infektion mit SARS-CoV\u20112 k\u00f6nnen Krankheitssymptome noch \u00fcber l\u00e4ngere Zeit persistieren und werden unter dem Begriff Long COVID zusammengefasst. Die h\u00e4ufigsten Beschwerden sind andauernde M\u00fcdigkeit (Fatigue), anhaltendes Krankheitsgef\u00fchl  mit eingeschr\u00e4nkter k\u00f6rperlicher und geistiger Leistungsf\u00e4higkeit, Kurzatmigkeit, Geruchs- und Geschmacksst\u00f6rungen und andere Symptome wie Gelenk- und Muskelschmerzen  bezeichnet. Ausgehend von der Beobachtung des deutschen Milit\u00e4rarztes Hans Reiter, dem w\u00e4hrend des 1.\u00a0Weltkriegs auffiel, dass Harnwegsinfekt, Augenentz\u00fcndung und Entz\u00fcndung der Gelenke mitunter begleitet von typischen Hauterscheinungen (Reiter-Dermatose) h\u00e4ufig gemeinsam auftreten. Bis vor wenigen Jahren wurde bei Vorliegen von zumindest 3\u00a0der 4\u00a0Symptome der Begriff Reiter-Syndrom oder Reiter-Trias  verwendet. Die Bezeichnung ReA wurde 1963 eingef\u00fchrt, 1983 vorl\u00e4ufige Diagnosekriterien formuliert und 1991 die ReA als Untergruppe den Spondylarthritiden zugeordnet [Arthritiden, die nach bekannten Infektionen auftreten, k\u00f6nnen in Abh\u00e4ngigkeit vom Nachweis im Gelenkpunktat in 3\u00a0Gruppen eingeteilt werden. Eine infekti\u00f6se Arthritis liegt vor, wenn im Gelenkpunktat ein Erreger nachgewiesen werden kann, eine postinfekti\u00f6se Arthritis bei Nachweis von mikrobiellen Bestandteilen, aber Fehlen eines intakten Erregers, und eine ReA bei eindeutiger Anamnese, aber Fehlen von Erregern oder Erregerbestandteilen Tab.\u00a0.GruppeInBez\u00fcglich der Bezeichnung einer Arthritis nach COVID-19-Infektion besteht noch kein Konsens. In den meisten Arbeiten wird von einer ReA nach COVID-19-Infektion berichtet . Die ReADie Richtigkeit der Bezeichnung ReA im Zusammenhang mit SARS-CoV\u20112 wird aber immer wieder hinterfragt und diskutiert, ob anstelle von ReA die Bezeichnung virale Arthritis, SARS-CoV\u20112 assoziierte Arthritis oder Post-COVID-Arthritis nicht eher zutreffen w\u00fcrde \u201318. Aus Die reaktive Arthritis zeigt sich typischerweise als asymmetrische Oligoarthritis mit Dominanz der gro\u00dfen Gelenke. Die Post-COVID-Arthritis neigt eher zu diffuser Verteilung mit Befall von Hand- und Sprunggelenken sowie Finger- und Zehengelenken. Die ReA findet man eher bei j\u00fcngeren Patienten, eine Post-COVID-Arthritis vermehrt bei Patienten in der zweiten Lebensh\u00e4lfte. Typischer Ausl\u00f6ser einer ReA sind Bakterien, bei Post-COVID Viren. Eine ReA wird h\u00e4ufiger bei M\u00e4nnern gesehen, eine Post-COVID-Arthritis scheint in beiden Geschlechtern etwa gleich verteilt zu sein. Patienten mit ReA sind \u00fcberwiegend HLA-B27-positiv, bei Patienten mit einer Post-COVID-Arthritis ist eine Anf\u00e4lligkeit von HLA-B27-positiven Patienten bisher noch nicht beobachtet worden , 16. In Das klinische Bild einer ReA wurde auch bei anderen Viruserkrankungen  wie HIV Das Auftreten einer Arthritis nach einer COVID-Infektion wird in zahlreichen Publikationen und Fallberichten belegt. Eine Suche in PubMed mit den Begriffen \u201earthritis\u201c und \u201eCOVID\u201c ergab 1958\u00a0Treffer.n\u202f=\u200911), Fu\u00df (n\u202f=\u20092), Sprunggelenke (n\u202f=\u20099), H\u00fcfte (n\u202f=\u20093), Handgelenke (n\u202f=\u20096), H\u00e4nde (n\u202f=\u20093), Ellenbogen (n\u202f=\u20093) und Schultern (n\u202f=\u20094). Vereinzelt wurde \u00fcber Tendinitis und Daktylitis berichtet [Eine Literatursuche von Dezember\u00a02019 bis Dezember\u00a02021 nach klar definierten Suchkriterien ergab 68\u00a0Artikel, aus denen nach entsprechenden Selektionen 25\u00a0F\u00e4lle aus 22\u00a0Artikeln extrahiert und zusammengefasst werden konnten . Von denerichtet .Eine weitere \u00dcbersichtsarbeit, in der eine Fallbeschreibung erg\u00e4nzt wird durch einen komprimierten Literatur\u00fcberblick bis Juli 2021, ergibt nicht unerwartet \u00e4hnliche Ergebnisse. Die Dauer von der Diagnose COVID bis zur Diagnose ReA war 7\u201390\u00a0Tage (Median\u00a018). Die am meisten betroffenen Gelenke waren Knie, Sprunggelenke und Interphalangealgelenke .In einem zusammenfassenden Bericht \u00fcber 23\u00a0eigene Patienten und 21\u00a0Patienten aus vorangehenden Berichten war die durchschnittliche Zeit von Beginn der COVID-Symptome bis zum Beginn einer Arthritis 23\u00a0Tage (Mittelwert). Die am h\u00e4ufigsten befallenen Gelenke waren Knie, Sprunggelenke und Interphalangealgelenke .Die Prognose der Arthritis nach COVID scheint gut zu sein. Die Therapie war in den meisten F\u00e4llen rein symptomatisch mit NSAR und Steroid lokal oder systemisch. Basistherapeutika wie Salazopyrin, Hydroxychloroquin, Methotrexat oder Biologika wurden nur selten verabreicht.Eine COVID-Infektion scheint auch Autoimmunerkrankungen zu verursachen. Berichtet wurde \u00fcber Guillain-Barr\u00e9-Syndrom (GBS), autoimmun h\u00e4molytische An\u00e4mie, Antiphospholipidsyndrome (APS), systemischen Lupus erythematodes (SLE), multiple Sklerose (MS), akute disseminierte Encephalomyelitis, Multisystem-Entz\u00fcndungssyndrom bei Kindern  und rheumatoide Arthritis (RA) . VermuteEinige Studien vermuten, dass Patienten mit Autoimmunerkrankungen vermehrt zu Hospitalisierung, schwereren Komplikationen und h\u00f6herer Mortalit\u00e4t durch COVID neigen. Dieser Vermutung liegt die \u00dcberlegung zugrunde, dass Patienten mit Autoimmunerkrankungen im Vergleich zur gesunden Normalbev\u00f6lkerung empf\u00e4nglicher sind f\u00fcr eine SARS-CoV-2-Infektion als Folge des geschw\u00e4chten Immunsystems, der Anwendung von immunmodulierenden Medikamenten und der bereits eingetretenen Organsch\u00e4den. Demgegen\u00fcber lassen andere Studien vermuten, dass die regelm\u00e4\u00dfige Medikamenteneinnahme Patienten mit Autoimmunerkrankungen vor schweren COVID-Komplikationen sch\u00fctzt .Neben der am h\u00e4ufigsten beschriebenen COVID-assoziierten ReA werden auch andere chronische entz\u00fcndlich-rheumatische Erkrankungen als COVID-bedingt vermutet und beschrieben. Berichtet wurde \u00fcber den Beginn unterschiedlicher Formen einer Vasculitis nach COVID-Infektion , 24, wobNicht nur \u00fcber vereinzelt nach COVID begonnene chronische entz\u00fcndlich-rheumatische Erkrankungen, die allgemein als Autoimmunerkrankung gelten, wird berichtet, sondern auch \u00fcber autoinflammatorische Syndrome wie die systemische juvenile idiopathische Arthritis  des Kindes , 28 sowiSeit Beginn der Pandemie bestand die Sorge, dass Patienten mit Autoimmunerkrankung in Bezug auf COVID verst\u00e4rkt gef\u00e4hrdet seien und ein h\u00f6heres Risiko f\u00fcr schwerwiegende Komplikationen h\u00e4tten. Die bisherige Erfahrung hat das aber nicht wirklich best\u00e4tigt \u201334.Die Impfskepsis ist hoch unter der Allgemeinbev\u00f6lkerung und unter Patienten mit chronischen entz\u00fcndlich-rheumatischen Erkrankungen. Die internationalen rheumatologischen Gesellschaften haben von Anfang an empfohlen, die Impfung gegen SARS-CoV\u20112 anzunehmen, da sie den besten Schutz bietet vor schweren oder fatalen Verl\u00e4ufen \u201337. BishNach COVID-19-Impfung kann es zu \u00e4hnlichen rheumatischen Krankheitsbildern kommen wie nach COVID-Infektion \u201344. BescTrotz der zunehmenden Berichte \u00fcber Autoimmunsyndrome nach COVID-Impfung ist die Gesamtzahl in Bezug auf die Zahl der Geimpften sehr gering und spricht nicht gegen die Vorteile einer Impfung.Vereinzelt reagieren Patienten nach COVID-Infektion oder Impfung mit dem akuten Symptomen passend zu einer chronisch-autoimmunen oder chronisch-inflammatorischen Erkrankung. Der Zusammenhang zwischen COVID und rheumatischen oder muskuloskelettalen Beschwerden oder Erkrankungen ist unklar . Auch beBei neu einsetzenden Symptomen einer autoimmunbedingten oder autoinflammatorischen Erkrankung sollte man auch an eine Reaktion nach COVID-Infektion oder -Impfung denken. Die Prognose ist eher gut."}
+{"text": "To examine the relationships between polyunsaturated fatty acids (PUFAs) dietary intake and asthma in children.In this cross-sectional study, a total of 14,727 participants from the United States National Health and Nutrition Examination Survey (NHANES) database in 1999\u20132018 were included, and the baseline characteristics of all participants were gathered. The description analysis was used to explore the possible covariates. Weighted multivariate logistic regression models were adopted to assessed the association between PUFAs dietary intake and asthma in children. In addition, we also performed subgroup analysis based on gender, age, and maternal smoking during pregnancy to investigate this relationship.n\u00a0\u2212\u00a03 PUFAs , and eicosapentaenoic  dietary intake were negatively associated with asthma in children. The subgroup analysis described that when children were male , or were 5\u20137\u00a0years , were 7\u201312\u00a0years , or their maternal smoking during pregnancy , docosahexaenoic dietary intake was negatively related to childhood asthma.The prevalence of asthma approximately was 15.38% in the present study. The result of weighted multivariate logistic regression indicated that, docosahexaenoic , total Docosahexaenoic dietary intake was negatively associated with the asthma in children, especially if children were male, or were 5\u201312\u00a0years, or their maternal smoking during pregnancy. Asthma, as a type of chronic airway inflammation, is widely recognized as the most prevalent pulmonary disease among children . While tn\u00a0\u2212\u00a03 and n\u00a0\u2212\u00a06 fatty acids were an essential nutrient found in many foods, including fish, fruits, soybean oils, purslane, and nuts  were used to express the measurement data of non-normal distribution, while the Mann\u2013Whitney U rank-sum test was employed for intergroup comparison. The categorical data were presented as weighted number of cases and the composition ratio [n (%)], and group comparisons were conducted using \u03c72 test.Considering the complex sampling design of the NHANES database, we conducted a weighted analysis. The measurement data of normal distribution were described as the weighted mean\u2009\u00b1\u2009standard deviation (Mean\u2009\u00b1\u2009SD), and group comparisons were conducted using P\u2009<\u20090.05 was considered as statistically significant difference.First of all, we performed the description analysis of baseline information in the study, which aimed to explore the possible covariables, which had effect on the outcome. Then, we interpreted the association between PUFA's dietary intake and asthma in children by using weighted multivariate logistic regression models. Covariate adjustments: Model 1 adjusted for energy; Model 2\u2009adjusted for energy, total fat, maternal smoking during pregnancy, weight at birth, age, BMI, race, gender, and family PIR. In addition, the association between PUFA's dietary intake and asthma in children was assessed based on the gender, age, and maternal smoking during pregnancy population. SAS (version 9.4) software was used for statistical analyses. Weighted odds ratio (OR) and 95% confidence interval (CI) were calculated in the study. n\u2009=\u200970,113) and had the missing information of asthma , mother's age , weight at birth (n\u2009=\u2009376), maternal smoking during pregnancy (n\u2009=\u200968), family PIR , BMI , and total fat intake , a total of 14,727 eligible participants were included eventually. These eligible participants were divided into the asthma group  and non-asthma group . The prevalence of asthma approximately was 15.38% in this study. Characteristics of all eligible participants were shown in Table P\u2009<\u20090.05)  and eicosapentaenoic acid  were related to the decreased odds of asthma in children after adjusting for energy, total fat, maternal smoking during pregnancy, weight at birth, age, BMI, race, gender, and family PIR. These results suggested that n\u00a0\u2212\u00a03 PUFAs might be related to asthma.As shown in Table n\u00a0\u2212\u00a03 PUFAs, docosahexaenoic acid and eicosapentaenoic acid) and the odds of asthma in children. The findings described , eicosapentaenoic acid , and docosahexaenoic acid  dietary intake on asthma were observed.We conducted a subgroup analysis based on the gender, age, and maternal smoking during pregnancy to explore the association between PUFA's dietary intake  we cannot determine a causal relationship between PUFA's dietary intake and the odds of children\u00a0asthma in this cross-sectional study. We only found that docosahexaenoic acid dietary intake was related to the asthma in children; (2) the diagnosis of asthma in NHANES database relied on self-reported problems from the interview; therefore, the study lacks testing modalities on diagnosis, and the interviews may be subject to recall bias; (3) this data on PUFAs intake was the first day dietary recall interviews, which only reflects the short-term intake of participants and cannot account for the association of long-term dietary changes and asthma in children. The information on the assessment of the incorporation of the fatty acids into cell membranes is lacking in the database. Also, the influence of children's dietary patterns can be influenced by their parents, but this information was not available from the database; (4) some potential confounders might not have been accounted for in the study, such as use of asthma medications. More researches about the association should be conducted in the future.This study indicated that docosahexaenoic acid dietary intake may be negatively associated with asthma in children, especially if children were male, or were 5\u201312\u00a0years, or their maternal smoking during pregnancy. However, more prospective studies are still needed to confirm these findings."}
+{"text": "As recreational genomics continues to grow in its popularity, many people are afforded the opportunity to share their genomes in exchange for various services, including third-party interpretation (TPI) tools, to understand their predisposition to health problems and, based on genome similarity, to find extended family members. At the same time, these services have increasingly been reused by law enforcement to track down potential criminals through family members who disclose their genomic information. While it has been observed that many potential users shy away from such data sharing when they learn that their privacy cannot be assured, it remains unclear how potential users\u2019 valuations of the service will affect a population\u2019s behavior. In this paper, we present a game theoretic framework to model interdependent privacy challenges in genomic data sharing online. Through simulations, we find that in addition to the boundary cases when (1) no player and (2) every player joins, there exist pure-strategy Nash equilibria when a relatively small portion of players choose to join the genomic database. The result is consistent under different parametric settings. We further examine the stability of Nash equilibria and illustrate that the only equilibrium that is resistant to a random dropping of players is when all players join the genomic database. Finally, we show that when players consider the impact that their data sharing may have on their relatives, the only pure strategy Nash equilibria are when either no player or every player shares their genomic data. Of these, approximately 62% of DTC-GT consumers have sought interpretations of raw genomic data using TPI services3. These services support a wide variety of applications, including, but not limited to, trait analysis, personalized nutrition and diet recommendations, genealogy and ethnicity analysis, and finding relatives. Though these services are exciting, they also pose risks to consumers that can limit their uptake and adoption of such services.Over the past decade, direct-to-consumer genetic testing (DTC-GT) has dramatically grown in its popularity. As the amount of personal genomic data has grown, numerous companies have emerged to provide third-party interpretation (TPI) services. This is driven by the number of potential DTC-GT consumers\u2014as of 2022, 23andme and Ancestry DNA, the two largest personal genomics companies had over 12 million and 18 million consumers, respectively4 by exploiting GEDMatch, a TPI website that, at that time, maintained a publicly available genomic database with approximately 1.5 million individuals\u2019 DNA profiles5. In this case, law enforcement officers uploaded crime-scene DNA to GEDMatch and found the suspect\u2019s third cousin, suggested by similarity in their DNA. Law enforcement officers were then able to reconstruct a family tree, trace down the suspect, and confirm the suspect\u2019s identity by another genomic test. While this case highlighted the forensic uses of personal genomic databases, it also raised the public\u2019s concerns over privacy with respect to this long-range familial search technique6. In addition, Hazel et al. pointed out that forensic investigations leveraging publicly available genomic database are unfair, underregulated, and haphazard7.For example, in 2018, the FBI arrested a suspected serial murderer known as the Golden State Killer8. Erlich et al. further analyzed the potential for identifying an individual through their genomic data using a technique similar to that adopted by law enforcement. They showed that at the time of their investigation in 2018, approximately 60% of individuals of European descent were at risk of being identified even if they were not in the genomic database9, which exhibits the negative externalities incurred through online genomic data sharing. The model we introduce in this paper relies on their methods to estimate the probability of finding relatives and being re-identified.There has been a growing body of research responding to the case of the Golden State Killer. Edge and Coop, for instance, calculated the expected number of genetically detectable cousins one can find in the genomic database10. More specifically, the regulation of TPI services remains uncertain. For example, Guerrini et al. analyzed the potential oversight for TPI services by four US agencies and showed that the main governance of TPI services are contracts between users and TPI service providers11. However, when Hazel and Slobogin surveyed the privacy policies of DTC-GT companies, they discovered that most fail to comply with the Fair Information Practice Principles and the Privacy Framework proposed by the U.S. Federal Trade Commission12. In addition, Wan et al. appraised threats to genomic data privacy and existing sociotechnical safeguards and concluded that there is no simple solution to provide appropriate levels of genomic privacy13. These studies highlight the lack of protection over the genomic privacy of DTC-GT and TPI consumers.Meanwhile, various studies have considered the extent to which current law and regulatory frameworks can address genomic privacy risks. Clayton et al. examined regulations that are applicable for genomic privacy and concluded that few, if any, are sufficient to protect genomic privacy comprehensively14. Wang et al. reported that users are highly motivated to use TPI services for ethnicity analysis and personal health implications3. While the majority of respondents were satisfied with the interpretation they received, 35% of the respondents were confused by the interpretation instead14. Besides, there are many ethical concerns on the TPI services, including inadequate informed consent, questionable clinical validity and utility, and lack of medical supervision15. Many concerns on DTC-GT services also apply to TPI services, including privacy, emotional toll, and general misuse of their genomic data by the company16. More specifically, Guerrini et al. probed public opinion on law enforcement\u2019s access to genetic genealogy databases and found that the majority of respondents supported the access17. By contrast, Slobogin and Hazel also surveyed the public\u2019s attitude, but observed that participants thought this kind of access intrusive18. Given the variability in users\u2019 attitudes towards the service, as well as the existence of the privacy paradox, which describes the dichotomies between privacy attitudes and actual behavior20, it is challenging to predict users\u2019 behavior simply through surveys.In recognition of such challenges, numerous surveys and vignette studies have been conducted to learn more about consumers\u2019 behaviors, motivations, and concerns with regards to the adoption of TPI services. Nelson et al. found that approximately 84% of DTC consumers used at least one TPI tool21. According to Humbert et al., the interdependent privacy risks are either caused by the direct sharing of information involves others or the sharing of information that is correlated between individuals. The two typical sources of correlation are (1) homophily for friends on either real-life social networks or online social networks and (2) genetic inheritance. To formalize the interdependence of privacy on online social network, researchers have considered game theoretic frameworks. Bicz\u00f3k and Chia first proposed an Interdependent Privacy Game (IPG) model to study the adoption of third-party tools in online social networks22. The third-party tools often collect information that involves its users\u2019 friends, which demonstrates the first kind of cause for the interdependent privacy risks. Subsequently, Pu and Grossklags investigated the adoption of third-party applications and generated a scale-free network to approximate the structure of real social networks23. Besides, Olteanu et al. investigated a more specific kind of interdependent privacy risks\u2014the sharing of co-location information on online social network and took the time dimension into account24.It should further be recognized that the aforementioned studies focus only on an individual\u2019s perspective. However, there are circumstances under which an individual\u2019s privacy depends not only on their own decision but also the decisions made by others. The interdependence of privacy has been studied by different research communities under different terminologies. Humbert et al. systematically summarized these research and categorized the interdependent privacy risks based on the data types28. Under the observation that genomic data is highly correlated among family members, an individual\u2019s genomic data can be inferred through their family members. Humbert et al. quantified the genomic privacy risks27, and developed a tool for laymen to evaluate kin genomic privacy and required no real genomic data28. Besides, Humbert et al. modeled the data sharing and management behaviors within a family via a game theoretic framework26.Humbert and colleagues first studied the interdependence of privacy in genomic data sharing8. As the number of users of TPI services increases, people start to worry about the re-identification attack enabled by the sharing decisions made by distant relatives. Besides, in reality, most TPI websites, if not all, do not provide direct access to users\u2019 genomic data but provide genetic matching results instead. Thus, the probability of the inference attacks goes low and the re-identification attacks become the primary concern for TPI websites. Hence, in this paper, we specifically focus on the re-identification risk, which is a main difference from Humbert et al.\u2019s model26.It is worth noticing that the aforementioned research focus on the risks that stem from value inference attacks on genome data. As the relationship between family members becomes distant, little information about the target\u2019s SNP values will be revealed even the SNP values of his relatives have been observed. Thus, the value inference risks significantly decrease as the degree of relatedness decreases. However, with the development of long-range familial searches, an individual can be identified by distant family members that they do not necessarily know. For example, the police have traced the Golden State Killer through 10 to 20 third-to-fourth cousins of him, most of whom he had probably never met26, we consider the data sharing behavior in the society instead of a single family. On one hand, we assume that the benefit of TPI comes from using this service to find relatives. While players in this game do not know the number of relatives they will find, they can consider the expected number based on genealogical information taken together with the population of other TPI users. Consequently, their benefit is an increasing function of the number of players who have chosen to use this service, which exhibits the first notable feature in our model, a network effect. On the other hand, privacy risks arise due to the possibility of the collection and use of the information in TPI by law enforcement or other third parties. Another notable feature of our model is the negative externality of participation decisions create for the privacy risks of others. In particular, the re-identification risks depend a great deal on a single player\u2019s decision to join and whether one\u2019s relatives used TPI services, as an individual can be identified by law enforcement by first identifying their relatives in TPI, as was indeed the case for the Golden State Killer and other recently solved cold cases. Nevertheless, a player can compute their privacy risks based on the number of other participants in the service as well. To the best of our knowledge, this is the first approach that captures these particular features of the decisions about whether or not to join TPI.In this paper, we introduce a game theoretic approach to characterize how potential users of TPI services may act as they weigh the tradeoff between benefits received from TPI with the privacy risks incurred in sharing one\u2019s data. In our model, both the benefits and privacy risks of each player in this game\u2014that is, a potential user of TPI\u2014depend on the population of other TPI users. Different from Hembert et al.\u2019s model29 and we find no significant differences in the simulation results. We observe that the only equilibrium that is resistant to a random dropping of players is when all players share their data. Finally, we observe that social welfare (the sum of player utilities) is negative in every pure strategy Nash equilibrium except when no one shares the data (which results in zero utility). This conclusion follows from our observation that, in our model, the amount of privacy risk  rises quickly and approaches the maximum when only a small proportion of all players share their data. This is due predominantly to the negative externality arises from long-range familial genomic inference and, consequently, is nearly independent of an individual user\u2019s decision.Through extensive simulations, we provide insights into user behavior in online genomic data sharing settings. We find that there exist three types of pure-strategy Nash equilibria in our model, which reflects what may happen in the real world: (1) no user shares genomic data with TPI (2) a small fraction of all potential users share, and (3) all users share. A Nash equilibrium is notable because it defines a solution to a game, such that no player has any incentives to change their strategy. The Nash equilibria provide us with intuitions into how the network effects and negative externalities shape players\u2019 aggregated behaviors. We further show that our simulation results are consistent as we vary the settings of user parameters. Specifically, in our simulations, we vary users\u2019 privacy preferences to be consistent with Westin\u2019s distributions of privacy pragmatists, unconcerned, and fundamentalists in different yearsIn this section, we first introduce a game theoretic model of online genomic data sharing in a TPI service and then describe our approach to analyzing this model using simulation. Table P of p players. We assume the players are potential users of TPI services. We denote the decision by player i on whether to participate in the TPI service by a binary strategy s to denote a profile of strategies, or strategy profile, of all players, while i.The TPI genomic data sharing game has a set i\u2019s net valuation30.There are three components in a player\u2019s utility function. The first component is a player i, then the expected number of relatives in the database is i who choose to join the database. Consequently, this component of the utility function can be formally represented as The second component is the utility gained from finding relatives in the database. Let i or i\u2019s relatives are in the database. We associate this privacy risk with a cost i and i is identified  when i shares genomic data, they can be identified with probability 1, and the associated cost is then i can be identified even if they choose not to join the database  increases by i joins, it requires i joins, only We represent the set of family members for player i who are in and out of the database as s as To evaluate the efficiency of Nash equilibria, we calculate the social welfare associated with each Nash equilibrium. In our model, we define social welfare in equilibrium 9. The assumptions we rely upon during the calculation are summarized as follows: We do not consider the influence of half-siblings or half-cousins.We assume generations are discrete and non-overlapping. For individuals in the current generation, their parents are chosen via random sampling with replacement from the previous generation.There are r children  segments, which are segments of DNA shared by people with common ancestors. We assume To have sufficient information to identify the target, we need to find at least Individuals are diploid , and only autosomal genomes are considered .Our calculation of P(relativeP(a player is from the current generationP(a player is from the current generation) P..9, \\docu in Eqs. , the muli and any other player share at least g generations ago. We assume they must share at least j IBD segments, where g generations ago, they have approximately i and any other player share j IBD segments is The multiplier in Eq.  calculati and the other player are cousins once removed and we assume player i is always from the later generation, we assume their shared ancestral couple is i and g generations from the other player. g generations ago, and player i has In Eq. , the mulg, Given the values of matching parameters and i can be identified through other players that use TPI services, where i required to be in the database in order to successfully identify player i. Our calculation of 9. When i equals one minus the probability that less than k relatives of player i are found in the database, where k relatives found, we need to calculate the probability that r children and people from the two generations have a equal probability to use TPI services, we have i is i is i in the database follows Binomial distribution. Thus, the probability that the target player i has i has entclass1pt{minimapure-strategy Nash equilibria (PSNE). Formally, a strategy profile s is a pure-strategy Nash equilibrium if for all type structure of our game. We say that a strategy profile s is a type-symmetric Nash equilibrium if it is a Nash equilibrium and for all types i and j with type t, Our analysis of the game defined above hinges on the ability to compute Nash equilibria in this game. We focus on 31 and tailor it for the binary strategy space. In this method, since the number of pure strategy profiles can be extremely large, their algorithm exhaustively searches over the partitions of strategies and examines if the partition will lead to a Nash equilibrium. A partition represents the number of players who choose each strategy. When the strategy space is binary, we can simply search among the possible number of players who choose to use TPI services.To calculate PSNE in our simulations, we follow the method proposed by Daskalakis and Papadimitrioup is on the order of hundreds of millions and it is extremely time consuming to search every possible K between 0 and p. However, below . Let i. Suppose that s is a PSNE, j\u2019s best response is We prove by contradiction. Let mentclass2pt{minimFor anyk, there is at most one pure-strategy Nash equilibriumswiths and t and k players other than i join in We prove by contradiction. Consider two type-symmetric PSNE mentclass2pt{minimAlgorithm\u00a01 returns all pure-strategy Nash equilibria.k that are viable for type-symmetric pure-strategy Nash equilibria. Since by Lemma\u00a0k is associated with at most one PSNE, it suffices to show two things for an arbitrary k: k is added to the set of Nash equilibria by the algorithm . Suppose that k, there is a PSNE s with s, by Lemma\u00a0i in each type t, it must be the case that if i,\u00a0t with t for which s. Let s is a PSNE, it must be the case that for each t, both k others join. However, this is ruled out by Lemma\u00a0Next, we consider completeness (condition 2). Suppose that for a given mentclass2pt{minims be a TSPNE, and suppose we flip the strategies of players of t and player i with this type, set C (returning to a previously visited profile l). Let BRD(s) returns either C. We can now define stability fully formally.In addition to the existence and distribution of Nash equilibria, we are interested in determining which Nash equilibrium is more stable. The notion of stability we adopt here is robustness to small perturbations of player strategies. Precisely, let s is r-stable if s of A strategy profile r specified in context.Typically, we will simply refer to profiles as stable or unstable, with p equal to 32.4 million, as in reality, only a small portion of people have gotten their genomic data tested and ready to share. We set the number of players in the game to be 10% of the population, which approaches the current number of DTC-GT customers. According to the most recent statistics, there are more than 26 million people who have taken at-home genomic tests by the start of 201932. In addition, we set We performed three sets of simulations. In the first set of simulations, we aim to approximate the real-world scenario and learn about whether there exists a Nash equilibrium. Furthermore, in the event that there are Nash equilibria, we aim to determine how they are distributed. In these simulations, we set 33. Specifically, Westin categorized consumers as \u201cprivacy fundamentalists\u201d, \u201cprivacy pragmatists\u201d, and \u201cprivacy unconcerned\u201d based on their privacy preferences. The fundamentalists value privacy most while the unconcerned care little about privacy protection. The pragmatists make their decisions based on the privacy risk and the value of their information in different scenarios. Though there are a number of critiques of Westin\u2019s segmentation, it is widely adopted when evaluating users\u2019 privacy attitudes35. Since surveys from different years yield different distributions of Westin\u2019s categories36, we aim to learn if the change in privacy preferences over time affects the distribution of Nash equilibrium outcomes. We use the statistics on Westin\u2019s categories summarized by Woodruff et al.29 and conduct 50 simulations for distribution of Westin\u2019s categories in 2001, 2003 and 2014, respectively, as summarized in Table Regarding the user parameters, we set entclass1pt{minimaIn the above simulations, we assume players\u2019 valuations for finding a relative are always p equal 324 million to approximate the size of U.S. population. This is because with the existence of long-range familial search, everyone in the U.S. faces the risk of being identified through their relatives, including people who do not use TPI services and even people who do not get their genomic data tested. In other words, regardless of whether people realize the risk or not, they become players in the game. Therefore, we consider the players of this game to contain the entire population in the U.S. and set the number of players to be 324 million. It is worth mentioning that the change in number of players does not affect In the second set of simulations, we examine the influence of user parameters on the distribution of Nash equilibria by varying the distribution of To conduct the simulation, we first assume In the third set of simulations, we adopt an uninformative parameter setting and aim to learn the distribution of Nash equilibrium when there is no prior knowledge on user preferences. More details on simulation setup and simulation results will be introduced in p; consequently, marginal cost of joining is negligible, whereas benefit is significant, since one can find relatives by joining TPI services.Figure interior Nash equilibria, that is, equilibria in which the number of players joining TPI services is In addition to the two extreme equilibria, we also identify a number of others joining approaching its maximum value with a relatively small number of joining players. Specifically, in our model, when K equals three million, the probability of being identified is 97.6% for a player who is not in the database. Thus, the privacy loss for this player, K increases, so that the extreme equilibrium at Next we consider the social welfare of the game for the different pure strategy equilibria. Since different set of simulations are based on different parametric settings, the values of social welfare are not comparable among different set of simulations. Figure Finally, our stability analysis reveals that both of the extreme equilibria are stable. In contrast, we observe that all interior equilibria are unstable. This is somewhat surprising, and indicates that the interior equilibria are unlikely to be persistent phenomena of the system.N in Algorithm 1 from one simulation where the parametric setting follows the distribution of Westin categories in 2001. It can be seen that N varies with changes in In the variation of our model in which the players are altruistic, we find that all interior equilibria are eliminated entirely, and we only observe the two extreme equilibria. To gain intuition into this result, Fig. N increases quickly and reaches p under a small Additionally, we observe that K that leads to a Nash Equilibrium increases. This is because as We consider the situation in which a player\u2019s valuation for the benefit of finding a relative discounts as the degree of relatedness decreases. While the value of K. We know a player will choose to join the database when K value.Figure For the simulations where we vary This investigation yields three main findings. First, we provide insights into the existence of Nash equilibria in the model of genomic data sharing through simulations based on a game-theoretic framework. First, we find that both extreme situations where either everyone, or no one, joins TPI, constitute Nash equilibrium outcomes in every setting we considered. In addition, we observe that interior equilibria tend to occur when the number of users of TPI services is a small fraction of the US population\u2014specifically, around 3 million. While many factors can affect the number of users of such services, in our model, this appears to be mainly caused by the negative externality incurred. Specifically, as the number of other players who use TPI services grows, the negative externality quickly increases to a point where players suffer from the same amount of risk they would incur if they fail to use such services. In such a situation, it is clear that the best strategy for potential users is to contribute their genomic data and join TPI. Second, we observe that the optimal Nash equilibrium is realized when no one uses TPI services. Notably, when everyone is in the system uses TPI services, the social welfare is still negative. This, too, is a consequence of the negative externality of players joining the service in leaking privacy of others who have not. Third, we observe that both extreme equilibrium outcomes (everyone and no one joins) are stable, whereas interior equilibrium outcomes (Our study has a number of limitations. First, for ease of computation, we assign players to a limited number of types. While this assignment significantly narrows the space needed to search for the pure-strategy Nash equilibrium, it clearly limits the diversity of preferences in the population and may thereby neglect potential Nash equilibria. We choose 18000 types in our first set of simulations, and 6000 when we vary the parametric settings. By conducting pilot studies, we are able to choose the number of types that balances the ability to capture interior Nash equilibria and the time consumption for one simulation. Second, the parameters we set for players in the game are conceptual rather than measured based on empirical observations or surveys. This is because, without performing behavioral experiments with humans, it is challenging to learn about a user\u2019s valuations of the benefits of using TPI services and their perceived costs of privacy risks. We believe, however, that this is a crucial area for future research in the behavioral economics of privacy. Moreover, the remarkable stability of our results as we vary parameters of the utility function suggests that our overall observations are relatively robust with respect to our modeling framework. Third, we model the online genomic data sharing as a one-shot game, which lacks the ability to capture the dynamics in user interactions. In reality, users can change their decisions as their valuations for the service change. Finally, we focus specifically on the re-identification risks in our model. There are other kinds of privacy risks can be induced by the genomic data sharing on TPI websites, such as attribute inference risks. The attribute inference attack can be carried out either by insiders who have authorized access to user-shared genomic data or outsiders who obtain access through a security breach. Attackers can infer a target\u2019s genomic data or traits by analyzing the genomic data of their relatives. As the attribute inference risks also depend on the distance between relatives and can be measured without the disclosure of actual genomic data, it can be smoothly integrated in our model and this is definitely one direction for our future work.Supplementary Information."}
+{"text": "This study focuses on identifying the key factors associated with ergonomic behaviors (ERBE) among women workers on assembly lines (WwAL) to prevent musculoskeletal disorders (MSDs) caused by repetitive motions and unfavorable body postures. To achieve this objective, this study employed Bayesian networks (BN) analysis based on social cognitive theory (SCT).A cross-sectional study was conducted to examine the predictive factors of ERBE among 250 WwAL from six different industries located in Neyshabur, a city in northeastern Iran. The study used a two-stage cluster sampling method for participant selection and self-report questionnaires to collect data on demographic characteristics, variables associated with SCT, ERBE, and the standard Nordic questionnaire. The collected data were analyzed using Netica and SPSS version 21, which involved statistical analyses such as independent t-tests, Pearson correlation, and ANOVA tests at a significance level of p\u2009<\u20090.05. BN analysis was conducted to identify the important factors that impact ERBE.The majority of individuals reported experiencing chronic pain in their back, neck, and shoulder areas. Engaging in physical activity, consuming dairy products, and attaining a higher level of education were found to be significantly associated with the adoption of ERBE p\u2009<\u20090.05. Among the various SCT constructs, observational learning, intention, and social support demonstrated the highest levels of sensitivity towards ERBE, with scores of 4.08, 3.82, and 3.57, respectively. However, it is worth noting that all SCT constructs exhibited a certain degree of sensitivity towards ERBE.The research findings demonstrate that all constructs within SCT are effective in identifying factors associated with ERBE among WwAL. The study also highlights the importance of considering education levels and variables related to healthy lifestyles when promoting ERBE in this specific population. Musculoskeletal disorders (MSDs) are not only one of the most common work-related health problems reported in the world but also a prevalent occupational health concern among women in specific occupations , 2. WorkMSDs can be attributed to a range of factors comprising ergonomic, physical, chemical, and psychological elements. These factors encompass aspects such as incorrect posture, repetitive motions, rapid work pace, non-ergonomic tools, unsuitable workstations, and exposure to certain equipment during work , 6. AsseUtilizing preventive measures for MSDs is essential in managing the considerable direct and indirect expenses associated with WMSD . To mitiUnderstanding individuals\u2019 beliefs and perceptions regarding factors that influence behavior can assist in the creation of impactful interventions aimed at fostering healthy behavior . SCT is Bayesian networks (BN), also referred to as Bayesian belief networks and belief networks, utilize directed acyclic graphs (DAGs) to represent variables. In these graphs, nodes represent the variables, and edges indicate their direct probabilistic dependencies. The field of medicine extensively employs BN due to their interpretability and their facilitation of inference . A BN moWMSDs have a significant impact on workers\u2019 health and work capacity, resulting in absenteeism, disability pensions, reduced productivity, functional limitations, and disruption of women\u2019s social roles. Therefore, addressing this issue is crucial, as it is recognized as a major challenge in the workplace .In promoting health within the primary healthcare system, health education plays a vital role. Hence, it is crucial for researchers to identify predictors of ergonomic behaviors, particularly for female assembly line workers, to develop more effective interventions . This apMoreover, health, safety, and environment (HSE) experts in the industry can contribute significantly to the healthcare system by utilizing the effective constructs of this theory in applying ergonomic behaviors.Previous studies that investigated the effectiveness of ergonomic behaviors (ERBE) have only used non-industrial environments \u201323, whilFrom February 11 to March 28, 2023, a cross-sectional study was conducted in Neyshabur, a city located in the northeastern region of Iran. The study focused on women employed in industrial assembly lines.The study employed a two-stage cluster sampling approach to select participants. In the initial stage, a list was compiled of all the electronic industries  with assembly lines where women were employed. Subsequently, a random sampling lottery method was utilized to select eligible women from each industry in proportion to the number of assembly line workers, ensuring an equal representation across industries.Taking into account an average total of approximately 500 female workers in assembly lines, the sample size was determined to be 217 individuals using Cochran\u2019s formula and considering a type I error of 0.05. However, factoring in a potential dropout rate of 15%, the estimated sample size was adjusted to 250 individuals.The inclusion criteria for this study encompassed providing consent to participate, being employed in the assembly line, having an age of over 20 years, and possessing the ability to read and write. On the other hand, no specific exit criteria were taken into account for this research.The study utilized two questionnaires: the Nordic standard questionnaire, which allows each woman to report pain in all nine body regions simultaneously, enabling respondents to aptly express the complexity of their musculoskeletal experiences; and the Ergonomic Behaviors Evaluation Tool (EBET) questionnaire, specifically designed to assess ERBE of WwAL based on SCT. The reliability and validity of the EBET questionnaire were assessed through comprehensive psychometric analysis, which demonstrated favorable psychometric properties. However, the reference to the original source of the questionnaire is currently under review and can be found in the citation information. The questionnaire exhibited an average CVR of 0.92, a CVI of 0.97, and an overall Cronbach\u2019s alpha coefficient of 0.79. Exploratory factor analysis successfully identified all dimensions of the questionnaire, explaining 65.25% of the variance, and confirmatory factor analysis demonstrated a good fit of the model. The psychometric properties of this tool were confirmed in previous study .All the women in our study were employed on the assembly lines of the electronics industry, where their work was characterized by repetitive movements, the frequent need to adopt non-optimal postures, and extended periods of sitting during their tasks. Demographic questions, covering age, height, weight, educational status, marital status, work experience, physical activity status, dairy consumption status, and financial status. When choosing the demographic variables for this research, we took into account factors that have previously been identified as potential influencers of musculoskeletal disorders and ergonomic practices which supported by pertinent literature , 31, 32.The researcher-made questionnaire aimed to evaluate ERBE based on SCT constructs and consisted of 11 dimensions, including outcome expectations, normative beliefs, perceived barriers, social support, observational learning, reinforcement, behavioral skills, self-efficacy, intention, and knowledge. Respondents used a 5-point Likert scale to indicate their opinions, and data collection was done through self-reporting methods .The behavior section included questions assessing the engagement in stretching exercises and the adherence to proper body posture during work. Participants rated their responses on a 3-point Likert scale, with options ranging from never to always, assigned values of 1 to 3 points, respectively.The questionnaires were distributed to WwAL based on their availability during a 20-minute break time, and they were completed through self-reporting. Prior to completing the questionnaire, the researcher provided an explanation of the study\u2019s purpose and the questionnaire\u2019s structure. During the questionnaire completion, the researcher was present in the environment to offer guidance if any difficulties arose. Following the completion of the questionnaires, team members were assigned the task of quality control, reviewing the questionnaires for accuracy and completeness. Ultimately, after removing any incompletely filled-out questionnaires, a total of 250 questionnaires were included in the study.We constructed the BN model for predicting ERBE based on SCT by identifying the relevant variables and their relationships. The target node was ERBE, and the co-constructs of the theory were the effective factors. We then parameterized the network by assigning weights to each of the parameters and quantified the model. This allowed us to predict the probability of ERBE given the values of the input variables, which can be useful in designing interventions to improve ergonomic behaviors in the workplace. BN serve as visual depictions that illustrate the directional connections between variables, determined by conditional probabilities. The nodes within these networks represent variables and can encompass various potential states, such as high, medium, or low. Associations between variables are depicted through directed edges, with the edges pointing from the independent (parent) variable to the dependent (child) variable , 34 Fig and 3. OThe questionnaire data were entered into SPSS 21 software, and the information was coded to ensure anonymity and confidentiality. The reliability of the SCT criteria was assessed using Cronbach\u2019s \u03b1 test. Descriptive statistics methods, such as frequency, percentage, mean, and standard deviation, were employed to characterize the studied population.To analyze the relationships between the dependent and independent variables, univariate analyses such as independent samples t-tests or ANOVA tests were conducted based on the characteristics of the variables. Additionally, correlation analysis was performed to examine the relationship between the variables of SCT and ERBE.To examine the factors that influence ERBE, BN analysis was conducted using Netika software. ERBE was considered as the dependent variable (target node), while the constructs of SCT were treated as the independent variables (parent nodes).The women who took part in this study can be characterized, on average, as being in the middle-aged category. They tended to have a moderately higher body mass index (BMI) and a moderate level of work experience. A majority of the women in the study were married, with 56.4% reporting no intentional physical activity. Furthermore, only 12% of the participants consumed one or more servings of dairy products per day . This finding suggests that if women prioritize and consistently practice ERBE in the workplace, the overall level of ERBE can be significantly improved. Additionally, Table\u00a0Utilizing the BN methodology, this study aimed to uncover the key factors influencing the adoption of ERBE among women employed in assembly line work, leveraging SCT. The findings revealed significant associations between women\u2019s level of education, physical activity, and consumption of dairy products, and their ERBE. Moreover, all variables within the SCT framework demonstrated sensitivity to women\u2019s ERBE, with observational learning, intention, and social support displaying the highest sensitivity compared to other factors.Our findings indicate a significant prevalence of musculoskeletal pain in the neck, shoulder, and back among WwAL. This is consistent with a recent study conducted by Yang et al., which highlights that electronic assembly line workers face a higher risk of MSDs. This increased risk is attributed to the adoption of poor posture, prolonged periods of sitting or standing, and frequent repetitive actions . WorkersIn our study, we observed that individuals who engaged in higher levels of physical activity exhibited a higher average score of ERBE. According to a European study, individuals who engage in low levels of physical activity are more likely to have poor posture. The study found that both a sedentary lifestyle and low physical activity levels have a significant impact on postural parameters . It appeObservational learning displayed the highest degree of sensitivity toward ERBE in this study. Specifically, as observational learning increased, there was a corresponding increase in women\u2019s engagement in ERBE. Observational learning encompasses various impacts, including the acquisition of skills and fostering confidence in one\u2019s capability to execute a particular behavior, known as self-efficacy. Notably, when individuals observe a \u201ccoping\u201d model who effectively tackles and overcomes obstacles, their self-efficacy in learning complex new behaviors tends to increase . IntentiThe study on WMSDs among WwAL possesses several notable strengths. Firstly, participants from six different industries offer diverse perspectives, enabling a comprehensive examination of the factors influencing WMSDs. Secondly, the study focuses specifically on a vulnerable group, acknowledging and addressing the unique challenges faced by these individuals. Thirdly, the study utilizes a comprehensive tool based on SCT, enabling the identification and assessment of multiple individual, cognitive, and environmental factors that influence behavior. Fourthly, the study utilizes BN Analysis, a powerful statistical technique that enables the identification and modeling of multiple individual, cognitive, and environmental factors. Collectively, these strengths highlight the study\u2019s methodological rigor, its relevance to a vulnerable population, and its comprehensive exploration of influential factors, thereby making a valuable contribution to the field of WMSDs among WwAL.Given that the data relied on self-reporting, potential biases such as recall and social desirability could have influenced the findings. Additionally, the use of a cross-sectional design limits the ability to establish causal relationships, warranting caution in drawing definitive conclusions. It is recommended to expand the scope of future research to include diverse age and gender groups, as this study exclusively focused on women employed in the assembly line.All constructs of SCT prove effective in identifying the factors associated with the involvement of WwAL in ERBE. Given the established correlations between education levels, variables related to a healthy lifestyle, and ERBE, it is clear that these factors should be considered when investigating the adoption of ergonomic practices among this population."}
+{"text": "This laboratory study determined the surface, mechanical and chemical properties of polymethyl methacrylate (PMMA) denture resin reinforced with micron-sized Gum Arabic (GA) powder in different weight ratios.This laboratory study was conducted at the Dental Health Department of the College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia from November 2022 to February 2023. Three experimental denture resins were prepared by incorporating GA powder in heat-polymerized PMMA powder using different wt.% . While pristine PMMA served as the control group. A total of ten bar-shaped specimens with dimensions of 65 mm \u00d7 10 mm \u00d7 3.5 mm were prepared for each study group. The surface properties , mechanical properties  and chemical properties (FTIR) were conducted. The data were statistically analyzed using the one-way analysis of variance and Tukey\u2019s post hoc tests (p<0.05).The surface and bulk properties of experimental GA-reinforced PMMA resin materials deteriorated while the mechanical properties were also negatively altered using GA-based PMMA denture resin. A linear correlation was observed between weak mechanical properties and increasing wt.% of GA in denture resin.The incorporation of GA powder in denture resin might not be a viable option. The surface and mechanical properties of experimental PMMA composites were adversely affected compared to the control group. There have been many materials introduced for the fabrication of denture bases. However, polymethyl methacrylate (PMMA) resin is a widely accepted and frequently used material.4Inadequate surface hardness and flexural strength of PMMA denture base material leave room for further development. Many other approaches have been experimented with for enhancing the mechanical properties such as utilizing substitute polymers i.e., polystyrene, polyethylene, and urethane dimethacrylate,Gum Arabic (GA) is a naturally occurring polymer having antibacterial properties. It is also a non-toxic natural compound utilized in the sustained release of medications to deliver bioactive.12A recently published study showed that the binding affinity between GA and glass ionomer cement (GIC) is improved, thus enhancing the mechanical properties such as flexural strength, fracture toughness and tensile strength of the set GIC.14Therefore, this laboratory study aimed to incorporate the varying weight ratios of micron-sized GA powder in a commercially available heat cure acrylic resin. The alternative hypothesis was that GA would increase the mechanical properties of denture base material.This laboratory study was performed over three months at the Dental Health Department, College of Applied Medical Sciences, King Saud University Saudi Arabia, i.e., from November 2022 to February 2023. The study received exemption letter from the Institutional Review Board because no human or animal subjects were involved in this research. Pure GA  in very a fine grade powder form (100-150 \u00b5m) was selected. The GA powder was soaked in 3-Methacryloxyproyltrimethoxysilane (MPS) using a commercially available dental silane coupling agent, ESPE\u2122 Sil . Thirty milliliter of MPS was used to agitate five grams of GA powder for two minutes. The extra solution was then decanted. Additionally, ethanol was used to rinse the GA powder twice. The GA powder was then dried for 24 hours at room temperature.The dried GA powder was dispersed robustly in heat cure PMMA powder for five minutes using a vacuum mixer. The heat cure denture base resin  was used with a powder: liquid ratio of 21g/10 ml according to the manufacturer\u2019s recommendation. The mixture was manually stirred with a stainless steel spatula and a rubber bowl until it reached the dough-like stage.\u00d710 mm \u00d7 35 mm. A load of 100 N was applied to the flask for one minute to remove any excess resin material. For polymerization, the flask was placed in a water bath for eight hours at 73\u00b0C before being heated to 100\u00b0C for one hour again.\u00d710 mm \u00d7 3.5 mm) for evaluating the flexural strength of denture base polymersFour different experimental groups with varying wt.% of GA powder incorporated in PMMA powder were fabricated: (A) Control group with 0 wt.% of GA powder, (B) 5 wt.% of GA, (C) 10 wt.% of GA, and (D) 20 wt.% of GA. Next, the dough was packed in a gypsum mould of a dental flask with inner dimensions of 65 mm A single bar-shaped specimen randomly selected from each study group was examined for porosity and agglomeration of GA powder using micro-computed tomography  at 100 kV voltage, 50 \u03bcA current, and 14.2 \u00b5m voxel size to characterize the agglomeration and pores in a three-dimensional structure. 360\u00b0 rotation around the vertical axis was used for scanning the specimens. Using the porosity tool in the proprietary software , the overall porosity values were determined.A randomly selected single specimen from each study group was evaluated for surface morphology using scanning electron microscopy (SEM)  in secondary electron mode at an accelerating voltage of 15 kV. SEM pictograms of the representative specimens were obtained using proprietary software at magnifications of 50 x.A micrometre-scale surface roughness of the specimens (n=10/group) was quantified with a non-contact surface profiler  as described previously.Nanoindentation tests were conducted on the specimens utilizing a nanomechanical instrument , fitted with a Berkovich diamond indenter nanotip (n=10/group). The experiments were performed at room temperature, employing loading and unloading rates of 0.5 mN/s and a 10 s dwell time. The maximum applied load was 20.0 mN.\u22121 using a NICOLET iS5 spectrometer . The system had a KBr beam splitter and a DTGS detector. With the aid of a monolithic diamond attenuated total reflectance (ATR iD7) accessory, spectra were recorded with a resolution of 2 cm\u22121.To identify the functional linkages and observe the compositional analysis, a randomly selected single specimen from each study group was measured for the FTIR spectrum. The targeted wavelength range was between 500 to 4000 cm3 (n=10/group). The specimens were subjected to a three-point bending flexural strength test using a universal testing machine . The proprietary software (Bluehill software version 2.6) of the testing machine recorded the flexural strength in megapascal (MPa). A load cell of 5 kN and a crosshead speed of 1mm/min was used for flexural strength evaluation.The specimens for the flexural strength test were prepared per ANSI/ADA specification No.12 for denture base polymers with dimensions 65 \u00d7 10 \u00d7 2.5 mmpost hoc Tukey\u2019s HSD multiple comparison tests (p=0.05) were employed in inferential statistics. The statistical program, SPSS ver. 23.0  was used for the statistical analyses.The obtained data were evaluated for descriptive and inferential statistics. The mean and standard deviation values were calculated in descriptive statistics while one-way analysis of variance (ANOVA) followed by The 3D images of the study specimens are presented in The nanohardness (in GPa) and elastic modulus (in GPa) values of the tested groups are represented in The surface roughness (in \u00b5m) and flexural strength (MPa) values of the tested groups are shown in -1 were due to C\u2013H vibrations. While the stretching vibrations of the ester carbonyl C=O between 1700-1750 cm-1, CH2 aromatic group in the range 1400-1450 cm-1, the C-O deformation between 1150-1200 cm-1, and the C-O-C vibration at 1149 cm-1. The original PMMA\u2019s structure was unaffected by the inclusion of different wt.% GA.The FTIR spectra of the study groups is presented in The hypothesis of this investigation is rejected: incorporation of varying wt.% of GA powder revealed a deleterious effect on the tested properties. The surface topography data suggest incorporation of GA powder increased the surface roughness of the PMMA composite, and the deleterious effect on surface roughness had a direct relationship with the wt.% of GA powder incorporated in PMMA. The higher filler loading as well as the poor distribution of GA powder in resin acrylic might have caused increased surface roughness.3Hardness is a vital property that can determine suitable material for a denture base.Although the GA powder was silanized before mixing in PMMA powder, the decrease in flexural strength with the rate of GA powder addition could be due to the poor adhesion of GA powder with the PMMA matrix. Also, the use of GA powder created intrinsic voids (as shown in The SEM images confirmed the presence of space/voids on the surface of the experimental composites. Whereas, microCT images further affirmed the formation of voids in the bulk materials. Here it is notable that tiny bubbles, during the polymerization process, grow larger either through amalgamation or expansion in an exothermic reaction.Laboratory studies often use controlled and simplified conditions that might not fully mimic the complex oral environment. Laboratory studies often focus on evaluating one specific variable or material property at a time. In future research, it would be appropriate to use low filler loading of GA powder in PMMA resin. Additionally, improved silane or other surface treatments should be considered to improve the bonding between the GA and acrylic. Accelerated aging studies to assess the durability and stability of GA-modified denture acrylic over extended periods would be interesting also.Considering the outcome of this laboratory findings, it can be concluded that the addition of GA powder increased the surface topography of the GA-reinforced PMMA composite. Nanohardness, elastic modulus and flexural strength of the PMMA composites were compromised with the increasing wt.% of GA powder. GA powder might not be chemically compatible with the PMMA denture resin.AAK performed data collection, data interpretation, and manuscript writing and is responsible for the accuracy and integrity of the research.AAA conceived, supervised the study project and approval of manuscript writing.MSA did statistical analysis, tabulation of the data, revision.LSJB Specimens test, formatting and editing of the final draft."}
+{"text": "Objective: Understanding the discernment of individuals about their health is crucial during public health situations such as the COVID-19 pandemic. Within this theme of study is how older adults perceive their vulnerabilities because it can relate to subsequent disease preventing behaviour.Materials and methods: The analysis explored optimism bias, or the perception of infection avoidance, regarding COVID-19 among lower-income Thais aged 60 and over. The study utilized an analytic sample of 2,139 individuals from the 2021 Survey on Housing and Support Services for Poor Older Adults. Logit regression model analysis was conducted, using optimistic bias as the outcome variable.Results: Increasing age and residing in urban areas were associated with a higher likelihood of bias. On the other hand, higher educational attainment was found to decrease the association with optimistic bias, indicating higher perception of risks. Adherence of older individuals to the residence-in-place policy might have contributed to perception of lower infection risks. Urban residents had better access to welfare benefits and medical facilities, which led to reduced worry and greater optimistic bias.Conclusion: Greater understanding of the disease and preventive strategies offer insights on how higher education levels lead to perceiving possible risks surrounding COVID-19. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the coronavirus disease COVID-19) [9 [1]. ItUnderstanding the breadth of individual and social psychology is crucial during unprecedented times like the current pandemic . One parIn the context of COVID-19 outbreak, health behaviour is influenced by social context . PoliticThailand was among the first countries to detect a positive COVID-19 case in January 2020, which subsequently led to the implementation of a national lockdown policy by March 2020 . After tThe exploration of optimism bias, or unrealistic optimism, in the context of older adults in Thailand remains unexplored. This is a significant issue due to Thailand\u2019s ageing population. This study focuses on lower-income older Thais and investigates the association between social factors, unrealistic bias, and susceptibility to COVID-19. The lifestyle and context of individuals in the lower-income category are often perceived as homogeneous, but it is crucial to comprehend their situation from a multidimensional perspective . UnderstThe economic context of many older people in Thailand can be described as precarious, with 40% reported to be living below the poverty line in 2017 . Filial During the COVID-19 pandemic, the older population faced significant challenges due to mobility restrictions in their respective communities. This age group was identified as among the most vulnerable to the disease and, therefore, had to shelter in place . HoweverOther sources of risk and insecurity during the pandemic were already present in society before the events occurred. One such challenge was limited access to healthcare facilities for rural residents prior to the pandemic . AlthougThe aforementioned geographic and socioeconomic factors interact and contribute to disparities in the health and well-being of the older population. Similar factors have been recognized in the literature to influence the attitudes and behaviour of individuals during the pandemic in other societies ,26. One In this study, the dataset analysed was the 2021 Survey on Housing and Support Services for Poor Older Adults. The respondents were individuals aged at least 55\u2009years old from five regions of Thailand . The socFor the analytic sample used in the current study, certain criteria were implemented. These criteria included: 1) excluding individuals who were bedridden, 2) cases where a proxy answered all the survey items on behalf of the older respondent, and 3) including individuals who were at least 60\u2009years old to align with most studies concerning the older population. The resulting analytic sample consisted of 1,775 participants. Selectivity bias was tested due to the aforementioned exclusion criteria, and no statistical differences were observed in the distribution of basic demographic characteristics, such as age, sex, residence, living arrangement, employment, and education attainment, between the total sample and the analytic sample.An item in the survey asked respondents about their perception of the risk of contracting COVID-19. The response options were \u2018yes\u2019 and \u2018no\u2019. Therefore, individuals who responded with the latter option were categorized as having an optimistic bias.The covariates in the sample were grouped into sociodemographic characteristics, dimensions of poverty, and health status measures. These categories were based on previous studies that had observed their effects on the health attitudes and behaviours of older adults in Thailand ,16,28. SSelected poverty dimensions focused on events within the scope of the pandemic period. These dimensions included employment status, income adequacy, skipping meals due to economic limitations, and perception of housing conditions. The perception of housing conditions is particularly relevant during the pandemic as households can be conducive to transmission when overcrowded . The per2; normal weight as BMI between 18.5\u2009kg/m2 and 24.9\u2009kg/m2; and overweight as BMI \u226525\u2009kg/m2 [Health status measures were all self-reported and included self-rated health during the past year and the presence of at least one chronic condition, such as neurological disease, osteoarthritis, chronic kidney disease, stroke, hypertension, diabetes, and hyperlipidaemia. Body mass index (BMI) was used in the analyses, calculated based on self-reported height and weight. Underweight was defined as BMI < 18.5\u2009kg/m25\u2009kg/m2 . Lastly,25\u2009kg/m2 ,31. ThesCharacteristics distribution was conducted initially, followed by a bivariate analysis. The \u03c72 test was used to determine if there were statistical differences between non-optimistic and optimistic biases for each independent variable. Subsequently, the logit regression model analysis was employed with the optimistic bias as the outcome variable. Three models were utilized to examine the effects of different groups of covariates on the dependent variable . Model 1The analytic sample comprised a larger proportion of individuals in the young-old age group, specifically aged 60 to 69. This group predominantly consisted of females residing in Bangkok and urban areas. Moreover, the majority were not currently married and lived with their grandchildren . Additiop value of <.05) in terms of optimism bias related to age, regional and urban-rural residence, and marital status, among other sociodemographic factors. Among individuals in the oldest age group, approximately 44% exhibited optimism bias, compared to 32% of those in their 60s. When comparing regions, nearly half of the residents in Bangkok displayed optimism bias, while around 39.2% of urban residents showed such bias, in contrast to approximately 30% of older people in rural areas.Bivariate analysis is also presented in Regarding educational attainment, individuals with lower than primary education exhibited the highest degree of optimism bias, at around 42%. Similarly, having a positive perception of one\u2019s housing conditions was associated with a bias towards optimism, with approximately 37% displaying such bias. In terms of health status measures, self-rated health showed a correlation, where individuals with good self-rated health tended to be biased towards optimism. On the other hand, the experience of emotional distress was correlated with not having optimism bias.Being in the older age groups and residing in urban areas were observed to increase the odds of exhibiting optimism bias. Conversely, the opposite trend was observed for the measures of residence. Older individuals residing in regions other than Bangkok had a lower likelihood of displaying optimistic bias. Similarly, living with a spouse and children was associated with decreased odds of exhibiting this outcome. Furthermore, higher levels of educational attainment were shown to decrease the likelihood of bias, with primary education attainment having an odds ratio (OR) of 0.70, while attainment beyond primary level had an OR of 0.79. Individuals perceiving a need for housing improvements also had lower odds, with an OR of 0.67. Experiencing emotional distress was also observed to have an indirect association with optimism bias where the OR was 0.35. On the other hand, having good self-rated health (SRH) and being underweight increased the odds of optimism bias, with respective odds ratios of 1.24 and 1.46.In the current study, the perception of susceptibility to COVID-19 among Thai older individuals was found to be associated with various socioeconomic, poverty-related, and health factors. These factors include older age, residing in rural areas, higher educational attainment, and having a positive perception of one\u2019s health status, among others. However, these individual characteristics do not consistently exhibit the same direction of association, as they can be related to either realistic or non-realistic optimism.Belonging to older age groups was observed to be associated with a lesser degree of optimism bias regarding vulnerability to COVID-19. This finding aligns with a similar result reported among older adults in Sweden during the early stages of the pandemic . Public In relation to the previous point, rural older adults were also observed to exhibit better adherence to protective behaviours against COVID-19 . It is cFurthermore, the observation suggests that individuals with higher levels of educational attainment display less optimism bias regarding susceptibility to COVID-19. Similar differentiations based on education levels have been noted in another study on health-related risk perception . It was As emotional distress has been observed here to be associated with less optimism bias, it produces an ambivalence in desirability. Caution was heightened among low-income people in the country because it exacerbated their problematic condition pre-pandemic ,25. TheiSeveral caveats must be mentioned regarding this study. Firstly, due to the use of cross-sectional data, causation cannot be established. Health-related information, such as height and weight for BMI, relied on self-reporting with no utilisation of biomarker data. Additionally, the operationalization of variables was constrained by the available information in the survey data. It is important to note that variables like housing dissatisfaction and inadequate food consumption were measured differently; therefore, comparing the presented results with other studies requires attention. Another limitation of this study is related to the data. As the aim of the survey used here was to assess the needs of lower-income people, the sample was limited to this group of individuals only. Comparing individuals from different income levels was not possible. In lieu of the information in the survey, characteristics of the people surrounding the older individuals who can influence the latter\u2019s perception and mind-set about the pandemic could not be established.Despite the aforementioned limitations, understanding patterns of health behaviour during emergency situations remains crucial. Individuals with diverse social characteristics and backgrounds will think and act based on their perceptions of the prevailing conditions. Older adults, in particular, are more vulnerable during such circumstances, necessitating efficient government planning and response. The implementation of programs that can provide timely and easily comprehensible information to the public is of paramount importance. These programs and approaches should also take into account the varying backgrounds of older individuals."
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