Lymnaea stagnalis  has been widely used as a model organism in neurobiology, ecotoxicology, and parasitology due to the relative simplicity of its central nervous system (CNS). However, its usefulness is restricted by a limited availability of transcriptome data. While sequence information for the L. stagnalis CNS transcripts has been obtained from EST libraries and a de novo RNA-seq assembly, the quality of these assemblies is limited by a combination of low coverage of EST libraries, the fragmented nature of de novo assemblies, and lack of reference genome.The pond snail L. stagnalis genome, we generated an RNA-seq library from the adult L. stagnalis CNS, using a combination of genome-guided and de novo assembly programs to identify 17,832 protein-coding L. stagnalis transcripts. We combined our library with existing resources to produce a transcript set with greater sequence length, completeness, and diversity than previously available ones. Using our assembly and functional domain analysis, we profiled L. stagnalis CNS transcripts encoding ion channels and ionotropic receptors, which are key proteins for CNS function, and compared their sequences to other vertebrate and invertebrate model organisms. Interestingly, L. stagnalis transcripts encoding numerous putative Ca2+ channels showed the most sequence similarity to those of Mus musculus, Danio rerio, Xenopus tropicalis, Drosophila melanogaster, and Caenorhabditis elegans, suggesting that many calcium channel-related signaling pathways may be evolutionarily conserved.In this study, taking advantage of the recent availability of a preliminary L. stagnalis transcriptome and provides insights into differences between vertebrates and invertebrates in CNS transcript diversity, according to function and protein class. Furthermore, this study provides a complete characterization of the ion channels of Lymnaea stagnalis, opening new avenues for future research on fundamental neurobiological processes in this model system.Our study provides the most thorough characterization to date of the The online version contains supplementary material available at 10.1186/s12864-020-07287-2. Lymnaea stagnalis is a widely used model organism in neurobiology, development, ecotoxicology, and parasitology. Its central nervous system (CNS) consists of large singly identifiable neurons and simple, well-characterized neural circuits, which allows detailed electrophysiological, biochemical, and molecular analyses of the cellular basis of behavior, including sensorimotor integration [L. stagnalis also exhibits notable traits absent in other orders of organisms, such as anoxia-tolerance [L. stagnalis has recently been adopted as the model species of a new OECD test guideline [L. stagnalis is relatively closely related to the land snail Biomphalaria glabrata, a vector for Schistosoma mansoni, the parasite that is the cause for schistosomiasis [S. mansoni elicits significant changes in neuropeptide dynamics in the B. glabrata CNS [L. stagnalis is shown to be resistant to S. mansoni infection [S. mansoni transmission by B. glabrata. In each case, thorough molecular characterization of the L. stagnalis CNS is key to realizing the translational potential of this model organism.The pond snail egration , learninegration , 3, centegration , and neuegration . In addiolerance  and centolerance . Elucidauideline . It has uideline , 10, watuideline , and pesuideline , 13. Finsomiasis , a promirata CNS . Interesnfection , suggestL. stagnalis.As ion channels are the fundamental units of excitability and synaptic transmission in the nervous system, characterization and identification of ion channels is enormously important to the field of neuroscience. Mutations in the genes encoding ion channels, or encoding proteins that regulate ion channels, have dramatic effects on normal functioning, resulting in a wide range of debilitating diseases , 18. TheA receptors [Animals have evolved a broad diversity of ion channels. In mammals, approximately 80 genes encode potassium channel subunits, forming 4 families and 7 subfamilies of potassium channels –21. Thoueceptors , nicotineceptors , ionotroeceptors , and pureceptors .Drosophila, the Shaker K+ channel, now known as Kv1.3, shares 82% sequence homology with the rat homologue [Drosophila Shaker, Shal, Shab, and Shaw K+ channels, and in humans as well, with the Drosophila K+ channels all having been shown to be expressed in human cardiac tissue [Though the kinetics, pharmacology, permeability, and conductivity of the various ion channels are diverse, many ion channels are widely evolutionarily conserved. One of the earliest potassium channels to be characterized in omologue . This rec tissue . On the c tissue . This inL. stagnalis was the GABAA receptor, an ionotropic receptor and ligand-gated ion channel [L. stagnalis, including glutamate receptor subunits [Aplysia californica, an immensely important model organism for neuroscience, has been used extensively to study ion channels [2+ and Na+ channels, several families of K+ channels, amiloride-sensitive Na+ channels, cyclic nucleotide-gated channels, and inositol trisphosphate/ryanodine receptors [Biomphalaria glabrata, has been shown to express transcripts encoding various ion channels as well, including Na+, K+, Cl−, Ca2+, and TRP cation channels, as well as glutamate, acetylcholine, and GABA receptors [D. melanogaster has been used to identify the molecular determinants of the newest family of ion channels, calcium release-activated current (CRAC) channels [L. stagnalis, continue to be a rich resource of discovery for evolutionarily conserved fundamental neuronal signaling processes applicable to all organisms.The first ion channel to be cloned from  channel . Since tsubunits –36, acetsubunits , L-type,subunits –40, T-tysubunits , and P2Xsubunits . Fellow channels . Study oeceptors . Anothereceptors . Furthereceptors , leadingchannels , 48. ThiL. stagnalis CNS is obtained from EST libraries [L. stagnalis CNS transcriptome. The recent availability of a preliminary L. stagnalis reference genome [L. stagnalis transcripts and characterization of whole protein classes, particularly of ion channels and ionotropic receptors, which have not yet been fully characterized. In this study, we employed a combination of genome-guided and de novo assembly programs to create an assembly that improves upon the completeness and coverage of existing resources. We have also identified transcripts encoding ion channels and ionotropic receptors and compared them to other species. This provides the most thorough characterization to date of the CNS transcriptome of this important molluscan model organism and builds a crucial foundation for leveraging the unique advantages of L. stagnalis in a wide range of research fields. Furthermore, as this study characterizes ion channels in an invertebrate molluscan species, where essential neurobiological processes are likely to be evolutionarily conserved across species, this study serves as a potential starting point for the identification of novel ion channel families which may be evolutionarily conserved in mammals.Recent advances in high-throughput RNA sequencing (RNAseq) have much accelerated the search for novel ion channel and receptor families. Current sequence information for the ibraries  and a deibraries . Howevere genome  has provL. stagnalis is a widely used model organism in understanding the fundamental mechanisms of neural function due to its simple and well-characterized CNS. A de novo assembly of the L. stagnalis CNS transcriptome from 100 bp single-end reads has previously been published [L. stagnalis genome, here we employed a combination of genome-guided and de novo approaches to assemble reads from four 150 bp paired-end libraries that we prepared from four adult central ring ganglia and the aforementioned published 100 bp single-end library  to create a new and improved L. stagnalis CNS transcriptome assembly  library  and de nL. stagnalis CNS protein-coding transcriptome.Comparison of the amino acid sequence length across the three sets of predicted protein coding sequences Fig. a found tL. stagnalis CNS.As shown in Fig. L. stagnalis CNS transcriptome provides an unprecedented opportunity to carry out comparative studies of CNS gene expression in a number of commonly used vertebrate and invertebrate neuroscience model organisms, so as to gain insights into commonalities and differences that may inform future studies. To this end, we analyzed publicly deposited RNA-seq libraries to characterize gene expression in the CNS of adult Mus musculus (mouse) [Xenopus tropicalis [Danio rerio (zebrafish) [Drosophila melanogaster (fruit fly) (BioProject: PRJNA320764), and Caenorhabditis elegans [L. stagnalis  , Xenopusopicalis , Danio rbrafish) , Drosoph elegans   ion channel or ionotropic receptor protein domains and 2) transmembrane helices to characterize the complement of ion channels and ionotropic receptors expressed in the L. stagnalis CNS. A proportion of sequences in this set were substantially shorter or longer than their best matched sequence in the Nr database. However, for the sake of completeness, we have included all in our analysis as some may be informative as splice isoforms. Sequence alignment-based phylogenetic analysis of these transcripts showed that they encode a wide range of ionotropic neurotransmitter receptors , where bitscores lower and higher than the average are shown in blue and red, respectively  by the absolute sum of standardized bitscores, such that the species with the highest degree of sequence similarity with L. stagnalis is on the left of the graph, we observed that putative L. stagnalis ion channel/ionotropic receptor transcripts showed the most sequence similarity with their homologs in fruit fly and X. tropicalis, and the least with mouse and C. elegans , we found that L. stagnalis transcripts encoding Ca2+ channels showed the highest degrees of sequence similarities with their homologs as compared to all other classes  by their absolute bitscore sum and focused on the top 20 transcripts , inositol 1,4,5-trisphosphate receptors, and voltage-gated Ca2+ channels (both L- and non-L-type). However, there were also a diversity of cation, K+ and TRP channels in this list. Taken together, these findings provide not only a resource for investigating the molecular mechanisms of CNS neural transmission and neuronal excitability in L. stagnalis, but also a foundation for better understanding the conservation and evolution of these processes across species.We further examined the degrees of protein sequence similarity between ion channel/ionotropic receptor transcripts identified in ely Fig. . Each roans Fig. A. Then, ses Fig. A. Finallpts Fig. B. IndeedL. stagnalis possesses a simple and well-characterized CNS that has been widely employed to elucidate key mechanisms of neural function [L. stagnalis CNS transcriptome and integrated it with existing sequence databases to create the most comprehensive to-date transcriptome resource.function , 59–63, function , 13, andfunction . Howeverhttp://lymnaea.org). Indeed, we have shown that this transcript set exceeds existing resources in terms of sequence length, completeness, and diversity  GPCRs, which are not present in vertebrates [L. stagnalis may be thus of interest for future study.Finally, we clustered all protein-coding transcripts expressed in the CNS of these six species into orthogroups, each of which was predicted based on sequence similarities to be descended from a common gene in the last common ancestor of these six species. As orthologous genes have been shown to exhibit similar functions , 91 and tebrates . Additiotebrates . A full L. stagnalis had more orthogroups in common with the three vertebrate species  than D. melanogaster or C. elegans, suggesting that it may be an advantageous system in which to leverage the simplicity of an invertebrate CNS but also maintain more conserved molecular mechanisms with higher organisms. To the best of our knowledge, there have been few systematic comparisons of CNS physiology in commonly used vertebrate and invertebrate model organisms. Such comparisons can provide insights into the evolutionary history of various cellular processes and protein families. Our findings in this study provide a starting point for better understanding of the functional commonalities and specializations of the vertebrate and invertebrate CNS, which is critically needed to inform future studies in terms of the suitability of the chosen model and generalizability of future findings.We have shown that L. stagnalis were dissected according to established protocols [L. stagnalis RNA. This phenomenon occurs due to a thermolabile hidden break in the molecule, as described in various invertebrates, which leads to its cleavage into two sub-parts of size similar to that of 18S RNA [The central ganglia ring from four adult rotocols , 98, 99 rotocols . RNA qua 18S RNA . For thi 18S RNA : 1) adap 18S RNA  were remL. stagnalis CNS transcriptome is summarized in Fig. https://github.com/harvardinformatics/TranscriptomeAssemblyTools/blob/master/FilterUncorrectabledPEfastq.py), and processed and assembled in the same manner, except using a modified custom script to remove unfixable single-end reads after rCorrector analysis. This resulted in five additional assemblies: four genome-guided assemblies and one de novo assembly of unmapped reads. These 22 assemblies  were merged and processed through the EvidentialGene tr2aacds pipeline [L. stagnalis predicted gene set . BUSCO analysis [Workflow for assembling and identifying protein-coding transcripts in the pipeline  to identpipeline , 112. Thpipeline  to allowanalysis  was perfL. stagnalis CNS were obtained from the European Nucleotide Archive and processed in the same manner as described above to predict protein-coding sequences. The amino acid sequences were annotated against Nr database using BLASTP . Finally, the amino acid sequences of predicted protein-coding sequences from these two libraries and the current assembly were clustered and deduplicated (100% similarity cut-off) separately using CD-HIT [For comparison with the current assembly, the previously published EST library  and de ng CD-HIT . The resM. musculus [D. rerio [X. tropicalis [D. melanogaster (PRJNA320764), and C. elegans [Published RNA-seq libraries of musculus , D. reriD. rerio , X. tropopicalis , D. mela elegans  CNS were elegans  was then elegans . The sof elegans  was used elegans  to ident elegans . Gene ID elegans  for func elegans , where I elegans .pfam.xfam.org) . The heatmap of standardized BLAST bitscores was visualized using Clustergrammer [Pfam IDs containing the word “channel” in their descriptions were obtained from Pfam  Table S. PredictT-Coffee , trimmedT-Coffee , and phyT-Coffee  in MEGA T-Coffee . InitialT-Coffee .M. muscurgrammer .Additional file 1: Table S1. Published adult CNS RNA-seq libraries from selected model organisms used in this study. Table S2. Pfam IDs used to identify transcripts encoding ion channels and ionotropic receptors in the L. stagnalis CNS.Table S3. Summary of L. stagnalis CNS RNA-seq library metrics before and after reads correction and filtering. Table S4. Mapping statistics of the CNS RNA-seq libraries to L. stagnalis genome assembly. Table S5. Transcript assembly statistics for L. stagnalis CNS RNA-seq libraries. Table S6. Proportion of transcripts containing complete and fragmented ORFs as identified by the Evigene pipeline in the “okay” and “okalt” sequence sets. Table S7. Top 20 expressed transcripts in the adult mouse brain. Table S8. Top 20 expressed transcripts in the adult X. tropicalis brain. Table S9. Top 20 expressed transcripts in the adult zebrafish brain. Table S10. Top 20 expressed transcripts in the adult fruitfly brain. Table S11. Top 20 expressed transcripts in adult C. elegans neurons. Table S12. Enriched GO terms of mouse genes in orthogroups shared amongst vertebrate and invertebrate species. Table S13. Enriched Reactome pathways of mouse genes in orthogroups shared amongst all the species examined. Table S14. Enriched KEGG pathways of mouse genes in orthogroups shared amongst all the species examined. Table S15. Transcript factors whose binding motifs are enriched in the set of mouse genes in orthogroups shared amongst all the species examined. Table S16. Enriched GO terms of mouse genes in orthgroups shared amongst only the vertebrate species examined. Table S17. Enriched Reactome pathways of mouse genes in orthgroups shared amongst only the vertebrate species examined. Table S18. Enriched KEGG pathways of mouse genes in orthgroups shared amongst only the vertebrate species examined. Table S19. Enriched GO terms of fruit fly genes in orthgroups shared amongst only the invertebrate species examined.Additional file 2: Table S20. Transcripts encoding ionotropic acetylcholine receptors in the L. stagnalis CNS. Table S21. Transcripts encoding ionotropic GABA or glycine receptors in the L. stagnalis CNS. Table S22. Transcripts encoding ionotropic glutamate receptors in the L. stagnalis CNS. Table S23. Transcripts encoding K+ channels in the L. stagnalis CNS. Table S24. Transcripts encoding Ca2+ channels in the L. stagnalis CNS. Table S25. Transcripts encoding Na + channels in the L. stagnalis CNS. Table S26. Transcripts encoding Cl- channels in the L. stagnalis CNS. Table S27. Transcripts encoding cation channels in the L. stagnalis CNS. Table S28. Transcripts encoding TRP channels in the L. stagnalis CNS.


Hereditary hearing loss is a heterogeneous group of complex disorders with an overall incidence of one in every 500 newborns presented as syndromic and non-syndromic forms. Cadherin-related 23 (CDH23) is one of the listed deafness causative genes. It is found to be expressed in the stereocilia of hair cells and in the retina photoreceptor cells. Defective CDH23 have been associated mostly with prelingual severe-to-profound sensorineural hearing loss (SNHL) in either syndromic (USH1D) or non-syndromic SNHL (DFNB12) deafness. The purpose of this study was to identify causative mutations in an Omani family diagnosed with severe-profound sensorineural hearing loss by whole exome sequencing technique and analyzing the detected variant in silico for pathogenicity using several in silico mutation prediction software.A novel homozygous missense variant, c.A7436C (p. D2479A), in exon 53 of CDH23 was detected in the family while the control samples were all negative for the detected variant. In silico mutation prediction analysis showed the novel substituted D2479A to be deleterious and protein destabilizing mutation at a conserved site on CDH23 protein.In silico mutation prediction analysis might be used as a useful molecular diagnostic tool benefiting both genetic counseling and mutation verification. The aspartic acid 2479 alanine missense substitution might be the main disease-causing mutation that damages CDH23 function and could be used as a genetic hearing loss marker for this particular Omani family. With the high rate of consanguineous marriages, several inherited diseases have been diagnosed among the Arab population including syndromic and non-syndromic deafness. A survey conducted in 2016 indicated that up to 49% of Omani marriages were consanguineous [https://sph.uth.edu/Retnet/sum-dis.htm). Among these genes is cadherin-related 23 (CDH23), causing Usher syndrome type 1D (USH1D) [There are no exact statistical figures of syndromic or non-syndromic hearing loss in Oman. However, worldwide studies revealed that approximately 466 million people 5.0%) of the world’s population were suffering from hearing loss .0% of th–10. So f (USH1D) , 12. Stu (USH1D) . More th (USH1D) , 15. Def (USH1D) –19. In t (USH1D) .Next generation sequencing (NGS) made a big leap in genome DNA sequencing. A whole exome and a gene panel can be rapidly sequenced, and the abnormality and specificity of the genome can be detected in a short period. However, the Sanger principle remains a useful technique for sequencing a short DNA fragment and for the confirmation of the NGS findings.In this study, we genetically analyzed an Omani family diagnosed clinically with severe to profound hearing loss. Mutation detection was performed by Illumina HiSeq2000 platform  NGS technique. DNA of an affected family member was sequenced to identify the family-specific mutated gene loci. The Sanger sequencing technique (ABI 3130 xl) was then applied for the whole family and control samples to confirm the NGS findings. A homozygous missense mutation in exon 53 of the cadherin-related 23 gene (CDH23) was detected in all affected members but was absent in the normal family members and controls. Subsequently, in silico genetic testing was used to verify the pathogenicity of the identified mutation.This study was conducted by the Department of Biochemistry, College of Medicine and Health Sciences, Sultan Qaboos University, and the Department of ENT, Al Nahdha Hospital, Ministry of Health, Oman, with collaboration from the Medical Genetics Unit, Polyclinic Sant’Orsola-Malpighi, Bologna, Italy.Clinical examination: Four affected members, two males and two females, from one Omani family of consanguineous marriage  were enrolled in this study.Clinical history and audiological evaluation were done at the ENT department, Al Nahdha Hospital. Clinical examination was conducted using standard pure tone audiometry (PTA), optoacoustic and acoustic emittance tests. Blood samples from patients and their close relatives were collected in EDTA tubes. Samples from 130 male and female individuals without any hearing or visual disorders were used as normal controls.DNA extraction and sequencing: Qiagen kit  was used to extract the genomic DNA from peripheral blood of all collected samples. DNA from an affected member was analyzed using the Illumina HiSeq2000 platform  NGS technique. The Sanger sequencing method  was used to sequence 442 base pairs including the variant site to confirm the NGS finding. The rest of the family members and 130 controls were also tested for the detected variant. Polymerase chain reaction (PCR) forward (5′TCAGTGTCAAATCTCCAGAG3′) and reverse (5′TTGGCAAAGATTTCTCCCAG3′) primers were designed to amplify and confirm the NGS detected variant.http://sift.jcvi.org/www/SIFT_enst_submit.html) [http://www.mutationtaster.org) [http://mutationassessor.org/r3/) [http://genetics.bwh.harvard.edu/pph2/) [http://mutpred2.mutdb.org/index.html) [https://rostlab.org/services/snap2web/) [http://mupro.proteomics.ics.uci.edu/) [http://folding.biofold.org/i-mutant/i-mutant2.0.html) [http://biosig.unimelb.edu.au/duet/) [In silico analysis: In order to evaluate the putative pathological nature of the detected missense variant, physical properties of the amino acids such as change in hydrophobicity, and the impact of the substitution on protein structure and function, were analyzed using the online software SIFT (it.html) , Mutatioter.org) , Mutatioorg/r3/) , PolyPheu/pph2/) , Mutpredex.html) , and SNAap2web/) . Additioci.edu/) , I-Mutan.0.html) , and DUEu/duet/)  were usehttp://polyview.cchmc.org/) [https://zhanglab.ccmb.med.umich.edu/TM-score/tmp/339254.html) were used to calculate the atomic deviation and the degree of similarity by superimposing models of native and mutant proteins [http://www.ncbi.nlm.nih.gov) and Ensemble genome browser (https://asia.ensembl.org/) online programs [http://genepipe.ncgm.sinica.edu.tw/ primerz/beginDesign.do) and BioEdit (http://www.mbio. ncsu.edu/ BioEdit/bioedit.html) were used for DNA primer design and sequence alignment [http://mordred.bioc.cam.ac.uk/ ~rapper/rampage.php) [http://swissmodel.expasy. org) and visualized by Swiss-PDBViewer [https://www.cgl.ucsf.edu/chimera/download.html) [http://jmol.sourceforge.net) [http://consurf.tau.ac.il/2016/) [https://www.ebi.ac.uk/Tools/msa/muscle/) [http://www.ebi.ac.uk/Tools/msa/clustalo/) [The protein secondary structure was visualized and annotated using the online POLYVIEW (mc.org/) . RMSD  . AttribuDBViewer , Chimeraad.html) , and Jmol/2016/) , NCBI mumuscle/) , and Cruustalo/) .The genetic abnormality of the affected family members diagnosed with hearing loss was detected by next generation sequencing whole exome technology. A novel homozygous missense variant, g.A71800709C, c.A7436 C, replacing the negatively charged aspartic acid residue with a nonpolar aliphatic amino acid alanine at position D 2479A in exon 53 of CDH23 gene was confirmed and verified by Sanger sequencing  was selected for further analysis to identify functional protein domains using the online SMART program. The program detected one signal peptide, 26 cadherin repeats (CA), also known as extracellular cadherin (EC) domains, one transmembrane region, and one low complexity region. The variant was found on domain 23 of the 26 CA  was calculated using MUpro, I-Mutant 2.0, and DUET online tools. Models of native and mutant proteins were superimposed to predict the level of similarity between the two protein structures using the template modeling score (TM-score) and the root-mean-square deviation (RMSD) online software  for the wild and mutated types was analyzed by the Swiss model program to build up possible protein templates and models. 5szn.1.A was selected to be the template and building model for CDH23 because of its similar identity with the wild and mutated types . The D2479A ensemble position moved to D2484A on the 5szn model. Jmol package and Ramachandran plots were used to align and validate the two 3D structures in order to predict the possible impact of the mutated amino acid on CDH23 protein structure  [CDH23 is an adhesive protein expressed in the neurosensory epithelium of the inner ear hair cells and encodes the transmembrane Ca (CDH23) . It is t (CDH23) . Using i (CDH23) , 43. It  (CDH23) . More th (CDH23) –19. Rece (CDH23) , 46. Pre (CDH23) , 48. The (CDH23) , 19, 49. (CDH23) , 51. The (CDH23) . The var (CDH23) , 46.In this study, we genetically analyzed an Omani family who was diagnosed clinically with severe to profound hearing loss. The analysis revealed a missense variant on CDH23 (c.A7436C) which was detected by NGS technology and confirmed by Sanger DNA sequencing methodology. Affected family members had the CC genotype, while all 120 normal control samples had the wild-type genotype of AA. The identified variant was subjected to various in silico functional prediction algorithm software to evaluate the pathogenicity level, functionality, and protein stability. The evaluation was based on criteria such as variant location on the genome, sequence homology, conservation level, and physicochemical and structural properties. Sorting Intolerant from Tolerant (SIFT), Polymorphism Phenotyping 2 (PolyPhen 2), PROVEAN , MutPred2 (Mutation Prediction), screening for non-acceptable polymorphisms (SNAPs), and Mutation Assessor protein function prediction software were used, and all agreed on the pathogenicity of the variant. The results from these predictors classified the variant as damaging, deleterious, and disease-causing, and boosted the variant damaging level on the mutant protein structure.Prediction of the variant’s influence on the stability of protein structure is a crucial aspect for studying the function of the protein. The unfolding Gibbs free energy change (ΔG) of the native and mutant structures was calculated by subtracting the free energy change of the mutant protein from the free energy change of the native protein  (DDG or ΔΔG)  = ΔG mutant – ΔG wild type. Above zero value of DDG predicts high stability of the mutant protein and a score below zero predicts low stability , 54. Str+ affinity, and that may play an important role in the interactions of CDH23 molecules either with CDH23 or with other proteins. Since calcium provides rigidity to the elongated structure of cadherin molecules and enables hemophilic lateral interaction, the mutation is likely to result in a decreased affinity for calcium and, in turn, impairs the whole process of protein interaction [Alignment and proteins similarity are important factors assessing generated protein models of related identity. The template modeling score (Tm-score) was used to determine the topological deviation of native from mutant model structures, whereas RMSD was used to calculate the average distance of the alpha carbon backbones between the two models . Both preraction . A Phi/Peraction . Conserveraction . Due to One limitation of this study is that the detected mutation was identified by next generation sequencing technology, which requires sequencing of the whole human exon. The technique is outside the routine daily assays, and the running cost is high. However, the mutation confirmation assay by Sanger DNA sequencing technology is more economical.In this study, we used various in silico mutation prediction programs to analyze a substituted variant on CDH23 protein. The variant was typed on an Omani family diagnosed clinically with hearing loss. The analysis predicted the novel substituted D2479A to be deleterious and protein destabilizing mutation at a conserved site on CDH23 protein. This mutation might lead to a major disruption in CDH23 protein structure that may cause disturbance of stereocilia organization and hair bundle formation affecting the mechano-transduction process and, in turn, hearing loss. In silico mutation prediction analysis might be used as a useful molecular diagnostics tool benefiting both genetic counseling and mutation verification in the governmental and private sectors. The affected family might benefit from the outcome of this research by considering the potential risk of consanguineous marriage.


Aldrichina grahami (Aldrich) , a necrophagous insect with forensic significance, to reveal the composition and expression feature of OBPs, CSPs, ORs, IRs and SNMPs genes at transcriptome level.The time-length between the first colonization of necrophagous insect on the corpse and the beginning of investigation represents the most important forensic concept of minimum post-mortem inference (PMImin). Before colonization, the time spent by an insect to detect and locate a corpse could significantly influence the PMImin estimation. The olfactory system plays an important role in insect food foraging behavior. Proteins like odorant binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (ORs), ionotropic receptors (IRs) and sensory neuron membrane proteins (SNMPs) represent the most important parts of this system. Exploration of the above genes and their necrophagous products should facilitate not only the understanding of their roles in forging but also their influence on the period before PMImin. Transcriptome sequencing has been wildly utilized to reveal the expression of particular genes under different temporal and spatial condition in a high throughput way. In this study, transcriptomic study was implemented on antennae of adult A. grahami was performed using next-generation deep sequencing on the platform of BGISEQ-500. The raw data were deposited into NCBI (PRJNA513084). All the transcripts were functionally annotated using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Differentially expressed genes (DEGs) were analyzed between female and male antennae. The transcripts of OBPs, CSPs, ORs, IRs and SNMPs were identified based on sequence feature. Phylogenetic development of olfactory genes of A. grahami with other species was analyzed using MEGA 5.0. RT-qPCR was utilized to verify gene expression generated from the transcriptome sequencing.Antennae transcriptome sequencing of A. grahami. Phylogenetic development suggested that some olfactory genes may play a role in food forging, perception of pheromone and decomposing odors.In total, 14,193 genes were annotated in the antennae transcriptome based on the GO and the KEGG databases. We found that 740 DEGs were differently expressed between female and male antennae. Among those, 195 transcripts were annotated as candidate olfactory genes then checked by sequence feature. Of these, 27 OBPs, one CSPs, 49 ORs, six IRs and two SNMPs were finally identified in antennae of A. grahami. Such differences are likely to greatly influence insect behavior around a corpse. In addition, candidate olfactory genes with predicted function provide valuable information for further studies of the molecular mechanisms of olfactory detection of forensically important fly species and thus deepen our understanding of the period before PMImin.Overall, our results suggest the existence of gender and spatial expression differences in olfactory genes from antennae of  Forensic entomology uses insects to help in determining origin, location and time of death of a human. The estimation of the postmortem interval by forensic entomologists is based on the development of the insects that colonize the corpse. The time elapsed between the first insect colonizing on the corpse to the start of forensic examination represents the most important forensic concept of minimum post-mortem inference (PMImin). When and how the first insect detects and colonizes the corpse could obviously affect the beginning of the PMImin. The length of time between the time point of death and the first insect colonization on corpse was defined as the pre-colonization interval (Pre-CI), which would account for large part of decomposition time depending on various conditions . Thus, tThe insects’ olfactory system is a highly specific and extremely sensitive chemical sensory nervous system, formed during long-term evolution. Antennae are the main olfactory organs in insects. They have critical roles in detecting environmental chemical signals and subsequently affecting insect behaviors , such asAntheraea polyphemus   is a common necrophagous insect of forensic importance. It feeds on corpses or feces and mainly distributes in the Palearctic and the partial Oriental regions. Research reported its intrusion into other parts of the world in the past decades  to identify the genes of olfactory family from A. grahami. Differences between female and male gene expression were analyzed. A set of putative OBPs, CSPs, ORs, IRs and SNMP in A. grahami was annotated and the expression level was verified. Additionally, different expression profiles of the olfactory related proteins between female and male organs were explored. Finally, predicting function of olfactory genes was discussed based on phylogenetic analysis.In this study, the first antennal transcriptome analysis of A. grahami was captured using pork as baits in Changsha, Hunan province, China. Adults were identified based on the description of Fan . The quality of RNA was confirmed using a NanoVue UV–Vis spectrophotometer , and RNA integrity was verified using a standard 1% agarose gel electrophoresis. Purification of RNA was carried out using DNase I as per manufacturer’s instructions .Total RNA was treated by enriching poly-A tail mRNA with magnetic beads with OligodT, and the desired RNA was obtained after purification. Subsequently, the RNA was fragmented with a break buffer, and reverse transcribed with random N6 primers to synthesize cDNA double strands to obtain double stranded DNA. The synthesized double-stranded DNA was flattened at the end and phosphorylated at the 5′ end, flattened at the 3′ end with sticky’ A′, and connected with adaptor. The ligation product was amplified by two specific primers then denatured by heat. Single-stranded DNA was cyclized with a bridge primer to obtain a single-stranded circular DNA library. Finally, the cDNA libraries were sequenced on the BGISEQ-500 sequencing platform .A. grahami genome assembly (NCBI: PRJNA513084) by using HISAT2. (Parameter: —dta —phred64 unstranded —new-summary -x index -1 read_r1 -2 read_r2 (PE).)Primarily, the raw reads were filtered by removing reads that contain adapters, poly nitrogen and low quality. The remaining high quality clean reads had a base quality 20% lower than Q20. Consequently, we calculated the Q20, Q30, GC-content and sequence duplication levels of the clean data. All subsequent analyses were performed using high quality clean reads. Clean reads were mapped to the p-value < 0.05 and FDR < 0.05 as thresholds.The quantity of gene expression levels from male and female groups were performed using FPKM (Fragments per kilobase of transcript per million mapped reads). It was calculated with NCBI gtf file through gene length annotation. The count calculation was performed using the HTSeq . The DifA. granhmi, respectively. All candidate OBPs, CSPs, ORs, IRs and SNMPs were manually checked by the BLASTp (http://blast.ncbi.nlm.nih.gov/Blast.cgi) search application. Soon after, the prediction opening reading frame (ORF) of the candidate OBPs, CSPs, ORs, IRs and SNMPS genes was identified by the ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/). Conserved domain was predicted utilizing Batch CD-search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). The transmembrane domains (TMDs) of IRs, ORs and SNMPs were predicted by TMHMM server (v2.0) (http://www.cbs.dtu.dk/services/TMHMM/). The signal peptide of putative OBPs and CSPs were predicted using Signa1P (v5.0)  server version with the default parameters.The tBLASTn program was performed with available sequences of OBPs, CSPs, ORs IRs and SNMPs from other species as a “query” to identify candidate genes that encoded putative OBPs, CSPs, ORs, IRs and SNMPs in A. grahami, the reads number of each olfactory-related gene was converted to FPKM  binding to double stranded DNA at the end of each elongation cycle. Primer sequences were designed by the Primer Premier 5.0 program , leg (FL), wing (FW), head (FH) and male antennae (MA), leg (ML), wing (MW), head (MH) were collected from adult Drosophila melanogaster, Calliphora stygia, Musca domestica and Lucilia cuprina were obtained from Uniprot (Protein sequences of  Uniprot . The phy Uniprot .A. grahami genome (SRA: PRJNA513084). The percentage of unique mapping reads was from 49.82% to 51.92% in each sample. Pearson Correlation Coefficient between three biological replicates from the two groups  had high repeatability .An average of 6.46 GB raw data were generated from each sample by reading the male and the female’s antenna samples. After removing adaptor sequences, low quality sequences, and N-containing sequences, a mean of 48.35 Mb clean reads were obtained for each sample . The meaA total of 14,193 genes were annotated, including 11,327 known genes and 2,866 predicted novel genes. A total of 16,995 new transcripts were found, of which 9,460 belonged to newly alternative splicing subtypes of known protein coding genes, 2,951 belonged to transcripts of new protein coding genes, and the remaining 4,584 were long-noncoding RNA.A total of 9,667 genes were enriched by GO annotation. GO divided genes into three categories, representing the molecular functions, cellular component, and biological processes, respectively. The first three categories that contain the largest number of genes are represented in In total, 7,243 were functionally clustered into six KEGG categories including cellular processes, environmental and genetic information processing, and diseases and metabolism. Among the 44 sub-categories, “signal transduction”  and “global and overview maps”  were the most enriched .Moreover, 195 candidate olfactory related transcripts were found based on the results of annotation, including 36 OBPs , 70 ORs A. grahami OBPs transcripts shared structural characteristics of OBPs  with other insects  (A. grahami OF05479 and OF07379) which contained two TMDs with 450–600 aa were identified as SNMPs (Seventy candidate ORs transcripts were found in the results of annotation . Of thesriptomes  includinOF01513)  had a sias SNMPs .2(Aldrich_M/Aldrich_F) ranged from 1.00 to 11.01 in the up-regulated genes. Meanwhile, in the down-regulated genes, the log2(Aldrich_M/Aldrich_F) ranged from −1.00 to −10.64.Based on the RNA-Seq by Expectation Maximization selection, 740 DEGs were chosen between female and male antennae. In total, 357 up-regulated and 383 down-regulated genes were found in male antennae. Among the DEG results, 21 differentially expressed olfactory genes, including four OBPs, eight ORs and nine other genes (which were enriched in the term of olfactory transduction function in KEGG) were found between the females and the males. The logBased on the transcriptome results, 76 genes exhibited sex-specific expression, among which 37 genes only found in male antennae and 39 genes only present in female antennae. In addition, among the 195 candidate olfactory genes, 10 genes were male-specific and seven were female-specific genes. These include five OBPs and three ORs male-specific and two ORs female-specific.A. grahami OF03173, OF08934 and BGI_novel_G000488 in males, OF12341 in females, respectively. A total of 10 DEGs were selected for qRT-PCR verification  in male antennae were significantly upregulated in qRT-PCR results and consistent with those obtained by RNA-seq  in both females and males r organs . The OF0In addition, the six ORs  genes were significantly upregulated in antennae, compared with other organs . Among tA. grahami OF12624 was mainly expressed in the head and antennae of male, BGI-novel-G000012 was expressed in the head and wing of female, and BGI-novel-G000010 were expressed in the leg of females and males. However, for A. grahami OF04198 expression, there was no difference between male and female.The three of four non-olfactory genes showed no difference in female and male antennae . HoweverA. grahami with D. melanogaster (40), C. stygia (20), M. domestica (11) and L. cuprina (10) . A. grahami OF03991 was clustered with D. melanogaster 83a/83b (a class of OBPs that were co-expressed with LUSH and related to pheromone detection), and OF06321 with D. melanogaster 28a.A phylogenic tree of OBPs was constructed using OBPs of ina (10) . Some paA. grahami ORs with D. melanogaster, C. stygia, M. domestica and L. cuprina showed that ORs were clustered into multiple groups. But the A. grahami OF07668 was clustered into the type of ORCo. All ORCos were grouped with high support value (99%) (The phylogenetic tree of ue (99%) .A. grahami OF11318 was clustered into a group of IR8a/25a  between the point of death happened and the beginning of PMImin represent a non-negligible factor which may greatly influence the PMImin estimation. Exploration on the mechanism beneath the corpse locating behavior of necrophagous insect should facilitate the understanding of pre-CI, which will definitely improve the estimation of PMI. It has been well demonstrated that insects’ olfactory system plays a major role in their food foraging behavior. Related genes and their products like OBPs, ORs, CSPs, IRs and SNMPs, were responsible for the odor detection and signal transduction. inactive . TherefoA. grahami was found similar to other invertebrates A. grahamtebrates , except tebrates . In additebrates .A. grahami antennae, three OBPs namely, OF08228, OF03173 and OF08934 were sex-specifically expressed in male. Interestingly, these three genes were also highly expressed in other organs compared to antennae, indicating that those genes were not solely involved in olfactory transduction. In fact, study had reported that part of OBPs could function in the gustatory system of the insect  was the atypical co-receptor ORCo. ORCo was previous reported highly conserved between species  of membranes  were differently expressed when male and female antennae were compared. Based on the annotation results, these non-olfactory genes could be participant in both sensory system and signal transduction in A. grahami olfactory system . The y axis is KEGG Pathway, and the size of the bubble indicates the number of genes annotated to a certain KEGG Pathway.Click here for additional data file.10.7717/peerj.9581/supp-3Supplemental Information 3D. melanogaster: Drosophila melanogaster (red); C. stygia: Calliphora stygia (orange); M. domestica: Musca domestica (green); L. cuprina: Lucilia cuprina (blue); A. grahami: Aldrichina grahami (black).Click here for additional data file.10.7717/peerj.9581/supp-4Supplemental Information 4D. melanogaster: Drosophila melanogaster (red); C. stygia: Calliphora stygia (orange); M. domestica: Musca domestica (green); L. cuprina: Lucilia cuprina (blue); A. grahami: Aldrichina grahami (black). The upper parts list the eight motifs discovered in the CSPs of above species. The numbers in the boxes correspond to the numbered motifs in the upper part of the figure, where a small number indicates high conservation.Click here for additional data file.10.7717/peerj.9581/supp-5Supplemental Information 5D. melanogaster: Drosophila melanogaster (red); C. stygia: Calliphora stygia (orange); L. cuprina: Lucilia cuprina (blue); A. grahami: Aldrichina grahami (black).Click here for additional data file.10.7717/peerj.9581/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.9581/supp-7Supplemental Information 7Sample: Sample name; Total Raw Reads (Mb): The reads amount before filtering; Total Clean Reads (Mb):The reads amount after filtering; Total Clean Bases (Gb): The total base amount after filtering; Clean Reads Q30 (%): The rate of bases which quality is greater than 30 value in clean reads; Clean Reads Ratio (%): The ratio of the amount of clean reads; Total Mapping (%): Compared with the clean reads ratio of the reference genome; Uniquely Mapping (%): Compare the clean reads ratio of the unique position of the reference genome.Click here for additional data file.10.7717/peerj.9581/supp-8Supplemental Information 8FPKM, fragments per kilobase of transcript per million mapped reads.Click here for additional data file.10.7717/peerj.9581/supp-9Supplemental Information 9TMDs, transmembrane domains; FPKM, fragments per kilobase of transcript per million mapped reads.Click here for additional data file.10.7717/peerj.9581/supp-10Supplemental Information 10TMDs, transmembrane domains; FPKM, fragments per kilobase of transcript per million mapped reads.Click here for additional data file.10.7717/peerj.9581/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.9581/supp-12Supplemental Information 12Click here for additional data file.


Puccinia striiformis f. sp. tritici (Pst), threats world wheat production. Resistance to Pst is often overcome by pathogen virulence changes, but the mechanisms of variation are not clearly understood. To determine the role of mutation in Pst virulence changes, in previous studies 30 mutant isolates were developed from a least virulent isolate using ethyl methanesulfonate (EMS) mutagenesis and phenotyped for virulence changes. The progenitor isolate was sequenced, assembled and annotated for establishing a high-quality reference genome. In the present study, the 30 mutant isolates were sequenced and compared to the wide-type isolate to determine the genomic variation and identify candidates for avirulence (Avr) genes.The stripe rust pathogen, P ≤ 0.001) to avirulence phenotypes. Eight of the SP genes were identified as avirulence-associated effectors with high-confidence as they met five or six criteria used to determine effectors.The sequence reads of the 30 mutant isolates were mapped to the wild-type reference genome to identify genomic changes. After selecting EMS preferred mutations, 264,630 and 118,913 single nucleotide polymorphism (SNP) sites and 89,078 and 72,513 Indels (Insertion/deletion) were detected among the 30 mutant isolates compared to the primary scaffolds and haplotigs of the wild-type isolate, respectively. Deleterious variants including SNPs and Indels occurred in 1866 genes. Genome wide association analysis identified 754 genes associated with avirulence phenotypes. A total of 62 genes were found significantly associated to 16 avirulence genes after selection through six criteria for putative effectors and degree of association, including 48 genes encoding secreted proteins (SPs) and 14 non-SP genes but with high levels of association (Pst. Since the avirulence gene candidates were identified from associated SNPs and Indels caused by artificial mutagenesis, these avirulence gene candidates are valuable resources for elucidating the mechanisms of the pathogen pathogenicity, and will be studied to determine their functions in the interactions between the wheat host and the Pst pathogen.Genome sequence comparison of the mutant isolates with the progenitor isolate unraveled a large number of mutation sites along the genome and identified high-confidence effector genes as candidates for avirulence genes in  Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe (yellow) rust, is a threat to wheat production worldwide xC motif xC is a conserved motif of effectors in the genus Puccinia. RXLR and RXLR-like motifs, which have been commonly found in oomycetes effector, were detected in eight Pst avirulence gene candidates in the present study. Also, motif [L/I]xAR previously reported from M. oryzae effectors was detected in two of the Pst avirulence gene candidates. It is still not clear whether these effector motifs from oomycetes and ascomycetes are related to avirulence genes in Puccinia of basidiomycetes. This information is important in characterizing effectors and understanding evolutionary mechanisms of plant pathogenic fungi and oomycetes. Functional verification of more effector candidates in Puccinia species and other fungi or oomycetes, conserved motifs could be identified in the near future.Previous studies failed to identify conserved sequence motifs like oomycetes motif RxLR in fungal effectors . HoweverPgt , 47 and ndidates . Due to Effectors can be classified into apoplastic, cytoplasmic and nuclear categories based on their subcellular localization and action inside hosts . Since sPst Avr candidates through comparative genomics and association analyses. With the identification of more effectors in Puccinia species in recently years VY[L/I]R xC R  in Bgh, Y/F/W]xC  in the wY/F/W]xC  in some n 0.0001 . The subequences . The worAdditional file 1: Table S1. Infection types of the progenitor isolate 11–281 and derived 30 mutants of Puccinia striiformis f. sp. tritici on 18 wheat Yr single-gene differentials. Table S2. Reads, rates and qualities of 30 Puccinia striiformis f. sp. tritici mutants mapped to the reference genome. Table S3. The distribution and density of SNPs and Indels on each scaffold. Table S4. Statistics of Indels of each mutant isolate in terms of insertions, deletions, and different lengths. Table S5. Number of SNPs, Indels, insertions and deletions of each mutant isolate identified from the isolate 11–281 haplotigs. Table S6. Genes highly associated to avirulence with P-value < 0.001.Additional file 2: Figure S1. Phylogenetic trees of Puccinia striiformis f. sp. tritici mutant isolates based on the EMS-induced SNPs. Figure S2. Types and frequencies of SNP effects detected from the reference genome haplotigs. A: The number and percentage of all EMS-induced SNPs for each type of effects. B: The types and percentages of deleterious SNP effects. Figure S3. Types and frequencies of Indel effects detected from the reference genome haplotigs. A: The number and percentages of all EMS-induced Indels for each type of effects. B: The types and percentages of deleterious Indel effects. Figure S4. Phylogenetic tree of the progenitor isolate 11–281 of Puccinia striiformis f. sp. tritici (Pst) together with other 12 fungal isolates. The phylogenetic tree was developed from 13 protein sequences using a hill-climbing algorithm with the GTRGAMMA model. 13 fungi isolates are Verticillium dahlia 12,008, Fusarium oxysporum f. sp. cepae FoC_Fus2, Fusarium graminearum ITEM_124, Blumeria graminis f. sp. hordei RACE1, Botrytis cinerea B05.10, Melampsora larici-populina 98AG31, Puccinia striiformis f. sp. tritici 104E137A-, 93–210, 11–281, Puccinia striiformis f. sp. hordei 93TX-2, Puccinia coronata f. sp. avenae 12SD80, Puccinia graminis f. sp. tritici CRL75–36–700-3 and Puccinia triticina BBBD Race 1. The numbers indicate the distances between nodes. Figure S5. Characterization of Puccinia striiformis f. sp. tritici Avr effector candidates in the Upset plot. A: 48 effector candidates of secreted protein (SP) genes. B: 14 effector candidates from non-SP genes. Figure S6. Percentages (%) of the subcellular localization sites of effector candidates of Puccinia striiformis f. sp. tritici in the fungal cells. A: Genes identified from secreted protein (SP) genes. B: Genes identified from non-SP genes. C: All 62 genes associated to avirulence genes. Figure S7. Percentages (%) of the subcellular localization sites of effector candidates of Puccinia striiformis f. sp. tritici inside the host. A: Genes identified from secreted protein (SP) genes. B: Genes identified from non-SP genes. C: All 62 genes associated to avirulence genes.Additional file 3: Table SE1. 62 genes considered as candidates for avirulence genes and their genome positions and derived amino acids. Table SE2. Orthogroups of Puccinia striiformis f. sp. tritici (Pst) avirulence gene candidates in isolate Pst 11–281 detected in other 12 fungal isolates. Table SE3. Search results of candidate genes identified in Puccinia striiformis f. sp. tritici (Pst) isolate Pst 11–281 against four P. striiformis protein databases using NCBI BLAST.


Mycoplasma bovis (M. bovis) causes various chronic inflammatory diseases, including mastitis and bronchopneumonia, in dairy and feed cattle. It has been found to suppress the host immune response during infection, leading to the development of chronic conditions. Both in vitro and in vivo studies have confirmed that M. bovis can induce proinflammatory cytokines and chemokines in the host. This consists of an inflammatory response in the host that causes pathological immune damage, which is essential for the pathogenic mechanism of M. bovis. Additionally, M. bovis can escape host immune system elimination and, thus, cause chronic infection. This is accomplished by preventing phagocytosis and inhibiting key responses, including the neutrophil respiratory burst and the development of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) that lead to the creation of an extracellular bactericidal network, in addition to inhibiting monocyte and alveolar macrophage apoptosis and inducing monocytes to produce anti-inflammatory factors, thus inducing the apoptosis of peripheral blood mononuclear cells (PBMCs), inhibiting their proliferative response and resulting in their invasion. Together, these conditions lead to long-term M. bovis infection. In terms of the pathogenic mechanism, M. bovis may invade specific T-cell subsets and induce host generation of exhausted T-cells, which helps it to escape immune clearance. Moreover, the M. bovis antigen exhibits high-frequency variation in size and expression period, which allows it to avoid activation of the host humoral immune response. This review includes some recent advances in studying the immune response to M. bovis. These may help to further understand the host immune response against M. bovis and to develop potential therapeutic approaches to control M. bovis infection. Mycoplasma is a global issue with consequences for cattle health and economics. M. bovis is responsible for bronchopneumonia, pharyngitis, laryngitis, otitis, keratoconjunctivitis, meningitis, and mastitis, as well as hormonal diseases such as metritis, infertility, and abortion. All age groups are susceptible to M. bovis infection, and the pathogen can be shed and survive for years in contaminated herds [M. bovis has emerged as a significant pathogen in Europe [M. bovis prevents lymphocyte proliferation due to its immune-suppressive characteristics [M. bovis infections, both in vivo and in vitro, there are conflicting findings regarding whether these same processes take place during bona fide M. bovis infection. Several research assessments have revealed that M. bovis adheres to surfaces  and erythrocytes while evading immune reactions. Some studies have indicated the M. bovis-induced activation and development of different cytokines from bovine macrophages, such as IFN-γ, interleukin-4 [M. bovis has been implicated by numerous studies (including those listed above) in the regulation of immune response both in vivo and in vitro [Mycoplasma bovis strain Mb1 invades and adheres to bovine PBMCs, preventing their proliferation, but does not appear to alter developmental responses in functional cytokines such as IFN-γ. In a relatively short time, M. bovis can invade any type of PBMC, resulting in the induction of lymphocyte differentiation and spreading to any host tissue. Adhesion is the primary step of infection with mycoplasma [M. bovis dissemination in hosts [M. bovis in host cells are available [M. bovis have been obtained regarding apoptosis induction, cytotoxic effects, and the host cells’ viability [Mycoplasma bovis has been found to inhibit the development of PBMCs [Infection of dairy and feed cattle by ed herds . Such died herds ,3. In thn Europe , and Japn Europe , resultin Europe . The usen Europe . In addieristics , and proeristics . Althougsurfaces , is locarophages ,12,13; trophages ,15 when rophages . Mycoplaleukin-4 , TNF-α, leukin-4 . This isin vitro . Van dercoplasma . A few rcoplasma ,20. Howecoplasma ,22,23 thin hosts . In vivovailable ,26,27. Iiability ,28,29,30of PBMCs ,32.M. bovis; however, these findings are difficult to transfer to in vivo conditions. Moreover, only a few experiments have been carried out on the local immune reaction in M. bovis-induced pneumonia. Cytometry tests were carried out on an experimental calf infection model to estimate the effects of an M. bovis strain in the field on changes in the peripheral blood leukocyte reaction, including phagocytic activity and oxygen metabolism. Immunohistochemical staining has been used to assess the local lung immunity of experimentally infected calves. The general stimulation of phagocytic activity and the killing mechanism of peripheral blood leukocytes in response to M. bovis infection indicate the upregulation of cellular antimicrobial pathways. In infected lungs, local immune responses are characterized by T- and B-cell activation but with more enhanced lymphocytic T-response. The activation of local lung immunity has also been confirmed by the high expression of phagocytes and antigen-presenting cells post-infection. Stimulation does not seem to be effective in eliminating M. bovis from the host and preventing specific lung lesions, suggesting the pathogen’s ability to avoid the host immune reaction—either by peripherals or by local cells—in M. bovis-induced pneumonia [Dudek et al., (2020) explored the leukocytes characterizing the host antimicrobial resistance mechanisms. Several in vitro experiments have been carried out on the effect of leukocytes on neumonia .M. bovis immune responses and sustain self-tolerance by negatively delivering Th1 and Th2 cells by developing the cytokines IL-10 and TGF-β. This review discusses the displayed antigens of naive T-cells in dendritic cells (DCs) by large histocompatibility complex–T-cell receptor interaction, which causes antigen-specific T-cells to be activated. T-cells differentiate into four sub-types, such as T-cells Th1, Th2, and Th17, or regulatory T-cells (Tregs), based on the microenvironmental cytokine environment . Th1 cells activate macrophages or CD8 T-cells by releasing IFN-γ. CD8 T-cells either degrade pathogens or cause apoptosis of infected cells by producing perforins and granzymes. Th2 cells activate B-cells by synthesizing cytokines and separating B-cells from plasma cells, modifying the antibody class and promoting the affinity to antibodies involved in pathogenic neutralization, opsonization, and phagocytosis. In neutrophil activation and immune regulation, Th17 cells are involved in the production of IL-17A, which is essential for protection against extracellular and specific intracellular pathogens. Regulatory T-cells (Tregs) control the reg), which, after the immune response, suppress the development of CD8 T-cells and memory T-cells [M. bovis from the host [M. bovis infiltrating mucosal sites are eliminated before the onset of the disease.The immune response can be divided into humoral immunity and cellular immunity, with cellular immunity regulated by T-cells and B-cells regulating humoral immunity. The immune system develops cytokines, including interleukins, which balance humoral and cell-mediated immune responses. T-cells have a specific antigen (antibody)-binding receptor molecule on the cell surface, which is called a T-cell receptor (TCR). Many TCRs recognize parts of major histocompatibility complex (MHC) molecules, especially the antigenic peptides linked to those molecules. T-helper cells (Th CD4 cells) recognize processed foreign peptides on the surface of antigen-presenting cells (APCs) complexed with MHC class II molecules and induce an immune response by secreting cytokines that stimulate CD8 T-cells and B-cells. Killer or cytotoxic T-cells (CD8) participate in surface interactions with other cells carrying processed foreign peptides complexed with MHC class I molecules ,34. Cell T-cells . Immune  T-cells . In spec T-cells . The act T-cells . It has the host . A critiM. bovis mainly colonized the upper respiratory tract and the tonsillar mucosa, at the autopsy, both colonized the palatine and pharyngeal tonsils with M. bovis in all inoculated calves. The tonsils of orally inoculated cattle had the largest amounts of Mycoplasma. These results confirm previous reports of naturally occurring M. bovis infection in calves indicating the initial colonization site in the upper respiratory tract [M. bovis is primarily anti-inflammatory. Simultaneously, it was found that M. bovis primarily infects the mucosal and serosal surfaces and live bacteria in infected cattle brains. Previous studies saw bacterial antigens also in the synovial membranes and infected cattle’s liver and kidneys [M. bovis reside on mucosal and extracellular surfaces but can invade and survive in various host cells. M. bovis can be seen living within the bovine alveolar macrophages. M. bovis infection in bovine epithelial cells occurs in many epithelial cell types, including embryonic bovine tracheal cells, embryonic bovine lung cells, and primary bovine mammary cells [M. bovis typically produces a heavy local mucosa characterized by high serum IgG and IgA reactions. Similarly, M. bovis mammary gland inoculation results in IgG serum and local IgG and IgA serum mucosa. However, to prevent infections, systemic antibodies are critical, and serum IgG M. bovis titers are associated with arthritis defense. Local antibodies are likely to be more critical on mucosal surfaces. For example, concentrations of anti-M. bovis antibodies correlated in milk but not in serum with resistance to reinfection in cows after M. bovis mastitis. IgG concentrations bronchoalveolar lavage fluid (BAL) are linked to resistance to respiratory disease associated with M. bovis. Mycoplasma respiratory infections are commonly recognized to have significant immunopathological components, characterized by large accumulations of lymphocytes in infected tissues, cytokine output, and inflammation of the lungs [Mycoplasmas, including M. bovis, may also modulate some inflammatory responses. However, in the lungs of calves with M. bovis infections, little is known about the cytokine environment. In response to the M. bovis antigen, peripheral blood mononuclear cells from M. bovis-infected calves secreted IFN-γ and IL4 and developed a strong systemic IgG1 response with little IgG2. These results suggest that M. bovis induces a mixed Th1-Th2 cytokine response, although the lack of development of IgG2 was more consistent with a Th2-biased response [The mucosal immune system includes digestive, gastrointestinal, urogenital, and exocrine glands. The predominant isotype formed in bovine species at mucosal sites is IgG1. The antigen-specific immune responses of Peyer’s patches dictate mucosal immune development through the production of cytokines following immune cell interactions. Since oral and transtracheal inoculation of ry tract . Consequry tract . With hi kidneys . Differery cells . M. bovihe lungs . MycoplaM. bovis blocks the ability of neutrophils to produce a chemiluminescent reaction after certain species have been ingested [M. bovis inhibits the blasts of respiratory neutrophils at or distal to the point of protein kinase C activation. A direct interaction between M. bovis cells and the neutrophil membrane is thought to be mediated by this effect; thus, it is essential to investigate the exact inhibition mechanism as well as the association between Mycoplasma spp. and innate immune cells typically containing proinflammatory cytokines such as IL-1, IL-6, and TNF-α. Furthermore, chemokines such as MIP-lα, MIP-1β, and ENA-78 are also produced through the evolution of IL-1, IL-6, and TNF-α; these, along with other epithelial and fibroblast cells, support other innate cells and adaptive immune cells. Chemokines contribute to the normal perivascular and peribronchiolar accumulation of mononuclear cells during mycoplasma respiratory infections [M. bovis using alveolar macrophages led to the production of two essential mediators of the immune response: NO and TNF-α. Antigens derived from M. bovis can also activate bovine γδ T-cells in vitro. Although T-cells are traditionally called adaptive immune cells, it has been recognized that in cattle with innate immunity, peripheral γδ T-cells are regularly activated  are involved in such M. bovis triggers. Compared to macrophages, neutrophils seem to be more inhibited by M. bovis [M. bovis by bovine neutrophils and macrophages is based on opsonization [M. bovis has various mechanisms to combat opsonization  and another that clings to IgG heavy chains, are expressed in some forms of mycoplasmas  [M. bovis, several copies of the MIB and MIP genes have been entirely sequenced in all M. bovis genomes [M. bovis with a different strategy to resist antimicrobial phagocytosis  are associated with immunization against bovine respiratory infection. Early response cytokines, including interferons (IFNs) and those of the complement system, are other vital components of the innate immune system . M. boviingested . It has fections . The funantigens , which cM. bovis . Efficienization . M. bovinization : First oormation . Third, se; MIP) . Althoug genomes . This meocytosis .M. bovis has many pathways to resist opsonization, thereby preventing and eliminating phagocytosis. Opsonization of the antibody or substitution considerably improves phagocytic bacteria’s chances being destroyed by phagocytic cells . M. bovis after an initial response of innated cells during Mycoplasma infection [Innate immunity is available or easily induced in a natural manner; however, these components are not antigen-specific and, more importantly, there is no immunological memory. On the other hand, memory’s extraordinary property through adaptive immunity has been demonstrated . Initialnfection .Mycoplasma often release cytokines known for their adaptive immune response, such as IL-13, to assess the type of adaptive immune response that occurs during M. bovis infection [M. bovis seems skewed against IgG1, a less effective phagocytosis challenger and neutrophil killing than IgG2 [M. bovis is an infectious organism that affects the host in both an immunostimulatory and immunosuppressive manner. Previous studies have shown an improvement in the percentage and subsets of T lymphocytes  in reaction to M. bovis experimental challenge in calves. Leukocytosis and a substantial increase in the percentage of WC4 (B lymphocytes) have also been observed under these conditions, suggesting the activation of CD4, CD8, and γδ T-cells due to M. bovis antigen incubation [M. bovis; however, M. bovis antigen levels of proliferation are not pronounced and have been absent in some studies. Aside from these variables and serological experiments, serology assessment has been used for the determination of the adaptive immune response to M. bovis. In lung disease, the immune response to M. bovis infection has been biased towards a type-2 immune response by analyzing peripheral lymphocytes and serum . Immunohistochemical studies have suggested that many IgGl cells are generated, consistent with serological trials (IgGl indicates a Th2 response in infected cattle’s lungs) [M. bovis continues to proceed as a chronic infection in terms of both the innate and adaptive immune cells, with a rebound in post-clinical animals for several months. It has been speculated that one of the reasons facilitating the chronic nature of bovine mycoplasma infection is that the serological reaction may not be that of the right phenotype, such as IgM. In addition, IgGl is an inferior complement activator relative to other IgG isoforms [Cells that come into contact with nfection . The humhan IgG2 ,49,50,51cubation . Blastogs lungs) . The infisoforms  such as M. bovis could evade the host immune response. M. bovis has immunosuppressive properties that prevent lymphocyte proliferation in response to mitogens [A previous study found that mitogens .M. bovis [M. bovis has been shown to be active in proliferative blood lymphocytes under mitogen inhibition [M. bovis was reported to suppress the naive activation of lymphocytes [Mycoplasma infections, indicating that this lymphocyte-inhibiting peptide may act as a mediator of immune evasion. Vanden et al. [M. bovis is responsible for, or causes, cell apoptosis. M. bovis has been identified as a pathogen responsible for the development of respiratory and arthritis diseases. There is a mass infiltration of lymphocytes and non-specific activation in arthritis, with no death of lymphocytes. Therefore, these authors indicate that Mycoplasma might not cause T-cell apoptosis. The apoptotic effects do not inhibit the infiltration of T-cells or cytokine production in other cell types [Lymphocyte proliferation inhibition has been seen as a supposed mechanism for immune evasion by M. bovis . M. bovihibition . DNA synphocytes . It has n et al.  also stall types . M. bovis can induce the expression of important cytokine  genes in bovine PBMCs at a multiplicity of infection (MOI) of 1000, as recorded by Gondaira et al. [M. bovis, which would engage the immune response. The upregulation of IFN-γ, which is essential for host immunity against intracellular pathogens, has been demonstrated by IL-12. Thus, IL-12 and IFN-γ play crucial roles in M. bovis infection. Previous studies have shown that heat-killed M. bovis did not induce expression and cytokine production of bovine PBMC cytokine mRNA, and have indicated the potential of live M. bovis to play a pathogenic role in the bovine PBMC immune response [Mycoplasma bovis induction was delayed by the inflammatory response [Mycoplasma bovis has been detected in natural milk secreted following M. bovis intramammary (IMI) infusion (106 to 1010 CFU/mL) for days prior to the appearance of clinical signs [M. bovis intramammary infusion [M. bovis was intracellularly detected in bovine PBMCs [M. bovis. Significant variations in the expression of IL-1α, IL-1β, IL-2, IL-6, and IL-8 were observed between infected vs. normal lungs of control calves [M. bovis can release cytokines [M. bovis infection for Th1 reaction [Mycoplasma bovis delays apoptosis in monocytes and suppresses the development of IFN-γ and TNF-α [M. bovis infection resulted in increased expression and a comparatively lower IL-1 response of the proinflammatory cytokine IL-6. Proinflammatory cytokines are essential for M. bovis pathogenesis. Although IL-6 was upregulated following co-incubation of PBMCs with live M. bovis at 100:1 MOI, there was no increase in IL-1 and TNF-α levels. It can be assumed that a high MOI is required for producing a consistent inflammatory response [a et al. . These fresponse . TNF-α iresponse . Previouresponse . Mycoplaal signs . Previouinfusion . M. bovine PBMCs . As effene PBMCs . This cane PBMCs ,60. Rodrl calves . The celytokines . This isreaction . Mycopland TNF-α , which presponse .M. bovis-induced mastitis has increased expression of immune exhaustion-related genes and decreased expression of innate mononuclear cells (MNC)-related immune response genes in cow quarters. These results showed that the immune function of bovine mononuclear cells (MNCs), linked to a prolonged duration of infection with M. bovis, was impaired by mastitis caused by M. bovis [Anaplasma marginale [Plasmodium berghei [Mycobacterium avium subsp. paratuberculosis [M. bovis infection. The differently modulated subpopulations of CD8 and CD4 T-cells with intermediate and robust expression of PD1 after M. bovis infection of cattle PBMC have also been studied. T-cells showed a substantial decrease in CD4 PD1 and CD8 PD1 subsets and a significant increase in CD4 PD1 and CD8 PD1 subsets. Furthermore, the use of blocking PD-1 antibodies results in the restoration of M. bovis strain Mb1 after PD-1/PD-L1 destruction as well as the proliferation of M. bovis strain Mb1 in contaminated PBMCs [Mycoplasma bovis pathogenesis and related immune exhaustion in inhibitory pathways, resulting in compromised host immune responses [In chronic infection, immunosuppressive factors such as programmed cell death 1 (PD-1), lymphocyte activation gene 3 (LAG3), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), T-cell immunoglobulin, and mucin-containing domain-3 (Tim3), are expressed in lymphocytes. These proteins bind to their respective ligands to cause immune suppression of the effector cells. These immune exhaustion-associated proteins cause a marked decrease in cell proliferation and cytotoxic activity. Newly identified M. bovis . Based oM. bovis , and manarginale , Plasmod berghei , Mycobacrculosis , and bovrculosis ,68. Suleed PBMCs . A recenesponses . Previouesponses .M. bovis infection and immunity, as well as the strategies by which M. bovis evades the innate and adaptive immune response, immune response exhaustion, and cytokines in addition to the proliferative response of bovine PBMC cells to M. bovis. M. bovis induces the host to produce an inflammatory response, causing pathological immune damage—its essential pathogenic mechanism. From the current literature, it can be concluded that M. bovis can induce the host to produce γδ, CD4, and CD8 T-cells as well as influence the host to produce IgM, IgG, and IgA antibodies in a time-dependent manner. Compared with IgG2, the IgG1 produced by the host induced by M. bovis is dominant. The IgG1 antibody has a low opsonin effect, leading to an inadequate humoral immune response and long-term infection of M. bovis in the host. Mycoplasma bovis can cause a wide variety of immune-modulatory products by directly activating cytokine secretion from lymphocytes, macrophages, and neutrophils. Surface antigen variation, potential intracellular invasion, and biofilm formation also allow M. bovis to impair the host immune response. However, further research on the host and microbial factors regulating M. bovis infection in the upper respiratory tract, contributing to disease development or the effective immune response, is required to facilitate the creation of better control mechanisms and preventative strategies for the pathogen. Further studies of host immune responses to M. bovis infection are needed to control the resulting disease. This review summarized some recent advances in the immune response relating to


Mycobacterium tuberculosis causes pulmonary tuberculosis, a deadly infection of which the clinical expression and prognosis are not fully understood at the individual level, apart from genetic susceptibility traits. We investigated whether individual gut microbiota may correlate with pulmonary tuberculosis status. Culturomics investigations of gut microbiota in two pulmonary tuberculosis patients and two controls in Burkina Faso found 60 different bacterial species in patients and 97 in controls, including 45 in common. Further analysis of the results at the individual level indicated seven bacteria, including Enterococcus mundtii and Enterococcus casseliflavus, which were exclusively cultured in controls. Blind quantitative PCR-based exploration of faeces samples in two cohorts in Burkina Faso and in France confirmed a nonsignificant association of E. mundtii and E. casseliflavus with controls. Further in vitro explorations found four E. mundtii and E. casseliflavus strains inhibiting the growth of M. tuberculosis strains representative of four different lineages as well as Mycobacterium africanum, Mycobacterium canettii, and Mycobacterium bovis, in an inoculum-dependent manner. Heat-killed E. mundtii or E. casseliflavus were ineffective. These unprecedented observations of direct interactions between gut E. mundtii and E. casseliflavus with M. tuberculosis complex mycobacteria suggest that gut microbiota may modulate the expression of pulmonary tuberculosis. Mycobacterium tuberculosis complex (MTBC), and is responsible for a high burden of morbidity and mortality in the most countries worldwide [Tuberculosis is a deadly bacterial infection due to mycobacteria forming the orldwide . In 2018M. tuberculosis inoculum, genotype and antibiotic susceptibility profile may play in part the clinical expression and prognosis of pulmonary tuberculosis [M. tuberculosis genotypes with certain populations has been clearly established in some geographic areas [The interplay between host and pathogen determining the clinical expression and prognosis of tuberculosis is not fully understood at the individual level. Indisputably, the rculosis ,4. Host rculosis . Such gerculosis ,7. Accoric areas ,7,8,9.However, the factors mention above may not explain all the variations observed in the clinical spectrum of tuberculosis and the hypothesis that individual microbiota may also govern part of the clinical expression of tuberculosis, has emerged . A few sIn order to advance this knowledge, we herein embarked on a preliminary study of the digestive tract microbiota in individuals with and without pulmonary tuberculosis using culturomics, in order to discover whether some living microorganisms detected in the digestive tract microbiota, including bacteria and archaea, were significantly correlated and anticorrelated with pulmonary tuberculosis prior to anti-TB treatment.A total of 150 stool samples collected from 150 individuals were included in this study. Fifty stools specimens from fifty individuals were collected as part of a tuberculosis diagnosis kit in patients being routinely investigated for medical suspicion of pulmonary tuberculosis in the IHU Méditerranée Infection, Marseille, France . MicrobiTo analyze the differences between the digestive microbiota of patients with pulmonary tuberculosis and subjects without pulmonary tuberculosis, culturomics was performed on 4 stool samples collected in Burkina Faso, 2 stool samples randomly selected from samples collected from individuals positive for pulmonary tuberculosis and 2 stool samples randomly selected from samples collected from individuals negative for pulmonary tuberculosis .https://www.dsmz.de/microorganisms/medium/pdf/DSMZ_Medium1611.pdf) . The blood culture bottles and YCFA medium were previously supplemented with 3 mL of filtered rumen fluid and 3 mL of sheep blood (Becton Dickinson). The inoculated blood culture bottles and negative controls, represented by blood culture bottle inoculated with DPBS, were incubated at 37 °C. Inoculations on solid agar were performed at three hours, six hours, nine hours, twenty-four hours, three days, seven days, and 14 days post-incubation. For each inoculation, 1 mL of supernatant was collected and diluted in 900 µL of DPBS in serial dilutions from 1/10 to 1/1010. These dilutions were inoculated on 5% sheep blood agar  incubated at 37°C for 48 h, respecting the strict atmospheric conditions of aerobic and anaerobic culture. All colonies were sub-cultured and identified by matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS), as previously described [http://bioinformatics.psb.ugent.be/webtools/Venn/).The culture was performed as previously described with modifications . In brieescribed . The bacE. casseliflavus and E. mundtii) in the stools of TB-positive and TB-negative patients, 150 stools specimens were tested with molecular PCR-based assays as previously described [® 480 Probes Master , 0.5 µL of each primer, 0.5 µL of probe, 3 µL of distilled water, 0.5 µL of Uracil-DNA glycosylase (UDG) and 5 µL of DNA. Analyses were performed using the thermal cycler CFX96 Touch™ . DNA extraction, PCR inhibitors and total bacterial richness was measured using real-time PCR targeting the universal bacterial 16S rRNA gene in the presence of negative controls , M. tuberculosis lineage 2 strain Beijing CSUR Q1316 [M. tuberculosis lineage 3 strain CSURP7204, M. tuberculosis lineage 4 strain CSUR P7739 [M. africanum strain CSURP7201, M. canettii strain CIPT140010059, and M. bovis strain CSURP7222 [7 CFU/mL, as previously described [M. tuberculosis growth inhibition by dead bacteria, the bacterial suspensions were inactivated by one-hour incubation at 100°C and the potential inhibitory effect of inactivated bacteria was tested as described above. All these manipulations were carried out three times in the presence of DPBS as a negative control.The antituberculosis activity of seven bacteria specifically found in the gut microbiota of two tuberculosis-free patients was assessed using the agar well diffusion method, with modifications ,21. BactUR Q1316  M. tuberUR P7739 , M. afriSURP7222 . All strescribed . This suhttps://www.xlstat.com). Statistical tests were performed on the website biostaTGV (https://biostatgv.sentiweb.frS) including Student’s T test to compare DPBS and the bacteria inhibition diameter and the ANOVA test was used to compare the susceptibility of mycobacteria to enterococci. Differences were considered to be statistically significant when p < 0.05.Data was entered into Microsoft Excel for Office 365 to calculate median, average and standard deviation. A clustering heatmap was used to visualise the potential clustering of cultured gut bacterial repertoires in positive and negative pulmonary tuberculosis patients. The heatmap and the principal component analysis were performed using XLSTAT v2019.1 statistical and data analysis solution  than in the TB-negative patients . Furthermore, we observed a reduction in the number of Bacteroides, Enterococcus, Streptococcus and Staphylococcus members in the TB-positive patients compared to the TB-negative patients and saw that the number of bacterial species was lower in the TB-positive patients. Preparing out a heatmap  were cultured in both the two TB-negative patients and were not cultured in both the two TB-positive patients, while five bacterial species  were shared by the two TB-positive patients and were absent from the two TB-negative patients  faeces samples: five faeces collected from TB-positive patients and six from TB-negative patients were negative. Accordingly, in the very same 150 individuals, blind quantitative real-time PCR targeting E. mundtii yielded 28 (18.22%) positive individuals with a median Ct of 35.47, an average Ct of 34.8, and Ct standard deviation of 2.61, including nine out of 64 (14%) TB-positive patients and 19 in 86 (22%) TB-negative patients (p = 0.21). All 28 E. mundtii-positive faeces specimens had been collected from Burkinabe individuals and none from French individuals. In TB-positive patients, E. mundtii was detected with a median Ct of 34.5, an average Ct of 34.2, and standard deviation of Ct 2.5 , no different from Ct values obtained in TB-negative patients with, respectively, a median Ct of 34.48, an average Ct of 34.23, and standard deviation of Ct 2.6 . Quantitative real-time PCR targeting E. casseliflavus yielded 78 in 150 (52%) positive individuals with a median Ct of 34.8, an average Ct of 34.1, and a Ct standard deviation of 2.85, including 28 in 64 (43%) TB-positive patients and 50 in 86 (58%) TB-negative patients (p = 0.08). In TB-positive patients, E. casseliflavus was detected with a median Ct of 34.45, an average Ct of 35.06, and a standard deviation Ct 2.06 , no different from Ct values obtained in TB-negative patients with, respectively, a median Ct of 34.11, an average Ct of 34.9, and a standard deviation Ct 2.85 .In order to further explore the prevalence of , France  and 100 M. tuberculosis strain Beijing CSUR Q1316 . The M. tuberculosis growth inhibition effect was observed on all tested strains of the MTBC including Mycobacterium canettii, Mycobacterium bovis and Mycobacterium africanum with diameters of growth inhibition being on average three times larger for M. canettii than for the other species . The M. tuberculosis growth inhibition effect was not merely due to changes in the pH, as the pH of the suspension was increased from 5 to 6, however M. tuberculosis also reduced the pH of the suspension to 6. However, a few colonies growing in the M. tuberculosis growth inhibition zone around either E. mundtii or E. casseliflavus were confirmed to be colonies of M. tuberculosis by whole genome sequencing. Genome sequences have been deposited in the European Molecular Biology Laboratory (EMBL).The mixture of these seven bacteria lowered the local pH of the Middlebrook culture medium to 5 and inhibited growth of the UR Q1316 . Further confusa . Inhibit species   to overcome some limitations of the metagenomics studies, and confirmed the decreased prevalence in Bacteroidetes in TB-positive patients compared to TB-negative ones [Clostridium members. This was in line with observations made in experimental severe tuberculosis in macaques [E. casseliflavus and E. mundtii exclusively in the faeces of TB-negative patients. These two Firmicutes were further qPCR-detected with an increased prevalence in the faeces of TB-negative patients in two independent cohorts. These observations were extended by experimental observations showing these two Firmicutes inhibited the growth of M. canettii, regarded as the MTBC species most closely related to the common ancestor of the complex and a pathogen responsible for tuberculosis in exposed individuals in the Horn of Africa [M. tuberculosis and M. bovis responsible for tuberculosis in humans and animals [M. tuberculosis [E. mundtii, all previous studies have dealt with the antibiotic activity of environmental isolates against opportunistic pathogens, mainly Listeria monocytogenes, such as E. mundtii fish isolates LP17 and LP18 [E. mundtii and E. casseliflavus against MTBC was beyond the scope of the present study, yet the E. mundtii and E. casseliflavus strains here investigated all encode for bacteriocin, as anticipated by their whole genome sequence.In this study, we used culturomics  cases, while five in 251 (1.99%) of the children died of unrelated causes. Although 18 neonates showed no signs of tuberculosis, 72 died. Follow-up of the 174 surviving children revealed only six sick children, while the others’ symptoms were spontaneously resolved. Such a heterogeneous natural history of tuberculosis in neonates in the Lübeck series of cases may partly be explained by different gut microbiota maturation in the neonates [M. canettii and M. tuberculosis has been shown to lead to both disseminating in the lymphatic and lung tissues after experimental gavage in mice [E. mundtii and E. casseliflavus interactions with M. tuberculosis in a mouse model, to observe interference with M. tuberculosis gut survival, translocation, and recirculation in the lungs. Complementary, indirect interactions may be mediated by inflammation and immune cells in the exact model of what is now established in immune cancerology [Prevotella, the prevalence of which was increased in TB-positive children and adults [Clostridium members that were found to be more abundant in TB-positive patients, induce Treg production in mice guts and an increased number of Venus+CD4+ cells in the liver and lungs [Clostridium members are short-chain fatty acid (SCFA) producers inducing anti-inflammatory production (IL-10) [Interactions between ive ones . In the neonates . Indeed, in mice . It woulcerology . As an ed adults ,30, wered adults . Likewisnd lungs . Clostri (IL-10) , and SCF (IL-10) .M. tuberculosis. First, E. mundtii was detected only in faeces samples collected from Burkinabe individuals and not from French ones, suggesting that gut carriage of E. mundtii is population-dependant and is probably linked to diet, given that E. mundtii has been found in several plants and meat entering the human food chain [M. tuberculosis colonies that were resistant to E. mundtii-inhibition, suggesting that the mechanism of growth inhibition is not absolute in one M. tuberculosis population.This is a preliminary study involving by culturomics only in four individuals, so that the generalized of data here reported are not possible today and will required additional studies in larger population. Also, this study disclosed potential limitations in the enterococci interactions with od chain ,44. SecoE. casseliflavus and E. mundtii against pulmonary tuberculosis. Further studies may evaluate the relative recombination of innate immunity in these observations such as macrophage and cytokine expression induced by enterococci and E. mundtii, in particular during the intracellular phase of tuberculosis. Depending on the elucidation of the underlying mechanisms and topography of the inhibition and extent of M. tuberculosis complex, some of these Firmicutes may be considered as probiotic adjuvants to conventional antibiotics in the fight against deadly tuberculosis.In conclusion, the data reported here indicate a higher bacterial diversity in non-tuberculous individuals versus pulmonary tuberculosis patients. In addition, we confirm a protective effect of certain gut microbiota and, particularly, of the Firmicutes


Raphidiopsis curvispora strain GIHE-G1, a coiled filamentous heterocyst-forming cyanobacterium isolated from a drinking water reservoir in Korea, are reported here. The genome information is expected to improve understanding of this species.The complete genome and plasmid sequences of  Raphidiopsis curvispora strain GIHE-G1, a coiled filamentous heterocyst-forming cyanobacterium isolated from a drinking water reservoir in South Korea, are reported here. The genome information is expected to improve understanding of this species.The complete genome and plasmid sequences of  Raphidiopsis curvispora is a coiled filamentous nitrogen-fixing cyanobacterium that is morphologically and genetically similar to R. raciborskii. This species was first detected in a Japanese reservoir in 1995  were generated. De novo assembly was performed through a three-step process  using HGAP v3.0  database  were retrieved. The database  with a tdatabase . DefaultR. curvispora is known to be potentially toxic , and 42 tRNA, 9 rRNA, and 4 noncoding RNA (ncRNA) sequences. Eleven CRISPR arrays were also detected via PGAP. ly toxic , but no R. curvispora GIHE-G1 have been deposited in NCBI under the accession numbers CP060822 and CP060823 (GenBank), PRJNA616171 (BioProject), SAMN15834505 (BioSample), and SRR12567980 (SRA).The complete genome and plasmid sequences of


Capnocytophaga canimorsus is a facultative anaerobic, slow-growing, capnophilic, Gram-negative bacillus, that is commonly found in the microflora of canine and feline oral cavities. Capnocytophaga infections are an emerging zoonotic disease that can cause fatal systemic infections in immunocompromised individuals. Localized ocular Capnocytophaga infections, including keratitis, blepharitis, and endophthalmitis, can lead to severe eye threatening situations. To our knowledge, there is currently no documented case of Capnocytophaga canimorsus blebitis with bleb perforation after trabeculectomy.Capnocytophaga blebitis that occurred after trabeculectomy, associated with close dog contact (i.e. face licking). The patient had underwent trabeculectomy 10 years prior and presented with conjunctival injection, perforated bleb, and hypotony. Overall, patient was medically treated subconjunctival vancomycin, gentamicin and moxifloxacin drops. Trabeculectomy revision was performed with good visual outcome. Bacterial cultures grew Capnocytophaga canimorsus.Our case report and literature review features a novel case of Capnocytophaga infections. At-risk patients with ocular infections should be asked about close contact with dogs and cats; and treated promptly with the proper antibiotic regimen.We discuss the strategies for diagnosis, treatment, and common risk factors for ocular  Capnocytophaga canimorsus is commonly found in canine oral microflora, and less commonly in cats. In certain studies, C. canimorsus could be cultured from oral secretions of approximately 26% of dogs and 18% of cats [C. canimorsus can cause a wide variety of infections, with a majority being systemic infections and less than 10% being localized infections [C. canimorsus. We present a case of C. canimorsus blebitis with bleb perforation, which highlights the virulence of C. canimorsus and the need for pet owners with predisposing risk factors to be vigilant of ocular exposures.Pet ownership has many benefits, with some studies reporting a lower risk of cardiovascular disease and mortality with dog ownership . This co of cats . Recent  of cats . C. canifections . Many ofAn 81-year-old man was referred by an outside ophthalmologist with bacterial blebitis and perforated bleb, 10 years after trabeculectomy. Patient’s medical history was unremarkable. His ocular history includes radial keratotomy, penetrating keratoplasty (PKP), and the aforementioned trabeculectomy 10 years prior. Upon presentation, patient’s exam was notable for significant conjunctival injection, elevated cystic bleb, positive Seidel’s test with significant flow, and intraocular pressure of 4 mmHg , with articles published in the 1980s referring to Capnocytophaga canimorsus as DF-2. The first human infection was reported in 1976, and there has only been approximately 500 reported cases worldwide [C. canimorsus is notoriously difficult to culture, and can also result in subclinical infections [C. canimorsus can be transmitted through bites, scratches, and close animal contact (e.g. licking) [fections . C. caniCapnocytophaga canimorsus into two different species. The proposed name of the subclinical strain is Capnocytophaga canis, and maintaining the same name of Capnocytophaga canimorsus for the clinically symptomatic strain. However, recent case reports have isolated C. canis from patients with sepsis [C. canimorsus infections. However, at-risk individuals  can have systemic C. canimorsus infections like endocarditis, meningitis, and even fatal bacteremia leading to multi-organ system failure [With recent advances in ribosomal DNA sequencing, some researchers have proposed splitting h sepsis . The strh sepsis . Healthy failure –15.C. canimorsus infections have been mostly identified in the eye; keratitis being the most common presentation. Capnocytophaga keratitis is an aggressive cornea infection; in a study of ten patients, 50% required enucleation and 30% required corneal transplant [Capnocytophaga has a predilection for the deeper layers of the corneal stroma. Patients often present with corneal edema, endothelial lesions, ring-shaped infiltrates, and corneal perforation. Like our presented case, patients with Capnocytophaga keratitis report frequent and close interactions with dogs (e.g. face licking) [Capnocytophaga keratitis are often immunocompromised—for example, status post bone marrow transplant, or rituximab infusions. Professions that are regularly exposed to canine and feline mouths (i.e. veterinarians) are also at risk [Capnocytophaga resides.Localized ansplant . Given ilicking) . Patient at risk . Topical at risk , 16, 17.Capnocytophaga blebitis is a late BAI and stage II . Diagnostic workup can include aqueous/vitreous stain and culture, polymerase chain reaction (PCR), biome representational in silico karyotyping (BRiSK), or metagenomic deep sequencing (MDS) [Staphylococcus, and late onset BAI is Streptococcus. There is currently no consensus on the most effective treatment for BAI. However, commonly used treatment regimens are fortified aminoglycosides (e.g. tobramycin (14 mg/mL) with vancomycin (q30 min) or 4th generation fluoroquinolone (q1 hour) [Capnocytophaga’s susceptibility to aminoglycosides and vancomycin varies widely between studies; but is typically susceptible to clindamycin, penicillin, cephalosporin, imipenem, and beta-lactamase inhibitor combinations [The reported incidence of bleb-associated infections (BAI) range from 1.5 to 4.8% at 5 years follow up. BAIs range from stage I with no cell in the anterior chamber and little to no visual impairment to stage IIIB with cell in the anterior chamber and vitreous with an obscured view of the fundus . Early Bng (MDS) –18. The q1 hour) . These ainations , 20. OurCapnocytophaga canimorsus is a member of the complex microflora of canine and feline oral cavities. In immunocompromised patients, C. canimorsus has been known to cause severe systemic infections like bacteremia and multi-organ failure. In rare cases, C. canimorsus can cause eye-threatening ocular infections ranging from keratitis to endophthalmitis. Our novel case of Capnocytophaga blebitis contributes to the scarce clinical information available regarding ocular Capnocytophaga infections. At-risk patients with ocular infections should be asked about close contact with dogs and cats; and treated promptly with the proper antibiotic regimen.