Grapholita molesta, is an extremely important oligophagous pest species of stone and pome fruits throughout the world. As a host-switching species, adult moths, especially females, depend on olfactory cues to a large extent in locating host plants, finding mates, and selecting oviposition sites. The identification of olfactory genes can facilitate investigation on mechanisms for chemical communications.The oriental fruit moth, G.molesta using the next-generation sequencing technique, and assembled transcripts from RNA-seq reads using Trinity, SOAPdenovo-trans and Abyss-trans assemblers. We identified 124 putative olfactory genes. Among the identified olfactory genes, 118 were novel to this species, including 28 transcripts encoding for odorant binding proteins, 17 chemosensory proteins, 48 odorant receptors, four gustatory receptors, 24 ionotropic receptors, two sensory neuron membrane proteins, and one odor degrading enzyme. The identified genes were further confirmed through semi-quantitative reverse transcription PCR for transcripts coding for 26 OBPs and 17 CSPs. OBP transcripts showed an obvious antenna bias, whereas CSP transcripts were detected in different tissues.We generated transcriptome of female antennae of Antennal transcriptome data derived from the oriental fruit moth constituted an abundant molecular resource for the identification of genes potentially involved in the olfaction process of the species. This study provides a foundation for future research on the molecules involved in olfactory recognition of this insect pest, and in particular, the feasibility of using semiochemicals to control this pest. The sensitive olfactory system plays a predominant role in insect behavior, such as seeking host plants, finding mates, selecting oviposition sites, recognizing kins, and escaping from predators and toxic compounds . AntennaOBPs belong to a group of small water-soluble proteins that are secreted by the accessory cells around the ORNs and impregnated in the sensilla lymph . OBPs arDe novo assembly of transcripts provides a workable solution to transcriptome analysis. At present, a lot of de novo transcriptome assemblers available are designed for Roche 454, Illuminia Solexa, and SOLID. SOAPdenovo, SOAPdenovo-Trans, Velvet Oases, ABySS, trans-ABySS, and trinity have been successfully applied to de novo transcriptome assembly form short-read RNA-Seq data of model and non-model organisms [The development of Next Generation Sequencing (NGS) technologies has greatly improved the efficiency and speed of gene discovery in recent years . De novorganisms ,14–18.Grapholita molesta Busck is an economically important oligophagous pest species of stone and pome fruits throughout the world, causing substantial losses in fruit yields [Prunus persica L) is considered the primary host, and pear (Pyrus communis L.) and apple  are secondary hosts [G.molesta, that can annually survive and reproduce on different hosts, are confronted with hight-variability in the volatile blends emitted by different host plant species at specific periods, as well as by the same host plant species across a growing season [G.molesta as the natural blend from peach shoots [G. molesta males [G.molesta females as good as natural blends [G.molesta females attraction to host plants [G. molesta females are attracted to butyl hexanoate at intermediate dosages [The oriental fruit moth t yields . Peach . After pupation, male and female pupae were placed in separate glass tubes and maintained in the conditions described previously. The adults were fed with a cotton swab dipped in 5% honey solution and changed daily. Antennae of 3–4 day-old female moths were dissected after eclosion, immediately frozen in liquid nitrogen, and stored at –80°C until RNA was extracted.Frozen antennae were immediately transferred into a 1.5 mL Eppendorf tube immersed in liquid nitrogen and ground with a pestle. Total RNA was extracted using RNAiso Plus Total RNA extraction reagent  following the manufacturer’s instructions. The residual genomic DNA in total RNA was removed by DNase I . Total RNA was dissolved in RNase-free water and RNA integrity was measured using Agilent 2100 bioanalyzer . The high quality RNA (RIN number: 9.3) was used for cDNA library construction and Illumina sequencing.Poly (A) mRNA was isolated from 12 μg of total RNA extracted from approximately 1200 antennae of 3–4 days-old adult female moths using the PolyA+Tract mRNA Isolation System , and further purified using the RNeasy MinElute Clean up Kit  following the manufacturer’s protocol. Fragmentation buffer was added to cleave mRNA into short fragments, and then, these fragments were used to synthesize first-strand cDNA using random hexamer primers, which was further transformed into double stranded cDNA with RHase H and DNA polymerase I. A paired-end library was constructed from the cDNA synthesized using the Genomic Sample Prep Kit (Illumina). Fragments larger than 375 bp were purified with QIAquick PCR Extraction Kit (Qiagen), end-repaired, and linked with sequencing adapters. AMPureXP beads were used to remove the unsuitable fragments, and then, the sequencing library was constructed with PCR amplification. After being validated using Pico green staining  and fluorospectrophotometry, and quantified using Agilent 2100 ), the library was sequenced using Illumina Miseq platform . For subsequent analysis, 1/2 run data was generated.de novo assembly. The reads were screened from the 39 to 59 to trim the bases with a quality score of Q<20 using 5 bp windows, and the reads with final length less than 50 bp were removed. In order to accurately discover and reduce false positive olfactory gene detection, we evaluate the performance of de novo transcriptome assembly using SOAPdenovo-Trans, Trans-ABySS, and Trinity form short-read RNA-Seq data of G.molesta antennae. The de novo transcriptome assembly was further analyzed using DETONATE  [http://hibberdlab.com/transrate/index.html). All derived transcript sequences were used to search NCBI non-redundant (NR) database (ftp://ftp.ncbi.nlm.nih.gov/blast/db/) with the BLASTn program , and the top-hit transcripts were selected as unigenes. For the unigenes that failed to be aligned with any sequence in the databases, the software GetORF was used to predict their open reading frames (ORFs) and ascertain their coding orientations with default settings.Raw reads were filtered using a stringent process and subsequent luation)  and Tranhttp://www.ncbi.nlm.nih.gov/gorf/gorf.html) based on the results given by BLASTX. ClustalX version 1.83 and MAFFT version 6.0  were used to conduct multiple sequence alignments. Signal peptides of the protein sequences were identified using SignalP 4.0  with default parameters. The transmembrane-domains (TMDs) of annotated genes were predicted using TMHMM version 2.0 (http://www.cbs.dtu.dk/services/TMHMM), while the ORFs were translated to amino acid sequences using ExPASy (http://www.expasy.org/translate/).The annotation of all derived sequences were executed using the BLASTX program against the NCBI non-redundant database (NR) and SwissProt protein sequences with e-value<1e-5. The BLASTX results were then imported into Blast2GO suite for GO Annotation. Open reading frames (ORFs) of the unigenes were predicted using ORF Finder  were also included in the data set, and the final data set contained 175 sequences. Amino acid sequences of proteins used in building the phylogenetic tree are listed in To verify the annotation of the candidate olfaction genes and to search for orthologs, phylogenetic analyses were conducted among se genes ,18,34–37lexippus , 16 sequirescens , 13 sequarmigera , and 13 miferana , 17 sequarmigera , nine seirescens , 14 sequttoralis , and 17 ttoralis  and 69 f[B. mori ). The OR B. mori , M. sextttoralis . Since Inogaster , A. mellellifera  and T. cTo confirm and compare the tissue expression of putative GmolOBPs and GmolCSPs identified from the transcriptome, semi-quantitative reverse transcription PCR was performed using cDNAs template prepared from male antennae, female antennae, and remaining bodies (without antennae) of the moth. Each experiment contained two biological repeats, three technical duplications, and controls were PCR with no template. Total RNA was extracted as described previously, treated with DNAse , and corresponding cDNAs were synthesized using the First Strand cDNA Synthesis Kit  following the recommended protocol. Primers were designed using the Primer Premier 5 software and sequences are available in A total of 5.6 million raw reads (average read length 251 bp) were obtained from the libraries of female antennae. After removing low quality, adaptor, and contaminating sequence reads and reads shorter than 50 bp, about 5.2 million clean-reads comprised the 2.2 gigabases were generated. In total, 114263, 79209 and 71086 transcripts, with the mean length of 630, 510, and 619 bp, were obtained from assembled with Trinity, SOAPdenovo-trans and Trans-Abyss . The rawde novo assemblers, we obtained the same number transcripts annotated to putative olfactory genes by the search against the non-redundant protein database. Most of the cover percent transcripts annotated to olfactory genes were greater than 70% among the three assemblers , Bombyx mori (6.5%), Tribolium castaneum (4.4%), Papilio xuthus (4.1%), and Acyrthosiphon pisum (1.5%)  . Fig 3 iG.molesta in NCBI, 23 sequences as new genes, including GmolPBP1, GmolOBP1, GmolOBP2, and GmolOBP4 to GmolOBP23. The information on the OBPs is listed in First, we used motif scanning to detect the conserved six cysteine residues pattern  of the candidate odorant-binding proteins . We thenG. molesta antennal transcriptome. Sequence analysis identified 15 unigenes with full-length ORFs and 16 unigenes with predicted signal peptide. One unigene without signal peptide since truncate at the 5'-end. The four conserved cysteine (with a pattern of C1-X6-8-C2-X18-19-C3-X2-C4) were found in all the 17 candidate GmolCSPs [Seventeen different sequences encoding putative chemosensory proteins were identified within the GmolCSPs  Fig 6)..G. molesG.molesta showed 95% identity to C. pomonella co-receptor, CpomOR2, one of the most conserved co-receptors in insect species. While similar with other insect ORs, most GmolORs were highly divergent and shared low similarity with other insect ORs, except for closely related species such as Cydia pomonella. Four candidate GmolORs sequences  tended to be pheromone receptors (PRs) as they are highly conserved with CpomOR fragments and BmorPRs amino acid sequences from other species [2 receptors. The information of ORs and GRs are given in Bioinformatic analysis identified 48 different sequences encoding putative ORs and four sequences encoding putative GRs. GmolORs and GmolGRs were named according to their similarities with previously annotated Lepidoptera ORs and the topology predictions from TMpred as observed from other insect ORs . Twelve  species . These f species ,47,48. AG. molesta antennal transcriptome. The alignment revealed that all the 24 sequences represent unique genes since they possessed overlapping regions without identity. Ten of the IRs appeared to contain a full length ORFs , and were longer than 1700 bp in general. In addition, it was predicted that three transmembrane domains existed in all 10 sequences by TMHMM 2.0, the typical characteristic of IRs. The remaining 14 sequences were truncate at either 5' or 3' terminus. Neighbor-joining tree analysis revealed that all putative IRs were found to have orthologoues from B. mori, M. sexta, C. pomonella, S. littoralis, D. melanogaster, A. mellifera, and T. castaneum. According to their positions in the phylogenetic tree and the strong bootstrap support, 15 of 24 putative G. molesta IRs were given names that are consistent with the number and suffix of the Dmel/Bmor/Slit/Cpom/Amel/Tcas IR orthologues in the same clade. Two of the remaining nine IR sequences, comp37980_c0_seq2 and comp40732_c0_seq6, clustered with their ionotropic receptor orthologues into N-methyl-D-aspartic acid receptor (NMDA receptors) clade, and these were named “GmolNMDAR1A” and “GmolNMDAR1B”. The other two sequences, comp5756_c0_seq1 and comp5467_c0_seq1, clustered with their ionotropic receptor orthologues of α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMAP) and Kainate receptors clade, and these were named “GmolGluIR1” and “GmolGluIR2”, respectively. The remaining five unigenes, comp52557_c0_seq1, comp36336_c0_seq3, comp41705_c0_seq15, comp43295_c0_seq6, and comp43552_c0_seq13, did not show meanful similarity with known IR encoding genes but with conserved structural features, and thus were named as “GmolIR2”, “GmolIR4”, “GmolIR5”, “GmolIR6” and “GmolIR7”, respectively , while GmolSNMP2 has 69% identity with SNMP2 of Ostrinia nubilalis (GenBank accession number: E5EZW9.1). GmolSNMP1 had a full ORF with two transmembrane domains at N-terminus and C-terminus, respectively, while GmolSNMP2 was incomplete due to truncation at the 3' terminus. In addition, we also identified an odor degrading enzyme gene (ODE). ODE is responsible in the signal inactivation step and it rapidly degrades the stimulatory odorant molecules. The information including the unigene reference, length, and best BLASTX hit of SNMPs and ODE was listed in Both SNMP1 and SNMP2 were obtained from The sex and tissue-specific expression of GmolPBP2 and GmolPBP3 had been studied previously . In thisG. molesta. The olfactory system genes identified in this work were comparable to recent reports from H. armigera , C. suppressalis , M. sexta , and C. pomonella.  [G. molesta olfactory genes available in NCBI were identified in the present antennal transcriptome.We used antennal transcriptomic sequencing to identify patutive olfactory genes and identified genes encoding 28 OBPs, 17 CSPs, 48 ORs, four GRs, 24 IRs, two SNMPs and one ODE in female antennae of nd 1 GR) ,16,17. OA. pernyi and A. polyphemus, the binding constants value of PBP1 for principal pheromone component E6,Z11-hexadecadienyl acetate was 1.83 and 0.63 μmol/L, respectively [The alignment of the predicted GmolOBPs showed low sequence identity among OBP family members . The preectively . These rCydia pomonella), which are closely related to G.molesta, three different transcripts were found to encode putative GOBPs. The GOBPs subfamily can be divided into GOBP1, GOBP2 and GOBP3 [G.molesta will need to be addressed in future studies.GOBPs are a subfamily of OBPs, consisting of two members, GOBP1 and GOBP2 in most Lepidopterans. But in tortricid moths and the codling moth  -8-dodecenyl acetate, (E)-8-dodecenyl acetate, (Z)-8-dodecenyl alcohol and dodecanol [Xenopus oocytes and electrophysiology. A distinct receptor of silkworm moth Bombyx mori, BmOR3, is expressed in the second ORN, and binds pheromone compound bombykal can inhibit male behavioural response [Heliothis virescens (HvORs), HvOR6 was found to be highly tuned to pheromone (Z)-9-tetradecenal, while HvOR13, HvOR14 and HvOR16 showed specificity for (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate and (Z)-11-hexadecen-1-ol, respectively [Apis mellifera ORs, AmOR11 responded specifically to the main “queen substance” component 9-oxo-2-decenoic that maintains the queen’s dominance in the colony and also acts as a long-distance sex pheromone [Drosophila OR, Or43a, has been found that only four odors molecules  can activate the receptor at a low micromolar concentration, as demonstrated using two-electrode voltage-clamp recording [G. molesta more sensitive to sex pheromone than females, Z8-12:Ac elicited the strongest antennal response in male [G. molesta is lacking.In  females . Conside females , by whicM. sexta , C. pomoomonella  and H. aarmigera . Phylogers (PRs) , and we odecanol . Howeverresponse . Candidaectively . A honeyheromone . The Droecording . ElectroB. mori fructose receptor (BmorGR9) and inositol receptor (BmorGR8) [myo-inositol and three sugars glucose, fructose, and sucrose, did stimulate the laying of eggs of codling female moths. Fructose, sorbitol and myo-inositol are important components of the stimulatory blend. The mated female of G. molesta prefer egg-laying to surfaces of ripe apple or peach fruit or secrete high carbohydrate on immature fruit. The function of these gustatory receptors seemed related to the recognition of these carbohydrates. In addition, one putative GR receptor (GmolGR1) was identified as a putative CO2 receptor, and the protein shares high sequence identity (79%) with the S. Iittoralis CO2 receptor, SlitGR3 [2 have been described only on labial palps [The gustatory receptors we identified here, including GmolGR2, GmolGR3 and GmolGR4, were found in a clade with putative sugar receptors . This clBmorGR8) ,47. In aBmorGR8) ,16. SugaBmorGR8) . An arti SlitGR3 . Until tal palps .D. melanogaster by bioinformatics screen genomic data for insect-specific genes with enriched expression in OSNs [D. melanogaster, 66 IRs were identified, 15 of which proved to be antenna-specific [S. littoralis [C. pomonella [C. suppressalis [H. armigera [G. molesta antennae, including two co-receptors, IR8a and IR25a. Compared to ORs, the IR family is relatively conserved in sequence. Among the 24 GmolIRs we discovered, GmolIR8a, GmolIR25a, GmolIR21a, GmolIR41a, GmolIR68a, GmolIR87a, GmolIR93a, GmolIR75d, GmolIR75p1, GmolIR75q.1, GmolIR75q.2, GmolIR76b, GmolIR7d, GmolIR1, and GmolIR3 were clustered in separate clades in neighbor-joining tree with Amel/Bmor/Cpom/Dmel/Msex/Slit/Tcas IRs, respectively. Considering the relatively high sequence conservation and similarities in expression, the functions of GmolIRs are probably conserved as IRs in other Lepidopterans.Ionotropic receptors represent a novel member of the chemosensory receptor family, which were first discovered in  in OSNs . These w in OSNs . In D. mspecific . Twelve omonella , 20 IRs ressalis , and 12 armigera . In our G. molesta. The number of OBPs, CSPs, ORs, IRs, GRs, and SNMPs genes that were identified in this species are close to the complete repertoire of olfactory genes from the antennae identified from other Lepidopteran species. The results demonstrated that Illumina Miseq sequencing was successful in the recovery of low-expressing putative olfactory genes, especially in a non-model pest species without an available genome sequence. Our findings made it possible for future research on the molecular level of olfactory system of G. molesta, and in particular, the discovery of receptor genes will also contribute to the identification of novel volatile host compounds, which would gain new options for controlling insects by mass trapping or disruption.The main purpose of this study was to identify the genes involved in the reception, processing, and degradation of volatiles by analyzing the antennal transcriptome sequence from S1 File(TXT)Click here for additional data file.S2 File(TXT)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSB)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


Sphingobacterium, Bacteroides, Flavobacterium and Pedobacter spp. Assembly of the RWS and TWS metagenomes generated 16,536 and 15,902 contigs of ≥10 Kb, respectively. Thirteen contigs, containing 39 glycosyl hydrolase genes, constitute novel (hemi)cellulose utilization loci with affiliation to sequences primarily found in the Bacteroidetes. Overall, this study provides deep insight in the plant polysaccharide degrading capabilities of microbial consortia bred from forest soil, highlighting their biotechnological potential.Based on the premise that plant biomass can be efficiently degraded by mixed microbial cultures and/or enzymes, we here applied a targeted metagenomics-based approach to explore the metabolic potential of two forest soil-derived lignocellulolytic microbial consortia, denoted RWS and TWS (bred on wheat straw). Using the metagenomes of three selected batches of two experimental systems, about 1.2 Gb of sequence was generated. Comparative analyses revealed an overrepresentation of predicted carbohydrate transporters , two-component sensing systems and β-glucosidases/galactosidases in the two consortia as compared to the forest soil inoculum. Additionally, “profiling” of carbohydrate-active enzymes showed significant enrichments of several genes encoding glycosyl hydrolases of families GH2, GH43, GH92 and GH95. Sequence analyses revealed these to be most strongly affiliated to genes present on the genomes of  The recent thrust to use plant waste such as straw for the production of compounds such as biofuel, plastics and intermediates for pharmaceuticals has raised major questions about the efficiency of lignocellulose deconstruction driven by microorganisms18Unfortunately, we hitherto understand very little about the intricacies of lignocellulose biodegradation processes in microbial consortia. A key assumption about the mechanisms in such consortia is that the Darwinian principle of survival of the fittest reigns. Thus, sets of “locally fit” microorganisms in relative abundances that are appropriate for efficient biodegradation are predicted to be obtained at each time point in the process. A “matured” consortium then presumably contains all of the microbial members that have been, or are, important for the biodegradative process. Based on such assumptions, analyses of such matured consortia via metagenomics101213et al.et al.Current analyses of metagenomics-based datasets rely strongly on alignment of sequences against those present in databases. In the “Carbohydrate-Active Enzyme database” (CAZy), enzymes that hydrolyze diverse carbohydrates are classified into different GH families on the basis of their amino acid sequences17i) untreated and ii) heat-treated wheat straw. Both consortia grew efficiently in each step and showed (hemi)cellulolytic enzymatic activities in the extracellular fractions24In previous work, we obtained two microbial consortia from forest soil by sequential transfers on To foster our understanding of the genetic make-up of the “phylogenetically-stable” lignocellulose-degrading consortia, two biodegradative consortia, denoted RWS (bred on untreated wheat straw) and TWS (bred on heat-treated wheat straw), were selected, each at three time points. In addition, the source community (forest soil-derived) was selected as a comparator. Based on preliminary results from Fourier transformed infrared spectroscopical (FTIR) analyses, we obtained evidence for slightly higher percentages of lignin, hemicellulose and cellulose in the RWS (untreated wheat straw) compared with TWS (heat treated) substrate (data not shown).8 bacterial cells/ml). A calculation of the extraction efficiency revealed that >90% of the bacterial cells in these had been captured into the extractsMetagenomic DNA from all microbial consortia was successfully obtained at a rate of, on average, about 0.75 μg/ml culture (containing roughly 102 = 0.480) between the RWS and the TWS consortia (Sphingobacterium sequences (35% versus 3% of Klebsiella) was found in the 16S rRNA-based classification, while Klebsiella-like sequences were most abundant in the protein-encoding gene approach (~15% versus 10% of Sphingobacterium) (MG-RAST-basedonsortia . They wecterium) .2 = 0.890). Thus, both consortia showed largely similar functional profiles at three time points, which were clearly different from those of the FS1 community . Based on principal components analyses (PCA) of the KEGG-based functional profiles, the TWS metagenome samples 1T, 3T and 10T all clustered together, suggesting stabilization and preservation of function as from transfer-1. Quite remarkably, these metagenomes still revealed considerable levels of similarity with those from RWS , at KEGG orthology level 2. These were membrane transport systems (mainly ABC transporters) next to systems for the metabolism of cofactors and vitamins, cell motility and glycan biosynthesis . ABC traComparative SEED-based analyses of the RWS and TWS consortium metagenomes versus that of the FS1 community showed that the most enriched specific genes (p < 0.005) in the 3W, 10W and 3T metagenomes were those for utilization of monosaccharides (2.06 ± 0.16 -fold RA increase) as well as iron acquisition (4.53 ± 0.26 -fold RA increase). In the 10T one, genes for proteins involved in iron acquisition were also strongly enriched (5-fold RA increase) . In the 2) between the RWS and TWS consortium metagenomes was 0.970. Specific enzyme families that abounded in the TWS and RWS metagenomes were glycosyl transferase (GT) families 2 (~8.5% RA) and 4 (~6.5% RA), followed by GH2, GH13 and GH3  in both derived consortia, albeit at rather low (≤0.3) RA levels. Specifically, these were genes for the GH117, GH50, GT8 and PL17 family proteins. The most abundant (approximately >1.5% RA) enriched (p <0.005) families in the two consortia were GH1, GH2, GH3, GH20, GH29, GH31, GH43, GH92, GH95 and CBM50. Remarkably, enrichment of these was already apparent as from transfer-1 . Moreover, sequences that belonged to the GH1, GH3, GH31 and CBM50 families were mostly affiliated with organisms from within the Enterobacteriaceae, especially within Klebsiella. The taxonomic affiliation of the GH retrieved from transfer-1 and -3, in both consortia, showed similar results, indicating that the enrichments selected for GH assigned to members of the Bacteroidetes and Enterobacteriaceae.The reads of overrepresented CAZy families at transfer-10 (stable community)  were cluSphingobacterium spp. However, the most abundant GH1, GH3 and GH31-encoding genes in 10W were affiliated with predicted proteins of Kluyvera ascorbata  of all reads in each overrepresented family. This approach yielded no contigs for the FS1 metagenome, indicating great heterogeneity. From the 10W metagenome, 17, 12, 9, 8 and 5 contigs were produced for the GH2, GH92, GH43, GH31 and GH29 families, respectively. Between 28.03 to 59.38% of the total retrieved reads, in each of these last families, were used. In contrast, in the 10T metagenome, 88, 86, 82, 81 and 79% of the total retrieved reads in GH29, GH92, GH31, GH2 and GH43 were used to construct 12, 15, 7, 13 and 15 contigs, respectively . Contigsscorbata .Sphingobacterium, Bacteroides, Parabacteroides, Flavobacterium and Pedobacter. However, the 13 contigs showed an ANIb < 72% and a correlation of TTNF less than 0.79, suggesting that these genomic fragments make part of as yet unknown (yet related) genomes , Pedobacter (contig 248) and Parabacteroides (contig 316), on the basis of BLAST analyses of the GH genes  and 15,902 (contig mean length 3.4 Kb) contigs were generated for the metagenomes obtained for the RWS and TWS consortia, respectively. Of these, 10,571 and 9,987 contigs, respectively for RWS and TWS, were >1,000 bp. Moreover, in RWS, 94 GH genes  were detected in 70 contigs (≥5 Kb). In TWS, 75 GH were identified in 58 contigs (≥5 Kb) . Thirtee genomes . Seven cGH genes . All conGH genes . In addiGH genes .In this study, we analyzed 576 (RWS) and 658 (TWS) Mb of sequence information from two lignocellulose-bred microbial consortia, in comparison to a source community from forest soil (FS1). This allowed a robust comparative metagenomic analysis to be performed. To avoid biases, we used two different methodologies. First, unassembled sequences were used for taxonomic and functional assignments , on the premise that such analyses would not “distort” the abundance and representation of the sequences of abundant species in the datasetComparative analyses of the consortium metagenome datasets versus those from the source community first revealed that organisms with genomes with relatively low G + C content had been strongly selected in the consortia. This indicated a selection of fast-growing decomposer organisms with, on average, lower G + C% genomes, e.g. Proteobacteria and Bacteroidetes, and concurrent deselection of the high G + C positive members of the soil microbiota, e.g. Actinobacteria. The fact that a majority (around 70%) of the predicted protein-encoding genes, or parts thereof, in both consortia had homologs (>50% homology) on published bacterial genomes supported the contention of selection of bacterially-dominated degrading consortia. The differences between the percentages of mappable reads between the bred microbial consortia (~70%) and the soil inoculum (~38%) could be explained by the fact that soil contains high microbial diversity and DNA novelty compared with most enrichment cultures. The percentage of annotated reads in FS1 was similar to that found in previous soil metagenomics-based studies3033Clostridium, Thermobifida and Streptomyces species in response to different lignocellulosic substrates3738et al.Enterobacter lignolyticus - the transport of xylose and lignin-derived aromatic compounds into the cells. Moreover, ABCT were also abundantly present in a metagenome of the gut of a wood-feeding beetle (Anoplophora glabripennis)With respect to the functional profiles, ABCT and phosphotransferase systems function in the cellular uptake of sugars . In addition, some ABCT can import key ions such as Sn, Fe and Co35Caulobacter crescentus, expression is induced by xyloseet al.et al.Gramella forsetii, revealed that TBR also could work as anticipatory polysaccharide sensorsClostridium cellulolyticum, TCSP perceive the presence of extracellular soluble sugars and regulate most GH and associated ABCTBacteroides, these TCSP act similarly and regulate (hemi)cellulose utilization loci (HULs)4748Regarding TonB-dependent receptors (TBR) , in The overrepresentation of genes for ABCT, TBR and proteins involved in lignocellulose degradation (e.g. β-xylosidases) in RWS and TWS is consistent with the hypothesis that enhanced capacities are required in these consortia in order to facilitate the utilization of complex plant materials (e.g. hemicellulose) and transport the products (e.g. xylose) of various structures into the cells. Bacterial consortia were thus selected that metabolize the partially degraded plant material. In addition, the genomic machineries in these consortia  apparently sense the activation of GH and ABCT in response to a wide range of environments, stressors and growth conditions, such the use of wheat straw as carbon source.The functional assignments, in conjunction with the taxonomic profiles based on genes encoding proteins, as well as previous 16S rRNA gene and ITS pyrosequencing analysis50The CAZy profile data were consistent with those previously reported based on the function predictor PICRUSt53Sphingobacterium, Bacteroides, Flavobacterium and Pedobacter) types at transfer-10 indicated these taxa as prime agents in the deconstruction of the hemicellulose part on the wheat straw. This stood in contrast to the presumed role of Enterobacteriaceae (i.e. Klebsiella and/or Kluyvera types) which may act mainly on the cellulose structures, and possibly also the ligninOn the other hand, based on “richness” values and the contigs constructed from the reads that came from enriched GH families in transfer-10, we hypothesize that strong selection, yielding “less niches or less complex and specific carbon sources”, occurred for the 10T and 10W systems, compared with FS1. In addition, a reduction of the functional “richness” values was more evident in 10T than in 10W, confirming that pretreatment reduces the complexity of the substrate and can introduce a stronger, or narrower, selection, which is possibly also based on the presence of furanic compoundsIn lignocellulolytic microbial consortia of reduced complexity, genome pieces and/or gene clusters might be assembled at the coverage levels used by us, allowing the description/detection of partial metabolic pathways related with plant waste deconstruction. Our metagenome assembly yielded thirteen putative novel Bacteroidetes HULs. This apparent “bias” towards Bacteroidetes is consistent with the reported relative dominance of this taxon in both our selected microbial consortiaBacteroides sp.: i) Sus-like regulators (or TBR), ii) TCSP, iii) extra cytoplasmic function (ECF) sigma/anti-sigma factors and iv) AraC family regulators46B. thetaiotaomicron, SusC is a member of the TBR family specialized in the transport of oligosaccharides from the outer membrane into the periplasmic spaceet al.Thermobifida fusca when grown on different lignocellulosic biomass. Interestingly, the heterologous expression of L-ribulose-5-phosphate 4-epimerase, L-arabinose isomerase, aldose-1-epimerase (found in contig 939) could improve sugar utilization and production of ethanol in Zymomonas and Saccharomyces species57Interestingly, the GH detected in the 13 retrieved HULs were mostly flanked by TBR, TCSP, ABCT and genes involved in sugar metabolism . Recently, four types of regulatory systems were proposed to be involved in transcriptional regulation of genes for polysaccharide degradation in Thus, our data demonstrate the presence of partial pathways related with plant biomass deconstruction and sugar  transport and metabolism. As a prospect, abundant and enriched enzymes, such as mannosidases GH92), arabinofuranosidases, xylanases (GH43), fucosidases (GH95 and GH29) and galactosidases (GH2) can be extracted from the HULs, custom-synthesized, codon-optimized and expressed in a suitable host. This will generate novel efficient enzyme cocktails that can enhance the activity of currently-used cellulolytic enzymes82, arabini) “raw” wheat straw (RWS) and ii) heat-treated  wheat straw (TWS). Subsequently, flasks were incubated at 25 °C with shaking (100 rpm). Once the systems reached 7 to 8 log cells/ml (between 6 and 8 days), aliquots (25 μl) were transferred to 25 ml of fresh MSM containing the same substrates. Samples from the soil suspension (FS1) and from the triplicate RWS and TWS flasks were taken after 1, 3 and 10 transfers (n = 21). The selection of the samples was based on previous bacterial PCR-DGGE resultsBreeding of the microbial consortia has been reported before-treated 0 °C, 1 h-treated 0 °C, 1 hA total of 5,298,311 unassembled sequences (reads) were uploaded to the MG-RAST v3.1.2 server5910 transformed.Genes, or parts thereof, involved in lignocellulose degradation were detected using as a starting point the reads  obtained by MG-RAST. Annotation was performed via BLASTX against the CAZy database v3 (downloaded from dbCAN site)61Unassembled sequences belonging to CAZy families CBM50, GH1, GH2, GH3, GH20, GH29, GH31, GH43, GH92 and GH95 were extracted from FS1, 10W and 10T (stable consortia), re-annotated (in-house) using BLASTX searches against the nr-NCBI database and clustered at 97% of nucleotide identity using the CD-HIT softwareData from MG-RAST and CAZy annotation were statistically analyzed using the STAMP packageThe metagenome assembly was performed using the CLC Genomics Workbench v4.0.3 with the following parameters: mismatch cost 2, insertion cost 3, deletion cost 3, length fraction 0.5 and similarity 0.96570How to cite this article: Jiménez, D. J. et al. Unveiling the metabolic potential of two soil-derived microbial consortia selected on wheat straw. Sci. Rep.5, 13845; doi: 10.1038/srep13845 (2015).


It is now well documented that natural products have played an important role in anticancer therapy. Many studies focus on the ability of these natural compounds to modulate tumor-related signaling pathways and the relationship of these properties to an anticancer effect. According to the World Health Organization (WHO), colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer death among men and women. Therefore, finding strategies to fight against CRC is an emergent health problem. CRC has a strong association with deregulation of Wnt/β-catenin signaling pathway. As some types of natural compounds are capable of modulating the Wnt/β-catenin signaling, one important question is whether they could counteract CRC. In this review, we discuss the role of flavonoids, a class of natural compounds, on Wnt/β-catenin regulation and its possible potential for therapeutic usage on colorectal cancer. The theory that natural molecules might inhibit cancer development started more than 100 years ago, when laboratories began to investigate the effect of small molecules extracted from plants and fungi on tumor development and growth ,3. CurreUncontrolled cell growth has been a feature of many types of tumors, in particular colorectal cancer which is, according to the WHO, the third most common cancer with 1,361,000 cases in 2012 . DespiteA great effort has been made to understand the molecular mechanism by which these natural compounds are acting to inhibit tumor growth ,6,10,15.Wnts are an evolutionary conserved family of secreted glycoproteins found as 19 distinct Wnt ligands in mammalians . The WntDuring canonical Wnt signaling, binding of Wnt ligands to Frizzled/low-density lipoprotein-related protein 5/6 (LRP5/6) receptor complexes causes stabilization of β-catenin. Activation of the Wnt pathway is controlled by regulation in disassembly of the destruction complex, which stabilizes β-catenin. The destruction complex, composed of APC (Adenomatous polyposis coli), Axin1, GSK-3β (Glycogen synthase kinase 3-β), and CK1 (casein kinase 1), phosphorylates serine residues in β-catenin leading to its ubiquitination by β-Trcp (F-box/WD repeat-containing protein 1A) and degradation by the proteasome. Stabilized β-catenin is then able to translocate to the nucleus and, through interactions with the T-cell factor (Tcf)/lymphoid enhancer factor 1 (LEF-1), modulates the expression of specific target genes that control developmental and cell cycle genes and oncogenes  20,21].,21.20,21Conversely, mutations in components of the Wnt/β-catenin are often associated with cancer, in particular in CRC ,25. The CRC is associated with polyps, not detected in early stages, leading to tumor development . Polyps in situ or intramucosal carcinoma [In the stage 0, cancer is in the earliest stage and it has not grown beyond the inner layer (mucosa) of the colon or rectum. This stage is also known as carcinoma arcinoma ,35. In sarcinoma ,35. WhenWnt, RAS2MAPK, PI3K, TGF-β, p53 and DNA mismatch-repair pathways [APC gene. Without APC, β-catenin accumulates to high levels, translocates into the nucleus, associates with TCF/LEF leading to its binding to DNA and consequent transcription of genes that are important for stem cell renewal and differentiation. However, when inappropriately expressed, APC can lead to cancer [A recent analysis published in 2012 by The Cancer Genome Atlas Network, has uncovered several critical genes and important pathways in the initiation and progression of CRC . These ipathways  They deso cancer . Anothero cancer . In addio cancer ,37. The in vitro and in vivo on APC mutant mice [Since Wnt signaling is critical for some carcinogenic development, many studies have been searching molecules capable of modulating this signaling pathway . Severalant mice . IWR-1 iant mice . G007-LKant mice . Interesant mice ,44.et al. showed ICG-001 as a selective inhibitor of Wnt/β-catenin signaling through specific binding of CREB-binding protein (CBP), disrupting its interaction with β-catenin leading to Wnt signaling inhibition [Emami KH hibition . While whibition . NSC6680hibition ,47,48,49Nuclear inhibitors of Wnt signaling are also very pursued, since upstream inhibition of Wnt signaling may have collateral effects on others signaling pathways. iCRT3, iCRT5, iCRT14, PKF118-310, PKF115-584 and CGP049090, BC21 are capable of inhibiting Wnt signaling by disrupting the interaction of β-catenin with its transcription factor TCF4 ,51. iCRTFlavonoids, polyphenolic compounds, constitute a very large group of natural products and one of the most characteristic classes of compounds in plants metabolism . More thin vitro [in vivo using Xenopus embryos as model system [Quercetin is one of the most studied and has been submitted to clinical trials. Quercetin has been pointed as a potential anti-cancer drug in colorectal cancer ,72 and iin vitro ,74. Morein vitro . This kiin vitro . Our grol system . Many adl system ,76,77,78wif-1 gene (Wnt inhibitory factor 1) expression through its demethylation. WIF-1 is a Wnt antagonist that directly binds to Wnt molecules, blocking signaling [−/+ mouse, a model for intestinal tumorigenesis, by reducing nuclear β-catenin levels, suggesting an inhibition of the Wnt mediator β-catenin translocation to the nucleus [SFRP1 gene in hepatoblastoma [Another vastly studied anticancer flavonoid is EGCG, which inhibited Wnt signaling in colorectal cancer cells ,79. The ignaling ,57,58,59 nucleus ,81. Receblastoma .O-β-d-glucopyranoside), a glycosylated derivative of quercetin, inhibits growth of different colorectal cancer cell lines (unpublished data). Genistein also suppressed β-catenin/TCF transcriptional activity in SW480 cells in a dose-dependent manner. This flavonoid affects upstream components of the β-catenin/TCF pathway by suppression of AKT phosphorylation, thus inhibiting GSK-3β dephosphorylation. Then, phosphorylated GSK-3β is able to phosphorylate β-catenin facilitating its ubiquitylation and degradation [APC gene. However, in HCT116 cells, which harbor wild-type APC but mutant β-catenin, silibinin has no effect, suggesting its selective effects on Wnt/β-catenin pathway [Scutellaria radix, a traditional Chinese medicine, has been shown to decrease expression of Wnt target genes Wnt3A, LRP6, Cyclin D1, c-Myc and to increase expression of Axin1 [et al. showed flavonone as an inhibitor of Wnt signaling by modulating TNKS activity [Ongoing studies of our group show that isoquercitrin (quercetin 3-radation . In 2010radation . They suradation . Interes pathway . Wogoninof Axin1 . Wogoninactivity . The autactivity , illustrDespite of the significant advances in diagnostic of CRC, current treatments consist mostly of surgery and conventional chemotherapy. These treatments confer limited benefit, which makes CRC the third leading cause of cancer death among men and women. CRC development and progression involve silencing of genes and alteration of regulatory mechanisms in one or more members of the Wnt/β-catenin signaling pathway. Thus, the identification of substances capable of modulating the Wnt/β-catenin signaling has been a major effort for many research groups. Notably several new compounds have recently been described as inhibitors of different components of the Wnt/β-catenin signaling pathway. Particular attention has been given to flavonoids since case-control, cell culture and animal studies have revealed a plethora of bona fide compounds with protective role against CRC malignancy. However a long way has still to be paved to achieve treatment success of CRC. One problem to overcome is the bioavailability of flavonoids during cellular metabolism. In this regard, chemical structure modification and the use of nanoparticles may be a good strategy to develop more effective flavonoids to target Wnt/β-catenin and fight CRC growth and progression.In summary, the recent advances in the understanding of the mechanisms of action of flavonoids targeting Wnt/β-catenin may shed light on future CRC therapies.


To investigate the transdifferentiation relationship between eight types of livercell during rat liver regeneration (LR). 114 healthy Sprague-Dawley (SD) rats were used in this experimental study. Eight types of liver cell were isolated and purified with percoll densitygradient centrifugation and immunomagentic bead methods. Marker genes for eight typesof cell were obtained by retrieving the relevant references and databases. Expressionchanges of markers for each cell of the eight cell types were measured using microarray.The relationships between the expression profiles of marker genes and transdifferentiation among liver cells were analyzed using bioinformatics. Liver cell transdifferentiationwas predicted by comparing expression profiles of marker genes in different liver cells.PROM1, KRT14 and LY6E), but also express biliary epithelial cell (BEC) markers (including KRT7 and KRT19); BECs express both HOC markers  and HC markers such as CPS1, TAT, KRT8 and KRT18; both HCmarkers  and BEC markers (KRT7 and KRT19) were detected inHOCs. Additionally, some HC markers were also significantly upregulated in hepatic stellate cells ( HSCs), sinusoidal endothelial cells (SECs) , Kupffer cells (KCs) and dendriticcells (DCs), mainly at 6-72 hours post partial hepatectomy (PH). During LR hepatocytes (HCs) not only express hepatic oval cells (HOC) markers(including Our findings indicate that there is a mutual transdifferentiation relationshipbetween HC, BEC and HOC during LR, and a tendency for HSCs, SECs, KCs and DCsto transdifferentiate into HCs. ALB, AFP, G6P, HNF4a, KRT18)and BEC markers gradually increase  were gradually lost. In turn,some bile duct-specific proteins (such as KRT7and KRT19) began to be expressed, indicating thatHCs have the potential to trans-differentiate intoBEC. Additionally, transdifferentiation of matureHCs into biliary cells has been shown to occurin rat liver  and examined theirtranscriptional profiles with Rat Genome 230 2.0Array. We also emphatically analyzed expressionchanges in the marker genes of the above livercell types during the regeneration process, and thepotential transdifferentiation relationships amongthese cell types.Mammalian liver is almost unique amongstbody tissues in its regenerative capacity . Thecapincrease . An in va et al. showed tat liver . It has s et al. showed tc) was calculated using the followingformula: Lc=regenerating liver weight (g)/body weight (g)×100%  rats that are obtained from theAnimal Center of Henan Normal University. A totalof 114 cleaning-grade adult rats, aged 10-12 weeksand weighing 190 ± 20 g were randomly divided intonine PH groups, nine sham-operation (SO) groupsand one control group with 6 rats per group. Rats inthe PH groups underwent an operation for 2/3 PHaccording to the guideline described by Higgins andAnderson ((g)×100% . All pro8 cells/mL. Six mL of the mixed cell suspension was spread onto the surface of 4 mL 60% percoll  in a 10 mL tube for a single centrifugation at 200 g for 5 minutes at 4˚C. The centrifuged pellets and supernatant were the purified HCs and nonparenchymal cells-enriched supernatant fractions, respectively. The supernatant was mixed with an equal volume of PBS, centrifuged at 400 g for 2×2 minutes at 4˚C. The mixed nonparenchymal cell-rich pellet was collected and adjusted to a concentration of 1×108 cells/mL, then mixed with 10 μL/mL of rat anti-THY1, -GFAP, -CD31, -CD68, CD161a, and -CD11c PE-antibodies , respectively. HOCs, HSCs, sinusoidal endothelial cells, KCs, PCs and DCs were identified using the immunomagnetic bead method  and incubated at 37˚C for 30 minutes. Finally, 3,3΄-diaminobenzidine  was added for staining and the results observed under an optical microscope   for 30 minutes, then smeared onto glass slides. Microwave antigen retrieval was undertaken once the cell samples on the glass slide had dried. In relation to HCs, for instance, the slides were incubated separately with anti-, China) . SimilarTotal RNAs from eight types of liver cell at each recovery time point were extracted one by one, according to the Trizol reagent manual  and then purified following the RNeasy mini protocol . The quality of the total RNA samples was assessed by agarose electrophoresis  with a 2:1 ratio of 28S rRNA to 18S rRNA intensities and optical density measurement at 260/280 nm prior to cDNA synthesis . RNAs poThe data for each microarray were normalizedby scaling all signals to a target intensity of 200using GCOS 2.0 software .Each probe set used in the Affymetrix GeneChipproduces a detection call, with present expression indicating good quality,marginal expression  indicating intermediate quality andabsent expression  indicatingrelatively low reliability. Therefore, probe sets thatresulted in A calls were removed to filter out falsepositives.Each array was analyzed based on the P, M, or Adetection call for probes. The relative values (foldchange) in gene expressions were evaluated accordingto the ratio of the normalized signal valuefor the surgical groups (including SO groups andPH groups) at different time points to those for thecontrol groups, e.g., genes with a relative value≥3were regarded as upregulated expression; geneswith a relative value≤0.33, as downregulated expressionand genes with a relative value between0.33~2.99 as insignificant expression changes.Relative values of three independent chip analysesat each time point were averaged as effectivevalues.To look for those genes whose expressionchanges are truly induced by the LR process, thisstudy compared the fold change in gene expressionin the PH groups with that in the SO groupsusing the F test. In this study, genes showing thesame or similar expression trends at the same timepoints in three independent chip assays, at least a3-fold change in expression level, and a significant(P≤0.05), or even extremely significant (P≤0.01),difference between the PH groups and SO groupsare referred to as genes differentially expressedduring LR.G6PC, GGT1, OC2, GFAP,CD14, LYZ, CD56, CD86 for HC, BEC, HOC,HSC, SEC, KC, PC and DC, respectively . Total RNA was reverse transcribedwith random primers using a reverse transcriptionkit . cDNA was amplified usingSYBR® Green I on Rotor-Gene 3000 . Standard curve and copy numberwere evaluated according to the protocol describedby Wang and Xu  and regeneratingliver weight (g) were determined and the Lectively  and suggectively .In this study, we employed an immunocytochemistry staining approach to identify eight types of hepatic cells isolated from rat liver regenerating after PH using corresponding specific protein markers, as described previously. Purity of the eight liver cell fractions were statistically analyzed according to the marker-positive cell rate. Results showed that the marker-positive rates at each time point were over 96% for HCs, over 94% for BEC, and over 95% for HOC, HSC, SEC, KC, PC and DC , suggestG6PC, GGT1, OC2, GFAP, CD14, LYZ, CD56 and CD86 for assessing the reliability of the microarray analysis. As shown in OC2, LYZ and CD56, increased mRNA expression of GGT1 at 12-30 hours and CD14 during almost the whole LR, respectively; decreased mRNA expressions of G6PC at 24-72 hours, GFAP between 2-120 hours after PH, and CD86 during almost the whole process, except 30 hours. On the whole, based on the data obtained from RT-PCR, with the exception of LYZ, CD56 and CD86 whose expression patterns detected by qRT-PCR were not always consistent with those in the array experiments. Expression trends for the other five genes as detected by qRT-PCR were consistent with that detected by microarray, despite the difference in mRNA abundance between the approaches, demonstrating the reliability of the chip results.In this study, quantitative real-time PCR was used to quantify the expression of eight target genes AFP, ALB), 10 BEC markers , 25 HOC markers ,19 HSC markers , 31 SECmarkers , 13 KC markers, 13 PC markers  and 41 DC markers . Correspondingly, therewere 15, 4, 15, 13, 12, 5, 6 and 12 markers  present on the rat genome 2302.0 array. Based on the stringent standards describedin the "Materials and Methods" section,out of the above 79 genes, 14, 18, 13, 25, 20,23, 11 and 33 markers were significantly differentlyexpressed in the above eight types of livercell, respectively. As shown in Through referring to a large number of scientificarticles, we found at least 23 HC markers and BEC (KRT7and KRT19) at 12-72 hours post-PH. After 12hours biliary lineages began to express oval cellmarkers GABRP, PCNA and THY1, and up to 30hours there appeared a remarkable increase inmRNA levels of HC markers  in BECs. According to thetranscriptional profiles of oval cells, the expressionsof marker genes for HCs (KRT18 andKRT8) and that for BECs (KRT7 and KRT19)were detected at 6-36 hours and at 2-168 hourspost-surgery, respectively. The observations indicatethat HC, HOC, and BEC have, at leastlimited, multi-differentiation potentials duringrat LR. In addition, some markers for HCswere detected in another four liver cell types at6-36 hours post-surgery, including HC makersKRT18, TAT, TRFR2 in HSC, HC makers CPS1,G6PC, KRT18, KRT8, PCK1, TAT and TRFR2in SEC, and HC makers KRT18, KRT8 and AFPin KC, and HC makers CPS1, G6PC, KRT18,KRT8 and Tat in DC, which might give somesigns of differentiation of these liver cells towardsHCs  category method, we analyzed the biological functions and processes of these 79 significantly expressed markers among 8 liver cell types during LR. The result showed that the above 79 genes were involved in many biological activities, such as response to stimuli, signaling pathways, immunity and inflammation, cell migration and adhesion, differentiation and development, cell proliferation, cellular metabolism, and so on. Contrastingly, amongst them, cell proliferation, differentiation, development, and apoptosis were the predominant biological processes. Notably, genes involved in responses to stimuli, signaling pathways, immunity and inflammation, cell adhesion, and differentiation and development included the largest proportion of genes , respectively . Genes iPROM1, KRT14, LY6E) and BEC . According to studies by others, the threeHOC markers have the effect of inducing cell differentiation or insignificant expression. Based on theabove analysis, it can be inferred that HC have thepotential to differentiate toward HOC and BEC,consistent with Nishikawa’s report of transdifferentiationof HC to BEC and four HC markers were strongly expressed in BEC cells whose ownmarkers were not significantly expressed or wereeven lower than in the control group. GO analysisshowed that PCNA contributes to cell cycle progressthrough promoting DNA replication  were obviously upregulatedin HSC whose own marker genes showed no significantchanges in mRNA levels, suggesting the tendencyfor HSC transdifferentiation towards HC.Somewhat differently, the results of the study by Chen et al.  were upregulated in SEC whose own markers did not obviously change. In addition, between 6-36 hours KCs and DCs predominantly expressed three  and six specific genes  for HC, respectively. As described above, these genes either accelerate cell differentiation or embody the biological functions specific to HCs replicate to restore the original size and function of the liver after PH in rats. Of eight liver cell types, HCs make up 70-80% of the liver mass and 65% of the total cell number. Therefore, recovery of the HC number is of primary importance after PH. While it is well-known that a majority of HCs enter into cell proliferation to compensate for the lost HCs immediately after PH, our analysis suggests that the compensatory HC production may originate partly from the transdifferentation of non-parenchymal cells. This may be the first report on the transdifferentiation relationship between non-parenchymal cells and HCs.In addition, some HC markers were significantlyexpressed in other liver non-parenchymal cellsduring LR. At 2-72 hours PH, only the HC markers were detected to strongly express HC markers, indicating some signs of differentiation in these four liver cells towards HCs. However, genechip can only test the transcriptional profiles of genes; it is not able to reflect the more exact or direct transdifferentiation activities of different liver cells. In future, we will further test these transdifferentiation relationship using methods such as immunohistochemistry, RNA Interference etc.


Adaptation of mammals to terrestrial life was facilitated by the unique vertebrate trait of body hair, which occurs in a range of morphological patterns. Keratin associated proteins (KRTAPs), the major structural hair shaft proteins, are largely responsible for hair variation.We exhaustively characterized the KRTAP gene family in 22 mammalian genomes, confirming the existence of 30 KRTAP subfamilies evolving at different rates with varying degrees of diversification and homogenization. Within the two major classes of KRTAPs, the high cysteine (HS) subfamily experienced strong concerted evolution, high rates of gene conversion/recombination and high GC content. In contrast, high glycine-tyrosine (HGT) KRTAPs showed evidence of positive selection and low rates of gene conversion/recombination. Species with more hair and of higher complexity tended to have more KRATP genes (gene expansion). The sloth, with long and coarse hair, had the most KRTAP genes (175 with 141 being intact). By contrast, the “hairless” dolphin had 35 KRTAPs and the highest pseudogenization rate . Unique hair-related phenotypes, such as scales (armadillo) and spines (hedgehog), were correlated with changes in KRTAPs. Gene expression variation probably also influences hair diversification patterns, for example human have an identical KRTAP repertoire as apes, but much less hair.We hypothesize that differences in KRTAP gene repertoire and gene expression, together with distinct rates of gene conversion/recombination, pseudogenization and positive selection, are likely responsible for micro and macro-phenotypic hair diversification among mammals in response to adaptations to ecological pressures.The online version of this article (doi:10.1186/1471-2164-15-779) contains supplementary material, which is available to authorized users. Terrestrial life in extant vertebrates was accompanied by the formation of diverse and rigid body coverings , along with other cornified appendages , that evolved in response to strong selective pressures. These body coverings helped protect vertebrates and allowed them to successfully adapt to environmental pressures like heat, ultra violet radiation, water loss, and mechanical forces , 2. SincHair is a dynamic mini-organ formed by ectodermal-mesodermal interactions –19 and iThe current diversity of hair in extant mammals is due to innovations and changes in numerous genes and their corresponding proteins. Humans have 54 functional alpha-keratin genes comprising 28 type I and 26 type II keratins , 31, 13 Vicugna pacos) low-coverage 2.51×, assembly, vicPac1, Jul 2008, (2) armadillo (Dasypus novemcinctus) low-coverage 2×, assembly, dasNov2, Jul 2008, (3) bushbaby (Otolemur garnettii) low-coverage 1.5×, assembly, otoGar1, May 2006, (4) cow (Bos taurus) coverage 7×, assembly Btau_4.0, Oct 2007, (5) dolphin (Tursiops truncatus) low-coverage 2.59×, assembly, turTru1, Jul 2008, (6) elephant (Loxodonta africana) coverage 7×, assembly, Loxafr3.0, Jul 2009, (7) gibbon (Nomascus leucogenys) whole genome coverage 5.6×, assembly, Nleu1.0, Jan 2010, (8) gorilla (Gorilla gorilla) gorGor3, Dec 2009, (9) guinea Pig (Cavia porcellus) high-coverage 6.79×, assembly, cavPor3, Mar 2008, (10) hedgehog (Erinaceus europaeus) low-coverage 1.86×, assembly, eriEur1, Jun 2006, (11) horse  coverage 6.79×, assembly, Equ Cab 2, Sep 2007, (12) marmoset , NCBI build 1.1, (13) megabat (Pteropus vampyrus) low-coverage 2.63× assembly, pteVam1, Jul 2008, (14) mouse Lemur (Microcebus murinus) low-coverage 1.93×, assembly, micMur1, Jun 2007, (15) orangutan (Pongo abelii) NCBI build 1.2, (16) panda (Ailuropoda melanoleuca) high-coverage, assembly, ailMel1, Jul 2009, (17) pig (Sus scrofa) from NCBI build 3.1, high-coverage, assembly, Sscrofa10, Jun 27, 2011, (18) rabbit (Oryctolagus cuniculus) high-coverage, assembly, oryCun2, Nov 2009, (19) sloth (Choloepus hoffmanni) low-coverage 2.05×, assembly, choHof1, Sep 2008, (20) tarsier (Tarsius syrichta) low-coverage, 1.82× assembly, tarSyr1, Jul 2008, (21) tree shrew (Tupaia belangeri) low-coverage 2×, assembly, tupBel1, Jun 2006, and (22) wallaby (Macropus eugenii) coverage 2×, assembly, Meug_1.0, Dec 2008, available at Ensembl and NCBI websites http://www.ensembl.org/[http://www.ncbi.nlm.nih.gov/[Advances in genome sequencing have made it easier to explore multigene families across different genomes. Expansion, contraction and pseudogenization, along with genomic/chromosomal organization (gene clusters) of gene families, are important mechanisms driving genome evolution and influencing fitness within lineages or species , as suggmbl.org/, 53 and nih.gov/.The KRTAP multigene family consists of ~100-180 gene members divided into two major classes, High Cystine (HS) and High Glycine/Tyrosine (HGT), which in turn are divided into many subfamilies with unique motifs and sequence repeats. We assigned all KRTAP multigene family members to their respective subfamilies following previously published guidelines . We builTo prove the absence of KRTAP genes in high coverage genomes with intact gene clusters, we performed synteny analysis and searched for human orthologs that should be flanking the missing KRTAP genes. For example, in pig the 5’ and 3’ human orthologs flanking the KRTAP cluster 5 was missing, indicating that this region has most-likely not been sequenced and that further research and/or higher genomic coverage is needed for confirmation. We verified the synteny of conserved orthologs flanking the missing genes for the subfamily KRTAP25 in the callitrix, cow and elephant, along with KRTAP25, KRTAP19 and KRTAP29 in cavia, and KRTAP12 in rabbit.Species-specific subfamily differences, changes in the total number of genes, functional genes, pseudogenes, amino acid content  and size polymorphism in genes within subfamilies may be responsible for the species-specific hair characteristics and the marked variability found in hair patterns among mammalian species.The KRTAP gene family consists of 30 subfamilies, 24 of which are high cysteine and six are high glycine-tyrosine. The complete KRTAP gene family is arranged into five clusters at five different genomic locations , a nocturnal hairy mammal with long, coarse and shaggy fur that serves as a host for different microorganism [Cavia porcellus), and 38 genes copies in the marsupial Wallaby (Macropus eugenii). Subfamily 28 has expanded in Rabbit (Oryctolagus cuniculus) (23 genes copies), which belongs to order lagomorpha. We typically observed functional genes in (HS-KRTAPs) subfamilies 11, 16, 17, 24-27 and 29 varying from a minimum of one to a maximum of three. The subfamilies 11, 16, 17 and 25 have a maximum of one functional gene member, subfamilies 24 and 29 have a maximum of two functional genes , and subfamily 26 has a maximum of three members (present in sloth and elephant). Subfamily 7, belonging to the high glycine-tyrosine group, has a maximum of one functional gene member . This aquatic mammal is almost hairless, with only a few hairs (bristles) on the upper lip of the rostrum, which are shed soon after birth, leaving hairless pits on the rostrum of adults that have specialized sensory function [For example, we observed high rates of pseudogenization Figures   that may be required during a precise stage of cell cycle . Gene coUsing Geneconv  and RDP3Sequences with higher synonymous substitution rates (dS) had higher overall GC content (GC%) and third-codon GC content (GC3%), and lower synonymous substitution rates in the high-cysteine genes than the high glycine-tyrosine genes. The negative correlation between GC content and synonymous substitution rate (dS) is consistent with the higher rates of concerted evolution observed in high cysteines Figure A and B. Gene expansion provides the essential raw material for the positive selection to act , which iThe KRTAP multigene family has experienced dynamic evolution and diversification within and among genomes as observed in the 30 diverse subfamilies of high cysteine and high glycine-tyrosine subfamilies. These two groups have evolved differently, with the high cysteine group showing high rates of gene conversion within subfamilies, with some exhibiting characteristic differences in copy number, while others have been more conserved. This may be an adaptive mechanism promoting a high order of amplification of similar copies to meet the high demand for the structural proteins required to adapt to changing environmental conditions (e.g. sloth have extensive hairs that can harbor symbiotic microorganism communities while the dolphin is “hairless” in response to a more-predictable and constant environment and to create less resistance when swimming). We also compared the differential evolutionary patterns between high cysteine and high glycine–tyrosine genes using the Pearson correlation coefficient for the number of genes in each subfamily between species. The coefficient value for high cysteine is significantly higher than for high glycine-tyrosine Figure A and theThe KRTAP family is widely grouped into three major categories based on amino acid composition: (i) high sulfur (<30% cysteine content), including subfamilies 1, 2, 3, 10-13, 16, 24-27, 29, 31, 34 and 35; (ii) ultrahigh sulfur (>30% cysteine content), including subfamilies 4, 5, 9, 17, 28, 30, 32 and 33 subfamilies; and (iii) high glycine/tyrosine, including subfamilies 6, 7, 8, 19, 20 and 21. The amino acid composition is shown in Table Gene families are formed by gene duplication, a process that provides important raw material for functional innovation and adaptive selection. Gene families vary in size from a few to thousands of gene members, which makes it difficult to identify and characterize them without sufficient genome sequences. The genome sequencing projects have made it possible to explore complex gene families involved in different phenotypes. Here we explored the mammal-specific KRTAP gene family, which is the major constituent of the hair proteome and plays a primary role in hair formation and thus long been associated with phenotypic differences in hair and wool. This study assessed patterns of variation using comparative genomic approaches in the KRTAP gene family. Our study used 22 diverse mammalian genomes that encompassed closely related species, e.g. family Hominidae of primates, comparing apes with dense hair cover with human with much less hair cover, along with species with diverse hair related characteristic, e.g. alpaca (fibre), armadillo , hedgehog (spines), sloth (hosting hair symbionts) and dolphin (mostly hairless and aquatic), to obtain greater insights into the KRTAP gene family evolution relative to mammalian hair and phenotypic variations.We found high molecular diversity within the KRTAP gene family, with 30 subfamilies (24 belonging to high cysteine and six belonging to high glycine-tyrosine KRTAP)  was found in sloth, which has long, dense and coarse body hair cover, which also serves as a host for symbiotic microorganisms  in this arboreal mammal. By contrast, we have detected a reduced number of functional KRTAP genes and high percentage of KRTAP pseudogenes (74%) in dolphin , highlighting the much lower KRTAP gene requirement in this smooth-skinned species that only has a few hairs (bristle) at the rostrum. These are lost soon after birth and in adults the hairless pits are adapted for sensory functions  provides extraordinary combinatorial complexity  for inteThe present study explored KRTAP gene family evolution in various mammalian species inhabiting diverse terrestrial and aquatic environments. The two groups of the KRTAP gene family, high cysteine and high glycine-tyrosine KRTAP genes, have evolve differently, resulting in species-specific diversification of this multi-gene family and leading wide morphological diversity in hair characteristics in extant mammals. We conclude that differences in KRTAP gene family repertoires, together with changes in expression patterns, are responsible for shaping unique hair characteristics in diverse mammalian species. These differences are more pronounced between aquatic and terrestrial species and demonstrate the important adaptive role of hairs in terrestrial colonization and the radiation of mammals from water to land. Future studies comparing the KRTAP repertoire in key model organisms, e.g. Alpaca and sheep, may provide insights to understanding the role of KRTAP gene variations in hair fibre traits and its use in textile industry.http://www.ensembl.org/Multi/Tools/Blast and NCBI genome database http://www.ncbi.nlm.nih.gov/blast/Blast.cgi using BLASTN algorithm [http://www.ncbi.nlm.nih.gov/gorf/gorf.html and Mobyle [All KRTAP genes are relatively small (ca. 1 kb) and generally have single exon . Some KRlgorithm  and E-valgorithm  TFASTX algorithm  and ORF d Mobyle . The ideWe employed phylogenic tree building method to further classify the identified KRTAP gene repertoire to their respective subfamilies. For each species the intact genes were used for building phylogentic tree. All intact KRTAP genes were translated to amino-acid and aligned using ClustalW incorporated in MEGA4.0 , 105 withttp://www.math.wustl.edu/~sawyer/geneconv/[We used the program Geneconv eneconv/ to deteceneconv/ to detecThe evolutionary distance between genes can be calculated with synonymous substitution, which are immune to selection and are not decreased by negative selection . The seqIn order to study the differential evolutionary pattern of high cysteine and high glycine-tyrosine KRTAP genes, we compared the pairwise-pearson correction coefficient Figure  of the nAll the supporting data are included as additional files.Additional file 1: Figure S1-S24: The phylogeny of high cysteine KRTAP genes in 22 mammalian species. Neighbor-joining method with P-distance and interiors branch test with 1,000 replications (shown on the branches) was employed to build the trees. Figures S23 and S24 shows loss of, one to one orthologous relationship between two species due to concerted evolution. The KRTAP members are labeled with species abbreviation, Gene ID and KRTAP subfamily Additional file 3: Table S3: Gene pairs under significant gene conversion, as detected by GeneConv program. (TXT 49 KB)Additional file 4: Table S4: Results of RDP3 showing unique recombination events with statistical significance P value of less than 0.01 employing Bonferroni correction. (XLSX 11 KB)


Objective. To explore the pathological feature and immunoprofile of immunoprofile accompanied with upper urinary tract obstruction and the immunoprofile in various types of glandular cystitis. Methods. Pathological sections from 31 cases of cystitis glandularis with upper urinary tract obstruction and 34 cases of cystitis glandularis without upper urinary tract obstruction were observed as pathological feature on microscopy. Meanwhile, an immunohistochemical analysis was employed to determine the expression of p53, Ki67, p21, MMP-9, MUC1, MUC2, and COX-2. Results. In the two groups, main pathological type was transitional epithelial, followed by intestinal epithelial; other types were a few, and the difference between the two groups was not significant. All immunohistochemical expressions of p53, Ki67, p21, MMP-9, MUC1, MUC2, and COX-2 were positive in varying degrees, and there was no significant difference between the groups. Transitional epithelial type was compared with mixed type; the difference of COX-2 was significant, P < 0.05. The differences of immunohistochemical expression among other different pathologic types were not significant. Conclusions. It is suggested that glandular cystitis accompanied with upper urinary tract obstruction shares the same pathological feature and immunoprofile as that without upper urinary tract obstruction. No significant differences of immunohistochemical expression in tissue are in cystitis glandularis with different pathological types. Cystitis glandularis is a change that occurs in the tissue that lines the bladder. This particular type of tissue change is benign, but it can be a symptom of another health problem that needs to be treated. In addition, some cases of this condition precede the development of cancerous growths. For this reason, this finding can be a cause for concern. Our previous study found that cystitis glandularis could be induced by upper urinary tract obstruction and cystitis glandularis accompanied with upper urinary tract obstruction is associated with upper urinary tract obstruction , 2. SeveThe pathological sections of 65 cases diagnosed as cystitis glandularis from February 2006 to August 2011 in our hospital were evaluated. The biopsy specimens were obtained by cystoscopy. Patient age was from 28 to 86 years, with a mean age of 62.97 years. Among them, 13 cases were male and other 42 cases were female. Thirty- one cases were accompanied with upper urinary tract obstruction and 34 cases were accompanied without upper urinary tract obstruction. Pathological types were observed under light microscope; meanwhile, an immunohistochemical analysis was employed to determine the expression of p53, Ki67, p21, MMP-9, MUC1, MUC2, and COX-2.μm thickness, and then stained with hematoxylin and eosin (H-E) for examination under a light microscope and the pathological type was divided by transitional epithelial, intestinal epithelial, prostatic epithelial, and mixed type.Tissue blocks were conventionally resected from the inflammatory lesions on bladder for preparation as microscopic specimens. The tissue blocks were fixed in 10% neutral formalin and conventionally dehydrated. Blocks were embedded in paraffin, cut at 3-4 Primary antibodies of p53 (clone:D0-7), Ki67 (clone:sp6), p21 (clone:DSC-60.2), MMP-9 (clone:2C3), MUC1 (clone:ZM35), MUC2 (clone:Ccp58), and COX-2 (clone:cx-294) were purchased from Gene Tech (Shanghai) Company Limited, which were used without dilution. The secondary antibodies, ChemMate Envision Detection Kit of goat against rabbit and mouse antibody, were obtained from DaKo.μm thickness, dried and deparaffinized in xylene for 3 times, every time 15 minutes, and then rehydrated in graded ethanol. Endogenous peroxidase was blocked with 3% hydrogen peroxide; subsequently the sections were washed by distilled water and heated in a citrate buffer (pH 6.0) using a steam cooker at 95°C for 30 min. After being cooled, the sections were mounted by fetal bovine serum for 60 minutes, and then primary antibody was added and kept in wet box at 4°C for 24 hours. Sections were washed by Tris Buffered Saline for three times, every time for 5 minutes [Standard two-step Envision method of immunohistochemical staining was used to assess the expressions of p53, Ki67, p21, MMP-9, MUC1, MUC2, and COX-2. Blocks were cut conventionally at 3-4  minutes .The secondary antibody was added and incubated for 30 minutes and then washed by Tris Buffered Saline for three times, every time for 5 minutes. Diaminobenzidine substrate solution was used as a chromogen and chromogenic time was controlled under a microscope. Then the slides were counterstained with hematoxylin, washed and dehydrated in ethanol, cleared in xylene, and finally mounted by gum. For the negative control, the buffer replaced the primary antibody. Positive staining patterns were compared with the known positive plates and other procedures follow the same step.Nuclear staining of p53, p21, and Ki-67, cytoplasmic staining of MMP-9, MUC2, and COX-2, and cytoplasmic staining or luminal membrane staining of MUC1 were considered positive .We used a semiquantitative analysis system to determine immunostaining score. In brief, the scoring system was established according to the percentage and intensity of positive cells. For percentage, 0~4 scores represent <5%, 5~25%, 26~50%, 51~75%, and >75% of labeled cells, respectively. For the intensity, 0~3 score indicates weak, middle, and strong staining, respectively. Multiplication of both scores decided the final quotation, ranging 0~12, negative (0), weak positive , positive , and strong positive  .A double-blind analysis was performed by two independent pathologists. Each sample was scored twice. All the slides were analyzed without knowledge of clinical data.The study was approved by the Medical Ethics Committee of Yangou Hospital, School of Medicine, Tongji University.χ2 test and Student's t-test. Difference was considered significant at a P value < 0.05.The differences between 2 analyzed data were compared using the In 32 cases with upper urinary tract obstruction, 26 cases (81.25%) were accompanied with ureteral calculi, 6 cases (18.75%) were accompanied with ureteral stenosis, 4 cases (12.50%) were accompanied with ureteropelvic junction obstruction, 2 cases (6.25%) were accompanied with ureteral deformity, 2 cases (6.25%) were accompanied with hematuria, and 6 cases (18.75%) were accompanied with positive urine culture. In 34 cases with upper urinary tract obstruction, 7 cases (20.59%) were accompanied with benign prostate hyperplasia, 3 cases (8.82%) were accompanied with urethra stenosis, 1 case (2.94%) was accompanied with prostate cancer, 1 case (2.94%) was accompanied with bladder stone, 1 case (2.94%) was accompanied with bladder tumor, 15 cases (44.11%) were accompanied with hematuria, and 8 cases (32.53%) were accompanied with positive urine culture.P > 0.05.On microscopy, main pathological type of cystitis glandularis was transitional epithelial, followed by intestinal epithelial; prostatic epithelial and mixed types were a few. In addition, squamous metaplasia, Von Brunn's nests, and inflammatory cell infiltration would appear among them in some pattern. Patient age was 57.25 ± 9.92 years in group with upper urinary tract obstruction and 68.35 ± 12.86 years in group without upper urinary tract obstruction, Cystitis glandularis is considered benign but with uncertain malignant potential. Several authors have described the progression to bladder adenocarcinoma during long-term follow-up, especially; intestinal metaplasia is most commonly considered as a risk factor and a putative precursor of adenocarcinoma, although others have disputed this finding , 2, 11. Ki67 antigen is a nonhistone protein, presented during the cell cycle phases G1, S, G2, and M, being an established indicator of tumor growth and aggressiveness. The well-known tumor suppressor p53, which functions primarily as a transcription factor, plays a vital role in protecting cells from a variety of cellular stresses, including DNA damage and oncogene activation. In previous studies, increased Ki67 and p53 expression was proven to be related to tumor grade, stage, recurrence, progression, and survival of bladder cancer –14. p53 MMP-9  is an enzyme that in humans is encoded by the MMP-9 gene. MMP-9 can be involved in the development of several human malignancies, as degradation of collagen IV in basement membrane and extracellular matrix facilitates tumor progression, including invasion, metastasis, growth, and angiogenesis . Mucins Cyclooxygenase-2 (COX-2) is regarded as induced inflammatory mediator involved in the development of tumors. It is an inducible enzyme  responsible for conversion of arachidonic acid to prostaglandins and other inflammatory mediators. It is not detectable in most normal tissues; however, it is induced at sites of inflammation by cytokines, growth factors, and tumor promoters. Also, prominent COX-2 expression has been described in bladder cancers including transitional cell and squamous cell carcinomas and this expression correlates with tumor grade and invasion –20. JianIt is suggested that glandular cystitis accompanied with upper urinary tract obstruction shares the same pathological feature and immunoprofile as that without upper urinary tract obstruction. Meanwhile, immunohistochemical expressions of P53, Ki67, p21, MMP-9, MUC1, MUC2, and COX-2 in cystitis glandularis with or without upper urinary tract obstruction have changed. The differences of immunohistochemical expressions in different types of cystitis glandularis and accompanied with or without upper urinary tract obstruction were not significant . It is h


Vibrio cholerae are responsible for endemic and pandemic outbreaks of the disease cholera. The complete toxigenic mechanisms underlying virulence in Vibrio strains are poorly understood. The hypothesis of this work was that virulent versus non-virulent strains of V. cholerae harbor distinctive genomic elements that encode virulence. The purpose of this study was to elucidate genomic differences between the O1 serotypes and non-O1 V. cholerae PS15, a non-toxigenic strain, in order to identify novel genes potentially responsible for virulence. In this study, we compared the whole genome of the non-O1 PS15 strain to the whole genomes of toxigenic serotypes at the phylogenetic level, and found that the PS15 genome was distantly related to those of toxigenic V. cholerae. Thus we focused on a detailed gene comparison between PS15 and the distantly related O1 V. cholerae N16961. Based on sequence alignment we tentatively assigned chromosome numbers 1 and 2 to elements within the genome of non-O1 V. cholerae PS15. Further, we found that PS15 and O1 V. cholerae N16961 shared 98% identity and 766 genes, but of the genes present in N16961 that were missing in the non-O1 V. cholerae PS15 genome, 56 were predicted to encode not only for virulence–related genes  but also genes involved in the metabolic biosynthesis of lipids, nucleosides and sulfur compounds. Additionally, we found 113 genes unique to PS15 that were predicted to encode other properties related to virulence, disease, defense, membrane transport, and DNA metabolism. Here, we identified distinctive and novel genomic elements between O1 and non-O1 V. cholerae genomes as potential virulence factors and, thus, targets for future therapeutics. Modulation of such novel targets may eventually enhance eradication efforts of endemic and pandemic disease cholera in afflicted nations.Pathogenic strains of  Vibrio cholerae, a Gram-negative, comma-shaped, facultative anaerobic bacterium [V. cholerae includes both pathogenic and non-pathogenic strains, and the bacteria responsible for pandemic outbreaks secrete the cholera toxin [V. cholerae was recorded in Southeast Asia, which quickly spread across the globe as the seventh pandemic [Cholera is an infectious disease characterized by profuse watery diarrhea and vomiting in humans, and the causative agent is acterium . V. cholra toxin . Since 1ra toxin ,4. In thra toxin . The mospandemic . In 2010pandemic .V. cholerae strains encode proteins that are directly or indirectly responsible for virulence. In many parts of the world, the O serogroups of V. cholerae are associated with diarrhea [The genomes of several pathogenic diarrhea . The mosdiarrhea . In addidiarrhea ,11. Othediarrhea .V. cholerae is EmrD-3, which belongs to the major facilitator superfamily (MFS) and is a drug/H+ antiporter with 12 transmembrane domains [V. cholerae is VceB [Unfortunately, wide spread use and misuse of these and other antimicrobials have resulted in selection of multidrug-resistant bacterial variants  which po domains . Another is VceB . Drug ef is VceB -21.Vibrio pathogenicity island (VPI), and the toxin co-regulated pilus (TCP) within the O1 serogroups of V. cholerae make these strains more virulent and pandemic than their non-O1 counterparts [ace, psh, PIIICTX, zot and cep, which are implicated in phage morphogenesis [Vibrio pathogenicity island-1 (VPI-1) confers toxin release, bioflim formation, attachment to disease vectors for transmission to humans, and are receptors of CTX. The Vibrio pathogenicity island-2 (VPI-2) helps the cholera toxin to gain entry into the intestinal epithelium by unmasking GM1 gangliosides in the lining of the human intestine. The absence of VPI-1 and VPI-2 in non-O1 serogroups of V. cholerae makes them less pathogenic than the O1 serogroups [The presence of the cholera toxin (CT), the terparts . A signiogenesis ,23,24. Trogroups .V. cholerae strains carry certain virulence genes, the severity of disease is less compared to O1/ O139 V. cholerae [V. cholerae are known as the non-agglutinating Vibrios (NAGs) because they lack the genes coding for CT and TCP [V. cholerae species [V. cholerae predictably transport phosphate molecules across the periplasm and may be essential for protein synthesis, amino acid exchange, and transport of fatty acids [Even though non-O1 cholerae . The non and TCP ,27. The  species . The ABCty acids .V. cholerae PS15 (GenBank Accession No. AIJR00000000) [V. cholerae PS15 is composed of 3,910,387 base pairs (bp) organized into 3,512 open reading frames with a G+C content of 47.55% [V. cholerae PS15 [V. cholerae El Tor N16961 because this latter genome was completely sequenced [V. cholerae bacterium possesses some virulence genes responsible for causing gastrointestinal infections, wound infections, septicemia and cellulitis in humans, little is known about the mechanisms that confer virulence in this microorganism. The aim of this work is to identify differences in the genetic elements between the genomes of virulent N16961 and non-virulent PS15 strains of V. cholerae in order to identify novel virulence mechanisms that may eventually serve as potential therapeutic targets for the ultimate purpose of fostering conditions that reduce dissemination of disease-causing virulent serotypes of V. cholerae through populations.We previously determined the genome nucleotide sequence of the non-O1 non-toxigenic 0000000) . Here, wf 47.55% . We chosrae PS15  using V.equenced . N16961 equenced . Even thV. cholerae PS15 environmental isolate (courtesy of Dr. Charles Kaysner) from sediment sampled in Puget Sound, WA [V. cholerae N16961 [V. cholerae microorganisms. The open reading frames (genes) encoding functional roles associated with a subsystem are referred as functioning parts, and a subsystem is referred as a set of predicted abstract functional roles [A function based genome comparison was performed between a non-toxigenic, non-O1 ound, WA ,31 and Oe N16961 , using te N16961  focusingal roles . The scral roles .V. cholerae PS15 genome sequence [Vibrio genome sequences in the database and is represented in Figure 1.The non-O1 sequence   with the complete DNA sequence of V. cholerae El Tor N16961  [The CGView server was used for comparative genome analysis . A graphe pairs) ,30.V. cholerae isolate from Puget Sound, strain PS15 [V. cholerae bacteria were highly related [V. cholerae PS15 with other microorganisms by constructing a phylogenetic tree using BLAST pair-wise alignment in order to represent genomes that are most closely related to V. cholerae non-O1 PS15 and to establish relatedness of PS15 to these microorganisms . Although the non-O1 V. cholerae PS15 genome sequence is most closely related to those of V. cholerae LMA 3984-4, O395, O1 strains 2010EL-1786, MJ-1236, O1 biovar El Tor strain N16961, IEC224, and M66-2, the non-O1 V. cholerae PS15 strain is, nonetheless, the most distantly related member within this cluster.We previously determined the whole genome sequence of a non-toxigenic, non-O1 ain PS15 . It had  related , possiblV. cholerae strain N16961 were elucidated [V. cholerae strain PS15 would implicate chromosome assignment in this bacterium as well. A circular genome representation was generated using the CGView server to plot the structural genome arrangement with BLAST analysis of the non-O1 V. cholerae PS15 genome with that of the O1 V. cholerae N16961 using their respective genomic nucleotide sequences in a FASTA format . Using the genome sequence data from V. cholerae N16961 to compare with the genome of V. cholerae PS15, chromosomes 1 and 2 were implicated for the non-toxigenic PS15 strain and are shown in Figure 2.Since the two chromosomes of the toxigenic O1 ucidated , we predV. cholerae PS15 genome is distantly related to the genomes of toxigenic O1 V. cholerae, the PS15 genome is still closely related to genomes of the Vibrio genus. This implies a striking similarity between the non-O1 and O1 genomes, specifically regarding the commonalities within the gene space. To test this, we used RAST Seed Viewer and UniProt to compare the genome sequences of O1 V. cholerae N16961 and non-O1 V. cholerae PS15, the general features of which are shown in Table 1. The O1 and non-O1 V. cholerae genomes shared 766 genes (open reading frames) that are predicted to code for proteins within functional categories pertaining to virulence, disease, defense, membrane transport, phages, prophages, transposable elements, plasmids, DNA metabolism, dormancy, sporulation and regulons. Interestingly, when compared to the N16961 genome, the V. cholerae PS15 genome appears to be truncated sporadically throughout by approximately 120 kbp . In Table 2 we listed 58 of 766 genes that share 98% identity between both genomes. The remaining genes are listed in Supplement Table S1. Even though non-O1 V. cholerae PS15 is believed to be non-pathogenic compared to the known virulent O1 V. cholerae N16961 strain, their genomes shared 90 genes in common that code for functions pertaining to virulence, disease and defense. Some of these genes included accessory colonization factor (acfD), TCP pilus virulence regulatory protein (tcpN), toxin coregulated pilus biosynthesis protein E (tcpE), TCP pilus virulence regulatory protein (toxT) and accessory colonization factor (acfC). In addition to these virulence-associated genes, both genomes shared 287 genes encoding functional properties in the DNA metabolism category, 8 genes encoding proteins for dormancy and sporulation, 366 genes encoding membrane transporters, 12 genes in the categories of phages, prophages, transposable elements and plasmids, and 3 genes pertaining to regulons. Among these shared genomic elements encoding membrane transporters are genes known to express multidrug resistance efflux pumps, including AcrA of the RND superfamily [We have shown above that although the non-O1 erfamily , SugE oferfamily , and Norerfamily .V. cholerae serotypes suggests that they harbor genomic elements that confer virulence. For instance, the cholera toxin of toxigenic V. cholerae strains is the primary virulence factor in endemic and pandemic cholera cases [V. cholerae PS15 genome [V. cholerae N16961, are in the categories including virulence, disease and defense, membrane transport, DNA metabolism, dormancy and sporulation (Table 3). The virulence genes which were present in O1 serotypes but largely absent in the non-O1 strains, including the PS15 strain, include the accessory cholera enterotoxin (ace), the cholera enterotoxin subunit B (ctxB), the cholera enterotoxin subunit A (ctxA), and the zona occludens toxin (zot). Comparison of the predicted proteins encoded of both PS15 and N16961 genomes using UniProt revealed the absence of other virulence genes in PS15, which include genes predicted to encode accessory colonization factors A and B (acfA and acfB), and the genes encoding VceA and VceB proteins shown to confer resistance to antimicrobial agents (Table 3) [V. cholerae PS15 genome [The pathogenicity of the O1 ra cases . Thus, i5 genome , 56 of tTable 3) . Notably5 genome ,21.V. cholerae PS15 bacterium . The most closely-related microorganisms that shared the DNA encoding the cholera toxin include V. cholerae IEC224, O1 biovar El Tor strain N16961, O395, MJ-1236 and the O1 strain 2010EL-1786.A phylogenetic tree, which was generated by BLAST for bacterial genomes that share the cholera toxin, indicated the absence of the cholera toxin gene in the non-O1 V. cholerae genome but present in O1, include genes that encode glycerolipid and glycerophospholipid metabolism, and genes that code for VPI [Table 3). Additional genes that are absent in non-O1 V. cholerae PS15 include those coding for the Rst operon essential for the synthesis of phage related replication protein (RstA), phage related integrase (RstB), phage related antirepressor (RstC), phage related transcriptional repressor (RstR) [V. cholerae but absent in non-O1 include those coding for TsaE, a protein required for the synthesis of threonylcarbamoyladenosine in the presence of tRNA [Other genes that were absent in non-O1  for VPI  (Table 3r (RstR) , and sul of tRNA .V. cholerae PS15 environmental isolate is considered to be non-toxigenic [Table 4). The three known genes (characterized) that are present in PS15, but absent in N16961, include the oligopeptide ABC transporter called periplasmic oligopeptide-binding protein (OppA) [uvrA) [Table 4).Because the non-O1 oxigenic ,43, thisn (OppA) , a proten (OppA) , and the) [uvrA) , all of V. cholerae PS15 genome, a non-toxigenic strain, to that of an O1 V. cholerae N16961, a toxigenic strain, we found that of the 619 missing genes, 56 of these missing genomic elements encode dormancy, sporulation, ribosome modulation in persister cells, lipid metabolism, phage infection, nucleoside metabolism, and sulfur metabolism which in turn is essential for biosynthesis of amino acids, vitamins and prosthetic groups [V. cholerae lacks genes coding for metabolism of sulfur, the non-O1 serotype is predicted to be unable to convert naturally available sulfur to sulfide, which could then be incorporated into various sulfur containing metabolites. Sulfur is critical for the biosynthesis of many important compounds like amino acids (cysteine and methionine), vitamins , and prosthetic groups (Fe-S clusters) [V. cholerae pathogens through populations. These determinants, therefore, clearly also warrant further studies in order to elucidate the complete molecular mechanisms of pathogenesis in cholera infections.Upon comparison of the non-O1 c groups . As non-lusters) . These glusters) , in ordeV. cholerae PS15. We confirm, however, the presence of other genes predicted to encode distinct toxins and colonization factors, as previously shown for the non-O1 V. cholerae strain NRT36S [V. cholerae [V. cholerae [Not surprisingly, also among the 56 missing genes in the non-O1 PS15 genome are those that are known to confer virulence, such as the cholera toxin , colonizn NRT36S . This lacholerae , predictcholerae .V. cholerae PS15 strain harbors genes absent from the genome of its toxigenic counterpart, N16961. Such determinants mainly include still uncharacterized genetic elements that are predicted to encode proteins that confer virulence, disease, defense, membrane solute transport and DNA metabolism, suggesting that PS15 may be pathogenic to organisms excluding humans, perhaps in environments such as estuary waters [V. cholerae infections in animals other than humans.Interestingly, we found that the genome of the nontoxigenic y waters ,53. Amony waters , and Secy waters . We propV. cholerae PS15 shares >97% identity with El Tor O1 biovar V. cholerae strain N16961, as per BLAST analysis at the nucleotide level. Based on the alignment of the non-O1 PS15 genome with that of O1 N16961, chromosomes 1 and 2 were assigned to the PS15 genome . This tentative chromosome assignment will require confirmation with additional experimental work. Even though the genomes of both strains are highly similar to each other, the non-O1 PS15 microorganism is considered to be non-pathogenic, compared to the O1 N16961 strain, possibly due to the absence of the cholera toxin in PS15, which is responsible for endemic and pandemic diseases [V. cholerae microorganisms support both of these contentions. Further work with additional variants, such as atypical El Tor [V. cholerae.The genome of non-O1 diseases . More rediseases . Our phyl El Tor , NRT36S l El Tor , and CT-l El Tor , will beV. cholerae strains harbor a variety of genes that have previously been demonstrated to confer multidrug resistance via active drug efflux pump systems, such as AcrAB, NorM / VcmA, SugE, and VcaM [V. cholerae N16961 have been studied physiologically [V. cholerae PS15 was missing only one of these pumps, called VexA. Additionally, we found a shared but uncharacterized genetic element, VC_A0083 in the toxigenic strain and OSU_1537 in the non-toxigenic strain, tentatively called multidrug resistance protein D and predicted to encode an MFS drug efflux pump. These multidrug resistance mechanisms may be important because of their potential selection and maintenance in environments containing antimicrobial agents, their genetic mobility to other microorganisms, and dissemination within populations [Remarkably, we found that both of the toxigenic and non-toxigenic and VcaM . All sixogically , and ourulations -64.V. cholerae pathogenic strains and on the identification of novel housekeeping genes which may be equally significant in contributing towards the microorganisms’ pathogenicity [We conclude that the study and comparison of the genomic sequences between pathogens and their non-virulent counterparts will help discover genes encoding both the classical virulence factors and those encoding novel virulence factors. Future work will focus on the study of solute transport and antibacterial resistance mechanisms of genicity ,65,66. Supplemental Table S1