Background. Laboratory data suggests a reduction of Faecalibacterium prausnitzii (F. prausnitzii) is confirmed both in fecal samples in inflammatory bowel disease (IBD) patients. Numerous observational studies have suspected dysbiosis, an imbalance between protective and harmful bacteria to be relevant to the etiology and pathogenesis of IBD. Methods. Medline, EMBASE, Pubmed, and others. were searched by 2 independent reviewers. Of 48 abstracts reviewed, 11 studies met our inclusion criteria (subject N = 1180). Meta-analysis was performed with Review Manager 5.2. Results. The bacterial count of F. prausnitzii  in IBD patients was significantly lower (6.7888 ± 1.8875) log10 CFU/g feces than healthy controls (7.5791 ± 1.5812) log10 CFU/g feces; P < 0.0001. The Standardization Mean Difference of F. prausnitzii in IBD patients was −0.94 . Subgroup analyses revealed a trend toward a greater effect for CD  when compared to UC . Conclusions. The abundance of F. prausnitzii was decreased in IBD patients compared with healthy controls. Furthermore, the reduction of F. prausnitzii and misbalance of the intestinal microbiota are particularly higher in CD patients with ileal involvement. IBD is suspected to arise from the interaction between the host's genetic background, mucosal immunity, and the resident bacterial flora . Genome-F. prausnitzii belongs to the phylum of Firmicutes and is the major bacterium of the Clostridium leptum group. The Meta-analysis of the Human Intestinal Tract project have shown that F. prausnitzii is one of the most abundant anaerobic bacteria in the human gut microbiota, with a proportion of around 5% of total bacteria in faeces [ F. prausnitzii plays an important role in providing energy to the colonocytes and maintaining the intestinal health [ F. prausnitzii both in vitro and in vivo. And deficiency of F. prausnitzii might provoke and enhance inflammation [ F. prausnitzii was found in UC patients, even with quiescent disease [ F. prausnitzii was observed in patients with untreated CD but not in the patients with chronic diarrhea, suggesting a relationship in the pathomechanisms of CD [ F. prausnitzii are revealed in the fecal samples of patients with IBD. The F. prausnitzii level was much lower when the disease activity increased [l health . Furtherammation . Special disease . The depms of CD . Finallyncreased . F. prausnitzii reduction and IBD, we conducted a meta-analysis and systematic review to estimate the relative risk of F. prausnitzii reduction in patients with and without IBD. Given the laboratory data previously cited, we hypothesized a relationship between F. prausnitzii reduction and IBD.To further investigate the possible association betweenFaecalibacterium prausnitzii,” “F. prausnitzii,” and “FP.” Boolean operators  were used to widen and narrow the search results. The titles and abstracts from the search results were examined for potential inclusion. Also, the references from selected articles were examined as further search tools.This review was performed according to the standard guidelines for meta-analyses and systematic reviews of observational studies . To find F. prausnitzii counts measured by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), terminal restriction fragment length polymorphism (T-RFLP), or fluorescence in situ hybridization (FISH), (2) studies of human, (3) inclusion of a control group, (4) IBD and control groups were similar in age and sex and from the same catchment area, and (5) data were reported that were sufficient to calculate F. prausnitzii reduction in both the IBD and control groups. Studies were excluded if they used data from a previously published study.For inclusion in the systematic review, a study had to meet the following criteria established by the study team: (1) F. prausnitzii reduction was diagnosed, (i) number of patients with ulcerative colitis (UC), (j) number of patients with Crohn's disease (CD), (k) number of patients in the control group, (l) reported previous use of antibiotics, probiotics, or prebiotics in the IBD and control groups, and (m) reported previous use of steroids, 5-aminosalicylates (5-ASAs), and tumor necrosis factor-alpha (TNF-α) antibody medications in the IBD group. Studies were excluded if participants had used steroids, 5-ASAs, TNF-α antibody antibiotics, probiotics, or prebiotics in the last month preceding fecal sampling as this could influence the intestinal microbiota. If needed, authors were contacted regarding specific questions relating to their study. The independent reviewers conferred after data extraction was complete, discrepancies were identified, and review of the relevant article led to consensus.To reduce reporting error and bias in data collection, 2 independent reviewers extracted data from selected studies using standardized data extraction forms. These forms, created by the study team, included the (a) title, (b) authors, (c) journal, (d) year of publication, (e) study design, (f) inclusion and exclusion criteria, (g) methods by which IBD was diagnosed, (h) methods by which F. prausnitzii counts in IBD versus controls. Std. mean difference was used to describe the counts of the F. prausnitzii in IBD patients versus the controls. We calculated the SMD with a 95% confidence interval (CI) based on a random-effects model as described by Mantel-Haenszel. Meta-analysis was performed with the Review Manager 5.2. Analysis with a funnel plot used to assess publication bias. An I2 statistic was used to measure the proportion of inconsistency in individual studies that could not be explained by chance. Any heterogeneity identified would prompt subgroup analysis in an attempt to explain these findings.The primary outcome of this analysis was the Standardization Mean Difference (SMD) of F. prausnitzii counts measurement, and (5) whether F. prausnitzii reduction was the primary or secondary outcome of the study.Each study chosen for review was carefully assessed for study quality by the study team. Study quality was assessed using the following criteria: (1) study design, (2) method of IBD diagnosis, (3) method of patient enrollment (consecutive versus selected), (4) method of F. prausnitzii counts [Our initial search strategy yielded 58 potential articles for inclusion. After detailed analysis of selected articles, 27 articles were reviewed in detail. Subsequently, 16 articles did not meet inclusion criteria –35. The i counts , 29–35. i counts , 28. 2 si counts , 27. Thei counts –20 with  F. prausnitzii counts were expressed as log10 values per gram feces. The largest and earliest study examining the relationship between F. prausnitzii reduction and IBD was conducted in Germany by Swidsinski et al. [ F. prausnitzii with high concentration was counted within a 10∗10 μm area of the microscopic field representative of the region of interest. F. prausnitzii with uneven distribution or overall low concentrations was enumerated within larger areas of 100∗100 μm.The characteristics of the included studies are summarized in Tables i et al. . The aut F. prausnitzii counts after denaturing gradient gel electrophoresis (DGGE) by real-time PCR (RT-PCR) [ F. prausnitzii cannot be cultured owing to its requirement for a complex anaerobic environment [8 of the included studies confirmed the differences in the presence or intensity of(RT-PCR) –18, 20. ironment . By RT-Pironment . F. prausnitzii counts. Wang et al. found sharply decreased F. prausnitzii in the feces of active CD and UC patients [ F. prausnitzii in active CD patients compared to CD patients in remission [Three of the included studies commented on the activity of IBD andpatients . Sokol eemission , 19. F. prausnitzii counts before and after treatment by an element diet [1 of the included studies examined theent diet . It sugg F. prausnitzii population after relapse Varela et al. found low counts of F. prausnitzii were associated with less than 12 months of remission and more than 1 relapse/year [1 of the included studies reported the relationship between the maintenance of clinical remission and the recovery of the F. prausnitzii in IBD patients was significantly lower (6.7888 ± 1.8875) log10 CFU/g feces than healthy controls (7.5791 ± 1.5812) log10 CFU/g feces; P < 0.0001. The SMD of F. prausnitzii in IBD patients was −0.94  (I2 = 96%). Furthermore, analysis of the funnel plots for publication bias suggested a possible bias against small studies demonstrating high SMD  . Subgrouhigh SMD . F. prausnitzii reduction and IBD. The majority of recent studies find a higher rate of F. prausnitzii reduction in IBD patients as compared to controls. All of the 11 included studies found significantly lower F. prausnitzii counts in IBD patients versus controls. Our meta-analysis suggests a possible link with the reduction of F. prausnitzii andmisbalance of the intestinal microbiota and IBD patients, especially CD patients with ileal involvement. The levels of F. prausnitzii were extremely low in two studies [ F. prausnitzii counts compared to stools [ F. prausnitzii in CD [ Faecalibacterium in pediatric CD patients compared with controls, which suggested a more dynamic role for this organism than previously described in adult IBD. It is possible that the microbial signature of pediatric IBD is distinct from adult disease. Furthermore, the early host and microbiota response to IBD may induce proliferation of F. prausnitzii to reverse the inflammatory change, which still remains to be explained.Our systematic review and meta-analysis of the literature has identified recent studies examining the relationship between studies , 28. Wano stools . Howeverii in CD . They re F. prausnitzii against IBD have been proposed. Duncan et al. demonstrated that the major end products of glucose fermentation by F. prausnitzii strains are substantial quantities of butyrate [κB activity and hyperacetylation of histones [ F. prausnitzii could induce relatively low amounts of IL-12 and large amounts of IL-10 and Tregs in epithelial and PBMC models to restrain the progression of inflammation [ F. prausnitzii led to significantly lower IL-12 and IFN-γ production levels and higher secretion of IL-10 in vitro peripheral blood mononuclear cells [Mechanistic theories of microbial etiopathogenesis between the possible protective benefit ofbutyrate . Butyratbutyrate . Butyratbutyrate . Therefohistones . Furtherammation . While Sar cells . F. prausnitzii reduction and IBD found in the literature has several limitations. Most of the studies did not comment on the participants' previous history of treatment such as antibiotics, probiotics, or prebiotics which may influence the intestinal microbiota. It is therefore possible that study participants had been treated for dysbiosis prior to entering the study, thereby producing a false F. prausnitzii reduction. Additionally, we did not evaluate confounding factors including diet and smoking in our study, even though these factors might reduce F. prausnitzii levels and faecal butyrate values [The data on the incidence ofe values . Further F. prausnitzii counts would be initiated. In patients found to have F. prausnitzii reduction, probiotics or prebiotics may be used to restore the “ecological balance” of intestinal microbiota. Dörffel et al. reported rifaximin was associated with an increased level of F. prausnitzii [ F. prausnitzii [ F. prausnitzii reduction and the initiation and perpetuation of inflammatory bowel disease has yet to be defined. Healthy controls who are age- and sex-matched to the IBD group would be selected from the same area as the IBD group and tested for F. prausnitzii by the same method. In both groups, a thorough history examining previous treatment such as antibiotics, probiotics, or prebiotics, steroids, 5-aminosalicylates (5-ASAs), and tumor necrosis factor alpha (TNF-α) antibody would be obtained. Prideaux et al. reported that regardless of ethnicity or geography, Crohn's disease resulted in reduced bacterial diversity. However, in ulcerative colitis, diversity was reduced in Chinese subjects only. It suggested that ethnicity might also play an important role in the pathogenesis of IBD [Future studies should address these limitations. After confirming the diagnosis of IBD through the endoscopic and histological findings, PCR-DGGE, T-RFLP, or FISH forusnitzii . Other susnitzii . The mecs of IBD . F. prausnitzii against the development of IBD. However, significant variation among the studies and the possibility of publication bias limit the certainty of this association. Therefore, further treatment such as probiotics or prebiotics to increase the levels of F. prausnitzii in IBD are lead to attempts. If F. prausnitzii is found to indeed protect against IBD, we can approach the treatment such as supplementing the microorganisms that produce butyric acid.In summary, our meta-analysis and systematic review suggest a possible protective benefit of


Allochromatium vinosum formerly Chromatium vinosum is a mesophilic purple sulfur bacterium belonging to the family Chromatiaceae in the bacterial class Gammaproteobacteria. The genus Allochromatium contains currently five species. All members were isolated from freshwater, brackish water or marine habitats and are predominately obligate phototrophs. Here we describe the features of the organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of a member of the Chromatiaceae within the purple sulfur bacteria thriving in globally occurring habitats. The 3,669,074 bp genome with its 3,302 protein-coding and 64 RNA genes was sequenced within the Joint Genome Institute Community Sequencing Program. Gammaproteobacteria whereas chemotrophic sulfur-oxidizing bacteria are found in four classes  of the Proteobacteria. Strain DSM 180T (= ATCC 17899 = D = NBRC 103801) is the type strain of the species Allochromatium vinosum, which belongs to the Chromatiaceae, one of currently five families in the order Chromatiales. Species belonging to the families Chromatiaceae and Ectothiorhodospiraceae are mainly anoxygenic photolithoautotrophic bacteria, which are able to oxidize various sulfur compounds. Anoxygenic purple sulfur bacteria like A. vinosum flourish wherever light reaches sulfidic water layers or sediments and often occur as dense accumulations in conspicuous blooms in freshwater as well as in marine aquatic ecosystems. Here, they are major players in the reoxidation of sulfide produced by sulfate-reducing bacteria in deeper anoxic layers. In contrast to anoxygenic purple sulfur bacteria of the family Ectothiorhodospiraceae and the only very distantly related green sulfur bacteria, members of the family Chromatiaceae like A. vinosum store sulfur globules inside of the cells when oxidizing sulfide or thiosulfate. They have this trait in common with a large number of environmentally important chemotrophic sulfur oxidizers like Beggiatoa or the sulfur-oxidizing bacterial symbionts of marine animals like Riftia pachyptila or Olavius algarvensis. Anoxygenic purple sulfur bacteria are also important primary producers of fixed carbon (up to 83% of primary production in lakes can be anoxygenic) [2 is fixed at the expense of the energy derived from the virtually unlimited and environmentally safe source of sunlight. Simultaneous with the large scale conversion of CO2 into organic compounds, purple sulfur bacteria oxidize reduced sulfur compounds and use these as photosynthetic electron donors [Alphaproteobacteria outnumber purple sulfur bacteria. In addition to their environmental importance, purple sulfur bacteria like A. vinosum have a long tradition of biotechnological application not only in waste remediation and removal of toxic compounds, e.g. odorous sulfur compounds like sulfide or even explosives [Anoxygenic purple sulfur bacteria are plosives , but alsplosives -8 and thAllochromatium species were isolated from freshwater, brackish water and marine habitats. A. vinosum is environmentally abundant and occurs not only in pelagic communities but also in littoral sediments like sandy beaches, salt marches or intertidal mud flats. Here we present a summary classification and a set of features for A. vinosum strain DSM 180T, together with the description of the complete genomic sequencing and annotation.Strains of all Allochromatium [A. vinosum, A. minutissimum,A. warmingii, A. phaeobacterium and A. renukae. A. vinosum DSM 180T in a 16S rRNA based maximum likelihood phylogenetic tree. Based on 16S rRNA gene sequences the closest related type strain is A. minutissimum DSM 1376T with 98.4% sequence identity, while the other type strains of the genus Allochromatium share 93.8-97% sequence identity.There are five described species currently belonging to the genus romatium , Table 1A. vinosum are Gram stain negative, rod shaped and about 2.0 µm x 2.5–6 µm in size [Allochromatium. For example the G + C content of A. vinosum (64.3%) and A. warmingii (55.1-60.2%) differs up to 10 mol % G + C content of the DNA. Cells of all species are motile and contain internal membrane structures of a vesicular type. The main carotinoid synthesized by A. vinosum and A. minutissimum belongs to the group of spirilloxanthins, whereas A. phaeobacterium and A. warmingii produce rhodopinals and A. renukae lycopenes, respectively. Optimal growth of A. vinosum is achieved within a temperature range between 25-35 °C and a pH range between 7.0-7.3, respectively [Cells of  in size . There iectively .A. vinosum is an ecologically significant, typically dominant inhabitant of intertidal sediments, i.e. a fluctuating environment in which redox conditions change rapidly within hours. A. vinosum has adapted to this environment and developed high metabolic versatility. While A. vinosum and A. minutissimum are capable of growing both photolithotrophically and chemolithotrophically the remaining species are obligate phototrophs. Photolithoautotrophic growth of A. vinosum occurs with hydrogen, sulfide, polysulfide, thiosulfate, sulfur and sulfite as electron donors. Light energy is used to transfer the electrons of these different compounds to the level of the more highly reducing electron carriers NAD(P)+ and ferredoxin for reductive carbon dioxide fixation. Photoorganoheterotrophic growth occurs with formate, acetate, propionate, butyrate, pyruvate, fumarate, succinate, malate and glycolate as organic electron donors. At reduced oxygen partial pressure even chemoorganoheterotrophic and chemolithoautotrophic growth in the dark is possible with oxygen as the terminal electron acceptor [A. vinosum and A. minutissimum assimilate sulfate. This versatility is not shared by other anoxygenic phototrophic organisms like the green sulfur bacteria (Chlorobiaceae).Most purple sulfur bacteria grow preferentially by photolithoautotrophic oxidation of reduced sulfur compounds. However, acceptor . Under sThis organism was selected for sequencing on the basis of its environmental abundance and importance, its capability to produce hydrogen and its accessibility by manipulative genetics for biotechnology. The genome project is deposited in the Genomes OnLine Database  and the A. vinosum DSM 180T was grown anaerobically in the light in RCV medium [V medium  at 30°C.V medium . BrieflyAllochromatium vinosum DSM 180 was sequenced at the Joint Genome Institute (JGI) using a combination of Illumina [The genome of Illumina  and 454 Illumina . An IlluIllumina . IlluminIllumina , and theIllumina  was usedIllumina -37 in thIllumina , or sequGenes were identified using Prodigal  as part The 3,669,074 bp genome consists of a 3,526,903 bp chromosome and two plasmids of 102,242 bp and 39,929 bp, respectively. The genome exhibits an overall G + C content of 64.19% . Of the A. vinosum contains two extrachromosomal elements of 102,242 bp (pALVIN01) and 39,929 bp (pALVIN02), respectively, in accordance with previous analyses performed in our laboratory [et al. 1995 is not present [boratory . A third present . None ofA. vinosum employs type II reaction centers to convert light energy into electrochemical energy. Three subunits of the photosynthetic reaction center, pufC, pufM and pufL are clustered and co-transcribed together with three sets of pufA and pufB genes encoding for light-harvesting complex (LH1) apoproteins (Alvin_2547-2555) [pufH, is located upstream, next to two genes encoding for photosynthetic complex assembly proteins and a hypothetical protein that probably constitutes an additional complex assembly protein (Alvin_2634-2637). Blast searches indicate that the genome contains six individual sets encoding for LH2 apoproteins, Alvin_0703-0706 and Alvin_0708-0709, which constitute one cluster together with lux genes, Alvin_2576-2579 and Alvin_2759-Alvin_2760. High copy numbers of different light harvesting subunits might allow adapting to alternating growth conditions such as light intensity or temperature [A. vinosum belong to the group of spirilloxanthins. Genes necessary for spirilloxanthin biosynthesis starting from the C-5 compounds dimethylallyl-PP and isopentyl-PP are located next to each other from Alvin_2564 to Alvin_2570. A. vinosum utilizes only one type of bacteriochlorophyll (BChl), namely BChla. Genes necessary for conversion of the heme biosynthesis intermediate protoporphyrin IX into Bchla are partly distributed over the genome .47-2555) . Subunitperature . The mai1 complex back to the reaction center. In most cases this feature is mediated by mobile, soluble electron-carrying proteins located in the periplasm. In non-sulfur purple bacteria, the high potential cytochrome c2 represents the main diffusible electron carrier. However, in A. vinosum cytochrome c2 function is replaced by other high potential c-type cytochromes and a high potential iron sulfur protein (HiPIP) [A. vinosum [Chromatiaceae [A. vinosum reveals the genetic information for several high potential c-type cytochromes. Alvin_1694 encodes for cytochrome c8 (c551) that is involved in transferring electrons to the photosynthetic reaction center [8 is slightly higher under autotrophic conditions with Na2S and Na2S2O3 than in presence of organic compounds [Rubrivivax gelatinosus [A. vinosum appears at least one cytochrome c8 isoenzyme, namely Alvin_1093 encodes for a cytochrome that is part of flavocytochrome c. The protein expressed by Alvin_2879 reveals high similarities to the flavocytochome c diheme subunit [4 of Thiocapsa roseopersicina [A. vinosum [8 and blast searches reveal most similarities to a cytochrome c553 from Magnetospirillum gryphiswaldense MSR-1.During photosynthesis, diffusible electron carriers maintain light-driven cyclic electron flow by channeling electrons from the cytochrome bc (HiPIP) . HiPIP i (HiPIP)  and is a vinosum ,53 and oatiaceae -56. It oatiaceae . Besidesn center . It appeatinosus  in A. vi subunit  and its ersicina . It is r vinosum . Cytochr vinosum  with no  vinosum  but also vinosum  in order vinosum . BesidesA. vinosum possesses two complete sets of genes encoding for large and small RuBis-CO subunits namely RbcA/ RbcB and RbcS/ RbcL represented by Alvin_1365/1366 and Alvin_2749/2750, respectively [A. vinosum under standard photolithoautotrophic growth conditions [rbcAB belong to form IAq RuBis-CO genes that are typically associated with cbbQ genes that encode for proteins affecting activity of RuBis-CO [rbcAB [rbcSL represent form IAc RuBis-CO genes that are always associated together with a cluster of α-carboxysome genes [Hydrogenovibrio marinus revealed a preferential expression of RuBis-CO form IAc at low CO2 concentrations, whereas expression of form IAq predominates at high CO2 concentrations. Thus RuBis-CO RbcSL in co-occurrence with carboxysomes might allow A. vinosum to grow at very low CO2 concentration. But so far carboxysomes have never been reported for A. vinosum. Interestingly, downstream of rbcL a second gene for a small subunit of RuBis-CO is found but its role during carbon dioxide fixation still has yet to be determined. Besides the RuBis-CO genes dedicated to carbon fixation, A. vinosum harbors a gene (Alvin_2545) encoding for a form IV RuBis-CO-like protein (RLP). These types of RuBis-CO enzymes do not catalyze ribulose 1,5-bisphosphate dependent CO2 fixation but might be involved in sulfur metabolism and stress response [A. vinosum is still not resolved.In a wide range of autotrophic organisms, the ability to grow autotrophically with carbon dioxide as the sole carbon source is due to the enzyme ribulose 1,5-biphosphate carboxylase/oxygenase (RuBis-CO). ectively . RuBis-Cnditions . AccordiRuBis-CO . UpstreaO [rbcAB . RuBis-Cme genes . Expressresponse ,73 as weresponse . The rolA. vinosum during photolithoautotrophic growth. Light energy is used to transfer electrons from reduced sulfur compounds to the level of the more highly reducing electron carriers NAD(P)+ and ferredoxin for reductive carbon dioxide fixation. The most abundant reduced sulfur component in the habitat of A. vinosum is sulfide but up to the time of publication of this report, it is still not clear which enzymes are responsible for sulfide oxidation in A. vinosum. One possibility is the oxidation via a soluble periplasmic flavocytochrome c. The protein is a heterodimer consisting of a 21 kDa di-heme cytochrome c subunit (FccA) and a 46 kDa flavin-binding subunit (FccB) [A. vinosum by Alvin_1093 and Alvin_1092, respectively. Flavocytochrome c catalyzes the oxidation of sulfide to sulfur or polysulfides in vitro using soluble c-type cytochromes as electron acceptors [in vivo is still not resolved. Mutants in which the genes fccAB are inactivated by a kanamycin cassette oxidize sulfide with rates similar to the wild type [Chlorobium luteolum (formerly Pelodictyon luteolum), and purple sulfur bacteria, e.g. Thiocapsa roseopersicina, Thiococcus pfennigii (formerly Thiocapsa pfennigii) and Allochromatium warmingii (formerly Chromatium warmingii), do not produce flavocytochrome c, which is an additional hint that flavocytochrome c is not essential for sulfide oxidation [The capability to oxidize reduced sulfur compounds is the central feature of cceptors  but the ild type . Some suA. vinosum [in vitro for Rhodobacter capsulatus [A. vinosum (Alvin_1195 and Alvin_2145). Alvin_2145 represents a SQR of type IV [Chlorobaculum tepidum [sqrD genes are present in members of the Chromatiaceae  while they are absent in those species of the family Ectothiorhodospiraceae that produce exclusively extracellular sulfur globules [C. tepidum is important for growth at high sulfide concentrations [Other candidate proteins for oxidizing sulfide are sulfide:quinone oxidoreductases (SQRs). This type of enzyme catalyzes the oxidation of sulfide with an isoprenoid quinone as electron acceptor. SQR activity was shown to occur biochemically in membrane-bound form in  vinosum . Polysulpsulatus  that migpsulatus  or enzympsulatus  to sulfu type IV , of whic tepidum ,80. A coglobules . The enztrations .sox gene cluster of the chemolithoautotrophic Alphaproteobacterium Paracoccus pantotrophus comprises 15 genes that encode proteins involved in the oxidation of thiosulfate, whereas the protein products of 7 genes soxXYZABCD are sufficient for oxidizing thiosulfate in vitro [A. vinosum, sox genes are separated into three gene clusters. Cluster one comprises Alvin_2108 to Alvin_2112. However, only Alvin_2011 and Alvin_2012 encode for Sox proteins, namely SoxY and SoxZ. The second gene cluster extends between Alvin_2165 and Alvin_2167 with Alvin_2167 encoding for SoxB. The third gene cluster includes Alvin_2168 to Alvin_2182, where SoxX, SoxA, SoxK and SoxL are encoded by Alvin_2168 to Alvin_2171. The additional genes of cluster three mainly encode for cytochrome c biogenesis proteins. The Sox protein complex is localized in the periplasm. In A. vinosum SoxCD activity of P. pantotrophus probably is replaced by sulfur transferase activity of SoxL transferring the sulfane atom for storage to sulfur globules, thus regenerating the cysteine residue of SoxY [The in vitro ,82. In A of SoxY . SoxK acUnder slightly acidic conditions thiosulfate is increasingly oxidized to tetrathionate via thiosulfate dehydrogenase encoded by Alvin_0091 . This pe-) and SO32-in vitro. In addition to SoxL, six other genes are annotated that might encode rhodaneses, namely Alvin_0258, Alvin_0866, Alvin_0868, Alvin_1587, Alvin_2599 and Alvin_3028. While Alvin_0258 encodes a signal peptide for periplasmic localization Alvin_3028 is proposed to encode a transmembrane protein. The remaining proteins lack both a signal peptide and transmembrane domains and are therefore expected to be cytoplasmic. Roles for these proteins in dissimilatory sulfur metabolism are currently unclear.Rhodaneses (thiosulfate sulfurtransferases) are enzymes that catalyze the transfer of the sulfane sulfur atom of thiosulfate to cyanide producing thiocyanate , a member of the heme-copper oxidase family. Cytochrome 3cbb oxidase is characterized by its ability to maintain catalytic activity under low oxygen concentration and the capability to translocate protons. The genomic arrangement corresponds to the well characterized 3cbb oxidase of Rhodobacter capsulatus [Besides phototrophic growth, ntration . The genBacteria . The funubunit I . Furtherpsulatus .A. vinosum reveals the presence of four individual NiFe hydrogenases. The typical protein structure of NiFe hydrogenases is heterodimeric and the correct folding, maturation and incorporation of ligands require accessory proteins. The membrane bound hydrogenase of A. vinosum DSM 185 encoded by Alvin_2036 and Alvin_2039 in strain DSM 180T is well characterized [hydS and hydL are separated by an intergenic sequence encoding a membrane-bound b-type cytochrome and a soluble iron-sulfur protein, respectively, with similarities to DsrM and DsrK [hynSL (former hydSL) in the photosynthetic bacterium Thiocapsa roseopersicina BBS, also belonging to the family Chromatiaceae [A. vinosum encodes for a second membrane associated hydrogenase that is highly similar to T. roseopersicina HupSLC. In A. vinosum, hupC (Alvin_2307) encodes for a b-type cytochrome, hupL (Alvin_2308) and hupS (Alvin_2309) for large and small hydrogenase subunits, respectively. While the accessory hupDHI genes are required for hydrogenase biosynthesis in T. roseopersicina [A. vinosum only hupD (Alvin_2306) is available. Hup hydrogenase of T. roseopersicina is also membrane -bound facing the periplasm, inducible and is involved in the oxidation of H2 occurring in the environment or resulting from nitrogenase activity. The b-type cytochrome represented by HupC participates in channeling electrons into the quinone pool [T. roseopersicina and A. vinosum Hup hydrogenases might result in at least a similar localization and function. A soluble hydrogenase was recently characterized in A. vinosum DSM 185 [T. roseopersicina [A. vinosum DSM 180T, Hox hydrogenase is encoded by the genes Alvin_1864 to Alvin_1868 followed by a gene (Alvin_1869) encoding for a hydrogenase maturation protein, namely HoxW. In T. roseopersicina, HoxEFUYH seems to be connected to sulfur metabolism and dark and photofermentative processes [Thermodesulfovibrio yellowstonii DSM 11347 and Chlorobium tepidum, but the exact function and localization still has to be determined. Besides hydrogenase gene clusters, there are several genes annotated for hydrogenase maturation distributed all over the genome such as Alvin_0054, Alvin_1451, Alvin_1958, Alvin_2766 and Alvin_2797 to Alvin_2799. A. vinosum is able to use hydrogen both for phototrophic and chemotrophic growth [Hydrogenases are oxidoreductases with the ability either to oxidize hydrogen as energy source or to produce hydrogen as an electron sink. According to their metal content, hydrogenases are divided into NiFe, FeFe and iron sulfur free hydrogenase classes. The genome of cterized . The strand DsrK ,106. Genatiaceae . Hyn hydatiaceae ,109. Besersicina , in A. vone pool ,109. Hig DSM 185  belonginersicina ,113. Theersicina . In A. vc growth , but theA. vinosum belongs to the diazotrophic bacteria encoding for iron-molybdenum dinitrogenase (nifDK) and dinitrogenase reductase (nifH). Genes involved in nitrogen fixation are spread all over the genome but always aggregated in gene clusters. The genome reveals that regulation takes place at least on two levels. Level one comprises the Ntr system consisting of the two component system NtrB/ NtrC (Alvin_2998/ Alvin_2997), nitrogen regulatory protein P-II (Alvin _0793) and the bifunctional uridylyltransferase/ UMP-removing enzyme of P-II GlnD (Alvin_2042), that is located in the same gene cluster as the membrane-bound hydrogenase HydSL and might reflect a role of this hydrogenase in metabolizing H2, that is released during nitrogen fixation. The NtrB/ NtrC system regulates transcription of the NifA protein (Alvin_1291) required for transcription of all nif genes [Bradyrhizobium japonicum [A. vinosum, which is in agreement with observations made for Halorhodospira halophila [H. halophila the A. vinosum genome reveals three distinct rnf gene clusters . The Rnf complex was shown to play a role in nitrogen fixation as overproduction of the rnf operon increased nitrogenase activity in Rhodobacter capsulatus [Acetobacterium woodii [A. vinosum is entirely unclear at this point in time. Gene arrangement in cluster one with a gene encoding for a sodium/ hydrogen exchanger (Alvin_0576) for example does not exclude a function similar to that in A. woodii in generating a sodium gradient across the membrane. There are no obvious genes present in A. vinosum mediating dissimilatory or assimilatory nitrate reduction despite a single gene encoding for nitric oxide reductase subunit B (Alvin_0931) and the two component regulatory system NarX and NarL (Alvin_1057 and Alvin_1058) for sensing nitrite and nitrate.if genes . Usuallyaponicum  or NifA aponicum . Neitheralophila . Perhapsalophila ,119. Intpsulatus . Furtherm woodii . The rolChromatiaceae and Ectothiorhodospiraceae, A. vinosum is able to grow photoorganoheterotrophically and therefore is able to assimilate sulfate as sulfur source. Assimilatory sulfate reduction starts with the uptake of sulfate from the environment by an ABC transporter encoded by Alvin_2443 to Alvin_2441 and annotated as cysTWA [cysDN [cysH) and sulfite reductase (cysI) (Alvin_2447 and Alvin_2446) for incorporation in cysteine for further utilization or shuttled into a second pathway. Here, APS is initially phosporylated to phosphoadenosine phosphosulfate (PAPS) via APS kinase (CysC) encoded by Alvin_1127 [Assimilatory sulfate reduction occurs in a variety of phototrophic purple bacteria. In contrast to more specialized species within the s cysTWA . DownstrA [cysDN . APS is vin_1127 .


Homologous recombination, together with selection, laid the foundation for traditional plant breeding. The recombination process that takes place during meiotic cell division is crucial for the creation of novel variations of highly desired traits by breeders. Gaining control over this process is important for molecular breeding to achieve more precise, large-scale and quicker plant improvement. As conventional ubiquitous promoters are neither tissue-specific nor efficient in driving gene expression in meiocytes, promoters with high meiotic activities are potential candidates for manipulating the recombination process. So far, only a few meiotically-active promoters have been reported. Recently developed techniques to profile the transcriptome landscape of isolated meiocytes provided the means to discover promoters from genes that are actively expressed in meiosis.Arabidopsis. Characterization of regulatory regions revealed that these meiotically-active promoters possessed conserved motifs and motif arrangement. Some of the promoters unite optimal properties which are invaluable for meiosis-directed studies such as delivering specific gene expression in early meiosis I and/or meiosis II. Furthermore, the examination of homologs of the corresponding genes within green plants points to a great potential of applying the information from Arabidopsis to other species, especially crop plants.In a screen for meiotically-active promoters, we examined ten promoter sequences that are associated with novel meiotic candidate genes. Each promoter was tested by expressing a GFP reporter gene in We identified ten novel meiotically-active promoters; which, along with their homologs, are prime candidates to specifically drive gene expression during meiosis in plants and can thus provide important tools for meiosis study and crop breeding. Arabidopsis and rice . -26. cis-We first analyzed known plant regulatory elements in these promoter sequences by using the PLACE collection. We considered CREs within one kb upstream of these genes, since the effective regions of individual promoters are still unknown, and the normalized length also facilitated a better comparison between different sequences. The names of 17 broadly distributed CREs which appeared in all 12 promoters, and their occurrence in each promoter are shown in Figure Several enriched PLACE motifs are universal or structural CREs that seem also common in meiotically-active promoters, such as TATABOX5 , POLASIGp < 0.1, Figure To complement searches for possible motifs that have a statistically overrepresented frequency in the genome, we computationally analyzed the twelve promoters using Pscan . As a rep = 1.52751e-02) and MA0045.1 (p = 4.32294e-02) are binding sites for the chromatin-associated proteins HMG-1 and HMG-I/Y .Furthermore, we used MEME software to search for possible unknown CREs . Three cArabidopsis thaliana alone while AT3G07250 has at least a putative homolog in Arabidopsis lyrata. The other 8 genes are broadly distributed in different taxa, mostly found in flowering plants (Angiosperm), including many important crop species such as soybean (Glycine max), maize (Zea mays), rice (Oryza sativa) and Sorghum (Sorghum bicolor). Therefore, we assume that for these genes, there is a higher possibility that similar regulatory networks may be shared among different flowering plants.To know whether these meiotically-active genes have homologous sequences in other plant species, especially in crops, we investigated the “family history” of the 12 genes whose promoters were characterized in this study. As is shown in Figure cis-regulatory elements that are present in all promoters. Results for meiosis-I, during which homologous recombination occurs, are of special interest to predict homologous recombination-related promoters in crop plants. We have tested 15 promoters that are associated with candidate meiosis genes that were discovered by a previous RNA-Seq experiment on isolated meiocytes . Howeveng cells . Furtherr system , which ing roots . Thus, tThe novel candidate meiotically-active promoters from this study should provide more powerful tools for a strict or specified meiotic expression. In our experimental setup, we first chose genes that are highly expressed in meiocytes . We thenpAT1G15320:GFP only showed a specific fluorescence signal in dissociated meiocytes but not in meiocyte cell columns  or meiosis-II (pAT1G15320). For molecular engineering, expressing genes during prophase-I, the stage of recombination, will be of utmost interest.Interestingly, we observed diversified expression patterns in different cell types of meiocytes (cell columns and dissociated meiocytes) resulting from the examined promoters Table . Transgecolumns  , which sBrowser, ]. There Given the complexity and a relatively long duration of meiosis  , the temAtDMC1 and MS5. 1000 bThe authors declare that they have no competing interests.cis-element studies and drafted the manuscript. J.L. and C.C. performed the GFP reporter assay. A.D.F, I.E.L. and J.M. performed the homologs study. S.D. performed the endoplasmic reticulum marker assay and helped to draft the manuscript. T.L. helped in plant transformation and plant care. All authors read and approved the final manuscript.C.C. and E.F.R. conceived the project, designed the experiments and helped to draft the manuscript. J.L. carried out the molecular genetic studies, Figure S1.GFP signals in meiocytes driven by meiotically-active promoters. (PDF 2536 kb).Click here for fileFigure S2.The position of GFP spots and nuclear DNA/chromosomes in pMS5: GFP transformants. (PDF 636 kb).Click here for fileFigure S3.GFP signals in both somatic and meiotic cells driven by a constitutive promoter d35S. (PDF 388 kb).Click here for fileTable S1.PLACE motifs enriched in promoters of meiotically-active genes. (PDF 12 kb).Click here for fileTable S2.Statistically overrepresented motifs in the promoters of meiotically-active genes. (PDF 8 kb).Click here for fileFigure S4.Sequence logos of novel motifs in meiotically-active promoters found by MEME. (PDF 213 kb).Click here for fileTable S3.A list of homologous recombination-related genes for comparative promoter analysis. (PDF 7 kb).Click here for fileFigure S5.Sequence logos of novel motifs in promoters of homologous recombination-related genes found by MEME. (PDF 68 kb).Click here for fileTable S4.Motifs enriched specifically in 15 known meiosis-related promoters. (PDF 7 kb)Click here for file


Schistosoma mansoni genome and predicted proteome sequences were recently published providing the opportunity to identify new drug candidates. Eukaryotic protein kinases (ePKs) play a central role in mediating signal transduction through complex networks and are considered druggable targets from the medical and chemical viewpoints. Our work aimed at analyzing the S. mansoni predicted proteome in order to identify and classify all ePKs of this parasite through combined computational approaches. Functional annotation was performed mainly to yield insights into the parasite signaling processes relevant to its complex lifestyle and to select some ePKs as potential drug targets.Schistosomiasis remains an important parasitic disease and a major economic problem in many countries. The S. mansoni predicted proteome, through sequence similarity searches using HMMs (Hidden Markov Models). Amino acid sequences corresponding to the conserved catalytic domain of ePKs were aligned by MAFFT and further used in distance-based phylogenetic analysis as implemented in PHYLIP. Our analysis also included the ePK homologs from six other eukaryotes. The results show that S. mansoni has proteins in all ePK groups. Most of them are clearly clustered with known ePKs in other eukaryotes according to the phylogenetic analysis. None of the ePKs are exclusively found in S. mansoni or belong to an expanded family in this parasite. Only 16 S. mansoni ePKs were experimentally studied, 12 proteins are predicted to be catalytically inactive and approximately 2% of the parasite ePKs remain unclassified. Some proteins were mentioned as good target for drug development since they have a predicted essential function for the parasite.We have identified 252 ePKs, which corresponds to 1.9% of the S. mansoni ePKs through combined similarity and phylogenetic-based approaches. As we continue this work, we will highlight the biochemical and physiological adaptations of S. mansoni in response to diverse environments during the parasite development, vector interaction, and host infection.Our approach has improved the functional annotation of 40% of  Schistosoma genus, remains an important parasitic disease and a major health economic problem in many tropical and subtropical countries. Schistosomes have a complex life cycle that includes six different stages  in different environments: water, definitive host  and intermediate host (snail). During parasite development, signals from the environment are sensed and stimulate physiological, morphological and, biochemical adaptations. Oils are shown to stimulate cercarial penetration; hormones and exposure to the snail haemolymph trigger specific physiological adaptations [Human schistosomiasis caused by blood fluke parasites of ptations -3. The fptations ,5. It haptations  and the ptations -8. ThereSchistosoma mansoni require specific extracellular signals that must be integrated to generate an appropriate response from the sensory receptor via intracellular proteins [2+ and diacylglycerol.The maintenance of homeostasis and complex cellular adaptations in proteins . Signal Protein kinases (PKs) play a central role in mediating intracellular signals by adding a phosphate group from ATP or GTP to an amino acid residue leading to a conformational change in the target protein that will switch its activation status . Most PKPKs are classified into two superfamilies containing the eukaryotic or conventional protein kinases (ePK) that share a conserved catalytic domain, and the atypical protein kinases (aPKs) Table . The cat®, that induces a conformational change in PTK and mimics substrate binding and therefore prevents activation by upstream kinases [S. mansoni [PKs are considered druggable targets from the medical and chemical viewpoints as a growing number of PKs inhibitors have been developed and approved for treatment of different human disease . An exam kinases . Beyond  mansoni -21.Schistosoma resistant strains is a real possibility [S. mansoni genome and large-scale transcriptome projects have yielded crucial information to the identification of new candidate drugs [S. mansoni predicted proteome data in order to identify all ePKs encoded in the genome of this parasite. For this purpose, we combined computational approaches such as sequence similarity searches using Hidden Markov Models (HMMs) and distance-based phylogenetic analyses. The functional annotation was performed mainly to yield insights into the signaling process related to the complex lifestyle of S. mansoni.The current schistosomiasis treatment frequently does not cure 100% of those treated in high-risk communities and the emergence of sibility -25. Thuste drugs -29. UndeS. mansoni, defined as the ePKinome, was identified by searching the parasite predict proteome with a HMM profile of the ePK catalytic domain of five selected organisms. This analysis revealed 252 ePKs in the S. mansoni predicted proteome, representing 1.9% of the total proteins encoded in the parasite genome. Although the total number of protein kinases found across the analyzed species varies greatly (from 82 to 503), the percentage values in respect to the genomes of protozoan and helminth parasites as well as other eukaryotes from KinBase range only between 1.5 to 2%  function, metabolic pathways, structural information, PDB structures, and links to external databases such as the TDR database [S. mansoni genes like antigenicity, essentiality, phenotypes and associated compounds (druggability).Amino acid sequences corresponding to the conserved catalytic domain of ePKs were aligned by MAFFT  and furtlsewhere ,13,32,32database . Schistodatabase . The TDRS. mansoni proteins have representatives in the main ePK groups. ePKs that do not fall into these groups are categorized as "Other" in which multiple families have been defined. The S. mansoni largest ePK group is CMGC, a feature unique to this parasite, and the smallest group is RGC, a common feature shared with many of the analyzed organisms .function ,12,34. AS. mansoni ePK remain unclassified once they do not have similarity to any known PK family. All these proteins have a truncated catalytic domain probably because of an incorrect protein prediction. The unclassified ePKs from C. elegans, D. melanogaster, H. sapiens and S. cerevisiae range from 19% to about 38% their kinomes.Approximately 2% of the S. mansoni, most AGC proteins belong to PKA (Protein Kinase A) (5 proteins), DMPK (Myotonic Dystrophy Protein Kinase) , PKC (Protein Kinase C) (4 proteins) and PKG (4 proteins) families. Other S. mansoni proteins have only one representative in the remaining AGC families  family  and catalytic domains are usually present in the same polypeptide . The inaxoplasma  and Eime[Eimeria  and PKG asmodium ,39 are earasites . This re2+; PKC Eta and Delta subfamilies containing the novel PKCs  which are regulated by DAG alone; and PKC Iota subfamily, that contain the atypical PKCs (ζ and ι), and are insensitive to both compounds [S. mansoni has representatives in the three main PKC subfamilies mentioned above  but lacks homologs in the Delta subfamily, present in C. elegans, D. melanogaster, M. musculus, and H. sapiens. The two PKC Alpha proteins found in S. mansoni (Smp_128480 and Smp_176360), belong to the PKCβI isoform and were recently characterized [PKC belongs to a large protein family that is classified into four important subfamilies: PKC Alpha subfamily, that contain the conventional PKCs  and are sensitive to diacylglycerol (DAG) and Caompounds ,42. PKC ompounds -45. S. mcterized -48. Bothcterized -48.One protein in AGC group, Smp_157370, remains unclassified. In the phylogenetic tree, this protein appears more closely related to the GRK (G-protein coupled Receptor Kinase) family  is one of the ions most widely used as a second messenger in cellular signaling. A significant portion of calcium-mediated signaling is controlled by calmodulin-binding kinases. Some members of the CaMK group are dependent on the binding of Ca2+/CaM [S. mansoni ePKinome, 32 proteins were classified as CaMK with the vast majority (18 proteins) belonging to the CaMKL  - like family. A similar number was found in other organisms analyzed here , MAPKAPK (MAPK associated protein kinase), MLCK (myosin light chain kinase), and PHK (phosphorylase kinase) families in the CaMK group , although no homologs were identified for the non-smooth muscle vertebrate MLCK through our phylogenetic analysis. This likely reflects the absence of a striated muscle in this parasite.MLCK is a Caht chain . The priht chain . Two disS. mansoni  is a protein that regulates the microtubule cytoskeleton and in the chick is specifically expressed in the developing brain ,53. CASKS. mansoni genome  family is encoded by four genes in humans, only a single CaMK2 gene, with two predicted alternative spliced transcripts, was identified in the ogaster. . The funlization .S. mansoni, a feature that can be explained by the requirement to control cell proliferation and to ensure correct replication and segregation of organelles, which together are essential mechanisms for parasites with a complex life cycle. In the CMGC group, all of the main families are conserved between S. cerevisiae, C. elegans, M. musculus, H. sapiens, and S. mansoni, including CDK (Cyclin Dependent Kinase), MAPK (Mitogen Activated Protein Kinase), GSK (Gycogen Synthase 3 Kinase), CLK (CDC-Like Kinase), SRPK (SR Protein Kinase), CK2 (Cell Kinase 2), and DYRK  , including homologs of all subfamilies   was selected as putative target for drug development after comparative chemogenomics approach [C. elegans [GSK family is represented by 3 proteins in approach . GSK pro elegans .S. mansoni contains nine MAPKs, compared to seven in D. melanogaster and 14 in C. elegans. As shown in Figure D. melanogaster and C. elegans, the MAPK pathways are involved in critical cellular and developmental processes [S. cerevisiae has four distinct MAPK signaling pathways that are likely mediators of responses to pheromone, nutritional starvation, and cellular or osmotic stress [S. mansoni  subfamily. JNK proteins play key roles in human cell function [C. elegans worms [The MAPK signaling pathways are some of the best characterized signaling systems. rocesses ,60. S. cc stress . The MAPi Figure , includii Figure . Each sufunction  and in tns worms . JNK mayS. mansoni, the STE group includes seven STE7 (MEK or MAPKK), two STE11 (MEKK or MAPKKK), and 13 STE20 (MEKKK) kinases  is a Ste20 family protein, recently characterized in S. mansoni, which is able to activate protein MAPK/JNK in human embryonic kidney (HEK) cells as well as in Xenopus oocytes. In addition, imunofluorescence showed that SmSLK was abundant in the tegument of adult schistosomes [S. mansoni predicted proteome  functions in spermatogenesis, and at least half of the proteins in this group are selectively expressed in C. elegans sperm as shown by microarray analysis [S. mansoni is unclear.The two smallest groups found in the C Figure . In conts Figure . The CK1s Figure . S. mansmutagens . One CK1analysis ,73. The S. mansoni kinome contains 15 RTKs and 19 CTKs. The 15 RTK include two InsRs (Insulin Receptors), four EGFRs , two VKRs (Venus Flytrap Kinase Receptors), a representative for Ephs (Ephrin receptors), Ror, CCK4 (Colon Carcinoma Kinase 4), and MUSK (Muscle_Specific kinase) families, besides three unknown receptors.PTKs can be classified, based on the presence or absence of transmembrane domains, into receptor tyrosine kinase (RTK) that relay intracellular signals , and cytS. mansoni, SmIR-1 (Smp_009990) and SmIR-2 (Smp_074030) present distinct functions during parasite development. These two receptors are well clustered within the InsR families but showed to be more divergent than the mammalian and D. melanogaster proteins  was localized in the parasite muscle and perhaps related to muscle development or function [S. mansoni EGFR and the downstream classical ERK pathway , was recently studied [Five  studied . We iden studied .S. mansoni are represented by 11 different families  were recently studied using a Abl specific inhibitor . The results showed an important morphological alteration in adult worms of S. mansoni that led to the death of the parasites [C. elegans contains 42 members of the Fer family, while only a single member, SmFes, was found in S. mansoni. The Fer gene of S. mansoni  exhibits the characteristic features of Fes/Fps/Fer  PTKs. By immunolocalization assays it was shown that SmFes is particularly expressed at the terebratorium of miracidia and tegument of cercaria and schistosomula skin-stage. These findings suggest that SmFes may play a role in signal transduction pathways involved in larval transformation after penetration into intermediate and definitive hosts [The CTKs in arasites . C. elegve hosts ,86.C. elegans that has a large expansion of these proteins and S. cerevisiae that has no protein with similarity to the TK catalytic domain  found in proteins of the TK group . The RGCn Figure . Only thormation . Other pGC group ,89. The rocesses ,90.S. mansoni, the TKL group includes MLK (Miked Lineage Kinases), LISK (Family containing closely related LIMK and TESK sub-families), Raf, RIPK (Receptor Interacting Protein Kinase), STKR (Serine/threonine kinase Receptors for activin and TGFβ ligands), and LRRK (Leucine Rich Repeat Kinase) (type 1 and type 2) families. Of the 19 TKL proteins found in S. mansoni, 15 display greater similarity to the serine/threonine catalytic domain and four (of Raf and MKL/ILK families) to the tyrosine catalytic domain. S. mansoni has no homologous proteins of the IRAK (interleukin-1 (IL-1) receptor-associated kinase) family that is present in C. elegans, B. malayi, D. melanogaster, Homo sapiens, and M. musculus  in C. elegans by RNAi, generate a sterile phenotype, which supports the hypothesis of the involvement of Raf protein in the germline development, somatic gonad development, oogenesis, spermatogenesis, ovulation or fertilization  and it was found to be localized in the parasite surface [S. mansoni predicted proteome and was not experimentally characterized so far  is a membrane receptor that can be divided into two subclasses (Type I and Type II). The type II receptor binds TGFβ and then recruits the type I receptor. The TGFβ type I receptor was cloned in  surface ,92. OtheS. mansoni ePKinome do not fall into any of the eight major groups, but include 20 smaller and conserved families.The Other group consists of a mixed collection of kinases with representatives in higher eukaryotes, including SCY1, NEK , PEK, Haspin, WEE, NAK (Numb-Associated Kinase), ULK (Unc-51 Like Kinase), IRE (Inositol Requiring), PLK (Polo Like Kinases), AUR (Aurora Kinase), and CDC7 (Cell Division Control 7) families  is sufficiently divergent to enable the discrimination of groups, families, and subfamilies Figure .Most ePKs also have a second domain that is involved in protein-protein interaction and allosteric regulation of the catalytic domain . In thisS. mansoni ePKs are multi-domain proteins containing various regulatory and signaling domains tethered to catalytic kinase domains , generating specificity towards cognate substrates and regulators in this parasite.The analysis of the sequence domain data from Pfam  showed t Figures  and 7. IS. mansoni kinases are Pkinase_C (Pfam: PF00433) all found in the AGC group; C1_1 (Pfam: PF00130) found in the AGC and TKL groups; SH2 (PF00017) all found in the TK group; and SH3 (Pfam: PF00018) found in TK and TKL groups. These domains are commonly found in protein kinase families as we observed in other species from KinBase [The most common Pfam accessory domains found in  KinBase .S. mansoni AGC group have the PKinase_C domain associated with the catalytic domain. The C1_1 domain is conserved in N-terminal regions of all PKC proteins of S. mansoni  has an unusual domain combination  are conserved in higher eukaryotes. However, the UPF0061 (Pfam: PF02696) is uncharacterized [S. mansoni CASK protein  and C-terminal CNH domain are usually found in the STE20 families Figure . PBD binS. mansoni CTKs. Fer PTK is usually composed of three domains, FHC domain, SH2, and C-terminal kinase domain as it occurs in Fer proteins of H. sapiens, M. musculus, and D. melanogaster. However, the S. mansoni Fer protein (SmFes - Smp_164810) [C. elegans seems to have lost the N-terminal FHC domain  in the Ephrin recptors (Eph) and Ig domains (Pfam: PF00047) in CCK4 proteins  domains are common found in CTK proteins. SH2 function as regulatory modules of intracellular signaling cascades and it was found in eight out of 19 _164810)  and the In conclusion, the protein architecture, including the accessory domains, may indicate potential protein partners. Signaling roles of schistosome specificities or unusual architectures are of special biological interest.S. mansoni. Together, these proteins represent 1.9% of the proteome and indicate that protein phosphorylation is an important mechanism for regulating the complex life cycle of the parasite. We improve the functional annotation of 40% of S. mansoni ePKs  as described below. Also based on sequence similarity, each predicted protein kinase was manually annotated by integrating data from InterProScan [Functional classification of protein kinases into groups, families, and subfamilies followed the proposed hierarchy described elsewhere ,13,32. PrProScan  and reverProScan  output srProScan . FurtherrProScan . FunctioS. mansoni, amino acid sequences of known protein kinases of five model organisms  were selected. A total of 68 diverse amino acid sequences corresponding to the kinase catalytic domain and sharing less than 50% sequence identity were aligned in MAFFT [http://hmmer.janelia.org from multiple sequence alignments and used for sensitive searches against the S. mansoni proteome [In order to identify potential homologs in in MAFFT  and manuproteome .seqboot with default parameters. For each dataset, it was calculated a distance matrix under the JTT model with gamma-distributed sites by protdist. Next, phylogenies were estimated from distance matrix data adopting the Fitch-Margoliash criterion as implemented in fitch. Finally, the results from the random datasets were summarized by consense, which computes consensus trees by the majority-rule consensus tree method. Phylogenetic trees were visualized and edited using the Tree Figure Drawing Tool - FigTree, version 1.3.1 [Amino acid sequences corresponding to the conserved catalytic domain (Pfam: PF0069 or PF07714) of each group of protein kinases were separately aligned using the default parameters of MAFFT . Multiplon 1.3.1 . Nodes wATP: adenosine triphosphate; cAMP: cyclic adenosine monophosphate; GTP: guanosine triphosphate; NCBI: National Center for Biotechnology Information; RNAi: RNA interference.LFA: carried out the functional annotation and phylogenetic studies, and drafted the manuscript. LAN: designed the phylogenetic studies, performed preliminary phylogenetic studies, coordinated the functional prediction, and co-wrote this manuscript. LGAV and LLS carried out the functional annotation based on sequence similarity search using HMMs. AZ wrote the Perl scripts for data manipulation and provided computational support for this study. JCR designed and performed the HMMs analysis. GO participated in the design and coordination of this study, and co-wrote the manuscript. All authors read and approved the final manuscript.Schistosoma mansoniEukaryotic protein kinases of .Click here for filePhylogenetic analysis of the AGC group. Amino acid sequences of the catalytic domain of the AGC proteins of S. mansoni (blue labels), C. elegans, D. melanogaster, S. cerevisiae, H. sapiens, and B. malayi were used to construct a distance-based phylogenetic tree using PHYLIP programs. Bootstrap values (1000 replicates) equal or higher than 80% are indicated (●) as cutoff values to support the family/subfamily classification. Functional classification is indicated individually in the protein labels. Protein families are highlighted by the vertical bars and include: RSK , PKA (Protein Kinase A), PKG (Protein Kinase G), PDK1(phosphoinositide-dependent protein kinase 1), NDR (nuclear Dbf2-related kinases), DMPK (Myotonic Dystrophy Protein Kinase), MAST (Microtubule Associated Serine/Threonine Kinase), RSKL (RSK-like); PKC (Protein Kinase C), PKN (Protein Kinase N), Akt , SGK (Serum and Glucocorticoid Resposive Kinase), GRK (G-protein coupled Receptor Kinase), YANK (Yet Another Novel Kinase).Click here for fileS. mansoni and model organismsDistribution of some ePKs Families in . S. mansoni proteins were classified according to KinBase [C. elegans, D. melanogaster, S. cerevisiae, and H. sapiens is shown. KinBase  by combiClick here for filePhylogenetic analysis of the CaMK group. Amino acid sequences of the catalytic domain of the CaMK proteins of S. mansoni (blue labels), C. elegans, D. melanogaster, S. cerevisiae, H. sapiens, and B. malayi were used to construct a distance-based phylogenetic tree using PHYLIP programs. Bootstrap values (1000 replicates) equal or higher than 80% are indicated (●) as cutoff values to support the family/subfamily classification. Functional classification is indicated individually in the protein labels. Protein families are highlighted by the vertical bars and include: MAPKAPK (MAP Kinase Associated Protein Kinase), CAMK1 (CAMK family 1), PHK (Phosphorylase Kinase), Trio, DAPK (Death Associated Protein Kinase), MLCK (Myosin Light Chain Kinases), CASK, CAMK2 (CAMK family 2), DCAMKL (Doublecortin and CaMK-Like), PKD (Protein Kinase D), RAD53, Trbl, CAMKL (CAMKL-like).Click here for filePhylogenetic analysis of the CMGC group. Amino acid sequences of the catalytic domain of the CMGC proteins of S. mansoni (blue labels), C. elegans, D. melanogaster, S. cerevisiae, H. sapiens, and B. malayi were used to construct a distance-based phylogenetic tree using PHYLIP programs. Bootstrap values (1000 replicates) equal or higher than 80% are indicated (●) as cutoff values to support the family/subfamily classification. Functional classification is indicated individually in the protein labels. Protein families are highlighted by the vertical bars and include: GSK (Gycogen Synthase 3 Kinase), MAPK (Mitogen Activated Protein Kinase), CK2 (Cell Kinase 2), CDKL (Cyclin Dependent Kinase Like), CDK (Cyclin Dependent Kinase), SRPK , DYRK  Regulated Kinase), CLK (CDC-Like Kinase).Click here for filePhylogenetic analysis of the STE group. Amino acid sequences of the catalytic domain of the STE proteins of S. mansoni (blue labels), C. elegans, D. melanogaster, S. cerevisiae, H. sapiens, and B. malayi were used to construct a distance-based phylogenetic tree using PHYLIP programs. Bootstrap values (1000 replicates) equal or higher than 80% are indicated (●) as cutoff values to support the family/subfamily classification. Functional classification is indicated individually in the protein labels. Protein families are highlighted by the vertical bars and include: STE11 (MAP3K (MAP kinase kinase kinase) genes), STE20 (MAP4K (MAP kinase kinase kinase kinase) genes), STE7 (MAP2K (MAP kinase kinase) genes).Click here for filePhylogenetic analysis of the TK group. Amino acid sequences of the catalytic domain of the TK proteins of S. mansoni (blue labels), C. elegans, D. melanogaster, S. cerevisiae, H. sapiens, and B. malayi were used to construct a distance-based phylogenetic tree using PHYLIP programs. Bootstrap values (1000 replicates) equal or higher than 80% are indicated (●) as cutoff values to support the family/subfamily classification. Functional classification is indicated individually in the protein labels. Protein families are highlighted by the vertical bars and include: JAK, Ack (Activated Cdc42-associated tyrosine kinase), Syk, FAK , VKR, Fer, Sev, ALK (Anaplastic Lymphoma Kinase), InsR (Insulin Receptor), DDR (Discoidin Domain Receptor kinase), Musk (Muscle-Specific Kinase), Ror, Lmr (Lemur Kinase), Eph (Ephrin receptors), Axl ), Ryk, EGFR , FGFR (Fibroblast Growth Factor Receptor), PDGFR, VEGFR, Kin16, Jak (Janus Kinases), Tec, Abl (Abelson murine leukemia homolog), and Src.Click here for filePhylogenetic analysis of the RGC group. Amino acid sequences of the catalytic domain of the RGC proteins of S. mansoni (blue labels), C. elegans, D. melanogaster, S. cerevisiae, H. sapiens, and B. malayi were used to construct a distance-based phylogenetic tree using PHYLIP programs. Bootstrap values (1000 replicates) equal or higher than 80% are indicated (●) as cutoff values to support the family/subfamily classification. Functional classification is indicated individually in the protein labels.Click here for filePhylogenetic analysis of the TKL group. Amino acid sequences of the catalytic domain of the CK1 proteins of S. mansoni (blue labels), C. elegans, D. melanogaster, S. cerevisiae, H. sapiens, and B. malayi were used to construct a distance-based phylogenetic tree using PHYLIP programs. Bootstrap values (1000 replicates) equal or higher than 80% are indicated (●) as cutoff values to support the family/subfamily classification. Functional classification is indicated individually in the protein labels. Protein families are highlighted by the vertical bars and include: IRAK (IL1 Receptor Associated Kinase), RIPK (Receptor Interacting Protein Kinases), LISK (Family containing closely related TESK and LIMK sub-families), MLK (Miked Lineage Kinases), LRRK (Leucine Rich Repeat Kinase), RAF, STKR (Serine/Threonine Kinase Receptors - receptors for activin and TGFb ligands).Click here for file


Mycobacterium tuberculosis, has highly conserved epitope sequences. This raises the question whether there is also a difference in the ability of these pathogens’ epitopes to bind diverse HLA alleles, referred to as an epitope’s binding promiscuity. To address this question, we compared the in silico HLA binding promiscuity of T cell epitopes from pathogens with distinct infection strategies and outcomes of human exposure.The HLA (human leukocyte antigen) molecules that present pathogen-derived epitopes to T cells are highly diverse. Correspondingly, many pathogens such as HIV evolve epitope variants in order to evade immune recognition. In contrast, another persistent human pathogen, M. tuberculosis. We also analyzed promiscuity of epitopes from Streptococcus pyogenes, which is known to exhibit epitope diversity, and epitopes of Bacillus anthracis and Clostridium tetani toxins, as these bacteria do not depend on human hosts for their survival or replication, and their toxin antigens are highly immunogenic human vaccines.We used computer algorithms to predict the binding affinity of experimentally-verified microbial epitope peptides to diverse HLA-DR, HLA-A and HLA-B alleles. We then analyzed binding promiscuity of epitopes derived from HIV and B. anthracis and C. tetani epitopes were the most promiscuous of the group that we analyzed. However, there was no consistent difference or trend in promiscuity in epitopes contained in HIV, M. tuberculosis, and S. pyogenes.We found that Our results show that human pathogens with distinct immune evasion strategies and epitope diversities exhibit equivalent levels of T cell epitope promiscuity. These results indicate that differences in epitope promiscuity do not account for the observed differences in epitope variation and conservation. MHC (major histocompatibility complex) molecules recognize and bind epitopes derived from foreign and self proteins in order to initiate and maintain adaptive immune responses. The HLA (human leukocyte antigen) alleles that encode MHC molecules are extremely diverse: over 2000 HLA class I, and over 600 HLA class II alleles have been identified . In a moMycobacterium tuberculosis, a highly successful persistent human pathogen, has highly conserved epitope sequences [M. tuberculosis employs unique approaches to achieve success as a pathogen.We recently reported that equences . This apIt has also been shown in a variety of pathogens that, even though the adaptive immune response is highly specific, individual HLA class II-restricted peptides -13 and HM. tuberculosis and HIV, since these human-specific pathogens vary in their epitope diversity [Streptococcus pyogenes, which undergoes antigenic variation [Bacillus anthracis and Clostridium tetani.We compared epitope promiscuity in iversity ,19 yet bariation , but is ariation , and we B. anthracis and C. tetani are consistently the most promiscuous, and that there is no consistent pattern of promiscuity between M. tuberculosis, HIV and S. pyogenes. Therefore we find similar levels of epitope promiscuity in very different pathogens, and we discuss the impact of promiscuity on host–pathogen coevolution.We used the computer algorithms NetMHCpan-2.0 and NetMHCIIpan-2.0 to predict the binding affinity of experimentally determined microbial epitopes to HLA-DR, HLA-A, and HLA-B alleles ,23. We thttp://www.cbs.dtu.dk/services/NetMHCpan-2.0/) uses an artificial neural network (ANN) to predict the binding affinity of peptide-MHC (HLA)-I molecule interactions [The NetMHCpan-2.0 algorithm  uses an ANN to predict the binding affinity of peptide-MHC (HLA)-II molecule interactions [The NetMHCIIpan-2.0 algorithm  contains peptidic and non-peptidic antibody and T cell epitope data for humans and other animal species. The database contains HLA binding data from diverse antigenic sources, including allergens and multiple human pathogens. It also provides tools for epitope prediction and analysis.The IEDB  contains data on HIV T cell epitopes, and provides access to tools for epitope prediction and analysis.The HIV Molecular Immunology Database (http://www.ncbi.nlm.nih.gov/projects/gv/mhc/main.fcgi?cmd=init) contains DNA seqeuence and other data related to the human MHC, including anthropology resources regarding the distribution and frequency of specific HLA alleles in multiple populations.The dbMHC , where aff is the IC50 value in nM. Thus the scores range from 0 to 1, with higher scores indicating higher affinity. We used a receiver operating characteristic (ROC) curve to obtain a threshold prediction value to specify which epitope/alleles combinations were predicted to interact. To construct the ROC curves we ran NetMHCpan-2.0 and NetMHCIIpan-2.0 against their published validation datasets. We plotted the true positive rate (TPR) against the false positive rate (FPR) at different thresholds of binding from 0 to 1. We chose a FPR of 0.05, which corresponded to thresholds of 0.29 for netMHCpan and 0.585 for netMHCIIpan, and TPRs of 0.89 for HLA class I and 0.24 for HLA class II.Binding prediction values were obtained from NetMHCIIpan-2.0 for HLA-DR alleles, and NetMHCpan-2.0 for HLA-A and HLA-B alleles. The two methods give the output binding scores as 1 – log curves for both the methods and confirmed that both methods performed significantly better than random guessing (see Methods). Based on the ROC curve we chose threshold levels for netMHCpan and netMHCIIpan such that the false positive rate (FPR) of detecting epitope binding was 0.05 . This coBacillus anthracis and Clostridium tetani, Streptococcus pyogenes, Mycobacterium tuberculosis complex, and Human immunodeficiency virus (HIV). B. anthracis and C. tetani were grouped together in all analyses because we found no significant differences between them in mean epitope promiscuity across all alleles (data not shown). These groups were selected for their biological diversity as indicated above, and because epitope information was readily available for each in the IEDB. We included all epitopes available for each pathogen in each database at the time of analysis. We analyzed all HLA-A and HLA-B, and HLA-DR alleles covered by netMHCpan and netMHCIIpan. We analyzed microbe-derived epitopes in four groups: B. anthracis and C. tetani were most promiscuous across all human HLA-DR, HLA-A and HLA-B alleles  is plotted against the false positive rate (FPR) at different thresholds of binding from 0 to 1. For subsequent analyses we chose a FPR of 0.05, which corresponded to thresholds of 0.29 for netMHCpan and 0.585 for netMHCIIpan, and TPRs of 0.89 for HLA class I and 0.24 for HLA class II.(TIF)Click here for additional data file.Figure S2B. anthracis and C. tetani , S. pyogenes and , HIV (green) and M. tuberculosis complex  across HLA-DR (A), HLA-A (B) and HLA-B (C) alleles. Differences in mean promiscuity are indicated with black bars . Error bars represent the standard error of the mean.Kernel density estimates of epitope promiscuity and graphs of mean HLA allele promiscuity (see Methods) of (TIF)Click here for additional data file.Figure S3B. anthracis and C. tetani (BA and CT), S. pyogenes and (SP), HIV and M. tuberculosis complex (MTBC) across alleles within HLA-A supertypes  and within HLA-B supertypes . For simplicity, Tukey post-tests are not shown because no trend was found between the groups.Graphs of mean epitope promiscuity (see Methods) of (TIF)Click here for additional data file.Table S1The first column refers to the IEDB reference number for bacterial epitopes, and amino acid sequence for HIV epitopes. The second column refers to the microbe group from which the epitope was derived. The third column refers to the protein from which the epitope was derived. The fourth column refers to the class of HLA alleles that the epitopes have been shown to bind, and the class in which they were analyzed in this study.(PDF)Click here for additional data file.Table S2(PDF)Click here for additional data file.Table S3(PDF)Click here for additional data file.Table S4(PDF)Click here for additional data file.


H. pylori colonization. We prospectively evaluated the effect of H. pylori eradication on meal-associated changes in serum ghrelin and leptin levels, and body weight.Appetite and energy expenditure are regulated in part by ghrelin and leptin produced in the gastric mucosa, which may be modified by H. pylori status and body weight were ascertained. During the first visit in all subjects, and during subsequent visits in the initially H. pylori-positive subjects and controls, blood was collected after an overnight fast and 1 h after a standard high protein meal, and levels of eight hormones determined.Veterans referred for upper GI endoscopy were evaluated at baseline and ≥8 weeks after endoscopy, and H. pylori-negative, 44 H. pylori-positive, and 10 were indeterminate. Among 23 H. pylori-positive subjects who completed evaluation after treatment, 21 were eradicated, and 2 failed eradication. After a median of seven months following eradication, six hormones related to energy homeostasis showed no significant differences, but post-prandial acylated ghrelin levels were nearly six-fold higher than pre-eradication (p = 0.005), and median integrated leptin levels also increased (20%) significantly (p < 0.001). BMI significantly increased  over 18 months in the initially H. pylori-positive individuals, but was not significantly changed in those who were H. pylori-negative or indeterminant at baseline.Of 92 enrolled subjects, 38 were H. pylori eradication, providing direct evidence that H. pylori colonization is involved in ghrelin and leptin regulation, with consequent effects on body morphometry.Circulating meal-associated leptin and ghrelin levels and BMI changed significantly after  The healthful regulation of energy homeostasis in humans, depends on centrally-acting hormones such as ghrelin and leptin ,2. SerumHelicobacter pylori, which colonizes the human stomach and interacts with host tissues  [H. pylori after further confirmation with the 13C Urea Breath Test while off antisecretory medications. One penicillin-allergic patient received metronidazole [500 mg twice a day] instead of amoxicillin. In accordance with current guidelines   . Seven pidelines , H. pyloand PPI] . The 13CA multi-hormone EIA panel  was used to quantify eight gut hormones that are important regulators of food intake ,3,23, ent-test, or ANOVA method, and pair-wise analyses  were performed using non-parametric tests , as appropriate. Data are expressed as mean ± SD, or median and interquartile range (25th- 75th percentile). Categorical variables were compared using the Chi-squared test with Yates' correction or using Fisher's exact test. Spearman correlation coefficients were calculated for the relationship of leptin and ghrelin to BMI. Corrections were made in instances of multiple comparisons using techniques such as Tukey's range test. Based on previous findings showing 75% increase in fasting ghrelin after H. pylori eradication , non-pain scores , or satisfaction scores . Thus, the observed increase in BMI following eradication  9 2-2 vs. 6  had an endoscopic evaluation prior to the first postprandial measurement of hormones, and not prior to the second postprandial measurement, the observed post-eradication changes could not be explained by the potential effect of the endoscopic examination alone. We measured acyl-ghrelin, which also may be relevant in energy homeostasis following H. pylori eradication, as opposed to total ghrelin as others have done [H. pylori status determination using multiple methods for all patients that improve sensitivity and diminish falsely negative categorization [H. pylori-negative group for comparison. We planned to analyze patients who were not successfully treated to eradicate H. pylori, however few individuals failed eradication therapy and thus that control group was not sufficiently populated. In addition, our ability to further analyze H. pylori-negative patients was limited by the fact that only 7 completed follow-up evaluations.Our findings are limited by the study setting at a veteran's hospital where most of the evaluated patients were older men. Measurement of ghrelin is not standardized,  and may ave done ,29. We drization , measureH. pylori eradication. Although the number of subjects is limited, using patients as their own controls and having multiple measurements allowed us to both confirm previous published data, and to use a standard meal technique to extend the findings. This study provides further evidence that gastric H. pylori is involved in the physiologic regulation of these hormones, and supports the rationale for randomized controlled H. pylori eradication trials to focus on the role of inflammation and endocrine cross-talk in explaining these findings.In conclusion, our study indicates that leptin and ghrelin physiology change and that BMI increases following The authors declare that they have no competing interests.All authors read and approved the final manuscript. FF participated in the design of the study, patient recruitment, sample procurement, statistical analysis, and manuscript preparation. JR participated in patient recruitment, sample processing, and manuscript preparation. NJ contributed with patient recruitment and with manuscript preparation. ZP was involved in review of pathology samples and manuscript. AC participated in patient recruitment and manuscript review. JRS participated in sample processing and manuscript preparation. AZO was involved in sample procurement and processing as well as manuscript review. GIP participated in sample processing and manuscript preparation. MJB participated in study design, analysis, and manuscript preparation.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/11/37/prepubSupplemental methods. Provides information regarding exclusion criteria for the study population, symptom evaluation, H. pylori evaluation, test meal, and metabolic tests.Click here for fileSupplemental figures. Provides information regarding the enrollment and classification of study participants , the relationship of baseline BMI and baseline pre-meal leptin according to H. pylori status , and the correlation of changes in ghrelin and BMI post-H. pylori eradication .Click here for fileSupplemental tables. Provides information regarding the indications for upper GI endoscopy among study subjects (Table S1), findings during upper endoscopy (Table S2), Histologic score at three gastric sites according to H. pylori status (Table S3), levels of eight hormones related to energy homeostasis at the baseline evaluation of subjects according to H. pylori status and in relation to the test meal (Table S4), test-meal induced change and normalized change in hormone profile according to H. pylori status at baseline (Table S5), comparison of baseline and post-eradication meal-associated ghrelin profile in 21 originally H. pylori-positive subjects according to severity of baseline histologic gastritis (Table S6), and meal-associated changes in ghrelin profile in 28 subjectsa who had follow-up evaluation according to anatomical distribution of histologic gastritis at baseline (Table S7).Click here for file


Eupolyphaga sinensis Walker (Blattaria) is a sexually dimorphic insect, which is also an important source of material used in traditional Chinese medicine. The male E. sinensis have shorter lifecycles and go through fewer instars than the female. Furthermore, the males have forewings, while the females are totally wingless.MicroRNAs are now recognized as key post-transcriptional regulators in animal ontogenesis and phenotypic diversity. E. sinensis. 19,097,799 raw reads were yielded in total: 7,817,445 reads from the female library and 11,280,354 from the male, respectively. As a result, we identified 168 known miRNAs belonging to 55 families as well as 204 novel miRNAs. Moreover, 45 miRNAs showed significantly different expression between the female and the male fourth-instar larvae, and we validated 10 of them by Stem-loop qRT-PCR. Some of these differentially expressed miRNAs are related to metamorphosis, development and phenotypic diversity.We used the Illumina/Solexa deep sequencing technology to sequence small RNA libraries prepared from the fourth-instar larvae of male and female E. sinensis. The results provide a useful resource for further in-depth study on molecular regulation and evolution of miRNAs. These findings not only enrich miRNAs for hemimetabolans but also lay the foundation for the study of post-transcriptional regulation on the phenomena of sexual dimorphism.This is the first comprehensive description of miRNAs in  MicroRNAs (miRNAs) are tiny (∼22 nucleotides), endogenous, and non-coding RNAs that negatively regulate gene expression by binding to mRNA target sequences in eukaryotes Drosophila, Anopheles. gambiae, Aedes aegypti, Culex quinquefasciatus), Hymenoptera , Coleoptera (Tribolium castaneum), Orthoptera (Locusta migratoria), Lepidoptera , and Homoptera (Acyrthosiphon pisum) (http://www.mirbase.org/).In recent years, next generation sequencing technology, such as solexa sequencing and 454 sequencing have become robust methods to identify numerous miRNAs in animals and plants Eupolyphaga sinensis Walker) is a kind of insect used in traditional Chinese medicine. In folk medical practices, it could enhance immune response and promote blood circulation by removing blood stasis. In recent years, researchers have discovered antitumor and immunomodulatory effects of extracts from E. sinensisE. sinensis is a species with sexual dimorphism. In general, the lifecycles of male ground beetles are shorter than the female. The male usually go through 7 instars while 9–11 instars in the female when they reach sexual maturity from their first instar larvae. Furthermore, phenotypic differences are also significant between both sexes. The male adults have forewings while females are totally wingless and have a bigger body size. Given that the scarcity of genetic information for E. sinensis, we thus largely focus on these differences in post-transcriptional level. Previous studies discovered a number of sex difference-related microRNAs from gonads of mammals E. sinensis of both sexes. We totally obtained 160 known miRNAs, 8 known miRNA*s and 204 novel miRNAs. Among these, 45 miRNAs showed significantly different expression between the female and the male. The result will provide a better understanding of miRNA-mediated regulation in different sexes.The ground beetle , according to the manufacturer's protocol. Quantity and integrality of the total RNA was evaluated by Agilent2100 .Two libraries for male and female larvae of The sequencing procedure was previously described by Ma et al. http://www.ncbi.nlm.nih.gov/), RFam (http://rfam.janelia.org/), and Repbase (http://www.girinst.org/repbase/) were discarded. Then, we clustered the remaining reads based on sequence similarity and they were analyzed by BLAST against miRBase 19.0. Sequences in our libraries with identical or related (one mismatch) from metazoan mature miRNAs were identified as known miRNAs. The residual sequences after known miRNAs identification were aligned to the T. castaneum genome (ftp://ftp.bioinformatics.ksu.edu/pub/BeetleBase/3.0/) in order to identify potentially novel miRNAs. Certain target sequences around the small RNA were used to explore the secondary structure and folding energy  using Mfold software  and Vienna RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi).After masking of vector and adaptor sequences and removal of contaminated reads, the clean reads were then filtered for miRNA prediction with the proprietary ACGT101-miR v3.7 package . First, raw sequences that matched metazoan mRNA, rRNA, tRNA, snRNA, snoRNA, repeat sequences, and other ncRNAs deposited in the NCBI databases  were remained, respectively. We further discarded certain known types of RNA sequences, such as mRNA, rRNA, tRNA, snRNA, snoRNA and repetitive sequences as well. After these screening processes, 2,278,581 and 4,229,351 high-quality reads  from the female and male samples were used for miRNA identification . Among which, 102 miRNAs corresponding to known insect pre-miRNAs were mapped to the T. castaneum genome  , which were only previously identified in one or two insect species.The mappable sequences were aligned to all mature animal miRNA and miRNA precursor sequences in miRBase 19.0  . FailureL. migratoriaBlattella germanicaB. mori and D. melanogasterDrosophila,miR-184, strongly expressed in the germline, regulates several distinct steps during oogenesis and early embryogenesis In our dataset, a large proportion of known miRNAs were ubiquitously expressed (>10 reads) in both libraries. The most abundant miRNA was miR-10-5p with a total of over 300,000 reads, followed by miR-100, miR-263a-5p, miR-184-3p and miR-276 . These mIn most cases, miRNA*s are undetectable by conventional methods because miRNA*s will be degraded soon after being exported to cytosol. During the process of identifying known miRNAs, 8 miRNAs that matched to the same precursor sequences with their mismatched complementary mature miRNAs were also detected, which might be miRNA* sequences . In fact, these 8 miRNA*s only accounted for a small portion of known miRNAs. On the whole, the abundance of miRNA*s was much lower than that of their corresponding miRNAs except for miR-281*, which was over 40 times higher. This kind of phenomenon was consistent with the instability of miRNA* during biogenesis. Various miRNA* sequences with high amounts have also been identified in other libraries by deep sequencing T. castaneum genome to forecast potentially novel miRNAs, since the E. sinensis genome has not been sequenced yet. Based on the criteria that the extended sequences at the aligned genome locations have the propensity of forming hairpin structures, a total of 204 novel miRNAs were predicted  and length difference still existed. Then the sequences of known miRNAs were analyzed in our libraries and they were divided into several categories : (1) 5′ http://telethon.bio.unipd.it/bioinfo/ IDEG6_form/), which performs a normalization calculation to adjust for the two libraries. The expression levels of miRNAs in the two libraries were normalized using the following formula: Normalized expression  =   ×1,000,000. Results of the Audic and Claverie test, Fisher exact test, and Chi-squared 2×2 test with a Bonferroni correction for multiple comparisons and a p value <0.0001 indicated that differences in the miRNA reads were statistically significant. As a result, 45 miRNAs showed significant differential expression between the female and male libraries Click here for additional data file.Table S2Sequences and number of reads for known candidate miRNAs found in E. sinensis.(XLS)Click here for additional data file.Table S3The most 20 abundant miRNAs identified in E. sinensis.(XLS)Click here for additional data file.Table S4Sequences and number of reads for novel miRNAs predicted in E. sinensis.(XLS)Click here for additional data file.Table S5Variation pattern and alignment of mature miRNA sequences among different species.(XLSX)Click here for additional data file.Table S6Comparison of the differentially co-expressed miRNAs between the female and male E. sinensis.(XLS)Click here for additional data file.


Nectonemertes cf. mirabilis and Zygeupolia rubens.The mitochondrial genome is important for studying genome evolution as well as reconstructing the phylogeny of organisms. Complete mitochondrial genome sequences have been reported for more than 2200 metazoans, mainly vertebrates and arthropods. To date, from a total of about 1275 described nemertean species, only three complete and two partial mitochondrial DNA sequences from nemerteans have been published. Here, we report the entire mitochondrial genomes for two more nemertean species: N. cf. mirabilis and 15513 bp for Z. rubens. Each circular genome contains 37 genes and an AT-rich non-coding region, and overall nucleotide composition is AT-rich. In both species, there is significant strand asymmetry in the distribution of nucleotides, with the coding strand being richer in T than A and in G than C. The AT-rich non-coding regions of the two genomes have some repeat sequences and stem-loop structures, both of which may be associated with the initiation of replication or transcription. The 22 tRNAs show variable substitution patterns in nemerteans, with higher sequence conservation in genes located on the H strand. Gene arrangement of N. cf. mirabilis is identical to that of Paranemertes cf. peregrina, both of which are Hoplonemertea, while that of Z. rubens is the same as in Lineus viridis, both of which are Heteronemertea. Comparison of the gene arrangements and phylogenomic analysis based on concatenated nucleotide sequences of the 12 mitochondrial protein-coding genes revealed that species with closer relationships share more identical gene blocks.The sizes of the entire mitochondrial genomes are 15365 bp for The two new mitochondrial genomes share many features, including gene contents, with other known nemertean mitochondrial genomes. The tRNA families display a composite substitution pathway. Gene order comparison to the proposed ground pattern of Bilateria and some lophotrochozoans suggests that the nemertean ancestral mitochondrial gene order most closely resembles the heteronemertean type. Phylogenetic analysis proposes a sister-group relationship between Hetero- and Hoplonemertea, which supports one of two recent alternative hypotheses of nemertean phylogeny. Cephalothrix hongkongiensis  , Lineusemertea) , and Parnemertea). Nearly hrix sp.  and Cephufifrons . Thus, cNectonemertes cf. mirabilis and Zygeupolia rubens are summarized in Figure N. cf. mirabilis and Z. rubens are circular DNA molecules of 15365 bp and 15513 bp, respectively. Lengths of the two nemertean mitochondrial genomes are within the range of previously sequenced nemertean mtDNAs - 14558 bp in Paranemertes cf. peregrina to 16296 bp in Cephalothrix hongkongiensis )trnC, trnG and trnM, having the highest levels of nucleotide conservation (%INUC > 50), followed by TrnE, trnL2, trnQ, trnS2, trnV and trnY at 40 ≤ %INUC ≤ 50  were found in the acceptor and/or the discriminator arms, without regard to the overall level of conservation of the tRNAs. As recently pointed out by Negrisolo et al.  for arthAmong the most conserved tRNAs in nemerteans, as in insects , nucleotrnC acceptor arm of P. cf. peregrina, which was mirrored by G-C in all other nemerteans , while other taxa exhibited the G-C pair  gene in N. cf. mirabilis, P. cf. peregrina and three Cephalothrix species is GCT, but it is TCT in L. viridis and Z. rubens. Cameron et al. )Phylogenetic trees were estimated under maximum likelihood (ML) and Bayesian inference (BI). ML analysis on the combined nucleotide dataset alignments was performed in RAxML 7.2.7 ,75 availAmino acid sequences were aligned with both Clustal X  and MAFFN. cf. mirabilis and Z. rubens are deposited in GenBank under the accession numbers HQ997772 and HQ997773.The mitochondrial genome sequences of atp6 and atp8: ATP synthase subunits 6 and 8; cob: cytochrome b; cox1-3: subunits I-III of cytochrome c oxidase; nad1-6 and nad4L: NADH dehydrogenase subunits 1-6 and 4 L; rrnL and rrnS: the large and small subunits of ribosomal RNA; trnX: genes encoding for transfer RNA molecules with corresponding amino acids denoted by the one-letter code and codon indicated in parentheses (xxx) when necessary; DHU: dihydrouridine loop; MtDNA: mitochondrial DNA; NC: non-coding region; PCR: polymerase chain reaction; Kb: kilobase; bp: base pair; nt: nucleotide; nucleotide symbol combination: R = A/G; Y = C/T; W = A/T; K = G/T; N = A/G/C/T.The authors declare that they have no competing interests.HXC performed the majority of the molecular experiments and analyzed the data, and drafted the manuscript. SCS supervised the research. PS contributed to the analysis of the data. WCR performed part of the study, and provided technical assistance. JLN collected specimens, conceived, designed the research plan and did significant revisions of the manuscript draft. All authors read and approved the final manuscript.Figure S1. Mitochondrial gene order (protein-coding genes and rRNAs only) of Nemertea and selected lophotrochozoan species and the putative bilaterian ground pattern . Gene s [Lineus , Paranemratulina , Kathariatharina , PhoroniPhoronis , PerionyPerionyx , Urechis[Urechis , Sipuncupunculus .Click here for fileTable S1. Pairwise breakpoint distance matrix of mitochondrial gene orders of nemerteans, the bilaterian ground pattern and six other lophotrochozoans*Click here for fileTable S2. Pairwise reversal distance matrix of mitochondrial gene orders of nemerteans, the bilaterian ground pattern and six other lophotrochozoans*Click here for file