Helicobacter pylori has been found to be effective for gastric cancer prevention, but uncertainties remain about the possible adverse consequences such as the potential microbial dysbiosis. In our study, we investigated the association between gut microbiota and H. pylori-related gastric lesions in 47 subjects by deep sequencing of microbial 16S ribosomal RNA (rRNA) gene in fecal samples. The dominant phyla in fecal samples were Bacteroidetes, Firmicutes, and Proteobacteria with average relative abundances of 54.77, 31.37 and 12.91%, respectively. Microbial diversity analysis showed that observed species and Shannon index were increased in subjects with past or current H. pylori infection compared with negative subjects. As for the differential bacteria, the average relative abundance of Bacteroidetes was found to significantly decrease from H. pylori negative (66.16%) to past infection group , as well as from normal (76.49%) to gastritis (56.04%) and metaplasia subjects . For Firmicutes and Proteobacteria, the average relative abundances showed elevated trends in the past H. pylori infection group  compared to negative group , and similar increased trends were also found from normal  to gastritis  or metaplasia subjects . These findings suggest that the alterations of fecal microbiota, especially the dominant phyla of Bacteroidetes, Firmicutes and Proteobacteria, may be involved in the process of H. pylori-related gastric lesion progression and provide hints for future evaluation of microbial changes after H. pylori eradication.Eradication of  Helicobacter pylori plays a crucial role by inducing inflammation and degradation of the gastric epithelium from early stage  is a major health problem in China, accounting for over 40% of the new GC cases annually worldwide  gene in feces. Our results provide baseline associations between gut microbiota and H. pylori-related gastric lesions, which will be important for further evaluation of eradication treatment and gastric carcinogenesis.In the present study, we analyzed the gut microbiota of subjects presenting gastric lesions associated with or independent of 13C-urea breath test (13C-UBT) and standardized upper endoscopy examination. The exclusion criteria were as follows: contraindication for biopsy sampling, risk of conscious sedation due to severe comorbidities, and inability to provide an informed consent due to psychiatric illness. In the final week of the endoscopic examinations, participants were invited to provide stool samples. A total of 47 participants agreed and provided samples, which were collected and frozen immediately at −80°C until DNA extraction. Besides, each participant completed a physical examination and a questionnaire on age, gender, cigarette smoking, alcohol consumption, and antibiotics treatment history under the supervision of a well-trained interviewer before the endoscopic examination. This study was approved by the Institutional Review Boards of Peking University Cancer Hospital and Institute and written informed consent was obtained from all of the participants.In November 2014, an endoscopy study was conducted in Linqu County, China. A total of 218 asymptomatic individuals (aged 40–69 years) from two villages of Yishan Township were selected for H. pylori infection, gastritis, activity of inflammation and intestinal metaplasia . The gastric mucosa was examined, and five or more biopsies were obtained from antrum, corpus, angulus, cardia, esophagus and any suspicious lesions. All gastric mucosa specimens were reviewed blindly by two pathologists (1 Chinese and 1 German) according to the Updated Sydney System  after 30 min were regarded as positive. Current H. pylori infection was defined when bacteria were histologically detected in any one of the biopsies or it was positively indicated by 13C-UBT. Non-current infection subjects were defined when both bacterial and 13C-UBT detection indicated negative results.H. pylori eradication therapy or histological diagnosis of non-active gastritis with basal lymphoid aggregates and reactive changes of the surface epithelium in antrum and corpus, indicating a prior H. pylori infection. Non-current infection subjects without eradication treatment history and specific histological diagnosis were considered negative H. pylori infection subjects.From the non-current infection subjects, past infection subjects were identified by record on previous Fecal DNA was extracted using the Qiagen Fast DNA Stool Mini Kit  according to the protocol provided by the manufacturer. Briefly, 180–220 mg sample was re-suspended in InhibitEX Buffer to adsorb DNA-damaging substances and PCR inhibitors. After thoroughly homogenizing, the suspension was heated to 95°C for 5 min to maximize the lysis of bacteria. The quantity and quality of total DNA were verified by NanoDrop-2000 spectrophotometer (Thermo Scientific) and 1% standard agarose gel electrophoresis.For analysis of microbial composition, the hypervariable region V4 of microbial 16S rRNA gene was amplified using Phusion® High-Fidelity PCR Master Mix (New England Biolabs) and the universal primers   . Rarefaction curves were displayed with R software (Version 2.15.3). Comparisons of microbial communities were visualized by heat-map and Principal Co-ordinates Analysis (PCoA) based on weighted unifrac distances between clinical parameters and gut microbiota, and the significance of differences was evaluated by the analysis of similarity (ANOSIM) depending on the Bray-Curtis dissimilarity distance matrices. Taxa with relative abundance more than 0.1% in at least one group  were included in identification of differential taxa. Welch's p < 0.05 were considered statistically significant.Continuous variables, such as age, body mass index (BMI) and fasting plasma glucose, were reported as mean ± standard deviation, while observed species and Shannon index were reported as median . Categorical variables  were described as frequencies and proportions. Comparisons of continuous parameters were performed by Mann-Whitney test between two groups or Kruskal-Wallis test among three groups. Fisher's exact test was conducted for comparisons between categorical variables. Statistical analysis was carried out using SPSS 13.0 , and H. pylori infection and 23 subjects as non-current infection, of which 15 were H. pylori negative and 8 showed signs of past infection . No significant differences were found among the three groups in age, gender, BMI, smoking, drinking, fasting plasma glucose and antibiotics use .From 47 subjects who provided fecal samples, 24 subjects were identified as having current A total of 2,827,158 paired-end reads were generated from 47 fecal samples including 2,557,652 quality-filtered reads with an average of 54,418 ± 5,665 (standard deviation) reads per sample. After aligning to the SILVA database and removing non-bacterial sequences, 2,518,760 (98.48%) of the quality-filtered reads were finally retained for subsequent analysis with an average of 53,591 ± 5,625 reads per sample, which generated 817 OTUs at 97% similarity level. The rarefaction curves showed a reasonable sequencing depth Figure .Bacteroidetes, Firmicutes, and Proteobacteria with average relative abundances of 54.77%, 31.37% and 12.91%, respectively. The three dominant phyla accounted for 99.05% of all fecal bacteria  of observed species  and Shannon index  between non-current H. pylori infection and current infection group , and increased for Gemella and Erysipelotrichaceae_UCG_004  in the current infection group when compared to the non-current infection group  and past infection (n = 8) groups according to antibiotics treatment history and histological assessment to further compare the fecal microbiota among the different groups. Observed species [median (IQR) 317.00 (289.00–328.00)] and Shannon index [4.74 (3.99–5.27)] were increased from negative, to past infection  or current infection group , but the differences were not statistically significant (p = 0.317 for observed species and 0.696 for Shannon index) (Table Non-current x) Table .H. pylori infection and negative groups  was significantly decreased from 66.16% in the H. pylori negative group to 31.01% in the past infection group, p = 0.007. The average relative abundance of Bacteroidetes was also found increased from 31.01% in the past H. pylori infection group to 55.57% in the current infection group, p = 0.043. Especially Bacteroides uniformis species showed significantly decreased relative abundance in the past H. pylori infection group (1.50%), when compared to the current infection  and the negative groups , respectively.We further studied differential bacteria among the three different s Figure . The avep > 0.05]  , 4.68 (3.85–4.84)], gastritis  and metaplasia groups  Table . When co Figures .R = −0.029, p = 0.704, Table No significant difference in fecal microbial community structure was found among the different gastric lesions groups by ANOSIM  to 56.04% (gastritis), p = 0.009, and Firmicutes with increased average relative abundance from 18.23 to 35.31%, p = 0.016. When we compared metaplasia cases with normal subjects, similar significant differences in average relative abundances were also found for Bacteroidetes  and Firmicutes . In addition, Proteobacteria showed significantly increased average relative abundance from normal (5.05%) to metaplasia subjects  and from gastritis (7.23%) to metaplasia . Finally, the average relative abundance of Enterobacteriaceae, a family of Proteobacteria, was significantly increased with the severity of the gastric lesions analyzed .The differential bacteria observed in the different gastric lesions groups are shown in Figure H. pylori-related gastritis, we further analyzed the association between activity of gastritis and fecal microbiota. Of the 18 subjects diagnosed with gastritis, 12 presented non-active gastritis and 6 showed active gastritis. Analyses of observed species and Shannon index showed that the microbial richness and evenness in fecal samples were slightly increased in non-active gastritis subjects  compared to normal subjects , although p values were 0.098 and 0.184, respectively  were detected when comparing the fecal microbiota community structure by ANOSIM (Table Ruminococcaceae_NK4A214_group was found decreased in active gastritis (0.06%) compared to non-active gastritis subjects   Figure .H. pylori-related gastric lesions, we finally analyzed relevant microbial alterations in relation to H. pylori infection and progression of gastric lesions. In the three dominant phyla  of fecal microbiota  to gastritis (56.04%) and metaplasia , as well as from H. pylori negative (66.16%) to past infection . For Firmicutes and Proteobacteria, the average relative abundances were increased from normal  to gastritis  or metaplasia , respectively. Similarly, the average relative abundances of these two phyla were found elevated in past H. pylori infection  compared to the negative group , although the p values (0.068 and 0.246) showed no significant differences.To further identify associations between fecal microbiota and a Figure , the aveH. pylori infection status and gastric lesions. Three genera  were found to have similar alterations in H. pylori infection and gastric lesion groups, although most of the p values were higher than 0.05 among groups with different H. pylori infection status or gastric lesions  decreased with the severity of gastric lesions , as well as H. pylori infection status . In addition, Faecalibacterium (belonging to the phylum of Firmicutes) showed a trend to higher average relative abundance both in the gastritis (3.45%) and the past H. pylori infection groups (4.04%) compared to their reference , respectively.At the genus level, a total of 19 genera with average relative abundances of more than 0.01% were analyzed in relation to s Figure . The aveH. pylori-related precancerous gastric lesions is very important for evaluation of overall benefits and adverse effects of eradication treatment and for the optimization of GC prevention strategies. In the present study, we profiled fecal microbiota in subjects with different H. pylori infection status. Diversity analysis showed slightly increased microbial richness and evenness in subjects presenting signs of past H. pylori infection or gastritis compared to controls. In addition, the relative abundances of the dominant phyla Bacteroidetes, Firmicutes, and Proteobacteria in fecal microbiota were similarly altered in subjects presenting with H. pylori infection and gastric precancerous lesions. These observations suggest that fecal microbiota may be associated with the progression of H. pylori-related gastric lesions.Deep understanding of the association between gut microbiota and H. pylori infection status. We found that the dominant phyla present in feces were Bacteroidetes, Firmicutes, and Proteobacteria, accounting for 99.05% of fecal microbiota, which is consistent with previous reports   in feces was found associated with current H. pylori infection. No significant differences in fecal microbial diversity and structure were found between subjects showing current or non-current H. pylori infection groups. These observations are in line with the results of a previous small sample size study showing no remarkable alterations of the fecal microbiota composition and structure after H. pylori eradication  showed no significant differences. Especially, the recent antibiotics use (within 1 month) was not significantly different among negative (33%), past infection (25%), and current infection groups (25%). Consistent with a mouse model study  were included in the gastric lesion groups than in the normal mucosa group. The significantly different taxa observed between different lesion groups suggest a possible role of intestinal microbiota in the progression of precancerous gastric lesions. Altered microbiota observed include the dominant phyla of Bacteroidetes and Firmicutes, as well as relevant gut bacteria such as Enterobacteriaceae and Faecalibacterium previously reported to be related to inflammatory diseases such as primary biliary cholangitis , further suggest that alterations in intestinal microbiota may be involved in the progression of H. pylori-related precancerous gastric lesions and carcinogenesis.The mechanisms by which gut microbiota contribute to carcinogenesis are still not clear. Dysbiosis of microbiota can affect the host immune system, inducing inflammation and thereby the development of cancer due to the tight interplay between bacteria and epithelial cells. Innate bacteria-sensing receptors such as Toll-like receptors (TLRs) and NOD-like receptors (Rakoff-Nahoum and Medzhitov, H. pylori infection status and the availability of tissue samples with a broad spectrum of gastric lesions from an ongoing intervention trial for H. pylori eradication. Exploration of the associations between fecal microbiota and H. pylori infection and gastric lesions builds a solid basis for our future study on the changes in microbiota after H. pylori eradication. A limitation of our study is that we only had fecal samples from 47 subjects after endoscopy examination. Therefore, the small sample size limited the feasibility to adjust possible confounding factors or correct multiple comparisons for the identification of differential taxa among groups with different H. pylori infection status or gastric lesions. However, possible factors influencing the intestinal microbiota were acquired in detail at baseline, including BMI, fasting plasma glucose and antibiotics use, which were equally distributed among different groups. Confirmation with a larger sample size including participants of the intervention trial as well as subjects showing precancerous lesions or GC is still needed. In addition, the composition of gastric microbiota and its association with H. pylori-induced gastric carcinogenesis will be further studied.A major strength of our study lies in the detailed information on H. pylori infection and gastric lesion status. The differential bacteria identified, including Bacteroidetes, Firmicutes and Proteobacteria, showed similar alterations associated with H. pylori infection and severity of the gastric lesions. Our results provide novel insights important for future studies on H. pylori-related carcinogenesis and alterations in the gut microbiota after H. pylori eradication.In conclusion, our study profiled the composition of fecal microbiota and assessed its association with The raw data of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.K-FP and W-CY contributed design of the study. MB and SS conducted upper endoscopy examination. LZ, J-LM, W-DL, and Z-XL contributed subject's recruitment and samples' collection. MV, KU, MQ, RS, and MC completed histological diagnosis. YZ, J-JG, and TZ carried out experiments. J-JG analyzed experiments' results. YZ and J-JG wrote the draft of the manuscript. MG, RM-L, W-QL and K-FP revised the manuscript. All authors read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


DNA replication starts from a specific point on the chromosome that is called the origin of replication. In contrast to prokaryotes in which DNA replication starts from a single origin, eukaryotic DNA replication starts from many origins scattered along the chromosomes. Budding yeast contains 300 origins, whereas fission yeast has 1,100, and the numbers of replication origins for human increase to over 20,000. These origins are fired in a coordinated manner, and there are spatial and temporal disciplines for this process, which happens in the S phase of the cell cycle. It was known that eukaryotic cells prepare all these potential origins during the G1 phase of the cell cycle but utilize only a portion of these origins during S phase. Furthermore, firing some of these origins are delayed until the mid and late phases of the S phase. Coordinated activation of these origins occurs under “Replication Timing Program”. The segments of the chromosome containing co-regulated origins that fire simultaneously are named “Replication Timing Domains”, ranging in size from 100 kb to 1 Mb. Replication timing is determined at a specific time in the early G1 phase that is called Timing Decision Point (TDP). Studies have shown that major chromosome repositioning occurs at TDP. Generally, replication timing domains are classified into three classes including Early, Mid and Late.There are three suggested mechanisms by which cell can distinguish the Early and other types of replication origins.First mechanism: The origins of replication are marked by the pre-RC components through specific covalent modification.Second mechanism: Firing of Mid/Late origins is prevented by Rif1 via two mechanisms. The first is done by recruiting phosphatase that inhibits the firing of origins. The second is carried out by creating chromatin structures that represent replication-suppressive domains.Third mechanism: Early firing is promoted by open chromatin structure or by euchromatic marks. Fkh1 is a protein known to recruit Cdc7 kinase, specifically to early-firing origins.Recent studies have found many factors regulating the replication timing. One of the most important factors is the Rif1 protein that plays a key role in the regulation of replication timing in yeast and higher eukaryotes. Rif1 protein may regulate the replication timing domains by organizing the chromatin loop structures. A C-terminal domain of Rif1 is able to bind DNA on its own and also become oligomerized. Through its ability to hold many chromatin fibers together by the DNA binding and oligomerization activities, C-terminal domain of Rif1 creates chromatin compartments that are inhibitory for origin firing. Rif1 capability to regulate the chromatin architecture and consequently to regulate the DNA replication timing could have versatile impacts on other chromosome functions, including recombination, transcription, and repair.Replication timing is amongst the cellular phenomena being modified during some diseases including cancer . The deregulation of the replication timing has been demonstrated to result in chromosomal disorders and genomic instabilities. More importantly, cellular changes that can cause tumors are accompanied by replication timing program disturbances. These observations could lead to a suggestion that Rif1, one of the key factors in replication timing, could be a diagnostic biomarker or a drug target for various malignancies. Studies have shown that changes in the replication timing are not limited to cancer but are also linked to some of the inheritable diseasesOverall, considering the multifunctional nature of Rif1 and its connection with some diseases including cancer, the design and set up of a platform for full-length Rif1 expression or each of its domain seems to be necessitated. At the same time, analysis of its interaction with DNA and other cellular proteins may provide a suitable target for future practical studies like drug discovery.More details in:Anatomy of mammalian replication domains. S Takebayashi et al. 2017. MDPI; Vol. 8, 1-12.Temporal and spatial regulation of eukaryotic DNA replication: From regulated initiation to genome-scale timing program. C Renard-guillet et al. 2014. Semin Cell Dev Biol; Vol. 30, 110-120.DNA replication timing influences gene expression level. CA Müller et al. 2012; J Cell Biol; 1-8.DNA replication origin activation in space and time. M Fragkos et al. 2015. Nature; Vol. 16, 360-374.Rif1 times replication replication. P Strzyz. 2015. Nat Rev Mol Cell Biol 2016; Vol. 17, 2662.A conserved chromatin factor regulating DNA replication, DNA repair, and transcription. In: The Initiation of DNA Replication in Eukaryotes Springer International Publishing. N Yoshizawa-Sugata et al. 2016. pp. 143-158.Forkhead transcription factors establish origin timing and long-range clustering in S. cerevisiae. SR Knott et al. 2012. Cell; Vol. 148, 99-111.Replication timing regulation of eukaryotic replicons: Rif1 as a global regulator of replication timing. S Yamazaki et al. 2013. Trends Genet; Vol. 29, 449-460.Alterations in replication timing of cancer-related genes in malignant human breast cancer cells. Fritz AJ. 2013. J Cell Biochem; Vol. 114, 1074-1083.DNA replication timing, genome stability and cancer. N Donley et al. 2013. Semin Cancer Biol; Vol. 23, 80-89.Alterations in replication timing of cancer related genes in malignant human breast cancer cells. AJ Fritz et al. 2013. J Cell Biochem; Vol. 114, 1074-1083.Perturbations in the replication program contribute to genomic instability in cancer. B Blumenfeld B et al. 2017. Int J Mol Sci; Vol. 18, 1138.Abnormal developmental control of replication-timing domains in pediatric acute lymphoblastic leukemia. T Ryba T et al. 2012. Genome Res; Vol. 22, 1833-1844.Human RIF1 encodes an anti-apoptotic factor required for DNA repair. H Wang et al. 2009. Carcinogenesis; Vol. 30, 1314-1319.Epigenetic abnormalities associated with a chromosome 18(q21-q22) inversion and a Gilles de la Tourette syndrome phenotype. MW State et al. 2003. Proc Natl Acad Sci USA; Vol. 100, 4684-4689.Altered replication timing of the HIRA/ Tuple1 locus in the DiGeorge and velocardiofacial syndromes. S D’Antoni et al. 2004. Gene; Vol. 333, 111-119.Hypomethylation of subtelomeric regions in ICF syndrome is associated with abnormally short telomeres and enhanced transcription from telomeric regions. S Yehezkel et al. 2008. Hum Mol Genet; Vol. 17, 2776-2789.


Clostridium difficile. More than ten functional type I TA modules have been identified in the genome of this emerging enteropathogen that could potentially contribute to its fitness and success inside the host. Despite the absence of sequence homology, the comparison of these newly identified type I TA modules with previously studied systems in other Gram-positive bacteria, i.e., Bacillus subtilis and Staphylococcus aureus, revealed some important common traits. These include the conservation of characteristic sequence features for small hydrophobic toxic proteins, the localization of several type I TA within prophage or prophage-like regions and strong connections with stress response. Potential functions in the stabilization of genome regions, adaptations to stress conditions and interactions with CRISPR-Cas defence system, as well as promising applications of TA for genome-editing and antimicrobial developments are discussed.Type I toxin-antitoxin (TA) modules are abundant in both bacterial plasmids and chromosomes and usually encode a small hydrophobic toxic protein and an antisense RNA acting as an antitoxin. The RNA antitoxin neutralizes toxin mRNA by inhibiting its translation and/or promoting its degradation. This review summarizes our current knowledge of the type I TA modules identified in Clostridia species focusing on the recent findings in the human pathogen  Toxin-antitoxin loci (TA) are widespread in prokaryotes and encode a stable toxin component and an unstable antitoxin to repress the function or expression of the toxin . The toxTA families are divided into six types depending on the nature and action of the antitoxin . In the Clostridium difficile. C. difficile is a Gram-positive, strictly anaerobic spore-forming bacterium that became one of the major nosocomial enteropathogens in the industrial countries [C. difficile-associated diarrhoea is currently the most frequently occurring nosocomial diarrhoea worldwide. For the last approximately ten years, the proportion of severe infection forms rose due to the emergence of a hypervirulent and epidemic 027/NAP1/BI lineage [C. difficile infection are age and antibiotic exposure. Indeed, antibiotic therapy causes alterations in colonic microflora known as dysbiosis that facilitates implantation or development of C. difficile, after contamination by spores or in the presence of endogenous C. difficile in healthy carriers [C. difficile infection is the high rate of recurrent infections that can even increase after a second and third recurrence [C. difficile infection [In this review, we will focus on the recently described type I TA modules in the human pathogen ountries ,11. C. d lineage . Two majcarriers . This pacarriers ,15. Thiscarriers . Despitecurrence . The abinfection . NeverthC. tetani genome [toxIN locus in Clostridial species, i.e., Clostridium botulinum, Clostridium cellulovorans, Clostridium nexile, Clostridium phage D-1873, and Clostridium sp.; a potential cptIN locus in Clostridium hiranonis and Clostridium sp.; and a potential tenpIN locus in C. hiranonis awaiting further characterization [C. difficile with a MazF-cd toxin that exhibits selective mRNA cleavage [C. difficile and their possible implication in recurrent infections together with sporulation and biofilm formation has been discussed [TA modules are generally abundant in all prokaryotes but have not been largely studied in Clostridia. As TA systems have been potentially implicated in the persistence after antibiotic treatment and stress responses, some recent efforts have been made to search for TA modules in Clostridial pathogens and commensal species. A recent genomics study revealed the presence of TA systems in the i genome . Exhaustrization . A countcleavage . The in iscussed .Clostridium bolteae and a Fst-like sequence in Clostridium asparagiforme [Clostridium acetobutylicum (4 proteins), Clostridium beijerinckii (3 proteins), C. botulinum (from 4 up to 14 proteins depending on the strain), C. difficile (9 proteins), Clostridium kluyveri (5 proteins), Clostridium novyi (1 protein), Clostridium perfringens (from 3 to 5 proteins depending on the strain), and Clostridium thermocellum (1 protein) awaiting further studies.The large in silico search for homologs to known type I toxins in bacterial genomes could not find such proteins in the majority of Clostridial species with the exception of TxpA-like and Ldr/Fst-like sequences in agiforme . Neverthagiforme . In ClosC. difficile  are located within the homologous phiCD630-1 and phiCD630-2 prophage regions in the C. difficile 630 strain (CD1233.1-SQ808 is located within the sigK intervening (skin) sequence element, a prophage-like element integrated into the sigK gene encoding the late sporulation sigma factor, which is excised from the chromosome during sporulation. This location is reminiscent of the txpA-RatA type I TA module in the Bacillus subtilis skin element [Interestingly, these TA modules are associated with CRISPR arrays from CRISPR -Cas (CRISPR-associated) systems for defence against foreign nucleic acids ,27,28,29 strains . In addicultures . Such cocultures . Two of 0 strain . Another element , but no C. difficile prophage regions and provided experimental evidence of their possible contribution to these genomic regions’ stability [CD0977.1-RCd11 and CD2889-RCd12, located in the homologous phiCD630-1 and phiCD630-2 prophage regions in the C. difficile 630 strain, are composed of the toxic protein of 47 amino acids in length and an antitoxin RNA associated with type I riboswitches responding to c-di-GMP signalling molecule: cdi1_4 and cdi1_5  and the core genome , raising the question on the possible cross-regulation between homologous systems and their redundancy [S. aureus Newman strain, an unusual condensed sRNA cluster could contain a novel type I TA system with the srn_9343 gene, encoding a secreted peptide that presents sequence similarities to RelE type II toxin and Srn_9344 cis-antisense RNA that could function as an antitoxin [S. aureus type I TA modules were related to stress adaptation with specific functions that could be suggested since they respond to different stress factors, including osmotic shock, nutrient starvation, and oxidative and acidic stresses [C. difficile, each SprF antitoxin specifically neutralizes its cognate SprG toxin; however, heterologous cross-regulations between the RNA from non-cognate pairs were also observed. Similarly, only specific action in trans of cis-encoded antitoxin to prevent the translation of its cognate toxin was demonstrated for SprA modules [In the human pathogen dundancy ,58,59,60ntitoxin . As for stresses ,53,59. Tstresses ,53,60,62txpA-RatA TA system and adjacent mazEF type II TA system in another human pathogen Enterococcus faecalis [ratA transcription, where RatA in turn controls the type I toxin mRNA txpA levels affecting its stability.A coordinated regulatory crosstalk has been recently described between type I faecalis . In addiB. subtilis [C. difficile, the RNA antitoxins from type I TA systems shared a long complementary region with convergently transcribed toxin mRNA suggesting an RNA degradation mechanism for antitoxin action [E. coli and for AapA1 in Helicobacter pylori [C. difficile and could be important for the regulatory processes [txpA-RatA system within the B. subtilis skin element [E. coli [B. subtilis [Two major mechanisms involved in the regulation of toxin expression and RNA decay within type I TA systems have been defined ,56,60,64subtilis . In C. dn action ,24. The r pylori ,66,67. Trocesses . Among arocesses ,56. Even element ,42. The [E. coli  and RNassubtilis ,50.C. difficile produce a rather stable toxin mRNA  and a less stable antitoxin RNA  [CD2907.1 and RCd10-CD0956.2 TA pairs, the stable duplex formation was reported in vitro and these full TA duplexes could serve as a substrate for the efficient degradation by E. coli RNase III [In accordance with the data for other bacteria, the measurements of transcript half-lives showed that type I TA modules in f RCd11) ,24. The f RCd11) ,24. For Nase III .trans-encoded sRNA action supporting their interactions with mRNA targets in Gram-negative bacteria [CD0977.1 toxin mRNA and antitoxin RCd11 RNA, and in a slight decrease in antitoxin RCd9/RCd10 half-life in C. difficile [ralR-RalA in E. coli requires Hfq for antitoxin function that appears to stabilize RalA [B. subtilis [C. difficile were also enriched in an RNA sample associated with Hfq in a co-immunoprecipitation assay  suggestDespite the continuous efforts on the study of TA systems, their biological role remains unknown in most cases. Among multiple functions discussed in the literature, only three have received experimental validation. This includes the stabilization of mobile genetic elements, abortive phage infection, and persister cell formation .C. difficile [B. subtilis [V. cholerae, a proteinous TA module mosAT promotes the maintenance of an integrative conjugative element STX conferring resistance to multiple antibiotics [Shewanella oneidensis, a type II TA system ParESO-CopASO stabilizes the prophage CP4So [C. difficile, as in other bacterial pathogens, the role of prophages in the physiology and virulence is being actively discussed [C. difficile pathogenesis [Initially, TA systems were shown to be important for the maintenance of plasmids through post-segregation killing mechanism ,5. So faifficile , a possisubtilis . In V. cibiotics , while ige CP4So . Anotherge CP4So . In C. discussed ,77. It hogenesis ,79.C. difficile chromosome suggests potential functional link between these systems for the defence against bacteriophage infection [In addition to the stabilization of genomic regions, other functions could be suggested for chromosomal TA modules. Among other mechanisms, abortive infection provides the resistance to bacteriophage infection to the bacterial population through the altruistic suicide of infected cells induced by TA module activation ,7. Abortnfection . In relanfection .E. coli in the formation of persisters induced under stress conditions, have been suggested [Streptococcus mutans and the role in persister cell formation has been suggested for this Fst-Sm-SRSm system [B. subtilis is the bacteriostasis induction helping dormant cells to cope with stresses [grtABC-AsgR is involved in the stress response in Corynebacterium glutamicum [C. difficile, the identification of Sigma B-dependent promoters upstream of type I toxin and antitoxin genes also suggests their potential role in stress response mechanisms in this pathogen [Bacterial TA systems, including type I TA, were shown to influence persister cell and biofilm formation, as well as general stress response . Persistuggested ,86,87,88m system . One hypstresses . A recenutamicum . In C. dpathogen ,24.skin element has been suggested regarding the elimination of defective cells to preserve symmetry in B. subtilis biofilms [C. difficile, we have shown that type I TA systems are induced in biofilm conditions suggesting the control of these systems via community-behaviour associated factors [The induction of TA modules and their potential bactericidal action within biofilm communities have been discussed ,91. For biofilms . In C. d factors .Salmonella enterica serovar Typhimurium uses distinct type I and type II TA modules to regulate its intracellular lifestyle [S. aureus, a selective endoribonuclease activity affecting virulence factor mRNA stability has been suggested for type II TA toxin MazF in C. difficile [Increasing evidence highlights the key roles of TA systems specifically in bacterial pathogens ,93,94. Oifestyle . For exaifficile .Unique features of bacterial TA modules provide a great potential for development of new biotechnological and therapeutic applications including genome editing, plasmid maintenance tools, selective reporter genes, new antimicrobials, and eukaryotic cell killing agents ,96,97,98B. subtilis type I TA module bsrG-SR4 has been recently used to design a new antitoxin RNA-guided gene repression system in this model bacterium [Several efficient strategies have been already described to reprogram the TA systems for the bacterial genome editing based essentially on the type II TA systems ,99. For acterium .C. difficile, despite the continuous efforts, the efficient genetic tools are still needed to accelerate the research on this major enteropathogen. Based on our recent characterization of a functional CD2517.1-RCd8 type I TA module in C. difficile [C. difficile.In ifficile , we haveifficile . InducibS. aureus can trigger cell lysis of both bacterial and human cells and was optimized via chemical modifications to increase the antibacterial potential and its stability, and to reduce the human cell toxicity [E. coli has been considered as a potential anti-cell-envelope antibiotic against pathogenic E. coli infections [C. difficile strains opens promising perspectives on their use for development of alternative or synergistic strategies against the C. difficile infections.Accumulating knowledge on TA modules from pathogenic bacteria led to the innovative strategies for design of new antimicrobials specifically targeting these pathogens ,96,97,98toxicity ,62. Anotfections . The ideC. difficile attracted the attention of the scientific community due to the increased incidence and severity of infections and high rate of recurrences. The recent identification of more than ten type I TA modules distributed throughout its genome suggests their potential contribution to the success of this emerging human enteropathogen. Similar to other bacterial TA modules, the role of C. difficile type I TA in the stress response, biofilm structure, and genomic region stabilization could be hypothesized. As observed in well-studied Gram-positive bacteria, i.e., B. subtilis and S. aureus, many of these newly identified type I TA modules are located within prophages and prophage-like regions in the C. difficile chromosome. The intriguing association of some of them with CRISPR arrays related to defence capacities against foreign DNA elements raised the question regarding the potential functional link between these systems. The abundant chromosomal type I TA modules could thus help bacteria to keep the important genomic regions, including prophages that could shape the expression of virulence determinants and the adaptation capacities inside the host. Further studies will define the exact functions of these enigmatic chromosomal type I TA modules and specify the molecular mechanisms of their action and regulations involved. For the moment, most of the available information on the type I toxins is based on the overexpression experiments validating their toxic nature at high levels. However, their role at endogenous physiological levels remains unclear. The specific induction of small toxic proteins from type I TA modules could promote a reversible decrease in growth rate and help cells to survive under various stressful conditions. This could happen in the large proportion of cells or could be restricted to only limited bacterial populations, as suggested during the induction of persister formation. Depending on the level of toxin induction, the bactericidal function could be also considered, for example, for the elimination of defective cells from biofilm.Despites recent efforts, multiple facets of bacterial TA systems remain to be unveiled. In particular, the roles of chromosomal TA modules found in great numbers in different bacterial species need further investigations. In bacterial pathogens, this increasing knowledge could lead to better understanding of their stress management and adaptation strategies for the development of a coordinated pathogenesis program. Among clostridial pathogens, C. difficile, the inducible toxicity of type I toxins has been already successfully used for the elimination of plasmid-harbouring cells during the genome editing procedure. Further development of promising antimicrobial strategies based on TA systems could be considered, for example, by interfering with the TA component balance in favor of toxin expression. Future studies will provide new insights into the functioning of type I TA systems essential for further biotechnological and therapeutic applications in major pathogens.The small size of toxins and unique properties of type I TA systems make them potential interesting tools for numerous applications. In


Background: Immunotherapy provided unprecedented advances in the treatment of several previously untreated cancers. However, these immunomodulatory maneuvers showed limited response to patients with glioma in clinical trials. Our aim was to depict the immune characteristics of glioma with immune cytolytic activity at genetic and transcriptome levels.Methods: In total, 325 gliomas from CGGA dataset as training cohort and 699 gliomas from TCGA dataset as validation cohort were enrolled in our analysis. We calculated the immune cytolytic activity for 1,000 of gliomas. The characteristics of immune cytolytic activity in gliomas were interpreted by the corresponding clinical, molecular genetics and radiological information.Results: We found that immune cytolytic activity was highly associated with molecular, clinical, and edema extent. High cytolytic activity gliomas were more likely to be diagnosed as glioblastoma and might be a potential marker of mesenchymal subtype. Moreover, those gliomas exhibited significantly increased copy number alterations including recurrent focal amplifications of PDGFA and EGFR, as well as recurrent deletions of CDKN2A/B. Subsequent biological function analysis revealed that the immune response and immune checkpoints expression were significantly correlated with the cytolytic activity of gliomas. Immune cytolytic activity was significantly positively associated with the extent of peri-tumor edema and was independently correlated with reduced survival time.Conclusion: Our results highlighted the immunoregulatory mechanism heterogeneity of gliomas. Cytolytic activity, indirectly reflected by the extent of peri-tumor edema, may provide a potential index to evaluate the status of immune microenvironment and immune checkpoints in glioma, which should be fully valued for precision classification and immunotherapy. Diffuse gliomas, accounting for the majority of adult malignant brain tumors , are maiThe immune system is a crucial regulator to facilitate or inhibit tumor biological functions , 6. ReceA simple and quantitative formula to estimate the immune cytolytic activity (CYT) of glioma was proposed by Rooney et al. . The CYTwww.cgga.org.cn) as training cohort. The RNA sequencing data from the Cancer Genome Atlas  of 699 glioma patients, ranging from grade II–IV, and corresponding clinical information were collected as validation cohort. Moreover, we collected another 20 patients for independent clinical validation. The OS was calculated from the data of initial diagnosis until death or the last follow-up. Thus, in total 1,024 glioma patients were evaluated in this study. Information on these patients are available from corresponding data portal.This study retrospectively enrolled RNA sequencing data of 325 glioma patients, ranging from WHO grade II–IV, and corresponding clinical information from the Chinese Glioma Genome Atlas (CGGA) database . The mutation results in a loss of normal enzymatic function and the abnormal production of 2-hydroxyglutarate (2-HG) . And theWe obtained Fragments Per Kilobase Million (FPKM) values of glioma samples from two datasets. The FPKM values was transformed into Transcript Per Million (TPM) values with following formula:http://david.abcc.ncifcrf.gov/home.jsp) for functional annotation of the genes.The CYT was calculated as the geometric mean of the GZMA and PRF1 expression in TPM . Signifihttp://www.cgga.org.cn) and the Cancer Imaging Archive (http://www.cancerimagingarchive.net), respectively. Tumor contrast enhancement was assessed by two experienced neuroradiologists blinded to the patients' clinical information. A third senior neuroradiologist re-examined the images and determined which should be used if the types of enhancement identified by the first two neuroradiologists were inconsistent. Briefly, a small (or no) region of edema (-) was defined as edema extending ≤1 cm from the margin of the tumor based on T2-weighted images; otherwise edema was graded as moderate to severe (+) (MR images of patients from the CGGA and TCGA databases were obtained from the CGGA imaging database (vere (+) .t-test or chi-squared test, as appropriated. The Kaplan-Meier curves were employed to estimate overall survival distribution. Log-rank test was applied to compare the statistic difference of survival curves between two groups. All figures and statistical analysis were conducted using R software  and GraphPad Prism 7.0 software. A p < 0.05 was considered as statistically significant. All statistical tests were two-sided.Difference in variables between groups were tested by the Student's r = 0.57, p < 0.0001) and TCGA  datasets. Due to the intratumor heterogeneity and plasticity of glioma, we investigated the relationship of CYT and previous widely accepted prognostic and predictive factors, including grade, IDH status, and 1p/19q status. In CGGA dataset, cytolytic activity was significantly increased with increasing grade and the CYT exhibited the highest value in glioblastoma (p < 0.001) when comparing to other four subtypes of glioma in both CGGA  and TCGA datasets (p < 0.0001). Meanwhile, we found CYT value was significantly upregulated in 1p/19q intact glioma group than 1p/19q co-deleted group in both CGGA (p < 0.0001) and TCGA datasets (p < 0.0001). These findings indicated that cytolytic T-cell-related immune activities were more prevalent in these relatively malignant subtypes of glioma.In addition, we also explored that CYT was highly enriched in IDH wild-type glioma group in both CGGA (p < 0.0001) in mesenchymal subgroup (p < 0.0001)  . Then Re 0.0001)  and TCGA 0.0001) , respectp < 0.05) significantly mutated in CYT-high or CYT-low cohorts. Generally, 91% glioma samples harbored IDH1 mutation, which mostly concurrent with either TP53 and ATRX mutations in astrocytoma samples or with CIC and FUBP1 mutations in oligodendroglioma samples in CYT-low cohort. Meanwhile, 16 genes were founded with significantly higher frequencies of nonsense, splice-site and frameshift mutations (p < 0.05) in CYT-high cohort, which could affect RTK-RAS-PI3K pathway, blood cell hereditary, ATPase activity, ERK pathway, immune system, SWI/SNF complex, and chromatin remodeling.To reveal the molecular mechanism underlying the cytolytic T-cell activity of glioma, we divided gliomas of TCGA dataset into CYT-high cohort (tumors in the top 25th percentile) and CYT-low cohort (tumors in the bottom 25th percentile). We identified the diversity mutational profiles characteristic of gliomas . OverallBesides coding mutations, we also estimated the copy number alterations information of gliomas between CYT-high and CYT-low cohorts . The mosp < 0.001. We found 354 down-regulated genes and 1,166 up-regulated genes strongly correlated with CYT in CGGA dataset (https://david.ncifcrf.gov/). We found that genes positively correlated with cytolytic T-cell activity were more involved in immune response, regulation of angiogenesis and cell adhesion, dendritic cell chemotaxis, leukocyte migration, monocyte chemotaxis, neutrophil chemotaxis, T cell activation, and negative regulation of B cell activation  | >1 and adjusted  dataset , while 5 dataset . To yieltivation . MeanwhiTo our knowledge, immune checkpoints were crucial for suppress unwanted and harmful self-directed activities and modulating the duration of immune system. However, in cancer, immune checkpoints could shut down the T-cell powerful fighting activities, allowing tumor to grow undisturbed. Then we explore the relationship of CYT and checkpoints expression . The findings were intriguing, both in CGGA and TCGA datasets, all checkpoints were significantly positively associated with T-cell cytolytic activity . These rp = 0.0003) and TCGA dataset (p = 0.0002), respectively. And we validated this intriguing phenomenon in an independent validation group (p = 0.04). Those results suggested peritumoral brain edema probably was a reflection of CYT and tumor immune response.Edema, a reflection of inflammation, was a common pathophysiological entity surrounding gliomas. The secretory products of macrophages were reported might contribute to the formation of glioma edema. Then, we compared the edema extending differences between CYT-low and CYT-high group to investigate the potential association of CYT and peritumoral edema. As shown in p < 0.0001) and GBM (p = 0.021) (p < 0.0001) and GBM (p = 0.036) (p < 0.0001) after adjusting other factors, such as age at diagnosis (p = 0.54), grade (p < 0.001), IDH status (p = 0.09), and 1p19q status (p < 0.001). In TCGA dataset (p = 0.04) was an independent prognostic indicator. These results demonstrated that CYT value was an independent unfavorable predictor for patients with glioma.To further analysis the prognostic value of CYT, we employed dichotomization to separate cases for depicting the Kaplan Meier survival curves according to the cutoff value . In CGGA dataset, we found that patients with higher CYT value had a significant shorter overall survival than their counterparts in glioma (= 0.021) . Simulat= 0.036) . Next, w= 0.036) , the res dataset , the resThe immune system played pivotal roles in preventing and combating tumor . RecentlThen we performed an integrated analysis the mutation profile of glioma in the context of CYT value. We found that mutations of IDH, ATRX, TP53, CIC, FUBP1, and 1p/19q co-deletion were more common in CYT-low group. IDH mutations was considered as the truncal event and occurred in the vast majority lower grade glioma and secondary GBM. ATRX and TP53 were reported as the driver mutations for astrocytoma , 27, wheThe gene ontology analysis showed a positive correlation of CYT and immune response, regulation of cell angiogenesis and adhesion, and immune cells activation. Meanwhile, we found that the expression level of several immune-checkpoint molecules, including CTLA-4, IDO1, LAG3, CD274, PD-1, PD-L2, TIGIT, and HAVCR2 were significantly positively associated with cytolytic activity in glioma. CYT could be a useful biomarker for glioma patients to predict the responsiveness to immune checkpoint blockade therapy . Such imPeri-tumoral edema, a common symptom caused by inflammation response, caused mass effect and was associated with poor prognosis in glioma patients. We found that patients in CYT-high cohort were significantly associated with greater extend of edema than in CYT-low cohort. Our result indicated that the MRI image may potentially provide a simple approach to reflect the host immune status and the effective of immunotherapy.Previously pan-cancer research shown that higher expression of CYT value was correlated with improved prognosis. However, we found that CYT value was an independent unfavorable prognostic factor for glioma patients. This prognostic value was also observed when taking GBM as a separated type of glioma. This controversial result may be the reflection of tumor heterogeneity . The posIn summary, we explored the clinical roles and immune biological processes of CYT in more than 1,000 diffuse gliomas. Calculating CYT may help us elucidate the responses driven by the immune microenvironment more completely and extend our understanding of immunotherapy strategies in glioma.ZLW made substantial contributions to conception, design, and wrote the manuscript. ZW, GZL, ZSB, and QWW gave suggestions on study design, discussed, and interpreted the data. TJ and CBZ designed and supervised study. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium parasites can further enhance the development of treatment and prevention recommendations as well as outbreak investigations. Here, we explore the utility of using the full Plasmodium mitochondrial genome to classify Plasmodium species, detect mixed infections, and infer the geographical origin of imported P. falciparum parasites to the United States (U.S.). Using the recently developed standardized, high-throughput Malaria Resistance Surveillance (MaRS) protocol, the full Plasmodium mitochondrial genomes of 265 malaria cases imported to the U.S. from 2014–2017 were sequenced and analyzed. P. falciparum infections were found in 94.7% (251/265) of samples. Five percent (14/265) of samples were identified as mixed- Plasmodium species or non-P. falciparum, including P. vivax, P. malariae, P. ovale curtisi, and P. ovale wallikeri. P. falciparum mitochondrial haplotypes analysis revealed greater than eighteen percent of samples to have at least two P. falciparum mitochondrial genome haplotypes, indicating either heteroplasmy or multi-clonal infections. Maximum-likelihood phylogenies of 912 P. falciparum mitochondrial genomes with known country origin were used to infer the geographical origin of thirteen samples from persons with unknown travel histories as: Africa (country unspecified) (n = 10), Ghana (n = 1), Southeast Asia (n = 1), and the Philippines (n = 1). We demonstrate the utility and current limitations of using the Plasmodium mitochondrial genome to classify samples with mixed-infections and infer the geographical origin of imported P. falciparum malaria cases to the U.S. with unknown travel history.The ability to identify mixed-species infections and track the origin of  Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi [P. falciparum parasites resistant to first-line artemisinin-combination therapies (ACTs) from further spreading from the Greater Mekong Region [P. falciparum drug resistance markers greatly facilitates tracking of drug-resistant parasites.Malaria is a significant public health problem, with over 3.3 billion individuals at risk of infection worldwide . In 2016Plasmodium species and drug-resistance detection include real-time polymerase chain reaction (PCR) and Sanger sequencing, which are time consuming and costly. Recently, we developed a standardized next-generation sequencing and bioinformatics pipeline for Malaria Resistance Surveillance (MaRS), which deep sequences and analyzes, in a single assay, all validated P. falciparum drug-resistance associated single-nucleotide polymorphisms (and novel variants) in the major drug resistance genes , as well as the Plasmodium mitochondrial genome [In 2014, there were 1,724 domestically-imported, travel-related cases of malaria in the United States (U.S.) reported to CDC, of which 137 samples were sent to the Centers for Drug Control and Prevention (CDC) for drug-resistance testing [Plasmodium mitochondrial genome can be used for such purposes, as an additional marker for surveillance. The 6-kb mitochondrial genome is inherited solely from the female gametocyte and does not undergo recombination, making the mitochondrial genome a desirable candidate marker for pathogen surveillance [P. falciparum from sub-Saharan Africa with multiple introductions to South America and Southeast Asia, depicting distinct population structure [et al. [P. falciparum isolates from five regions including South America, West Africa, East Africa, Southeast Asia, and Oceania.While the primary purpose of MaRS is to identify drug resistance, the sequence data generated could provide additional information about imported parasites. For example, sequence data could be used to identify mixed infections and could also help determine the geographic origin of drug-resistant parasites, which would be useful to refine chemoprophylaxis and treatment recommendations as well as for case and outbreak investigations. The eillance –7. The ftructure ,9. FurthPlasmodium mitochondrial genome using a targeted-amplicon deep sequencing approach for malaria surveillance purposes. MaRS was used to sequence full Plasmodium species mitochondrial genomes from U.S. domestically-imported malaria samples, from 2014–2017, as part of an ongoing effort for anti-malarial drug resistance surveillance  were obtained from U.S. domestic, imported malaria cases during 2014–2017 from the CDC domestic malaria surveillance network and tested in accordance with protocol 2017–309 approved by the Office of the Associate Director of Science, Center for Global Health, Centers for Disease Control and Prevention as a surveillance activity.crt, mdr1, k13, dhfr, dhps, and cytochrome b genes and full mitochondrial genome were amplified, normalized, and pooled into one amplicon pool per patient. Libraries were constructed using the Nextera XT kit  and indexed at the patient level. Each indexed library was pooled and sequenced on the MiSeq (Illumina) using a 2 x 250 bp read length.Detailed methods for DNA extraction, targeted gene enrichment, library preparation, and sequencing were previously reported by Talundzic et al.  , using medium sensitivity and a minimum mapping quality score of 30. Variants were identified in a region 50 bp downstream and upstream from the 5’ and 3’ ends of the reference sequence, respectively, using a threshold of ≥ 5X read depth and ≥ 10% frequency. The variant calling region was designated to reduce low-confidence variant calls in regions with lower read depth.Fastq files were imported into Geneious v11.0.5 (Biomatters) for analysis. Paired-end sequences were paired, and BBduk Trimmer v37.28 [P. falciparum infected samples were aligned to the following Plasmodium mitochondrial reference genomes: Plasmodium vivax, NCBI accession NC_007243; Plasmodium ovale curtisi, accession HQ712052; Plasmodium ovale wallikeri, accession HQ712053; Plasmodium malariae, accession LT594637; and Plasmodium knowlesi, accession NC_007232. Variants were identified as stated above.Suspected multi-species mixed- and/or non-P. falciparum infections in samples with all variants that had greater than 95% frequency. Samples with genotype allele frequencies less than 95% were considered mixed infections and were excluded during the construction of haplotypes. Sequence data (raw fastq files) can be accessed through NCBI BioProject accession PRJNA428490 [P. falciparum reference genome was constructed using Geneious v11.0.5.Mitochondrial haplotypes were identified for NA428490 . Fig 1 dP. falciparum full mitochondrial genome sequences (n = 745) were downloaded from NCBI Nucleotide [Maximum likelihood phylogenetic trees comprised of domestic, imported samples with unknown travel history, known travel history, and publicly available sequences with known country of origin were constructed to infer potential origin or phylogenies of samples with unknown geographical origin. Publicly available cleotide , with gecleotide ,9,14–16.P. falciparum mitochondrial reference genome were hard-trimmed 50 bp on the 5’ and 3’ ends to remove low-quality bases. Multiple sequence alignments (MSAs) were constructed using MUSCLE v3.8.425 [Consensus sequences from alignments to the v3.8.425  for eachv3.8.425  and iTOLv3.8.425 .A median-joining network was constructed using 408 mitochondrial genomes with known geographical origin (n = 343) and samples with unknown travel history (n = 65). The number of samples was reduced  for better visualization by removing singleton haplotypes , removing samples with multiple country origins, and reducing the number of samples per region per haplotype to a maximum of 10 samples. Countries/regions of origin of known samples were reclassified into 12 different geographical regions .Multiple sequence alignments (MSAs) were constructed using MUSCLE v3.8.425 . HaplotyP. falciparum mitochondrial reference genome for 256 P. falciparum infected samples , with a mean of 5394 (±4831) reads aligned. The average read depth for the full mitochondrial genome ranged from 15.76X to 877.10X with a mean of 176X (±152.15) per sample. A total of 135 variants, including 133 SNPs and 2 indels (insertions), were identified , were aligned to mitochondrial reference genomes for P. falciparum, P. vivax, P. malariae, P. ovale curtisi, P. ovale wallikeri, and P. knowlesi.Full mitochondrial genomes generated by MaRS were used for species identification and to resolve mixed-species infections for all 265 samples used in this study. Samples suspected of mixed infections or non-P. falciparum and P. ovale curtisi, P. ovale wallikeri, or P. malariae as a co-infector. All mixed-infections were able to be deconvolved into their major and minor species contributors based on read count ratios  to 159.5X (±78.7). The minor Plasmodium species for all samples ranged from 2.6% to 42.7% abundance and reads aligned to 32.9% to 93.7% of the reference mitochondrial genomes with mean read depths ranging from 0.8X (±1.3) to 107.4X (±88.3).Six samples were identified as mixed-species infections containing t ratios . Five ofP. falciparum infections  to 491.2X (± 205.4). P. ovale wallikeri infected samples, 17–012 and 5909, had an identical mitochondrial haplotype  as did P. malariae infected samples, 16–209 and 5910 .Eight samples were identified as non-fections . Reads aP. falciparum infected samples with unknown travel history based on the phylogenetic relationship of each unknown sample to 912 P. falciparum mitochondrial genomes with known or reported geographical origin  Geographical origin was resolved for 13/89 samples to clades unique to Africa (n = 10), Ghana (n = 1), Southeast Asia/Oceania (n = 1), and the Philippines (n = 1) –S4 Figs.A median-joining network also was used to analyze the relationships of the more common haplotypes  based on varying geographical regions to better visualize the number of mutational steps and connections between the haplotypes observed . Forty-nPlasmodium species mitochondrial genome. One of the advantages of deep sequencing the Plasmodium mitochondrial genome is that is allows for species identification, including mixed-species infections. Targeted-amplicon deep sequencing provides a more comprehensive approach for molecular characterization of Plasmodium species infection by providing a high-throughput, quantitative method for species and mixed-species infection identification and improved detection of minor alleles present within a sample [Here, we describe the utility of using the MaRS targeted-amplicon deep sequencing assay  to sequea sample .Plasmodium spp. for single-species and mixed-species infections and characterize all P. ovale samples down to the subspecies level . Minor contributing species could also be detected in mixed-infections even when more than 97% of sequencing reads were from the major infecting Plasmodium species. Future studies will be needed to determine the limit of detection of a targeted-amplicon deep sequencing approach and to further assess the comparison of sensitivity and specificity of molecular targets within the MaRS assay to other molecular diagnostic methods.Sequenced full mitochondrial genomes were used to be able to classify Plasmodium species from each sample and use them for downstream analyses to infer geographical origin of the parasites. P. falciparum infected samples with more than one mitochondrial genome, however, present a more challenging task in deconvolving mixtures.Additionally, a targeted-amplicon deep sequencing approach provides a high-throughput, quantitative method to measure ratios of mixed sequences within a given sample and enables extraction of sequence reads from mixed-species infection. In contrast, an analog method, such as Sanger sequencing, would require at least 10 separate PCRs and sequencing reactions (~600 bp in size) to produce two sequence reads for each sample. Alternatively, using the deep sequencing approach used in this study can produce 5394 (±4831) reads at 176X (±152.15) depth per sample for complete coverage of the mitochondrial genome for a single PCR reaction and next-generation sequencing library preparation. For all mixed-species infections tested, we were able to extract full mitochondrial genome haplotypes of the major Multi-clonal infections may present challenges for effective treatment when drug-resistant clones may be at low levels. More than 18% of the samples in this study contained at least two or more mitochondrial haplotypes, indicating potential multi-clonal infections; however, it is important to note that multiple mitochondrial genomes could be indicative of heteroplasmy instead of multi-clonal infections due to multi-copy number mitochondrial genomes up to ~22 copies) per cell  copies p. AdditioP. vivax [Plasmodium falciparum mitochondrial haplotypes, 772C and 772C-1692A, which are present worldwide and in Africa and India, respectively, making geographical origin prediction for the majority of samples imported into the U.S. impossible using the mitochondrial genome alone, a finding previously reported by Preston et al. [et al. [et al. [Another advantage to capturing the entire mitochondrial genome for molecular surveillance of malaria is to potentially infer geographical origin of the parasite. In this study we employed a phylogenetic and median-joining network approach using the full mitochondrial genome to infer geographical origin of samples imported to the U.S. with unknown travel history, a strategy previously employed using mitochondrial genomes from P. vivax . We were [et al. , whom de [et al. .et al. [Plasmodium mitochondrial genome and 18 SNPs from the Plasmodium apicoplast genome (~35kb), to differentiate parasites specific to regions of South America, West Africa, East Africa, Southeast Asia, and Oceania. The 772C and 772C-1692A mitochondrial genome haplotypes from this study correspond to the 772C-853T-973T-1283A-2383G mitochondrial SNP barcode haplotype found worldwide by Preston et al. [et al. identified as the most informative, containing the highest Fst compared to the other SNP loci evaluated, to differentiate isolates from Southeast Asia/Oceania compared to the other three regions [et al. [et al. [P. falciparum parasites.Previously, Preston et al.  develope [et al. –31, and  [et al.  may be nPlasmodium mitochondrial genomes as part of domestic surveillance in the U.S. and to link this data with additional genetic markers that are collected with MaRS.The ability to predict the geographical origin of parasites for molecular surveillance of drug-resistance is imperative to be able to effectively track the spread of drug-resistant parasites. Of the 265 samples used in this study, only 65% had reported travel history. While we can report on samples with unknown origins sharing the same haplotypes in samples from particular countries or regions, we are limited in our confidence due to the numbers of samples representative from these countries in the database. In order to have increased confidence in geographical origin predication we need to have larger, more deliberate samplings from countries in order to infer origin at the country level. Public databases of sequences from parasites with different geographical origins are necessary to be able to effectively track parasites. While such databases, for this purpose, are not readily available, we hope to build a database of S1 File(CSV)Click here for additional data file.S2 File(CSV)Click here for additional data file.S3 File(CSV)Click here for additional data file.S1 FigP. falciparum mitochondrial genomes with known geographical origin to infer the geographical origin of Sample 16–003 with unknown travel history. Grey triangles represent collapsed clades for visualization purposes. Blue = clade of origin.A maximum-likelihood phylogenetic tree was constructed using 912 (TIF)Click here for additional data file.S2 FigP. falciparum mitochondrial genomes with known geographical origin to infer the geographical origin of Sample 5884 with unknown travel history. Grey triangles represent collapsed clades for visualization purposes. Blue = clade of origin.A maximum-likelihood phylogenetic tree was constructed using 912 (TIF)Click here for additional data file.S3 FigP. falciparum mitochondrial genomes with known geographical origin to infer the geographical origin of Sample 16–071 with unknown travel history. Grey triangles represent collapsed clades for visualization purposes. Blue = clade of origin.A maximum-likelihood phylogenetic tree was constructed using 912 (TIF)Click here for additional data file.S4 FigP. falciparum mitochondrial genomes with known geographical origin to infer the geographical origin of Sample 5923 with unknown travel history. Grey triangles represent collapsed clades for visualization purposes. Blue = clade of origin.A maximum-likelihood phylogenetic tree was constructed using 912 (TIF)Click here for additional data file.


Eucommia ulmoides is a model representative of the dioecious plants with sex differentiation at initiation. Nevertheless, the genetic mechanisms of sexual dimorphism and sex determination in E. ulmoides remain poorly understood. In this study de novo transcriptome sequencing on Illumina platform generated >45 billion high-quality bases from fresh leaves of six male and female individuals of E. ulmoides. A total of 148,595 unigenes with an average length of 801 base-pairs (bp) were assembled. Through comparative transcriptome analyses, 116 differentially expressed genes (DEGs) between the males and the females were detected, including 73 male-biased genes and 43 female-biased genes. Of these DEGs, three female-biased genes were annotated to be related with the sexually dimorphic gutta content in E. ulmoides. One male-biased DEG was identified as putative MADS box gene APETALA3, a B class floral organ identity gene in the flowering plants. SNPs calling analyses further confirmed that the APETALA3-like gene was probably involved in the sex determination in E. ulmoides. Four other male-biased DEGs were potential sex-associated genes as well with segregated SNPs in accord with sex type. In addition, the SNPs density was 1.02 per kilobase (kb) in the expressed genes of E. ulmoides, implying a relatively high genetic diversity. Eucommia ulmoides Oliver is a dioecious tree species of the family Eucommiaceae, in the asterid lineage of the angiosperms [trans-1,4-polyisoprene (TPI) in E. ulmoides has attracted broad attention [E. ulmoides therefore has the potential to replace the commonly known para-rubber tree  because of its wide distribution in the subtropical and temperate ecozones and good resistance [E. ulmoides is difficult due to its dioecious sexual system and long-life cycle, which constrains its breeding process [E. ulmoides trees [E. ulmoides to assist the breeding progress for improving the yield of gutta or other target traits.iosperms , that isiosperms . It is aiosperms . Recentlttention ,6. E. ulsistance ,8. Howev process . Moreovees trees , making n = 34, 1C DNA value per haploid genome = 723.7 megabases (Mb)) [E. ulmoides has a heteromorphic sex chromosome(s) or not is still uncertain [E. ulmoides in Nature both suggest that the sex of this species is most likely controlled by a genetic mechanism [Cucumis sativus) [Silene latifolia) [Rumex hastatulus [Asparagus officinalis) [E. ulmoides remain unknown to date.About 6% flowering plants  are dioecious and dispersed in 987 genera belonging to 175 families . Among tes (Mb)) ,14, whetncertain ,15. Neveechanism ,15,16. Dechanism ,19. The echanism ). NGS-basativus) , white ctifolia) , Rumex hcinalis) . NeverthE. ulmoides there is no occurrence of stamen primordia in the female trees neither is there pistil primordia in the male trees [E. ulmoides belong to type II, like in its closely-related taxa Aucuba chinensis and Garrya elliptica of the family Garryaceae [Historically two types of unisexual flowers have been recognized, i.e., type I and type II ,26. The le trees , causingrryaceae ,29.Arabidopsis with the former two as B class genes whereas the latter one as C class gene [Spinacia oleracea [Thalictrum dioicum [E. ulmoides and probably express constitutively, in line with the hypothesis proposed by Diggle et al. [In the ABC model of flower development, the B and C class organ identity genes are responsible for determining stamen and carpel initiations . The genass gene . All theass gene ,33,34. Ioleracea  and Thal dioicum . We thuse et al.  stating trans-structure [E. ulmoides [E. ulmoides, which is essential to further detect. In the present study, large-scale NGS transcriptome data were generated from the fresh leaves of the male and female samples in three independent full-sib families of E. ulmoides by RNA-seq method in combination with comprehensive comparative analyses. Through these investigations we aimed to: 1) detect the DEGs related to the sexual dimorphism of gutta yield in the male and female leaves of this economically important tree species; and 2) identify potential sex-associated genes in E. ulmoides.Sexual dimorphism such as differences in the morphology and physiology is commonly present in the dioecious plants, which may be related to the DEGs ,38 or ottructure . Pyruvattructure ,40. Sinculmoides  we thereE. ulmoides from three independent full-sib families with each sex having three individuals were sequenced according to Illumina PE (paired-end) sequencing protocol. About eight to ten gig abases (Gb) raw sequencing data were generated for each individual, reaching to a total of 327,129,846 raw reads in all the samples. After all the sequenced reads were quality-filtered by removing reads containing adapter sequences, ambiguous nucleotides, low-quality sequences, and all possible contaminations, c. eight Gb clean data were obtained for each individual. A total number of 311,326,872 clean reads (46.71 Gb) were eventually retained, of which 161,269,666 were from the male trees and 150,057,206 were from the female trees  were assembled. The N50 contig size, the minimum and maximum contig length were 705, 201, and 12,536 bp, respectively. These contigs were further assembled into 148,595 unigenes with an N50 length of 1064 bp and average length of 801 bp. The transcript with the longest length of each unigene was selected as the reference sequence for further analysis. The total length of all the unigenes reached to 117,298,413 bp . Furtherwww.ncbi.nlm.nih.gov; Protein family ; euKaryotic Ortholog Groups ; Swiss-Prot ; KEGG Ortholog database ; and Gene Ontology . Among the total 148,595 unigenes, 84,271 (56.71%) unigenes were annotated successfully in at least one database  and NCBI non-redundant nucleotide sequences (Nt) on the website of database . There wdatabase .Furthermore, the unigene sequences were characterized by assigning to the GO terms a. In totp-value (padj) < 0.05 and a minimal twofold difference in expression i.e., |log2 | ≥ 1, we found that 116 genes were differentially expressed in the male and female leaves, including 73 (62.9%) male-biased (up-regulated) and 43 (37.1%) female-biased (down-regulated) genes  ≥ 5) or down-  ≤ −5) regulated. Among these DEGs, sex-specific genes that expressed in either males or females were further investigated. We used the following criteria: DEGs had an estimated abundance of zero read counts in one sex but contained a certain expression in the other sex i.e., fragment per kilobase per million (FPKM) value >0.03. The result showed that 71 DEGs were identified as sex-specific genes, including 40 male-specific genes and 31 female-specific genes  genes . All theic genes .E. ulmoides were further analyzed by K-means clustering method using the pheatmap R package. The result revealed that the six individuals were clustered according to sex types i.e., males in a clade and females in another one  having homology with the MLS gene  of Solanum was enriched in pyruvate metabolic process (GO:0006090)  epigenetic pathway  . Similar(Solanum . Anothertabolism . In additabolism . This ge pathway . There wE. ulmoides (N-methyltransferase) was highly expressed in females, while LPCAT1_2  was highly expressed in males. For “starch and sucrose metabolism” (ko00500), the expression of TPP gene was only detected in females , q-value and gene number . The topulmoides . These tulmoides . For “gl females . Two mal females . One mal females  may to sE. ulmoides identified 100 putative MADS box unigenes  (The search of MADS box TFs in the assembled transcriptome of unigenes . Of thesunigenes . Furthermilarity . The othmilarity . In addid (>90%) .E. ulmoides (Eucommiaceae) was revealed to be located in eudicots correlating to species from Solanaceae and Amaranthaceae with low support values (64/0.89). Brassicaceae, Fabaceae, Malvaceae and Rosaceae clustered together in a clade with medium level supports (79/0.95), which then sistered to a clade comprising of Salicaceae and Vitaceae (72/0.91). However, the relationships of species from large families like Poaceae and Brassicaceae, were not well resolved here.The maximum likelihood (ML) and Bayesian inference (BI) analyses based on 58 AP3 orthologues from 38 angiosperms produced a congruent tree as shown in E. ulmoides, including 16 male-biased genes and eight female-biased genes  occurrence in the DEGs revealed that a total of 24 DEGs were polymorphic in ed genes . Furthered genes . There wed genes . The fivg plants . Neverthg plants .E. ulmoides genes was plentiful (E. ulmoides was 1.02 per kilobase (kb), amounting to a total of 119,411 SNPs . It is likely that the female-specific expression of MLS lead to higher accumulation of malate, which produced more pyruvate, and finally higher content of gutta is yielded [E. ulmoides [www.genome.jp/kegg) was preferentially expressed in the male leaves. This is suggestive of a fine dynamic regulation process of pyruvate concentration in the E. ulmoides leaves. Furthermore, Acetyl-CoA produced through the glycolysis/gluconeogenesis metabolism is a precursor of mevalonic acid in the MVA pathway [E. ulmoides could induce a higher concentration of glucose , which likely further regulates the downstream metabolisms to accumulate more gutta [H. brasiliensis where the efficiency of sugar metabolism seemed to be positively associated with the rubber yield [Two distinct pathways i.e., the MEP pathway and the MVA pathway play key roles in the synthesis of IPP which is the precursor of polyisoprene ,39. Pyru pathway . Malate  yielded . It may ulmoides . It is n pathway ,44. Herere gutta . This iser yield . Arabidopsis  that is homologous to the gene RDM1 in bidopsis . RDM1 isnthesis” . The difE. ulmoides [Aucuba [Eucommia [Garrya [E. ulmoides has diverged from its close lineages for >48 million years [The sex determination of plant has long been an intriguing and intractable issue since Darwin’s time . Sex detulmoides  are the ulmoides . In angi [Aucuba , EucommiEucommia  and Garr [Garrya  in the Gon years ,50, indiActinidia chinensis) [Salix suchowensis) was recorded to express significantly higher in the female flower buds than in the males [A. officinalis) also expressed differentially in the two sex types of flower buds [E. ulmoides transcriptome data (Solanum lycopersicum) [E. ulmoides  . One MADhe males . Severalwer buds . In thisome data , this nuersicum) . We founulmoides . One of larities . Furtherent tree . MonocotAquilegia, homologs of B class genes AP3 and PI are necessary for stamen identity [T. dioicum, the B and C class genes are differentially expressed in the male and female flowers early in development, resulting in gender determination [S. oleracea (SpAP3 and SpPI) is only detected throughout the stamen development in the male flowers, confirming the role of B class genes in sex differentiation [E. ulmoides and the SpAP3 gene of S. oleracea have c. 70% similarity  using RAD-seq approach [Populus balsamifera and P. trichocarpa) discovered that hundreds of SNPs were significantly associated with sex and located in ten different genomic regions [E. ulmoides.In dioecious plants, sex-linked genes are generally polymorphic and segregate between genders ,64. SNPsapproach . The who regions . We condCarica papaya [S.viminalis [Fragaria chiloensis [Mercurialis annua [S. latifolia [R.hastatulus [Populus [E. ulmoides and other species were detected. Distant phylogenetic relationship among E. ulmoides and those above species [E. ulmoides genome. We tend to believe that those putative sex-associated genes in the leaves detected here might be differently expressed in the reproductive organs as well, e.g., flower buds [Moreover, another four non-MADS box DEGs  segregated within the six selected individuals according to sex . Two Cl. Anothera papaya , S.vimiiminalis , Fragariiloensis , Mercuriatifolia , R.haststatulus  and two [Populus . However species ,71. The wer buds , given twer buds ,37. HoweE. ulmoides was reported based on SSR (simple sequence repeats) markers  gene regions (one SNP per 5.2 kb) [E. ulmoides may be one of the factors contributing to the high level of its genetic diversity [E. ulmoides compared to the previously published ones [E. ulmoides. In short, the results obtained in this study provided first insights into the sexual dimorphism of gutta content and the genetic mechanism of sex determination in E. ulmoides. These data generated here will facilitate further comprehensive research on the sex differentiation of E. ulmoides and the other dioecious plants in the future.A high level of genetic diversity at the population level of = 0.716, ). Here iulmoides . In tota 5.2 kb) . The dioiversity ,75. Furthed ones ,77 to imE. ulmoides growing on the campus of the Northwest A&F University in Yangling, Shanxi, China were selected for this study. In each family one mature male and female individual determined by their distinct male and female flowers in early spring (April) 2016 were sampled. A total of six individuals were sampled with three replicates for each sex. Fresh healthy leaves were collected from each individual before being immersed into liquid nitrogen immediately, and then stored at −80 °C until RNA isolation.Three independent full-sib families of ® spectrophotometer . The integrity of RNA was further assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system . The concentration of the RNA was subsequently measured with a Qubit 2.0 Fluorometer .Total RNA was isolated from the leaf samples using a TRIzol Reagent  following the manufacturer’s instructions. The quality of RNA was monitored on the 1% agarose gels and checked using the NanoPhotometer® UltraTM RNA Library Prep Kit for Illumina®  was used to generate one library for each of the examined individuals according to the manufacturer’s instructions. The cDNA fragments with 150–200 bp in length were selectively purified with AMPure XP system . Finally, the PCR enriched cDNAs were sequenced on the Illumina HiSeq 2500 platform at Novogene , to generate PE reads with 150 nucleotides in length.For each individual a total of c. 1.5 µg RNA was used for subsequent RNA-seq experiment. NEBNextRaw reads generated from the RNA-seq were filtered by removing reads containing adapter or ploy-N, and low quality reads to obtain high-quality clean reads. In the meanwhile, the sequencing error rate, Q20, Q30, and GC content of clean data were calculated for assessing the sequencing quality. All the subsequent analyses were based on the clean data.E. ulmoides [−5 [Since there is no publicly available genome of ulmoides , all theulmoides  was usedides [−5 . The seqides [−5  was usedp-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate [padj < 0.05 and |log2 | ≥ 1 were considered as DEGs [Gene expression level was estimated for each sample by RSEM . Clean rery rate . Genes w as DEGs , i.e., mhttps://cran.r-project.org/web/packages/pheatmap) was applied for K-means cluster analysis [http://www.genome.jp/kegg/, [The pheatmap R package . We usep/kegg/,  softwareE. ulmoides iTAK program [http://hmmer.org/) to identify differentially expressed MADS box TF genes between the males and females based on the plant TF database .To investigate the sex differentially expressed MADS box genes in  program  was usedhttp://www.geneious.com, [E. ulmoides differentially expressed in the two sexes was further examined across other 37 angiosperm species. 57 AP3 orthologues in total were collected from EnsemblPlants database (http://plants.ensembl.org/) and aligned with the identified AP3-like gene of E. ulmoides. Amborella trichopoda was defined as outgroup according to the updated APG IV system [All the obtained MADS box-like genes were aligned using MUSCLE . An unroous.com, ), with 1V system  under thV system . Two runActinidia chinensis [Bryonia dioica [Hippophae rhamnoides [E. ulmoides. Picard-tools v1.41 (http://broadinstitute.github.io/picard/) and samtools v0.1.18 [Given the sex-associated genes in dioecious plants are polymorphic and segregate along with sex, e.g., in hinensis , Bryoniaa dioica , and Hipamnoides , we here v0.1.18  were use v0.1.18  was furtE. ulmoides were surveyed. Then the SNPs in DEGs i.e., sex-biased expressed genes were examined to investigate their most likely associations with the dioecy of E. ulmoides. To obtain a more solid result more stringent cutoff values were applied as follows: (1) at least five unique reads in each individual covered the same nucleotide position; (2) the genes must be polymorphic (in terms of SNPs) in the six individuals; (3) the SNPs must be common in each sex type, i.e., males shared one SNP and females shared the other one. The SNPs that satisfied the above criteria were considered as potential sex-associated SNPs and the corresponding genes were identified as the putative sex-associated genes.The frequency and distribution of SNPs occurrence in the expressed genes of E. ulmoides, a representative of dioecious woody plant with sex differentiation at initiation. Our results revealed 116 DEGs between the male and female leaves. A number of candidate genes related to the sexual dimorphism of gutta yield in the leaves were characterized. Five putative sex-associated genes including a MADS box TF gene involved in the male organ identity were identified. High frequency of SNPs was also detected in the expressed genes. Collectively, this study will shed light on our understanding of the genetic regulation of sex expression in E. ulmoides and will also facilitate further comprehensive research on more dioecious species in the future.NGS-based transcriptome sequencing was applied to investigate the genes showing sex-biased expression patterns in the vegetative leaf tissues of


Xenoturbella, Nemertodermatida, and Acoela differ extensively in their central nervous system anatomy, the reconstruction of the xenacoelomorph and bilaterian neuropeptide complements may provide insights into the relationship between nervous system evolution and peptidergic signaling. Here, we analyzed transcriptomes of seven acoels, four nemertodermatids, and two Xenoturbella species using motif searches, similarity searches, mass spectrometry and phylogenetic analyses to characterize neuropeptide precursors and neuropeptide receptors. Our comparison of these repertoires with previously reported nephrozoan and cnidarian sequences shows that the majority of annotated neuropeptide GPCRs in cnidarians are not orthologs of specific bilaterian neuropeptide receptors, which suggests that most of the bilaterian neuropeptide systems evolved after the cnidarian–bilaterian evolutionary split. This expansion of more than 20 peptidergic systems in the stem leading to the Bilateria predates the evolution of complex nephrozoan organs and nervous system architectures. From this ancient set of neuropeptides, acoels show frequent losses that correlate with their divergent central nervous system anatomy. We furthermore detected the emergence of novel neuropeptides in xenacoelomorphs and their expansion along the nemertodermatid and acoel lineages, the two clades that evolved nervous system condensations. Together, our study provides fundamental insights into the early evolution of the bilaterian peptidergic systems, which will guide future functional and comparative studies of bilaterian nervous systems.Neuropeptides are neurosecretory signaling molecules in protostomes and deuterostomes (together Nephrozoa). Little, however, is known about the neuropeptide complement of the sister group of Nephrozoa, the Xenacoelomorpha, which together form the Bilateria. Because members of the xenacoelomorph clades  Neuropeptides are a diverse group of signaling molecules that play a crucial role in the function of the nervous system in most metazoan animals . These sXenoturbella, Nemertodermatida, and Acoela, with the latter two forming the clade Acoelomorpha  and in the nemertodermatid Nemertoderma westbladi. In both Xenoturbella species we also identified GPH beta, and in X. bocki the GPH-related bursicon beta precursor. In acoels, we detected a single sequence in Isodiametra pulchra, which shows no clear similarity with a specific type of GPH-related peptide in our phylogenetic analysis , were also annotated as bilaterian neuropeptide GPCRs. These neuropeptides include neuropeptide FF, neuropeptide Y, orexin, cholecystokinin-, qRFamide-, RYamide, substance-K-, bombesin-, gastrin-, galanin-, and somatostatin-GPCRs. In order to resolve the inconsistencies between previous studies and to better reconstruct the ancestral cnidarian–bilaterian neuropeptide complement, we reanalyzed the automatically annotated sequences, as well as GPCR sequences that were predicted from the genome of Nematostella vectensis as galanin-like, tachykinin/SIFamide-like, RFamide/neuropeptide FF/GnIH/neuropeptide Y-like, and GnRH/vasopressin-like receptors , while the main diversification into the different bilaterian GPCR groups occurred after the cnidarian–bilaterian evolutionary split, with a parallel diversification in the cnidarian lineage.While a previous large analysis of cnidarian and bilaterian taxa showed that only relaxin-like and GPH-related GPCRs are plesiomorphic to cnidarians and bilaterians , other s pallida , Acroporgitifera , and Orbeceptors . Our anaXenoturbella species, whereas only nine homologs were identified in Acoela  were those related to GnRH and PTH signaling, whereas GPCRs related to achatin and allatostatin C signaling were found in both Acoelomorpha clades, but not in Xenoturbella. Receptors related to prokineticin, neuropeptide Y/F, TRH, ETH, and CCHamide signaling were restricted to Nemertodermatida. The only GPCRs exclusively detected in acoels were receptors that are related to CCAP signaling, and a single sequence that shows similarity to a large cluster that diversified greatly in protostomes into proctolin, arthropod FMRFamide, myomodulin, RGWamide, and myoinhibitory peptide receptors  g, SSxxxFN. westbladi ELamide peptides possess aromatic amino acids on position 3 and 5 from the C-terminus, which makes them somewhat similar to arthropod allatostatin A peptides  was mainly used to confirm our bioinformatic predictions of the neuropeptide precursors. From the predicted peptide precursors, the presence of processed LWDY peptides, AWDF peptides, SSxxxF peptides, FxxxFamide peptides, and amidated as well as nonamidated MxGFG peptides was confirmed by LC-MS/MS in at least one species , C. macropyga (Acoela), D. gymnopharyngeus (Acoela), D. longitubus (Acoela), Eumecynostomum macrobursalium (Acoela), H. miamia (Acoela), I. pulchra (Acoela), Ascoparia sp. (Nemertodermatida), M. stichopi (Nemertodermatida), N. westbladi (Nemertodermatida), Sterreria sp. (Nemertodermatida), X. bocki (Xenoturbella), X. profunda (Xenoturbella). Most of the transcriptomes were generated from several whole adults, except for X. profunda, and should thus include neuronal expressions .GPCR sequences were clustered using CLANS2 , and a srthologs . We analrthologs  with therthologs , nonconsrthologs , and phyrthologs  and Fastrthologs  using the-value cutoff of 1. In addition to sequence similarity search, we employed a Perl script that uses PROSITE pattern syntax and flat files to detect specific protein sequence motifs. We screened translated transcriptomes for the presence of recurrent cleavage and amidation sites that are commonly found in multicopy neuropeptide precursor sequences, including x-G-[KR]-[KR]-x-G-[KR]-[KR]-x-G-[KR] and x-[KR]-[KR]-x-[KR]-[KR]-x-[KR]-[KR]-x-[KR]-[KR]. For possible RFamides with alternative monobasic cleavage sites, we searched for the following protein sequence motif: x-R-F-G-[KR]-x-R-F-G-[KR]-x-R-F-G-[KR]. This approach gave us candidate neuropeptides that were further analyzed manually for recurrent peptide sequences between the cleavage sites. All sequences from the motif and similarity search with complete 5′-ends were tested for the presence of a signal peptide with Signal-3L 2.0   . A C-terC. macropyga, I. pulchra, and H. miamia following the protocol described by 18 StageTips  fragmentation of the seven most intense precursor ions. High-resolution HCD MS/MS spectra were acquired with a resolution of 60,000. The target values for the MS scan and MS/MS fragmentation were 3 × 106 and 105 charges with a maximum fill time of 25 and 110 ms, respectively. Precursor ions were excluded from sequencing for 30 s after MS/MS. MS/MS on singly charged precursor ions was enabled. The acquired MS raw files were processed using the MaxQuant software suite v.1.5.2.8  coupled with a Q Exactive HF mass spectrometer (Thermo Scientific). Separations of the peptide mixtures were performed as previously described  with sli.1.5.2.8 . Extract.1.5.2.8  to query.1.5.2.8 . Spectrahttps://doi.org/10.6084/m9.figshare.c.4064639.v1; last accessed August 23, 2018 and https://doi.org/10.6084/m9.figshare.6287759; last accessed August 23, 2018. The script for the pattern searches is available on https://github.com/faguil/protein-motif-searching_neuropeptides; last accessed August 23, 2018.Amino acid sequences of xenacoelomorph neuropeptide precursors and neuropeptide GPCRs are available in the Supplementary DataClick here for additional data file.