Live imaging studies give unparalleled insight into dynamic single cell behaviours and fate decisions. However, the challenge of reliably tracking single cells over long periods of time limits both the throughput and ease with which such studies can be performed. Here, we present NucliTrack, a cross platform solution for automatically segmenting, tracking and extracting features from fluorescently labelled nuclei. NucliTrack performs similarly to other state-of-the-art cell tracking algorithms, but NucliTrack’s interactive, graphical interface makes it significantly more user friendly.http://nuclitrack.readthedocs.io/en/latest/ A video guide can be viewed online: https://www.youtube.com/watch?v=J6e0D9F-qSU Source code is available through Github: https://github.com/samocooper/nuclitrack. A Matlab toolbox is also available at: https://uk.mathworks.com/matlabcentral/fileexchange/61479-samocooper-nuclitrack-matlab.NucliTrack is available as a free, cross platform application and open source Python package. Installation details and documentation are at: Bioinformatics online. Live imaging studies are now allowing us to explore the previously shrouded world of mammalian cell signalling dynamics. For example, imaging populations of live single cells over several days has revealed how signalling dynamics can control key cell fate decisions . HoweverTo date, the majority of cell tracking software relies on ‘frame-to-frame linking’ of segmented nuclei or cells . AdditioThe identification and segmentation of fluorescently labelled objects in NucliTrack is based on marker controlled watershed segmentation, built with the scikit image Python library  colour-coded tracked segments; (ii) the original video; and (iii) feature values over the full length of a selected track (in the form of a line plot over time), allow the user to easily analyze and inspect tracks for errors, or anomalous results. Moreover, using a set of tools with keyboard hotkeys the user can efficiently navigate the video, manually correct errors in tracking, and add flags to mark specific events, before exporting results. Throughout NucliTrack we have also incorporated data management steps, such that intermediate steps are saved in the HDF5 file format, through the Python h5py package . ParametTo characterize the performance of the global optimization approach to automated cell tracking we tested NucliTrack against a benchmark set of fluorescently labelled nuclei videos . The perNucliTrack is an easy to use package for tracking and extracting data from fluorescently labelled cells or nuclei, in significantly higher throughput than manual tracking can achieve. Importantly, by allowing users to quickly inspect and correct tracking data, near 100% accuracy can be achieved, which is critical in quantifying cell fate decisions. For example, NucliTrack allowed us to study how DNA damage influences the proliferation-quiescence decision in thousands of cells, tracked for hundreds of frames, over multiple days (S.C. is supported by the NIHR Imperial BRC, and the STRATiGRAD graduate training program. C.B. and A.R.B. are funded by a BBSRC Strategic LoLa grant [BB/M00354X/1], C.B. is also funded by a Cancer Research UK Programme Foundation Award [C37275/1A20146]. Conflict of Interest: none declared.Supplementary DataClick here for additional data file.


Pisolithus microcarpus (Cooke & Massee) G. Cunn is a gasteromycete that produces closed basidiocarps in symbiosis with eucalypts and acacias. The fungus produces a complex basidiocarp composed of peridioles at different developmental stages and an upper layer of basidiospores free of the hyphae and ready for wind dispersal upon the rupture of the basidiocarp pellis. During basidiosporogenesis, a process that takes place inside the basidiocarp peridioles, a conspicuous reserve of fatty acids is present throughout development. While several previous studies have described basidiosporogenesis inside peridioles, very little is known about gene expression changes that may occur during this part of the fungal life cycle. The objective of this work was to analyze gene transcription during peridiole and basidiospore development, while focusing specifically on cell cycle progression and lipid metabolism.p-value < 0.05) corresponding to 3.49% of the genes present in the P. microcarpus genome. We identified three clusters among the regulated genes which showed differential expression between the peridiole developmental stages and the basidiospores. During peridiole development, transcripts for proteins involved in cellular processes, signaling, and information storage were detected, notably those for coding transcription factors, DNA polymerase subunits, DNA repair proteins, and genes involved in chromatin structure. For both internal embedded basidiospores  and external free basidiospores , upregulated transcripts were found to involve primary metabolism, particularly fatty acid metabolism (FA). High expression of transcripts related to β-oxidation and the glyoxylate shunt indicated that fatty acids served as a major carbon source for basidiosporogenesis.Throughout different developmental stages of the peridioles we analyzed, 737 genes were regulated between adjacent compartments  contains supplementary material, which is available to authorized users. Pisolithus microcarpus (Coker & Mass.) Cunn. is an ectomycorrhizal mutualistic symbiont able to interact with economically important plants for the forestry sector, such as eucalyptus and acacias [P. microcarpus has a worldwide distribution and forms ectomycorrhizal associations with eucalypts and acacias growing in native Australian forests  and exotic forest plantations throughout the world  [P. microcarpus first originated in Australia and was subsequently introduced to other locations by human activities [P. microcarpus genome [The basidiomycete fungus  acacias . P. micr Africa) . Phylogetivities , 3. Reces genome , the facP. microcarpus. The so-called gasteromycetation, the development of hypogeous fruiting bodies from mushroom-shaped fungi is a striking change in fruiting body morphology during evolution. It was explained by secotioid intermediary shapes [Pisolithus spp. basidiocarps show wide variation in morphology, including stipe size, shape, length, peridium features, and type and color of spore masses produced inside the fruit body [P. microcarpus, the main morphological features of basidiospores and basidiosporogenesis are well known. The fungus produces a complex basidiocarp which serves as a structure within which the peridium (or “nest”) contains small capsules known as peridioles (or “eggs”) in various developmental stages. Enclosed in these peridioles are immature basidiospores and an upper layer of free basidiospores ready for dispersion upon rupture of the basidiocarps cellular cortical layer known as the pellis [Fungi classified in the phylum Basidiomycota form reproductive structures or fruiting bodies called basidiocarps (mushrooms). The production of spores in closed basidiocarps is a common trait of the Sclerodermataceae, a family which includes y shapes , 6 and iy shapes , 7. The uit body –10. For e pellis , 12  has been used for the transcriptomic analyses of fruiting body development in model fungi such as d others –26. Thesd others , 27–30. idiocarp , 31, andidiocarp , 12, 14.P. microcarpus basidiocarp formation and for determining factors that affect fatty acid metabolism in the basidiocarps and basidiospores. Overall, transcriptome information will contribute to better characterize the different developmental stages of the basidiospores, from cell individualization to maturation and germination, shedding light on the degree of preparedness and the biochemical activities required for subsequent basidiospore germination.These transcriptome data is essential for a better understanding of the genes involved in basidiosporogenenis during P. microcarpus basidiocarp [P. microcarpus. Our analysis of differentially expressed genes (DEGs) focused on those that may contribute to processes responsible for basidiosporogenesis and mobilization of reserve compounds.In this work, transcriptome analyses of the stages of peridiole development in idiocarp  were perP. microcarpus basidiocarps, including three peridiole stages as well as internal and free basidiospores  were found expressed in all compartments. For those remaining (6443), transcripts could not be found in any of the developmental stages analyzed , the sequenced strain and used here as reference genome, indicating that this result is not due to sequence polymorphism. These genes could be either expressed in specific situations or tissues or could represent pseudogenes or misannotations. About 15% of these undetectable transcripts corresponded to genes with predicted functions, such as cellular processes and signaling (4.8%), information storage and processing (5.16%), metabolism (2.12%) while the majority showed no similarity to known domains or functions.Illumina-RNA sequencing was used to study gene expression in five developmentally different compartments within the pts 13,83 were foup-value < 0.05)   between the different compartments. The greatest difference was found between mature peridioles and free spores, with 387 differentially expressed transcripts. The EuKaryotic Orthologous Groups (KOG groups) functional classification of the induced transcripts for each compartment  revealed a predominance of information, storage and processing-related transcripts in the peridiole compartments while metabolism-related transcripts were dominant in basidiospore compartments  Fig. . Clusternts Fig. .Fig. 2GeP. microcarpus up-regulated transcripts in each compartment compared to the number of specific genes in its genome. Between 15 and 32% of the differentially expressed transcripts are P. microcarpus - specific compared to about 25% in the P. microcarpus genome. Both the differentially expressed genes in young and mature peridioles were significantly enriched for P. microcarpus specific genes .Table 2 sum of RPKM/KOG class) within each compartment. In all peridiole compartments, cell cycle control, cell division and replication-related transcripts were highly expressed compared to transcripts in spores. Up-regulated and highly expressed transcripts for mainly young peridioles, but also mature peridioles, were related to chromatin structure and dynamics as well as to posttranslational modification, protein turnover and catabolism. Cell wall/membrane/envelope biogenesis related transcripts were highly expressed and regulated during the transition from peridioles to spores . In the basidiospore compartments, lipid transport and metabolism related transcripts were highly induced, as well as transcripts related to amino acid transport and metabolism. Transcripts for posttranslational modification, protein turnover and catabolism, carbohydrate metabolism, secondary metabolism, energy production and transcription-related transcripts showed to be highly expressed and regulated in all compartments. In general, genes showed similar expression in the peridiole stages    Fig. . These g05) Fig. , respectMost of the up-regulated transcripts in internal and free spores (57 and 196 respectively) Fig.  appearedP. microcarpus [P. microcarpus was annotated using the KEGG (Kyoto Gene and Genomes) pathway database and was highly expressed in the peridiole compartments  showed its highest expression in peridioles compared to the spores. Transcripts for meiosis induction protein kinase (ime2) were more abundant in unconsolidated peridioles and internal spores than in the other developmental stages. Following DNA replication, transcripts of genes involved in checkpoint mechanisms for meiosis/mitosis progress, such as rad17, rad24, rad53, chk1, and cdc14 were detected. These transcripts were highly expressed in all P. microcarpus compartments, particularly during peridiole development. A set of genes involved in spindle checkpoint process, mad1 and mad2, and a control APC/C function were more highly expressed especially in the peridioles. As for chromosome segregation, transcripts to cdc7 were up-regulated only in mature peridioles, while cdc14 and cdc20 transcripts were accumulated in peridiole compartments. Among the transcripts involved in the structural maintenance of chromosomes, smc1 and smc3 also appeared to accumulate in the peridiole compartments.Transcriptome analyses along peridioles and basidiospore compartments revealed two main pathways as highly expressed and up-regulated: cell cycle and lipid metabolism . The most abundant were TFs with HMG_box (5), homeobox and zinc binding (Zn_cluster) domain (3)  gene expression in between the basidiocarp compartments Fig. . The genors Fig. . During (3) Fig. . The hea(3) Fig. .Fig. 7DiP. microcarpus basidiosporogenesis, in the base of basidiocarp, unconsolidated peridioles consist of an agglomeration of vegetative hyphae that differentiates into basidia. Campos et al. [This work provides the first glimpse into the transcriptome of a basidiosporogenesis from a gasteromycete fungus with its typical compartments reflecting different developmental stages of peridioles and basidiospores Fig. . These ds et al. , 12 showIn young peridioles, each hyphal cell forms one to several basidia before the final stage where spore primordia are formed inside the mature peridioles. These processes were visible by differential expression of gene families related to signal transduction, replication, chromatin structure, cytoskeleton, and cell cycle control activation. The mature peridioles presented the highest number of regulated genes and in addition, genes coded for clade-specific proteins were significantly enriched in these two compartments. It was not possible, however, to assign functions to these orphan genes with no known domains. This high transcriptional activity, particularly the increase of cell cycle related transcripts, supports the fact that major cell division processes take place in these compartments as Campos and Costa were able to show using fluorescence microscopy and staining with calcofluor white and SYBR Green .P. microcarpus spores when they are mature and ready for dispersion.Following the differentiation of hyphal cells into basidia , basidioP. microcarpus basidiosporogenesis, conjugated division, karyogamy, meiosis, post-meiotic mitosis, and nuclear migration events were observed in unconsolidated, young, and mature peridioles [spo11 has been reported as the main protein responsible for DNA double strand breaks in homologous chromosomes in fungi, such as C. cinerea [P. microcarpus. For other fungi, such as Neurospora sp., transcriptome analyses also revealed correlations between the morphological changes observed during sexual development and cell cycle transcripts [C. cinerea, numerous microtubules are oriented longitudinally in the sterigmata and Golgi vesicles carry carbohydrates to the developing spore and spore wall [C. cinerea, microtubule formation is regulated and necessary for early fruiting events [During ridioles . In thisridioles . Genes c cinerea . Our resnscripts . The up-ore wall . In C. cg events . OverallP. microcarpus possibly indicates that these compounds may be important factors for basidiospore germination, a process currently not well-understood and which may limit P. microcarpus inoculation.Regulation of a large number of genes coding for proteins involved in primary metabolism, particularly in lipid transport and metabolism, was also observed. During peridiole development, genes coding for phosphatidylserine decarboxylase were consistently expressed, but the expression was approximately seven times higher in free spores than in the other compartments. This enzyme is responsible for converting phosphatidylserine into phophatidylethanolamine, a compound known to accumulate in un-germinated spores of fungi , 39. In P. microcarpus both organelles are involved in fatty acid oxidation. The increase in gene transcripts of the glyoxylate shunt, such as malate synthase in free spores, also confirmed activation of this pathway. This activation of the glyoxylate cycle was also observed in fruiting bodies of other fungi, such as Tuber borchii, Fusarium graminearum, and F. verticillioides [C. cinerea, 38% of the genes encoding proteins involved in cell metabolism were up-regulated at the primordium stage in this fungus [A. aegerita, genes for this pathway were also induced in fruiting bodies [P. microcarpus.Histochemical stains showed that lipid reserves are particularly important in internal and free spores, in that they take up most of the cellular space and push the organelles and the nucleus against the cell wall . Consistllioides , 29, 40.s fungus . In A. ag bodies . Our resP. microcarpus are embedded in a gelatinous matrix and the presence of hydrophobins in the spore cell walls also exhibited high concentrations [P. microcarpus peridiole development, TFs involved in general cellular processes like zinc binding domain-containing (PF10533 and PF00096 for C2H2 type) and HMG-box domains containing TFs were highly expressed in all compartments. Moreover, C2H2-type TFs had the highest expression in primordia and mature fruiting bodies of S. commune and Agaricus bisporus [P. microcarpus basidiocarp [P. microcarpus.Hydrophobins have previously shown to have high expression levels during fruiting body formation , 37. Thetrations . Both fitrations , 41. Tratrations , 42, 43.bisporus , 32, 44.idiocarp . In all idiocarp . The disP. microcarpus basidiocarp. The presence of gene coding for proteins involved in lipid metabolism, mainly in fatty acid metabolism, during the last two stages of peridiole development confirm the important role of lipid metabolism during basidiospore production and subsequent germination. These data will contribute to further our understanding the formation and development of fruiting bodies in basidiomycetes, particularly in gasteromycetes, for which most of the genetic, physiological, and morphological processes involved remain poorly characterized.This is the first work describing the transcriptional landscape within the different compartments of an ectomycorrhizal gasteromycete fungus. We were able to show that genes related to cell cycle regulation and signaling are induced in the peridiole compartments in P. microcarpus basidiocarps (UFV-VIC 26495) were collected in March 2013 from two eucalypt plantations belonging to Universidade Federal de Viçosa in Viçosa, Minas Gerais State (MG), Brazil . The collected basidiocarps were immediately transported to the laboratory and superficially cleaned with 70% ethanol. The basidiocarps were opened and the gleba representative of the different stages of peridiole development were collected according to [Intact rding to , 12. TheP. microcarpus 441 reference transcripts available at the JGI database http://genome.jgi.doe.gov/Pismi1/Pismi1.home.html [P. microcarpus 441 was used to fish out ITS reads from FS and MP samples of each of the three basidiocarps and to extract their ITS consensus sequences in order to compare them to the one from P. microcarpus 441  method . Assays ome.html  using CLFor read mapping the CLC genomic workbench parameters were the following: minimum length fraction 0.9, minimum similarity fraction 0.8, Mismatch cost = 2, insertion cost = 3, Deletion cost = 3 and the maximum number of hits for a read was set to 10. The unique and total mapped reads number for each transcript were determined and then normalized to RPKM (Reads Per Kilobase of exon model per Million mapped reads). Intact pairs were counted as two, broken pairs as one. Additional file It should be noted that UP, YP and MP represent dikaryotic tissues with unknown degree of allelic expression while IS and FS are a mix of (post-meiotic) monokaryotic spores of unknown polymorphism.p-value <0.05 were used.To identify differentially regulated transcripts in the different fruiting body compartments pairwise comparison by the Baggerley test  implemenFor Additional file http://genome.jgi.doe.gov/Pismi1/Pismi1.home.html) [P. microcarpus (Pismi) (http://genome.jgi.doe.gov/Pismi1/Pismi1.home.html) [Pisolithus tinctorius (Pisti) (http://genome.jgi.doe.gov/Pisti1/Pisti1.home.html) [P. microcapus specific proteins. If a protein cluster only contained predicted proteins from P. microcarpus, these were considered as Pismi-specific.GO, KEGG, KOG and InterPro (IPR) annotations were downloaded from the Joint Genome Institute (JGI) website (me.html) . A Markome.html) , includime.html)  and of tme.html)  was usedhttp://www.bioinf.ebc.ee/EP/EP/EPCLUST/) [For hierarchical clustering EPCLUST (PCLUST/)  or GENESPCLUST/)  was usedP. microcarpus was conducted using the respective pathways (based on KEGG) from P. microcarpus and when the gene was not in the automatic annotation provided by the JGI orthologous genes from Laccaria bicolor were used. The annotated sequences in L. bicolor were used as query sequences for BLASTP searches for similar proteins in the P. microcarpus genome. The BLAST output was sorted and top hits ranked by BLAST scores. The e-value cutoff used to assign homologues was 1e−5.A search for homologues of genes involved in cell cycle and lipid metabolism in http://www.transcriptionfactor.org) [P. microcarpus proteins containing these PFAM domains with HMMER package.To identify transcription factors we established a list of 330 PFAM domains linked to transcription factor activity, based on the EMBL file available (tor.org)  and pfamhttp://crn2m.univ-mrs.fr/pub/amplifx-dist) [Assays for relative quantification were conducted using a Chromo 4TM Detector . The primers were designed using the Primer3 software  and Amplfx-dist)  (see Addfx-dist) . Tests ffx-dist) . Primers


AbstractPlatygasteringeniosus Matsuo & Yamagishi, sp. n. and P.urniphila Matsuo & Yamagishi, sp. n. (Hymenoptera: Platygastridae) are described from Japan. The former species is an egg-larval solitary parasitoid of Masakimyiapustulae Yukawa and Sunose (Diptera: Cecidomyiidae). The latter species is an egg-larval gregarious parasitoid of Rhopalomyialongitubifex (Shinji) (Diptera: Cecidomyiidae). Platygaster Latreille (Hymenoptera: Platygastridae) contains 601 species, of which approximately 300 species have been described from various biogeographic regions during the last two decades   that induces leaf galls on Euonymusjaponicus (Celastraceae)  (Diptera: Cecidomyiidae) that induces axillary bud galls on Artemisiaindicavar.maximowiczii (Asteraceae)  . In addie larvae . One spetraceae) . This Pl thicker . Anothereraceae) .Cynipidae (Hymenoptera), Diptera: Cecidomyiidae) into two groups, early and late attackers, according to their parasitic strategies. Early attackers (koinobionts) are host-specific endoparasitoids that oviposit into host eggs or younger host larvae before galls start to develop  (Diptera: Cecidomyiidae). Rabdophagarosaeformis Kovalev (Diptera: Cecidomyiidae) parasitized by Platygasterstimulator Yamagishi mature in summer whereas unparasitized R.rosaeformis larvae pass through the summer as first instars. In terms of reproductive strategy, some species including P.robiniae Buhl and Duso are known to be gregarious parasitoids  (As demonstrated by  develop . In contttackers . Host spasitoids . In addi (Myers) .Platygaster and to provide information on their host range and reproductive strategies.In order to contribute to further taxonomic and ecological studies of platygastrid parasitoids, we intend in this paper to identify the two undescribed species of PageBreakMasakimyiapustulae and Rhopalomyialongitubifex were collected from Kyushu, Japan, in 2007–2017 to rear sufficient numbers of adults of Platygaster species for taxonomic study. In rearing Platygaster species that attacks Rhopalomyialongitubifex, the number of males and females emerged from one host larva were recorded to confirm its gregarious parasitism.Galls of For morphological observation, adult parasitoids were preserved in 70–75% ethanol and subsequently dried from ethanol using the method described in Platygastergifuensis (Ashmead) that has been kept in the Smithsonian National Museum of Natural History, Washington, DC, USA. Adult morphological terminology follows To compare morphological characters between known and the two Japanese species, we referred to original descriptions, redescriptions or keys for 512 (85.2%) out of 601 known species ; posterior margin of median lobe of mesoscutum not reaching base of mesoscutellum (reaching base of mesoscutellum in P.urnicola). Platygastergifuensis was described based on a single male from Japan, from which P.urniphila can be distinguished by having A5–A9 quadrate (approximately 1.5 times as long as wide in P.gifuensis). Platygasterurniphila is quite similar to P.sublongicornis Buhl because they share the following morphological characteristics: vertex between ocelli smooth; frons smooth medially; mesopleuron with a few setae anteriorly, with a coriaceous area below tegula; mesoscutellum distinctly convex; T2 weakly striated in basal half, with shorter striae medially. However, Platygasterurniphila can be distinguished from P.sublongicornis based on the following characters: A4–A5 subquadrate (distinctly elongate in P.sublongicornis); OOL as long as LOL (1.6 times as PageBreaklong as LOL in P.sublongicornis); sides of pronotum smooth along posterior margin ; hind wing approximately 5.3 times as long as wide (4.5 times in P.sublongicornis).Platygasterurniphila is an egg-larval gregarious parasitoid of Rhopalomyialongitubifex that induces axillary bud galls on Artemisiaindicavar.maximowiczii in Japan (R.longitubifex: long jar-shaped, jar-shaped, and globular jar-shaped (see figures 1–5 of P.urniphila has been reared only from globular jar-shaped galls. Various sorts of galls induced by Rhopalomyia spp. have been found on Artemisia spp. (e.g. P.urniphila has been reared only from galls of Rhopalomyialongitubifex on A.indicavar.maximowiczii in Japan. Future intensive studies are needed to confirm the host range of P.urniphila.in Japan . Gall popp. e.g. , but P.Platygastervernalis, a polyembryonic species, occasionally laid male and female eggs into a single host egg. Thus, P.vernalis has both polyembryonic and gregarious reproductive strategies. Our rearing experiments indicated that P.urniphila is a gregarious parasitoid because males and females were reared from a single host larva (Table P.urniphila, we need histological survey or MIG-seq analysis (va Table . To confanalysis  that dis


Adenosine deaminase-deficient severe combined immunodeficiency disease (ADA-SCID) is a primary immune deficiency characterized by mutations in the ADA gene resulting in accumulation of toxic compounds affecting multiple districts. Hematopoietic stem cell transplantation (HSCT) from a matched donor and hematopoietic stem cell gene therapy are the preferred options for definitive treatment. Enzyme replacement therapy (ERT) is used to manage the disease in the short term, while a decreased efficacy is reported in the medium-long term. To date, eight cases of lymphomas have been described in ADA-SCID patients. Here we report the first case of plasmablastic lymphoma occurring in a young adult with ADA-SCID on long-term ERT, which turned out to be Epstein–Barr virus associated. The patient previously received infusions of genetically modified T cells. A cumulative analysis of the eight published cases of lymphoma from 1992 to date, and the case here described, reveals a high mortality (89%). The most common form is diffuse large B-cell lymphoma, which predominantly occurs in extra nodal sites. Seven cases occurred in patients on ERT and two after haploidentical HSCT. The significant incidence of immunodeficiency-associated lymphoproliferative disorders and poor survival of patients developing this complication highlight the priority in finding a prompt curative treatment for ADA-SCID. The development of immunodeficiency-associated lymphoproliferative disorders (IALDs), especially related to Epstein–Barr virus (EBV), is a life-threatening phenomenon. The incidence of IALD in primary immune deficiencies due to various gene defects ranges from 0.7 to 18% of patients . Since AEight cases of lymphoma have been reported to date in ADA-SCID patients, and half of them are EBV associated –13.ex vivo with a gammaretroviral vector encoding ADA  and multiple infusions of genetically modified T cells. The patient was diagnosed with ADA deficiency at 3 months of age due to pneumonia and failure to thrive . PEG-ADAding ADA  (NCT0059ding ADA . Despiteding ADA  EBV sero2 in two doses) were started before pathological diagnosis was confirmed. Histopathological examination showed an EBV-related plasmablastic B-cell lymphoma  and splenomegaly Figure . Intersta Figure . BM evals. Therapy consisted of continued rituximab weekly infusions and a cycle of APO  with no response. Accordingly, cyclophosphamide (300 mg/m2 BID for 3 days) and dexamethasone were started. Twelve days after the initiation of chemotherapy, the patient died for a hemorrhagic alveolitis eventually causing worsening respiratory failure, refractory to continuous positive airway pressure and biphasic positive airway pressure mechanical ventilation.The disease was classified as stage IV B ETo investigate the potential relationship with PB gene therapy, T cells and B cells were purified from BM, PB, and lymph node, and vector transduction measured by quantitative PCR . T cellsn = 2) and PB T-cell gene therapy. Unfortunately, all patients died despite rescue attempts with several lines of treatments, with the exception of a Burkitt’s hip lymphoma that persisted in complete remission after 20 months from the onset of the malignant disease  were not encouraging at that time  . ERT oft T cells –29.ADA has emerged as a safe and efficacious therapeutic strategy for patients lacking HLA-identical sibling donor . Written informed consent was obtained from parents for treatment, biological sample collection, and data publication.M.M., AndreaA., M.P., R.P, F.B., F.G., F.F., M.F., I.B., F.D., R.N., M.C., M.G.R., C.D., J.P., F.C., M.P.C, AlessandroA. participated to clinical care of the patient and related clinical data. M.M. and AlessandroA. designed the study, analyzed results and wrote the paper.AlessandroA is the Principal investigator of gene therapy studies sponsored by GlaxoSmithKline (GSK), which holds the licence for the medicinal product based on autologous CD34+ cell gene therapy for ADA-SCID. All other 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.


Oncomelania hupensis.The South-to-North Water Diversion (SNWD) project is designed to channel fresh water from the Yangtze River north to more industrialized parts of China. An important question is whether future climate change and dispersal via the SNWD may synergistically favor a northward expansion of species involved in hosting and transmitting schistosomiasis in China, specifically the intermediate host, O. hupensis  were estimated, and niche conservatism tested among each pair of subspecies. Fine-tuned Maxent (fMaxent) and ensemble models were used to anticipate potential distributions of O. hupensis under future climate change scenarios. We were largely unable to reject the null hypothesis that climatic niches are conserved among the four subspecies, so factors other than climate appear to account for the divergence of O. hupensis populations across mainland China. Both model approaches indicated increased suitability and range expansion in O. h. hupensis in the future; an eastward and northward shift in O. h. robertsioni and O. h. guangxiensis, respectively; and relative distributional stability in O. h. gangi.In this study, climate spaces occupied by the four subspecies of O. h. hupensis in 2050–2060; its suitable areas will also expand northward along the southern parts of the Eastern Route by 2080–2090. Our results call for rigorous monitoring and surveillance of schistosomiasis along the southern Central Route and Eastern Route of the SNWD in a future, warmer China.The southern parts of the Central Route of SNWD will coincide with suitable areas for  Oncomelania h. hupensis in the future; an eastward and northward shift in O. h. robertsioni and O. h. guangxiensis, respectively; and relative stability in O. h. gangi. The southern Central Route of SNWD will coincide with suitable areas for O. h. hupensis in 2050–2060; its suitable areas will also expand northward along the southern Eastern Route in 2080–2090. Our results call for rigorous monitoring and surveillance of schistosomiasis along the southern Central Route and Eastern Route of the SNWD in a future, warmer China.The South-to-North Water Diversion (SNWD) project is designed to channel fresh water from the Yangtze River north to more industrialized parts of China. An important question is whether future climate change and dispersal via the SNWD may synergistically favor northward expansion of schistosomiasis in China. Our models indicated increased suitability and range expansion in  Schistosoma japonicum, a parasitic flatworm that completes its life cycle through one intermediate (i.e. the snail Oncomelania hupensis) and diverse definitive  hosts. Over the past five decades, China has made remarkable progress in reducing S. japonicum infections in humans through a combination of chemotherapy and snail control, but schistosomiasis has re-emerged in recent years owing to changes in ecological and socio-economic factors, together with termination of the World Bank Loan Project on schistosomiasis control in 2001 [Schistosomiasis is a neglected tropical disease that is known to have affected people in China for more than 2100 years, with presently ~800,000 infected and ~65 million people at risk of infection [ in 2001 . Given tBased on the environmental variables that associated with species’ occurrence records, ecological niche modeling (ENM) seeks to characterize environmental conditions suitable  for a particular species and then identify where suitable environmental habitats are distributed in the space , it is aENM tools, however, are also subject to issues including the need to balance goodness-of-fit against model complexity , and theOncomelania hupensis is the sole intermediate snail host of S. japonicum in China, which thus depends entirely on this snail species for transmission [O. hupensis in mainland China has been debated in view of marked morphological variation. Liu et al. recognized 5 subspecies [O. h. guangxiensis from O. h. hupensis based on molecular characters, and recognized 4 subspecies in mainland China [O. h. hupensis and O. h. robertsoni dominate transmission of S. japonicum, as control measures have reduced O. h. guangxiensis and O. h. tangi considerably [S. japonicum [smission . Howeverbspecies , whereasbspecies . Howevernd China , which wnd China ,18. Herend China ,16,18,19iderably . These faponicum .S. japonicum [O. hupensi [S. japonicum transmission have been identified, including distance to snail habitat and wetlands, seasonal land surface temperature, and seasonal variation of vegetation indices [hupensis is thus crucial [Previous attempts to predict spatial dimensions of transmission risk of schistosomiasis have characterized transmission environments of aponicum –22 or ec hupensi ,24; thes indices ,22. Clim indices ,26. Unde crucial , and yet9 m3 fresh water annually from the Yangtze River in southern China to the more arid and industrialized north via two routes  project is a multi-decade mega-project in China. It is the biggest inter-basin transfer scheme in the world, aiming to channel 25 × 10nd China ; howeverO. hupensis presences from more than 5 thousand villages to explore ecological dimensions and potential distributions of O. hupensis in mainland China. The aims of this study were to (1) compare climate spaces occupied by the four subspecies of O. hupensis, to (2) test whether climate niches were conserved during the four subspecies’ divergence , to (3) predict their potential distributions using state-of-the-art modelling techniques, to (4) investigate the potential impacts of future climate change and the SNWD project on O. hupensis. The overall purpose was to predict the S. japonicum transmission risk at present and under climate change in mainland China.In this study, we used a unique dataset of O. hupensis were assembled from Qian [O. hupensis [O. h. hupensis, 470 O. h. robertsoni, 64 O. h. guangxiensis, and 60 O. h. tangi  and ensemble approaches [O. hupensis as a whole.To forecast climate change effects, we used proaches ,12 to caproaches ). Howeveproaches . We usedproaches  to fine-proaches . In all,fMaxent model described above were included in ensembles. Individual GAM, GBM, GLM, and RF models were developed using BIOMOD2 [Ensemble models are used commonly in forecasting climate change effects, seeking to generate a consensus estimate that reduces individual model uncertainty by reflecting the central tendency of multiple models ,12,41. H BIOMOD2 , as impl BIOMOD2 ; GARP mo BIOMOD2 . Details BIOMOD2 ). Howeve BIOMOD2 ,45. Ther BIOMOD2 ,11. The  BIOMOD2 ,11,12.E = 5%; [The occurrence data used to fit the niche models were split randomly into two datasets, for calibration (70% of points) and interpolation evaluation (30% of points). Performance of individual and consensus models was evaluated via a partial ROC (receiver operating characteristic) approach . CompariE = 5%; ).E = 5% was applied to fMaxent model predictions for O. hupensis and O. h. hupensis to generate binary predictions. We did not generate threshold predictions in ensemble future projection because such predictions are not applicable and hard to interpret .Final model runs incorporating all point data were used for visualizations and risk assessments. A modified least training presence threshold based on Future climate variables were downloaded from WorldClim , the ConfMaxent and ensemble models based on present predictions were applied to these future conditions. Future climate models applied to the intermediate scenario of representative concentration pathways of 4.5  in whic“RCP45”; . Climati“RCP45”; .Oncomelania hupensis hupensis and O. h. robertsoni occupied similar temperature and precipitation dimensions in terms of annual mean temperature (bio1), mean diurnal temperature range (bio2), and annual precipitation (bio12), but not isothermality (bio3), temperature seasonality (bio4), mean temperature of warmest quarter (bio10), or precipitation of driest month (bio14); O. h. guangxiensis and O. h. tangi occupied similar temperature and precipitation regimes in terms of annual mean temperature (bio1), temperature seasonality (bio4), mean temperature of warmest quarter (bio10), annual precipitation (bio12), and precipitation of driest month (bio14), but not mean diurnal temperature range (bio2) or isothermality (bio3) (Different degrees of overlap were observed in the eight climate dimensions among the four subspecies . Oncomely (bio3) . The fouO. h. hupensis and O. h. robertsoni occupying larger volumes than O. h. tangi and O. h. guangxiensis. Niche overlaps between pairs of subspecies also corresponded roughly to their genetic distances estimated by 16S sequence (O. h. hupensis and O. h. tangi coincided with the highest climatic niche overlap (D = 0.215) among all pairs (O. h. tangi and O. h. guangxiensis (D = 0.147), but to a lesser extent  generally showed better discriminant ability than regression models ; GARP showed unstable performance  differed from default settings , O. h. guangxiensis, O. h. hupensis, O. h. robertsoni and O. h. tangi, respectively, with fMaxent models of O. h. hupensis (AUCdiff = 0.09) and O. h. tangi (AUCdiff = 0.02) showing less overfitting than the other three  to forecast climate change effects. Our results have important implications regarding genetic divergence of O. hupensis and likely climate change effects on schistosomiasis transmission in mainland China.Limitations on materials and methodologies employed in this study need to be addressed here. While this paper focused on climate drivers, these factors occur in a complex milieu of other non-climatic drivers of snail distribution and parasite endemicity ,22, althO. hupensis were not identical, but we were unable to reject the null hypothesis that climatic niches are similar (except O. h. robertsoni versus O. h. tangi). Although failure to reject the null hypothesis does not assure that the climatic niche has been conserved, no evidence indicates that they have not been conserved, and broad climate spaces overlapped among the four subspecies ; the mountainous region of Sichuan and Yunnan Provinces (O. h. robertsoni); the hilly, littoral part of Fujian province (O. h. tangi); and the karst landscape of Guangxi Autonomous Region (O. h. guangxiensis). This landscape-level segmentation of the four subspecies is generally consistent with the foundational work of Liu et al. [O. h. robertsoni and O. h. tangi might relate to the long geographic distance separating them.The ecological niches of the four subspecies of bspecies . The relbspecies . The sig ecology , with pou et al. : indeed,u et al. ,16). CliS. japonicum in mainland China, but most such risk assessments have relied solely on mechanistic approaches . ,48. S. jes Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableThe bold values underline the six models for which PC1s were highest; these six modelling techniques were selected for the “PCA(median)” consensus method. Climate model abbreviations refer to (DOCX)Click here for additional data file.S1 FigWarm-colored areas indicate high variation among individual climate models. Bioclimatic variable abbreviations refer to (DOCX)Click here for additional data file.S2 FigBioclimatic variable abbreviations refer to (DOCX)Click here for additional data file.S3 FigRed diamonds indicate actual niche overlap distributed in the frequency of simulated overlaps (gray volume), * indicates the significance of niche identity and similarity tests.(DOCX)Click here for additional data file.S4 FigOncomelania hupensis as a whole.Maxent models were run with regularization multipliers ranging from 0.5 to 6.0 (increments of 0.5) using six different feature combinations . Black arrows denote the AICc-selected models . AICc-selected models settings are given in each box, together with performance metrics. Models were generated for the four subspecies separately, and for (DOCX)Click here for additional data file.S5 FigWarm colors indicate high variation of individual model predictions.(DOCX)Click here for additional data file.S6 FigOncomelania hupensis as a whole.Areas with different degree of similarity (grays) and strict extrapolation (black) were assessed between present and future climate datasets for the four subspecies and (DOCX)Click here for additional data file.S1 File(CSV)Click here for additional data file.


Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus helveticus, Bifidobacterium breve and B. animalis subsp. lactis (two distinct strains). Comparative genomics revealed that the draft genomes of the S. thermophilus and L. helveticus strains were predicted to encode most of the defence systems such as restriction modification and CRISPR-Cas systems. Genes associated with a variety of potential probiotic functions were also identified. Thus, in the three Bifidobacterium spp., gene clusters were predicted to encode tight adherence pili, known to promote bacteria-host interaction and intestinal barrier integrity, and to impact host cell development. Various repertoires of putative signalling proteins were predicted to be encoded by the genomes of the Lactobacillus spp., i.e. surface layer proteins, LPXTG-containing proteins, or sortase-dependent pili that may interact with the intestinal mucosa and dendritic cells. Taken altogether, the individual genomic characterization of the strains present in the VSL#3 product confirmed the product specifications, determined its coding capacity as well as identified potential probiotic functions.Several probiotic-marketed formulations available for the consumers contain live lactic acid bacteria and/or bifidobacteria. The multispecies product commercialized as VSL#3 has been used for treating various gastro-intestinal disorders. However, like many other products, the bacterial strains present in VSL#3 have only been characterized to a limited extent and their efficacy as well as their predicted mode of action remain unclear, preventing further applications or comparative studies. In this work, the genomes of all eight bacterial strains present in VSL#3 were sequenced and characterized, to advance insights into the possible mode of action of this product and also to serve as a basis for future work and trials. Phylogenetic and genomic data analysis allowed us to identify the 7 species present in the VSL#3 product as specified by the manufacturer. The 8 strains present belong to the species  There has been a steady interest in functional food products consisting of live lactic acid bacteria (LAB) and bifidobacteria that are marketed as probiotics, defined as ‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host’ . The majLactobacillus and Bifidobacterium spp. have been extensively characterized and used in numerous well-performed trials. When the number of publications is taken as an indicator, Lactobacillus rhamnosus GG is the most studied strain marketed as a probiotic. The L. rhamnosus GG genome has been characterized and used for comparative genomics studies with other isolates, resulting in the identification of mucus-binding sortase-dependent pili [L. acidophilus NCFM has been well-characterized for its genome and functional properties, including the S-layer protein and associated proteins that have been found to interact with the DC-SIGN receptor of dendritic cells [L. plantarum WCFS1 has been the first Lactobacillus strain to be genomically characterized and served as paradigm for several studies aiming to understand the interaction with the host [Bifidobacterium animalis subsp. lactis BB-12 has also been genomically characterized and compared to other Bifidobacterium species [B. breve that are involved in intestinal persistence and the host-microbe dialogue [B. breve UCC2003 were also shown to be involved in host-microbe cross-talk [The limited knowledge of many marketed probiotics contrasts with the fact that several strains of ent pili –4. Theseent pili ,6. Similic cells –9. L. plthe host –13. The  species –16. A spdialogue ,18. Surfoss-talk .L. casei or B. animalis share a recent common ancestor [Streptococcus salivarius subsp. thermophilus (now known as S. thermophilus), L. acidophilus, L. casei, L. plantarum, L. helveticus (first described as L. bulgaricus subsp. delbrueckii), and three strains of bifidobacterial species [B. breve, B. longum and B. infantis [B. longum and B. infantis have reported to be a single species and mislabelling of bifidobacteria in commercial products has been reported to occur often [Genomic characterizations have been instrumental in discovering the molecular basis of the host-interaction of industrial strains as well as providing an overview of their future capacities ,21. Moreancestor ,23. Thesancestor –26. Seve species ,28. Baseinfantis . Howeverur often . Genome-ur often .9 bacterial cells per dose, the multispecies VSL#3 product is typically used in treatments at doses of 450–900 109 cells per day. Significant improvement by VSL#3 of disease symptoms were observed in the treatment of ulcerative colitis and pouchitis, an inflammation of the ileal pouch in colectomy patients [via increasing GLP-1 production, and correcting the inflammatory status of obese adults [in vitro studies that supported the therapeutic effects and have shown the capacity of the VSL#3 product to affect the immune function associated with specific transcriptional response [In contrast to conventional marketed products that are consumed in a daily dose of approximately 10patients ,32. Moree adults –35. Thesresponse –38. HoweoC. Genomic DNA was then isolated from cell lysates with a RSC Blood DNA kit AS1400 according to the manufacturer’s protocol on a Promega Maxwell RCS instrument . DNA was quantified using Quanti-iT™ Pico Green dsDNA Assay .All eight bacterial strains that make up the VSL#3 product were individually provided by the manufacturer , coded and cultivated as listed in S. thermophilus, in LSMb  for lactobacilli, and in LSMc  for bifidobacteria. The reference strain L. paracasei LMG12586 (ATCC334) reported in the ISO10932/IDF233 2010 document [Minimum inhibitory concentration (MIC) was determined using the standard microdilution method for drug susceptibility testing ,40. Datadocument  was inclhttp://www.vicbioinformatics.com/software.barrnap.shtml), tRNA prediction by Aragorn v1.2.36 [Purified genomic DNA was sent for paired end  whole genome sequencing using the next-generation sequencing platform  at BaseClear (The Netherlands) with an expected coverage of 150x and an output of approximatively 100 contigs. The quality of the FASTQ sequences was enhanced by trimming off low-quality bases using the “Trim sequences” option of the CLC Genomics Workbench version 9.5.1. The quality filtered sequence reads were assembled to contigs using the “De novo assembly” option of the CLC Genomics Workbench version 9.5.1 (Qiagen). Mis-assemblies and nucleotide disagreement between the Illumina data and the contig sequences were corrected with Pilon version 1.20 . Subsequ v1.2.36 , and pCD v1.2.36 , and con v1.2.36 .http://greengenes.lbl.gov/) [Two different methods were used to determine the species of the sequenced strains. First, 16S rRNA gene sequences were compared using the blast option of the greengenes database (bl.gov/) . SecondlFor the phylogenetic (core genome) tree, the genomes of the strains from the product were compared to a set of relevant reference and representative genomes (NCBI genome database sept 25th 2017) using OrthoMCL . The amii.e. insertion elements, transposases and phages, were detected using keyword searches against the genome annotation. LPXTG proteins were initially identified using the method as previously described by Boekhorst and colleagues [The annotated genome sequence of all eight bacterial strains were analyzed for the presence of antibiotic resistance genes using the resfams database  and was lleagues , followelleagues , to remoThe genome sequences of all eight strains present in the multispecies product VSL#3 were deposited in NCBI's Sequence Read Archive (SRA) under the following biosample accession numbers  and under the following genome accession numbers .Lactobacillus helveticus BT08, which had 132 scaffolds possibly due to a large number of transposons in the genome , except for e genome . The str strains . In line strains , the genS. thermophilus had fewer tRNAs genes compared to other draft genomes sequences. This is, however, possibly due to the genome assembly based on gene context and comparison with the strain S. thermophilus JIM8232 [Subsequently, the predicted core proteomes of the 8 VSL#3 strains were compared to a set of relevant reference and clearly positioned in the phylogenetic tree next to the genome of the same species . This fi JIM8232 .Bifidobacterium strains and Lactobacillus strains of the VSL#3 product were predicted to have a relative low number of transposons, Streptococcus thermophilus strain BT01 and Lactobacillus helveticus strain BD08, in contrast, were estimated to harbor the most, respectively 27 and 24 IS transposons , peptidase activity and also carbohydrate, metal and amino acid transport , glycosyl-transferases and stress response (acid stress)  .Lactobacillus acidophilus BP05 and Bifidobacterium breve BB02 were devoid of CRISPR-Cas locus. Remarkably, Streptococcus thermophilus BT01 harbored three distinct CRISPR-Cas loci , which is naturally competent [S. thermophilus BT01 may be naturally competent. The spacers found in L. paracasei BP07 CRISPR-Cas locus were similar to some from several well-studied strains, including the probiotic strain L. casei BD-II [L. casei strain W56 [L. casei group [L. plantarum BP06 and L. plantarum CLP-0611 isolated from kimchi [Bifidobacterium animalis subsp. lactis BI03 or BL04 with B. animalis subsp. lactis strain BF052, a fecal isolate from healthy infants [L. helveticus BD08 gave significant hits with sequenced L. helveticus, L. amylovorus and L. gallinarum strains (10) and, some known L. helveticus plasmids and phages (3) but the majority (19) could not be matched against the tested phage or plasmid databases, suggesting that the strain derives from a poorly characterized habitat but has encountered some shared environmental DNAs with other lactobacilli.There are a variety of defense mechanisms that protect bacteria against foreign mobile DNA elements, such as plasmids or bacteriophage DNAs. plasmids . In someompetent . This suei BD-II  and L. crain W56  that belei group . Similarm kimchi  as well  infants . Blast aS. thermophilus BT01 and L. helveticus BD08 were predicted to harbor up to 11 and 8 R/M systems in their respective genome. Interestingly, these two particular strains had CRISPR-Cas loci with a relatively wide repertoire of spacers . The co-existence of R/M and CRISPR-Cas systems was shown to provide an increased resistance to phages (additive protection) [Enterococcus faecalis [While some of the strains harbor only few genes encoding the R/M system, others such as tection)  and alsofaecalis .Bifidobacterium spp. and L. acidophilus. The tetW gene identified in the Bifidobacterium genomes  and a potential aminoglycoside aminotransferase gene in strain BI04 were located in the vicinity of a putative transposon gene cassette. The presence of tetW gene, associated with a putative transposon, is generally found in other bifidobacteria, such as the widely-consumed Bifidobacterium animalis subsp. lactis BB-12 [L. acidophilus BA05 and B. animalis subsp. lactis BL04 and BI04. The AAC3 of L. acidophilus BA05 was annotated as being of prophage origin and was also present with complete identity in the genome of the widely consumed strain L. acidophilus NCFM [B. animalis subsp. lactis BL04 and BI04 could be also found in Bifidobacterium animalis subsp. lactis BB-12 [L. plantarum BP06 similar to the one in L. plantarum WCFS1 (99% homology) [L. plantarum BP06 was also found in many other L. plantarum genomes based on blast searches, but not in L. plantarum WCFS1.Using the resfams database , genome is BB-12  and otheis BB-12 . Two genlus NCFM . Similaris BB-12 . The genomology) . The claL. paracasei BP07, L. acidophilus BA05 and L. helveticus BD08 used by EFSA [To better address the safety of VSL#3 strains, the antibiotic sensitivity was performed according to the recommendations of the European Food Safety Agency (EFSA) using a micro-dilution method . The dat by EFSA . However by EFSA , as illu by EFSA .Summarizing, the present comparative analysis revealed that some potential antibiotic-associated genes found in the strains of the multispecies probiotic product VSL#3 were also present in widely-marketed probiotic strains or well-characterized LAB and bifidobacteria. However, the possible transfer of these potential antibiotic resistance genes to other species remains to be demonstrated.clpC, clpE) or cell wall/CPS biosynthesis . In addition, only single genes were found in specific strains, indicating that the complete pathway required for biosynthesis was absent or at least different from the ones found in pathogenic strains.Our initial analysis also predicted the presence of putative virulence genes in the different genomes by comparative analysis with the VFDB. However, the VFDB is rather broad database and also includes genes that may have an indirect role in virulence. Further inspection of these virulence gene candidates showed that they were related to stress response . All predicted LPXTG proteins were compared in details with those of other LAB or bifidobacteria since many have been studied in details for their host interaction capabilities  gene cluster. Recently, we have shown that homologous genes in an Asian L. plantarum strain are involved in inuline degradation, suggesting that L. paracasei BP07 may also degrade this fructose polymer [Cell-surface associated proteins harboring LPXTG motifs are sortase substrates and many, often large ones, with predicted sizes of up to 3,515 amino acid residues, were found to be encoded by all strains with bilities . A numbeg others . The LPXei group . Its gentemplate   and one sortase gene. However, in B. breve BB02 these pili genes were also detected but no sortase gene was found. A very recent study showed these sortase-dependent pili to be ubiquitous in Bifidobacterium spp. and some coded for FimB subunits that were able to bind starch, xylan and pectin, indicative of a luminal location, while others showed interaction with the host [Along the same lines, all sortase-substrates of xosamine . Its genunctions . A sortathe host ,88.L. paracasei BP07 and is highly conserved with the well-characterized spaCBA-srtC1 pilus gene cluster from L. rhamnosus GG [in vitro and signal the host [spaCBA-srtC1 PGC is not flanked with an insertion element as observed in L. rhamnosus GG. In the latter, the IS element contains a constitutive promoter. Therefore, the expression of spaCBA-srtC1 in L. paracasei BP07, if any, may be controlled and induced by some unknown signaling. A second pilus gene cluster was present in L. paracasei BP07 with an identical gene order, i.e. three pilin genes and one sortase gene. Interestingly, the second PGC was highly related to the spaFED-srtC2 PGC present in L. rhamnosus GG (One sortase-dependent pilus gene cluster (PGC) was also found in nosus GG  that hasnosus GG , form bithe host ,89. Howenosus GG .L. helveticus BD08 and L. paracasei BP07 and possibly play a similar role as in other LAB, i.e. signaling dendritic cells and T-cell functions, bacterial adherence or enzymatic functions [L. acidophilus BA05 and L. helveticus BD08 for example, S-layer associated proteins PrtX and SlpA respectively may be involved in bacteria-host interactions and stimulation of the immune response, as previously described in L. acidophilus NCFM and L. helveticus MIMLh5 [S. thermophilus BT01 genome showing high identity with ure operons identified in S. thermophilus strains and available in the GenBank database. Although urease is associated with the pathogenesis of several bacteria, the human gut microbiota urease is considered a health-related factor [Lactobacillus reuteri [S. salivarius [Genes coding for S-layer proteins were also found in the genomes of unctions . In L. as MIMLh5 ,92. A cod factor  by moduld factor . Moreove reuteri  and S. slivarius . An actiLactobacillus, Streptococcus and Bifidobacterium spp. in the VSL#3 product suggests possible complementary and synergistic effects in the gut that will need to be further investigated using both functional genomic approaches and experimental studies.In recent years, the globally-marketed multispecies product VSL#3 has been subject to numerous clinical trials and studies that demonstrated its health beneficial properties for the human host. The lack of genomic information relating to the strains composing the VSL#3 product, however, limited a further understanding of its mode of action and efficacy in the human gut. In the present work, we genomically characterized the 8 different strains present in VSL#3. Our results confirmed and extended the species designation as specified previously and provided a rational basis for future studies of probiotic functions, safety, and ecological fitness. The genome sequences will be instrumental in understanding the mechanisms by which the different strains may interact with each other, other intestinal bacteria and the human host. Several candidate genes involved in these processes have been identified and discussed here, such as Tad pili, sortase-dependent pili, mucus binding proteins, some even glycosylated, and S-layer proteins that were previously studied in related organisms. It is of interest that some of the strains analyzed in this work showed high genomic similarities with well-characterized industrial strains or model strains where particular probiotic traits or modes of action towards the host have been investigated in detail. In most cases, these studies were conducted on single species, and hence the original and unique combination of S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.


Catalase is an essential antioxidant enzyme that is well characterized from microbial and animal sources. The structure of plantcatalase is unknown. Therefore, it is of interest to understand the functional and structural characteristics of catalase from an Indiangooseberry, Phyllanthus emblica . Hence, catalase from P. emblica was cloned in pUC18 plasmid, sequenced andsubmitted to GenBank with the accession numbers "MF979112" and "ATO98311.1". InterProScan showed that the coding sequence ismonofunctional and haem-dependent catalase-like superfamily. Multiple sequence alignment (MSA) followed by phylogeneticanalysis showed that the P. emblica catalase groups with soybean catalase. We further report the characteristics of structural model ofthe enzyme for functional characterization. Free radicals including reactive oxygen species (ROS) areregularly generated as byproducts of various metabolic reactionsin a cell. Excessive release of ROS damage proteins, lipids, andDNA, which causes oxidative stress that eventually, leads tofunctional loss of a cell and apoptosis . To counCatalases have been purified and structurally characterized fromvarious microbial , 8, 9 anCloning vector (pUC18) and E.coli strain DH5α, DNA ladder,protein molecular weight marker and restriction endonucleases(EcoR1 and HindIII) were obtained from Genei laboratories Pvt.Ltd., India. RNA isolation and cDNA synthesis wereaccomplished using RNAsolTM and the first strand cDNAsynthesis kits respectively from Chromous Biotech, India. Allother chemicals of analytical reagent grade from HiMedia, Indiawere used.Polymerase chain reactions (PCR) were performed withpeqSTAR96 universal gradient thermal cycler, Avantor, U.S.A.Other instruments used were BioRad Mini-Protean Tetra System for gel electrophoresis, U.S.A and BioRad Gel Doc EZ imager,U.S.A for capturing the images of gel. Sequencing was done withABI 3500 Genetic analyzer at Chromous Biotech, India. Thecomputational work was done on Intel(R) core, 2.20 GHz, 32-bitoperating system.RNA was isolated from the freshly plucked young leaves of ahealthy P.emblica plant and used to synthesize the first strand ofcDNA. This cDNA was used to amplify the catalase codingsequence (CDS) or the catalase gene with PCR, using catalasespecific primers  at initiThe coding sequence obtained from P. emblica was confirmedusing BLAST and translated into protein sequence using theExPASy translate tool .Protein annotation was done by InterProScan protein domainidentifier  by scannThe secondary structure features of P. emblica catalase wasanalyzed by Self-Optimized Prediction method with Alignment(SOPMA)  and its The energy of 3D models was minimized using GalaxyRefineweb server , 19. TheThe agarose gel image presented in BLASTP of translated CDS revealed a pretty high 96% identitywith other homologous sequences . InterPrClustal Omega (1.2.4) was used for multiple sequence alignment(MSA) and active site identification. An alignment of thetranslated CDS of P. emblica catalase with catalases from othersources is shown in A phylogenetic tree was also constructed using MEGA 6.06 toknow the evolutionary relatedness of the P. emblica catalase CDSwith all other isozymes of plant catalases available at UniProtKBdatabase. Though, the translated catalase CDS clustered with abranch of several plants, including soybean, pea, and mung beanbut was found to be phylogenetically closest to the catalase(CATA1) from soybean .The secondary structure features as predicted by Self-OptimizedPrediction method with Alignment (SOPMA) shows that randomcoils (35.88%) dominated among secondary structure elementsfollowed by extended strands (28.82%), beta-turn (18.82%) andthe alpha helix (16.47%). The predominance of coils points to thefact that catalase from P. emblica might not be a very stableenzyme .The 3D model of the P. emblica partial catalase sequence was builtby I-tasser server is depicted in The surface analysis of the model was done using solventaccessibility score and electrostatic potential map. Solventaccessibility (SA) prediction score, ranging from 0 (buriedresidue) to 9 (highly exposed residue) was calculated using Itasserserver . The SA To visualize the charge distributions of molecules, electrostaticpotential maps are very useful. To make the electrostaticpotential energy data easy to interpret, a color spectrum, with redas the lowest electrostatic potential energy value and blue as thehighest, is employed by chimera 1.5.1 to convey the varyingintensities of the electrostatic potential energy values. Here, thered color binding cleft  shows thIt is of interest to understand the functional and structuralcharacteristics of Phyllanthus emblica. We deposited the catalasescoding sequence (CDS) at GenBank. InterProScan shows thesequence is of a mono functional haem-containing catalase.Conserved key residues involved in substrate catalysis wereshown using multiple sequence alignment grouped with thecatalase from P. emblica after phylogentic analysis. A structuralmodel of the plant catalase and its surface analysis was reportedfor further functional characterization.


Enantia jethys complex that has been studied for over a century; many debates exist regarding the species composition of this complex. Currently, three species that live sympatrically in the Gulf slope of Mexico  are recognized in this complex . Where these species live in sympatry, some cases of interspecific mating have been observed, suggesting hybridization events. Considering this, we employed a multilocus approach  to study hybridization in sympatric populations from Veracruz, Mexico. Genetic diversity parameters were determined for all molecular markers, and species identification was assessed by different methods such as analyses of molecular variance (AMOVA), clustering, principal coordinate analysis (PCoA), gene flow, and PhiPT parameters. ISSR molecular markers were used for a more profound study of hybridization process. Although species of the Enantia jethys complex have a low dispersal capacity, we observed high genetic diversity, probably reflecting a high density of individuals locally. ISSR markers provided evidence of a contemporary hybridization process, detecting a high number of hybrids (from 17% to 53%) with significant differences in genetic diversity. Furthermore, a directional pattern of hybridization was observed from E. albania to other species. Phylogenetic study through DNA sequencing confirmed the existence of three clades corresponding to the three species previously recognized by morphological and molecular studies. This study underlines the importance of assessing hybridization in evolutionary studies, by tracing the lineage separation process that leads to the origin of new species. Our research demonstrates that hybridization processes have a high occurrence in natural populations.Hybridization events are frequently demonstrated in natural butterfly populations. One interesting butterfly complex species is the  Pygaera pigra and P. curtula were chromosomally incompatible and short-lived. Current studies have shown that hybridization is not a rare process in nature and is evidence of the evolutionary speciation process, wherein population splitting lead to the subsequent divergence of entire lineages . C. CEnanties (COI)  and studes (COI) –74, likeousquets  suggesteE. albania to E. jethys and E. mazai. Some field observations  combined with molecular information support this hypothesis. First, in the field, interspecific mating events were observed between E. albania and E. jethys, and between E. mazai and E. jethys, but no genetic profiles corresponding to a combination of E. jethys and E. mazai were identified among the 117 hybrid individuals detected in this study. Second, very few hybrids were observed in the E. albania group; and third, all hybrid individuals from the two other morphospecies contained a genetic component of E. albania. These results could suggest some reproductive barriers among species of the complex and differences in the viability of hybrids, with the existence of a prezygotic barrier  that present premating reproductive isolation [Our molecular results suggest a directional pattern for hybridization from lization ) betweenterflies . Many stterflies  for revipulatory , or betwsolation .Enantia jethys complex, considering that closely related species are more likely to hybridize [Enantia jethys complex are subject to the same environmental pressures as they share the same oviposition and larval host plants [Many cases of interspecific hybridization have been reported for Lepidoptera .ybridize , and thaybridize . Furthert plants , which mEnantia jethys complex did not show intermediate morphology, however E. mazai males have a wide variation of morphotypes  than non-hybrids, probably a reflection of sample size. Second, hybrid individuals of E. jethys presented a low but significant decrease in genetic diversity value (h), which could reflect a reduction in hybrid fitness. Finally, hybrid individuals of E. mazai showed an important and significant increase in genetic diversity value (h). This is not surprising because non-hybrid E. mazai individuals presented the lowest value of genetic diversity. Therefore, when they cross with individuals with higher genetic diversity, hybrids presented higher genetic diversity values (h), which could result in novel adaptations. Nevertheless, additional ecological, behavioral, chromosomal, and genomic studies will be necessary to test our hypothesis.A study on Lepidoptera showed that hybrid individuals may present intermediate morphology . Hybrid photypes . The hybEnantia jethys complex using morphological data supported the existence of three distinct species: E. albania, E. jethys, and E. mazai, subsequently subdivided in subspecies E. mazai mazai and E. m. diazi [COI barcode) [E. albania, E. jethys, and E. mazai, without any evidence for subspecies or deep intraspecific differentiation. However, these authors discovered a sister clade of E. mazai composed of specimens that were morphologically indistinguishable from E. albania, suggesting the existence of a potential cryptic species originating in a specific geographic area . Using a multilocus approach , we confirmed the existence of three clades in the Enantia jethys complex in agreement with the results reported by Jasso-Matínez and colleagues [Wg marker   validatelleagues . The difS1 FigWg, B) RpS5, and C) COI. Black circles are the mutational steps.A) (TIF)Click here for additional data file.S2 FigCOI, RpS5, and Wg for the Enantia jethys complex. E. albania (red), E. jethys (green), and E. mazai (blue).(TIF)Click here for additional data file.S3 FigE. albania (red), E. jethys (green), and E. mazai (blue).(TIF)Click here for additional data file.S4 FigE. albania (red), E. jethys (green), and E. mazai (blue).Obtained after removing hybrid individuals from the dataset. (TIF)Click here for additional data file.S5 FigE. albania; B, C, and D) Common phenotypes; E) Phenotype very similar to E. jethys males.Pictures are organized according to a gradient of brown wing pigmentation, from lower to higher extent of brown spots. A) Phenotype very similar to a male of (TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 TableCOI) and nuclear (RpS5 and Wg) DNA sequences.Evaluated by analysis of molecular variance using mitochondrial ((DOC)Click here for additional data file.S3 Table(XLS)Click here for additional data file.


Lysinibacillus sphaericus, one of the most popular and effective entomopathogenic bacteria, was a highly heterogeneous group. Many authors have even proposed it comprises more than one species, but the lack of phenotypic traits that guarantee an accurate differentiation has not allowed this issue to be clarified. Now that genomic technologies are rapidly advancing, it is possible to address the problem from a whole genome perspective, getting insights into the phylogeny, evolutive history and biology itself.Early in the 1990s, it was recognized that L. sphaericus OT4b.49 was sequenced, assembled and annotated, obtaining 3 chromosomal contigs and no evidence of plasmids. Using these sequences and the 13 other L. sphaericus genomes available on the NCBI database, we carried out comparative genomic analyses that included whole genome alignments, searching for mobile elements, phylogenomic metrics  and pan-genome assessments. The results support the hypothesis about this species as a very heterogeneous group. The entomopathogenic lineage is actually a single and independent species with 3728 core genes and 2153 accessory genes, whereas each non-toxic strain seems to be a separate species, though without a clear circumscription. Toxin-encoding genes, binA, B and mtx1, 2, 3 could be acquired via horizontal gene transfer in a single evolutionary event. The non-toxic strain OT4b.31 is the most related with the type strain KCTC 3346.The genome of the Colombian strain L. sphaericus is actually a sensu lato due to a sub-estimation of diversity accrued using traditional non-genomics based classification strategies. The toxic lineage is the most studied with regards to its larvicidal activity, which is a greatly conserved trait among these strains and thus, their differentiating feature. Further studies are needed in order to establish a univocal classification of the non-toxic strains that, according to our results, seem to be a paraphyletic group.The current The online version of this article (doi:10.1186/s12864-016-3056-9) contains supplementary material, which is available to authorized users. Bacillus thuringiensis in the 1960s, many bacteria with insecticidal activity have been described. Isolates of B. thuringiensis and Lysinibacillus sphaericus are frequently reported [Culex and Anopheles spp. and more persistent in polluted aquatic environments than B. thuringiensis var. israelensis [Lysinibacillus sphaericus is a gram-positive and spore-forming bacteria isolated for the first time from fourth-instar larvae of Culiseta incidens near Fresno, California [Since the discovery of entomopathogenic activity in aelensis , 3. Lysilifornia . Howeverlifornia  and it wL. sphaericus are toxic against mosquitoes. Nowadays, it is well known that a plethora of insecticidal toxins are responsible for the entomopathogenic activity of the toxic strains. Binary prototoxin (Bin) is the major insecticidal protein produced by L. sphaericus; it is contained inside the parasporal crystal and comprises two proteins: BinA (42 kDa) and BinB (51 kDa). After being ingested by larva, these proteins are solubilized in the gut and undergo proteolysis to active lower molecular weight derivatives [B. thuringiensis and Bin family toxins, respectively [L. sphaericus may also produce insecticidal toxins during vegetative stage; this is the case of Mtx proteins [In spite of being widely used in biological control programs, not all strains of s: BinA 4 kDa and ivatives , 8. Othend BinB 5 kDa. Aftproteins , 10 whosBacillus sphaericus, L. sphaericus is characterized by having a spherical terminal spore and by its inability to utilize carbohydrates, except N-acetylglucosamine [Formerly known as cosamine . Insteadcosamine . This spcosamine –14, phoscosamine , among ocosamine .meso-diaminopimelic acid, the major component of Bacillus cell wall [L. sphaericus as species and as mosquito-control agent.In 2007, this species was reclassified to a new genus according to phenotypic traits, mainly based on differences in peptidoglycan composition which includes lysine and aspartic acid instead of ell wall . No genoell wall . HoweverL. sphaericus OT4b.49, a previously isolated Colombian strain [L. sphaericus genomes previously uploaded to the NCBI database, were used in the current study  were detected for the strain OT4b.49. As reported by Hu and coworkers, the identified GIs comprise sequences of mobile genetic elements as prophages and transposons, and several recombination-involved proteins as integrase, recombinase, and transposase . InteresL. sphaericus could compromise of more than one species in a single sensu lato [L. sphaericus type strain) as the most divergent ones.Since some researchers have proposed that nsu lato –20, we cnsu lato . In the nsu lato . All the. In concordance with results mentioned above, little functional diversity was found in 4 representatives of toxic lineage since no unique genes were detected for any strain  from protein sequences comparisonspipeline . Three tL. sphaericus [L. sphaericus sensu stricto whereas toxic lineage should be considered as a new species.The phylogenetic tree constructed based on core genes supports the hypothesis about toxic strains as separate species from non-toxic strains, the latter without a clear circumscription Fig. . Intereshaericus . Thus, tL. sphaericus as it is described nowadays (comprising toxic and non-toxic strains). The pan-genome of the toxic lineage of L. sphaericus was composed of 3728 core genes and 2153 accessory genes, whereas the pan-genome of the whole L. sphaericus species was larger, with 391 core genes and 19,826 accessory genes, which provides evidence for high intra-species diversity  and mtx2 (or cry) toxin-encoding genes is a good indicator of the feasibility for using a round-spored bacilli as mosquito-control agent. This could be easily assessed, for instance, by a PCR assay. In addition, these genes also would indicate the presence of other interesting core genes from this lineage, such as those that encode for S-layer protein and metal efflux pumps [Since the toxic lineage are made up by nearly clonal strains, the sole presence of ux pumps , 31.L. sphaericus genomes with one another by using ANIb, TETRA, and digital DDH, in order to achieve a clearer species circumscription [L. sphaericus as a single and independent species  in some non-toxic strains.Herein, we compared 14 cription , 26, 32.L. sphaericus genomes indicated a pan-genome with frequent rearrangements, revealing the striking genomic heterogeneity inside this group. When performing the same comparative analysis on the 8 entomopathogenic strains, an open but smaller pan-genome as well as highly syntenic regions and less frequent genomic rearrangements were found , dispensable (genes present in two or more strains), and unique (genes present in single strains) genomes . As Tettund Fig. .Finally, it is important to take into account that frequent gaps and sequencing errors might cause underestimation in genome annotation and therefore, errors in the estimation of the core- and pan-genome . Only 4 L. sphaericus OT4b.49 and carried out an analysis of the full repertoire of L. sphaericus available genomes in order to assess intraspecific diversity.We generated a draft genome for the Colombian mosquitocidal L. sphaericus as a new species. Historically, many round-spored mesophilic bacilli have been grouped under L. sphaericus classification, leading to the formation of a heterogeneous sensu lato. We assessed taxonomic composition by means of overall genome relatedness indices and phylogenomic analysis based on core genes. We found that toxic strains form a well-defined lineage that should be considered as a novel species. The differentiating feature of this species is the presence of toxin-encoding genes such as binA, B and mtx1, 2, 3, which might be acquired by HGT.The current study provides strong evidence for considering the toxic-lineage of L. sphaericus strains did not show a clear circumscription and are, indeed, a paraphyletic group. Further studies are needed in order to establish a univocal classification, though this is still challenging in the light of the absence of an unambiguous species definition for bacteria.On the other hand, the remaining L. sphaericus OT4b.49 was carried out using Pacific Biosciences technology with 1 SMRT cell, P4-C2 chemistry, and a mixed library (CCS and subreads). This service was provided by McGill University and Génome Québec Innovation Centre. Contig assembly was done using the HGAP 2.0 workflow [The genome sequencing of workflow . Sequencworkflow  to obtaiworkflow  and thenL. sphaericus OT4b.49 was annotated using the NCBI Prokaryotic Genome Annotation Pipeline [The genome of Pipeline  and RASTPipeline . In addiPipeline , which lBacillus subtilis 168T (X60646), Bacillus licheniformis DSM 13T (X68416), Bacillus megaterium IAM 13418T (D16273), Bacillus sp. BD-87 (AF169520), Bacillus sp. BD-99 (AF169525), Bacillus sp. NRS-1691 (AF169531), Bacillus sp. NRS-1693 (AF169533), Solibacillus silvestris StLB046 (NR_074954), Bacillus sp. NRS-250 (AF169536), Bacillus sp. B-1876 (AF169494), Bacillus sp. NRS-1198 (AF169528), Bacillus sp. B-4297 (AF169507), Bacillus sp. NRS-111 (AF169526), Bacillus sp. B-183 (AF169493), Lysinibacillus sphaericus B-23268T (AF169495), Lysinibacillus sphaericus JG-A12 (AM292655), Bacillus sp. B-14905 (AF169491), Lysinibacillus sphaericus ZC1 (NZ_ADJR01000054.1:1-1487), Bacillus sp. B-14865 (AF169490), Lysinibacillus fusiformis ATCC-7055 (AJ310083), Bacillus sp. B-14957 (AF169492) and Bacillus sp. B-23269 (AF169496).The 16S rDNA sequences obtained with Prokka, together with the 16S rDNA sequences from other bacilli listed below, were aligned using MEGA 6.0  with theMAUVE software  was usedL. sphaericus strains using Island Viewer 3 [Genomic islands were predicted in the complete genomes of toxic Viewer 3 . This toL. sphaericus genomes. DDH estimates were obtained from Genome to Genome Distance Calculator 2.1, which transforms the distances from the high-scoring segment pairs to values analogous to DDH using a generalized linear model. This model is inferred from an empirical reference dataset comprising real DDH values and genome sequences [The values for ANIb, ANIm and Tetra were calculated by JSpecies  for all equences . All of equences .L. sphaericus as well as for the toxic and non-toxic strains were obtained using OrthoMCL [The pan- and core-genomes for all strains of OrthoMCL  and RoarOrthoMCL . Roary uOrthoMCL . The resOrthoMCL  as a phyOrthoMCL .


True ventricular aneurysm is a scarred wall that most commonly results after an unrevascularized ST elevation myocardial infarction. Patients usually present with heart failure, angina, ventricular arrhythmia, systemic embolization, or ventricular rupture. Diagnosis can be achieved via echocardiography, left ventricle angiogram, cardiac computed tomography, or cardiac magnetic resonance. A 63‐year‐old gentleman with diabetes presented with dyspnea. Electrocardiography showed Q waves in the inferior leads Fig. . EchocarYS: involved in management of the patient, wrote the first draft. AA: involved in management of the patient, performed major revision, and approved the final manuscript. OA: involved in management of the patient, approved the final manuscript. MA: involved in management of the patient, approved the final manuscript. BH: involved in management of the patient, approved the final manuscript.None declared.Video S1. Echocardiogram showing reduced systolic function and an inferior wall aneurysm.Click here for additional data file.Video S2. Coronary angiogram showing proximal 90% lesion in the left circumflex artery.Click here for additional data file.Video S3. Coronary angiogram showing 70% lesion in the proximal left anterior descending artery.Click here for additional data file.Video S4. Coronary angiogram showing proximal total occlusion of the right coronary artery.Click here for additional data file.Video S5. Left Ventricular Angiogram showing a large inferior wall aneurysm.Click here for additional data file.


Bats are a highly successful, globally dispersed order of mammals that occupy a wide array of ecological niches. They are also intensely parasitized and implicated in multiple viral, bacterial and parasitic zoonoses. Trypanosomes are thought to be especially abundant and diverse in bats. In this study, we used 18S ribosomal RNA metabarcoding to probe bat trypanosome diversity in unprecedented detail.Trypanosoma cruzi I, T. cruzi III/V, Trypanosoma cruzi marinkellei, Trypanosoma rangeli, and Trypanosoma dionisii, and seven were identified as novel genotypes monophyletic to basal T. cruzi clade types of the New World. Another OTU was identified as a trypanosome like those found in reptiles. Surprisingly, the remaining OTU was identified as Bodo saltans–closest non-parasitic relative of the trypanosomatid order. While three blood samples featured just one OTU (T. dionisii), all others resolved as mixed infections of up to eight OTUs.Total DNA was extracted from the blood of 90 bat individuals (17 species) captured along Atlantic Forest fragments of Espírito Santo state, southeast Brazil. 18S ribosomal RNA was amplified by standard and/or nested PCR, then deep sequenced to recover and identify Operational Taxonomic Units (OTUs) for phylogenetic analysis. Blood samples from 34 bat individuals (13 species) tested positive for infection by 18S rRNA amplification. Amplicon sequences clustered to 14 OTUs, of which five were identified as This study demonstrates the utility of next-generation barcoding methods to screen parasite diversity in mammalian reservoir hosts. We exposed high rates of local bat parasitism by multiple trypanosome species, some known to cause fatal human disease, others non-pathogenic, novel or yet little understood. Our results highlight bats as a long-standing nexus among host-parasite interactions of multiple niches, sustained in part by opportunistic and incidental infections of consequence to evolutionary theory as much as to public health. Bats make up a mega-diverse, intensely parasitized order of volant mammals whose unique behavioural and physiological adaptations promote infection by a vast array of microorganisms. Trypanosomes stand out as ancient protozoan parasites of bats. As cryptic morphology, low parasitaemia and selective growth in culture have recurrently biased survey, we used 18S ribosomal RNA metabarcoding to resolve bat trypanosomatid diversity in Atlantic Forest fragments of southeast Brazil. Next to several unknown species, our deep sequence-based detection and assignment protocol recognized multiple known human-pathogenic trypanosomes, another linked to reptile hosts as well as a non-parasitic kinetoplastid in the blood of various phyllostomid bats. The striking permissivity exposed here, in a region where bat trypanosomes recently featured in a fatal case of Chagas disease, compels further research on bats’ role in the dispersal and spill-over of various microorganisms among humans and wildlife. Trypanosoma cruzi is the etiological agent of Chagas disease, a complex zoonosis that continues to take dozens of human lives each day [Trypanosoma cruzi marinkellei in the Schizotrypanum subgenus, this important protozoan flagellate belongs to a broader, inter-continental group (the “T. cruzi clade”) of ancient endoparasites found to infect the mammalian fauna far and wide [T. cruzi sensu stricto, i.e., T. cruzi, and to T. rangeli).each day . Alongsiand wide –3. Infecand wide , marsupiand wide  and a muand wide , but mosT. cruzi clade diversity is found in bats. Chiroptera are known to carry both generalists such as T. cruzi and T. rangeli as well as multiple bat-restricted species—some abundant , others rare (e.g. T. livingstonei and T. wauwau) [The majority of  wauwau) , 7–8. Ch wauwau) . Several wauwau)  with vir wauwau) –12, bact wauwau) –14, fung wauwau) –16, prot wauwau) –18 and h wauwau) –20, seve wauwau) .T. cruzi-clade trypanosomes throughout the New and Old Worlds, many now accredit the Chiroptera with a fundamental role in the evolution of this parasite group [T. cruzi clade suggests a common ancestral lineage of bat-restricted trypanosomes that diversified into several independent lineages that on rare occasion switched into other terrestrial mammal hosts [Given the diversity of bat-infecting te group . In factal hosts . Bats’ ral hosts .T. cruzi-like” in the past [Many trypanosomes from bats are morphologically indistinguishable, often described simply as “the past . As mixethe past –25 to gethe past –27. Thesthe past –30.T. cruzi infection. A fatal case of human T. cruzi (I-IV) and T. dionisii coinfection [In this study, we applied next-generation metabarcoding methods to the most bat-diverse (per area) biome of Brazil . We focunfection  immediatThe sampling procedures reported herein were authorized by the Brazilian Institute of the Environment and Renewable Natural Resources (IBAMA) under license no. 19037–1 (23-05-2009). Euthanasia and blood collection met guidelines set by the Federal Council of Veterinary Medicine, Resolution 1000 (11-05-2012), in accordance to Federal Law 11.794/2008. All procedures followed protocols approved by the Oswaldo Cruz Foundation (Fiocruz) Ethics Committee for Animal Research (L0015-07).Bat captures were carried out in two periods of 2015: June (dry season) and November (rainy season). Mist nets were opened upon sunset for four hours on two consecutive nights at each study location. A total of 108 bats were captured using ten mist nets  placed along forest edges near banana and coffee crops at three different rural locations in Guarapari municipality, ES state, southeast Brazil: Rio da Prata (350 m a.s.l.), where a fatal case of Chagas disease occurred in 2012; Buenos Aires (250 m a.s.l.), where reports of triatomine invasion have increased in recent years; and Amarelos (at sea level), where triatomines have not been reported from the domestic zone  .Taxonomic identification by morphology followed  and a maDNA was purified from 90 guanidine-EDTA blood lysates in DNeasy mini spin columns (Qiagen), with each of nine extraction rounds including one negative control. Purified DNA samples were then PCR-amplified with primers 5’-TGGGATAACAAAGGAGCA-3’ (forward) and 5’-CTGAGACTGTAACCTCAAAGC-3’ (reverse) for 30 cycles of 94°C (30 s), 55°C (60 s) and 72°C (90 s) to target a trypanosome-specific, ~556 bp region of the 18S rRNA gene as established in . SterileTrypanosoma spp. reference collection from SILVA v119 [in silico testing on trypanosomatid 18S rRNA sequences from NCBI. Samples were clustered to OTUs de novo at 98% sequence similarity and assigned to extant species with a confidence threshold of 80%. Unassigned clusters were considered valid OTUs only if present at > 300 reads in any single sample and present at > 600 reads across all samples of the dataset.Amplicon sequences were filtered to retain only full-length reads of ≥ 99.9% base call accuracy by windowed trimming in Sickle , verifieLVA v119  using BoLVA v119 . OperatiLVA v119  and BLASLVA v119 , with ruFollowing OTU establishment, sequence read pairs from one representative per OTU were merged and aligned in Clustal W . Phylogenies were inferred in Mega 6  by maximMyotis nigricans) in the Vespertilionidae family. Species and their abundances are listed in Of the 108 bats captured at Amarelos, Buenos Aires and Rio da Prata study sites, 105 individuals represent 16 species in the Phyllostomidae family, and three individuals represent one species (T. cruzi I (OTU 3), T. cruzi III/IV (OTU 5), T. c. marinkellei (OTU 6), T. rangeli lineage D (OTU 10) and T. dionisii (OTU 2). A further seven OTUs did not assign to any known species of the T. cruzi clade. Phylogenetic analyses placed these seven OTUs  within a monophyletic group that includes trypanosome species from bats of the New World. Finally, two OTUs showed greater homology outside of the T. cruzi clade—OTU 4, similar to a trypanosomatid species found in reptiles, and OTU 9, nearly identical to the eubodonid Bodo saltans  bat blood samples. The 34 positive samples derived from 13 bat species  and comprised 14 distinct kinetoplastid OTUs. Five OTUs were assigned to isii OTU . A furthans Figs .D. rotundus, G. soricina and R. pumilio) contained a single OTU . The bat species A. lituratus, C. perspicillata, D. rotundus and P. recifinus presented greatest trypanosome diversity, with seven to eight OTUs per species  and into the (primarily insectivorous) Vespertilionidae.Unprecedented as they may be as complex co-infections, the diversity of individual kinetoplastids we report is not unexpected. Every recent trypanosome survey of bats has revealed novel parasite genotypes, host- and/or geographic range , 43–50, mid bats . This exT. cruzi reservoirs in ES state. Trypanosoma cruzi I and III/V found in bats of this study correspond to Discrete Typing Units (DTUs) associated with a recent fatal T. cruzi–T. dionisii mixed infection and occur in Triatoma vitticeps at the study site [Triatoma vitticeps is thought to have poor stercocarian vector competence [en masse from bat to human host via ingestion of the vector [T. cruzi transmission elsewhere in South America. For example, recent molecular surveys rank bats as top feeding sources of synanthropic T. cruzi-infected triatomines throughout Colombia, emphasize bats’ bridging of domestic and sylvatic transmission cycles in rural areas of Ecuador [T. cruzi genotype associated with anthropogenic bats (TcBat) is also accumulating from around the continent [Our study provides strong, if circumstantial, evidence for the role of bats as udy site . These Dudy site . Triatommpetence  and orale vector  supportse vector . Furthere vector , bats ma Ecuador  (where n Ecuador –54) and  Ecuador . Evidencontinent , 55–58. T. rangeli lineage D in bats, a strain initially isolated from Phyllomys dasythrix in southern Brazil [Rhodnius spp. complex is thought to drive lineage divergence in T. rangeli [R. domesticus [A. caudifer) for pollination [Here, we also provide first report of n Brazil . As the  rangeli –61, an emesticus  occurs tmesticus  that rellination .T. rangeli comes as little surprise, the presence of trypanosomes (OTU 4) with reptilian affinities in our study population is perhaps more intriguing. Nonetheless, bats and reptiles do commonly co-occur in an arboreal niche. Ecological host-fitting, involving opportunistic host switching mediated by vectors' feeding patterns within an ecological niche, is thought to be a prevailing mode of trypanosome evolution [Viannamyia tuberculate [Evandromyia evandroi [Whilst the expansion of the range of volution . Reptilivolution –66, witherculate ) as wellevandroi ). SharedMost trypanosomatid diversity observed in this study was associated with complex mixed infections, a likely consequence of bats’ gregarious way of life. Tolerance of intracellular pathogens in the Chiroptera  suggestsD. rotundus), was assigned to B. saltans, considered the closest free-living relative of the parasitic trypanosomatids. This observation joins others in unsettling assumptions about putatively free-living, yet seldom studied protist taxa. For example, 18S rRNA analysis  found an apparent case of babesiosis in China to involve erythrocytic colpodellids, the closest “free-living” relatives of the parasitic Apicomplexa [Although our study demonstrates the power of the metabarcoding approach, several caveats are relevant. Sensitivity to contamination and errors from amplification and sequencing are of foremost concern , 75. We complexa .Bodo saltans belongs to the most widely adapted, physiologically tolerant zooflagellates on Earth [B. saltans may withstand some iodine-based disinfection , though Giardia ). In anyT. dionisii to a human from a bat, as well as the presence of reptile-infecting and putatively non-parasitic kinetoplastids in the same bat population, highlights the role of bats as keystone species in parasite spill-over events. Many questions remain on how the role of sylvatic hosts in pathogen dispersal varies in space and time, upon change to environment and at the evolutionary scale. Research into these intricacies of complex zoonosis will require much further innovation with high-sensitivity, high-throughput tools. We point to the power of next-generation metabarcoding strategies in studies of trypanosomatid ecology and evolution and strongly commend their future complementation with non-molecular methods.In this section of Atlantic Forest, where a rural Chagas disease fatality in all likelihood involved a bat-feeding triatomine , our deeS1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.