Understanding epidemiological variables affecting gametocyte carriage and density is essential to design interventions that most effectively reduce malaria human-to-mosquito transmission.Plasmodium falciparum and P. vivax parasites and gametocytes were quantified by qPCR and RT-qPCR assays using the same methodologies in 5 cross-sectional surveys involving 16,493 individuals in Brazil, Thailand, Papua New Guinea, and Solomon Islands. The proportion of infections with detectable gametocytes per survey ranged from 44–94% for P. falciparum and from 23–72% for P. vivax. Blood-stage parasite density was the most important predictor of the probability to detect gametocytes. In moderate transmission settings , parasite density decreased with age and the majority of gametocyte carriers were children. In low transmission settings , >65% of gametocyte carriers were adults. Per survey, 37–100% of all individuals positive for gametocytes by RT-qPCR were positive by light microscopy for asexual stages or gametocytes .Interventions to reduce human-to-mosquito malaria transmission in moderate-high endemicity settings will have the greatest impact when children are targeted. In contrast, all age groups need to be included in control activities in low endemicity settings to achieve elimination. Detection of infections by light microscopy is a valuable tool to identify asymptomatic blood stage infections that likely contribute most to ongoing transmission at the time of sampling. Plasmodium vivax and Plasmodium falciparum cause the vast majority of all human malaria cases. Across all transmission settings, a large proportion of infections of the two species remain asymptomatic. These infections are not diagnosed and treated by control programs focusing on clinical cases. They can carry gametocytes, the sexual stage of the parasite that establishes infections in mosquitos, thus asymptomatic infections contribute to transmission. In order to determine who is likely to contribute to transmission, gametocyte densities were measured by sensitive molecular methods in afebrile individuals in four countries. The proportion of infections with gametocytes varied greatly among surveys, and was higher in regions that had experienced low transmission for extended periods of time. In moderate-high transmission settings, gametocyte densities were particularly high in children below six years, highlighting the importance that interventions to reduce transmission include this age group. The majority of gametocyte carriers was positive by light microscopy. The comprehensive data on gametocyte carriage presented here lays the foundation for the development of more effective screen and treat activities to reduce malaria transmission. A variety of malaria control interventions aim to reduce the transmission of parasites from the human to the mosquito host. Vector control tools such as bed nets and indoor residual spraying  lower thInterventions that reduce human-to-mosquito transmission are most effective when they target individuals within a population who contribute most to transmission. Not all individuals with blood-stage parasitemia are equally infectious to mosquitos. Only a small fraction of all parasites in the human host develop into sexual stages termed ‘gametocytes’; parasite development in the mosquito relies on uptake and subsequent mating of male and female gametocytes .P. falciparum gametocytes are sequestered in extravascular sites such as bone marrow for 1–2 weeks [P. falciparum endemic countries. As a result of the continued release of sequestered gametocytes after treatment, and their circulation for days to weeks, individuals can carry gametocytes for several weeks after treatment [P. falciparum gametocytes. Low-dose primaquine has been shown to reduce duration of gametocytemia after treatment [Developing –2 weeks , 8. Game–2 weeks . Mature –2 weeks –12. Mostreatment . Primaqureatment , 16.P. vivax gametocytes infective for mosquitoes appear within 2–3 days after blood-stage asexual parasitemia commences and might circulate for only 2–3 days [P. vivax gametocytes are sensitive to common drugs such as chloroquine or artemether-lumefantrine. In drug trials, up to 90% of patients were gametocyte-free one day after treatment, and virtually none carried gametocytes at day 7 [2–3 days , 18. Gam2–3 days –21. P. vat day 7 –22.P. vivax [In most malaria endemic regions the vast majority of infections are asymptomatic, i.e. not associated with fever. 50–80% of infections are not detectable by microscopic inspection of blood smears , 24. SinP. vivax –33. It iP. falciparum and P. vivax gametocyte carriage across a range of transmission settings, five cross-sectional surveys involving a total of 16,493 individuals were conducted in Brazil, Thailand, Papua New Guinea (PNG) and Solomon Islands [P. vivax being the predominant parasite . Blood samples were collected from the general population irrespective of symptoms of malaria illness. Total blood-stage parasites and gametocytes were quantified by LM and sensitive qPCR and RT-qPCR assays using the same methodology across all studies, allowing for the first direct comparison across transmission intensities.To advance our understanding of  Islands –31. The The study was approved by the PNG Institute of Medical Research IRB (1116/1204), the PNG Medical Research Advisory Committee , the WEHI Human Research Ethics Committee , the Case Western Reserve University Hospitals of Cleveland Medical Center , the Solomon Islands National Health Research Ethics Committee (HRC12/022), the Ethics Committee of the Faculty of Tropical Medicine, Mahidol University, Thailand , the Brazilian National Committee of Ethics (CONEP) (349.211/2013), and by the Committee of Ethics for Clinical Investigation from Barcelona Hospital Clinic (7306/2012). Prior to sample collection, the aims of the study were explained to all individuals and informed written consent was obtained from participants or, in case of minors, from their guardians.Details on study sites are given in The study sites experience little or moderate seasonality in transmission. In PNG, the rainy season is from December to April. Both surveys were conducted in May to July, i.e. after the rainy season. There is minimal seasonal variation in Solomon Islands; samples were collected in May to June. In Thailand, the peak transmission season is from April to July, and samples were collected in September and October. In Manaus, Brazil, highest incidence occurs from May to September. Half of the samples were collected in September to early January, and the other half in August to September.Form each participant, 250 μL blood was collected by finger prick into 2 mL EDTA microtainers (BD). Hemoglobin levels were determined using the HemoCue handheld meter. For RNA extraction and gametocyte detection, 50 μL blood was transferred into tubes containing 250 μL of RNAprotect (Qiagen) in the field. Samples in RNAprotect were kept on ice packs in the field and transferred to -80°C storage every evening and kept there until RNA extraction. The remaining 200 μL blood were kept in the EDTA microtainer, also kept on ice packs, and transferred to -20°C storage until DNA extraction.pfs25 and pvs25 [DNA was extracted from 200 μL whole blood kept in EDTA microtainers and eluted in 200 μL elution buffer. Parasites were quantified by qPCR using the 18S rRNA gene as target . The assnd pvs25 .pvs25 and pfs25 copy numbers, were included in the analysis. Expert microscopy was conducted in PNG, Solomon Islands, and for parts of the survey in Brazil. To determine multiplicity of infection (MOI), P. falciparum infections were genotyped by msp2 [P. vivax infections were genotyped by msp1F3 and MS2 [Procedures for sample collection, qPCR, and RT-qPCR were standardized between all sites. Results of individual cross-sectional surveys have been published previously –31, 35.  by msp2 , and P.  and MS2 .The following definitions are used: ‘proportion gametocyte-positive infections’ describes the number of gametocyte carriers divided by the number infected with asexual parasites and/or gametocytes; ‘population gametocyte prevalence’ is the prevalence of gametocytes among all individuals, infected and non-infected .10 transformed for all calculations. To correct for imperfect detection of gametocytes and include low-density infections without gametocytes detected in multivariable models, +0.1 was added to all gametocyte density values prior to log10-tranformation. For multivariable analyses, individuals were grouped into age classes ≤6, >6–12, >12–20, and >20 years. Survey and age group were included as fixed effects. The ratio of pfs25 or pvs25 transcripts per parasite genome was assessed, representing the proportion of gametocytes among all parasites. Infections  with densities at the technical limit of detection of 0.25 copies/μL blood (i.e. 1 DNA/RNA template per PCR) were excluded from correlation analysis as quantification is imprecise at very low densities, and including them at a set density of 0.25 copies/μL would artificially increase correlation. All data is available in supplementary file Multivariable regression models were used to predict factors associated with the proportion gametocyte-positive infections and gametocyte density. Parasite densities were logP. falciparum by qPCR ranged from 0.14%  to 18.5% , and P. vivax prevalence from 3.3%  to 19.7%  , with no clear trend between population parasite prevalence and mean parasite densities  were asymptomatic, i.e., not accompanied by measured or reported febrile illness in the preceding 48 hours.Prevalence of 96/2517) . Light m by qPCR . Mean paensities . In eachpfs25 RT-qPCR, the proportion of gametocyte-positive infections differed significantly between surveys .By PNG 2010 . In univpvs25 RT-qPCR, P. vivax gametocytes were detected in 22.6% and 23.5% of infected individuals in PNG 2014 and Solomon Islands, and in 48.9%, 53.7%, and 71.5% of infections in PNG 2010, Brazil, and Thailand .By P<0.001, . This repfs25 or pvs25 transcripts per P. falciparum or P. vivax genome among gametocyte-positive samples was plotted , while no significant change was observed for P. vivax . A febrile episode and/or antimalarial treatment in the preceding 2 weeks increased the proportion gametocytes significantly for P. falciparum , with 9.0-fold more gametocytes per 10-fold increase in genome density.In multivariate analysis across all surveys, parasite density was a strong predictor for the probability that a sample was gametocyte-positive . Each 10ensities . The cor density . ConcordP. falciparum positive individuals, the odds to detect gametocytes was 54% lower (P<0.0001) and gametocyte densities were 69% lower (P<0.0001) in individuals co-infected with P. vivax. Reported malaria or anti-malarial treatment in the past 2 weeks was associated with higher P. falciparum gametocyte prevalence and densities . In Thailand and Brazil, for both species, and for P. vivax in PNG 2014, no other factors than parasite density were significantly associated with gametocyte density.Factors affecting gametocyte density were assessed independently in PNG 2010 (moderate-high transmission), PNG 2014 (recently decreased transmission), and Thailand and Brazil pooled (long-time low transmission) . For PNGAs a result of the correlation of parasite and gametocyte density and the decreasing parasite densities with age in both PNG surveys, for both species the majority of individuals with detectable gametocytes were children. 48–78% of gametocyte carriers were <12 years . In contP<0.0001, Pf: n = 676, P = 0.471; Pv: n = 1501, P = 0.512). Likewise, P. falciparum and P. vivax gametocyte densities were not affected by an interaction between DNA copies and survey .When including parasite density in multivariable analysis to correct for differences in mean density among surveys, the proportion gametocyte positive infections, and gametocyte densities of both species differed significantly between surveys  of LM-positive individuals. Among LM-negative individuals, gametocytes were detected in 40.2% (170/422) of individuals. Pfs25 transcript densities were nearly 10-fold higher in LM-positive individuals . The ability of LM to detect P. falciparum gametocyte carriers differed significantly between surveys , P<0.001, , and appectively .P. vivax gametocytes were detected by RT-qPCR in 64.9% (235/362) of LM-positive individuals, but in only 16.7% (163/976) of LM-negative individuals. Mean gametocyte densities in LM-positive individuals were almost twice as high compared to LM-negative individuals . Thus, approximately 50% of P. falciparum and 59% of P. vivax gametocyte carriers were positive by light microscopy.P. falciparum and P. vivax infections carrying detectable gametocytes in 5 cross-sectional surveys representing distinct malaria-epidemiological contexts. The proportion of gametocyte-positive infections in community surveys is heavily impacted by the sensitivity of the assays used for parasite and gametocyte detection [We observed substantial differences in the proportion of etection . The usePlasmodium species (as determined by RT-qPCR) were positive by LM for asexual parasites. A lower proportion of gametocyte carriers was LM-positive in Madang 2014 (both species) and Solomon Islands (P. vivax), where transmission had declined in the years prior to the surveys [P. falciparum in Solomon Islands), expert LM or other tools such as RDT remain sufficiently sensitive to identify the majority of gametocyte carriers.Blood-stage parasite densities were a strong predictor for gametocyte positivity and could largely explain differences between surveys. In most surveys, the majority of gametocyte carriers of both  surveys , 30, andP. vivax. This contrasts findings from P. falciparum in Africa, where school-age children were proposed to contribute most to transmission densities [Pronounced age trends in gametocyte carriage were evident in PNG and Solomon Islands. Prevalence of infection peaked in children or adolescents, and parasite densities decreased rapidly with increasing age, most likely due to the acquisition of immunity. As a result, the vast majority of gametocytes were detected in children below 6 years, especially for ensities , 41. In P. vivax, a constant proportion of gametocytes among all parasites was observed irrespective of parasite density. In the case of P. falciparum, high proportions of gametocytes were observed in a subset of infections with low-to-moderate densities The 2-week sequestration of developing P. falciparum gametocytes results in a temporal lag of peak gametocytemia following peak parasitemia [P. falciparum gametocyte density reflects parasite density in the preceding 2 weeks.Apart from parasite density, limited effects of transient factors on gametocyte densities were observed. Multiple clone infection or hemoglobin levels did not affect gametocyte carriage of either species. For asitemia . Thus, iasitemia . This isasitemia . LongituP. falciparum gametocyte positive infections and lower gametocyte densities were observed in individuals co-infected with P. vivax. It is not known whether co-infection results in an adjustment of the gametocyte conversion rate, or whether multi-species infection is a surrogate marker for higher exposure, and thus, higher levels of gametocyte-specific immunity.As an exception to the limited impact of transient factors, a lower proportion P. vivax infectivity in Brazil [P. vivax, in Thailand a steep increase in infectivity was found at densities (by LM) of 10–100 parasites/uL, with little effect if densities increased further [Mosquito feeding assays have repeatedly shown a correlation between parasite density and infectivity. Few studies have included asymptomatic individuals and individuals negative by LM for gametocytes and asexual stages –50. Withn Brazil , individ further . This depfs25 and pvs25 RT-qPCR, is expected to allow for better predictions of infectivity [Studies assessing gametocyte densities and infectivity over time will be required to determine what proportion of low-density infections will rise in density and become highly infective. The importance of male gametocyte densities to predict infectivity is increasingly recognized. In low density infections, male gametocytes might be the limiting factor for onward transmission . Measuriectivity . Due to ectivity , it is uP. falciparum in Brazil and Solomon Islands compared to PNG. Recent malaria control activities have resulted in significant changes in vector composition and biting behavior in the study sites [P. falciparum populations in East Africa [gametocyte development gene 1 (gdv1), which is essential for early gametocyte development, was found to be under strong selection in P. falciparum populations in regions of different endemicity in West Africa [P. vivax [Even after correcting for different mean parasite densities between surveys, a higher proportion gametocyte positive samples were found for both species in Thailand and for dy sites , 53. A rt Africa , and thet Africa . In CambP. vivax . The preThe probability to detect gametocytes was closely correlated to blood-stage parasitemia in different transmission settings. The vast majority of all infections with high gametocyte densities (as determined by RT-qPCR) could be diagnosed by microscopy. Pronounced age trends of gametocyte carriage in areas of moderate to high transmission was observed. Even though the contribution to transmission is influenced by vector exposure and transmission blocking immunity in addition to gametocyte prevalence and density, the age trends suggest that interventions to reduce transmission will have the greatest effect when targeted towards children. In contrast, in order to achieve elimination in low transmission settings individuals of all ages need to be protected from vector contact, which is often not the case .S1 Data(TXT)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(DOCX)Click here for additional data file.


Inferelator. This approach produces genome-wide gene regulatory networks that include contributions by both transcription factors and sRNAs. We show the benefits of estimating and incorporating sRNA activities into network inference pipelines using available experimental data. We also demonstrate how these estimated sRNA regulatory activities can be mined to identify the experimental conditions where sRNAs are most active. We uncover 45 novel experimentally supported sRNA-mRNA interactions in Escherichia coli, outperforming previous network-based efforts. Additionally, our pipeline complements sequence-based sRNA-mRNA interaction prediction methods by adding a data-driven filtering step. Finally, we show the general applicability of our approach by identifying 24 novel, experimentally supported, sRNA-mRNA interactions in Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis. Overall, our strategy generates novel insights into the functional context of sRNA regulation in multiple bacterial species.Small noncoding RNAs (sRNAs) are key regulators of bacterial gene expression. Through complementary base pairing, sRNAs affect mRNA stability and translation efficiency. Here, we describe a network inference approach designed to identify sRNA-mediated regulation of transcript levels. We use existing transcriptional data sets and prior knowledge to infer sRNA regulons using our network inference tool, the IMPORTANCE Individual bacterial genomes can have dozens of small noncoding RNAs with largely unexplored regulatory functions. Although bacterial sRNAs influence a wide range of biological processes, including antibiotic resistance and pathogenicity, our current understanding of sRNA-mediated regulation is far from complete. Most of the available information is restricted to a few well-studied bacterial species; and even in those species, only partial sets of sRNA targets have been characterized in detail. To close this information gap, we developed a computational strategy that takes advantage of available transcriptional data and knowledge about validated and putative sRNA-mRNA interactions for inferring expanded sRNA regulons. Our approach facilitates the identification of experimentally supported novel interactions while filtering out false-positive results. Due to its data-driven nature, our method prioritizes biologically relevant interactions among lists of candidate sRNA-target pairs predicted in silico from sequence analysis or derived from sRNA-mRNA binding experiments. Because transcription factors (TFs) and sRNAs share targets or even regulate each other  are key regulators of global bacterial gene expression –5. Via c–es only) , 6. Herech other , a comprch other .Escherichia coli is currently the bacterial species with the highest number of well-characterized sRNA-mRNA interactions, where 22 sRNAs and their targets form an intricate network of 102 experimentally supported interactions  or exploit gene coregulation; however, the recall of known sRNA-mRNA interactions is limited, and the accuracy of novel predictions is not systematically evaluated (–Bacillus subtilis)  . Moreoveubtilis) , 19.In this work, we address the complexity of sRNA-mediated regulation by estimating sRNA regulatory activities to generate models of gene regulation for four bacterial species. We show that our pipeline outperforms previous efforts, detects novel sRNA-mRNA interactions, and complements RNA-RNA interaction prediction methods by discriminating between true and false targets. By identifying the most likely sRNA targets, our strategy can help researchers to select promising interactions for experimental validation.Inferelator , sRNA transcript levels exhibited only a weak linear relation with their mRNA targets, implying that sRNA transcript levels are less than optimal proxies for sRNA regulatory activity in network inference procedures.Transcriptional profiles of regulators  commonly serve as proxies for their regulatory activity , 15, eve10.1128/mSystems.00057-20.1FIG S1E. coli CyaR is expressed when levels of cyclic AMP (cAMP) are high (E. coli GcvB regulates genes involved in amino acid transport and amino acid biosynthesis (E. coli MicA is a stress-related sRNA (E. coli OmrA is important in the response to membrane stress (E. coli RybB is a stress-related sRNA (B. subtilis (E. coli RyhB. (G) RsaE regulates TCA cycle and arginine catabolism in S. aureus  is indicated. In all cases, a stronger relation is observed between the estimated sRNA activities and the average expression profile of their dependent genes (right panels) than between the expression profile of the sRNAs and the average expression profile of their targets (left panels). (A) are high . (B) E. ynthesis , 76. (C)ted sRNA . (D) E. e stress , 82. (E)ted sRNA . MicA ansubtilis . FsrA is. aureus . DownloaCopyright © 2020 Arrieta-Ortiz et al.2020Arrieta-Ortiz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the To estimate SRA, we used transcriptional profiles of experimentally supported sRNA targets. Thus, estimated SRAs can account for multiple mechanisms of sRNA-mediated control of mRNA stability. Conceptually, this is analogous to relying on a reporter gene, except that every presumed target of the sRNA is considered in the estimation, as previously done for TF activity (TFA) estimation . We checInferelator  and a mixed CLR (a modified version of CLR) (E. coli sRNAs) made by the Inferelator with SRA (BBSR.SRA), 28 were experimentally supported (25 for mixed CLR). In contrast, the Inferelator without SRA predicted only eight experimentally supported targets (three for mixed CLR). The BBSR.SRA performed best for Spot 42 (10 supported targets in the top 20) and GcvB (9 supported targets). In general, we observed that incorporation of SRA consistently improved the detection power of both Inferelator and CLR (We compared the performance of the  of CLR) , with an of CLR) . The ran of CLR) . Genes u of CLR)  or when  of CLR) . Among t10.1128/mSystems.00057-20.7DATA SET S1E. coli, P. aeruginosa, B. subtilis, and S. aureus. Download Data Set S1, XLSX file, 0.03 MB.List of sRNA-mRNA interactions inferred using manually curated sRNA priors for Copyright © 2020 Arrieta-Ortiz et al.2020Arrieta-Ortiz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli sRNA network inferred using BBSR.SRA is shown in marA was regulated by five TFs and one sRNA (FnrS) in the prior network. Only the interactions between marA and FnrS and one TF (AcrR) were recalled into the final model. Nevertheless, TFs were commonly the most influential regulator, and on average, the influence of sRNAs is subtler than the one exerted by TFs (Inferelator run (where sRNAs were not considered as regulators), 90% of the genes exclusively regulated by sRNAs in our combined network   . To conf priors) . Althoug priors) , the ave priors) . Thus, t priors) , 24.10.1128/mSystems.00057-20.2FIG S2Inferelator. Each dot is the mean value of ten Inferelator runs . Each colored symbol corresponds to one of seven sRNAs with recovered priors. Black lines indicate the medians of the average proportions for all seven sRNAs. The number of true targets for each sRNA is shown in parentheses. (A) Ratio between the number of recovered priors in the Inferelator runs with noisy sRNA priors and the total number of recovered priors in the Inferelator run without false-positive interactions. (B) Ratio between number of recovered priors with experimental support (true priors) in Inferelator runs with noisy sRNA priors and the total number of recovered priors in the Inferelator run without false-positive interactions. Download FIG S2, PDF file, 0.02 MB.Presence of false sRNA-mRNA interactions reduced the number of sRNA priors included in the networks inferred by the Copyright © 2020 Arrieta-Ortiz et al.2020Arrieta-Ortiz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Inferelator to noisy priors to separate true positive from false-positive interactions among computationally predicted sRNA-mRNA interactions  and likely include many false-positive interactions.We exploited the robustness of the ractions . For anyCopraRNA , a stateCopraRNA . Standar10.1128/mSystems.00057-20.4TABLE S2Inferelator filters CopraRNA-derived priors and predicts novel sRNA-mRNA interactions with experimental support. Download Table S2, DOCX file, 0.01 MB.The Copyright © 2020 Arrieta-Ortiz et al.2020Arrieta-Ortiz et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli sRNAs RyhB, GcvB, and Spot 42, since each may directly or indirectly regulate dozens of genes  targets associated with enriched functional terms, (set iv) the intersection of sets ii and iii, (set v) the union of sets ii and iii, and (set vi) the union of the top 15 targets based on P value  and setInferelator dramatically shrank the initial set of priors  are listed in cheY interaction is supported in E. coli by physical binding data and significant upregulation of cheY in an S. Typhimurium strain missing one of its two RyhB genes , the tricarboxylic acid (TCA) cycle (sucA-sucB-sucD), and nucleoside metabolism (amn) were among the novel predictions.For RyhB, best results were obtained with a set of priors containing 38 genes associated with enriched functional terms . The infP values of ≤0.01. Eleven priors (out of 46) were recalled. Ten of these 11 priors were previously validated or experimentally supported . Among these, hisJ and hisQ are in the same operon as argT, a known GcvB target. Similarly, dppB, dppD, and dppF all belong to the same operon, involved in peptide transport , and dcyD were supported by physical binding data  that have been validated and three more with experimental support  controls sugar metabolism . Since a physical interaction between Spot 42 and maeB mRNA was recently reported  is a known Spot 42 target  and was predicted as a target by CopraRNA  or because Spot 42 promotes faster degradation of the sdh polycistronic transcript in a still unidentified condition. Overall, all five of the Spot 42 novel targets were experimentally supported.For five selected sRNAs, we show how our approach identified 30 novel sRNA-mRNA interactions with experimental support in . aureus . Spot 42tabolism , and Figrogenase . It had rogenase ; however  is shown in P value of <1e−07) , although not supported by transcriptomic data, were detected as physically interacting with GcvB in vivo (thrC and cysC) were indirectly supported. The leader sequence of the thr operon, thrL, interacts with GcvB and is a confirmed GcvB target in S. Typhimurium  (in vivo UV cross-linking with RNA deep sequencing (GcvB is known to regulate amino acid biosynthesis and transport . An expa <1e−07) . Notably in vivo , 32. Twohimurium , 34, whesC, CysB . The Gcvultocida . A challRIL-Seq) , in vivoquencing , and MS2quencing , 43. OurP. aeruginosa are functional analogs of E. coli RyhB (B. subtilis FsrA (antR and sdhC) are experimentally validated targets  were significantly upregulated in P. aeruginosa cells grown under high- versus low-iron conditions, as well as in the prrF1-prrF2 deletion mutant versus wild-type (WT) cells  cells , 46. PA2T) cells . Similarn mutant  and for n mutant . Unexpecn mutant . This man mutant . Further are also among the FsrA targets predicted by CopraRNA  in the fsrA-fur deletion mutant compared to the fur deletion mutant , or ppnKB were supported by upregulation under the same conditions  (sucC was downregulated (0.5 mean fold change) in the fur single deletion mutant compared to the WT  . Furthero the WT . Interacn target , 49. In subtilis . This masubtilis . Follow-S. aureus involved in the regulation of arginine degradation (rsaE) and in vitro trapping of sRNA targets  were significantly upregulated in the rsaE deletion strain and downregulated in an overexpression strain (versus WT). One target (SAOUHSC_00405) was significantly upregulated only in the rsaE deletion strain. Six novel targets  were significantly downregulated in a rsaE overexpression strain. The set of novel targets was thus significantly enriched with genes downregulated in the rsaE overexpression strain . In summary, the inferred RsaE regulon includes new candidates and some of the RsaE targets discovered in reference RsaE is a partially characterized sRNA of radation . Rochat  targets . We infe targets  using a  targets . Seven pP. aeruginosa and RsaE in S. aureus.Estimated SRA profiles can be directly used to determine the physiological context associated with highest sRNA activity. For any sRNA of interest, conditions of highest SRA can be compared to reveal commonalities. As an example, we analyzed the activity profiles of PrrF in Pseudomonas quinolone signal (PQS) in the growth medium (orange circles). This observation is explained by the iron chelator properties of PQS . This discrepancy is epitomized by the fact that the experiment with the highest RsaE activity (linezolid-treated S. aureus in stationary phase) had the lowest RsaE RNA level in the transcriptomic compendium . We selected bacterial species for which we could comprehensively assess the quality of the inferred models; however, there is a much larger group of species, including  species , and exp species . In addiDrosophila , TF-gene interactions. The prior networks were used for estimating the regulatory activities of TFs included as potential regulators, inferring the corresponding transcriptional network, and defining the final model of the inferred networks (see below). Sources for each species are shown in X) where each row corresponded to the transcriptional profile of a gene. Then, we applied a network component analysis (NCA)   to decomInferelator Bayesian best subset regression (BBSR), as detailed in reference Inferelator with incorporation of TFAs and SRAs can be summarized as:iX,j is the mRNA level of gene i in condition j, Â is the matrix of estimated activities generated with NCA (as described above), and βi,k indicates the effect (positive or negative) and strength of regulator k’s activity on gene i. β is the main output of the Inferelator. To model the sparsity of biological networks, BBSR solves for a matrix β where most values are zero. More details about BBSR solution can be found in reference Inferelator is a confidence score matrix generated as explained in reference mi_and_clr.R script in the Inferelator release, available at https://sites.google.com/a/nyu.edu/inferelator/home.TF and sRNA networks were simultaneously inferred using We ranked the set of all potential regulator (TF/sRNA)-gene interactions based on the associated confidence scores. We used a 0.5 precision cutoff  for each species. A total of 383 candidate E. coli sRNA-mRNA interactions were retrieved from available literature (excluding binding data). This set of potential interactions was extended to 689 to include genes located in the same operons, as predicted in MicrobesOnline .To estimate the basal performance of the spf overexpression (GEO accession no. GSE24875) reported in reference E. coli transcriptomic data used as input for the Inferelator and excluded genes that were absent in any of the replicates. Genes with P values of ≤0.01 were considered differentially expressed. Adjusted P values were not used because only five and seven genes had adjusted P values of ≤0.05 or adjusted P values of ≤0.1, respectively. This would narrow down the set of potential targets , and it would leave out previously validated Spot 42 targets. We have successfully used a 0.01 raw P value threshold for analyzing B. subtilis transcriptomic data  were downloaded and used as priors. When predictions were not available for an sRNA of interest, a new run was submitted to the CopraRNA website. All CopraRNA predictions for E. coli sRNAs were downloaded between January and July 2016.For sRNAs that were conserved among multiple bacterial species, precomputed predictions from the CopraRNA website (MS2 affinity purification coupled with RNA sequencing (MAPS) technology to detect mRNAs that physically interact with RyhB and GcvB sRNAs (Lalaouna and collaborators have successfully developed and applied the vB sRNAs , 70. ForvB sRNAs .sRNA networks and regulons were visualized using Cytoscape v 3.4.0 .https://github.com/marioluisao/sRNA_networks.R scripts and necessary input files to infer the described sRNA regulons are publicly available in the following GitHub repository:


TM7 and lower abundances of Veillonella and Peptoniphilus in the HTNCI group than in the HTN without cognitive impairment (HTNnCI) group. We next clarified the link between GM and MoCA scores or HTNCI factors. KEGG analysis revealed the involvement of decreased bile secretion. An evident correlation showed up between HTNCI and Veillonella abundance (P = 0.0340). We concluded that some representative GM species, especially Veillonella, could predict cognitive impairment in hypertension patients, making them potential benchmarks of HTNCI.A connection exists between hypertension (HTN) and cognitive impairment (CI) or gut microbiota (GM) and neuropsychiatric disease. However, the link between GM and HTNCI has not been illustrated. This study endeavoured to profile the landscape of GM in HTNCI patients and evaluate the value of GM as HTNCI biomarkers. We recruited 128 patients with hypertension and assigned them to two groups of different MoCA scores. Clinical and biological data were recorded. GM composition was illustrated with 16S ribosomal RNA sequencing, and the dominant species were identified by linear discriminant analysis Effect Size (LEfSe). It showed higher abundance of  Dementia, often aging-related, features progressive and irreversible cognitive decline severe enough to impair the quality of life . About 5The human gastrointestinal tract populates a colony of symbiotic bacteria with abundant species and a dynamic balance. Gut microbiota (GM) is tightly involved in the physiology and pathology of humans . In receVerrucomicrobia and Proteobacteria increase, and Ruminococcus and Butyricicoccus decrease [GM dysbiosis has a link with neuropsychiatric disorders in human and animal studies. Studies have shown characteristics of fecal microbial diversity and composition in AD patients: more bacteria eliciting proinflammatory response and less bacteria synthesizing short-chain fatty acids (SCFAs) , 17. In decrease . Moreovedecrease –22. Howedecrease , 24 and decrease , 26. Othdecrease . Probiotdecrease . Oral SCdecrease . Fecal mdecrease . HoweverHere, we characterized the GM profile and its link with MoCA scores and HTNCI risk factors. Our findings may pluck potential diagnostic biomarkers out of GM for HTNCI.Patients, aged >60 years, confirmed with hypertension, and treated in the Xin'an community in Wuxi city from May to October 2018 were enrolled. The exclusion criteria included the following: severe vision, hearing and speaking impairments, use of antibiotics or probiotics within the previous 6 months, diet restriction, gastrointestinal surgery, infection, mental disorders (such as schizophrenia), and severe life-threatening illnesses. The study protocol was approved by the Ethics Committee of the Wuxi People's Hospital. Each patient provided written informed consent.https://www.mocatest.org/) at 3 months after hypertension onset. A score ≥ 26 was defined as normal. To control the bias in MoCA assessment due to the education background of the patient, one point was given to the patient having education of <12 years, but not if the total score of this patient exceeded 30 points.The neuropsychological function was scored by the Montreal Cognitive Assessment (MoCA) , and profiles of vitamin B12 and thyroid-stimulating hormone (TSH). We also prepared 128 fecal samples and stored them at -80°C.https://qiime.org/), followed by the generation of taxonomic profiles of each OTU at five levels . Principal component analysis was carried out on SIMCA 14.0 . Candidate biomarkers were filtered out through linear discriminant analysis Effect Size (LEfSe). With an alpha value of 0.05 for both the factorial Kruskal-Wallis test among classes and the pairwise Wilcoxon-Mann-Whitney test between subclasses, the threshold on the logarithmic LDA score for discriminative features was set at 2.0. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was employed to predict the gene function of gut microbiota between groups with and without cognitive impairment. Related pathways were determined using Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology. The processing of 16S rRNA sequencing data was implemented in LEfSe and PICRUSt online .DNA extraction from fecal samples was performed with the FastDNA Spin Kit , followed by amplification of V3–V4 16S ribosomal RNA with the primers 5′-CTCCTACGGGAGGCAGCA-3′ and 5′-GGACTACHVGGGTWTCTAAT-3′. Sequencing was conducted on an Illumina MiSeq PE300 platform . After analysis on the QIIME pipeline, only high-quality sequences were retained (score > 30 and length ≥ 200 bp). Those with >97% similarity were concentrated into operational taxonomic units (OTUs) by QIIME 1.9.1 , R software (V.3.4), and Adobe Illustrator CC 2015 . Categorical variables were subjected to a chi-squared test and continuous variables to Student's n = 60) and the HTNCI group (n = 68) , but not in age, body mass index (BMI), smoking, alcohol use, diabetes mellitus, coronary heart disease, and vitamin B12.Recruited were 234 patients with hypertension. Excluded were 36 for unwillingness to join the study, 42 for missing data, and 28 for exclusion criteria. The 128 eligible were assigned to the HTNnCI group ( 68) see . As showA total of 7825 OTUs were generated by 16S ribosomal RNA sequencing, including 14 phyla, 22 classes, 29 orders, 64 families, and 151 genera. Fecal microbiota alpha-diversity columns and the PCoA scatterplots showed no compositional difference between groups, but significant differences in the diversity of some taxa see .P = 0.014, see Synergistetes , Veillonella (P < 0.05), Firmicutes_Other (P < 0.01), and Peptoniphilus (P < 0.05) levels were positively associated with the MoCA score. Moreover, Paludibacter demonstrated a positive correlation with TSH (P < 0.05) and stroke (P < 0.05), but a negative correlation with vitamin B (P < 0.05). Prevotella was negatively correlated with TSH (P < 0.05) and hypertension (P < 0.05). In addition, Methanobrevibacter was negatively associated with diabetes (P < 0.05) and hypertension (P < 0.05). Prevotella was negatively correlated with TSH (P < 0.05), while Clostridium (P < 0.01), Paludibacter (P < 0.05), Oscillospira (P < 0.05), and Sutterella (P < 0.05) were all positively associated with TSH. Taken together, GM might harbor biomarkers sensitive to HTNCI.According to the results of Spearman's rank analysis see , LachnosVeillonella was detected in the HTNCI group, and its potential to predict cognitive impairment was verified in LEfSe analysis. Moreover, we associated GM profile with MoCA score and HTNCI risk factors, such as education, sex, stroke, hypertension, diabetes, vitamin B12, and TSH. Interestingly, Veillonella also showed an ability to discriminate HTNCI. It is suggested that GM might contain efficient biomarkers for HTNCI.We drew a profile of GM in HTNCI patients in the present study. GM exhibited no significant difference in GM diversity in HTNCI patients, but in abundances of various GM components. The low abundance of β-amyloid mediates neurotoxicity through various mechanisms, such as inhibiting acetylcholine activity in the cortex and hippocampus [In this study, sex, education, stroke, and TSH showed between-group differences and close associations with GM. Positive correlations between hypertension and cognitive impairment and GM dysbiosis have been determined , 32. Bespocampus , 40. It pocampus . Our resTM7 and TM7-3 were found in HTNCI patients than in HTNnCI patients. Another study has indicated that the enrichment of TM7 occurs with gut dysbiosis and contributes to inflammation [TM7 is associated with a compromised intestinal barrier and elevated intestinal immune infiltration [Proteobacteria participate in the pathogenesis of cognitive impairment [Paludibacter are positively associated with the course of Parkinson's disease [A previous study has hinted that GM may be associated with hypertension , 42–44. ammation . Moreoveltration . Evidencpairment , 48. Gen disease , which iBesides, we presented that HTNCI involved several KEGG pathways. Bile secretion is progressively reduced in HTNCI patients. Cholesterol metabolism in the liver is closely linked with AD . In factVeillonella abundance rose as the MoCA score dropped. Previous studies have revealed that the abundance of Veillonella decreases in the case of cognitive impairment [Veillonella species, as a harmless or even beneficial gram-negative anaerobic coccus, colonizes the mouth after one's birth [Veillonella is also the main species in the gut to produce propionic acid in human GM [β-amyloid plaques, and inactivate microglia [Bilophila belongs to Desulfovibrionaceae, a type of proinflammatory bacteria that induce LPS accumulation during inflammatory response [Desulfovibrionaceae is also implicated in psychiatric disorders [Bilophila with HTNCI. This study provided that some species in GM could predict HTNCI, with an accuracy expected higher if combined with other valuable biomarkers.A link was set up between GM and MoCA scores or HTNCI factors. Spearman's analysis showed that pairment . Veillon's birth . Veillonhuman GM . In animhuman GM , exert nicroglia . Chen eticroglia . FMT canicroglia . Howeverresponse . High-leisorders , 73, hinSeveral limitations also exist in this study. Our results might be biased by some unstandardized variables, and the significance of the MoCA score might be overemphasized. Clinical scales should be employed to testify our results. In addition, we did not evaluate the GM profiles over a long period. Besides, the sample size was still not enough. Future experiments with larger gender-matched samples are needed.The study has also its strengths. First, this is the first that reports the profile of the GM in HTNCI patients. Second, we constructed broader connections between GM and HTNCI risk factors. Third, this study laid a roadmap to explore reliable predictive biomarkers for HTNCI.Veillonella, might be adopted to predict HTNCI.In summary, the abnormal structure of GM is associated with HTNCI. Some species in GM, especially


Helicobacter pylori (H. pylori) carcinogenicity depends on three major factors: bacterial virulence constituents, environmental factors and host’s genetic susceptibility. The relationship between microenvironmental factors and H. pylori virulence factors are incontestable. H. pylori infection has a major impact on both gastric and colonic microbiota. The presence of non-H. pylori bacteria within the gastric ecosystem is particularly important since they might persistently act as an antigenic stimulus or establish a partnership with H. pylori in order to augment the subsequent inflammatory responses. The gastric ecosystem, i.e., microbiota composition in children with H. pylori infection is dominated by Streptoccocus, Neisseria, Rothia and Staphylococcus. The impairment of this ecosystem enhances growth and invasion of different pathogenic bacteria, further impairing the balance between the immune system and mucosal barrier. Moreover, altered microbiota due to H. pylori infection is involved in increasing the gastric T regulatory cells response in children. Since gastric homeostasis is defined by the partnership between commensal bacteria and host’s immune system, this review is focused on how pathogen recognition through toll-like receptors  on the surface of macrophages and dendritic cells impact the immune response in the setting of H. pylori infection. Further studies are required for delineate precise role of bacterial community features and of immune system components. Helicobacter pylori (H. pylori) is probably the most common bacterial infection worldwide. As it is well-known, this infection is particularly important in children since it is usually acquired during childhood and its seroprevalence increases with age. Moreover, if left untreated, H. pylori infection results in a chronic inflammation of the gastric mucosa and imminent long-term carcinogenesis. Thus, it was proved that 80% of all gastric cancers and 5.5% of all malignancies are related to the injuries produced by this bacterium [H. pylori as a class I carcinogen since gastric cancers associated to this infection are amongst the first three most common causes of death worldwide [H. pylori has developed a wide spectrum of mechanisms in order to survive and escape the host’s defense immune responses. Among these mechanisms, we recall the ability to regulate T-cell responses, the inhibition of cathepsin X or the altered structure of lipopolysaccharides, which are definitely involved in the mediation of resistance against drugs [H. pylori gastritis and even immune responses [H. pylori, gastric microbiota and innate immune responses shapes the pathophysiology of H. pylori-related disorders and the persistence of this infection during adulthood.acterium ,2. The Worldwide . Its carorldwide . In termst drugs . Moreovest drugs . As we ast drugs ,8,9. Gasesponses ,11. ThusH. pylori is a well-defined microaerophilic, Gram-negative bacterium that usually colonizes the human gastric mucosa during childhood and it might result in long-life inflammation and subsequent development of a wide spectrum of gastropathies like chronic gastritis, peptic ulcer disease and even certain types of malignant disorders [H. pylori infection pointing out a significant difference between 25.6% in 2005 and 12.8% in 2017 [isorders ,13. Rece in 2017 . Neverth in 2017 ,19,20.H. pylori infection remain unspecific including abdominal pain, nausea, vomiting, heartburn [H. pylori-positive gastritis [H. pylori related systemic inflammation should be a main goal in patients with this infection. The standard recommendation for diagnosing H. pylori infection implies only symptomatic children [The clinical spectrum of this infection in children varies from asymptomatic to life-threatening conditions, such as peptic ulcer disease ,22. Neveeartburn ,23, and eartburn . A wide astritis ,8. Takinchildren . Thus, nH. pylori infection, in association with amoxicillin and clarithromycin, and the eradication was successful in 85.1% of the cases [H. pylori infection [The recent guidelines no longer recommend a ‘test and treat strategy’ in pediatric patients . The stahe cases . The supnfection , but thenfection . Thus, Enfection . Similarnfection .H. pylori virulence factors and immune activation are incontestable [H. pylori-related gastropathies [H. pylori infection [H. pylori binding avidity is definitely another sine qua non condition for the severity and persistence of this infection since the strains identified in children with peptic ulcer disease were shown to express a higher binding avidity as compared to those encountered in pediatric patients with non-ulcer dyspeptic symptoms [cagA, vacA, babA2, homB and oipA genes [H. pylori infection [H. pylori identified in children with peptic ulcer disease were shown to express a higher binding avidity as compared to those encountered in pediatric patients with non-ulcer dyspeptic symptoms [H. pylori itself or host-defense mechanisms definitely require further attention in order to delineate their precise role in children.In terms of pathophysiology the reports on children are scarce. Nonetheless, the relationship between microenvironmental factors, testable . Specifiopathies ,30,31. Tnfection . Moreovesymptoms . Aside fpA genes . CagA isnfection . Moreovesymptoms . TherefoH. pylori [H. pylori infection has a major impact on both gastric and colonic microbiota and several studies have assessed the interaction between this infection and the diversity of bacteria colonizing the gastrointestinal tract.Human homeostasis is defined by the proper relationship between microbiota and host’s immune responses . The puz. pylori ,36 destr. pylori . H. pyloPrevotella and Streptococcus were the most frequently identified in the gastric samples, followed by Neisseria reported in 25 studies, as well as Veillonella, Haemophilus and Fusobacterium, identified in 24 of these studies [H. pylori related gastropathies or gastric cancer. Nevertheless, 6 bacterial genera were reported in 88 healthy subjects: Prevotella, Streptococcus, Megasphaerae, Oribacterium, Capnocutophaga and Propionibacterium [H. pylori colonization, when studies reported both a lower abundance of other bacteria and a decrease in microbial diversity [H. pylori expresses a negative impact on both abundance and diversity of gastric bacterial community.Further studies revealed that gastric microbiota is diverse and depends on both host’s underlying gastropathy and external factors, such as geographic area. A very recent meta-analysis that assessed 36 studies on this topic pointed out that  studies . Out of  studies . The infacterium ,38,39,40iversity . TherefoH. pylori infection that aimed to assessed both H. pylori and non-H. pylori strains, pointed out 114 H. pylori positive cases and identified 366 non-H. pylori genera from 189 gastric biopsy specimens, 247 Gram-positive and 119 Gram-negative [H. pylori strains: 86.4% for metronidazole, 36.4% for clarithromycin, 31.8% for levofloxacin, and 22.7% for both tetracycline and amoxicillin, as well as 10 multidrug-resistant strains [H. pylori bacteria identified in this pediatric cohort presented generally higher resistance rates to the same antibiotics: 94.8% for metronidazole, 46.7% for clarithromycin, 42.6% for amoxicillin, 26.2% in case of tetracycline and 15.3% for levofloxacin. Moreover, 63 isolates were found to be multidrug-resistant [H. pylori bacteria within the gastric ecosystem is particularly important since they might persistently act as an antigenic stimulus or establish a partnership with H. pylori in order to augment the subsequent inflammatory [H. pylori infection is dominated by Streptoccocus, Neisseria, Rothia and Staphylococcus [H. pylori bacteria were present in pediatric gastric biopsy samples even in the absence of H. pylori, such as Staphylococcus aureus and epidermidis, Pseudomonas plecoglossicida, Acinetobacter johnsonii, Neisseria mucosa, Neisseria flavescens, Neisseria meningitidis, Neisseria perflava, Rothia mucilaginosa, and Micrococcus luteus [Staphylococcus haemolyticus, Staphylococcus epidermidis, Neisseria mucosa, Micrococcus luteus, Rothia dentocariosa, and Actinomyces naeslundii are well-known for their nitrate-reducing function with further implication in carcinogenesis due to the accumulation of nitrite and N-nitroso compounds in the gastric microenvironment [In terms of pediatric patients, scarce information is available regarding the composition of gastric flora. A recent study performed on 346 children with symptoms suggesting negative . Moreove strains . Non-H. ammatory . Therefoironment . Thus, wH. pylori carriers. This situation is particularly found in pediatric subjects since as we already mentioned the infection is usually acquired during childhood although they are not deemed always to develop the disease. Thus, a study performed on Spanish children proved that 17 of the children enrolled in this study were detected with a low abundance of H. pylori, carrying an average abundance of 0.45% within the gastric microbiota [H. pylori negative were found to be healthy carriers after sequencing-based methods, with a low abundance varying between 0.04–0.67% [H. pylori as an infection and instead use colonization for a better approach of this topic since H. pylori might be considered a ‘core member’ or, according to some authors, the only ‘true resident’ of the gastric microbiota [Highly sensitive DNA-based detection methods suggest there may be many 04–0.67% . Therefocrobiota .H. pylori, increasing the risk of carcinogenesis [H. pylori infection were proven to harbor more diverse gastric bacterial communities when compared to infected adults [Firmicutes and increased abundance of Proteobacteria along with higher proportions of Gammaproteobacteria in comparison to infected adult patients [Rothia was found to be less abundant in H. pylori positive children, while they harbored increased abundance of Haemophilus, Neisseria and an unidentified genus in Neisseriaceae family [H. pylori infection in children might be a cornerstone for the development of preventive and therapeutic strategies in order to reduce the persistence of this infection during adulthood and decrease the risk for long-term complications.Studies performed on mice pointed out that gastric commensal bacteria have the ability to accelerate the inflammation caused by ogenesis . Concernd adults . The autpatients . Regardie family . Thus, tH. pylori-related gastropathy.The interrelation between gastric commensal bacteria and age is undoubtable, and gastric microbiota might play a dichotomous role in promoting or suppressing H. pylori is the most common inhabitant of the gastric environment, but another 6 genera were identified in patients with H. pylori associated disorders: Veillonella, Neisseria, Fusobacterium, Prevotella, Streptococcus and Haemophilus [H. pylori is influenced and influences the gastric microbial community, although when present it tends to predominate and the patient’s final outcome depends on the network of coexisting bacteria [H. pylori on the gastric microbiota is reversible according to Li et al., who proved that once eradicated, the gastric microbiota diversity can reach the levels of healthy uncolonized individuals [H. pylori and gastric microbiota is therefore a decisive cornerstone for gastric carcinogenesis, and especially non-cardia adenocarcinomas. Bacterial virulence represented by certain allotypes, i.e., CagA and VacA is one of the major factors involved in gastric carcinogenesis. Recent studies hypothesized that the eradication of this infection might not only prevent gastric cancer, but also minimize the risk of metachronous cancer in patients treated for early gastric cancer [H. pylori that also increase the risk for carcinogenesis due to their ability of nitrosamine forming with a well-documented oncogenic potential [H. pylori oncogenic potential.It is a well-established fact that mophilus . Most ofbacteria . This neividuals . The recc cancer . In termotential . Thus, wH. pylori, multiple hypotheses have been proposed in order to explain the co-occurrence or mutual exclusion of microorganisms, such as the secretion of 20-amino-acid-long cecropin-like antibacterial peptides leading to a pro-inflammatory response and subsequent local acidosis; gastrin release; initial reduction of gastric pH in the acute phase resulting in an abundance of H. pylori that tends to predominate the gastric environment; a further increase of gastric pH associated to H. pylori chronic infection, which has exactly the opposite effect resulting in a microbial bloom that further inhibits the growth of H. pylori [H. pylori chronic infection is a sign of atrophic gastritis that might influence itself the growth of certain microbes within the gastric environment. Thus, a study performed on patients with autoimmune chronic gastritis identified Gemella and Bosea as the main bacteria genera colonizing the stomachs of these patients [H. pylori influences and is influenced by the gastric microbial community.In terms of . pylori ,53,54,55patients , althougH. pylori eradication regimens were proved to impact the diversity and abundance of gastric microbial community. Proton pump inhibitors (PPIs), due to their ability to increase the gastric pH, definitely result in considerable changes of gastric microbiota composition. Thus, a study performed on Israeli patients before and after PPIs administration proved that major changes occurred in gastric microbiota after 8 weeks of treatment consisting of a significant decrease in Flavobacteriaceae, Moraxellaceae, Comamonadaceae and Methylobacteriaceae, along with an increase in Erysipelotrichaceae and a family from the order Clostridiales [Prevotellaceae as the most abundant bacterial family in control group, followed by Streptococcaceae, Paraprevotellaceae and Fusobacteriaceae; while in PPIs group, the most abundant was Streptococcaceae, followed by Prevotellaceae, Campylobacteraceae, and Leptotrichiaceae [Certain drugs that are also used in ridiales . Anotherchiaceae . Thus, PLactobacillus gasseri OLL2716 in patients with functional dyspepsia and healthy controls underlined that this therapy has the ability to change gastric fluid microbiota composition in these patients resulting in similar composition to that of healthy volunteers [H. pylori-positive patients undergoing standard regimens might be useful for an effective eradication.Another class of therapeutic agents that has an effect on gastric microbiota are probiotics, but this topic is far to be understood. Nevertheless, a study that assessed lunteers . Thus, pH. pylori, gastric microbiota and immune system is represented by the effects of gastric bacterial community on immune responses with further crucial impact on H. pylori persistence and its associated gastropathies. Gastric microbiome could be defined as a complex genomic content including microbial communities, host epithelium and host’s immune system elements [A less explored side of this balance between elements . Moreoveelements . Thus, delements  (Table 1H. pylori infection and the increase in their number is closely related to the disease severity and infection persistence [H. pylori owns the ability to activate macrophages and induce the synthesis of interleukins (IL) 1, 6 and 8, but also tumor necrosis factor alpha (TNF α), MIP-1 alpha and GRO-alpha, which further express a positive feedback on the recruitment and activation of additional macrophages and T cells [H. pylori infection, among which matrix metalloproteinases 9 and 2, which were proved to be elevated in patients with this infection, being involved in the destruction of gastric tissue [H. pylori activating adaptive immunity through IL 12 synthesis, which will further stimulate Th1 cytokines such as IFN γ [H. pylori plays a key role in favoring the migration of polymorphonuclear cells to the gastric mucosa via its virulence factor called H. pylori neutrophil-activating protein (HP-NAP) [H. pylori in modulating the activity of Th1 cells with a positive feedback on Th1 cytokines production. Th1 lymphocyte response is also positively influenced by gastric epithelial cells that act as antigen presenting cells activating CD4+ Th cells [H. pylori such as Th17, which mainly produce IL 17A [Macrophages are well-known to accumulate within the gastric tissue as a response to sistence ,82. Exce T cells ,84. In ac tissue . It was as IFN γ . Moreove(HP-NAP) . Another(HP-NAP) . Thus, wTh cells . Other Te IL 17A . Furthere IL 17A . Aside fe IL 17A . Thus, Ie IL 17A .H. pylori infection such as IL 6, 1β, 10 and 12 [H. pylori according to a study performed on cultured gastric epithelial cells as a result of both TLR 2 and 4 activation by H. pylori heat shock protein 60, and subsequent augmentation of nuclear factor-κB activity [H. pylori lipopolysaccharide, and that TLR2 also triggers a Th1 immune response since it binds to H. pylori neutrophil-activating protein [H. pylori-induced chronic gastritis result in an alteration of TLRs expression and subsequent downstream signaling. In normal settings, these TLRs have the ability to initiate fast and effective inflammatory responses for hindering microbial invasion [H. pylori is well-documented to impair their normal functioning and to escape their recognition for establishing its persistence within the gastric tissue [The activation of TLRs signaling pathways triggers the synthesis of inflammatory cytokines, chemokines, costimulatory molecules and antigen-presenting molecules . Two mai0 and 12 ,64,91. Iactivity . Studiesactivity ,64. It s protein ,92. It iinvasion , whereasc tissue .H. pylori due to an increase in T regulatory cells [H. pylori infection is involved in increasing the gastric T regulatory cell response in children pointing out that H. pylori infected pediatric patients with subsequent altered gastric microbiota have significantly increased levels of IL 10 as compared to healthy children and H. pylori-positive adults due to the enhanced FOXP3 response, a protein involved in immune response [H. pylori infection [H. pylori infection that commonly occurs during childhood is based on the development of T regulatory-mediated tolerance to this bacterium, and further suppression of the effector T-cell responses [H. pylori that carry the CagA pathogenicity island using coculturing of monocytic THP-1 cells [H. pylori infection during the acute phase of this infection since it is able to promote anti-inflammatory signaling [An association between TLR 4 signaling and T regulatory cells was underlined in mice, pointing out that the suppression of this signaling pathway results in an overcolonization of ry cells . Additiory cells ,95, and ry cells ,97. The response . The samnfection  since thnfection ,99. Baseesponses . TLR 5 iesponses . Moreove-1 cells . TLR 9 r-1 cells . Contrarignaling . Thus, tH. pylori induced gastritis was proved to be associated with considerable increase in β-defensin 2 as compared to non-H. pylori gastritis [H. pylori infection involving strain-dependent mechanisms and genetical factors, such as Cag pathogenicity island [H. pylori and immune system in pediatric patients is far to be fully understood, but both innate and adaptive immune response play a key role in the host’s response to this infection and might represent future targets for individualized therapies.Defensins, defined as short proteins belong to the family of mammalian antimicrobial peptides (AMPs) and they are known for their antimicrobial activity not only against bacteria, but also viruses, fungi and protozoa ,101. Theastritis . Innate astritis . AMPs haastritis . Neverthastritis . Multiply island . Gastricy island . Furthery island . The croH. pylori, the most common bacterial infection worldwide usually acquired during childhood, is influenced and influences gastric microbiota. Moreover, host’s immune system shapes the development of gastric microbiota and plays a decisive role in promoting or suppressing H. pylori infection. Nevertheless, H. pylori, gastric microbiota and host’s immune responses form a complex puzzle that is far to be properly assembled requiring further studies especially in pediatric patients taking into account the age-related differences reported so far.Gastric homeostasis depends mostly on the appropriate interplay between the local commensal bacterial community and host’s immune system.


Clostridioides difficile infection (CDI) is a large intestine disease caused by toxins produced by the spore-forming bacterium C. difficile, which belongs to Gram-positive bacillus. Using antibiotics treatment disturbances in the gut microbiota and toxins produced by C. difficile disrupt the intestinal barrier. Some evidence indicates fecal microbiota transplantation and probiotics may decrease the risk of CDI recurrence. This study aimed to evaluate the efficacy of fermented mango by using the lactic acid bacteria Lactobacillus acidophilus and develop innovative products in the form of fermented mango jam. L. acidophilus-fermented mango products inhibited the growth of C. difficile while promoting the growth of next-generation probiotic Faecalibacterium prausnitzii. Both supernatant and precipitate of mango-fermented products prevented cell death in gut enterocyte-like Caco-2 cells against C. difficile infection. Mango-fermented products also protected gut barrier function by elevating the expression of tight junction proteins. Moreover, L. acidophilus-fermented mango jam with high hydrostatic pressure treatment had favorable textural characteristics and sensory quality. Clostridioides difficile (formerly Clostridium difficile) infection (CDI) is 10-fold higher in older adults than in young adults ) and b* (yellowness/blueness [+/−]) ) .The appearance, odor, texture, flavor, and overall acceptability of each sample were evaluated by panelists. For a trained sensory panel, panelists were trained to isolate each factor and to focus on each independently of the others for evaluation of the property that is of interest to the researcher. There are 20 panelists in this study, including 9 men and 11 women. Assign a rating to each parameter on a 9-point descriptive hedonic scale, in which 9 is reserved for the highest-quality sample .p value < 0.05 were considered statistically significant.The data were expressed as means ± SD. The statistical analysis was performed by one-way analysis of variance (ANOVA) to compare among groups through Duncan’s multiple-range test . Statistic differences with a L. acidophilus, L. plantarum subsp. plantarum, and L. paracasei subsp. paracasei have been used to ferment plant products to improve their antioxidation, anti-inflammation, and lipid metabolism, as well as abilities to prevent acute gastric ulcers, antiatherosclerosis, and antiobesity [L. acidophilus BCRC14079-fermented mango pulp (30%) after 1, 3, and 5 d of fermentation. The pH value decreased significantly (p < 0.05), as fermentation progressed from days 3 to 5 (L. acidophilus BCRC14079 produced lactic acid (L. acidophilus BCRC14079 growth in mango pulp (30%). The number of L. acidophilus BCRC14079 increased after mango pulp had been fermenting for 3 days, which can exceed approximately 1010 CFU/mL. However, the number of L. acidophilus BCRC14079 was lower on day 5, suggesting that the growth of L. acidophilus BCRC14079 in mango pulp as a fermentative material is limited.iobesity ,28. Moreiobesity . Titratas 3 to 5 A. An inctic acid B. FigureC. difficile develops antibiotic resistance to a number of antibiotics. Therefore, alternative treatment or prevention strategies are needed. The fermented products were centrifuged and divided into L. acidophilus BCRC14079-fermented mango supernatant and precipitate. The inhibition of C. difficile growth by L. acidophilus BCRC14079-fermented mango extracts was evaluated.The focus of CDI treatment guidelines is to stop continuing using antibiotics and switching to metronidazole and vancomycin ,31. HoweL. acidophilus BCRC14079-fermented mango supernatant and precipitate could inhibit the growth of C. difficile dose dependently (31.25–500 μg/mL). The suppressive effect of L. acidophilus BCRC14079-fermented mango supernatant was similar to that of L. acidophilus BCRC14079-fermented mango precipitate (24 h treatment). In addition, a dose of 250 μg/mL was used to evaluate the inhibitory effect in C. difficile treated with mango extracts, L. acidophilus BCRC14079-fermented mango supernatant, and L. acidophilus BCRC14079-fermented mango precipitate for 8, 12, 18, 24, and 36 h. As shown in L. acidophilus BCRC14079-fermented mango supernatant to inhibit C. difficile growth was greater than that of the mango extract and L. acidophilus BCRC14079-fermented mango precipitate during 18–36 h.As shown in C. difficile belongs to Gram-positive bacillus that produces spores. It is an important pathogenic bacterium in adults and children. C. difficile was first isolated from the intestinal tract of infants and was clinically important when it became one of the main causes of antibiotic-associated diarrhea [C. difficile to produce resistant spores, partly due to the inability to recover the gut microbiota after antibiotic treatment; importantly, 25% of CDI patients may occur recurrence, and the rate of secondary recurrence can be as high as 40–60% [diarrhea . Recurres 40–60% ,32.C. difficile. Toxins A and B of C. difficile are secreted and bound to host receptors, subsequently, entered into the cytoplasm of enterocytes [L. casei LBC80R and L. acidophilus CL1285 against CDI has been reported [L. reteri products on inhibiting C. difficile colonization has been evaluated [L. acidophilus BCRC14079-fermented mango can inhibit the growth of C. difficile.Cytotoxins toxin A and toxin B are cytotoxic proteins, which are the principal virulence factors of erocytes ,31, whicerocytes ,34. In areported . Moreovevaluated . We founC. difficile and reduces the risk of infection. The intestinal mucosa is the first site for the contact between host and pathogen  while infection. Intestinal epithelial cells are able to produce mucin, which can form a mucosal barrier to block tissue represent sits and avoid pathogenic infection.In a cell model, toxins A and B have been shown to induce cell necrosis and apoptosis in intestinal epithelial cells ,37. TherL. acidophilus BCRC14079-fermented mango. As shown in L. acidophilus BCRC14079-fermented mango supernatant and precipitate (250 μg/mL) treatment significantly increased the expression of mucin-13, ZO-1, and OCC in Caco-2 cells when compared with mango-extract treatment.Apart from mucin, epithelial integrity is associated with intestinal tight junction proteins such as zonula occludens-1 (ZO-1) and occludin (OCC). These proteins regulate cellular permeability and maintain intestinal barrier functions . We inveC. difficile-derived toxin A causes cell apoptosis and reduction of mucosal integrity in Caco-2 cells [C. difficile infection in Caco-2 cells, and their results showed that the cell viability was decreased after 60 min infection [C. difficile for 30, 60, 90, 120, 150, and 180 min. As shown in C. difficile treatment for 120 min. In addition, both L. acidophilus BCRC14079-fermented mango supernatant and precipitate extract treatment markedly alleviated the cytotoxic effect of C. difficile on Caco-2 cells  were suppressed by Rho signaling mediation, leading to enterocytes disruption [L. acidophilus BCRC14079-fermented mango has the potential to improve the intestinal barrier and attenuate cell death induced by C. difficile in Caco-2 cells.One study has found that -2 cells . Janvilinfection . We inve-2 cells B. A resesruption . In our L. acidophilus BCRC14079-fermented mango on the growth of F. prausnitzii  significantly increased growth of F. prausnitzii . Evidence suggests that some dietary factors may influence the abundance of F. prausnitzii [F. prausnitzii [F. prausnitzii.The population of ondition . Severalusnitzii . Severalusnitzii . Mango iF. prausnitzii produces short-chain fatty acid butyrate in the colon, which inhibits the growth of C. difficile. Moreover, some anti-inflammatory bacteria such as F. prausnitzii and lactic acid bacteria support the innate immune response and minimize bacterial burden and negative effects during antibiotic and C. difficile exposure [L. rhamnosus R0011) supplements have shown intestinal protection against CDI [F. prausnitzii-cultured supernatant showed the ability for inhibiting inflammatory cytokines production in Caco-2 cells [L. acidophilus BCRC14079-fermented mango can suppress the growth of C. difficile, increase the expression of tight junction proteins, and inhibit C. difficile by promoting the growth of F. prausnitzii and increasing the level of butyrate. These results show that L. acidophilus BCRC14079-fermented mango can be developed as a health product to improve the intestinal microenvironment and gut microbiota, and has the potential to be used in the adjuvant treatment of CDI.exposure . Antioxiinst CDI . Moreove-2 cells . SCFAs p-2 cells . For exa-2 cells . Taken tJam, a kind of medium-moisture food product, is prepared by boiling fruit pulp with sugar, acid, pectin, and/or other substances to achieve a suitable and strong consistency to maintain the texture of the fruit. Jam is usually made by mixing fruit and sugar in a ratio of 45%:55% by weight. Typically, jams are widely consumed for breakfast and incorporated into bakery products and confectionery. With the increasing attention to health and wellness, and the increasing incidence of obesity, metabolic syndrome, and diabetes, the interest in low-calorie foods has also increased . For exaL. acidophilus BCRC14079-fermented mango was investigated in this study for developing the fermented jam that contained probiotics. Browning and color loss were found to be higher in the heat-treatment group than the HPP-treatment group under all conditions  among all groups (C. difficile. This jam was fermented using lactic acid bacteria L. acidophilus BCRC14079 and treated with HPP to retain its physicochemical, textural, and rheological properties.We assessed textural characteristics, including hardness, adhesiveness, and cohesiveness . We founl groups . Many foL. acidophilus BCRC14079-fermented mango products exhibited beneficial effects by promoting the growth of F. prausnitzii and protected intestinal cells against C. difficile infection via preventing the cell death of Caco-2 cells. This enteroprotective role of L. acidophilus BCRC14079-fermented mango products is related to improve the gut barrier function through upregulating tight junction proteins expression and mucin secretion. A novel type of mango jam that was fermented by L. acidophilus BCRC14079 and treated with high hydrostatic pressure processing was produced, which had favorable textural characteristics and sensory quality, and had the potential to be developed as an innovative food product for inhibiting the growth of pathogens and improving the human health.In this study,


Helicobacter pylori eradication therapy in recent years has reduced the H. pylori infection rate, indicating that gastric cancer cases diagnosed in the future may be H. pylori-naïve. The typical endoscopic presentation of signet-ring cell carcinoma, which accounts for the majority of H. pylori-naïve gastric cancer cases, is a discolored, flat, or depressed lesion; it is rarely presented as an elevated lesion. In this study, we treated a patient with elevated signet-ring cell carcinoma in an H. pylori-naïve stomach. Histopathological testing after endoscopic submucosal dissection showed proliferation of fibromuscular tissue in the tumor, which may have caused the formation of the elevated lesion.The widespread use of  Helicobacter pylori [H. pylori eradication therapy in recent years has reduced the H. pylori infection rate, indicating that higher rates of H. pylori-naïve gastric cancer cases may be diagnosed in the future. H. pylori-naïve gastric cancer is broadly divided into three types: signet-ring cell carcinoma (SRCC), fundic gland-type gastric carcinoma, and foveolar-type gastric carcinoma [In the past, most cases of gastric cancer occurred in the stomach of people infected with r pylori . Howeverarcinoma . Amongstarcinoma .The World Health Organization Classification of Tumors classifies all carcinomas consisting of discohesive cells arranged singly or in small clusters and lacking well-formed glands as poorly cohesive carcinoma. Cancer that mainly exhibits a signet-ring morphology is designated as SRCC . GastricH. pylori serology result (IgG < 3) and fecal H. pylori antigen were negative. The patient had not undergone H. pylori eradication therapy.A 63-year-old female with no relevant medical history underwent upper gastrointestinal endoscopy for screening. She neither smoked nor drank alcohol and had no family history of malignancy. The patient's H. pylori-negative mucosa  to describe the phenomenon by which fibromuscular obliteration, induced by peristalsis, elevates both tumor and nontumor tissue towards the luminal side. Similar to the present case, there is a report that the same “FE” phenomenon was observed in some differentiated adenocarcinoma . Althoug


It is one of the most isolated inhabited islands on Earth and studies on the diatoms of Rapa Nui are very few.Rapa Nui (Easter Island = Isla de Pasqua) is of volcanic origin, best known for about 900 man-made stone statues known as Light (LM) and electron microscopic (SEM) observations of a single sample collected from Rapa Nui are presented. The samples (mix of soil and organic detritus) were collected from ground of cave entrance.Humidophila, Nitzschia, Angusticopula, Orthoseira, Tryblionella and Luticola. Whereas most of taxa of the enumerated genera were identifiable, only one among four Luticola species distinguished in the samples studied was identified. This taxon was L. ectorii, a cosmopolitan species known previously from South America (Brazil) and Asia (China). The three remaining species could not be assigned to any established species. Therefore, based on external and internal morphological features from light and scanning electron microscopic analysis, we describe here three species new to science, including: L. georgzizkae, L. rapanuiensis and L. moaiorum. All three taxa new to science are compared to established Luticola species and their significance for the global distribution of the genus is discussed.The samples were characterized by low diatom diversity and strongly dominated by terrestrial (soil) forms. Among the taxa present in the material studied were cosmopolitan forms of the genera  Studies on the diatoms of Rapa Nui are very few e.g., . These rFlorella pascuensis Navarro was also described at water depths of 30–40 m and epiphytic on Halimeda sp. and Padina sp.  D.G.Mann was selected as the generitype. Luticola is one of the few diatom genera which earned an extensive treatise with LM and scanning electron microscopy (SEM)  microhabitats  . These adwelling . Indeed, quality . Howeverhabitats .Luticola is interesting. Luticola taxa have been chiefly distributed in the Holarctic plant realm, especially in Eurasia (Navicula mutica Kützing) and North America  habitats from the Rapa Nui using high resolution LM and field emission SEM. Although only one sample turned out to be rich in diatoms with moderate species diversity, and many of the taxa present were cosmopolitan forms known from terrestrial and aerophytic habitats world-wide, with a few typical of South America. Of the 2. The climate on Rapa Nui is classified as subtropical marine. Annual mean temperature is 20.8 °C with an annual average daily variation of 6.3 °C. Annual mean precipitation recorded is nearly 1,217 mm (Rapa Nui (Easter Island = Isla de Pasqua) is located in the southeastern Pacific Ocean and is a special territory of Chile located 3,580 km off the coast of this country. It is one of the most isolated inhabited islands on Earth. The island is 21 km wide, with an area of 166 km1,217 mm . Because1,217 mm . The por1,217 mm moai. The vascular plant flora of Rapa Nui is extremely poor compared to other tropical islands, reflecting its young geological age, small size, and high degree of isolation (Rapa Nui is best known for about 900 man-made stone statues known as solation . Nearly solation . 23% of solation . Zizka (solation . The flosolation . Around solation . The hissolation . A seconsolation . A thirdsolation . A fourtsolation .2O2) for a few hours at 150 °C and then rinsed and settled 5 times with deionized water. The resulting cleaned diatom material was pipetted onto coverslips and dried, and then mounted on glass slides using Naphrax® mounting medium . LM observations of the cleaned material were made with a Zeiss Axio Imager A2 and Zeiss Axio Imager M2  using a × 100 Plan Apochromatic oil immersion objective (NA = 1.46) equipped with Differential Interference Contrast (DIC). Diatom images were captured with a Zeiss AxioCam ICc5 camera . 300 diatom valves were counted for to establish diatom species composition in the samples. For SEM examination, a few drops of cleaned material were put onto Whatman Nuclepore polycarbonate membrane filters . Once dried, the membranes were mounted onto aluminum stubs and coated with 20 nm of gold. SEM observations were performed at the University of Rzeszów, using a Hitachi SEM SU8010. Diatom terminology follows The samples were collected from cave number 436, on 30 April 1988 by Professor Dr. Georg Zizka during his work on flowering plants of Rapa Nui project see  Fig. 1)Fig. 1). Luticola species represented by numerous well-established populations, we also identified 24 diatom species. The genus Luticola was represented in this small sample by four species, three of which are described herein as new to science and Luticola ectorii Levkov, Metzeltin & Pavlov  areola per stria  in the native language and to the indigenous people of the island itself.Luticola moaiorumsp. nov. Peszek, M. Rybak, Witkowski & Lange-Bertalot , Humidophila gallica (W.Smith) Lowe, Kociolek, Q.You, Q.Wang & Stepanek (∼10%), Humidophila cf. pantropica  Lowe, Kociolek, J.R.Johansen, Van de Vijver, Lange-Bertalot & Kopalová (∼16%) and Tryblionella debilis Arnott ex O’Meara (∼4%). Additionally, various terrestrial species such as Angusticopula chilensis (Grunow) Houk, Klee & H.Tanaka, Achnanthes tumescens A.R.Sherwood & R.L.Lowe, Halamphora montana (Krasske) Levkov, Humidophila contenta (Grunow) Lowe, Kociolek, J.R.Johansen, Van de Vijver, Lange-Bertalot & Kopalová, Humidophila sp., Geissleria ignota (Krasske) Lange-Bertalot & Metzeltin and two unidentified Orthoseira species were common in the studied sample. In the studied sample, other taxa identified included: Cavinula sp., Denticula subtilis Grunow, Fallacia insociabilis (Krasske) D.G.Mann, Ferocia ninae Van de Vijver & Houk, Halamphora normanii (Rabenhorst) Levkov, Mayamaea permitis (Hustedt) K.Bruder & Medlin, Navicula veneta Kützing, Nitzschia inconspicua Grunow, Nitzschia cf. microcephala Grunow, Nitzschia vitrea G.Norman, Pinnularia borealis Ehrenberg, Rhopalodia brebissoni Krammer, Sellaphora atomoides (Grunow) Wetzel & Van de Vijver, Sellaphora saugerresii (Desm.) Wetzel & D.G.Mann and Staurosirella pinnata (Ehrenberg) D.M.Williams & Round. These taxa appeared much less frequently, often in the form of single specimens and many of them had already been reported from the Rapa Nui Island by In the sample from which the newly described observed  and 7. TLuticola can be found across a diverse range of habitats worldwide. As shown in literature most Luticola taxa have a distinct preference for wet limno-terrestrial environments including: edges of streams, wet soils or living on bryophytes  L.Denys & W.H.De Smet, L. frequentissima Levkov, Metzeltin & A.Pavlov, L. puchalskiana Kochman-Kedziora, Zidarova, T.Noga, Olech & B.Van de Vijver and Luticola andina Levkov, Metzeltin & A.Pavlov  D.G.Mann, but the shapes of their valves and their dimensions . Moreover, in the case of L. spinifera and L. lagerheimii, the spines are positioned only on the edge of the valve and support formation of ribbon-like colonies. In the case of L. georgzizkae sp. nov. conjoined valves were not observed and the spines are also present directly on the surface of the valve. Luticola spinifera suggested that spines present were capable of strong adhesion to the substratum. Colony formation might offer some compensation for the potential disadvantages of having small-sized frustules, such as the possibility of movement by wind. Colony formation also significantly reduces the area from which water may be quickly lost. Unfortunately growing in colonies limits the ability of individual cells to move, reducing the chances of finding more suitable environmental conditions in a constantly-changing terrestrial environment  Lowe, Kociolek, Q.You, Q.Wang & Stepanek . Mature valves of this species presented two morphotypes: one with a raphe system and no marginal spines, the other with marginal spines and without a raphe system   . Additioin 10 µm  composedntissima .Luticola moaiorum sp. nov. belongs to a group of small species that have elliptic-lanceolate valve outlines. From this group the most similar taxa are L. ipevii Van de Vijver & Levkov, L. puchalskiana Kochman-Kedziora, Zidarova, Noga, Olech & Van de Vijver and L. andina Levkov, Metzeltin & Pavlov. The newly described species can be easily distinguished from L. ipevi based on denser striae  and mainly shorter and narrower valves. Another important characteristic is that L. ipevii has distal raphe fissures that continue onto the valve mantle  in the crater lakes and this observation is documented with a line drawing. Subsequently, Luticola identified (as Navicula s.l.). These species included Navicula goeppertiana (Bleisch) H. L. Smith (=Luticola goeppertiana (Bleisch) D.G.Mann), Navicula goeppertiana var. monita (Hustedt) Lange-Bertalot  D.G.Mann) and Navicula mutica (=Luticola mutica). With the above taxa the number of Luticola taxa in Rapa Nui terrestrial and limnic waters increases to seven, three of which are new to science. It is worth comparing these figures with the closest continental mass which is the coast of South America and the Andes Mountains. Luticola species in samples representing one of the Earth’s longest mountain ranges taken from the Feuer Land up to Panama. Among the reported Luticola species only two had been described as new to science and an additional seven were identified to the genus level only implying, they may potentially also be species new to science. Furthermore, of numerous cosmopolitan Luticola species, only L. goeppertiana was observed in the Andes. Also, when we analyze the Subantarctic Islands the figures in terms of Luticola species diversity are similar even though, as a rule, more extensive sampling has been done. This is fairly different from the case of Rapa Nui, where in one sample we have been able (thus far) to identify and describe three species new to science.Diatom studies from Rapa Nui, though very few, do present some observations on Luticola (Navicula) mutica from limnic waters of Rapa Nui may be perceived as problematic, since this taxon is considered a mud-dwelling, brackish water species (L. mutica occurring in freshwaters of Rapa Nui comes from species composition of the surface waters and of the core from the crater lake. In the species lists from both sites diatom taxa which are considered brackish water forms are included. Examples in this group are: Cyclotella meneghiniana, Navicula cf. phyllepta, Nitzschia cf. vidovichii or N. vitrea. We interpret this as an indication of strong winds which transfer an aerosol and sea salts from the sea coastal zone to the freshwater habitats.The identification of  species . HoweverLuticola species, it seems that three new species described in this paper have a unique set of characters allowing their correct identification. So far, Luticola georgzizkae sp. nov., Luticola rapanuiensis sp. nov., and Luticola moaiorum sp. nov. are known only from Rapa Nui Island. Taking into account the fact that there is a high degree of endemism within Luticola (Taking into consideration all described Luticola , as well10.7717/peerj.11142/supp-1Data S1Click here for additional data file.