Toll-like receptors (TLRs) are powerful molecular regulators by which the immune system may “sense” the environment and protect the host from pathogens or endogenous threats. In mammalian cells, several TLRs were identified with a tissue and cell type-specific distribution. Understanding the functions of specific TLRs is crucial for the development and discovery of compounds useful to maintaining or re-establishing homeostasis in the gastrointestinal tract (GIT). Due to their relevance in regulating the inflammatory response in the GIT, we will focus here on TLR2, TLR4, and TLR5. In particular, we describe (a) the molecular pathways activated by the stimulation of these receptors with their known bacterial ligands; (b) the non-bacterial ligands known to interact directly with TLR2 and TLR4 and their soluble forms. The scope of this minireview is to highlight the importance of bacterial and non-bacterial compounds in affecting the gut immune functions via the activation of the TLRs. Being in proximity to the largest interface between the body and the external environment, the gut immune system has evolved to distinguish the tiniest molecular difference that exists between pathogenic and non-pathogenic microorganisms. It has developed the capacity to react rapidly to any harmful threats before the “enemy” can multiply or even before harmless bacteria may mutate into dangerous ones ,2. ThereThe immune reaction caused by an invading pathogen needs to be properly tackled, avoiding underestimations, but also without developing over-reactions deleterious for the host organism. Hence, the first signals the pathogen-sensing cells recognize are the most crucial ones. An early interaction point occurs between potentially pathogenic bacteria and the toll-like receptors (TLRs) present on the membrane of the gut immune cells, as well as on the cellular membrane of the gut epithelium ,4 Figur. In someTLRs belong to the pattern recognition receptors (PRRs) family. They recognize molecules broadly shared by pathogens collectively named as pathogen-associated molecular patterns (PAMPs) and microbe-associated molecular patterns (MAMPS), the latest usually expressed by resident microbiota ,8,9,10. Therefore, TLRs on one hand sense the potentially harmful molecules of pathogens that infect humans  and switUpon binding with the pathogenic ligands, TLRs form homodimers or heterodimers, and initiate signaling cascades leading to the release of pro or anti-inflammatory cytokines or antiviral compounds ) .In this minireview, we focus on TLR2, TLR4, and TLR5, since among all TLRs, these are the main extracellular receptors that interact with the microbiota in the human GIT. We will describe the ligands responsible for their activation and the induced signaling pathways. A specific section is also dedicated to the description of plant-derived compounds that may regulate the inflammatory response in the GIT via TLR activation. Understanding the dynamic equilibrium that exists between microorganisms in the gut and the activity of the immune system regulated by TLRs is crucial for the development and discovery of compounds critical to maintaining or re-establishing homeostasis in the GIT such as pre- and probiotics, as well as pharma- and nutraceuticals.Consistent with their roles in immune surveillance, TLRs expression is elevated in those tissues exposed to external environments such as lung and GIT  with patGenerally, TLRs that respond to bacterial products such as triacyl and diacyl lipoproteins, lipopolysaccharide (LPS), and flagellin are transmembrane receptors expressed on the cellular membranes of the immune and gut epithelium cells. TLR2, together with its heterodimer partners TLR1 and TLR6, and TLR5, belong to this category. By contrast, nucleic acid-sensing TLRs such as TLR3, TLR7, TLR8, TLR9, TLR11, TLR12, and TLR13 are exclusively expressed on the membrane of intracellular compartments. TLR4 instead, is expressed on the cellular membranes , as well as intracellularly  ,27,28.Using a combination of immunohistochemistry, enzymatic separation, and laser capture microdissection of the intestinal epithelium, it was shown that healthy human colon tissues contain lower levels of TLR 2, TLR4 in the intestinal epithelial cells (IECs), with higher levels of TLR5 ,30. TLRsThe expression pattern of specific TLRs extends also to other cell types of the intestinal epithelium such as Paneth cells, expressing TLR2, TLR4, and TLR5 , and celA different expression pattern for TLRs occurs in pathological conditions . ColonicEach TLR is composed of a leucine-rich ectodomain responsible for the recognition of the PAMPs, DAMPs, or MAMPs, a transmembrane domain, and a cytoplasmic Toll-IL-1 receptor (TIR) domain that initiate the signaling cascade ,37,38. TTLR2 recognizes a wide range of ligands  rather than rough LPS or lipid A [Several PAMPs including the lipopolysaccharide (LPS), as well as plants and viral motifs, can activate TLR4 ,66,67. L lipid A . Of note lipid A .TLR4 expression is upregulated in both Crohn’s disease and ulcerative colitis, and in pediatric IBD patients, higher levels of TLR4 mRNA and protein were found in the inflamed colonic mucosa when compared with non-inflamed controls ,70. MoreMYD88-dependent pathway: This pathway is the same as the one activated by TLR2 stimulation. Shortly, TLR4 homodimerization recruits MYD88 and the IRAK complex including TRAF6 that activates TAK1. Upon TAK1 and IKKβ activation, the transcription factor NF-κB translocates into the nucleus where, together with the MAPK activated AP-1, it regulates the expression of pro-inflammatory cytokines and related genes such as IL-6, TNF-α, IL-1, and others  that function as decoy receptors to prevent a direct interaction between TLRs and their bacterial ligands . ConsequAs for most soluble receptors, sTLR2 is generated from the transmembrane TLR2 ,87. At lSoluble forms of TLR2 have been detected in human fluids such as breast milk, amniotic fluid, saliva ,87, and The soluble forms of TLR4 are produced by other mechanisms  other than a post-translational modification. In mice, a shorter mRNA codes for sTLR4, which probably originates by differential splicing ,6. InterAn emerging field in nutritional science is the search for dietary compounds from the food chain with the goal to reduce risks and the incidence of gut-related diseases. At least 11 polysaccharides that interact with TLR4 have been identified in eatable plants such as apple or ginseng . In addiTLR-interacting polysaccharides contain mainly α-, α-, β-, and β- glyosidic bonds . Of inteβ--fructans, to which inulin belongs, are natural carbohydrate storages in many plants . They acIn addition, natural compounds that regulate the expression of TLRs are of interest to influence inflammation in specific conditions. Paeoniflorin, a monoterpene glucoside, is the principal bioactive constituent of peony root that recently has been associated with a reduced inflammation of the colon in mice models for colitis (dextran sulfate sodium (DSS)-induced colitis) . The mecThe saturated fatty acids (SFAs) are non-microbial activators of the TLR-signaling pathways ,101. LauAlthough TLRs are responsible for detecting and triggering an inflammatory response after recognition of specific pathogenic molecules, they also maintain inflammatory tolerance if the same molecule belongs to commensal bacteria ,111. In Enterococcus spp. and Bacteroides spp. accompanied by a decrease of Bifidobacterium spp. and Lactobacillus spp. levels [The relationship existing between TLRs and microbiota is therefore crucial for the onset and progression of specific pathological conditions of the GIT. IBD, for example, is characterized by dysbiosis of the gut microbiota, a general term that defines an abnormal or misbalanced composition of the bacterial ecosystems in the intestinal tract ,114. Pat. levels ,116,117.. levels ,118. Ano. levels .B. fragilis use TLR2 signaling to enhance the colonization of the gut and establish host-microbial tolerance [In chronic inflammation, the increased expression of TLR2 leads to an exaggerated immune response following NF-κB activation and inflammatory cytokine release . Moreoveolerance ,121.In IBD, the expression levels of TLR4 are also upregulated, and TLR4 signaling has been shown to affect the intestinal microbiota via alterations in gastrointestinal motility that drives clearance of pathogens and maintenance of commensal populations, differentiation of goblet cells, and expression of antimicrobial peptides ,122,123.Activation of TLR5 signaling induces differentiation of naive B-cells into plasma cells producing IgA, as well as interleukins (IL-17 and IL-22) promoting early defenses against pathogen invasion of host tissues .TLR2, TLR4, and TLR5 have important roles in maintaining intestinal homeostasis. In pathological conditions, the expression and activity of these TLRs is altered, and whether these changes initiate the disease, or whether they represent the effect resulting from pro-inflammatory cytokines release, needs to be verified in more detail .Since TLRs “sense” molecular features specific to pathogen and endogenous threats, it is not surprising that several pathologies develop or progress via activation or deregulation of these receptors.Elevated expression of TLR2 and 4 was found in the colonic mucosa of children with IBD . WhetherTLRs represent the first point of contact between environment and organism, and for this reason altering the activity or the expression of specific TLRs could reduce or prevent the incidence of developing diseases associated with an inflammatory status. To date, polysaccharides are the only compounds identified as non-bacterial ligands of TLR2 and 4 in particular, and therefore they are reported in this manuscript. Identifying novel ligands of TLRs is key to modulating the immune response in the GIT, as well as in other tissues.In this minireview we have focused on TLR2, TLR4, and TLR5. We have described their ligands, as well as the signaling cascade events through which these receptors may regulate the inflammatory response in the GIT primarily. Understanding the dynamic equilibrium that exists between microorganisms in the gut and the activity of the immune system regulated by TLRs is a crucial issue for the development and discovery of compounds that are critical to maintaining or re-establishing homeostasis in the GIT, as well as in other tissues of the organism.


Asthma caused substantial economic and health care burden and is susceptible to air pollution. Particularly, when it comes to elder asthma patient (older than 65), the phenomenon is more significant. The aim of this study is to investigate the Markov-based acute effects of air pollution on elder asthma hospitalizations, in forms of transition probabilities.  A retrospective, population-based study design was used to assess temporal patterns in hospitalizations for asthma in a region of Sichuan province, China. Approximately 12 million residents were covered during this period. Relative risk analysis and Markov chain model were employed on daily hospitalization state estimation. 2, and SO2, only SO2 was significant. When air pollution is severe, the transition probability from a low-admission state (previous day) to high-admission state (next day) is 35.46%, while it is 20.08% when air pollution is mild. In particular, for female-cold subgroup, the counterparts are 30.06% and 0.01%, respectively.  Among PM2.5, PM10, NO2 was a significant risk factor for elder asthma hospitalization. When air pollution worsened, the transition probabilities from each state to high admission states increase dramatically. This phenomenon appeared more evidently, especially in female-cold subgroup . Based on our work, admission amount forecast, asthma intervention, and corresponding healthcare allocation can be done. SO Asthma is a major public health issue in the USA, affecting over 23 million persons . In ChinAmbient air pollution has been linked to the development and exacerbation of asthma and its related diseases in Europe, North America, Korea, Japan, and Taiwan  threshold, were fairly consistent across time-series and case-crossover analyses and suggest that effect estimates based on linear models (without thresholds) may underestimate the true risk. Cai et al. [2 and NO2 were robust after adjustment for PM10. The associations appeared to be more evident in the cold season than in the warm season. Cho et al. [2, PM10 (particulate matter not greater than 10 mm in aerodynamic diameter), NO2, and CO were positively associated with ED visits for asthma.Many works have been done in this field and achieved specific findings. Schouten et al.  applied i et al.  exploredi et al.  exploredi et al.  applied o et al.  applied However, previous studies have serval limitations: (1) Although elder asthma patients were found to be more fragile to air pollution , sex andThis study aims to investigate the Markov-based acute effects of air pollution on elder asthma hospitalization, which is the key chain of forecasting admission amount, by measuring the acute effects of air pollution on elder asthma admission first. We take sex and seasonality factors into consideration to achieve a systematic research. The Markov model is particularly useful in analyzing risk factors in cohort studies and has been applied successfully to the study of lung cancer, HIV infection , and theOur study area covers a region of Sichuan province, China. We obtained inpatient records of asthma hospitalization for adult residents between January 1, 2014, and December 31, 2014 (365 days). Approximately 12 million residents were covered during this period. The main diagnoses of hospital admission were coded according to the International Classification of Diseases, Revision 10 (ICD-10): Asthma . The data was also classified by season and sex. Warm season is defined as a period from April to September, and cold season is defined as the rest of time period in a year. Elder person is defined as person older than 65 years.2), and nitrogen dioxide (NO2), from January 1, 2014, to December 31, 2014, were obtained from the website of the Environmental Monitoring Center (EMC) database. To allow adjustment for the effect of weather on hospital admission, meteorological data (daily min temperature) were obtained from the website of Meteorological Bureau.Daily (24 h) air pollution concentration data including particulate matter less than 2.5 mm in aerodynamic diameter (PM2.5), particulate matter less than 10 mm in aerodynamic diameter (PM10), sulfur dioxide . In this way, the pollutants which acutely effect elder asthma admission can be confirmed.Daily asthma hospitalization and air pollution levels were linked by date and therefore could be analyzed with a time-series design. Because daily hospital admission for asthma approximately follows a Poisson distribution , we utilhttp://cran.r-project.org/) with the mgcv and msm package.For the purpose of healthcare resource allocation and schedule, it is important to measure the association between air pollution and elder asthma admission amounts and make corresponding forecast. Markov chain is a useful way to describe the asthma admission amount evolution process; it can not only reliably reflect the transition situation but also build a bridge between healthcare management and healthcare resource scheduling optimization. For instance, assuming that Markov transition probability between each admission amount state and future distributions of air pollution condition is given, then, future distributions of the admission amount state are also known. Hence, healthcare resource scheduling according to future distributions of the admission amount state will achieve a better performance. Markov models allow the modelling of patient follow-up as a succession of transitions between states over time. They are quantified as the rate of transition and expressed in number of transitions. The model was considered to be homogeneous; that is, the transition forces are independent of time. To construct the elder asthma admission Markov chain, we use the Lorenz curve and OR analysis to determine admission amount states and severity of air pollution. In economics, the Lorenz curve is a graphical representation of the distribution of income or of wealth, while in this situation, the Lorenz curve is a graphical representation of the distribution of daily admission amount. In statistics, the odds ratio (OR) is one of three main ways to quantify how strongly the presence or absence of property A is associated with the presence or absence of property B in a given population; while in our research, OR is employed to quantify the association between environmental properties and admission amount states. Then, a multistate model (MSM) was used to calculate the Markov transition probabilities. All models were fitted using R software , 0.5 , 7.27 , and 3.26  increase in risk of asthma hospitalization. The presented values are in percentages. For all lags, the effects of PM2.5 were always bigger than those of PM10, which indicates that PM2.5 has a stronger association with asthma hospitalization. All four pollutants showed the pattern that the effect increases with lag firstly and then peaks at a certain lag.Generally, PM2.5, PM10, SOch other .  Table x-axis denotes the cumulative proportion of admission days, and the y-axis denotes the cumulative proportion of admission counts. The curve shows that the 70% admission days with the lowest admission amount takes 50% admission amount of total admission amount; therefore, the top 30% admission days (the days of which admission amount is not less than 5) are labelled as “high day.”To define different admission amount states, we used the Lorenz curve to analyze the total admission amount. In the framework of Markov chain, the transition probabilities between different states vary, when decision or situation changes. For instance, when air pollution converts from mild to severe, the transition probability from low-admission amount to high-admission amount increases evidently. In our research, we constructed two transition probability matrices between different states, for mild air pollution and severe air pollution, respectively. Results from 2, the appropriate index measures whether within recent 2 days, SO2 daily average concentration exceeded the primary standard not less than 1 time. For NO2, the appropriate index measures whether within recent 6 days, NO2 daily average concentration exceeded the primary standard not less than 1 time. Considering that SO2 has the biggest odds ratio ), severe air pollution is defined as whether within recent 2 days, SO2 daily average concentration exceeding 35 μg/m3 not less than 1 time.Odds ratio analysis is a vastly used method to select a related index. 2 in Milan are stronger than those in this study, which may lie in the fact that emergent visits are more sensitive to air pollution. However, the effect of NO2 in Milan is much weaker than that in this study; this gap needs further study.2, and NO2 are all significant in Shanghai [2 of those is significant. Besides, the RRs of Shanghai for the three pollutants mentioned above are 1.88 , 4.79 , and 9.38 , respectively, whereas those of this study are 1.44 , 25.81 , and 7.11 , respectively. The results indicate that (1) for PM10 and NO2, the effects are slightly weaker than those in Shanghai and (2) for SO2, the effects are dramatically stronger than those in Shanghai. These differences may lie when sexual and season factors were not considered.The acute effects of PM10, SOShanghai ; however2, all the male subgroups show to be insignificant, whereas the significant effects appeared in cold season or the whole for female subgroups. The largest and significant RR was 13.71 in the female-cold subgroup. NO2 also showed no significance in the male subgroup; however, the significance appeared in both cold and warm subgroups. We can find an interesting phenomenon that the male subgroup was only sensitive in warm season, especially for PM2.5 and PM10. For male, the effect was significant only during warm season with PM2.5 and PM10. However, for the female, every effect is significant during cold season. Due to the fact that the strongest effect for each pollutant was in the female-cold subgroup, which indicates that the female-cold subgroup is more fragile to air pollution, we decided to construct the Markov transition probability matrix only for the female-cold subgroup.2 and NO2) and particulate (PM10 and PM2.5) pollutants across all the different sex groups and season groups. PM2.5, PM10, and NO2 showed no significant effects on elders, whereas SO2 was evidently significant from lag0 to lag0–2.This study certified that air pollutants have adverse short effects on elder hospital admissions for asthma. Such effects were observed for both the gaseous  When compared with Shanghai (admission), for PM10 and NO2, the effects are slightly weaker than those in Shanghai, whereas for SO2, the effects are dramatically stronger than those in Shanghai. These differences may lie when sexual and season factors were not considered.We also made a comparison with the effects in other studies: (1) When compared with Milan (emergent visit), the effects of PM2.5, PM10, and SOSex- and season-specific analysis indicates that for male, the effect was significant only during warm season with PM2.5 and PM10; however, for the female, every effect is significant during cold season.Precise Markov transition probabilities between high-admission states and low-admission states are obtained by a multistate model. It was also shown that when air pollution gets worse, the transition probabilities from low-admission states and high-admission states to high-admission states increase dramatically. When we focused on the female-cold subgroup, this phenomenon appeared more evidently: the probability increasing due to air pollution worsening of the female-cold subgroup was much dramatic than that of full samples.When these transition probabilities were combined with the forecast of air pollution, we can obtain the distributions of asthma admission, with reference to asthma healthcare resource demand  for a long period. Further, based on these distributions, asthma healthcare resource allocation can be done by the operation research method.2, and NO2, only the increment of SO2 was significant with that of elder asthma hospitalization. The effects of PM2.5, PM10, and SO2 in Milan are stronger than those in this study, which may lie in the fact that emergent visits are more sensitive to air pollution. However, the effect of NO2 in Milan is much weaker than that in this study; this gap needs further study.Among PM2.5, PM10, SOFor male, the effect was significant only during warm season with PM2.5 and PM10. However, for the female, every effect is significant during cold season. The strongest effect for each pollutant was in the female-cold subgroup, which indicates that the female-cold subgroup is more fragile to air pollution and that is why we constructed Markov transition probability matrix only for the female-cold subgroup.The difference between full samples and the female-cold subgroup was quite evident: the probability increasing due to air pollution worsening of the female-cold subgroup was much dramatic than that of full samples. That is to say, air pollution matters for the female-cold subgroup more than full samples. This gap may lie when the acute effects between each air pollutant and elder asthma admission vary on sexual and season factors.There are three points that should be focused:2, and SO2) has a great impact on elder asthma admission. For different air pollution conditions, the index to forecast high admission differs. (2) Outputting an effective air pollution index was performed to associate it with elder asthma admission. (3) Outputting Markov transition probabilities between high-admission states and low-admission states was performed, which could be used to forecast asthma healthcare resource demand when combined with air pollution forecast and then lead to healthcare resource allocation optimization.In summary, this study mainly achieved three goals: (1) validating air pollution . Future work will be done in the aspect of forecasting admission amount, asthma intervention, and corresponding resource allocation.


Solanum melongena) using a recent high quality sequence of its whole genome. We found nearly 133,000 perfect SSRs, a density of 125.5 SSRs/Mbp, and also about 178,400 imperfect SSRs. Of the perfect SSRs, 15.6% were complex, with two stretches of repeats separated by an intervening block of <100 nt. Di- and trinucleotide SSRs accounted, respectively, for 43 and 37% of the total. The SSRs were classified according to their number of repeats and overall length, and were assigned to their linkage group. We found 2,449 of the perfect SSRs in 2,086 genes, with an overall density of 18.5 SSRs/Mbp across the gene space; 3,524 imperfect SSRs were present in 2,924 genes at a density of 26.7 SSRs/Mbp. Putative functions were assigned via ontology to genes containing at least one SSR. Using this data we developed an “Eggplant Microsatellite DataBase” (EgMiDB) which permits identification of SSR markers in terms of their location on the genome, type of repeat (perfect vs. imperfect), motif type, sequence, repeat number and genomic/gene context. It also suggests forward and reverse primers. We employed an in silico PCR analysis to validate these SSR markers, using as templates two CDS sets and three assembled transcriptomes obtained from diverse eggplant accessions.We have characterized the simple sequence repeat (SSR) markers of the eggplant ( Solanum melongena L., 2n = 2x = 24), also referred to as aubergine or brinjal, belongs to the Solanaceae family. After potato and tomato, eggplant represents the third most important solanaceous crop species and its bulk production is concentrated in China, India, Iran, Egypt and Turkey, with Italy and Spain representing the most important European Union producers1. Unlike tomato (S. lycopersicum) and potato (S. tuberosum), which belong to the Potato clade and are native to South America, eggplant belongs to the spiny Solanums (subgenus Leptostemonum) and it is native to the Old World. A minimum of two domestication events from the wild species S. insanum has been supported: one in India and one in southern China/SE Asia, with a possible additional and independent center of domestication in the Philippines (S. aethiopicum L.) and the gboma eggplant (S. macrocarpon L). Both of them can be inter-crossed with S. melongena producing hybrids with intermediate fertility  platforms, through multiplexed sequencing of barcoded samples in a single run, have driven the costs down and considerably reduced the time needed for the whole genome sequencing of plant species. Genomes provide opportunities to develop a huge amount of novel molecular markers, which can be used for assessing the genetic variations within cultivars and germplasms, for the development of molecular genetic and physical maps, for identifying genes and quantitative trait loci controlling economically important traits and to assist breeding for crop improvement. Among them, microsatellites or simple sequence repeat (SSR) markers represent one of the most informative, versatile, and practical DNA-based markers used in plant breeding programs, since they are easy to score and have wide genomic distribution, codominant inheritance and a multi-allelic nature . The firCT motif  and, moreggplant . In eggpeggplant . Recentlplant , along with all unmapped scaffolds, were chopped into manageable pieces using SciRoKo tool4. Perfect, compound, and imperfect SSRs were identified in silico using the SciRoKo SSR—search module (see footnote 4). A minimum of four repetitions together with a minimum length of 15 nt was requested. Any sequence was considered as a perfect SSR where a motif was repeated at least fifteen times (1 nt motif), eight times (2 nt), five times (3 nt) or four times (4–6 nt), allowing for only one mismatch. For compound repeats, the maximum default interruption (spacer) length was set at 100 bp. The coordinates (start/end position) of each SSR were matched with those of the gene space using Bedtools intersect5, using the default parameters with -loj (left outer join) option: where the overlap comprised at least 1 nt, the repeat was designated as a genic SSR. The putative function of genes carrying at least one SSR was defined by the GO (gene ontology) approach. Enrichment for GO terms was recognized by examining the set of genic SSRs upon the whole genome GO annotation dataset using the AgriGO suite6, collecting enriched GO terms with P-values < e-5 and a false discovery rate <0.01, and visualized through ReviGO 7.The high quality genome of the eggplant inbred line “67/3,” recently sequenced  and avaiSolanum lycopersicum, S. pimpinellifolium, S. pennellii, S. tuberosum, Capsicum annuum, C. chinense, C. baccatum, Nicotiana tabacum, N. attenuata, N. benthamiana, Petunia axillaris, P. inflata) and their closely related species Coffea canephora, were collected from the related public database and scanned for the presence of perfect SSRs, using the same procedures described above. The source of each of the above full genome sequences is given in Supplementary Table For comparison purposes, the draft genome sequence of the Asian eggplant cultivars ‘Nakate-Shinkuro’ , togetheEgMiDB) was developed to provide browsable access to the SSR data. This web application, based on a LAMP stack, comprises a client tier (client browser), a middle tier (Apache web server with PHP interpreter) and a database tier (MySQL DBMS). A user-friendly interface was developed using PHP, which is an open-source server-side scripting language. The set of in silico detected SSRs were stored in the MySQL database, using PHP scripts to parse the text file from SciRoKo. User need-based customized queries can be generated from the web interface and allow users to search the microsatellite marker information in MySQL database. A stand-alone version of Primer38 is also provided to design primer pairs for any given SSR: its output lists alternative sets of primer pairs, and the characteristics of the expected amplicon.The Eggplant Microsatellite DataBase  in three transcriptomes (An Table 1). The number of imperfect SSR motifs was 178,407 (Table 2). The content and distribution of SSRs in the genome sequence of the eggplant breeding line “67/3” were compared with those of 14 other plant genomes, related to different degrees  and retrieved from databases (Supplementary Table Table 1). This indicates that the high quality of the recently released eggplant genome sequence markedly influenced the number of detectable microsatellites.In the ∼1.06 Gb of the gap-free eggplant genomic sequence, we identified 132,831 perfect SSR motifs (125.5 SSR/Mb), which included 20,670 compound SSRs  and N. attenuata , but only half of those in N. tabacum  and C. baccatum . The cumulative length of the full collection of eggplant SSRs was 3.4 Mbp, which is 0.32% of the assembled genome, a percentage analogous to those of tomato (0.31%) and potato (0.28%), but considerably higher than found in C. chinense and C. annuum . Compound SSRs represented 15.6% of the eggplant perfect SSRs, a proportion exceeded only in S. lycopersicum (27.7%) and S. pennellii (37.6%) (Table 1).The “67/3” eggplant genome was also found to include almost twice the number of perfect microsatellites compared to the wild species R2 = 0.948, P < 0.01). However, as a general trend, species possessing larger genomes show lower SSR density (SSRs/Mb)  . This isS. pennellii and P. axillaris, and, in the context of this study, inferior only to that found in Coffea canephora (139.1 SSRs/Mb) (Table 1).However, although differences in genome size may contribute to the level of repetition of microsatellites, density of SSRs has been found not to be related to genome size . The denTable 2). Dinucleotide sequences were in the majority, amounting to 1.7 Mbp . Dinucleotides were also the most common type in tomato and in Nicotiana species. Among the imperfect SSR motifs the occurrence of mono- to trinucleotide motifs was low compared to that seen among the group of perfect SSRs. There were relatively larger amounts of longer motifs: together the penta- and hexanucleotide SSRs represented 21% of the total, and 17% of the cumulative length of all imperfect SSR motifs (Table 2). We found that the sum of di- and trinucleotides formed the majority of perfect SSRs in all the genomes of Solanum, Capsicum and Nicotiana species searched, ranging from 67% in potato to 81% in N. benthamiana. However, in Petunia species and Coffea canephora, mononucleotides were the most frequent type .Eggplant SSR motifs which predominated were the di- and trinucleotides , with lesser proportions of mono- (8%) and tetranucleotides (7%); the penta- and hexanucleotide repeats contributed <5% . As a consequence, the mean number of dinucleotide repeat units (15.3) was more than twice the number of trinucleotide repeat units (7.4), and about three times higher than for tetra-, penta- and hexanucleotides (5.0–5.5) (Table 2). Based on the length of the perfect repeat motifs, 19.7% of SSRs were considered to belong to hypervariable class I (≥30 nt). Another 22.7% were assigned as potentially variable class II (20–30 nt) types, while the remaining 57.7% represented variable class III (<20 nt) types . Most of the mononucleotides (83.6%) belonged to class III, while for hexanucleotides classes I and II preponderated  . Class I consisted mainly of di- (77.2%) and trinucleotides (17.2%), with all the other motifs accounting for <2% .The variation of perfect microsatellites in the eggplant genome with regard to the number of repeat units is shown in Table 2).Grouping of repeats into classes was carried out according to the method of Figure 3 and Supplementary Table We also evaluated in detail the individual repeat motifs for each type of SSR in the eggplant genome . Analogous motif type distributions have been found for all the other related species analyzed here  while the motifs AAAAT, ATATC, AAACC, and AATAT represented nearly half (49.0%) of the total pentanucleotide repeats. Only three hexanucleotide motifs AACAAT, AAAAAT, and AAGAGG were present with a relative frequency higher than 5% . The longest linkage group E01 contained the greatest number of SSRs , while the shortest  hosted the fewest. However, substantial differences among chromosomes in the densities of SSRs were observed, ranging from 116.9 (E12) to 166.6 (E09) perfect SSR/Mbp (Table 3). On the whole a general trend of higher SSR density for short LGs (and vice versa) was observed, with the exceptions of E06 and E10 (Table 3). The distribution of motif types within individual chromosomes was very similar to the pattern found over the whole genome , with di- and trinucleotide repeats the most frequent and penta-/hexanucleotides the scarcest. Mono- to trinucleotide SSRs exhibited maximum variation among LGs, with E03 and E09 showing, respectively, the lowest percentages for di- (38%) and tri- (32%) and the highest for mononucleotides (ca 12%). When considered the distribution of different motifs on a chromosome basis, the percentages of the most frequent mono- di- and tri-nucleotides were similar to those detected in the whole genome, while the relative contributions of the main tetra-, penta- and hexanucleotides varied between LGs . This represents 6–8% of the gene space. These eggplant genes were estimated to cover a total of 132.18 Mbp , similar to the gene space distribution detected in other species (Using data from assembled chromosomes of the eggplant breeding line “67/3” (see footnote 2), the genomic distribution of SSRs was compared to their association with individual genes. In all, 2,449 perfect SSRs  and 3,524 imperfect SSRs (1.98%) were associated with, respectively, 2,086 and 2,924 genes  and non-triplet repeats ; a higher frequency in gene sequences of triplet repeats was detected for both perfect (73.1%) and imperfect (68.6%) motifs . Trinucleotides were the most common, representing 65.4% (12.2 SSR/Mbp) and 49.8% (13.3 SSR/Mbp) of perfect and imperfect genic SSRs, respectively; the next most frequent group within perfect motifs was dinucleotides , while for imperfect SSRs hexanucleotides were the second most frequent group  . In analogy with other species . Similar patterns of motif distribution have been observed in other species. In the those compared by The most frequent genic SSR motif types were the trinucleotides AAG/CTT (22.2%), ATC/ATG (9.5%), AGG/CCT, and ACC/GGT. AG/CT  were the most common dinucleotides , “molecular functions” (MF) and “cellular components” (CC)]. Over-representation was found for a number of gene families .The public domain tabase 9 . In brieEgMiDB is highly user-friendly, optimized to be accessed on all mobile devices. It implements input checking with auto-correction of errors. It: (i) displays default values or ranges; (ii) auto-corrects data immediately on entry rather than when a query is run; (iii) provides comprehensible error messages; and (iv) indicates when a field is valid. It also provides a simple interface for use by administrators when importing data into the system, and facilitates conversion of SciRoKo output into data formatted for EgMiDB.EgMiDB, the standard linkage map’s marker density can be increased, and this will contribute to QTL and gene mapping studies. In order to facilitate the use of these markers, we have implemented several plugins to generate primers at user-defined chromosomal locations, which can be directly exported for use in genotyping assays. Additionally, the developed SSR markers can also be used in DUS testing for variety identification in multiplex mode. From our large marker dataset, the identification of markers with thermodynamic compatibility for multiplex designing can be also accomplished.With the marker information present in in silico validation exercise was carried out, taking advantage of available transcriptomic/genomic resources. Normal methods for validating SSR markers involve employing them to prime PCRs containing appropriate template DNA. Here, in silico validation was possible because of the sequence data available from five eggplant accessions. The CDS set derived from the inbred “67/3” line . By taking into account ambiguous amplification, validation percentages were substantially increased in fragmented sets .In addition to understanding the characteristics of SSR distribution, the development of molecular markers for eggplant based on these loci was one of the most important objectives in this study. An /3” line  provided/3” line  validateEgMiDB provides key information for genetics and breeding studies as well as the integration of un-mapped scaffolds for improvement and optimization of the eggplant genome sequence.By using the genes predicted from the two sequenced eggplant varieties, almost 32–39% of primer pairs were predicted to be functional. When data from the three different assembled transcriptomes was included, the validation level was reduced to 23–28%, which can be considered satisfactory, since in previous similar studies validation success rates ranging from 50%  to 11%  have beeThe main goal of the present study was to identify a large set of valid SSR markers in eggplant, by adopting key standards including: (i) an SSR length of not less than 15 bp and (ii) a minimum of four repeat units per SSR. These parameters were chosen since microsatellite polymorphisms and mutation rates correlate positively with increase in the number of repeat units  as this EgMiDB contains all the available information related to genomic and genic perfect/imperfect microsatellite loci, with unrestricted public access. Through an easy-to-use web interface and a highly customized primer-designing tool, it is optimized to cater for the needs of plant biologists and breeders. It is very flexible and allows access with user-defined options. The database will help in selection of microsatellites of a particular type and also in identification of all putative microsatellite sites within specific genomic regions, such as functional microsatellites present in gene sequences. Furthermore, it makes it possible the identification of markers with thermodynamic compatibility for multiplex designing by allowing a large number of samples to be screened in a given experiment. EgMiDB will thus contribute to uniform bioinformatics workflow strategy to achieve SSR genotyping tailored to eggplant, and represents a key tool for making progress in both basic genomics research and molecular breeding of the species. The database will be constantly updated as additional sequencing data for eggplant genotypes become available, allowing rapid and simple in silico identification of SSRs including those polymorphic.The database EP, SL, FP, GLR, and AA conceived and designed the research. EP, LB, LT, and AA analyzed the data. FP and LV designed and constructed the web database. EP and AA drafted the manuscript. SL, LT, and GLR reviewed and edited the manuscript. All authors read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with several of the authors EP, SL, LB, AA.


Aedes aegypti or Aedes albopictus. The liquid from shells of cashew nuts is attractive for its important biological and therapeutic activities, which include toxicity to mosquitoes of the genus Aedes. The present study evaluated the effects of a mixture of surfactants from natural cashew nutshell liquid and castor oil (named TaLCC-20) on the mortality of larvae and on the reproductive performance, embryonic and fetal development and genetic stability of Swiss mice. A total of 400 Ae. aegypti larvae  were treated with TaLCC-20 concentrations of 0.05 mg/L, 0.5 mg/L, or 5 mg/L (ppm). Twenty pregnant female mice were also orally administered TaLCC-20 at doses of 5 mg/kg and 50 mg/kg body weight (b.w.), and 10 animals were given only drinking water at 0.1 mL/10 g b.w. . The results of a larvicide test demonstrated that 5 mg/mL TaLCC-20 killed 100% of larvae within three hours, which is comparable to the gold standard indicated by the Ministry of Health. Overall, these results show that TaLCC-20 is an efficient larvicide that does not induce genetic damage. In addition, changes in reproductive performance and embryo-fetal development appear positive, and the formulation is cost effective. Therefore, TaLCC-20 is an important product in the exploration of natural larvicides and can assist in fighting mosquitos as vectors for dengue fever, chikungunya fever and Zika virus, which are emerging/re-emerging and require proper management to ensure minimal harm to the human population. Therefore, TaLCC-20 can be considered a key alternative to commercial products, which are effective yet toxigenic.Dengue fever, chikungunya fever and Zika virus are epidemics in Brazil that are transmitted by mosquitoes, such as  Dengue is an epidemic in Brazil and worldwide. Coupled with chikungunya fever and Zika virus, these three diseases have caused extensive public health problems. In 2016, there were approximately 1,426,005 probable cases of dengue, including 798 severe cases and 7,105 suspected cases. In addition, dengue was responsible for 509 deaths [In Brazil, 3,657 cases of chikungunya fever were diagnosed in 2014. In 2016, the number of cases increased to 216,102 autochthonous cases. Zika virus was first diagnosed in Brazil in 2015, when 1,248 cases were reported in the northeast, in addition to 739 cases of microcephaly in newborns, which characterizes the most serious form of the disease. In 2016, there were approximately 196, 976 probable cases of the disease [Aedes aegypti and Aedes albopictus after a bite from an infected female. There is currently no effective vaccine for these diseases. Thus, the prevention of these diseases depends exclusively on the elimination of mosquito foci [Transmission of these diseases occurs through the mosquitoes ito foci –6.According to Machado et al. , the mosAnarcadium occidentale L.) shell liquid (CNSL), which contains phenolic compounds with great biological potential, such as in treatments for asthenia, respiratory problems, genital infections, and skin diseases [Accordingly, there is a critical need for new larvicidal compounds that do not exhibit toxicity to humans or the environment. One attractive organic raw material is cashew nut , whereas isolated cardol and cardanol showed LC50 values of 14.2 and 32.9 ppm, respectively. Therefore, we show that cardol is the constituent primarily responsible for the activity demonstrated by technical CNSL [Larvicide activities against cal CNSL , 16.Ae. aegypti larvae, obtaining an LC50 of 55.47 ppm. Mukohopadhyay et al. [50 of 12 ppm and an LC50 of 38 ppm for larvae of the Anopheles subpictus, while Raraswati et al. [Sapindus rarak DC) against Ae. aegypti larvae in the 3rd instar stage and found an LC50 of 14.12 ppm.Although the phenolic lipids isolated from CNSL show larvicide activity, these compounds do not dissolve in water. To solve this problem, Farias et al.  used sody et al.  tested ci et al.  studied Ae. aegypti showed that all compounds were active. The product termed Tensoativo do Líquido da Casca da Castanha do Caju (TaLCC-20) (CNSL:Castor oil 20:80 w/w) exhibited more effective activity at a concentration of 0.2 ppm, killing 97% of larvae within the first 24 hours, while the surfactant from only castor oil showed the lowest activity. That mixture presented efficient larvicide action and resulted in the registration of a patent [Ae. aegypti, Rockefeller lineage (Ae-Rockefeller), and on the reproductive performance, embryo development and genetic stability of Swiss mice.In preliminary studies, Galdino; Beatriz  and Beata patent . Conside1H-NMR spectra were recorded in CDCl3  solution on a Bruker DPX300 spectrometer (300 MHz), and spectra was referenced to TMS using residual solvent signals as secondary standards. The ethanol employed for extraction of CNSL was AR grade , and the methanol used in HPLC was HPLC grade . HPLC-grade water (18 mW) was prepared using a Milli-Q system (Millipore). Solvents were filtered with spare membrane filters .Cashew nut shell (CNS) was donated by Kardol Indústria Química in 2014. The plant material was verified by Msc. Juliana Miron Vani, and a voucher specimen was deposited (No. 51838) in the herbarium of the Federal University of Mato Grosso do Sul (UFMS). The extraction of natural CNSL from CNS was performed using the procedures of Gandhi et al. [For these procedures, the following reagents were used: absolute ethanol VTEC, p.a., ACS reagent, 99% purity; castor bean oil EXP type 01, batch M-05-04 Celtic; sodium hydroxide Dinâmica, p.a., ACS reagent, 97% purity; distilled water; and natural CNSL obtained via Soxhlet extraction. The common procedures for producing soaps from vegetable oils were used . ApproxiAe. aegypti eggs (Rockefeller line) were supplied by the Animal Biology Department, UNICAMP, Campinas-SP and Control of Endemia Superintendency (SUCEN), Marilia-SP. The establishment and maintenance of the Ae. aegypti colony and the production of eggs were realized in a creation room of the LIVe laboratory  at a controlled temperature of ± 28°C, relative humidity of ± 60% and a programed photoperiod of 10 hours of dark and 14 hours of light.The prime matrices of Ae. aegypti eggs were supplied by LIVe.For biological assay execution, rd-stage Ae. aegypti larvae of the Rockefeller lineage (Ae-Rockefeller). First, eggs (on filter paper) were placed in plastic trays containing a volume of distilled water greater than 1 mL per larva and macerated fish feed for larval hatching (Alcon Basic® Lot 162). After five days, the larvae reached the 3rd developmental stage. With a polyethylene Pasteur pipette, 20 Ae-Rockefeller larvae were transferred to a 50-mL beaker containing 20 mL of solution and 20 larvae. Four-hundred larvae divided into four replicates were subjected to each treatment: a negative control containing only distilled water, a positive control containing temephos at a concentration of 0.012 mg/L, and three concentrations of TaLCC-20 .The larvicide bioassay was carried out with 3The residual effect of TaLCC was assessed for the larvicide concentration (5 ppm—5 mg/L) and for a 10× higher dose (50 ppm—50 mg/kg). Drinking water was used for the control group, which was the TaLCC dilution vehicle.Ae. aegypti larvae of the Rockefeller line (Ae-Rockefeller) in the 3rd stage.The tests were performed in triplicate. Beakers were used with 30 mL of each solution containing 20 Larval mortality was assessed every 24 hours of exposure. Daily larval counting and replenishment continued until complete loss of the larvicide effect in the solution.Mus musculus)  of reproductive age (8–10 weeks) with an average weight of 30 g, obtained from the State Bureau of Animal and Plant Health Protection , were used. This study was approved by the Ethics Committee for Animal Experimentation of the Federal University of Mato Grosso do Sul (No. 401/2012).Swiss mice  and a temperature of 22 ± 2°C. The mice were provided with commercial feed  and filtered water.Overnight mating was performed at a ratio of 1 male: 2 females, and detection of pregnancy was based on vaginal plug formation (considered day zero of gestation) –25.After vaginal plug formation, health and well-being of the animals were evaluated daily. The following clinical signs of toxicity were observed: mucosal dryness, walking alterations (locomotor hypo-activity and hyperactivity), behavioral changes, diarrhea, decreased food and water intake, eye opacity, hair bristling, tremors, and morbidity –28.st to 18th); gestational group animals received TaLCC-20 at doses of 5 mg/kg (Gest. D1) and 50 mg/kg (Gest. D2) b.w. orally on all days of gestation : control group animals received drinking water at 0.1 mL/10 g body weight (b.w.) orally (gavage) on each day of pregnancy  was based on the larvicide dose, and the security dose was defined as 10× greater than the indicated for guidelines, i.e., 50 mg/kg  ,30.th day of gestation, the animals were euthanized, followed by laparotomy, hysterectomy and oophorectomy. Analgesia was not used to euthanize mice, as analgesia could affect the frequency of damaged cells in the micronucleus assay according to studies of Hoerauf et al. [On the 18f et al. , Heine ef et al. , Kotani f et al.  and Souzf et al. . Thus, tThe fetuses were euthanized using isoflurane, as only the tissues and bones needed to be preserved for subsequent analysis. The micronucleus assay was not performed for the fetuses.The spleen, heart, liver, lungs and kidneys were collected and weighed, and the fetuses and placentas were also weighed. An external systematic analysis of the fetuses was performed to detect possible external malformations, and the sex of the fetuses was determined. The number of implantations, resorptions, and live and dead fetuses was recorded. Based on these data, fetal viability (number of live fetuses/number of implantations x 100), the post-implantation loss rate (number of implantations–number of live fetuses x 100/number of implantations), the resorption rate (number of resorptions x 100/number of implantations), the placental index  and the sex ratio  were obtained , 23–25. Subsequently, the fetuses were randomly divided into two subgroups. The first subgroup underwent visceral analysis, for which the fetuses were fixed in Bodian’s solution (distilled water (142 mL), acetic acid (50 mL), formaldehyde (50 mL) and 95% ethanol (758 mL)) for at least seven days. Visceral analysis was performed via microdissection with strategic cuts to examine the chest and abdomen, according to Barrow and Taylor , and to All analyses were performed under a stereomicroscope (NIKON SMZ745T).th gestational day  to assess whether TaLCC-20 had the ability to cause cumulative damage. The slides were stored in a freezer at -20°C for at least 15 days. A total of 2,000 cells/animal were analyzed under an epifluorescence microscope  at a magnification of 400×).The technique used for the micronucleus assay was based on Hayashi et al. , as modiThe data are presented as the mean ± standard error of the mean (SEM) and were evaluated according to the nature of their distribution . The chi-square test was used to compare frequencies (percents) between the control and experimental groups. The level of significance was set at p<0.05.1H NMR spectrum . This result is considered the gold standard and was therefore used as a positive control . TaLCC-2rd day of exposure. After the 4th day, a decreased larvicidal effect was observed, which was completely lost by the 13th day. The concentration 10× higher than the larvicide dose (50 ppm-50 mg/kg) caused 100% mortality of the larvae through the 22nd day of exposure. After this time, larval mortality decreased until no morality was noted on the 36th day  concentration, 100% of larval mortality was observed through the 336th day .Despite the random distribution of animals in the experimental groups, the animals of the group control corresponded to higher initial weights than animals of the experimental groups. This weight difference was maintained until the end of pregnancy. However, the weight gain and net weight gain were similar among all groups (p>0.05). A reduction in uterus weight (p<0.05) was observed in the Gest. D2 group compared with the control group .Regarding the relative weights of organs, an increased weight (p<0.05) was observed for the lungs and a decreased weight (p<0.05) for the kidneys and the liver in the Gest. D1 group compared with the control group .The number of implants, live and dead fetuses, mean number of fetuses, resorption rate, placental weight and sex ratio did not differ (p>0.05) among the experimental groups. However, there were decreases (p<0.05) in the fetal viability rate, post-implantation losses and fetal weight and increases (p<0.05) in the number of resorptions and placental index. Despite these differences, the weight of the fetuses in the experimental groups was considered suitable for the gestational age .Malformations of the external limbs and tail were observed in all experimental groups, though at a low frequency that did not significantly differ among groups .Visceral observed malformations included hydrocephaly and hydronephrosis. These conditions occurred at the same frequency in all experimental groups .Regarding the detected skeletal malformations, an absence of or reduced ossification was observed in the phalanges, metacarpals, metatarsals, sternal centers, palate, presphenoid, parietal and ribs. The occurrence of these malformations was similar in all experimental groups. However, there was an increase (p<0.05) in the degree of ossification reduction recorded in the palate and presphenoid in the TaLCC-20-treated groups compared with the control group .th gestational day , demonstrating that TaLCC-20 does not cause cumulative genetic damage.TaLCC-20 showed no mutagenic activity , as therLarvicides are generally used in fumigation and/or deposited in water tanks (regardless of whether they contain drinkable water) . Thus, iAccording to Machado et al. , the besIt is well established in the literature that the use of pesticides, insecticides, larvicides and/or growth inhibitors is directly linked to genetic/genomic instability, which can increase predisposition to cancer ,49–55, iAccording to Barros et al. , the afoFor a suitable product to be developed, the product must exhibit equal or better efficiency than commercially available products in combating larvae and/or mosquitoes/vectors in other life stages and be more selective .Developing such a product would solve important public health issues without causing harm to the population that could potentially require further investments to treat chronic non-degenerative diseases, such as cancer, subfertility and infertility.CNSL exhibits larvicidal activity, as previously described ,13,16. IAe. aegypti larvae within a mean time of three hours at a dose of 5 mg/L (ppm). These results are considered by the Ministry of Health to represent the gold standard for commercial compounds, such as temephos, which also induces the death of 100% of larvae within a few hours of exposure [New tests performed with TaLCC have demonstrated its ability to kill 100% of exposure ,59. In aexposure .The residual effect of TaLCC for maintaining lethality of 100% of larvae lasted for up to 13 days and 22 days at the used doses of 5 and 50 ppm, respectively. After these periods, larvicide action was gradually lost and ceased within 13 and 37 days for the doses of 5 and 50 ppm, respectively. These results demonstrate that a formulation of TaLCC not only improves the solubility of other products prepared based on cashew nuts but also improves the time of action. According to Guissoni et al. , TaLCC cIn addition to these benefits, TaLCC-20 presents strong commercial appeal because it is easily produced, exhibits good yield and is inexpensive. Thus, TaLCC-20 may provide and inexpensive solution for a public health issue that is associated with high costs in various regions of the world. In addition, this product can prevent many deaths and improve the quality of life of millions of people who are affected by the symptoms caused by dengue, chikungunya fever and Zika virus. These infections and their complications are also responsible for the absence of workers from their workplaces, causing major harm to the production sectors of various countries ,61.The toxic effects of xenobiotics on genetic material and/or reproductive health can be evaluated based on mutagenesis, reproductive performance, embryonic and fetal development and teratogenesis assays –64. ProdThe guidelines for reproductive toxicology  and geneAccording to Zhang et al.  and SallConsidering the above issues, the results of the present study were promising, including 1) the high larvicidal efficiency of the product, and2) the lack of changes in embryonic and fetal development caused by the product and no detected genetic instability. Such effects can be correlated with infertility, teratogenesis/congenital malformations and cancer ,53,56,57Regarding the mutagenic capacity of TaLCC-20, the micronucleus assay revealed no genetic damage. This result is important because some products used against mosquitoes, such as temephos and diflubenzuron, are mutagenic and/or cause changes in DNA ,51.The preclinical trial also indicated an absence of toxicity based on the biometric parameters that were evaluated. According to other studies, weight loss and changes in the absolute and relative weights of organs may be indicative of toxicity ,72. In tst to the 4th gestational day), organogenesis  [th to 18th gestational day) [Regarding reproductive performance and embryonic and fetal development, pregnant females may be exposed to a test compound in different stages to predict any interference with implantation   or fetalnal day) . The litnal day) ,75. In tThe reduction in fetal weight and the increase in the placental index are not worrisome because, despite these differences, the fetuses continued to show an adequate weight for their gestational age. In general, a low weight of fetuses after birth is associated with an increased placental index, which is an adaptation of the maternal body in an attempt to increase the provision of nutrients to the fetus and, thus, allow proper development ,77. The The observed visceral malformations  may be normal variations, as they were also present in the control group ,23,37. FThus, the cost-benefit relationship observed for issues related to reproductive performance and embryonic and fetal development is also positive and supports the use of TaLCC-20, especially because the available commercial products that are indicated for use by the government, such as temephos  and diflIn summary, TaLCC-20 is considered an effective larvicide that does not induce genetic damage. In addition, the cost-benefit relationship associated with changes in reproductive performance and embryonic and fetal development appears to be positive. These findings indicate that it is an important product to be explored for use as a natural larvicide that can be employed against the mosquitoes/vectors responsible for dengue, chikungunya fever and Zika virus. These are emerging and/or re-emerging diseases that require proper management while minimizing harm to the population and the environment. Therefore, CNSL is considered an important alternative to commercial products that are toxigenic, although effective.


Plasmodiophora brassicae, is a severe disease of cruciferous crops that causes large hypertrophic galls in the roots. The plant microbiome is important for growth promotion and disease suppression. In this study, using 16S rRNA and internal transcribed spacer (ITS) sequencing techniques, we compared the endosphere microbiome of symptomatic and asymptomatic B. napus roots infected with P. brassicae collected from the same natural clubroot field. The results showed that the microbial population and its relative abundance in the asymptomatic roots was far higher than that in the symptomatic roots, and that many microorganisms in asymptomatic roots have biological control and plant growth promotion functions that may be related to clubroot symptoms. These results suggest the importance of the endosphere microbiome in clubroot disease and provide potential bio-control resources for its prevention.Clubroot caused by  Although studies have been conducted for many years, the plant microbiome has gained public attention as a new concept only recently , 2. TurnMagnaporthe oryzae, Sclerotinia scleroterum, Botrytis cinerea, and Fusarium sp. In terms of benefits, among others, it contributes to the promotion of growth and resistance to disease [Azorhizobium, Bradyrhizobium, Ensifer, Mesorhizobium, Rhizobium, and Sinorhizobiumn [Trichoderma controls a broad range of diseases such as Fusarium wilt, grey mold, and white rot disease [Pseudomonas can be used to control cotton blight and other soil-borne diseases [Streptomyces has been useful in controlling Phytophthora root rot [The plant microbiome has been identified as an important determinant of plant health . In its  disease , 5, 6. T disease , 8. Someizobiumn , and mosizobiumn , 11. Man disease , 13; Psediseases , 15; androot rot . OverallPlasmodiophora brassicae, and is a significant disease in cruciferous crops [P. brassicae, we found that some rapeseed seedlings were seriously diseased with typical symptoms whereas some of the neighboring seedlings showed no obvious symptoms. One of the reasonable explanations for this phenomenon is the uneven distribution of the pathogen throughout the field; however, P. brassicae could be detected in the root cortical cells of asymptomatic plants using polymerase chain reaction (PCR) amplification. Therefore, symptoms manifestation may be associated with other factors. In previous studies, a higher frequency of Curtobacterium flaccumfaciens was observed in asymptomatic plants than that in symptomatic plants, which may contribute toward resistance to citrus variegated chlorosis (CVC) [Methylobacterium spp., C. flaccumfaciens, and X. fastidiosa) [Clubroot is caused by a protist, us crops , 18. It is (CVC) . Asymptotidiosa) . The fretidiosa) . Bruez itidiosa) . TherefoWhile 16S rRNA is the most suitable appraisal indicator for the systemic development and classification of bacteria, as the use of the internal transcribed spacer (ITS) region in fungal identification has also gradually increased , 24. SeqP. brassicae-infected B. napus samples were collected  from a natural field large approximately 3 Mu (1 Mu = 0.0667 hectares) in Dangyang County, Hubei Province, PR China , where clubroot disease had been occurring for five years. The plants were at the five- to ten-leaf stage. Two sample groups were collected: RS1  and RS2 . In each group, 30 samples were randomly collected and divided equally into three sub-groups. Each sub-group (including 10 plant roots) was regarded as a single sample for sequencing .P. brassicae in RS1 and RS2 samples. PCR amplification was performed using the primers PbITS6: 5′-CAACGAGTCAGCTTGAATGC-3′ and PbITS7: 5′-TGTTTCGGCTAGGATGGTTC-3′ [All roots were carefully washed with tap water before being peeled with a sterilized razor. The genomic DNA of all samples was extracted using the CTAB (hexadecyl trimethyl ammonium bromide) method . DNA conGGTTC-3′ . The conP. brassicae in RS1 and RS2 samples, q-PCR analysis was performed using the primers Pb actin-F: 5′-CACCGACTACCTGATGAA-3′ and Pb actin-R: 5′-CAGCTTCTCCTTGATGTC-3′, according to the method described by Chen et al. [B. napus actin gene (primers: Bn actin-F: 5′-TGAAGATCAAGGTGGTCGCA-3′ and Bn actin-R: 5′-GAAGGCAGAAACACTTAGAAG-3′) was used as the internal control for normalization.For quantitative detection of n et al. . The DNA5′-GTGCCAGCMGCCGCGGTAA-3′ and 806R: 5′-GGACTACHVGGGTWTCTAAT-3′ for 16S rRNA; ITS1-1F-F: 5′-CTTGGTCATTTAGAGGAAGTAA-3′ and ITS1-1F-R: 5′-GCTGCGTTCTTCATCGATGC-3′ for ITS) tagged with abarcode. PCR was conducted in a total reaction volume of 30 μL containing 15 μL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs), 0.2 μM forward and reverse primers, and approximately 10 ng of template DNA. The thermal cycling consisted of initial denaturation at 98°C for 1 min, followed by 30 denaturation cycles at 98°C for 10 s, annealing at 50°C for 30 s, elongation at 72°C for 60 s, and finally 72°C for 5 min. An equal volume of 1X loading buffer (containing SYBR green) was mixed with the PCR products and electrophoresed on 2% agarose gel for detection. Samples with bright main bands between 400 and 450 bp were chosen for further experiments. The PCR products were mixed in equidensity ratios and purified using the Gene JET Gel Extraction Kit (Thermo Scientific). Sequencing libraries were generated using the NEB Next® Ultra™ DNA Library Prep Kit for Illumina®  according to the manufacturer’s recommendations, and index codes were added. The quality of the library was assessed using the Qubit® 2.0 Fluorometer (Thermo Scientific) and the Agilent 2100 Bioanalyzer system. The library was sequenced on an Illumina HiSeq 2500 platform by Beijing Novogene Bioinformatics Technology Co. Ltd. The raw sequencing data for 16S rRNA and ITS sequencing results have been deposited at the Sequence Read Archive  under accession numbers PRJNA388067 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA388067) and PRJNA388081 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA388081), respectively.Using the genomic DNA as a template, the 16S rRNA gene and ITS fragments were amplified using specific primers  for Mosaic [Raw read splicing: the split data of each sample read were spliced using FLASH  using the UCHIME Algorithm  to test the chimeric sequences. Final valid data (effective tags) were obtained after removing the chimeric sequences [Chimera removal: the clean tags were contrasted with the Gold database , and the ITS sequences were assigned with the UNITE/INSDC database using QIIME  –40.After assigning the corresponding database, the OTUs were classified into different classification levels . The level into which more than 80% of the OTUs could be classified was chosen for microorganism community analysis. The relative microorganism abundance in each sample was determined according to the number of OTU reads.To determine the difference in the microorganisms between the two groups, microorganism community cluster analysis was performed based on the Z value using the software MeV 4.9. The Z value of one sample in a classification was equal to the value calculated as the difference between the relative abundance in the sample and the average relative abundance in all samples, divided by the standard deviation of all samples.Fig 1. The roots of RS1 samples were not obviously swollen and the lateral roots remained healthy, whereas the roots of RS2 samples were obviously swollen, and few lateral roots could be observed . PCR amplification results showed that P. brassicae could be successfully detected in both RS1 and RS2 samples . To evaluate the relative P. brassicae content in RS1 and RS2 samples, expression of the P. brassicae actin gene was measured using q-PCR. In the symptomatic (RS2) samples, the P. brassicae numbers were thousand-fold higher than those in the asymptomatic (RS1) samples .The symptoms of the clubroot samples are shown in S1 Table), 45471, 27897, 82715, 57064, 25939, and 20842 tags were generated as raw data for the six samples . For each sample, the percentage of effective tags in the raw data was up to 92%, and the Q30 values were above 96%. In the case of ITS sequencing (S2 Table), 66344, 66782, 55796, 44365, 44576, and 66344 tags were generated as raw data for the six samples . The percentage of effective tags in the raw data was up to 96% and the Q30 values were above 99% in each sample. This demonstrated that the data were suitable for subsequent analyses.Raw data were generated by Illumina sequencing. Impurities such as joint and primer sequences, and low-quality reads were first removed to obtain effective tags for subsequent analyses. Sequencing quality was determined using the number of effective tags, their proportion in the raw data, and the base percentage of the sequencing error rate, which is less than 0.1% (Q30). In the case of 16S rRNA sequencing . The number of OTU clusters formed for the RS1 samples  as a result of 16S rRNA and ITS sequencing were 114, 108, and 123, and 135, 133, and 148, respectively; and for the RS2 samples  the number of OTU clusters formed were 93, 63, and 88, and 87, 81, and 69, respectively .All effective tags from the six samples were clustered into OTUs according to sequence similarity (≥97%) using the UPARSE software . According to the OTU statistical results, most effective tags (Taxon Tags) could be clustered to OTUs, with the exception of only a few data (Unclassified Tags and Unique Tags) . In both RS1 and RS2 groups, three samples were used for sequencing. Shared and unique OTUs were found among the three samples. To ensure the accuracy and completeness of the data, OTUs that existed in at least two of the three samples were chosen for further study. In the RS1 group, 96 OTUs (49 shared and 47 in two samples) from 16S rRNA and 110 OTUs (61 shared and 49 in two samples) from ITS were used for subsequent studies . In the RS2 group, 58 OTUs (31 shared and 27 in two samples) from 16S rRNA and 54 OTUs (31 shared and 23 in two samples) from ITS were used for further studies . Comparison of the OTUs in at least two samples from each group showed that, in 16S rRNA 48 OTUs were common to both groups, 48 OTUs were only in the group RS1, and 10 OTUs were only in the group RS2 . In ITS, 48 OTUs were common to both groups, 62 OTUs existed only in the group RS1, and 6 OTUs were only in the group RS2 . Therefore, 106 OTUs from 16S rRNA and 116 OTUs from ITS were used for further analysis. The number of OTUs in the group RS1 was obviously greater than that in the group RS2, both from 16S rRNA and ITS sequencing results.Since the number of taxon tags in each sequencing sample was different, the OTU clusters formed above could not be directly used tocompare the two samples. The OTU clustering results were standardized for subsequent analysis on the basis of the minimal sequence read number. The standardized OTU information for RS1 and RS2 samples was rendered using the Wayne figure  and at the genus level from the ITS sequencing results ; thus, the family and genus taxa were used to analyze the bacterial and fungal communities. The 106 OTUs from 16S rRNA and 116 OTUs from ITS as obtained above were classified into 45 bacterial families and 71 fungal genera . To determine the differences in endosphere microorganism communities between the asymptomatic (RS1) and symptomatic (RS2) B. napus samples, the relative abundance of bacteria and fungi in each sample was calculated based on the number of OTU reads and used for analysis .After the OTU cluster analysis, the same OTU sequences were assumed to derive from a certain taxon, and a representative sequence for each OTU was classified into different classification levels ., 39. TheS3 Table). The two most abundant families  were excluded from the analysis. Except these two families, the relative abundances of the top 15 bacterial families in each sample is shown in Fig 3A. The most abundant family was Oxalobacteraceae, followed by Pseudomonadaceae, Comamonadaceae, Xanthomonadaceae, Methylophilaceae, Rhizobiaceae, Flavobacteriaceae. The relative abundance of these bacteria in the asymptomatic samples  was much greater than that in the symptomatic samples  (S3 Table). In symptomatic samples, the relative abundance of other bacteria was the highest , whereas in asymptomatic samples, the relative abundance of other bacteria was low .Based on the classification results, 39 and 28 bacterial families were found in the asymptomatic RS1 and symptomatic RS2 groups, respectively  was slightly higher than that in the asymptomatic samples . Based on the classification, 65 and 39 fungal genera were found in RS1 and RS2 groups, respectively (S4 Table). The most abundant fungal genera were Un—s-Tremellomycetes sp., Tetracladium, Un—s-Nectriaceae sp., Verticillium, Fusarium, Gibberella, Alternaria, Un—s-Sordariomycetes sp., and Mortierella. The relative abundance of these fungi in the asymptomatic samples was also much greater than that in the symptomatic samples . In the asymptomatic samples, other fungi  were present in greater amounts than those in the symptomatic samples .The ITS sequencing results indicated the endosphere fungal communities and Fig 4). Among the 16 families that were present only in the asymptomatic samples, Streptomycetaceae, Pseudonocardiaceae, Rhodospirillaceae, Sphingomonadaceae, and Verrucomicrobiaceae were present in all three groups , and the remaining families  were present in two of the three samples. The families present only in the symptomatic samples included Peptostreptococcaceae, Moraxellaceae, Acetobacteraceae, Deinococcaceae, and an unclassified bacterium (auto67_4W) .For further analysis of the differences in the endosphere communities between the asymptomatic and symptomatic samples, the relative abundance of all bacterial families and fungal genera was clustered. For the bacterial endosphere community, 16 families existed only in the asymptomatic samples, five families only in the symptomatic samples, and 21 families common to both samples . The abundance of Methylophilaceae, Oxalobacteraceae, and Microbacteriaceae was similar in all the samples . The remaining 15 families were more abundant in the asymptomatic samples than in the symptomatic samples, especially Xanthomonadaceae, Comamonadaceae, Flavobacteriaceae, Sphingobacteriaceae, Rhizobiaceae, Caulobacteraceae, and Bradyrhizobiaceae .Of the 21 shared families, the relative abundance of Nocardiaceae, Phyllobacteriaceae, and Enterobacteriaceae in the symptomatic samples was greater than that in the asymptomatic samples . Only four genera were detected in the symptomatic samples, namely Un—s-Pleosporales sp. REF107, Aspergillus, Un—s-fungal sp. DG16, and Un—s-Helotiales sp. r427 . Among the 37 shared genera, seven, including P. brassicae, were much more abundant in the symptomatic samples than in the asymptomatic samples . The abundance of the remaining 30 genera including Tetracladium, Fusarium, Alternaria, Un—s-fungal sp. 3 EO_2010, and Sporobolomyces was much greater in the asymptomatic samples than in the symptomatic samples .In the fungal endosphere community, 30 genera were unique to the asymptomatic samples, including Glomus was detected only in the asymptomatic samples, whereas Aspergillus was detected only in the symptomatic samples (Tables 2).Overall, the bacterial and fungal endosphere communities in the asymptomatic samples were more abundant than those in the symptomatic samples. Among the unique and highly abundant bacteria and fungi in the asymptomatic samples, six bacterial families and six fungal genera have been attributed with biological control functions, three bacterial families with plant growth activity, and four bacterial families and seven fungal genera causing plant disease. Interestingly, Rhizobiaceae and Bradyrhizobiaceae were detected in both the asymptomatic and symptomatic samples, and their relative abundance in the asymptomatic samples was much greater than that in the symptomatic samples. Furthermore, B. napus infested with P. brassicae using 16S rRNA and ITS sequencing, and tailored the sequencing quality to the requirement of the analysis. The 16S rRNA sequencing results revealed 106 OTUs (OTUs that existed in at least two of the three samples), and 116 OTUs were detected from the ITS sequencing results. The majority of OTUs (except OTU3) from the 16S rRNA sequencing results could be classified to the family level and most OTUs from the ITS sequencing results could be classified to the genus level .Recently, research on microbiome has attracted increasing attention. Previously, 16S rRNA and ITS sequencing have been widely used in plant microbiome studies –42. In tS2 Fig). This might suggest that new microorganisms were being hosted by P. brassicae-infected B. napus roots including unknown bacteria . There may be two possible reasons for this. First, our knowledge of microorganisms is still very limited especially for those that cannot be purified and cultivated. Secondly, only 500 bp sequences were created and analyzed in the 16S rRNA sequencing platform, which is a little short for bacterial identification. Moreover, it is difficult to overcome the chloroplast and mitochondrial sequence interference in 16S rRNA sequencing [Although the results showed that 16S rRNA and ITS sequencing work well in microbiome research, some difficulties persist. For instance, in the 16S rRNA sequencing results, 11, 15, and 10 OTUs in the asymptomatic samples  and 9, 6, and 7 OTUs in the symptomatic samples  could not be classified into any known family. In the symptomatic samples, OTU 3 was classified as an uncultured bacterial clone and its relative content was 70–90% of all the OTUs. The ITS sequencing results also showed 18, 23, and 25 OTUs in the asymptomatic samples  and 14, 8, and 8 OTUs in the symptomatic samples  that could not be classified into any known genera  was also related to the CVC asymptomatic or symptomatic presentation in citrus plants [C. flaccumfaciens abundance in asymptomatic plants was higher than that in symptomatic plants, and may contribute to CVC resistance [Bacillus sp. from the family Bacillaceae, was reported as a biocontrol agent for P. brassicae [Streptomyces spp. [Streptomyces griseoruber A316 [Flavobacteriumhercynim EPB-C313, an endophytic bacteria of the family Flavobacteriaceae, has been used to control Chinese cabbage clubroot [Pythium spinosum, whereas strains of Pseudomonadaceae efficiently control fusarium wilt disease [Sporobolomyces, Leucosporidium, and Sporidiobolus can be used to control gray mold [The main purpose of this study was to clarify the difference between the endosphere microbiomes of symptomatic and asymptomatic iwifruit . The mics plants . Besidess plants ; C. flacsistance . In our rassicae , 46, 47;ces spp. , 49 and ber A316  from theclubroot . In this disease , 15, 52. disease , 54. Yearay mold –57. In aB. napus root samples, a variety of pathogenic plant microorganisms from other crops were detected including Xanthomonadaceae, Fusarium, Gibberella, Alternaria, Sclerotinia, Phoma, Leptosphaeria, and Cylindrocarpon. The impact of these pathogens on non-host plants remains a topic for further study.Another purpose of this study was to exploit the endosphere microbiome analysis technique to mine microorganism resources for resistance to clubroot disease. Little is known about Rhodocyclaceae, Verrucomicrobiaceae, Pseudonocardiaceae, and “Un—s-” fungi, all of which were only present in the asymptomatic samples, along with the unknown bacteria (OUT 3) in the symptomatic samples. These microorganisms may be potential bio-control resources for preventing clubroot disease. Moreover, in the asymptomatic B. napus root samples, suggesting that P. brassicae might consume oxygen or need environmental hypoxia when growing in B. napus roots [Aspergillus are typical saprophytes or opportunistic pathogens [Aspergillus could only be detected in the symptomatic samples. Therefore, we postulate that P. brassicae needs to decay and disintegrate the host roots with the help of saprophytic microorganisms, in order to release its resting spores into the soil at the later stages of clubroot development. Furthermore, we also found an interesting phenomenon that bacteria from the family Rhizobiaceae and Bradyrhizobiaceae and fungi from the genus Glomus were detected in B. napus infested with P. brassicae, although B. napus is not a plant that hosts rhizobia and arbuscular mycorrhizal fungi. Further analysis on this phenomenon is underway.A few anaerobic or facultative anaerobic bacteria like Acetobacteraceae  were unius roots . Interesathogens , and AspB. napus roots collected from the same natural clubroot field using 16S rRNA and ITS sequencing. The microbe population and their relative abundance in asymptomatic roots are far greater than that in the symptomatic B. napus roots. Many microorganisms detected in the asymptomatic roots have biological control and plant growth promotion functions. Furthermore, we also found many pathogenic plant microorganisms to which B. napus is not a host, and the related mechanism underlying this observation needs further research. These results provide a new basis for studying the endosphere microbiomes of roots and some potential bio-control resources for clubroot prevention.In summary, this study was mainly conducted to compare the differences in the endosphere microbiomes of symptomatic and asymptomatic S1 Fig(a) and ITS sequencing results (b). Total Tags, the filtered splicing sequence numbers; Taxon Tags, the number of tags used to build the OTUs and gain classification information; Unclassified Tags, the number of tags used to build OTUs but which did not provide any classification information; Unique Tags, the number of tags for which the frequency is one and cannot be clustered into OTUs; OTUs, the final OTU numbers.The statistical figures show the number of Tags and OTUs for all samples from the 16S rRNA sequencing results (TIF)Click here for additional data file.S2 Fig(a), The OTUs were classified by the taxonomic information into different bacterial classification levels detected in the 16S rRNA sequencing results. The above row of numbers refers to the number of OTUs classified to the bacterial Kingdom level and the lower row of numbers refers to the number of OTUs classified to the bacterial Family level. (b), The OTUs were classified by the taxonomic information into different fungal classification levels detected in the ITS sequencing results. The above row of numbers refers to the number of OTUs classified into the fungal Kingdom level and the lower row of numbers refers to the number of OTUs classified into the fungal Genus level.(TIF)Click here for additional data file.S1 TableRaw data, the number of PE reads; Raw Tags, Tag number of patchwork sequence; Clean Tags: Tags taken off the low quality tag number; Effective Tags, Tag number for aftershock; Base, The number of basesof the Effective Data; AvgLen, The average length of the Effective Tags; Q30, Base percentage of the sequencing error rate is less than 0.1% in Effective Tags; Effective (%), Effective Tags/PE Reads.(DOCX)Click here for additional data file.S2 TableRaw data: the number of PE reads; Raw Tags: Tag number of patchwork sequence; Clean Tags: Tags taken off thelow quality tag number; Effective Tags: Tag number for aftershock; Base: The number of bases of the Effective Data; AvgLen: The average length of the Effective Tags; Q30: Base percentage of the sequencing error rate is less than 0.1%in Effective Tags; Effective (%): Effective Tags/PE Reads.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Congenital hypogonadotrophic hypogonadism (CHH) and Kallmann syndrome (KS) are caused by disruption to the hypothalamic-pituitary-gonadal (H-P-G) axis. In particular, reduced production, secretion or action of gonadotrophin-releasing hormone (GnRH) is often responsible. Various genes, many of which play a role in the development and function of the GnRH neurons, have been implicated in these disorders. Clinically, CHH and KS are heterogeneous; however, in 46,XY patients, they can be characterised by under-virilisation phenotypes such as cryptorchidism and micropenis or delayed puberty. In rare cases, hypospadias may also be present.Here, we describe genetic mutational analysis of CHH genes in Indonesian 46,XY disorder of sex development patients with under-virilisation. We present 11 male patients with varying degrees of under-virilisation who have rare variants in known CHH genes. Interestingly, many of these patients had hypospadias.PROKR2, PROK2, WDR11 and FGFR1 with CHD7, may contribute to under-virilisation phenotypes including hypospadias in Indonesia.We postulate that variants in CHH genes, in particular  Proper function of the hypothalamic-pituitary-gonadal (H-P-G) axis is essential for the development of the reproductive system. Gonadotrophin-releasing hormone (GnRH), secreted by the hypothalamus, stimulates the biosynthesis and the release of gonadotrophins from the anterior pituitary gland. These gonadotrophins (luteinising hormone (LH) and follicle-stimulating hormone (FSH)) both play distinct roles in the gonads during embryonic development. In males, FSH stimulates the proliferation of immature Sertoli cells and spermatogonia . FSH alsEstimates of the prevalence of CHH range between 1 and 10 in 100,000 live births, with approximately two thirds of cases arising from KS . CHH in Genetically, CHH is highly complex. More than 30 genes have been implicated in CHH and/or KS including nine genes that cause an overlapping syndrome , 12. VarT). Reference levels for FSH and LH are based on paediatric measurements depending on age [T levels were also measured following Leydig cell stimulation by human chorionic gonadotrophin (hCG). For more details on blood hormone analysis, see [Patients with 46,XY DSD were referred to the Center for Biomedical Research, Faculty of Medicine, Diponegoro University (FMDU), Semarang, Indonesia. The medical ethics committee of the Dr. Kariadi Hospital/FMDU approved this study, and informed consent was obtained from all participants, as well as their parents or guardians, prior to their participation in this study. Following informed consent, a detailed interview was performed at recruitment and data concerning medical history, age of initial presentation, sex of rearing, family history  and consanguinity were collected. Patients were clinically evaluated by a trained andrologist; a detailed description of the external genitalia was obtained and, in many cases, images taken. A blood sample was obtained for karyotyping, hormonal analysis and DNA extraction. Referral and data collection took place between 2004 and 2010. Eighty-eight of these patients have been described previously . A totalg on age . In somesis, see .CHD7, GNRH1, GNRHR, HESX1, LEP, PROKR2, PROP1, TAC3, FGFR1, KAL1, LHX3, FGF8, PROK2, KISS1R, WDR11, SPRY4, FSHB, CGA, SOX10). Library preparation and sequencing were carried out as detailed in [Genomic DNA was obtained from peripheral EDTA-blood samples using the salting out method . The DNAailed in . Raw datailed in .Following C-pipe analysis, variants were checked for quality and depth and were filtered for those less than 1% minor allele frequency (MAF) in both the ESP6500 and 1000 genome project. As non-affected controls from Indonesia were not included, variants that were found very frequently in our screen  were also discounted. We manually check variant frequency in EVS and extracted ExAC data on frequency in Asia (South Asia and East Asia). Variants were checked for previous implication in human disease via ClinVAR and HMGD. Predicted pathogenicity of each variant was analysed using a range of up-to-date in silico prediction tools . Effects on protein structure and function were predicted using the HOPE tool .androgen receptor (AR), SRD5A2, HSD17B3). Rare and damaging mutations in these genes were found in 19 patients [All forty-seven 46,XY DSD patients from Indonesia were first analysed for mutations in DSD genes that cause androgen insensitivity or reduced testosterone production , which can often be increased by hCG stimulation. Indeed, we found that all of the patients tested showed moderate to high increases in testosterone after hCG stimulation Table . In addiThe remaining 28 patients were then analysed for mutations in the exonic regions of CHH genes as previously detailed in . Eleven PROKR2 gene. Two patients (173 and 143) had the same variant—PROKR2:c.C563T:pS188L . This missense heterozygous variant was not found in any of the online databases; however, it was not predicted to be pathogenic (Table One patient (47) was found to harbour a variant in this gene  was not previously found in any online databases and was predicted to be damaging and highly conserved  . No other CHD7 variants were found.Curiously, two of the three patients who had a AR (data not shown). Of the remaining 28, we found a likely CHH variant(s) in 11 patients, making this a total of 25% of the total original cohort. In addition, while our targeted DSD panel has a comprehensive list of diagnostic DSD genes, it only covers 19 of approximately 24 genes that cause CHH/KS without an associated syndrome. Sequencing of the entire list of known CHH genes, including those that cause CHH in association with additional anomalies, may increase the diagnostic yield of a genetic screen like this. This will be important for future studies of this cohort.In this study, we have investigated variants in CHH-related genes in a cohort of Indonesian 46,XY DSD patients who had an under-virilisation phenotype. After excluding patients with mutations in known DSD genes, we found rare and damaging variants in CHH genes in 11 of the remaining 28 patients. CHH and KS can present at birth with under-virilisation phenotypes in males such as micropenis and cryptorchidism . Our stuPROKR2 variant previously shown to be deleterious in functional studies, and we have sequenced more than 100 individuals from Indonesia  who were not enriched for these or other rare variants in CHH genes (data not shown).Penile and urethral morphology is established before 14 weeks gestation meaning that the foetal pituitary-hypothalamic axis is typically thought to be unnecessary for normal penile development . However, after week 14, continued increase in penile length is dependent upon the hypothalamic-pituitary axis. Therefore, boys with hypogonadism will often have micropenis but normal phallic morphology. However, we found that many of individuals in our cohort had varying degrees of hypospadias. Given this, it is interesting to note that while rare, hypospadias in patients with CHH or KS has been described. A large study found two patients with CHH and hypospadias , and sevPROK2 and PROKR2 are thought to contribute to around 9% of patients with KS [PROKR2 and one with PROK2. We also had three patients with WDR11 variants, meaning this gene may also play a significant role in Indonesian 46,XY DSD patients. Overall, we have found eight variants in CHH genes that have not been previously described in this disorder. Of these, four are not present in online variant databases ExAC or EVS. The PROKR2 variant p.W352G lies within the cytoplasmic tail of this transmembrane receptor. Other variants have been described in this region  [PROK2 (p.R23H) that has not been previously described. The affected amino acid lies within the signal peptide region, and the mutant residue (histidine) is smaller than the wild-type residue and has a different charge . This may change the activity of the signal peptide, and a patient with a variant affecting the neighboring amino acid (p.A24P) has been described previously in CHH [Mutations in  with KS . In our p.R357W) . In thisy in CHH .in silico predictions of pathogenicity in our pipeline, we have chosen to report all rare variants in this manuscript regardless of these predictions.The PROKR2 p.V331M variant that we and others have found has been shown to have reduced functional activity  –23; howeWDR11 variants were found in our screen. WDR11 is predicted to exhibit two β propellers made up of WD domains. Protein structure modelling has predicted that WDR11 has 12 WD domains and that nine of them (second through tenth) participate in the genesis of two consecutive β propellers [Finally, two novel opellers . The p.Wopellers . Cysteinopellers .FGFR1 and CHD7 variants. Indeed, oligogenicity has been described to be a feature of CHH  were not associated with classical CHARGE syndromic features. Indeed, they show that rare deleterious SNVs in this gene contribute to the mutational burden of patients with both KS and CHH in the absence of full CHARGE syndromic features [FGFR1 and CHD7 can cause hypospadias and under-virilisation. However, several of the FGFR1 and CHD7 variants had a total MAF of around 0.2%, with a prevalence of 0.3 or 0.4% in East Asia indicating that they may be over-represented in the Indonesian population. Further studies to address the pathogenicity of these variants and the interaction between FGFR1 and CHD7 are required and are beyond the scope of this study.Interestingly, we also found two patients with both ary, see ). Speciffeatures . It may Hormonal analysis at the right age can be highly informative in a clinical diagnosis of CHH. This includes assays of the levels of the gonadotrophins FSH and LH, as well as testosterone levels before and after hCG stimulation. Diagnosis of KS and CHH in many of these patients has been limited by access to detailed blood hormone analysis (in particular as many are pre-pubescent children meaning that measuring LH and FSH is not informative). Most of our patients showed low levels of testosterone (consistent with their age), but these levels were stimulated by hCG. Nevertheless, the genetic results of this study suggest that boys presenting with under-virilisation phenotypes in Indonesian clinics should be tested for CHH or KS. The patients presented here will be monitored as they develop, and we recommend they have their gonadotrophin levels retested at a later date when reduced levels can be detected.A genetic diagnosis can inform family planning and fertility investigations, as well as direct clinical management. Treatments exist for many of the features of CHH. In early life, this can include low-dose testosterone or gonadotrophins for micropenis and stimulation of gonadal development. Later, during adolescence or adulthood, testosterone therapy can also induce puberty including psychosocial development . CHH-assPROKR2, PROK2, WDR11 and FGFR1 with CHD7, may contribute to under-virilisation phenotypes including hypospadias in Indonesian boys. We suggest that in this population, 46,XY DSD patients should be monitored for signs of CHH including hormonal and genetic analysis.We conclude that variants in CHH genes, in particular