Leuconostoc (Leu.) mesenteroides (11251), Lactobacillus (L.) brevis (B151), and Lactobacillus plantarum (LB41K) strains were tracked in yogurt, and L. plantarum (LB41P) was tracked in a commercial probiotic powder. The yogurt was intentionally inoculated with the selected bacterial strains. Two types of yogurt with known and unknown bacterial pools were utilized. The standard 16S rRNA gene sequencing was used to evaluate the initial screening. The molecular typing tools, random amplified polymorphic DNA (RAPD), repetitive element palindromic PCR (rep-PCR), and comparative gene sequence analysis of selected housekeeping loci were used to track the inoculated dubious strains. Out of 30 random selections for each inoculation, the developed method identified seven (11251), nine (B151), and five (LB41K) colonies in the yogurt. The validation was performed by identifying 7 colonies (LB41P) out of 30 in the probiotic powder. The DNA banding profiles and the gene sequence alignments led to the identification of the correct inoculated strains. Overall, the study summarizes the use of molecular tools to identify the deliberately inoculated LAB strains. In conclusion, the proposed polyphasic approach effectively tracked the intentionally inoculated strains: Leu. mesenteroides, L. brevis, and L. plantarum (LB41K) in yogurt and L. plantarum (LB41P) in probiotic powder. The study demonstrates how to track industrially relevant misused LAB strains in marketable food products.The present work aimed at tracking intentionally inoculated lactic acid bacteria (LAB) strains in yogurt and probiotic powder.  For over thousands of years, the intentional addition of bacteria to commercial products as starters or as food additives has been a continuous practice across the globe. These days, consumers are attracted more to food products with high quality, safety, and viable bacterial additives for societal health benefits . On the The accurate species-specific documentation of LAB is significant from technical and safety perspectives . A recenRAPD is a molecular tool based on the random amplification of DNA fragments using short arbitrary sequence primers that bind at different locations within the genome, thereby generating a fingerprint of the bacteria . The advLeuconostoc, Lactobacillus, Oenococcus, Lactococcus, Pediococcus, Streptococcus, and Enterococcus [Leuconostoc (Leu.) mesenteroides, Lactobacillus (L.) brevis, and Lactobacillus plantarum, have been selected to validate the tracking technology. Leuconostoc species are mostly found on vegetables and fruits, and play an important role in various food and industrial fermentations; they are responsible for the organoleptic properties [L. brevis has been reported in plants, fermented foods, and beverages, and its strains are characterized as probiotics [L. brevis strain was also reported for its oral probiotic properties [L. brevis strains have also been reported for the spoilage of beer [L. plantarum is a versatile and heterogeneous bacterium found in fermented foods and feed products, as well as in the human gastrointestinal tract (GIT) [L. plantarum strains also synthesize vitamins and induce the production of host immunomodulatory particles [Leu. mesenteroides, L. plantarum, and L. brevis in commercial yogurt by using various molecular tools. Further validation was assessed by tracing L. plantarum strains from a commercial probiotic powder.The industrialized food units are mostly interested in LAB species of the following genera: rococcus . Among toperties ,19. L. bobiotics ,21. Receoperties . Contrar of beer . L. planct (GIT) . It is uct (GIT) . Some L.articles ,27. In tLeu. mesenteroides 11251, L. plantarum LB41K, and L. brevis B151. The strains were procured from the Korean culture collection of probiotics (KCCP) and maintained on De Man Rogosa Sharpe  medium at 37 °C under aerobic conditions for 24 h. For the quantification of selected strains, the viable colonies were counted from 50 µL  cultures grown on MRS agar plates and expressed as colony-forming units (CFU) per mL.The three LAB strains used in this study were Leu. mesenteroides, whereas yogurts with known bacterial strains  were used for the inoculation of L. brevis and L. plantarum. The yogurts were inoculated  using selected strains, rigorously vortexed, and serially diluted in phosphate-buffered saline . The dilutions were spread plated on MRS agar plates and incubated for 24 h at 37 °C. The next day, cell viability was measured. For each experiment, 30 colonies were arbitrarily selected and labelled as Y1–30 for Leu. mesenteroides, YB1–30 for L. brevis, and YP1–30 for L. plantarum. For the final validation, a commercial probiotic powder  containing four bacterial strains  was used to track the L. plantarum (LB41P) strain. The industry also provided the pure culture of the target strain (LB41P) as a reference. Two types of yogurts were purchased from a supermarket in Korea for a better insight of the presented technology to identify the suspected LAB strains. A yogurt with an unknown bacterial pool  was used for the deliberate addition of 2, 40 mM KCl, and 1 U of Taq polymerase) [For each experiment, genomic DNA was isolated from 30 colonies using the AccuPrep Genomic DNA Extraction kit . The PCR reaction was carried out by using 20 μL of Bioneer premix  .Leu. mesenteroides and L. brevis were designed using the primer BLAST  from the National Center of Biotechnology Information database (NCBI) website (www.ncbi.nlm.nih.gov/BLAST). For L. plantarum, the primer sequences were used as described previously [Primers for eviously . The priThe detailed procedure was performed as reported by reference .5, enterobacterial repetitive intergenic consensus (ERIC), and repetitive extragenic palindromic (REP) for the analysis of 30 colonies for each experiment. The PCR procedure followed has been described previously [w/v) agarose  gel electrophoresis at 70 V for 5 h. The PCR amplification conditions for all the three rep-primers used in the present study are described in To obtain the genomic fingerprints, the second typing method rep-PCR was performed using three primers: (GTG)eviously . Each reK and LB41P) have been described in w/v) agarose gel at 70 V.The third tracing method was based on the sequencing and analysis of the housekeeping genes. The genes, their products, and other relevant information for 11251, B151, and LB41  using RAPD and Rep-PCR tools. Finally, the tracking was performed using comparative gene sequence analysis of the housekeeping genes through sequence trimming and alignments with the BioEdit  SequenceLeu. mesenteroides, L. brevis, and L. plantarum LB41K: MF540920 to MF541009 and MF541070 to MF541098 for L. plantarum LB41P. Housekeeping gene sequences of Leu. mesenteroides: MG003189 to MG003195 (atpA), MG003211 to MG003217 (pyrG), MG003299 to MG003305 (gyrB), MG003321 to MG003327 (groEL), MG003255 to MG003261 (pheS), MG003277 to MG003283 (rpoA), MG003233 to MG003239 (uvrC); L. brevis: MF988109 to MF988117(dnaK), MF988120 to MF988128 (groEL), MF988131 to MF988139 (gyrB), MF988142 to MF988150 (pheS), MF988153 to MF988161 (recA), MF988164 to MF988172 (rpoA), MF988175 to MF988183 (rpoB); L. plantarum LB41K: MF988186 to MF988227; L. plantarum LB41P: MF988228 to MF988283.The gene sequences from this study have been deposited at the GenBank, and their accession numbers are as follows: for 16S rRNA of Leu. mesenteroides-inoculated culture, 22 colonies were identified as S. thermophilus, 7  colonies were identified as Leu. mesenteroides, and 1 colony (Y6) was identified as L. plantarum  were identified as L. brevis  out of 30 colonies were identified as L. plantarum, whereas the remaining 25 colonies were identified as S. thermophilus  that were likely to be similar to the reference strain, with a prominent band at 1100 bp a. Howeve primers a,b.L. brevis showed that nine colonies with a matching fingerprint profile to B151 strain had bright bands at 1200 and 660 bp with primers 239 5, REP, and ERIC) were found to be identical to the pattern of the type strain for the seven identified colonies 5 primer a. rep-PC5 primer b. With t5 primer c. These 5 primer .5 primer, three prominent bands at 1000, 1100, and 1500 bp were observed both in the reference and suspected bacterial colonies colonies d. With Rcolonies e. Likewicolonies f. The ouum LB41K . With thnd LB41K g. The REcolonies h. The ERer bands i.Leu. mesenteroides (seven colonies), L. brevis (nine colonies), and of L. plantarum (five colonies) were compared and analyzed for the presence of SNPs with the reference strains. Alignment of the seven sequences with the respective partial gene sequences from the reference strain 11251 showed no SNPs in the seven identified colonies among groEL, gyrB, atpA, pyrG, pheS, rpoA, and uvrC housekeeping genes  genes. Thus, we concluded that the suspected colonies, Y3, Y7, Y10, Y14–16, and Y27, were the initially inoculated Leu. mesenteroides strain 11251, and the nine suspected colonies, YB2, YB4, YB9, YB15, YB18, YB21, YB23, YB25, and YB30, were the L. brevis B151 reference strain. Likewise, the comparative gene sequence analysis of ddl, gdh, gyrB, mutS, pgm, purK1, and tkt4 showed that each of the partial sequences matched with the sequence of LB41K without any SNP in the housekeeping gene sequences  was evaluated in a probiotic powder consisting of a mixture of four bacterial species. As with the other inoculations, 30 colonies were randomly picked and labelled as PP1–30. The 16S rRNA gene sequencing results  to meet consumers’ demand under the stringent guidelines of various regulatory bodies . TherefoLeu. mesenteroides in yogurt , which were identical to the reference strain, 11251. For the tracking of L. brevis B151 and L. plantarum LB41K in yogurt , nine and five colonies were identified, respectively. It is to be noted that Leu. mesenteroides is not generally used to produce commercial yogurt; however, some strains of Leu. mesenteroides have been used in the production of cheese [Leu. mesenteroides 11251. However, a study by Chun et al. explained that 16S rRNA gene sequencing is not a suitable tool for inferring the phylogeny of Leu. mesenteroides strains [B. longum, L. casei, and L. acidophilus) of the four species were not detected, which reflects that these species require supplemented growth media and different culture conditions to grow. Bifidobacterium spp. generally prefer to grow MRS/MRS-nalidixic acid, paromomycin, neomycin sulphate, and lithium chloride (NPNL) at 37 °C for 72 h under anaerobic conditions. L. acidophilus shows selective growth on MRS agar supplemented with maltose (MRSM) or 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) followed by anaerobic incubation. On the other hand, L. casei can grow on selective medium containing ribose (1% w/v) LC agar, anaerobic incubation at 27 °C. In addition, many other media have also been reported for the enumeration of these aforementioned bacterial species [S. thermophilus. Furthermore, the bacterial pool in the yogurt (Korea Yakult) inoculated with L. brevis and L. plantarum LB41K strains were already known (marked on the label), and it was certain that the yogurt was not made by utilizing any of the L. brevis and L. plantarum strains. Thus, the identified colonies should be the inoculated strains, B151 and L. plantarum LB41K.The routine use of LAB species in industrial fermentations represents a billion-dollar industry . Among df cheese . Hence,  strains . Usually strains ,37. In t species . The remLactobacillus genus using housekeeping gene sequences is superior and more sensitive than the 16S rRNA gene sequencing [Leu. mesenteroides and L. brevis from Korea using the multilocus sequence typing (MLST) molecular tool [Leu. mesenteroides 11251 and L. brevis B151 in yogurt [L. plantarum strains, the housekeeping genes were used from an earlier described MLST study [PCR-based fingerprinting tools such as RAPD  and rep-quencing . Previoular tool . The samn yogurt ,46. For L. plantarum is the most commonly used bacterial species as a microbial starter or probiotic bacteria [L. plantarum strain from a commercial probiotic powder was investigated by identifying the LB41P strain. All four molecular tools effectively identified seven colonies as the reference strain. These results confirm the utility of the developed technology for the tracking of desired bacterial strains.In industrial fermentations, bacteria . TherefoCapillary sequencing  is a fast and cost-effective technology that is suitable for a low number of targets such as cloned DNA fragments or PCR products. It suffers challenges such as low sensitivity, high cost per sample for large number of targets, and challenging to scale. On the other hand, using next-generation technologies (NGS), one can reach a better conclusion about the identification of the suspected strain. The important criteria of this technology include read length, quality of the sequence, and cost. Ideally, the sequence data should be with long read length and low error rates. However, there is no report of such a technology. This technology is favored where novel or unique variants of the bacteria are required. Moreover, NGS is a labor-intensive, costly affair that requires technical expertise with heavy and costly equipment. Moreover, approximately 4000 USD is required to sequence the complete genome of probiotic bacteria . MoreoveFlow cytometry (FC) is a quick and automated method for enumeration, detection, and microbial profiling. However, the identity of microorganisms depends upon the use of different probes and fluorescent dyes. Also, the cost of this sophisticated and expensive instrument and technical operator is much higher compared to the tools used in the present technology. On the other hand, analysis time for FC is faster, around 15–20 min compared to 2–5 days for culturing of bacteria . RecentlIn our research, all the molecular methods were proven to be practical tools in the tracing of intentionally inoculated strains. In addition, the yogurt utilized was not made from any of the LAB strains selected for the present study. Nonetheless, as discussed earlier, all the molecular tools have some disadvantages; therefore, the identification of the desired strain cannot depend on a particular method and should be followed by a combined approach. Notably, the selection of the typing method depends on the objective of the research, the availability of skilled personnel, and, most importantly, the resources in the laboratory. It is a fact that the comparison of housekeeping gene sequences is a measured approach. However, new alternatives such as whole-genome sequencing (WGS) and whole-genome MLST (wgMLST) are becoming popular (subject to affordability) for the description and identification of a bacterial species. Further investigations should be focused on the utilization of these technologies.The strength of the present study includes the use of practically viable molecular tools such as RAPD, rep-PCR, and comparative gene sequence analysis for the identification of three LAB species. Moreover, the technology was validated by identifying target bacterial species from a commercial product. In addition, the presented cost-effective technology can be easily performed in a basic molecular biology lab. It can be used for the identification of bacteria in most food products. The limitations of the study include the time-consuming culture dependency and the selection of food products without the presence of target reference strains.Leu. mesenteroides, L. brevis, and L. plantarum (LB41P) in commercial yogurt, and substantiated viability by identifying L. plantarum (LB41K) in a commercial probiotic powder. Overall, the union of these low-cost molecular tools would help users for the identification of suspected LAB or other probiotics strains.Molecular tools for identifying microorganisms have been emerging in recent decades. Nonetheless, many limitations such as high running costs, skilled workforce, and expensive equipment need to be overcome for many of these newly developed technologies. Herein, our approach describes a technology with low cost and simple instrumentation for the identification of LAB. Our analysis of the 30 random selections and corresponding reference strains showed the practical feasibility of the approach for identifying suspected LAB strains. These PCR-based molecular tools showed efficacy for the identification of suspected


Helicobacter pylori infection and/or gastric disorders and chronic kidney disease (CKD) has not been elucidated. We investigated the relationship between Helicobacter pylori and/or atrophic gastritis (AG) and chronic kidney disease. In total, 3560 participants (1127 men and 2433 women) were eligible for this cross-sectional study. We divided participants into four study groups: with/without Helicobacter pylori infection and with/without AG. The HP (+) AG (−) group demonstrated a significant association with CKD compared with the HP (−) AG (−) group . In contrast, the HP (+) AG (+) group showed significantly lower adjusted odds of CKD than the HP (−) AG (−) group . H. pylori infection without AG might be associated with CKD in these participants. Conversely, the HP (+) AG (+) group had lower odds of CKD. Uncovering an association between gastric and renal conditions could lead to development of new treatment strategies.The relationship between  Helicobacter pylori (H. pylori) infection is associated with the metabolic syndrome and abnormal lipid profiles [H. pylori infection and insulin resistance [H. pylori infection and AG are useful for risk assessment of osteoporosis [H. pylori infection and/or AG affect not only the stomach but also other organs.The stomach is reported to be associated with conditions affecting other organs. Such as, an association between atrophic gastritis (AG) and coronary artery disease has been described ,2, with profiles ,5, whilesistance ,7, liversistance  and ventsistance . In addioporosis . These sH. pylori had a higher risk of subsequent renal dysfunction than those not infected [H. pylori infection rate is lower in patients with peptic ulcer disease and concomitant chronic kidney disease (CKD) than in those without CKD [H. pylori infection and/or gastric disorders and CKD has not been elucidated. Therefore, in this study, we investigated the relationship between H. pylori and/or AG and CKD, in order to determine whether an association exists between the stomach and kidneys.A previous study reported that individuals infected with infected . Convershout CKD . HoweverH. pylori eradication therapy (n = 530) or who had undergone gastrectomy (n = 30). Further, participants who used a proton-pump inhibitor (n = 112), or who had missing medical information data (n = 105), were excluded. This left 3560 participants (1127 men and 2433 women) eligible for analysis. This cross-sectional study included 4337 individuals enrolled in the Japan Multi-Institutional Collaborative Cohort Study in the Kyoto area, from 2011–2013. The Japan Multi-Institutional Collaborative Cohort Study is a new cohort study, launched in 2005, to examine gene–environmental interactions in lifestyle-related diseases [2) = 194 × creatinine−1.094 × year−0.287 (for men) and eGFR (mL/min/1.73 m2) = 194 × creatinine−1.094 × year−0.287 × 0.739 (for women) [2). Anthropometry data was obtained during the clinical examination. Medical history and medication use were assessed by means of a questionnaire. Hypertension was defined as a resting systolic blood pressure ≥ 140 mmHg or if receiving medication, diabetes mellitus (DM) as HbA1c ≥ 6.5% or if receiving medication, dyslipidemia as LDL-cholesterol ≥ 140 mg/dL and HDL-cholesterol < 40 mg/dL or if receiving medication, and anaemia as haemoglobin ≤ 13.0 g/dL in men and ≤ 12.0 g/dL in women.The study evaluated medical information obtained via self-administered questionnaires. Metabolic equivalents (METs) were assessed, as previously reported . Furtherr women) . The preH. pylori antibody (HP) positivity and levels of serum PG. This method has recently been used in Japan for gastric cancer screening of high-risk individuals [H. pylori antibodies (HP) and PG levels. AG was defined according to the serum PG I and II criteria proposed: when a participant fulfilled the criteria of both serum PG I value ≤ 70 ng/mL and PG I/II ratio ≤ 3.0, he or she was diagnosed as having AG. Infection with H. pylori was diagnosed using a microplate enzyme immunoassay kit  [H. pylori, i.e., HP (+). The control group consisted of participants who tested HP (−) AG (−); while the other three groups consisted of participants who tested HP (+) AG (−), HP (+) AG (+), or HP (−) AG (+).Participants were divided into four study groups according to a combination of serum anti-ividuals ,17,18. I, Japan) . Serum sp values < 0.05 were considered statistically significant. Continuous variables were expressed as means ± standard deviations (SD), and categorical data were expressed as sums and percentages. Inter-group comparisons were performed using the one-way analysis of variance for continuous variables, or the chi-squared test for categorical variables. Categorical variables included sex, alcohol use, smoking, hypertension, DM, dyslipidemia, stroke, myocardial infarction and/or stenocardia, and anaemia. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated using logistic regression methods in which CKD was the dependent variable and year, sex, body mass index (BMI), METs, smoking, alcohol use, hypertension, DM, dyslipidemia, stroke, myocardial infarction and/or stenocardia, and anaemia were the independent variables.3.Analyses were performed using SPSS statistical software (PASW 25.0). For all analyses, H. pylori infection and AG. The mean age of the control group was 50.0 years, vs. > 54.0 years for the other groups. H. pylori infection and diagnosis of AG. Relative to the control group, the HP (+) AG (−) group had an adjusted OR for CKD of 1.465  adjusted for yeae and sex; 1.439  adjusted for year, sex, BMI, METs, alcohol use, and smoking; and 1.443  adjusted for year, sex, BMI, METs, smoking, alcohol use, hypertension, stroke, dyslipidemia, myocardial infarction and/or stenocardia. There was no significant difference in the adjusted odds of CKD between the control group and the HP (+) AG (−) group—adjusted for lifestyle factors  or medical history . In contrast, we compared the HP (+) AG (+) group and HP (−) AG (−) group, the HP (+) AG (+) group had lower an adjusted OR for CKD of 0.610  adjusted for year and sex. There was no significant difference in the odds of CKD between the control group and the HP (+) AG (+) group, adjusted for lifestyle factors or medical history. On the other hand, the HP (−) AG (+) group showed no significant association with CKD.H. pylori infection not only affects the stomach, but is associated with other risk factors for CKD: hypertension, metabolic syndrome, DM, cardiovascular disease, changes in lipid profile [H. pylori infection and/or AG.Associations between two or multiple organs are essential for the human body to maintain homeostasis and to function normally. Elucidating these associations may be important for clinical decision-making and the development of appropriate treatments for some diseases. With knowledge of interactions between organ systems, we can treat diseases focusing not only on the symptomatic organ, but also on the organ fundamentally causing the disease. Moreover, such knowledge should allow for prediction and prevention of other related-organ disorders when a patient suffers from a particular organ disorder. Indeed,  profile . In thisH. pylori infection and CKD and revealed no association between H. pylori infection and either non-dialysis [H. pylori infection in patients with CKD and concluded that there is a lower prevalence of H. pylori infection in patients with CKD [H. pylori infection is still conflicting. Given that H. pylori inhabits the stomach, it is necessary to consider the state of AG when demonstrating the relationship between H. pylori infection and CKD. Although it has been that the risk factor for peptic ulcer are H. pylori infection [H. pylori infection is lower in individuals with peptic ulcer disease and concomitant CKD than in those without CKD [H. pylori infection. Indeed, in the present study, the HP (+) AG (−) and HP (+) AG (+) groups demonstrated an inverse association with CKD. If we examine only the presence or absence of H. pylori infection and the risk of CKD without considering the presence or absence of AG, the results are inconsistent. Furthermore, in the 50–59-year-old age stratum, the HP (+) AG (−) group had a CKD prevalence approximately double that of the control group. CKD prevalence and H. pylori infection are strongly associated with age [H. pylori infection without AG may have been accelerated the onset of CKD in the 50–59-year-old age stratum.Previous meta-analyses have reported that association of dialysis  or dialydialysis . On the with CKD . The repnfection , the rathout CKD . Thus, iwith age ,25. ThisH. pylori infection reduces ghrelin secretion. Ghrelin, a gastrointestinal peptide hormone, is known to perform a plethora of central and peripheral actions in distinct areas, including gut motility, gastric acid secretion, and glucose metabolism [H. pylori infection is associated with decreased ghrelin production and with a reduction in the number of ghrelin-producing cells [H. pylori infection with/without AG. Furthermore, there was no difference in the relationship between CKD and lifestyle factors and medical history between the control and HP (+) AG (−) group. In sum, H. pylori infection may induce renal dysfunction via reduction of ghrelin, and further studies should be conducted to confirm this hypothesis  AG (−) status is higher in CKD and may have relationship with the risk of CKD, an effect that has been speculated by one mechanism: that tabolism . There atabolism ,28. The tabolism ,30. H. png cells ,32. In cng cells . This suH. pylori infection. A prospective study is warranted to assess the relationships between CKD and H. pylori. Although the study evaluated medical information obtained via self-administered questionnaires, some anti-diabetes drugs used for other reasons or for pre-diabetes, like metformin and we can’t detect diabetics from non-diabetics. Furthermore, we could not show direct evidence of an interaction between ghrelin levels and H. pylori infection and/or AG status. In addition, CKD is generally defined in terms of proteinuria and eGFR; however, we had limited data on proteinuria. Last, we diagnosed H. pylori infection via serum antibody detection, whereas the gold standard is gastric-based testing.The limitations of the present study are mainly related to the study design and lack of measured ghrelin and proteinuria. The present study was cross-sectional; thus, we could not investigate the incidence of CKD in relation to H. pylori, an important pathogenic factor in the stomach, is probably involved in the development of many other diseases. Uncovering the association between gastric and renal conditions could lead to the development of new treatment strategies. In theory, if a patient suffers from H. pylori infection, we could predict renal disorders and potentially prevent their development by treating the stomach, for example, by administering H. pylori eradication treatment.We demonstrated that the prevalence of HP (+) AG (−) status is higher in CKD and may have relationship with the risk of CKD.


Giardia intestinalis is an intestinal protozoan most commonly found in humans. It has been grouped into 8 assemblages (A-H). Markers such as the glutamate dehydrogenase gene, triose phosphate isomerase and beta-giardin (β-giardin) have been widely used for genotyping. In addition, different genetic targets have been proposed as a valuable alternative to assess diversity and genetics of this microorganism. Thus, our objective was to evaluate new markers for the study of the diversity and intra-taxa genetic structure of G. intestinalis in silico and in DNA obtained from stool samples. We analysed nine constitutive genes in 80 complete genome sequences and in a group of 24 stool samples from Colombia. Allelic diversity was evaluated by locus and for the concatenated sequence of nine loci that could discriminate up to 53 alleles. Phylogenetic reconstructions allowed us to identify AI, AII and B assemblages. We found evidence of intra- and inter-assemblage recombination events. Population structure analysis showed genetic differentiation among the assemblages analysed.  Giardia intestinalis , a single-celled eukaryotic protozoan, is the most common cause of parasitic diarrhoea in humans worldwide [orldwide . It infeorldwide . Transmiorldwide  and wildorldwide . The freorldwide . Still, G. intestinalis [Different molecular tools ,7,8 and stinalis ,13, basestinalis ,15. Thesstinalis ,17,18,19stinalis . Assemblstinalis ,22,23. Sstinalis ,25, havestinalis  or low rstinalis  when ideSome authors have proposed the study of new genes , which, G. intestinalis is needed to identify new markers for understanding its diversity and evolution. Such markers should possess sufficient discriminatory power to establish groupings related to epidemiological factors. Thus, investigation of new markers should focus on detection and typing, and allow additional inference on reproduction, evolution, zoonotic potential and population structure [Evaluation of additional regions of the genome of tructure ,31,32. Structure . Initialtructure ,34, and tructure ,36,37,38tructure ,40,41. TG. intestinalis are available that sought to address additional genetic markers. Yet, generating multilocus analyses is essential for understanding the genetic characteristics of circulating strains in different geographical regions and monitoring their evolution and adaptation. Such analysis will encourage the design of strategies to decrease infection incidence [G. intestinalis tested in publicly available whole-genome sequences (WGS) and subsequently analyse these markers in DNA from stool samples from some regions of Colombia. Our proposed new markers are capable of elucidating diversity, population structure and possible recombination events between and within G. intestinalis assemblages.Few studies on ncidence . In the Pyrophosphate-fructose 6-phosphate 1-phosphotransferase alpha subunit (PFP-ALPHA1), Fructose-bisphosphate aldolase (FBA), Phosphoglycerate kinase (PGK), enolase, Acetyl-CoA synthetase (ACS), NADP-dependent malic enzyme (NADP-ME), Serine palmitoyltransferase 2 (SPT), Glutamate dehydrogenase (GDH) and Triose phosphate isomerase (TPI) genes in 80 available WGS from G. intestinalis. , AII (DH), B (GS and GS_B) and E (P15)  for varitinalis. . We mappNADP-ME and SPT genes displayed the highest values for both nucleotide diversity (Pi: 0.107 and 0.108. respectively), Theta (per site) from Eta (0.060 and 0.059. respectively) and numbers of segregating polymorphic sites . The SPT showed the fewest haplotypes (h: 11) and haplotypic diversity (Hd: 0.686). In contrast, genes such as GDH, commonly used to type G. intestinalis, showed a nucleotide diversity value, Pi of 0.058 and a Theta (per site) from Eta of 0.034, both being the lowest among analysed loci . However, when analysing assemblages, results for all loci in AI and B assemblages were negative  and discriminatory power (DP), using MLSTest software  . Numbers alleles . We alsoS, 0.815 .G. intestinalis assemblages most commonly found in humans, the AI, AII and B  , for all evaluated alignments, using Dnasp software . The higctively) . This re, both by gene and concatenated sequence. . The preStatistics used for genetic differentiation between populations are shown in G. intestinalis, collected in different regions of Colombia, from Amazonas [We randomly chose 24 samples positive for Amazonas , CasanarAmazonas .G. intestinalis axenic culture. All primers adequately amplified corresponding regions with each locus. A single band of the expected size was obtained for each marker (n = 24) of positive samples. Of the 24 samples, 95.8% (n = 23) amplified GDH; 83.3% (n = 20) amplified TPI; 66.7% (n = 16), ACS; 29.2% (n = 7) SPT, and 50.0% (n = 12), enolase. Many other samples amplified the target genes, and a band of the expected size was evident. However, other bands of different sizes were also observed. Also, concentrations of products obtained after the polymerase chain reaction (PCR) were low in some cases, showing bands so thin that it was impossible to obtain results from these sequences or no amplification occurred. Poor quality electropherograms were obtained for the NADP-ME gene, and sequences for this gene were ignored. Finally, five genes  were evaluated with DNA from the 24 stool samples. At least three markers were amplified in each sample, and all five loci were amplified in a few samples. , compared to other loci, and the TPI locus the lowest number (S = 133), even though it displays the largest number of sites analysed. Also, nucleotide diversity, Pi, for the ACS gene was 0.210, followed by SPT and TPI loci with estimates of 0.114 and 0.109, respectively. Similarly, the ACS locus presented the highest value for Theta (per site) from Eta. Thus, locus that shows the greatest diversity is ACS. It is important to highlight that calculation of D Tajima as a neutrality test produced almost universally negative values. Not all values statistically significant, but still may indicate population expansion, at least for those loci, D Tajima for the TPI locus was positive, suggesting balancing selection. Another index calculated for this data set, corresponding to Rm, indicates a high value (Rm = 52) for the TPI locus compared to other target genes  [G. intestinalis [When comparing ML trees results with phylogenetic networks, each assemblage shows important divergence with respect to AII. Such divergences in notable for A and B, and to a lesser extent, AI. Further, we observed reticulation signals and possible genetic exchange among assemblages, mainly between A and E, and for AI and AII . InconsissurRNA) . Other gssurRNA) , based issurRNA) ,26,33,64stinalis , considestinalis . Also, cstinalis ,68.TPI gene from different continents. Genetic differentiation is reported between assemblage A and populations in Asia, Australia, and America. Moderate genetic differentiation is also seen with comparison using assemblages B and E. The latter case shows the dispersion of the same population of G. intestinalis [G. intestinalis, and for investigating diversity, evolution, genetic structure and plausible genetic exchange events.Further, admixture was observed among assemblages with the contribution of E assemblage alleles to AI, AII and B assemblages . Mixturestinalis . We consG. intestinalis in the EuPathDB database: The Eukaryotic Pathogen Genomics Resource (GiardiaDB) (https://giardiadb.org/) , Pyrophosphate-fructose 6-phosphate 1-phosphotransferase alpha subunit (PFP-ALPHA1), Fructose-bisphosphate aldolase (FBA), Phosphoglycerate kinase (PGK) and enolase. We chose Acetyl-CoA synthetase (ACS) from the end of the alcoholic fermentation process, NADP-dependent malic enzyme (NADP-ME) from the TCA cycle and Serine palmitoyltransferase 2 (SPT) from lipid synthesis  , we searorganism . Other eorganism . Specifiynthesis .https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and were chosen considering: (1) primers were located in conserved regions for all assemblages, (2) the final product had a size between 300 and 700 bp, (3) selected markers were single-copy genes in the G. intestinalis genome, and (4) information on dimers, formation of forks, melting temperature (Tm), per cent GC, size of initiator, size of the amplified region, and specificity were available. Single-copy genes were needed to estimate intra- and inter-assemblage allelic diversity, avoiding bias by recombination between copies of the genes. The fourth criterion was verified using Basic Local Alignment Search Tool (BLASTn).For bioinformatic design of primers, CDS sequences of orthologous and synthetic genes for these enzymes were downloaded for AI (isolate WB), AII (isolate DH), B (isolates GS and GS_B) and E (isolate P15) assemblages of eight genes . We alighttps://orthomcl.org/, was queried. The corresponding group of orthologs was searched for each protein to identify any alternate AA sequences. In the second, we used the CD-HIT comparative analysis algorithm, a tool for grouping and comparing biological sequences [Two approaches were used to verify single-copy genes. First, the Ortholog Groups of Protein Sequences (OrthoMCL DB) database, available at equences . SequencG. intestinalis genomes available online. Among these loci, we included genes commonly used for typing of G. intestinalis, glutamate dehydrogenase (GDH) and triose phosphate isomerase (TPI). Genes were used to evaluate genetic diversity, recombination, typing capacity, and discrimination of G. intestinalis in whole-genome sequencing (WGS) data available in the public database of The European Nucleotide Archive (ENA)  .G. intestinalis. All other genomes were discarded  tool  to extraInitially, consensus sequences of each gene and their concatenation were aligned using the multiple sequence alignment programme MAFFT v7  . Subsequhttp://itol.embl.de) [Phylogenetic trees were constructed from alignment of consensus sequences from WGS data . Subsequembl.de) . Additioembl.de) , with thOnce assemblages were established for each consensus sequence with each loci, an allelic plot schema was constructed with concatenated sequences and by gene. Each assemblage was represented in a different colour, the AI in blue, the AII in red, the B in turquoise, the E in green, and sequences that did not correspond to any assemblage were left in black. The scheme compares assemblages assigned to each consensus sequence with each gene, so that assemblage agreement among loci is represented. This scheme represents alleles found, such that the number of colours in the allelic plot represents the number of clusters discriminated by each marker, as reported in other studies .To verify the existence of recombination events, we performed an additional analysis with the Recombination Detection Programme version 4 (RDP4) , using thttp://www.ub.edu/dnasp) were sequences aligned for each marker. Indices used included nucleotide diversity (Pi)—the average number of nucleotide differences per site between a pair of DNA sequences; Theta (per site) from the total number of mutations (Eta); numbers of polymorphic (segregating) sites (S); numbers of haplotypes (h); and haplotypic diversity (Hd). The latter index indicates the probability that two random haplotypes are different. Tajima D was calculated to determine if sequences evaluated reflected neutral variation or were involved in a selection process. This index indicates a balancing selection for positive values and a purifying selection for negative values [All sequences in WGS data were used to calculate diversity indices, for each gene and concatenated sequence. Indices were also calculated with sequences grouped by assemblage. Input data in the DnaSP v.5  programme values . The minhttp://www.ub.edu/dnasp).Separate statistics for genetic differentiation among assemblages for each gene and concatenated sequence were also calculated. Assemblages that could not be defined for some sequences in some genes were assessed, as was the concatenated sequence from the E assemblage. Genetic differences were estimated using statistics based on haplotypes (Hs), nucleotide sequences (Ks), and several others that reflect gene flow from nucleotide sequences, including Wright’s F (Fst), Delta ST, Gamma ST, and Nst. Average number of nucleotide differences in pairs (Kxy), nucleotide substitutions per site (Dxy), net nucleotide substitutions per site (Da), and gene flow from haplotypes (Gst) were then calculated. DnaSP v.5 software  was usedAll subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the National University of Colombia  and the ethics committee of the Universidad del Rosario (registered in Act No. 394 of the CEI-UR). This project was conducted under the contract number RGE131 of access to genetic resources granted by the “Ministerio de Medio ambiente y Desarrollo sostenible”. G. intestinalis was performed as described in Sánchez et al. [G. intestinalis positive samples were taken at random to evaluate markers assessed in silico. The number of samples by location were: 12 from Amazonas, six from Bolívar, three from Casanare and three from Córdoba. Convenience sampling of human faeces samples was performed in the departments of Amazonas, Bolívar, Casanare, and Córdoba in Colombia. The collection, extraction and typing of samples positive for z et al.  for sampz et al.  for sampG. intestinalis axenic cultures. PCR was performed in a final volume of 25 µL, containing 2 µL of quenched DNA, 12.5 µL of Go Taq Master Mix Green (Promega) (cat. No. M7122) . at a final concentration of 1X and primers at a concentration of 1 µM each. Primers used for PCR are shown in G. intestinalis positive stool sample was amplified using the above conditions. All PCR products were verified by observation on 2% agarose gel, stained with SYBR Safe, Thermo Fisher Scientific (cat No. S33102) . Each PCR product was purified with ExoSAP-IT®, Affymetrix ™ (cat. No. 15513687)  following the manufacturer’s recommendations. Both chains of each product were sequenced with the Sanger method. Sequences were edited in MEGA 7.0 [We amplified each locus from DNA extracted from MEGA 7.0  to extraTrees and phylogenetic networks were constructed with sequences obtained for each locus. Consensus sequences from the SRST2 tool output were concatenated with sequences obtained from human faeces of Colombian origin. This process used the same procedures described above for phylogenetic reconstructions. Diversity indices were also calculated by gene with the Dnasp v.5 programme .G. intestinalis. These loci can also be used as alternatives to and supplements for genomic studies of Giardia diversity. We demonstrate intra-taxa diversity and show both genetic structures for established assemblages, and admixture among populations due to genetic exchange, apparently by recombination among individuals. Analysis of proposed loci should extend to future studies that include genomic sequences of additional assemblages to define their diversity and population structure. Further, future studies might focus on increasing the number of samples to evaluate markers on a large scale and extend sampling and analysis to other hosts and water sources that may be sources/reservoirs of infection. Such investigations will help elucidate transmission dynamics of the pathogen. Finally, future studies should assess a broader set of stool samples using nested PCR to examine the usefulness of genetic markers developed in the present study.We highlight loci useful of an MLST scheme for typing of


Aedes albopictus in larval resource competition was originally proposed as the cause of displacements of Aedes aegypti in the USA. However, satyrization, a form of reproductive interference, was later invoked as an alternative or complementary mechanism for the observed displacements of Aedes aegypti. This study tests the hypotheses that Ae. albopictus female activity is not altered by the presence of accessory gland extracts from conspecific and heterospecific males, and Ae. albopictus females remain receptive to conspecific males even after receiving accessory gland (AG) extracts from Ae. aegypti males. We performed experiments with a control group , a group of females injected with accessory gland extracts of Ae. aegypti males and a group of females injected with accessory gland extracts of Ae. albopictus males and measured the locomotor activity and the ability of inseminated females to copulate with conspecific males. Females injected with conspecific and heterospecific extracts showed significant decreases in total and diurnal activity. Females injected with heterospecific extracts showed significant decreases in nocturnal activity. A total of 83% of females injected with heterospecific and 10% of females injected with conspecific extracts copulated with conspecific males. These results considered together with our previous report on effects of interspecific mating and cross-species injections of AG products on Ae. aegypti females, show consistent depressions of locomotor activities between species, but the loss of sexual receptivity only in Ae. aegypti. We propose that different male seminal fluid proteins control these activities.The superiority of Ae. albopictus females is not significantly altered by the presence of accessory gland (AG) extracts from conspecific and heterospecific males, and that Ae. albopictus females remain receptive to mating with conspecific males even after receiving AG of Ae. aegypti males. Virgin Ae. albopictus females were injected with saline (control group), AG extracts of Ae. aegypti males (aegMAG) or AG extracts of Ae. albopictus males . Locomotor activity was evaluated under 12 h of light and 12 h of darkness at 25 °C. All live Ae. albopictus females were subsequently exposed to conspecific males for 48 h, and their spermathecae were dissected for the presence of sperm. Females injected with aegMAG and albMAG showed significant decreases in total, diurnal and diurnal without lights-on Period activities. Females injected with aegMAG showed significant decreases in nocturnal and nocturnal without lights-off period activities. Females injected with albMAG showed significant decreases in lights-off activity. A total of 83% of Ae. albopictus females injected with aegMAG and 10% of females injected with albMAG were inseminated by conspecific males. These results, coupled with our previous paper on MAG and interspecific mating effects on female Ae. aegypti, demonstrate contrasting outcomes on locomotor activities and loss of sexual receptivity, both conspecific and heterospecific MAGs capable of sterilizing virgin Ae. aegypti, but only conspecific MAGs sterilizing Ae. albopictus, whereas locomotor activities were depressed in females of both species after heterospecific and conspecific injections or treatments.This study tests the hypotheses that the locomotor activity of  Aedes aegypti (L.) and Aedes albopictus (Skuse) are arguably the two most important invasive species of mosquito vectors of public health concern. Although both belong to a common subgenus, Stegomyia, and share many behaviors, such as occupancy of containers during immature stages and diurnal feeding and reproductive activities as adults, the two species evolved independently in different regions of the world, and thus, did not meet one another until one or the other’s invasive range expansions led to interspecific encounters [counters .Ae. aegypti have been long known [Ae. aegypti from continental Africa into the Western Hemisphere in the 15–17th centuries was facilitated by intercontinental shipping and the slave trade [The African origins of ng known , but onlng known . The diave trade . InvasioAe. albopictus, by contrast, have mostly occurred in the 20th century, from its native range in tropical and temperate Asia [Ae. aegypti [The invasions of ate Asia ,6. Earli aegypti .Ae. aegypti and Ae. albopictus populations have met in invasive-invasive or invasive-native ranges: Competitive displacement of Ae. aegypti or habitat segregation between the two species [Ae. aegypti by Ae. albopictus in the SE USA between 1986–1994 was relatively complete, except for large, urban redoubts of Ae. aegypti, and persistent through at least two decades in Florida [Ae. albopictus about the same time this species became established in the southern USA [Two outcomes, not mutually exclusive, have been reported where  species . Displac species  and Berm species . The comhern USA .Ae. aegypti typically predominates in more urban environments and Ae. albopictus in more vegetated habitats, such as suburbs with trees [Ae. albopictus in larval, resource competition was originally proposed as the cause of competitive displacements of Ae. aegypti in the USA [Ae. aegypti [Ae. albopictus male with an Ae. aegypti female sterilizes the female recipient, which asymmetry is shown by population models to lead to the possible rapid extinction of the inferior competitor species [Ae. aegypti exposed to male Ae. albopictus is believed to facilitate the co-existence of these two species [Where the two species coexist in sympatry, such as in the southern USA and Brazil, th trees . The sup the USA . However the USA , was inv aegypti . Althoug aegypti , only th species . The rap species ,16,17.As products of the male accessory glands (MAG) of mosquitoes transferred during mating are known to prevent further matings by inseminated females , it is nAe. aegypti females injected with MAG products from Ae. albopictus did not differ from saline-injected controls in total locomotor activity during the diel cycle, but did differ in lights-on responses, despite the complete sterilizing effects of the MAGs from Ae. albopictus on Ae. aegypti females. Recognizing the potentials of such cross-insemination experiments to help decipher the complex roles of MAGs in controlling female behaviors [Ae. aegypti led to decreased locomotor activities [Ae. albopictus should lead to decreases in locomotor activities of these experimental females. However, based on the previously demonstrated unidirectional sterilizing effect of MAGs in interspecific crosses between these species [Ae. albopictus will remain receptive to future conspecific mating after their injection of MAGs from Ae. aegypti.In the first report of the effects of interspecific matings on mosquito activity rhythms, Lima-Camara et al.  describeehaviors , and bastivities ; we hypo species , we hypoAe. aegypti and Ae. albopictus eggs from Rio de Janeiro were obtained from colonies at Laboratório de Mosquitos Transmissores de Hematozoários, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, state of Rio de Janeiro (RJ), Brazil, as described in Lima-Camara et al. [®) and were maintained in a room with a photoperiod of light/dark (LD) 12:12, at 25 °C and 70% relative humidity. Pupae of both species were individually placed in small glass containers with 5 mL of tap water to ensure virgin males and females after emergence [Both a et al. . After emergence .Ae. aegypti and Ae. albopictus were dissected under a stereoscopic microscope to remove their accessory glands (Ags) [g [Twenty-five 5–7 day-old virgin male ds (Ags) . Twenty-(Ags) [g .Ae. albopictus females were separated into three groups of 64 females each. Females from the first group (control group) were individually injected by intrathoracic inoculation with 0.28 µL of saline; whereas, the second and third groups (MAG groups) were intrathoracically inoculated with 0.28 µL of male accessory gland extract (MAG) solution of Ae. aegypti and 0.28 µL of MAG solution of Ae. albopictus, respectively, using a Nanoject microinjector (Drummond Scientific). Non-injected virgin females of Ae. albopictus were not included as controls in the present study. Results of locomotor activity of virgin and naturally inseminated females of Ae. albopictus were published previously [Six to seven day-old virgin eviously . The voleviously ,23. We heviously , which wAe. albopictus female (control and both MAG groups) was individually placed in a cylindrical glass tube (1 cm × 10 cm) with a cotton plug soaked in 10% sucrose at one of the extremities for feeding. The glass tubes were sealed with Parafilm® and then placed in a larger version of the Drosophila Activity Monitor  as described in Lima-Camara et al. [Ae. albopictus female, the total locomotor activity during 30-min intervals was continuously recorded for six consecutive days after inoculation, using the DAM System Data Collection program . Two replicates were conducted, and only control and MAG-injected females that lived until the fourth day of the locomotor activity recordings (five days after inoculation) were considered for analysis.Each injected a et al. ,23. All Ae. albopictus females of each group were counted and transferred to three different cages (17 cm × 17 cm) containing conspecific virgin males in a ratio of 1 female: 2 males. After 48 h, all three spermathecae were dissected from live females for detection of Ae. albopictus sperm under a compound microscope at 100 × magnification.At the end of the locomotor activity experiment, all live w) as an estimate of the central tendency of activity during each time interval for exploratory analysis and figures [Ae. albopictus injected with saline versus females of Ae. albopictus injected either with Ae. aegypti male AG (aegMAG) or Ae. albopictus male AG , we calculated seven indices [For statistical analysis, we transformed the locomotor activity values of all mosquitoes to log (N + 1), in order to avoid the influence of very high or very low values frequently observed in the activity of mosquitoes and to accommodate the zero values observed in the data series . We calc figures ,23,28,29 indices ,23: (i) Ae. albopictus locomotor activity. Because we have repeated measures for each individual mosquito across four days of continuous experiments, we opted to use generalized linear mixed models (GLMM) with a Gaussian distribution [Ae. aegypti MAG; and Ae. albopictus MAG). We also included “block” (a dichotomous variable for experiments 1 and 2) and a two-way interaction between status and block. Assumptions of homoscedasticity and normality of residuals were checked via graphical evaluation [We were interested in testing the effect of the different accessory gland origins on female ribution , using tribution . We creaaluation . F-testsaluation . All anaaluation  and Rstualuation .Ae. albopictus females showed a bimodal rhythm, with peaks at lights-on and lights-off ; diurnal activity ; diurnal activity without lights-on ; and lights-on activity . We found borderline significance for nocturnal activity . Status was not significant for both nocturnal activity without lights-off  and lights-off activity   .1, 201 = 6.9824; p < 0.01). However, in both blocks, the lights-on peak of the control group was higher  than the females injected with conspecific MAG  and heterospecific MAG . No significant interactions between block and status were observed in any of GLMMs (p > 0.05)  .Ae. aegypti and with Ae. albopictus MAG was significantly lower  than the control group (0.264 ± 0.004). The same pattern of higher locomotor activity in the control group was observed for the diurnal activity (0.270 ± (0.007) vs. 0.199 ± (0.006) and 0.201 ± (0.006), respectively for control, aegMAG and albMAG), diurnal activity without lights-on (0.254 ± (0.007) vs. 0.186 ± (0.006) and 0.189 ± (0.006)) and lights-on activity (0.634 ± (0.043) vs. 0.503 ± (0.036) and 0.483 ± (0.037)) ) . No stat(0.037)) .Ae. albopictus injected with aegMAG and albMAG showed a significant decrease in the total activity group in relation to control females . Both MAG injected females also showed significant decreases in the diurnal activity  and in the diurnal activity without lights-on  groups in relation to control females , even when we did not consider the lights-off in the nocturnal activity without lights-off group (p < 0.05). For the lights-on activity group, a significant decrease was observed just in Ae. albopictus females injected with albMAG (p < 0.01), and no significant difference was reported for the lights-off activity group (p > 0.05)  .Ae. albopictus females that were exposed to conspecific males for 48 h at the end of the locomotor activity experiments. Most females of Ae. albopictus injected with saline had positive spermathecae (87%). A similar proportion of positive spermathecae was observed in females injected with heterospecific MAG (83%), that is, females of Ae. albopictus copulated with their conspecific males, even though they were injected with AG from Ae. aegypti males. However, only 10% of Ae. albopictus females injected with conspecific MAG had positive spermathecae  of an AG into each Ae. albopictus female. This dose is more than sufficient for females to respond as if they have been inseminated, since each male apparently could sterilize at least 64 females [As observed by Klowden ,38 and Ga et al.  evaluatetivities . These rtivities , the fem females .Ae. aegypti females are more likely to be inseminated interspecifically than Ae. albopictus females, supporting the competitive advantage of the former species [Ae. aegypti females inseminated by Ae. albopictus males produced more eggs than Ae. albopictus females inseminated by Ae. aegypti males, although no eggs were viable. Moreover, Bargielowski et al. [Ae. albopictus females and Ae. aegypti males in cages are significantly less common than between Ae. aegypti females and Ae. albopictus males. In nature, the frequencies of cross-insemination between Ae. aegypti and Ae. albopictus were estimated in collections from different countries, such as the United States [Ae. aegypti females were more likely to be inseminated interspecifically than Ae. albopictus females under uncontrolled conditions in the field [Ae. aegypti by Ae. albopictus may occur without evidence of successful insemination [Ae. aegypti females exposed to Ae. albopictus males may receive MAGs that inhibit subsequent conspecific mating even though their spermathecae contain no heterospecific sperm [Ae. aegypti females and Ae. albopictus males from several Brazilian and North American cities. These authors showed that Ae. albopictus male origin might be a key driver of their satyrization potential upon Ae. aegypti females, which might explain why the displacement of the former species was pronounced in North America, but undetected in Brazil [Ae. aegypti and Ae. albopictus from different origins on the behaviors and activities of inseminated females. As the insemination status of a female  is one of the key drivers of satyrization, our results show that this ecological interaction might have very complex effects on the life history of both species. Because of a higher proportion of Ae. albopictus females injected with heterospecific and a lower proportion of females injected with conspecific extracts copulated with conspecific males, satyrization plays an important role that shapes the distribution of Ae. aegypti and Ae. albopictus [Ae. albopictus females (the former impacting fewer activity indexes than the later) shows that important biological traits and behaviors might be modulated in a very complex manner and deserve further studies [2 and chemical compounds, to examine if the diurnal locomotor activity would be impacted by the presence of conspecific and/or heterospecific MAGs. Furthermore, other experimental conditions could be manipulated to produce further results, such as using different lighting conditions, photoperiods, temperature and/or humidity, etc.Previous laboratory experiments have indicated that  species ,40,41. Ni et al.  observedd States ,15, Venehe field ,15. Moremination . Thus, Aic sperm . Honórioic sperm  conducten Brazil . These fbopictus ,40,41. M studies . Some ofAe. aegypti and Ae. albopictus brought about by mosquito invasions, and their capacities to cross-inseminate, could play a role in helping to answer these questions. For the first time, we demonstrated that both MAGs of Ae. aegypti and Ae. albopictus decrease the locomotor activity of Ae. albopictus females. Moreover, this study confirms that MAG of Ae. aegypti does not make Ae. albopictus females refractory to mating with conspecific males. Thus, we believe that further studies to investigate the effects of cross-insemination, would illuminate finer details of reproductive interference between these species, as well as the control of female behaviors by male accessory gland products.Understanding how MAGs control female behaviors are important for vector physiology/molecular biology, because of their potential for contributing to vector and disease control. The fortuitous interspecific encounters of


The reproducibility of gene expression measured by RNA sequencing (RNA-Seq) is dependent on the sequencing depth. While unmapped or non-exonic reads do not contribute to gene expression quantification, duplicate reads contribute to the quantification but are not informative for reproducibility. We show that mapped, exonic, non-duplicate (MEND) reads are a useful measure of reproducibility of RNA-Seq datasets used for gene expression analysis.In bulk RNA-Seq datasets from 2,179 tumors in 48 cohorts, the fraction of reads that contribute to the reproducibility of gene expression analysis varies greatly. Unmapped reads constitute 1–77% of all reads ; duplicate reads constitute 3–100% of mapped reads ; and non-exonic reads constitute 4–97% of mapped, non-duplicate reads . MEND reads constitute 0–79% of total reads .Because not all reads in an RNA-Seq dataset are informative for reproducibility of gene expression measurements and the fraction of reads that are informative varies, we propose reporting a dataset's sequencing depth in MEND reads, which definitively inform the reproducibility of gene expression, rather than total, mapped, or exonic reads. We provide a Docker image containing (i) the existing required tools  and (ii) a custom script to calculate MEND reads from RNA-Seq data files. We recommend that all RNA-Seq gene expression experiments, sensitivity studies, and depth recommendations use MEND units for sequencing depth. Assessing the reproducibility of RNA sequencing (RNA-Seq) gene expression measurements has been a priority since the development of the assay ,2. The aHowever, RNA-Seq data are not homogenous. Of the tens of millions of sequences (reads) in a typical RNA-Seq dataset, some reads cannot be mapped back to the reference transcriptome. Others map to genome regions outside of exons or have been duplicated by PCR during the library construction process or sequencing. Nearly all methods for quantifying gene expression in bulk RNA-Seq data count reads that align to exons in a gene; thus, unmapped and non-exonic reads do not contribute to measurements and are consequently uninformative ,6. ThereDuplicate reads may be due to either highly abundant transcripts or technical artifacts. The process of preparing RNA-Seq libraries involves PCR amplification. This step can generate duplicated identical or nearly identical reads, especially if the input amount is low. While the original read represents gene expression in the experimental system, the artifactual duplicate reads do not. However, duplicate reads are also generated by very highly expressed genes because each gene has a finite number of unique read sequences that can be generated from it . PreviouHere we analyze 2,179 bulk, paired-end, polyA-selected RNA-Seq datasets to characterize the read types present in the datasets and evaluate what fraction of each dataset is unequivocally relevant to the reproducibility of gene expression measurements.RRID:SCR_000468)  of 25% [16–37%]. There are 330 datasets that have a fraction of non-exonic reads >50%. Therefore, the percentage of duplicate reads or non-exonic reads among all mapped, non-duplicate reads is not directly informative about the dataset's gene expression measurements.We recorded the time required to run our most recent pipelines on computers with 64 GB of memory and 12 VCPU. The 382 RNA-Seq datasets examined were obtained from SRA and EGA and had reads that were 100 bases in length. For datasets within 10 million total reads of the median total read size in our survey of 2,179 datasets , the median duration is 290 minutes (4.8 hours) for the expression pipeline and 143 minutes (2.4 hours) for the MEND pipeline.Researchers wish to know that their data are sufficient for making reproducible measurements. For RNA-Seq experiments, they often wish to know whether the dataset is sufficient for reproducibly measuring expression of known genes. Here we show that the fraction of relevant content of an RNA-Seq dataset (percent of MEND reads) varies substantially within and between cohorts, and thus should be measured in each dataset.This work was performed using data from pediatric tumor datasets as part of the development of our comparative RNA-Seq assay for patients with pediatric cancer ,16. BecaPrevious studies have shown that paired-end libraries contain relatively few artifactual duplicate reads ,9. HowevThere are several reasons why a survey of this breadth has not been previously performed. Obtaining and processing clinical datasets from multiple sources is an intensive effort . Access Measuring the number of MEND reads in a dataset is specific to the alignment parameters and gene model. We use Gencode v23, which is inclusive, defining >60,000 genes. By default, the aligner we use, STAR, defines reads that map to as many as 20 positions as mappable. If we changed our pipeline, asking STAR to exclude reads mapping to >2 positions and using a more conservative gene model with 30,000 genes, the same dataset would have fewer MEND reads owing to the loss of reads that map to too many places or map only to regions newly defined as non-exonic.In addition to being sensitive to reference files, MEND counts are slow to compute, increasing the duration of our RNA-Seq pipeline by 50%. It would be valuable to create a faster utility that takes raw reads rather than aligned reads as input. The reference-dependence could also be addressed by including a default set of references, with support for alternate ones.Researchers planning RNA-Seq experiments look for guidance on how much sequencing their experiment requires. For comparing gene expression measurements between datasets, ENCODE recommends a minimum of 30 million mapped reads . HoweverOn the basis of these results, we recommend that (i) publications reporting the results of an RNA-Seq study with gene expression applications should report the depth of sequence as the number of MEND reads present in each dataset; (ii) sensitivity studies should include read type fractions and report on the relationship between MEND reads and the measured outcome; and (iii) sequencing depth recommendations should be based on MEND reads rather than total or total mapped reads.Project name: MEND QChttps://github.com/UCSC-Treehouse/mend_qcProject home page: Operating system(s): Platform independentProgramming language: Bash and ROther requirements: DockerLicense: MITRRID:SCR_020934GigaScience GigaDB repository [Accession numbers, clinical data, and read counts for 2,179 publicly available, bulk RNA-Seq datasets are in pository .Supplementary Table S1. Accession numbers, clinical data, and read counts for 2,179 publicly available, bulk RNA-Seq datasets. The accession numbers are the definitive sources; the DOI links to citations are provided for convenience.Supplementary Table S2. Sequence data repositories, URLs, and abbreviations.Supplementary Table S3. Cohort names, code, repositories, and dataset counts.CDS: coding sequence; DOI: digital object identifier; EGA: European Genome-Phenome Archive; GEUVADIS: Genetic European Variation in Disease; GTEx: Genotype-Tissue Expression; IQR: interquartile range; med.: median; MEND: Mapped, Exonic, Non-Duplicate; QC: quality control; RNA-Seq: RNA sequencing; SRA: Sequence Read Archive; UTR: untranslated region; VCPU: virtual central processing unit.The UCSC Institutional Review Board (IRB) has determined that our use of previously released sequence data does not constitute human subject research, and therefore does not require an IRB review.The authors declare that they have no competing interests.This study was funded by American Association for Cancer Research NextGen Grant for Transformative Cancer Research Award (O.M.V.), Emily Beazley Kures for Kids Fund St. Baldrick’s Consortium Grant, Alex's Lemonade Stand Foundation for Childhood Cancer Research, Unravel Pediatric Cancer, Team G Childhood Cancer Foundation, California Initiative to Advance Precision Medicine, Live for Others Foundation, The Schmidt Futures Foundation (D.H.). D.H. is a Howard Hughes Medical Institute Investigator. O.M.V. holds the Colligan Presidential Chair in Pediatric Genomics.Analysis and manuscript authorship: H.C.B., J.M.R., M.A.C., L.T.M., D.K.A.T.MEND pipeline development and integration and manuscript review: R.C., D.L.L., J.V.Data access, data processing, and manuscript review: K.L., E.T.K., L.S., J.P., A.G.L., and I.B.Funding, scientific oversight, and manuscript review: D.H., S.R.S., and O.M.V.giab011_GIGA-D-20-00263_Original_SubmissionClick here for additional data file.giab011_GIGA-D-20-00263_Revision_1Click here for additional data file.giab011_GIGA-D-20-00263_Revision_2Click here for additional data file.giab011_Response_to_Reviewer_Comments_Original_SubmissionClick here for additional data file.giab011_Response_to_Reviewer_Comments_Revision_1Click here for additional data file.giab011_Reviewer_1_Report_Original_SubmissionAlexandra Sexton Oates -- 9/27/2020 ReviewedClick here for additional data file.giab011_Reviewer_2_Report_Original_SubmissionMark Ziemann, PhD -- 9/28/2020 ReviewedClick here for additional data file.giab011_Reviewer_2_Report_Revision_1Mark Ziemann, PhD -- 1/6/2021 ReviewedClick here for additional data file.giab011_Supplemental_TablesClick here for additional data file.


Their most severe side effects are hepatotoxicity, nephrotoxicity, and myelotoxicity. Whereas triazoles exhibit the most significant drug–drug interactions, echinocandins exhibit almost none. The antifungal resistance may be developed across most pathogens and includes drug target overexpression, efflux pump activation, and amino acid substitution. The experimental antifungal drugs in clinical trials are also reviewed. Siderophores in the Trojan horse approach or the application of siderophore biosynthesis enzyme inhibitors represent the most promising emerging antifungal therapies.We reviewed the licensed antifungal drugs and summarized their mechanisms of action, pharmacological profiles, and susceptibility to specific fungi. Approved antimycotics inhibit 1,3-β- Invasive fungal infections represent a global problem resulting in 1.7 million deaths every year . They arCandida genera [Aspergillus fumigatus and A. flavus [Aspergillus spp. [d-glucans located in the fungal cell wall. Echinocandins are fungicidal and fungistatic against Candida and Aspergillus spp., respectively [Currently, four antifungal drug classes are used by clinicians and veterinarians for systemic treatment . These ca genera  and Aspe. flavus . Second,lus spp. . Echinocectively . Finallyectively . The oveectively . The Worectively .In this work, we reviewed the important approved and selected experimental antifungal drugs. Immunomodulatory therapies ,16, coveSince the 1950s, more than 200 polyenes with antifungal activity have been discovered; however, amphotericin B remains the single polyene drug of choice in the treatment of invasive fungal infections . CaspofuStreptomyces nodosus. It has two mechanisms of action  and electrolyte glucose are released. The rapid depletion of intracellular ions results in fungal cell death. Second, AMB induces the accumulation of reactive oxygen species (ROS), resulting in DNA, protein, mitochondrial, and membrane damage [Amphotericin B is a cyclic heptaene A producef action . First, e damage . The stre damage .5-Flucytosine B is a syThe mechanism of action of triazoles D–H is ba0 (Aspergillus nidulans), caspofungin from pneumocandin B0 (Glarea lozoyensis), and micafungin from pneumocandin A0 (Coleophoma empetri). Echinocandins are targeted against 1,3-β-d-glucan synthase  and feces (43%) as the unchanged drug. The inherent limitations of AMB deoxycholate application consist in its infusion- and dose-related toxicity. The infusion-related toxicity arises from the induction of pro-inflammatory cytokines and chemokines such as interleukin 1β, tumor necrosis factor α, monocyte chemotactic protein 1, and macrophage inflammatory protein 1β. On the other hand, high doses of AMB cause nephrotoxicity due to the non-selective disruption of renal cells. To reduce these undesirable effects, AMB deoxycholate has been incorporated into lipidic formulations still retaining its fungicidal activity. Three lipidic formulations are commercially available: AMB lipid complex (ABLC), liposomal AMB (LAMB), and AMB colloidal dispersion (ABCD) .In contrast, 5-flucytosine, as a small hydrophilic molecule, is absorbed rapidly, and its bioavailability reaches almost 90%. Liver enzymes metabolize 5-FC minimally. It is eliminated exclusively via glomerular filtration with excellent antifungal activity in the bladder, and its plasma clearance is as fast as creatinine clearance. 5-FC is dosed frequently because of its half-life, which is up to four hours. Co-administration of 5-FC with the cytarabine (an effective drug for the treatment of acute myeloid leukemia ) competiThe pharmacological profiles of triazoles vary with their molecular weight, solubility, and protein binding. Selected pharmacological data are presented in On the other hand, genetic polymorphisms of CYP2C19 and CYP3A4 affect the metabolism of voriconazole. Generally, triazoles are well tolerated. The most common serious side effect, hepatotoxicity, occurs most often with VOR in 31% cases). Whereas ISV shortens the QT interval (the time between the start of the ventricular depolarization and the end of the ventricular repolarization), the rest of triazoles act in the opposite way. Unfortunately, triazoles have many potential drug–drug interactions due to their affinity for CYP450 isoenzymes [% cases. N-acetylation into two inactive metabolites. In contrast, anidulafungin has the longest elimination half-life. Its biotransformation results in an ineffective open-ring peptide. Finally, the metabolism of micafungin forms three metabolites by arylsulphatase, catechol-O-methyltransferase (COMT), and CYP3A side-chain hydroxylation  is a must for both the optimal treatment and the detection of possible antifungal resistance. AST is performed by both reference and commercial methods based on broth microdilution and agar-based (or disk diffusion) assays. These assays are standardized according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST)  and the Aspergillus and Candida species covering the period 2007–2019 from EUCAST only [To distinguish if the microorganism isolates are resistant to antifungal agents or not, EUCAST and CLSI have set clinical breakpoints expressed as the minimal inhibitory concentration (MIC), indicating the minimum drug concentration that inhibits fungal growth. According to the 2019 EUCAST definition , a microAST only .Aspergillus [Candida [Fusarium, and Scedosporium [A. terreus and A. niger. Furthermore, VOR should be used in both first-line and salvage therapy in immunocompromised patients suffering from fusariosis and scedosporiosis. In the case of candidemia and invasive candidiasis, the use of all three echinocandins (including caspofungin) is strongly recommended as an initial targeted treatment in the adult population.The European Society for Clinical Microbiology and Infectious Diseases (ESCMID) published guidelines for the management of ergillus , Candida[Candida , Fusariuosporium  infectioC. krusei [C. glabrata [Aspergillus species [A. fumigatus to triazole fungicides may explain their resistance in azole-naïve patients [Microbial resistance to antifungal drugs is a result of multiple factors and emerges by a series of molecular mechanisms . Whereas. krusei , C. glabglabrata , and Asp species , the acq species . Environpatients . Additiopatients  and humapatients .ERG enzymes [ERG3, ERG5, ERG11), gene deletion (ERG11), and harbored mutations  lead to the decreased AMB sensitivity in Candida species [C. tropicalis isolates showed a thickened cell wall due to the increased content of 1,3-β-d-glucans [A. terreus [Amphotericin B susceptibility depends on the ergosterol content in the fungal cell membrane. Ergosterol biosynthesis is regulated by 25 known  enzymes ; their a species . Further-glucans . On the  terreus .Candida spp. have emerged as a consequence of alterations in the FCY2, FCY1, and FUR1 genes responsible for 5-FC uptake and its conversion, respectively. Additionally, the susceptibility of C. glabrata to 5-FC increased after deletion of the CgFPS1 and CgFPS2 homologue encoding plasma membrane aquaglyceroporins that are required for osmotic stress resistance [A. fumigatus at pH 5. Furthermore, FCYB gene expression is repressed by the cytosine-cytosine-adenosine-adenosine-thymidine binding complex (CBC) and pH-response transcriptional factor PacC at pH 7, confirming intrinsic resistance [5-Flucytosine is known for its rapid development of resistance; therefore, it is used only in combination therapy with AMB and triazoles. Primary and secondary resistance in clinically relevant sistance . It has sistance .ERG11/CYP51A/CYP51B genes included in ergosterol biosynthesis. The overexpression of ERG11 is due to gain-of-function mutations in Upc2 in C. albicans. Furthermore, another transcriptional factor SrbA is required for azole resistance in A. fumigatus. The second mechanism of acquired azole resistance arises from the upregulation of ABC (adenosine triphosphate binding cassette) transporters as well as major facilitators responsible for higher drug efflux [C. albicans, and AtrF, Mdr3, and Mdr4 in A. fumigatus, respectively. Furthermore, intrinsic resistance to triazoles is often caused by Candida biofilm formation [The first mechanism of azole resistance involves amino acid substitutions near the heme binding site of 14-α-demethylase or overexpression of the g efflux . Cdr1, Cormation .FKS1 and FKS2 subunits of glucan synthase. The most frequent amino acid changes encompass Ser641 and Ser645 in C. albicans and Ser629, Phe659, and Ser663 in C. glabrata. Moreover, the elevated MIC values in C. parapsilosis are due to naturally occurring polymorphisms linked with the proline-to-alanine change in FKS1. FKS2-dependent resistance in C. glabrata can be reversed by treatment with the calcineurin inhibitor tacrolimus. Treatment with echinocandins is connected with higher biosynthesis of β-d-glucans and chitin in C. glabrata and C. parapsilosis. The higher molecular weight cell wall content may play a role in echinocandin resistance [The basis of echinocandin resistance consists of amino acid substitutions in the sistance . In addisistance .Fusarium spp.  are resocandins . The Scetion 695 . Mucormyreatment .The research in antifungal drug development focuses on both old and new fungal targets. In addition, antifungal susceptibility testing and ongoing clinical trials have modified already licensed antifungals, i.e., drugs with originally different purposes or novel synthesized substances .ERG11 and ERG24, respectively. Aminopiperidines show antifungal activity against fluconazole-resistant Candida spp. and A. fumigatus [C. albicans [In ergosterol biosynthesis, modified tetrazoles  or synthetically with antibiotics . Next, ixylation . Last, cxylation . Phosphoxylation ,97, eithAcremonium spp. and exhibits significant activity against Aspergillus spp. The mechanism of action of this compound is not known. Still, its selectivity for fungal cells derives from the fact that it is taken up by the fungal siderophore transporter Sit1, which is absent from mammalian cells [It is worth mentioning that specialized mammalian cells secrete lipocalins preventing iron hijack by catecholate-type siderophore sequestration . VL-2397an cells . Besidesan cells , and thean cells .


Synovial sarcoma (SS) is one of soft tissue sarcomas (STS), characterized by t chromosomal translocation. Clinical diagnosis of SS in groin is difficult owing to rarity and various manifestations. We reported a rare and interesting case of inguinal biphasic SS with initial impression of a vascular lesion.We reported a 72-year-old man who presented with a right inguinal progressively enlarged mass for 5 years. The mass became ulcerative with active bleeding after blunt trauma. Computed tomography (CT) showed a large heterogeneous tumor with focal vivid enhanced area, and an inguinal vascular lesion was impressed. Angiography revealed faint tumor stain without contrast extravasation or aneurysm lesion. Surgical resection of the tumor was performed. Final pathology and fluorescence in situ examination (FISH) demonstrated biphasic SS with SS18 gene rearrangement. No evidence of recurrence after one-year follow-up.SS constitutes 5%–10% of STS. The most primary sites are extremities (68.7%) and trunk (15.7%). Treatment options for SS include surgery, chemotherapy, radiotherapy, targeted therapy, and novel therapies. In our case, the initial impressions of large inguinal tumor were hematoma or pseudoaneurysm. Preoperative angiography assisted in differentiating the lesion, evaluating tumor vessels, and preventing bleeding by embolization. Our patient recovered satisfactorily after surgical excision without major complications.Primary inguinal SS is an extremely rare tumor which can present as a vascular lesion. Diagnosis is confirmed by pathology and FISH exam. Further studies are required to determine the manifestation and management of SS. SS can occur at any age, but it commonly occurs in teenagers and young adults aged 15–40 years . From a Numerous unusual locations of SS have been reported and confirmed by using molecular and immunohistochemistry techniques Cases of SS primarily originating in inguinal areas are rare in English language literature . Many le26 uL; hemoglobin level: 8.9 g/dL; and hematocrit level: 26.2%). Ultrasound examination revealed a heterogeneous mass, diameter up to 15 cm, with abundant blood supply, as assessed by Doppler mode. Computed tomography (CT) revealed a well-circumscribed right inguinal heterogeneous tumor, measuring up to 15 cm, with a vivid enhanced area, implying the possibility of vascular lesion  and trunk (15.7%) . The cel8.7% and Treatment options for SS include surgery, chemotherapy, radiotherapy, targeted therapy, and novel therapies. Wide-margin surgical resection remains the essential treatment for localized tumors. However, no consensus exists regarding the systemic treatment for SS. Recent studies have adequately discussed insight into neoadjuvant chemotherapy. Le Cesne et al. pooled two phase III clinical trials and revealed that adjuvant chemotherapy provided no overall survival or relapse-free survival benefits in completely resected SS . A phaseFour studies reporting inguinal SS in the literature were reviewed . Hiede eIn our case, the initial impressions of large inguinal tumor were hematoma or pseudoaneurysm. Preoperative angiography assisted in differentiating the lesion, evaluating tumor vessels, and preventing bleeding by embolization. We did not performed embolization in our patient due to high risk of nontargeted embolization to abdominal skin. Our patient recovered satisfactorily after surgical excision without major complications. We suggested adjuvant chemoradiotherapy, but the patient refused due to personal reasons. There was no evidence of recurrence for 12 months follow-up.4In conclusion, this is the first case report of biphasic SS in inguinal region. Various manifestations and rarity of SS lead us have the first impression of vascular lesion. Our study demonstrated diagnostic image, surgical views, and treatment options for SS. Complete surgical resection is main treatment. Additionally, we reviewed and summarized reported cases of inguinal SS. Additional case reports and studies are warranted to achieve a clearer understanding of diagnoses and treatments of SS in groin.The authors report no declarations of interest.This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.The research related to human use has been complied with all the relevant national regulations, institutional policies, and has been approved by the Research Ethical Committee of Taipei Medical University Hospital (Case number: N201910051).Written informed consent was obtained from the patient for publication of this case report and accompanying images.Data collection: Wei-Chieh Chen and Po-Chien Wu.Writing the paper: Po-Chien Wu and Ting-En Tai.Editing the paper: Wei-Chieh Chen and Chun-Yu Lin.1.Name of the registry: Research Ethical Committee of Taipei Medical University Hospital.2.Unique identifying number or registration ID: N201910051.Ting-En Tai.Not commissioned, externally peer-reviewed.


E. coli and H. pylori require nickel for [NiFe]-hydrogenases. H. pylori also requires nickel for urease. At high concentrations nickel can be toxic to the cell, therefore, nickel concentrations are tightly regulated. Metalloregulators help to maintain nickel concentration in the cell by regulating the expression of the genes associated with nickel import and export. Nickel import into the cell, delivery of nickel to target proteins, and export of nickel from the cell is a very intricate and well-choreographed process. The delivery of nickel to [NiFe]-hydrogenase and urease is complex and involves several chaperones and accessory proteins. A combination of biochemical, crystallographic, and spectroscopic techniques has been utilized to study the structures of these proteins, as well as protein-protein interactions resulting in an expansion of our knowledge regarding how these proteins sense and bind nickel. In this review, recent advances in the field will be discussed, focusing on the metal site structures of nickel bound to metalloregulators and chaperones.Nickel is essential for the survival of many pathogenic bacteria.  The nic2.1.E. coli, NikR controls the expression of the nik operon that encodes the E. coli Ni(II) importer NikABCDE . It. ItE. co changes .E. coli NikR DNA bound crystal structure shows that there are two distinct metal-binding sites, four “high affinity” metal binding sites located in the MBD and two “low affinity” metal-binding sites located near the interface of the C-terminus and the DBD (E. coli Ni(II) NikR bound to DNA shows that potassium ions are coordinated in a bidentate manner by Glu30 and Asp34, and by the backbone carbonyl oxygens of Ile116, Gln118, and Val121 /monomer with excess Ni(II) ions -hydrogenase. These studies are discussed in this section. ds and the pH at which the Kd was determined, the coordination number (CN #), and the ligands used to coordinate the metal ion in HypA, HypB, SlyD, and UreE.The nickel chaperones HypA, HypB, SlyD, and UreE have been characterized individually and in complexes from various organisms including 3.1.H. pylori it is involved in nickel delivery to urease -hydrogenases -hydrogenase and in the case of o urease . Homologogenases ,93. HypAogenases ,95, howeogenases . HypA progenases –100. Theogenases –100. Theogenases ,98,101. g-fura 2 . Mutagenactivity ,97–101 wactivity .H. pylori HypA binds Ni(II) with a Kd of ~1 μM at pH 7.2 and this binding is weaker at pH 6.3 . ITtructure  [97]. Innd His95 ,B. Mutatt pH 6.3  [97].H. pylori mutant strains determined that the dynamic zinc site in HypA is important for the acid viability of the bacterium, urease activity, and [NiFe]-hydrogenase activity -hydrogenase activity -hydrogenase activity and only had 2% of the wild-type urease activity -hydrogenase activity [Nickel binding to the MHE motif is also important for both [NiFe]-hydrogenase and urease activity ,95,105. activity . FurtherE. coli, H. pylori, and T. kodakarensis -hydrogenase maturation . S. SH. pylMethanocaldococcus jannaschii (M. jannaschii) with Zn(II)) and (GTPγS) bound -hydrogenase activity -hydrogenase activity  [A. fulgidus. A. fulgidus HypB is a monomer in the apo and the GDP-bound forms but the GTP bound form of the protein is a dimer [E. coli) mutant A. fulgidus hypB though the protein could still bind GTP. It was shown that a K22A mutant HypB protein could not recover [NiFe]-hydrogenase activity in a ΔhypB E. coli strain -hydrogenase activity in vivo . Ur. Ur28].  ~9.6 μM . XAS stu ~9.6 μM . Most of ~9.6 μM . H. pyloasteurii ) lack thectively ,140. Theectively . S. pastof 35 μM . The nicof 35 μM ,143. Stuo urease ,145. A Hresidues . Like thresidues . Urease residues .ds of 47 μM and 1.45 μM -hydrogenase and urease is a highly choreographed process that involves several accessory proteins. Protein–protein interactions between the metallochaperones HypA, HypB and SlyD have been observed experimentally, however, a ternary complex has not been detected . Work doE. coli [H. pylori [E. coli SlyD also forms a complex with a Strep-tagll variant of the large subunit of [NiFe]-hydrogenase 3, HycE . I. IH. pyl protein ,103. AddK. aerogenes the urease accessory proteins UreD, UreF, UreG, and UreE bind sequentially to the urease enzyme (H. pylori two different UreE–UreG complexes have been observed (H. pylori UreG bound to one H. pylori UreE dimer (UreE2–UreG2) with a Kd of 4.0 μM . IE. coli [NiFe]-hydrogenase  chaperones and accessory proteins. These studies have led to models for nickel insertion into rogenase  and H. pi urease . For nic unknown . Nickel 5.E. coli HypB, utilize the N-terminal amine to coordinate the nickel ion. The Ni(II)-responsive metalloregulators: RcnR, DmeR, and KmtR are also Co(II)-responsive.Over the past 20 years significant progress has been made towards elucidating the role that nickel plays in the proper functioning of metalloregulators and chaperones. Multiple themes have emerged from the studies conducted on nickel metalloregulators and chaperones regarding nickel binding and protein allostery. The proteins discussed in this review bind nickel in one of three different geometries: four-coordinate square planar, four-coordinate tetrahedral, or six-coordinate octahedral geometry. Four of the proteins, RcnR, NmtR, HypA, and Imidazole ligands and 1 S-dnonors does the correct structural changes occur in the protein that favors DNA binding -hydrogenase and urease activity in H. pylori  hydrogenase maturation  and is kori SlyD  and K. anes UreE  have hisnes UreE , but thenes UreE . All Ureing site ,142. It ing site .E. coli what is the oligomeric state of the HypA–HypB complex? What favors HypA delivering nickel to [NiFe]-hydrogenase versus UreE and vice versa? What ligands compose the HypA-UreE metal binding site? How does UreG deliver nickel to urease? What is the role of UreD? How does UreF stimulate the GTPase activity of UreG?The bioinorganic chemistry of nickel metalloregulators and chaperones is fascinating. Numerous studies have been conducted on individual chaperones and accessory proteins involved in [NiFe]-hydrogenase and urease maturation but some details regarding the sequential protein-protein interactions that are necessary to deliver nickel to [NiFe]-hydrogenase and urease still need to be elucidated. How does HypB get nickel? What is the exact role of SlyD in the delivery of nickel to hydrogenase? What role does HypB play in urease maturation? In H. pylori, studies involving animal models have determined that the pathogenicity of H. pylori is dependent on both [NiFe]-hydrogenase and urease [H. pylori [H. pylori infections [H. pylori is crucial.For d urease ,165,166.. pylori . To datefections . Prior tSupplementary Material