Polyphosphate kinase-1 regulates bacterial and host metabolic pathways involved in pathogenesis of Mycobacterium tuberculosis.

Inorganic polyphosphate (polyP) is primarily synthesized by Polyphosphate Kinase-1 (PPK-1) and regulates numerous cellular processes, including energy metabolism, stress adaptation, drug tolerance, and microbial pathogenesis. Here, we report that polyP interacts with acyl CoA carboxylases, enzymes involved in lipid biosynthesis in Mycobacterium tuberculosis. We show that deletion of ppk-1 in M. tuberculosis results in transcriptional and metabolic reprogramming. In comparison to the parental strain, the Δppk-1 mutant strain had reduced levels of virulence-associated lipids such as PDIMs and TDM. We also observed that polyP deficiency in M. tuberculosis is associated with enhanced phagosome-lysosome fusion in infected macrophages and attenuated growth in mice. Host RNA-seq analysis revealed decreased levels of transcripts encoding for proteins involved in either type I interferon signaling or formation of foamy macrophages in the lungs of Δppk-1 mutant-infected mice relative to parental strain-infected animals. Using target-based screening and molecular docking, we have identified raloxifene hydrochloride as a broad-spectrum PPK-1 inhibitor. We show that raloxifene hydrochloride significantly enhanced the activity of isoniazid, bedaquiline, and pretomanid against M. tuberculosis in macrophages. Additionally, raloxifene inhibited the growth of M. tuberculosis in mice. This is an in-depth study that provides mechanistic insights into the regulation of mycobacterial pathogenesis by polyP deficiency.

Identification and optimization of pyridine carboxamide-based scaffold as a drug lead for Mycobacterium tuberculosis.

New drugs with novel mechanisms of action are urgently needed to tackle the issue of drug-resistant tuberculosis. Here, we have performed phenotypic screening using the Pathogen Box library obtained from the Medicines for Malaria Venture against Mycobacterium tuberculosis in vitro. We have identified a pyridine carboxamide derivative, MMV687254, as a promising hit. This molecule is specifically active against M. tuberculosis and Mycobacterium bovis Bacillus Calmette-Guérin (M. bovis BCG) but inactive against Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Escherichia coli pathogens. We demonstrate that MMV687254 inhibits M. tuberculosis growth in liquid cultures in a bacteriostatic manner. Surprisingly, MMV687254 was as active as isoniazid in macrophages and inhibited M. tuberculosis growth in a bactericidal manner. Mechanistic studies revealed that MMV687254 is a prodrug and that its anti-mycobacterial activity requires AmiC-dependent hydrolysis. We further demonstrate that MMV687254 inhibits M. tuberculosis growth in macrophages by inducing autophagy. In the present study, we have also carried out a detailed structure-activity relationship study and identified a promising novel lead candidate. The identified novel series of compounds also showed activity against drug-resistant M. bovis BCG and M. tuberculosis clinical strains. Finally, we demonstrate that in contrast to MMV687254, the lead molecule was able to inhibit M. tuberculosis growth in a chronic mouse model of infection. Taken together, we have identified a novel lead molecule with a dual mechanism of action that can be further optimized to design more potent anti-tubercular agents.

IDSL.CSA: Composite Spectra Analysis for Chemical Annotation of Untargeted Metabolomics Datasets.

Poor chemical annotation of high-resolution mass spectrometry data limits applications of untargeted metabolomics datasets. Our new software, the Integrated Data Science Laboratory for Metabolomics and Exposomics─Composite Spectra Analysis (IDSL.CSA) R package, generates composite mass spectra libraries from MS1-only data, enabling the chemical annotation of high-resolution mass spectrometry coupled with liquid chromatography peaks regardless of the availability of MS2 fragmentation spectra. We demonstrate comparable annotation rates for commonly detected endogenous metabolites in human blood samples using IDSL.CSA libraries versus MS/MS libraries in validation tests. IDSL.CSA can create and search composite spectra libraries from any untargeted metabolomics dataset generated using high-resolution mass spectrometry coupled to liquid or gas chromatography instruments. The cross-applicability of these libraries across independent studies may provide access to new biological insights that may be missed due to the lack of MS2 fragmentation data. The IDSL.CSA package is available in the R-CRAN repository at https://cran.r-project.org/package=IDSL.CSA. Detailed documentation and tutorials are provided at https://github.com/idslme/IDSL.CSA.

Efficient Chemical and Enzymatic Syntheses of FAD Nucleobase Analogues and Their Analysis as Enzyme Cofactors.

Flavin adenine dinucleotide (FAD) is an essential redox cofactor in cellular metabolism. The organic synthesis of FAD typically involves coupling flavin mononucleotide (FMN) with adenosine monophosphate, however, existing synthesis routes present limitations such as multiple steps, low yields, and/or difficult-to-obtain starting materials. In this study, we report the synthesis of FAD nucleobase analogues with guanine/cytosine/uracil in place of adenine and deoxyadenosine in place of adenosine using chemical and enzymatic approaches with readily available starting materials, achieved in 1-3 steps with moderate yields (10-57 %). We find that the enzymatic route using Methanocaldococcus jannaschii FMN adenylyltransferase (MjFMNAT) is versatile and can produce these FAD analogues in high yields. Further, we demonstrate that Escherichia coli glutathione reductase is capable of binding and using these analogues as cofactors. Finally, we show that FAD nucleobase analogues can be synthesized inside a cell from cellular substrates FMN and nucleoside triphosphates by the heterologous expression of MjFMNAT. This lays the foundation for their use in studying the molecular role of FAD in cellular metabolism and as biorthogonal reagents in biotechnology and synthetic biology.

Dysregulated metabolites and lipids in serum of patients with acute hepatitis E: A longitudinal study.

Hepatitis E is a disease associated with acute inflammation of the liver. It is related to several dysregulated metabolic pathways and alterations in the concentration of several metabolites. However, longitudinal analysis of the alterations in metabolites and lipids is generally lacking. This study investigated the changes in levels of metabolites and lipids over time in sera from men with acute hepatitis E compared to healthy controls similar in age and gender. Untargeted measurement of levels of various metabolites and lipids was done using mass spectrometry on 65 sera sequentially sampled from 14 patients with acute hepatitis E and 25 serum samples from five controls. Temporal changes in intensities of metabolites and lipids were determined over different times at 3-day periods for the hepatitis E virus (HEV) group. In carbohydrate metabolism, glucose levels, fructose 1-6-bisphosphate and ribulose-5-phosphate were increased in the HEV-infected persons compared to the healthy controls. HEV infection is significantly associated with decreased levels of inosine, guanosine, adenosine and urate in purine metabolism and thymine, uracil and ß-aminoisobutyrate in pyrimidine metabolism. Glutamate, alanine and valine levels were significantly lower in the HEV group than in healthy individuals. Homogentisate of tyrosine metabolism and cystathionine of serine metabolism were increased, whereas kynurenate of tryptophan metabolism decreased in the HEV group. Metabolites of the bile acid biosynthesis, urea cycle (arginine and citrulline) and ammonia recycling (urocanate) were significantly altered. Co-enzymes, pantothenate and pyridoxal, and co-factors, lipoamide and FAD, were elevated in the HEV group. The acylcarnitines, sphingomyelins, phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysoPC and lysoPE tended to be lower in the HEV group. In conclusion, acute hepatitis E is associated with altered metabolite and lipid profiles, significantly increased catabolism of carbohydrates, purines/pyrimidines and amino acids, and decreased levels of several glycerophospholipids.

Role of Serology, Dietary Assessment, and Fecal Gluten Immunogenic Peptides for Predicting Histologic Recovery in Children with Celiac Disease.

BACKGROUND: A strict lifelong gluten-free diet (GFD) remains the only treatment of celiac disease (CD). Adherence to gluten-free diet is best reflected by mucosal healing. Noninvasive tools capable of predicting mucosal recovery in CD patients need to be identified. AIMS: To compare the ability of various modalities used to assess compliance to GFD, for predicting persistent mucosal damage in children with CD. METHODS: A prospective, single-center, observational study on children with CD on a GFD was conducted between January 2020 and April 2021. Children with CD on GFD were consecutively enrolled and various modalities used to assess adherence to GFD were compared. RESULTS: One hundred and fifty children (Mean age 12.2 ± 3.6 years, 58% Boys) on GFD (Mean duration 6 ± 3.1 years) were enrolled in the study. Persistent mucosal damage was seen in 88% of the enrolled. Fecal gluten immunogenic peptide (GIP) was positive in 87.8% (129/147). Antibodies to tissue transglutaminase (TGA-IgA) and/or deamidated gliadin peptide (DGP) were positive in 32% (48/150) whereas antibody to synthetic neoepitopes of TGA-IgA was positive in 24.8% (37/149). Non-compliance as assessed by local questionnaire, Biagi score, and dietitian detailed interview were 62.7%, 60%, and 75.3%, respectively. Serology had the highest specificity (83%) and fecal GIP had the highest sensitivity (89%). On logistic regression analysis, only non-compliance by Biagi score predicted poor mucosal recovery. CONCLUSION: Fecal GIP may be sensitive to detect only "one-point dietary transgression." None of the existing modalities used to assess compliance to GFD accurately predict persistent mucosal damage. A subset of patients may develop gluten tolerance.

Clinical spectrum & changing presentation of celiac disease in Indian children.

Background & objectives: Celiac disease (CD) is a genetic immune mediated disorder characterised by gluten intolerance. This single centre study, from north India was aimed to assess the clinical, serological and histological profile of CD in a large cohort of children and the changing trends in its presentation. Methods: A review of clinical details of CD children diagnosed between 2000 and 2019 and currently on follow up was performed. Information on demography, symptoms, associated conditions, serology, biopsy findings and gluten-free diet were analyzed. Results: The mean age (±standard deviation) of 891 children included in the study, at onset and at diagnosis was 4.0±2.7 and 6.2±3.1 yr, respectively. Growth faltering, abdominal pain, abdominal distension and diarrhoea were presenting symptoms in 70, 64.2, 61.2 and 58.2 per cent, respectively. A positive family history of CD was present in 14 per cent and autoimmune conditions in 12.3 per cent of children. Thyroid disorders were seen in 8.5 per cent of children and Type 1 diabetes mellitus (T1DM) in 5.7 per cent. The duration of breastfeeding had a weak positive correlation with age at onset and diagnosis of CD (P<0.001). Non-classical CD was significantly more common in children aged >10 yr and in those presenting after 2010 (P<0.01). T1DM and hypothyroidism occurred more frequently in non-compliant children. Interpretation & conclusions: This was the largest single centre study, pertaining to the presentation and follow up of CD in children. Infants and young children were more likely to present with classical symptoms of diarrhoea, abdominal distension and growth failure while older children presented with non-classical CD. There was a trend towards non-classical forms of CD in recent years.

Risk factors for clinically significant diffuse parenchymal lung abnormalities persisting after severe COVID-19 pneumonia.

Background & objectives: The risk factors for clinically significant diffuse parenchymal lung abnormalities (CS-DPLA) persisting after severe coronavirus disease 2019 (COVID-19) pneumonia remain unclear. The present study was conducted to assess whether COVID-19 severity and other parameters are associated with CS-DPLA. Methods: The study participants included patients who recovered after acute severe COVID-19 and presented with CS-DPLA at two or six month follow up and control group (without CS-DPLA). Adults volunteers without any acute illness, chronic respiratory illness and without a history of severe COVID-19 were included as healthy controls for the biomarker study. The CS-DPLA was identified as a multidimensional entity involving clinical, radiological and physiological pulmonary abnormalities. The primary exposure was the neutrophil-lymphocyte ratio (NLR). Recorded confounders included age, sex, peak lactate dehydrogenase (LDH), advanced respiratory support (ARS), length of hospital stay (LOS) and others; associations were analyzed using logistic regression. The baseline serum levels of surfactant protein D, cancer antigen 15-3 and transforming growth factor-ß (TGF-ß) were also compared among cases, controls and healthy volunteers. Results: We identified 91/160 (56.9%) and 42/144 (29.2%) participants with CS-DPLA at two and six months, respectively. Univariate analyses revealed associations of NLR, peak LDH, ARS and LOS with CS-DPLA at two months and of NLR and LOS at six months. The NLR was not independently associated with CS-DPLA at either visit. Only LOS independently predicted CS-DPLA at two months [adjusted odds ratios (aOR) (95% confidence interval [CI]), 1.16 (1.07-1.25); P<0.001] and six months [aOR (95% CI) and 1.07 (1.01-1.12); P=0.01]. Participants with CS-DPLA at six months had higher baseline serum TGF-ß levels than healthy volunteers. Interpretation and conclusions: Longer hospital stay was observed to be the only independent predictor of CS-DPLA six months after severe COVID-19. Serum TGF-ß should be evaluated further as a biomarker.

Molecular insights into the genome dynamics and interactions between core and acquired genomes of Vibrio cholerae.

Bacterial species are hosts to horizontally acquired mobile genetic elements (MGEs), which encode virulence, toxin, antimicrobial resistance, and other metabolic functions. The bipartite genome of Vibrio cholerae harbors sporadic and conserved MGEs that contribute in the disease development and survival of the pathogens. For a comprehensive understanding of dynamics of MGEs in the bacterial genome, we engineered the genome of V. cholerae and examined in vitro and in vivo stability of genomic islands (GIs), integrative conjugative elements (ICEs), and prophages. Recombinant vectors carrying the integration module of these GIs, ICE and CTXΦ, helped us to understand the efficiency of integrations of MGEs in the V. cholerae chromosome. We have deleted more than 250 acquired genes from 6 different loci in the V. cholerae chromosome and showed contribution of CTX prophage in the essentiality of SOS response master regulator LexA, which is otherwise not essential for viability in other bacteria, including Escherichia coli In addition, we observed that the core genome-encoded RecA helps CTXΦ to bypass V. cholerae immunity and allow it to replicate in the host bacterium in the presence of similar prophage in the chromosome. Finally, our proteomics analysis reveals the importance of MGEs in modulating the levels of cellular proteome. This study engineered the genome of V. cholerae to remove all of the GIs, ICEs, and prophages and revealed important interactions between core and acquired genomes.

Development of a Systematic Strategy toward Promotion of α-Synuclein Aggregation Using 2-Hydroxyisophthalamide-Based Systems.

Establishing a potent scheme against α-synuclein aggregation involved in Parkinson's disease has been evaluated as a promising route to identify compounds that either inhibit or promote the aggregation process of α-synuclein. In the last two decades, this perspective has guided a dramatic increase in the efforts, focused on developing potent drugs either for retardation or promotion of the self-assembly process of α-synuclein. To address this issue, using a chemical kinetics platform, we developed a strategy that enabled a progressively detailed analysis of the molecular events leading to protein aggregation at the microscopic level in the presence of a recently synthesized 2-hydroxyisophthalamide class of small organic molecules based on their binding affinity. Furthermore, qualitatively, we have developed a strategy of disintegration of α-synuclein fibrils in the presence of these organic molecules. Finally, we have shown that these organic molecules effectively suppress the toxicity of α-synuclein oligomers in neuron cells.

IDSL.IPA Characterizes the Organic Chemical Space in Untargeted LC/HRMS Data Sets.

Generating comprehensive and high-fidelity metabolomics data matrices from LC/HRMS data remains to be extremely challenging for population-scale large studies (n > 200). Here, we present a new data processing pipeline, the Intrinsic Peak Analysis (IDSL.IPA) R package (https://ipa.idsl.me), to generate such data matrices specifically for organic compounds. The IDSL.IPA pipeline incorporates (1) identifying potential 12C and 13C ion pairs in individual mass spectra; (2) detecting and characterizing chromatographic peaks using a new sensitive and versatile approach to perform mass correction, peak smoothing, baseline development for local noise measurement, and peak quality determination; (3) correcting retention time and cross-referencing peaks from multiple samples by a dynamic retention index marker approach; (4) annotating peaks using a reference database of m/z and retention time; and (5) accelerating data processing using a parallel computation of the peak detection and alignment steps for larger studies. This pipeline has been successfully evaluated for studies ranging from 200 to 1600 samples. By specifically isolating high quality and reliable signals pertaining to carbon-containing compounds in untargeted LC/HRMS data sets from larger studies, IDSL.IPA opens new opportunities for discovering new biological insights in the population-scale metabolomics and exposomics projects. The package is available in the R CRAN repository at https://cran.r-project.org/package=IDSL.IPA.

IDSL.UFA Assigns High-Confidence Molecular Formula Annotations for Untargeted LC/HRMS Data Sets in Metabolomics and Exposomics.

Untargeted liquid chromatography/high-resolution mass spectrometry (LC/HRMS) assays in metabolomics and exposomics aim to characterize the small molecule chemical space in a biospecimen. To gain maximum biological insights from these data sets, LC/HRMS peaks should be annotated with chemical and functional information including molecular formula, structure, chemical class, and metabolic pathways. Among these, molecular formulas may be assigned to LC/HRMS peaks through matching theoretical and observed isotopic profiles (MS1) of the underlying ionized compound. For this, we have developed the Integrated Data Science Laboratory for Metabolomics and Exposomics-United Formula Annotation (IDSL.UFA) R package. In the untargeted metabolomics validation tests, IDSL.UFA assigned 54.31-85.51% molecular formula for true positive annotations as the top hit and 90.58-100% within the top five hits. Molecular formula annotations were also supported by tandem mass spectrometry data. We have implemented new strategies to (1) generate formula sources and their theoretical isotopic profiles, (2) optimize the formula hits ranking for the individual and aligned peak lists, and (3) scale IDSL.UFA-based workflows for studies with larger sample sizes. Annotating the raw data for a publicly available pregnancy metabolome study using IDSL.UFA highlighted hundreds of new pregnancy-related compounds and also suggested the presence of chlorinated perfluorotriether alcohols (Cl-PFTrEAs) in human specimens. IDSL.UFA is useful for human metabolomics and exposomics studies where we need to minimize the loss of biological insights in untargeted LC/HRMS data sets. The IDSL.UFA package is available in the R CRAN repository https://cran.r-project.org/package=IDSL.UFA. Detailed documentation and tutorials are also provided at www.ufa.idsl.me.

Characterization, genome analysis and in vitro activity of a novel phage vB_EcoA_RDN8.1 active against multi-drug resistant and extensively drug-resistant biofilm-forming uropathogenic Escherichia coli isolates, India.

AIM: We aimed to study host range, stability, genome and antibiofilm activity of a novel phage vB_EcoA_RDN8.1 active against multi-drug resistant (MDR) and extensively drug-resistant (XDR) biofilm-forming uropathogenic Escherichia coli isolates. METHODS AND RESULTS: A novel lytic phage vB_EcoA_RDN8.1 active against UPEC strains resistant to third-generation cephalosporins, fluoroquinolones, aminoglycosides, imipenem, beta-lactamase inhibitor combination and polymyxins was isolated from community raw sewage water of Chandigarh. It exhibited a clear plaque morphology and a burst size of 250. In the time-kill assay, the maximum amount of killing was achieved at MOI 1.0. vB_EcoA_RDN8.1 belongs to the family Autographiviridae, has a genome size of 39.5 kb with a GC content of 51.6%. It was stable over a wide range of temperatures and pH. It was able to inhibit biofilm formation which may be related to an endolysin encoded by ORF 19. CONCLUSIONS: The vB_EcoA_RDN8.1 is a novel lytic phage that has the potential for inclusion into phage cocktails being developed for the treatment of urinary tract infections (UTIs) caused by highly drug-resistant UPEC. SIGNIFICANCE AND IMPACT OF THE STUDY: We provide a detailed characterization of a novel lytic Escherichia phage with antibiofilm activity having a potential application against MDR and XDR UPEC causing UTIs.

Antinuclear antibody pattern and autoantibody profiling of systemic sclerosis patients in a tertiary referral center in North India.

Antinuclear antibody (ANA) pattern and autoantibody (autoAb) profiling of 150 adult systemic sclerosis (SSc) patients concerning their clinical association and diagnostic significance were analyzed by indirect immunofluorescence (IIF), immunoblot, and fluorescence enzyme immunoassay. One hundred and forty-three (95.3%) patients had positive ANA: DNA topoisomerase I (topo I)-like pattern-84(56%); speckled pattern-44(29.3%);centromere pattern-7(4.6%); and nucleolar pattern-4(2.6%). Three distinct topo I-like immunofluorescence patterns were detected at 1:40 dilution. Topo I-like pattern (32/75-limited cutaneous systemic sclerosis (lcSSc) vs. 52/75-diffuse cutaneous systemic sclerosis (dcSSc); p < 0.001) was found to be associated with dcSSc subset and speckled pattern (lcSSc 28/75 vs. dcSSc 16/75; p < 0.03) with lcSSc subset. One hundred and thirty-eight (92%) patients were positive for SSc-associated autoAbs. The frequency distribution of autoAbs to topo I, centromere A (CENP A) and centromereB (CENP B), RNA polymerase III (RP11, RP155), fibrillarin (U3RNP), nucleolus organizer region (NOR)-90, Th/To, PM-Scl75, PM-Scl100, Ku, platelet-derived growth factor receptor (PDGFR) and Ro-52, were 87(58%), 9(6%), 8(5.3%), 6(4%), 9(6%), 0, 6(4%), 6(4%), 8(5.3%), 5(3.3%), 11(7.3%),0 and 46(30.6%), respectively. Topo I autoAb was strongly associated with dcSSc (35/75 lcSSc vs. 52/75 dcSSc; p < 0.004), Raynaud's (p < 0.003), interstitial lung disease (ILD) (p < 0.001) and pulmonary arterial hypertension (PAH) (p < 0.04). This study helps in defining SSc clinical subset, prognostic markers of disease severity, characterization of the topo I-like ANA pattern, and provides a definite association between the ANA patterns and corresponding autoAb.

Characterization of a Novel Mesophilic CTP-Dependent Riboflavin Kinase and Rational Engineering to Create Its Thermostable Homologues*.

Flavins play a central role in metabolism as molecules that catalyze a wide range of redox reactions in living organisms. Several variations in flavin biosynthesis exist among the domains of life, and their analysis has revealed many new structural and mechanistic insights till date. The cytidine triphosphate (CTP)-dependent riboflavin kinase in archaea is one such example. Unlike most kinases that use adenosine triphosphate, archaeal riboflavin kinases utilize CTP to phosphorylate riboflavin and produce flavin mononucleotide. In this study, we present the characterization of a new mesophilic archaeal CTP-utilizing riboflavin kinase homologue from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic Methanocaldococcus jannaschii homologue. We reconstitute the activity of MmpRibK, determine its kinetic parameters and molecular factors that contribute to its unique properties, and finally establish the residues that improve its thermostability using computation and a series of experiments. Our work advances the molecular understanding of flavin biosynthesis in archaea by the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to this unique structural fold that characterizes archaeal riboflavin kinase enzymes, with implications in enzyme engineering and biotechnological applications.

Interaction of chloramphenicol with titin I27 probed using single-molecule force spectroscopy.

Titin is a giant elastic protein which is responsible for passive muscle stiffness when muscle sarcomeres are stretched. Chloramphenicol, besides being a broad-spectrum antibiotic, also acts as a muscle relaxant. Therefore, it is important to study the interaction between titin I27 and chloramphenicol. We investigated the interaction of chloramphenicol with octamer of titin I27 using single-molecule force spectroscopy and fluorescence spectroscopy. The fluorescence data indicated that binding of chloramphenicol with I27 results in fluorescence quenching. Furthermore, it is observed that chloramphenicol binds to I27 at a particular concentration ([Formula: see text] 40 µM). Single-molecule force spectroscopy shows that, in the presence of 40 µM chloramphenicol concentration, the I27 monomers become mechanically stable, resulting in an increment of the unfolding force. The stability was further confirmed by chemical denaturation experiments on monomers of I27, which corroborate the evidence for enhanced mechanical stability at 40 µM drug concentration. The free energy of stabilization for I27 (wild type) was found to be 1.95 ± 0.93 kcal/mole and I27 with 40 µM drug was 3.25 ± 0.63 kcal/mole. The results show a direct effect of the broad-spectrum antibiotic chloramphenicol on the passive elasticity of muscle protein titin. The I27 is stabilized both mechanically and chemically by chloramphenicol.

Modulation of Insulin Resistance, Dyslipidemia and Serum Metabolome in iNOS Knockout Mice following Treatment with Nitrite, Metformin, Pioglitazone, and a Combination of Ampicillin and Neomycin.

Oxidative and nitrosative stress plays a pivotal role in the incidence of metabolic disorders. Studies from this lab and others in iNOS-/- mice have demonstrated occurrence of insulin resistance (IR), hyperglycemia and dyslipidemia highlighting the importance of optimal redox balance. The present study evaluates role of nitrite, L-arginine, antidiabetics (metformin, pioglitazone) and antibiotics (ampicillin-neomycin combination, metronidazole) on metabolic perturbations observed in iNOS-/- mice. The animals were monitored for glucose tolerance (IPGTT), IR (insulin, HOMA-IR, QUICKI), circulating lipids and serum metabolomics (LC-MS). Hyperglycemia, hyperinsulinemia and IR were rescued by nitrite, antidiabetics, and antibiotics treatments in iNOS-/- mice. Glucose intolerance was improved with nitrite, metformin and pioglitazone treatment, while ampicillin-neomycin combination normalised the glucose utilization in iNOS-/- mice. Increased serum phosphatidylethanolamine lipids in iNOS-/- mice were reversed by metformin, pioglitazone and ampicillin-neomycin; dyslipidemia was however marginally improved by nitrite treatment. The metabolic improvements were associated with changes in selected serum metabolites-purines, ceramide, 10-hydroxydecanoate, glucosaminate, diosmetin, sebacic acid, 3-nitrotyrosine and cysteamine. Bacterial metabolites-hippurate, indole-3-ethanol; IR marker-aminoadipate and oxidative stress marker-ophthalmate were reduced by pioglitazone and ampicillin-neomycin, but not by nitrite and metformin treatment. Results obtained in the present study suggest a crucial role of gut microbiota in the metabolic perturbations observed in iNOS-/- mice.

Integrated Transcriptomic and Proteomic Analyses Suggest the Participation of Endogenous Protease Inhibitors in the Regulation of Protease Gene Expression in Helicoverpa armigera.

Insects adapt to plant protease inhibitors (PIs) present in their diet by differentially regulating multiple digestive proteases. However, mechanisms regulating protease gene expression in insects are largely enigmatic. Ingestion of multi-domain recombinant Capsicum annuum protease inhibitor-7 (CanPI-7) arrests growth and development of Helicoverpa armigera (Lepidoptera: Noctuidae). Using de novo RNA sequencing and proteomic analysis, we examined the response of H. armigera larvae fed on recombinant CanPI-7 at different time intervals. Here, we present evidence supporting a dynamic transition in H. armigera protease expression on CanPI-7 feeding with general down-regulation of protease genes at early time points (0.5 to 6 h) and significant up-regulation of specific trypsin, chymotrypsin and aminopeptidase genes at later time points (12 to 48 h). Further, coexpression of H. armigera endogenous PIs with several digestive protease genes were apparent. In addition to the differential expression of endogenous H. armigera PIs, we also observed a distinct novel isoform of endogenous PI in CanPI-7 fed H. armigera larvae. Based on present and earlier studies, we propose potential mechanism of protease regulation in H. armigera and subsequent adaptation strategy to cope with anti-nutritional components of plants.

Serovar diversity of Salmonella among poultry.

Background & objectives: Salmonellosis due to the consumption of contaminated poultry products is a well-known public health concern, and assessing the distribution of Salmonella serovars among poultry becomes important for better prevention and control. The objective of the present study was to assess the distribution of Salmonella serovars among poultry. Methods: The isolates received at National Salmonella and Escherichia Centre during 2011-2016 were subjected to biochemical identification, followed by serological characterization to identify the Salmonella serovars, and the data were presented to exhibit the distribution of Salmonella serovars among poultry. Results: Salmonella was found to be present in poultry in all the regions included in the study. Salmonella Typhimurium, S. Gallinarum and S. Enteritidis were the most prevalent serovars accounting for 96.2 per cent of isolates. Salmonella was identified in poultry from all major egg-producing and egg-consuming States. Other serovars which were scantly identified included S. Infantis (2.7%), S. Montevideo (0.64%), S. Newport (0.26%) and S. Pullorum (0.13%). Interpretation & conclusions: Diverse distribution of Salmonella serovars in poultry in India, with known potential to infect human population and/or other poultry flocks, requires urgent nationwide stringent control measures.

Molecular characterization of diarrheagenic Escherichia coli pathotypes: Association of virulent genes, serogroups, and antibiotic resistance among moderate-to-severe diarrhea patients.

BACKGROUND: Diarrheagenic Escherichia coli (DEC) signifies as an important etiological agent of moderate-to-severe diarrhea. This study was primarily focused on molecular identification of DEC pathotypes; their association with serogroups and estimates of resistance profiles against different antibiotics regime. METHODS: Five hundred seventy-two stool specimens from diarrhea patients were investigated for DEC pathotypes. Molecular pathotypes were identified by amplification of virulence genes associated with distinct pathotypes followed by sequencing. Diarrhea is a self-limiting disease, however, severity and persistence of infection suggest antibiotic use. Therefore, AST and MIC were determined against common antibiotic regimen. Correlations between molecular pathotypes and serogroups were analyzed by somatic "O" antigen serotyping. RESULTS: The present findings reveal incidence of DEC as an etiological agent up to a level of 21% among all diarrheal age groups. DEC infection rate was higher in children. Enteropathogenic E. coliEPEC, a molecular pathotype of DEC, was found as a predominant pathotype with highest frequency of 13.7%. Two other molecular pathotypes enterotoxigenic E. coli (ETEC) and enteroaggregative E. coli (EAEC) accounted for 5.7% and 1.3%, respectively for all diarrhea incidences. Serological analysis deciphered somatic antigens O26, O2, and O3 as major serogroups identified among EPEC, ETEC, and EAEC pathotypes, respectively. All DEC pathotypes exhibited high levels of antibiotic resistance except for cotrimoxazole and norfloxacin. CONCLUSION: Comprehensive molecular characterization of DEC pathotypes, their incidence estimates, and antibiogram patterns will help in ascertaining better diagnostic and therapeutic measures in management of diarrheal diseases.