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.

Aged AG129 mice support the generation of highly virulent novel mouse-adapted DENV (1-4) viruses exhibiting neuropathogenesis and high lethality.

Dengue virus infection in humans ranges from asymptomatic infection to severe infection, with ∼2.5 % overall disease fatality rate. Evidence of neurological manifestations is seen in the severe form of the disease, which might be due to the direct invasion of the viruses into the CNS system but is poorly understood. In this study, we demonstrated that the aged AG129 mice are highly susceptible to dengue serotypes 1-4, and following the adaptation, this resulted in the generation of neurovirulent strains that showed enhanced replication, aggravated disease severity, increased neuropathogenesis, and high lethality in both adult and aged AG129 mice. The infected mice had endothelial dysfunction, elicited pro-inflammatory cytokine responses, and exhibited 100 % mortality. Further analysis revealed that aged-adapted DENV strains induced measurable alterations in TLR expression in the aged mice as compared to the adult mice. In addition, metabolomics analysis of the serum samples from the infected adult mice revealed dysregulation of 18 metabolites and upregulation of 6-keto-prostaglandin F1 alpha, phosphocreatine, and taurocholic acid. These metabolites may serve as key biomarkers to decipher and comprehend the severity of dengue-associated severe neuro-pathogenesis.

Impact of Anti-nuclear Antibody Seropositivity on Clinicopathological Parameters, Treatment Response, and Survival in Lymphoma Patients.

PURPOSE: Lymphoproliferative disorders and autoimmune diseases both are interrelated. The high incidence of lymphoma in autoimmune diseases and frequent antinuclear antibody (ANA) positivity in lymphoma patients have been observed. But the impact of ANA positivity on various clinical parameters and responses to therapy has not been elucidated properly. METHODS: In the present study, 73 treatment-naive lymphoma patients were recruited prospectively and samples were collected at baseline and after completion of therapy for evaluation of ANA. Comparative analysis was performed for various parameters between ANA-positive and ANA-negative groups. RESULTS: The prevalence of ANA at baseline was 27% in lymphoma patients which further increased to 35% after chemotherapy. The ANA-positive group had a significantly higher mean age (58±14.7 vs 47±19.9; p=0.01), early stage (77% vs 38%; p=0.02,) and infrequent B-symptoms (25% vs 52%; p=0.03) as compared to ANA-negative group. No significant difference was observed in the response to therapy and survival (both event-free and overall survival). The most frequent ANA pattern was speckled (50%) at baseline, and homogenous (42%) after the therapy. CONCLUSION: ANA is more frequent in lymphoma and increases further after chemotherapy. Higher mean age, early stage, and infrequent B symptoms were found to be significantly more frequent in ANA-positive lymphoma patients; however, only limited evidence supports its role as a prognostic marker or response to therapy. A wider study with appropriate follow-up data and molecular assay could shed light on the immunobiology of ANA production and its more defined clinical utility in lymphoma.

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.

Rv0495c regulates redox homeostasis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) has evolved sophisticated surveillance mechanisms to neutralize the ROS-induces toxicity which otherwise would degrade a variety of biological molecules including proteins, nucleic acids and lipids. In the present study, we find that Mtb lacking the Rv0495c gene (ΔRv0495c) is presented with a highly oxidized cytosolic environment. The superoxide-induced lipid peroxidation resulted in altered colony morphology and loss of membrane integrity in ΔRv0495c. As a consequence, ΔRv0495c demonstrated enhanced susceptibility when exposed to various host-induced stress conditions. Further, as expected, we observed a mutant-specific increase in the abundance of transcripts that encode proteins involved in antioxidant defence. Surprisingly, despite showing a growth defect phenotype in macrophages, the absence of the Rv0495c enhanced the pathogenicity and augmented the ability of the Mtb to grow inside the host. Additionally, our study revealed that Rv0495c-mediated immunomodulation by the pathogen helps create a favorable niche for long-term survival of Mtb inside the host. In summary, the current study underscores the fact that the truce in the war between the host and the pathogen favours long-term disease persistence in tuberculosis. We believe targeting Rv0495c could potentially be explored as a strategy to potentiate the current anti-TB regimen.

Comparative profiling of gut microbiota and metabolome in diet-induced obese and insulin-resistant C57BL/6J mice.

Diet-based models are commonly used to investigate obesity and related disorders. We conducted a comparative profiling of three obesogenic diets HFD, high fat diet; HFHF, high fat high fructose diet; and HFCD, high fat choline deficient diet to assess their impact on the gut microbiome and metabolome. After 20 weeks, we analyzed the gut microbiota and metabolomes of liver, plasma, cecal, and fecal samples. Fecal and plasma bile acids (BAs) and fecal short-chain fatty acids (SCFAs) were also examined. Significant changes were observed in fecal and cecal metabolites, with increased Firmicutes and decreased Bacteroidetes in the HFD, HFHF, and HFCD-fed mice compared to chow and LFD (low fat diet)-fed mice. Most BAs were reduced in plasma and fecal samples of obese groups, except taurocholic acid, which increased in HFCD mice's plasma. SCFAs like acetate and butyrate significantly decreased in obesogenic diet groups, while propionic acid specifically decreased in the HFCD group. Pathway analysis revealed significant alterations in amino acid, carbohydrate metabolism, and nucleic acid biosynthesis pathways in obese mice. Surprisingly, even LFD-fed mice showed distinct changes in microbiome and metabolite profiles compared to the chow group. This study provides insights into gut microbiome dysbiosis and metabolite alterations induced by obesogenic and LFD diets in various tissues. These findings aid in selecting suitable diet models to study the role of the gut microbiome and metabolites in obesity and associated disorders, with potential implications for understanding similar pathologies in humans.

Effect of herbal extracts and Saroglitazar on high-fat diet-induced obesity, insulin resistance, dyslipidemia, and hepatic lipidome in C57BL/6J mice.

We evaluated the effects of select herbal extracts (Tinospora cordifolia [TC], Tinospora cordifolia with Piper longum [TC + PL], Withania somnifera [WS], Glycyrrhiza glabra [GG], AYUSH-64 [AY-64], and Saroglitazar [S]) on various parameters in a diet-induced obesity mouse model. After 12 weeks of oral administration of the herbal extracts in high-fat diet (HFD)-fed C57BL/6J mice, we analyzed plasma biochemical parameters, insulin resistance (IR), liver histology, and the expression of inflammatory and fibrosis markers, along with hepatic lipidome. We also used a 3D hepatic spheroid model to assess their impact on profibrotic gene expression. Among the extracts, TC + PL showed a significant reduction in IR, liver weight, TNF-α, IL4, IL10 expression, and hepatic lipid levels (saturated triglycerides, ceramides, lysophosphocholines, acylcarnitines, diglycerides, and phosphatidylinositol levels). Saroglitazar reversed changes in body weight, IR, plasma triglycerides, glucose, insulin, and various hepatic lipid species (fatty acids, phospholipids, glycerophospholipids, sphingolipids, and triglycerides). With the exception of GG, Saroglitazar, and other extracts protected against palmitic acid-induced fibrosis marker gene expression in the 3D spheroids. TC + PL and Saroglitazar also effectively prevented HFD-induced insulin resistance, inflammation, and specific harmful lipid species in the liver.

Human leukocyte antigen association in systemic sclerosis patients: our experience at a tertiary care center in North India.

Introduction: Systemic sclerosis (SSc) is a chronic multisystem autoimmune rheumatic disease of unknown etiology. Several studies have established that SSc is triggered by a dynamic interplay between genetic factors and environmental stimuli. In the present study, we aimed to study the association of human leukocyte antigen (HLA) with familial and non-familial SSc patients [limited cutaneous SSc (lcSSc) and diffuse cutaneous SSc (dcSSc)] from North India. Methods: The HLA-A, B, DRB1, and DQB1 genotyping of 150 (70 lcSSc and 80 dcSSc) adult-onset SSc patients and 150 age-gender-matched healthy controls were performed with sequence-specific oligonucleotide (SSO) typing kits using the luminex platform. HLA typing for HLA class I (A, B, and C) and II (DRB1, DQB1, and DPB1) in five North Indian families consisting of parent-child/sibling pairs affected with SSc or overlap syndrome was performed by Next Generation Sequencing (NGS) with Illumina MiniSeq. Rseults: Among the non-familial SSc patients, HLA- DRB1*11 (P = 0.001, OR: 2.38, P c = 0.01) was identified as a risk allele, and DRB1*12 (P = .0001, OR: 0.00, P c = 0.001) as a protective allele. There was no statistical association found with HLA-DQB1*. Also, no significant association was observed between HLA antigens and different clinical subsets (lcSSc and dcSSc) of SSc. Two cases of familial SSc patients had the DRB1*11 allele. The DRB1*12 allele was absent in all the familial SSc patients. Discussion: HLA DRB1*11 (risk allele) and DRB1*12 (protective allele) were found to be strongly associated with non-familial SSc patients and partially explain the disease's familial clustering, supporting the susceptible genetic background theory for SSc development. The study also indicates the HLA allele as a common genetic risk factor in distinct autoimmune diseases contributing to overlap syndrome or polyautoimmunity.

DiabeticSense: A Non-Invasive, Multi-Sensor, IoT-Based Pre-Diagnostic System for Diabetes Detection Using Breath.

Diabetes mellitus is a widespread chronic metabolic disorder that requires regular blood glucose level surveillance. Current invasive techniques, such as finger-prick tests, often result in discomfort, leading to infrequent monitoring and potential health complications. The primary objective of this study was to design a novel, portable, non-invasive system for diabetes detection using breath samples, named DiabeticSense, an affordable digital health device for early detection, to encourage immediate intervention. The device employed electrochemical sensors to assess volatile organic compounds in breath samples, whose concentrations differed between diabetic and non-diabetic individuals. The system merged vital signs with sensor voltages obtained by processing breath sample data to predict diabetic conditions. Our research used clinical breath samples from 100 patients at a nationally recognized hospital to form the dataset. Data were then processed using a gradient boosting classifier model, and the performance was cross-validated. The proposed system attained a promising accuracy of 86.6%, indicating an improvement of 20.72% over an existing regression technique. The developed device introduces a non-invasive, cost-effective, and user-friendly solution for preliminary diabetes detection. This has the potential to increase patient adherence to regular monitoring.

Characterization of uropathogenic E. coli from various geographical locations in India.

Objectives: Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infection, accounting for more than 80% of cases worldwide. This study presents data on prevalent serotypes, resistance profiles, and colonization-aiding virulence characteristics of UPEC from different geographical regions in India. Methods: UPEC were serotyped through microtiter plate agglutination. Standard techniques were used to detect various virulence characteristics, i.e., biofilm formation (tissue culture plate method), siderophore production (screened on Chrome Azurol S agar and categorized with Csaky's and Arnow's methods), colicin release (agar overlay technique), gelatin hydrolysis (on gelatinase agar), and cell surface hydrophobicity (salt aggregation method). Antibiotic resistance profiles (against 20 antimicrobial agents) and extended-spectrum beta-lactamase (ESBL) were evaluated according to Clinical and Laboratory Standards Institute guidelines. Results: UPEC strains exhibited very high drug resistance rates to most of the commonly used antimicrobial agents; the highest resistance rates were observed for ampicillin (63.4%), nalidixic acid (63.4%), and cefotaxime (62.1%). High rates of multi-drug resistance (63.36%), ESBL-production (34.1%), and carbapenem-resistance (25.0%) were detected in UPEC strains from all geographical regions of India. Hydrophobicity (61.2%), biofilm production (62.5%), and siderophore production (67.7%) were the most common virulence characteristics of UPEC isolates. Co-expression of virulence characteristics was common (69.8%) in UPEC strains. Conclusion: UPEC strains with very high antimicrobial-resistance are in circulation in India, and have diverse serotypes and virulence characteristics.

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.

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.

Untargeted metabolomics and phenotype data indicate the therapeutic and prophylactic potential of Lysimachia candida Lindl. towards high-fat high-fructose-induced metabolic syndrome in rats.

The present study evaluated the therapeutic potential of the medicinal plant Lysimachia candida Lindl. against metabolic syndrome in male SD rats fed with a high-fat high-fructose (HFHF) diet. Methanolic extract of Lysimachia candida Lindl. (250 mg kg-1 body weight p.o.) was administrated to the HFHF-fed rats daily for 20 weeks. Blood samples were collected, and blood glucose levels and relevant biochemical parameters were analysed and used for the assessment of metabolic disease phenotypes. In this study, Lysimachia candida decreased HFHF diet-induced phenotypes of metabolic syndrome, i.e., obesity, blood glucose level, hepatic triglycerides, free fatty acids, and insulin resistance. Liquid chromatography-mass spectrometry-based metabolomics was done to study the dynamics of metabolic changes in the serum during disease progression in the presence and absence of the treatment. Furthermore, multivariate data analysis approaches have been employed to identify metabolites responsible for disease progression. Lysimachia candida Lindl. plant extract restored the metabolites that are involved in the biosynthesis and degradation of amino acids, fatty acid metabolism and vitamin metabolism. Interestingly, the results depicted that the treatment with the plant extract restored the levels of acetylated amino acids and their derivatives, which are involved in the regulation of beta cell function, glucose homeostasis, insulin secretion, and metabolic syndrome phenotypes. Furthermore, we observed restoration in the levels of indole derivatives and N-acetylgalactosamine with the treatment, which indicates a cross-talk between the gut microbiome and the metabolic syndrome. Therefore, the present study revealed the potential mechanism of Lysimachia candida Lindl. extract to prevent metabolic syndrome in rats.

ResR/McdR-regulated protein translation machinery contributes to drug resilience in Mycobacterium tuberculosis.

Survival response of the human tuberculosis pathogen, Mycobacterium tuberculosis (Mtb) to a diverse environmental cues is governed through its versatile transcription regulatory mechanisms with the help of a large pool of transcription regulators (TRs). Rv1830 is one such conserved TR, which remains uncharacterized in Mtb. It was named as McdR based on an effect on cell division upon its overexpression in Mycobacterium smegmatis. Recently, it has been implicated in antibiotic resilience in Mtb and reannotated as ResR. While Rv1830 affects cell division by modulating the expression of M. smegmatis whiB2, the underlying cause of its essentiality and regulation of drug resilience in Mtb is yet to be deciphered. Here we show that ResR/McdR, encoded by ERDMAN_2020 in virulent Mtb Erdman, is pivotal for bacterial proliferation and crucial metabolic activities. Importantly, ResR/McdR directly regulates ribosomal gene expression and protein synthesis, requiring distinct disordered N-terminal sequence. Compared to control, bacteria depleted with resR/mcdR exhibit delayed recovery post-antibiotic treatment. A similar effect upon knockdown of rplN operon genes further implicates ResR/McdR-regulated protein translation machinery in attributing drug resilience in Mtb. Overall, findings from this study suggest that chemical inhibitors of ResR/McdR may be proven effective as adjunctive therapy for shortening the duration of TB treatment.

Polymeric immunoglobulin receptor and galectin-3-binding protein are raised in biliary atresia: Reveals a proteomic-based study.

To identify and evaluate differentially expressed plasma proteins in biliary atresia (BA), we performed plasma proteome profiling using liquid chromatography with tandem mass spectrometry (LC-MS/MS) in 20 patients with BA and 10 control children. Serological assays validated the most significant and highly upregulated proteins in a cohort of 45 patients and 15 controls. Bioinformatics tools were used for functional classification and protein-protein interactions of differentially expressed proteins (DEPs). Of 405 proteins detected in patients and 360 in controls, 242 proteins, each with ≥2 unique peptides (total of 3230 peptides), were common in both groups. Compared to controls, 90 proteins in patients were differentially expressed and were dysregulated. Twenty-five were significantly upregulated with polymeric immunoglobulin receptor (PIgR), galectin-3-binding protein (Gal-3BP), complement C2, the most prominent, and 15 had low expression. The bioinformatic analysis revealed functional interaction between DEPs and their role in an inflammatory immune response. Enzyme immunoassay for PIgR and Gal-3BP in patients' plasma showed their levels raised significantly (p = 0.0021 and p = 0.0369, respectively). The PIgR and Gal-3BP are novel proteins upregulated in BA and may be tested further for their utility as potential circulating disease biomarker(s). SIGNIFICANCE: The study shows that plasma PIgR and GAL-3BP levels are significantly raised in infants with BA within the first 3 months of life. If tested in a larger cohort, these proteins may be found to have their diagnostic potential and utility as disease biomarkers. The study also provides valuable information on the involvement of several DEPs in innate immune response, chronic inflammation, and fibrosis. This strengthens the hypothesis that the immune-mediated inflammatory processes are responsible for the progressive nature of BA.

Characterization of lipid signatures in the plasma and insulin-sensitive tissues of the C57BL/6J mice fed on obesogenic diets.

Diet-induced obesity mouse models are widely utilized to investigate the underlying mechanisms of dyslipidemia, glucose intolerance, insulin resistance, hepatic steatosis, and type 2 diabetes mellitus (T2DM), as well as for screening potential drug compounds. However, there is limited knowledge regarding specific signature lipids that accurately reflect dietary disorders. In this study, we aimed to identify key lipid signatures using LC/MS-based untargeted lipidomics in the plasma, liver, adipose tissue (AT), and skeletal muscle tissues (SKM) of male C57BL/6J mice that were fed chow, LFD, or obesogenic diets (HFD, HFHF, and HFCD) for a duration of 20 weeks. Furthermore, we conducted a comprehensive lipid analysis to assess similarities and differences with human lipid profiles. The mice fed obesogenic diets exhibited weight gain, glucose intolerance, elevated BMI, glucose and insulin levels, and a fatty liver, resembling characteristics of T2DM and obesity in humans. In total, we identified approximately 368 lipids in plasma, 433 in the liver, 493 in AT, and 624 in SKM. Glycerolipids displayed distinct patterns across the tissues, differing from human findings. However, changes in sphingolipids, phospholipids, and the expression of inflammatory and fibrotic genes showed similarities to reported human findings. Significantly modulated pathways in the obesogenic diet-fed groups included ceramide de novo synthesis, sphingolipid remodeling, and the carboxylesterase pathway, while lipoprotein-mediated pathways were minimally affected. This study provides a tissue-specific comparison of lipid composition, highlighting the usefulness of DIO models in preclinical research. However, caution is warranted when extrapolating findings from these models to dyslipidemia-associated pathologies and their complications in humans.

Touch imprint smear: A prerequisite to obtain better quality and "true" tumor RNA in breast tissues.

BACKGROUND: RNA is the primary genetic material required for various molecular studies. RNA derived from breast tissue has low quality and quantity compared to that extracted from other tissues. Therefore, optimization of techniques for breast tissue RNA extraction is a challenging but essential requirement. METHODS: RNA was extracted from 60 samples of breast cancer after dividing them into 2 groups. Each tissue was divided into 2 halves for RNA extraction and histopathology respectively. In group 2 RNA was extracted after taking touch imprints whereas group1 was not subjected to any such procedure. Concentration and purity of RNA was assessed by using spectrophotometer and 1% agarose gel followed by RT-PCR for 18 S rRNA and CCND1 gene. RESULTS: Based on microscopic observations of imprints, group 2 samples were further subdivided into 2 subgroups. Group 2 A (n = 30) showing tumor in imprint smears were found to yield best concentration of pure RNA (1846.50 ng/µl and 1.92) as compared to group 2B (n = 15) with no malignancy in imprints (102.61 ng/µl and 1.53). The correlation of imprint smears with their corresponding H&E-stained slides further leads to grouping of each group in 2 groups. RT-PCR analyses showed better melting peaks and high relative expression of CCND1 in group 2 A. CONCLUSION: Touch imprints may provide valuable information regarding presence or absence of tumor in tissue samples being used for extraction of genetic material. This approach can be used as easy, cheap and fast strategy to resolve the doubts associated with RNA being truly representative of the tumor.

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.

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.

Comparison of freeze-thaw and sonication cycle-based methods for extracting AMR-associated metabolites from Staphylococcus aureus.

Emerging antimicrobial resistance (AMR) among Gram-positive pathogens, specifically in Staphylococcus aureus (S. aureus), is becoming a leading public health concern demanding effective therapeutics. Metabolite modulation can improve the efficacy of existing antibiotics and facilitate the development of effective therapeutics. However, it remained unexplored for drug-resistant S. aureus (gentamicin and methicillin-resistant), primarily due to the dearth of optimal metabolite extraction protocols including a protocol for AMR-associated metabolites. Therefore, in this investigation, we have compared the performance of the two most widely used methods, i.e., freeze-thaw cycle (FTC) and sonication cycle (SC), alone and in combination (FTC + SC), and identified the optimal method for this purpose. A total of 116, 119, and 99 metabolites were identified using the FTC, SC, and FTC + SC methods, respectively, leading to the identification of 163 metabolites cumulatively. Out of 163, 69 metabolites were found to be associated with AMR in published literature consisting of the highest number of metabolites identified by FTC (57) followed by SC (54) and FTC + SC (40). Thus, the performances of FTC and SC methods were comparable with no additional benefits of combining both. Moreover, each method showed biasness toward specific metabolite(s) or class of metabolites, suggesting that the choice of metabolite extraction method shall be decided based on the metabolites of interest in the investigation.