Persistent organic pollutants-environmental risk factors for diabetes mellitus? - A population-based study.

BACKGROUND: Globally, type-2 diabetes mellitus is increasing in epidemic proportions. A major cause of concern in India is the increasing incidence of cases, especially troubling is the observed increase in younger age groups with no risk factors. New evidence suggests that many environmental factors, such as air pollution, persistent organic pollutants (POPs), and environmental estrogens are implicated as risk factors for type-2 diabetes mellitus. Animal and human epidemiological studies have shown ubiquitous lipophilic substances, including POPs, are frequently associated with type-2 diabetes mellitus. Such studies have not been undertaken in Indian youth. METHODS: This is a cross-sectional study that explored the association between POPs and type-2 diabetes mellitus in Indian urban and rural population. About 7 ml of venous blood was collected from all consenting patients and serum was separated immediately and was transported to the lab for further analysis. Serum levels of POPs, including organochlorine (OC) compounds and organophosphorus pesticides, were estimated using sample gas chromatography-mass spectrometry (GC-MS). The fasting blood sugar values and the serum levels of POPS were tested using Pearson correlation coefficient. The magnitude of increase in blood sugar corresponding to increase in POPs was analyzed using linear regression analysis. The odds ratios (ORs) were expressed at 95% confidence intervals (CIs). RESULTS: Three OC pesticides and one organophosphate pesticide were strongly associated with increasing blood sugar levels after adjusting for age, sex, and body mass index - lindane (OR 4.95, 95% CI 1.03-23.73), DDT o, p' (OR 3.50, 95% CI 1.04-11.73), dimethoate (OR 19.31, 95% CI 4.22-88.37), and dichlorvas (OR 6.33, 95% CI 1.28-31.18).

The genetics of cardiac failure: Role of a G protein-coupled receptor polymorphism in therapeutic response in an Indian population.

BACKGROUND AND AIM: The incidence of heart failure (HF) is rising to epidemic proportions in developing countries like India. A lack of adequate Indian studies underscores the importance of pursuing research into HF in an Indian population. G protein-coupled receptor kinase 5 (GRK5) Gln41>Leu (rs2230345) polymorphism was reported as a genetic modifier associated with survival in HF patients. A prospective study was conducted to investigate the association of GRK5 Gln41>Leu polymorphism with response to ß-blocker therapy in Indian HF patients. METHODS: HF patients (n=584) were recruited for the study. The patients were genotyped by tetra-primer based allele specific polymerase chain reaction and confirmed with Sanger sequencing. The HF patients were evaluated for GRK5 gene expression and followed up for ~3 years. Drug dosages, cardiac output and hospitalization-free survival were evaluated as study outcomes. HF subgroups (i.e. systolic or diastolic dysfunction, biventricular dysfunction and pulmonary artery hypertension) were also analyzed in association with hospital-free survival. RESULTS: HF patients showed genotype frequencies of AT (15%) and TT (1%). AT/TT genotype carriers showed downregulated GRK5 gene expression and significant reduction in carvedilol drug dosage (p=0.0001). Moreover, AT/TT genotype carriers on ß-blockers showed improved ejection fraction from 27% to 36% (p=0.0007) and increased hospitalization-free survival in comparison to other HF patients. HF patients with AA genotype showed an increased rate of hospital admission in comparison with patients with the AT/TT genotype. HF subgroups with the AT/TT genotype showed an increased hospitalization-free survival versus subgroups with the AA genotype. CONCLUSIONS: GRK5 Gln41>Leu polymorphism in response to ß-blocker therapy improved cardiac function in HF patients. RELEVANCE FOR PATIENTS: This study presents a comprehensive clinicofunctional pharmacogenetic characterization of GRK5 Gln41>Leu polymorphism in a cohort of Indian HF patients. GRK5 Gln41>Leu polymorphism can confer improved cardiac function and reduce hospitalization, thus improving the quality of life in HF patients.

Correlation of the Expression of BRAF V600E Mutation With Various Phenotypic Expressions of Thyroid Neoplasms.

Aims We aimed to assess the incidence of the BRAF V600E mutation in thyroid neoplasms at a tertiary care center and its association with various phenotypic features. Methods and material We included all cases diagnosed as thyroid neoplasm in the past decade at the Department of Pathology of our institute and obtained their clinical details from the medical records department of the institute after obtaining permission from the authorities and due International Human Epigenome Consortium clearance. We included data on age, sex, clinical presentation, hormone status, and T and N status of the malignant neoplasms. Hematoxylin and eosin (H&E) slides of all cases were evaluated for the type of neoplasm, nuclear features, invasion into the capsule and vascular spaces, extrathyroidal extension, lymph node metastases, mitoses, necrosis, and presence/absence of amyloid. Paraffin blocks of sections with high tumor density and less normal tissue were chosen for evaluation after H&E staining. The slides showing tumors with large areas of hemorrhage, cystic change, or necrosis were excluded. Two primers were used to amplify a 339-bp fragment containing the V600E mutation in exon 15 of BRAF. Tissues were prepared from formalin-fixed paraffin-embedded (FFPE) blocks, and DNA was isolated using a standard protocol BRAF NF and BRAF NR Primer Standardized Protocol For FFPE Tissue DNA. Percentages and tables have been used for data presentation. Results Among 47 identified cases, 14 were positive for the BRAF V600E mutation and had papillary carcinoma (n = 9) or follicular neoplasms (n = 5; follicular adenoma, n = 3; follicular carcinoma, n = 2). In the BRAF-positive papillary carcinomas, five cases were aged 20-30 years, eight were female, eight (88.88%) were euthyroid, and one was hypothyroid. Furthermore, 55.55% (5/9 cases) of BRAF-positive cases were stage I, 33.3% (3/9 cases) were stage II, and 0.02% (1/9 cases) were stage III. Conclusions In our cohort, 31% of cases of papillary thyroid carcinoma (PTC) and 18.72% of follicular neoplasms expressed the BRAF V600E mutation. BRAF V600E mutation-positive papillary thyroid carcinomas consistently showed all characteristic nuclear features, such as nuclear crowding, overlapping, and grooves. Considering the greater prevalence in the younger age group, the importance of mutation surveillance in PTCs for a total thyroidectomy may be warranted in mutation-positive patients.

Cirrhotics with Monocyte Chemotactic Protein 1 Polymorphism are at Higher Risk for Developing Spontaneous Bacterial Peritonitis - A Cohort Study.

BACKGROUND: Spontaneous bacterial peritonitis (SBP) is a complication of liver cirrhosis and its occurrence portends poor patient survival. There is emerging evidence that genetic predisposition could significantly alter the occurrence and course of SBP. Monocyte chemotactic protein 1 (MCP1) is a potent chemokine that perpetuates the pro-inflammatory milieu in SBP. AIM: This study aimed at investigating MCP1 genotype polymorphism and its survival impact in patients with decompensated liver cirrhosis. METHODS: We recruited 107 individuals with decompensated liver cirrhosis and categorized them into two groups. Patients having SBP formed the cases (Group 1) and controls were patients without SBP (Group 2). MCP1 polymorphism (-2518A/G) was assessed in both groups by restriction fragment length polymorphism method. The Chi-square test was used to assess the differences in categorical variables and Kaplan-Meier analyses were used to assess the survival. RESULTS: Patients with SBP (36.5%) had higher frequency of G allele than patients without SBP (23%) (P=0.031; odds ratio=1.955, 95% confidence interval: 1.0553-3.6216). Kaplan-Meir analysis revealed that presence of SBP (P=0.030) and G allele (P=0.021) had significantly reduced the likelihood of survival among cirrhotics. CONCLUSIONS: Cirrhotic patients with MCP1 G allele have a higher risk for developing SBP. In general, the presence of the MCP1 polymorphic G allele (AG/GG genotype) reduced the likelihood of survival among patients with cirrhosis. RELEVANCE FOR PATIENTS: This study identifies a critical subgroup of patients with SBP and also predicts prognosis in these individuals. The presence of this genetic polymorphism in addition to the underlying clinical condition may prompt aggressive monitoring, treatment, and follow-up.

In-silico analysis of Covid-19 genome sequences of Indian origin: Impact of mutations in identification of SARS-Co-V2.

Covid-19 disease caused by SARS-CoV-2 is still being transmitted in developed and developing countries irrespective of healthcare setups. India with 1.3 billion people in the world is severely affected by Covid-19 with 11.3 million cases and 157 000 deaths so far. We have assessed the mismatches in WHO recommended rRT-PCR assays primer and probe binding regions against SARS-CoV-2 Indian genome sequences through in-silico bioinformatics analysis approach. Primers and probe sequences belonging to CN-CDC-ORF1ab from China and HKU-ORF1b from Hong Kong targeting ORF1ab gene while NIH-TH-N from Thailand, HKU-N from Hong Kong and US-CDCN-2 from USA targeting N genes displayed accurate matches (>98.3%) with the 2019 novel corona virus sequences from India. On the other hand, none of the genomic sequences displayed exact match with the primer/probe sequences belonging to Charité-ORF1b from Germany targeting ORF1ab gene. We think it will be worthwhile to release this information to the clinical and medical communities working in Indian Covid-19 frontline taskforce to tackle the recently emerging Covid-19 outbreaks as of March-2021.

Papillon-Lefèvre syndrome (PLS) with novel compound heterozygous mutation in the exclusion and Peptidase C1A domains of Cathepsin C gene.

Papillon Lefevre syndrome (PLS) manifests with palmoplantar keratoderma, combined with a rapidly progressive periodontitis associated with mutations in Cathepsin C (CTSC) gene. This article reports a 15-year old male proband with typical PLS traits having a novel compound heterozygote with p.Q49X mutation in exon 1 and p.Y259C missense mutation in exon 6 of CTSC gene respectively. The exon 1 mutation, p.Q49X, (found in proband's mother) was located in exclusion domain and exon 6 mutation, p.Y259C (found in proband's father), was present in peptidase C1A, papain C-terminal domain. Interestingly, missense mutation p.Y259C identified in this study was found to be not reported so far. Upon computational analysis, this missense mutation was found to be lethal. Moreover, our protein modelling approach using mutant protein revealed the presence of monomeric structure on contrary to the tetrameric structure of the wild type protein. In addition, in vitro functional characterization of mutant p.Y259C expressed in HEK293 cells showed a significant reduction in CTSC activity (0.015 ± 0.009 mU/ml) when compared with wild type protein (0.21 ± 0.008 mU/ml). Thus, in this study, we have demonstrated that the pathogenic missense mutant p.Y259C might cause PLS by impaired CTSC function.

In vitro ubiquitination of Mycobacterium tuberculosis by E3 ubiquitin ligase, MKRN1.

OBJECTIVES: Ubiquitination has a role as a host defense mechanism against pathogens. To channelize autophagic mycobacteria to destruction, ubiquitin ligase like, Makorin Ring Finger Protein 1 (MKRN1) was speculated to play a role in ubiquitinating M. tuberculosis. We have developed a flow cytometry based in vitro ubiquitin ligase assay to understand the role of MKRN1 in ubiquitinating mycobacteria and confirmed the results by western blotting. RESULTS: MKRN1 was cloned and expressed in E. coli BL21 (DE3) strain. The recombinant MKRN1 protein was solubilised, purified and refolded to restore the activity. In addition, through autoubiquitination assay, the activity of protein was confirmed. The corresponding E1 and E2 enzymes for MKRN1, UBE1 and UBE2D3 respectively, were selected using BioGrid tool. Surprisingly, flow cytometric assay revealed that at a concentration of 300 nM of MKRN1, 38% of M. tuberculosis was found to be ubiquitinated in vitro with 3.5% of the cells having bound MKRN1. Immunoblot results also substantiates the ubiquitination of M. tuberculosis. MKRN1 did not ubiquitinate B. Subtilis and therefore, we speculate that the E3 Ub ligase activity might be specific to M. tuberculosis. CONCLUSION: This clearly demonstrates that recombinant MKRN1 ubiquitinates M. tuberculosis which opens up a novel, potential role of MKRN1 against mycobacteria which has to be unfolded.

Comparison of Polymerase Chain Reaction (PCR), Microbiological and Histopathological Observations in the Diagnosis of Endometrial Tuberculosis.

BACKGROUND: Female genital tuberculosis often faces diagnostic challenges due to the asymptomatic nature of the disease. Our study aims at comparing the microbiological and histopathological results with PCR in diagnosing genital tuberculosis in endometrial curettage specimens. METHODS: Around 139 patients with diverse gynaecological complaints were recruited for the study, and endometrial curettage specimens were collected. The specimens were subjected to microbiological culture and staining, histopathological examination and PCR to look for the presence of M. tuberculosis. Statistical analyses of the PCR results include calculating sensitivity, specificity, positive and negative prediction values and positive and negative likelihood ratios. RESULTS: PCR yielded a detection rate of 41.7% (58/139) when compared to the microbiology (2.15%) and histopathology results (1.43%). PCR with hsp65 and cfp10, in combination, detected 20% of the cases. Statistical analyses were suggestive that PCR with hsp65 showed a higher sensitivity and specificity of 50% and 92.59% respectively. CONCLUSION: The results obtained in this study suggest that for a definitive diagnosis, combinations of the results from various diagnostics techniques can only be considered.

Evidence for a hyper-reductive redox in a sub-set of heart failure patients.

BACKGROUND: Oxidative stress has been linked to heart failure (HF) in humans. Antioxidant-based treatments are often ineffective. Therefore, we hypothesize that some of the HF patients might have a reductive stress (RS) condition. Investigating RS-related mechanisms will aid in personalized optimization of redox homeostasis for better outcomes among HF patients. METHODS: Blood samples were collected from HF patients (n = 54) and healthy controls (n = 42) and serum was immediately preserved in - 80 °C for redox analysis. Malondialdehyde (MDA; lipid peroxidation) levels by HPLC, reduced glutathione (GSH) and its redox ratio (GSH/GSSG) using enzymatic-recycling assay in the serum of HF patients were measured. Further, the activities of key antioxidant enzymes were analyzed by UV-Vis spectrophotometry. Non-invasive echocardiography was used to relate circulating redox status with cardiac function and remodeling. RESULTS: The circulatory redox state (GSH/MDA ratio) was used to stratify the HF patients into normal redox (NR), hyper-oxidative (HO), and hyper-reductive (HR) groups. While the majority of the HF patients exhibited the HO (42%), 41% of them had a normal redox (NR) state. Surprisingly, a subset of HF patients (17%) belonged to the hyper-reductive group, suggesting a strong implication for RS in the progression of HF. In all the groups of HF patients, SOD, GPx and catalase were significantly increased while GR activity was significantly reduced relative to healthy controls. Furthermore, echocardiography analyses revealed that 55% of HO patients had higher systolic dysfunction while 62.5% of the hyper-reductive patients had higher diastolic dysfunction. CONCLUSION: These results suggest that RS may be associated with HF pathogenesis for a subset of cardiac patients. Thus, stratification of HF patients based on their circulating redox status may serve as a useful prognostic tool to guide clinicians designing personalized antioxidant therapies.

Chronic Endurance Exercise Impairs Cardiac Structure and Function in Middle-Aged Mice with Impaired Nrf2 Signaling.

Nuclear factor erythroid 2 related factor 2 (Nrf2) signaling maintains the redox homeostasis and its activation is shown to suppress cardiac maladaptation. Earlier we reported that acute endurance exercise (2 days) evoked antioxidant cytoprotection in young WT animals but not in aged WT animals. However, the effect of repeated endurance exercise during biologic aging (WT) characterized by an inherent deterioration in Nrf2 signaling and pathological aging (pronounced oxidative susceptibility-Nrf2 absence) in the myocardium remains elusive. Thus, the purpose of our study was to determine the effect of chronic endurance exercise-induced cardiac adaptation in aged mice with and without Nrf2. Age-matched WT and Nrf2-null mice (Nrf2-/-) (>22 months) were subjected to 6 weeks chronic endurance exercise (25 meter/min, 12% grade). The myocardial redox status was assessed by expression of antioxidant defense genes and proteins along with immunochemical detection of DMPO-radical adduct, GSH-NEM, and total ubiquitination. Cardiac functions were assessed by echocardiography and electrocardiogram. At sedentary state, loss of Nrf2 resulted in significant downregulation of antioxidant gene expression (Nqo1, Ho1, Gclm, Cat, and Gst-α) with decreased GSH-NEM immuno-fluorescence signals. While Nrf2-/- mice subjected to CEE showed an either similar or more pronounced reduction in the transcript levels of Gclc, Nqo1, Gsr, and Gst-α in relation to WT littermates. In addition, the hearts of Nrf2-/- on CEE showed a substantial reduction in specific antioxidant proteins, G6PD and CAT along with decreased GSH, a pronounced increase in DMPO-adduct and the total ubiquitination levels. Further, CEE resulted in a significant upregulation of hypertrophy genes (Anf, Bnf, and ß-Mhc) (p < 0.05) in the Nrf2-/- hearts in relation to WT mice. Moreover, the aged Nrf2-/- mice exhibited a higher degree of cardiac remodeling in association with a significant decrease in fractional shortening, pronounced ST segment, and J wave elevation upon CEE compared to age-matched WT littermates. In conclusion, our findings indicate that while the aged WT and Nrf2 knockout animals both exhibit hypertrophy after CEE, the older Nrf2 knockouts showed ventricular remodeling coupled with profound cardiac functional abnormalities and diastolic dysfunction.

Genotype Phenotype Correlation of Genetic Polymorphism of PPAR Gamma Gene and Therapeutic Response to Pioglitazone in Type 2 Diabetes Mellitus- A Pilot Study.

INTRODUCTION: Pro12Ala polymorphism is a missense mutation at codon 12 in peroxisome proliferator-activated receptor γ gene (PPARG). This polymorphism is known to be associated with increased insulin sensitivity. Pioglitazone, a thiazolidinedione, is an anti-diabetic drug which acts as an agonist at PPAR γ receptor. AIM: To determine the association between Pro12Ala polymorphism of the PPARG and variation in therapeutic response to the PPARγ agonist, pioglitazone. MATERIALS AND METHODS: The study was done as a hospital based pilot project in 30 patients with type 2 diabetes mellitus, on treatment with sulfonylurea or metformin but without adequate glycaemic control. They were started on pioglitazone as add on therapy for a period of 12 weeks. The participants were categorized as responders and non-responders based on the change in HbA1C level after 12 weeks. Pro12Ala polymorphism was analysed by polymerase chain reaction-restriction fragment length polymorphism. STATISTICAL ANALYSIS: Logistic regression analysis was done to evaluate the associations between age, baseline body weight, BMI, waist circumference, waist-hip ratio and Pro12Ala variants with the response to pioglitazone. The p-value< 0.05 was considered significant. RESULTS: The frequency distributions of PPAR gamma genotypes were 80% for Pro/Pro and 20% for Pro/Ala in the study population. Among the study participants, 30% were non-responders and 70% responders to pioglitazone. A significantly higher frequency of the polymorphism was detected in the responders (p=0.005) compared to non-responders group. CONCLUSION: Our study suggests that there is a potential association between Pro12Ala polymorphism and glycaemic response to pioglitazone.

Ethics workshops-are they effective in improving the competencies of faculty and postgraduates?

INTRODUCTION: Responsible conduct of research requires a good knowledge about research ethics. With the recent changes in the clinical trial regulations and the proposed introduction of ethics in medical curriculum by the Medical Council of India, there is an urgent need to train the medical faculty and postgraduates in research ethics. METHODOLOGY: We wanted to measure the effectiveness of a one day program which was organized using didactic lectures and case scenarios on the knowledge, attitude and skills on ethics among faculty and postgraduates. This was done using a retropre questionnaire. We performed a Kolmogorov Smirnov test to measure the normality, Mann Whitney U-test to test the difference in scores between faculty and postgraduates and a Wilcoxin signed rank test to measure the prepost scores. RESULTS: The faculty showed better scores in knowledge and attitude (p<0.05) when compared to postgraduates both before and after the workshop. The overall scores for both faculty and postgraduates had increased after the workshop (p<0.05). CONCLUSION: This re-emphasizes the need for introduction of ethics training during undergraduate course and also the fact that even a short training program in research ethics could be effective.

Diagnostic testing by CFTR gene mutation analysis in a large group of Hispanics: novel mutations and assessment of a population-specific mutation spectrum.

Characterization of CFTR mutations in the U.S. Hispanic population is vital to early diagnosis, genetic counseling, patient-specific treatment, and the understanding of cystic fibrosis (CF) pathogenesis. The mutation spectrum in Hispanics, however, remains poorly defined. A group of 257 self-identified Hispanics with clinical manifestations consistent with CF were studied by temporal temperature gradient electrophoresis and/or DNA sequencing. A total of 183 mutations were identified, including 14 different amino acid-changing novel variants. A significant proportion (78/85) of the different mutations identified would not have been detected by the ACMG/ACOG-recommended 25-mutation screening panel. Over one third of the mutations (27/85) occurred with a relative frequency >1%, which illustrates that the identified mutations are not all rare. This is supported by a comparison with other large CFTR studies. These results underscore the disparity in mutation identification between Caucasians and Hispanics and show utility for comprehensive diagnostic CFTR mutation analysis in this population.