Socio-demographic determinants of cardiovascular risk in rural population of Central India.

Introduction: Raising trend of cardiovascular diseases (CVD) in developing countries created a platform for exploring the sociodemographic nexus in search of underlying cause. Aim and Objectives: The precise aim of the study is to detect any possible association of social determinants and metabolic derangement with CVDs risk, particularly focusing on comparative analysis of the data to decipher the most significant factor(s), if any among the studied parameters contributing toward prediction of such cardiometabolic risk in linked with insulin resistance. Results: In the present study, it was found that 2% of the studied population had high risk, and 13.3% had intermediate risk of developing cardiovascular events in next 10 years. Results also showed that estimated CVD risk was significantly higher in males with central obesity and age more >60 years as key determinants showing more insulin resistance at lower cut-off. Conclusion: This study also strongly suggests need to revise the cut-off values for HOMA index in defining insulin resistance to rural population with active the life style and need of redefining new targeted preventive health care planning.

Assessment of Oxidative Stress Biomarkers and Body Mass Index in Pulmonary Tuberculosis Patients: A Case-Control Study.

Background: Pulmonary tuberculosis (PTB) is a major public health concern in most underdeveloped and developing countries. PTB affects the nutritional status of the patients and influences the body mass index (BMI). There is tissue inflammation and free radical burst from activated phagocytes resulting in oxidative stress. The present study was designed to assess the relationship between oxidative stress and body mass index in newly detected pulmonary tuberculosis patients. Method: This was a case-control study designed to assess oxidative stress parameters such as nitric oxide (NO) and malondialdehyde (MDA) in 40 consecutives newly diagnosed PTB patients and compared with 40 age-matched healthy controls. The nutritional status of the study subjects was measured by calculating the BMI. Results: The mean BMI was 21.61±3.52 Kg/m2 in controls and 17.47±1.56 Kg/m2 in PTB patients and the difference was statistically significant (p <0.0001). The mean levels of MDA (7.65±0.65 nmol/ml) and NO (36.12±1.07 µmol/l) were significantly higher in PTB patients compared to controls (MDA 3.56±0.41 nmol/ml and NO 14.48±0.93 µmol/l). Conclusions: Increased levels of malondialdehyde and nitric oxide were observed in newly diagnosed PTB patients when compared to controls indicating oxidative stress in PTB. The BMI of these patients was significantly lower than the controls. Thus, it is concluded that there is an inverse relationship between oxidative stress and BMI in PTB patients and antioxidant supplementation in addition to nutritional intervention under the National Tuberculosis Elimination Program may help to improve the BMI and promote better recovery in these patients.

Immunological host responses as surveillance and prognostic markers in tubercular infections.

Background: Tuberculosis (TB) control is challenging due to failure of drug compliance and resistance. Mycobacterial antigen-induced cytokine secretions are helpful in detecting Mycobacterium tuberculosis infection and to determine prediction for the fate of TB infection and its cure. Considering immunological response to be a crucial factor in pathogenesis and cure of TB, it can be explored to determine clinical prospects in different categorical tubercular infections. This study was designed to compare serum levels of inflammatory (tumor necrosis factor [TNF]-alpha and interferon [IFN]-gamma) and anti-inflammatory cytokines/chemokines (interleukin [IL]-10 and IL-8) among different TB groups (freshly diagnosed, relapse, cases on antitubercular treatment [ATT], and healthy controls). Methods: This cross-sectional study included total 100 subjects. The study subjects were further divided into four study groups with 25 cases in each of freshly diagnosed TB, TB relapse cases, cases on ATT, and 25 healthy controls. Levels of serum cytokines/chemokines (TNF-alpha, IFN-gamma, IL-10, and IL-8) were measured by flow cytometry. Results: Data analysis observed statistically significant differences in serum levels of TNF-alpha and IFN-gamma among the studied groups with significantly low levels in subjects on ATT and markedly high levels in TB relapse subjects. No statistically significant difference was observed in IL-10 and IL-8 levels. However, subjects with relapse revealed low IL-8 and high IL-10 levels. Conclusion: TNF-alpha and IFN-gamma have important roles in immune response and might be considered as indicators for response to ATT. However, high levels of IL-10 with low IL-8 appear to be associated with poor outcome and possibility of relapse.

Oxidative stress and its impact on mitochondrial DNA in pulmonary tuberculosis patients- a pilot study.

BACKGROUND: Pulmonary tuberculosis (PTB) remains a major cause of morbidity and mortality all around the world. Recent studies have pointed out increased oxidative stress and also DNA damage in peripheral blood in PTB. Till date, to the best of our knowledge, no study has so far been conducted to show the mitochondrial DNA (mtDNA) deletions mapping in PTB patients. Therefore we performed the present study with the aim to investigate oxidative stress parameters along with mtDNA damage in newly diagnosed untreated PTB patients. MATERIAL AND METHODS: This is a prospective study carried out in Mahatma Gandhi Institute of Medical Sciences, Sevagram,Wardha, Maharashtra during september 2017 to september 2018.Thirty newly diagnosed untreated PTB patients and thirty age matched healthy controls were enrolled in the present study. Analysis of Oxidative stress parameters such as nitric oxide (NO) and malondialdehyde (MDA) were done by calorimetric methods. Assessment of mitochondrial DNA damage was carried out by mtDNA deletions mapping using primer shift long range polymerase chain reaction technique. RESULTS: There was significant increase in levels of oxidative stress parameters, nitric oxide and malondialdehyde, in PTB patients compared to controls (p < 0.01). Generally there are two common deletion sites of "13 bp direct repeats" (ACCTCCCTCACCA) in mtDNA. One at the junction sites from bp 8470 to 8482 bp and another from bp 13447 to 13460 bp which make mtDNA more prone for 4977bp deletion. Out of thirty cases of PTB, two cases showed mtDNA damage in the form of mtDNA deletion of 4977bp. There was no mtDNA deletion in any control which can be attributed to continuous generation of oxidative stress. CONCLUSION: This pilot study has been able to demonstrate that compared to controls, in newly diagnosed pulmonary tuberculosis patients some mtDNA damage did occur and was probably due to continuous generation of oxidative stress in tuberculous patients. However, sample size is too small to draw any conclusions but definitely a more comprehensive study, by recruiting more number of pulmonary tuberculosis patients is warranted to establish correlation between oxidative stress and mtDNA damage in PTB.

Identification of potential inhibitors for mycobacterial uridine diphosphogalactofuranose-galactopyranose mutase enzyme: A novel drug target through in silico approach.

Background: The Mycobacterium tuberculosis (MTB) uridine diphosphogalactofuranose (UDP)-galactopyranose mutase (UGM) is an essential flavoenzyme for mycobacterial viability and an important component of cell wall. It catalyzes the interconversion of UDP-galactopyranose into UDP-galactofuranose, a key building block for cell wall construction, essential for linking the peptidoglycan and mycolic acid cell wall layers in MTB through a 2-keto intermediate. Further, as this enzyme is not present in humans, it is an excellent therapeutic target for MTB. Thus, inhibition of this UGM enzyme is a good approach to explore new anti-TB drug. This study aims to find novel and effective inhibitors against UGM from reported natural phytochemicals and ZINC database using virtual screening approach. Methods: In this study, 148 phytochemicals with reported antitubercular activity and 5280 ZINC compounds with 70% structural similarity with the natural substrate of UGM (UDP-galactopyranose and UDP-galactofuranose) were screened against UGM. Results: In virtual screening, 19 phytochemicals and 477 ZINC compounds showed comparatively better binding affinity than natural substrates. Among them, best 10 compounds from each group were proposed as potential inhibitors for UGM based on the binding energy and protein-ligand interaction analysis. Among phytochemicals, three compounds, namely, tiliacorine, amentoflavone, and 2'-nortiliacorinine showed highest binding affinity (binding energy of -10.5, -10.4, and -10.3 Kcal/mol, respectively), while among ZINC compounds, ZINC08219848 and ZINC08217649, showing highest binding affinity (binding energy of -10.0 and -9.7 Kcal/mol, respectively) toward UGM as compared to its substrates. Conclusion: These selected compounds may be proposed as potential inhibitors of UGM and need to be tested in TB culture studies in vitro to assess their anti-TB activity.

Microalbuminuria: A Mere Marker or An Ominous Sign?

Microalbuminuria represents a condition wherein the urinary albumin excretion is in the range of 30 mg/24 hrs -300 mg/24 hrs. Recently microalbuminuria is gaining attention as more than just a surrogate marker of kidney damage. Although apparently representing passage of a rather trace amount of protein excretion, this has great implication in terms of clinico-pathological association with diabetic nephropathy and other cardiovascular complications in subjects with or without diabetes. Early detection of microalbuminuria is considered to be the aim of clinicians in context to patient's health perspective so as to change the treatment strategy accordingly. Therefore quite reasonably, understanding of exact patho-physiological significance of microalbuminuria and its prognostic impact in kidney diseases should be of immense help in providing better clinical outcome. To this end, an approach from the clinical biochemistry perspective might provide a better overview. The present commentary is an endeavor to address several relevant issues in this context for development of a better insight.

Study of mechanism of interaction of truncated isoniazid-nicotinamide adenine dinucleotide adduct against multiple enzymes of Mycobacterium tuberculosis by a computational approach.

OBJECTIVE/BACKGROUND: Isoniazid (INH) is one of the effective antituberculosis (TB) drugs used for TB treatment. However, most of the drug-resistant Mycobacterium tuberculosis (MTB) clinical strains are resistant to INH, a first-line antituberculous drug. Certain metabolic enzymes such as adenosylhomocysteinase (Rv3248c), universal stress protein (Rv2623), nicotinamide adenine dinucleotide (reduced)-dependent enoyl-acyl carrier protein reductase (Rv1484), oxidoreductase (Rv2971), dihydrofolate reductase (Rv2763c), pyrroline-5-carboxylate dehydrogenase (Rv1187) have been identified to bind INH-nicotinamide adenine dinucleotide (INH-NAD) and INH-nicotinamide adenine dinucleotide phosphate adducts coupled to Sepharose resin. These enzymes are reported to be involved in many important biochemical processes of MTB, including cysteine and methionine metabolism, mycobacterial growth regulation, mycolic acid biosynthesis, detoxification of toxic metabolites, folate biosynthesis, etc. The truncated INH-nicotinamide adenine dinucleotide (oxidized) adduct, 4-isonicotinoylnicotinamide, isolated from urine samples of human TB patients treated with INH therapy is proposed to have antimycobacterial activity. METHODS: To understand the mechanism of interaction of the truncated INH-NAD adduct, binding energy studies were carried out on the aforementioned six enzymes with known three-dimensional structures using AutoDock4.2. RESULTS: In silico docking analysis of these MTB enzymes with the truncated INH-NAD adduct showed favorable binding interactions with docking energies ranging from -5.29 to -7.07 kcal/mol. CONCLUSION: Thus, in silico docking study revealed that the INH-NAD adduct, which is generated in vivo after INH activation, may undergo spontaneous hydrolysis to form the truncated INH-NAD adduct and further binds and inhibits multiple enzymes of MTB, in addition to InhA, confirming that INH is an effective anti-TB drug acting at multiple enzymes. Further analysis of amino acid residues in the active site of INH-NAD-binding proteins showed the probable presence of catalytic triad in four enzymes possibly involved in INH binding to the enzyme.

Computational approach to understanding the mechanism of action of isoniazid, an anti-TB drug.

Tuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis (MTB), which remains a major cause for morbidity and mortality in several developing countries. Most drug-resistant MTB clinical strains are resistant to isoniazid (INH), a first-line anti-TB drug. Mutation in KatG, a catalase-peroxidase, of MTB is reported to be a major cause of INH resistance. Normally upon activation by KatG, INH is converted to an active intermediate which has antimycobacterial action in MTB. This INH intermediate in the presence of NADH forms INH-NAD adduct which inhibits inhA (2-trans-enoyl-acyl carrier protein reductase) of MTB, thus blocking the synthesis of mycolic acid, a major lipid of the mycobacterial cell wall. In this docking study, the high binding affinity of INH-NAD adduct towards InhA was observed in comparison with INH alone. In this study, two resistant mutants of KatG (S315T and S315N) were modeled using Modeller9v10 and docking analysis with INH was performed using AutoDock4.2 and the docking results of these mutants were compared with the wild type KatG. Docking results revealed the formation of a single hydrogen (H) bond between the secondary amine nitrogen (-NH) of INH with Thr or Asn residues in place of Serine at 315 position of KatG mutant strains respectively, whereas in the case of the wild type, there was no H-bond formation observed between INH and Ser315. The H-bond formation may prevent free radical formation by KatG in mutant strains thus the development of resistance to the drug. This in silico evidence may implicate the basis of INH resistance in KatG mutant strains.

Elisa protocol for rapid screening of potential anti-tubercular drugs based on antigenic reactivity of mycobacterial ES-31 serine protease - a drug target supported by axenic culture of Mycobacterium tuberculosis H37 Ra strain in the presence of inhibitor.

BACKGROUND: Mycobacterial ES-31 serine protease has been reported to be a drug target using protease and lipase inhibitors in axenic and macrophage cultures. Simple screening techniques are needed for rapid testing of anti-tubercular drugs. AIM: To demonstrate the usefulness of ELISA protocol based on antigenic reactivity of mycobacterial serine protease by indirect ELISA for detecting anti-tubercular activity. MATERIAL AND METHODS: Indirect ELISA for assessment of antigenic reactivity of mycobacterial ES-31 serine protease was standardized using ES-31Ag and anti-DSS-goat-serum and assessed the inhibition of the antigenic reactivity by isoniazid, an anti-tubercular drug and serine protease inhibitor and orlistat, a lipase inhibitor. RESULTS: Optimal antigenic reactivity of mycobacterial ES-31 serine protease was observed at 5 microg/well of ES-31 antigen and at 1:25 dilution of anti-DSS-goat-serum. Isoniazid showed 42% inhibition of ES-31 serine protease at 0.4 microg/well, while orlistat showed inhibition of 60% at 0.5 microg/well. Inhibition of Mtb H,37Ra bacilli is further confirmed in axenic culture. 35% and 29% inhibition by isoniazid at 0.4 microg/well and orlistat at 0.5 microg/well were observed respectively on bacterial growth. CONCLUSION: Simple ELISA protocol based on assay of antigenic reactivity of mycobacterial ES-31 serine protease, a drug target, has been standardized for rapid screening of potential anti-tubercular drugs.

Inhibitory effect of isoniazid and orlistat combination on mycobacterial ES-31 serine protease in vitro and on the growth of M.tb bacilli in axenic culture.

BACKGROUND: Isoniazid and orlistat were reported to have inhibitory effect on mycobacterial ES-31 serine protease in vitro and bacterial cell growth in axenic culture. AIM: To study the cumulative effect and understand drug - drug interaction, if any, when isoniazid and orlistat used in combination. MATERIAL AND METHODS: Inhibition of mycobacterial ES-31 serine protease by different combinations of orlistat and isoniazid together and individually were studied using azocasein assay. Inhibition of secretion of excretory secretory ES-31 antigen in Sautan culture medium was studied under axenic condition and growth of M. tuberculosis H37Ra bacilli by CFU count on LJ-medium. RESULTS: Orlistat and isoniazid both showed inhibitory activity of ES-31 serine protease in in vitro as well as in vivo. Individually, isoniazid showed 90% inhibition at 200 ng/ml while orlistat at 250 ng/ml showed 65% inhibition of mycobacterial ES-31 serine protease in vitro. A combination of orlistat (250 ng/ml) and isoniazid (200 ng/ml) showed 86% inhibition in vitro while 73% inhibition was observed by orlistat (25 ng/ml) and isoniazid (200 ng/ml) on bacterial growth in axenic culture. CONCLUSION: Significant inhibition by orlistat suggests that it could be tried in patients with intolerance to isoniazid or in those already developed isoniazid resistance. It may also be explored in the suspected TB patients as initial medication in place of antibiotics for clinical relief.