Genetic insights into obesity: in silico identification of pathogenic SNPs in MBOAT4 gene and their structural molecular dynamics consequences.

Membrane Bound O-Acyltransferase Domain-Containing 4 (MBOAT4) protein catalyzes ghrelin acylation, leading to prominent ghrelin activity, hence characterizing its role as an anti-obesity target. We extracted 625 exonic SNPs from the ENSEMBL database and one phenotype-based missense mutation associated with obesity (A46T) from the HGMD (Human Gene Mutation Database). These were differentiated on deleterious missense SNPs of the MBOAT4 gene through MAF (minor allele frequency: <0.01) cut-off criteria in relation to some bioinformatics-based supervised machine learning tools. We found 8 rare-coding and harmful missense SNPs. The consensus classifier (PredictSNP) tool predicted that the SNP (G57S, C: rs561065025) was the most pathogenic. Several trained in silico algorithms have predicted decreased protein stability [ΔΔG (kcal/mol)] function in the presence of these rare-coding pathogenic mutations in the MBOAT4 gene. Then, a stereochemical quality check (i.e. validation and assessment) of the 3D model was performed, followed by a blind cavity docking approach, used to search for druggable cavities and molecular interactions with citrus flavonoids of the Rutaceae family, ranked with energetic estimations. Significant interactions with Phloretin 3',5'-Di-C-Glucoside were also observed at R304, W306, N307, A311, L314 and H338 with (iGEMDOCK: -95.82 kcal/mol and AutoDock: -7.80 kcal/mol). The RMSD values and other variables of MD simulation analyses on this protein further validated its significant interactions with the above flavonoids. The MBOAT4 gene and its molecular interactions could serve as an interventional future anti-obesity target. The current study's findings will benefit future prospects for large population-based studies and drug development, particularly for generating personalized medicine.Communicated by Ramaswamy H. Sarma.

Understanding the impact of structural modifications at the NNAT gene's post-translational acetylation site: in silico approach for predicting its drug-interaction role in anorexia nervosa.

PURPOSE: Anorexia nervosa (AN) is a neuropsychological public health concern with a socially disabling routine and affects a person's healthy relationship with food. The role of the NNAT (Neuronatin) gene in AN is well established. The impact of mutation at the protein's post-translational modification (PTM) site has been exclusively associated with the worsening of the protein's biochemical dynamics. METHODS: To understand the relationship between genotype and phenotype, it is essential to investigate the appropriate molecular stability of protein required for proper biological functioning. In this regard, we investigated the PTM-acetylation site of the NNAT gene in terms of 19 other specific amino acid probabilities in place of wild type (WT) through various in silico algorithms. Based on the highest pathogenic impact computed through the consensus classifier tool, we generated 3 residue-specific (K59D, P, W) structurally modified 3D models of NNAT. These models were further tested through the AutoDock Vina tool to compute the molecular drug binding affinities and inhibition constant (Ki) of structural variants and WT 3D models. RESULTS: With trained in silico machine learning algorithms and consensus classifier; the three structural modifications (K59D, P, W), which were also the most deleterious substitution at the acetylation site of the NNAT gene, showed the highest structural destabilization and decreased molecular flexibility. The validation and quality assessment of the 3D model of these structural modifications and WT were performed. They were further docked with drugs used to manage AN, it was found that the ΔGbind (kcal/mol) values and the inhibition constants (Ki) were relatively lower in structurally modified models as compared to WT. CONCLUSION: We concluded that any future structural variation(s) at the PTM-acetylation site of the NNAT gene due to possible mutational consequences, will serve as a basis to explore its relationship with the propensity of developing AN. LEVEL OF EVIDENCE: No level of evidence-open access bioinformatics research.

In Silico Characterization of Withania coagulans Bioactive Compounds as Potential Inhibitors of Hydroxymethylglutaryl (HMG-CoA) Reductase of Mus musculus.

Hypercholesterolemia is a mediator for the etiology of cardiovascular diseases, which are characterized as the global leading cause of mortality. We aimed to investigate the inhibitory activity of Withania coagulans compounds against 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) of Mus musculus using an extensive in silico approach. The 3D structure of the Hmgcr protein is not yet known, so we performed the homology modeling using MODELLER and SWISS-MODEL tools, followed with structural validation and assessment. The PROCHECK web server showed that the top-ranked homology model from SWISS-MODEL has 93.4% of residues in the most-favorable region, the quality factor was 98%, and the Verify3D score was 91.43%, compared to the other generated models. The druggable protein-binding cavities in a 3D model of Hmgcr were investigated with the aid of commonly prescribed statin compounds using the CB-dock approach. We compiled a 3D compound library of W. coagulans, followed by drug-likeness evaluation, and found 20 eligible compounds. The pattern of consensus residues obtained from the CB-dock procedure was then used for grid-box docking of W. coagulans compounds and statin drugs using AutoDock 4.2, respectively. The results showed that withanolide R (-10.77 kcal/mol), withanolide Q (-10.56 kcal/mol), withanolide J (-10.52 kcal/mol), atorvastatin (-8.99 kcal/mol), simvastatin (-8.66 kcal/mol), and rosuvastatin (-8.58 kcal/mol) were promising candidates that bind Hmgcr protein. The key residues involved in protein-ligand (withanolide R) interactions were Y516, C526, V529, I530, M533, I535, and V537, and the formation of a H-bond was at C526, M533, and I535 residues. M533 was the consensus residue having a tendency to form a H-bond with withanolide Q, too. Molecular dynamics simulations were used to validate the top-ranked docked complexes for the stability of the modeled protein. We also predicted the pharmacokinetic properties of binding affinity-based top-ranked compounds and concluded that they could be used as potential inhibitors of Hmgcr. However, further in vitro and in vivo studies are essential to completing the drug development process.

Risk association of fasting insulin resistance index in non-diabetic heart failure patients: A case-control study at Karachi, Pakistan.

Present work investigates the risk association of insulin resistance (IResistance) in Non-Diabetic Heart Failure (NDHF) patients. Eighty (n=80) NDHF patients and same numbers of healthy controls were included to investigate with anthropometric measures, fasting blood glucose level (FBGL), serum insulin (SI), FIRI and ß-cells quantification was computed through HOMA-IR. Mean rank assessment of NDHF patients showed higher significant (p<0.0001) set of values in FBGL, SI, FIRI and HOMA-IR, when compared with controls. High (p<0.0001 & p<0.05) risk in NDHF patients was associated in SI status (OR=8.93-95% CI: 4.1-19.42) and also in HOMA-IR (OR=6.6-95% CI: 3.30-13.19), when compared for Pearson value based probability through Chi Square (χ2 Test) values estimates of probability, respectively. Area under the curve (AUC) of targeted NDHF patients showed higher set of estimation (FBGL-AUC =0.667, SI-AUC =0.763, FIRI-AUC=0.780 and HOMA-IR-AUC=0.776). Association of determinants through Pearson's (r) correlation was found significantly (p<0.0001) linked with HOMA-IR and FIRI. Regression coefficient shows that for every additional unit score in FBGL and SI can expect HOMA-IR to increase by an average of 0.883 (for FBGL) and 0.0368 (for SI), respectively. Findings concluded the association of IResistance with greater risk estimation in NDHF patients.

Risk assessment of insulin resistance with anthropometric measures in non-diabetic heart failure patients: A gender based case-control investigation at tertiary care hospitals of Karachi, Pakistan.

To assess the risk of anthropometric measures and serum markers of insulin resistance in non-diabetic heart failure (NDHF) patients and the difference among male and female subjects. 53 males, 27 females NDHF patients were enrolled and 80 healthy subjects were matched as control. Anthropometric measures, fasting blood glucose level (FBGL) and serum insulin (SI) were measured. Insulin's function (ß-cells quantification) was computed through Homeostasis Model Assessment of Insulin Resistance (HOMA-IR). Significant (p<0.05) difference in height was observed in NDHF male and female patients, whereas, weight was only significant in females. Higher mean values of FBGL, SI, and HOMA-IR in NDHF patients were observed. Highly significant (p<0.0001 & p<0.05) difference was also found in similar gender based case-control markers like FBGL, SI, and HOMA-IR. Phi coefficient for risk associations showed weak-positive correlation in both genders in FBGL, SI, and HOMA-IR. Anthropometric measures indicated lesser risk in both gender, especially in females' decreased risk in body height and waist circumference was observed. Risk measurement through odds ratios (OR) of FBGL and HOMA-IR in female subjects indicated significantly (p<0.001) double risk, whereas, in SI, three fold risks were measured in females as compared to male. Odds of exposure in cases were significantly (p<0.001) greater than in controls verified with significant Pearson and Fisher Exact Probability Test (FEPT) values with two-tailed estimates of probability in χ2 (Chi -Square) estimation test. Findings suggest increased risk of IR in female NDHF patients. Increased in FBGL IR and HOMA-IR and BMI were also found as distinguishing findings in NDHF cases.

Association of serum leptin with serum estradiol in relation to breast carcinogenesis: a comparative case-control study between pre- and postmenopausal women

Background/aim: The aims of this study were to observe the interhormonal relationship of serum leptin with serum estradiol and to compare pre- and postmenopausal women for possible implications in breast carcinogenesis. Materials and methods: This comparative case-control study encompasses 175 breast cancer patients and 175 age-, sex-, and BMI- matched healthy controls. For comparison, the subjects were divided into pre- and postmenopausal groups according to their menstrual history. Blood samples were analyzed for serum leptin and serum estradiol by the ELISA method. Results: Higher mean values of serum estrogen and leptin were observed in breast cancer cases among both the pre- and postmenopausal groups. While estimating the association of serum leptin with serum estradiol, a weak positive (r = 0.186) but statistically significant (P = 0.022) correlation was found among the premenopausal subjects, while a moderate positive association (r = 0.556) was found among the postmenopausal subjects, which had high statistical significance (P = 0.001). Furthermore, correlation coefficients (r) of the pre- and postmenopausal groups were compared using Fisher r-to-z transformation and found to have a statistically significant difference (z = 4.03, P = 0.0001). Conclusion: The findings of this study suggest the mitogenic effects of leptin on mammary tissue via augmenting peripheral estrogen production more significantly among postmenopausal subjects. Thus, the therapeutic modulation of leptin may be a potential adjuvant treatment for breast cancer patients with high levels of leptin along with high estrogen concentration as a consequence.

Ameliorative effect of hydro-methanolic extract from roots of Rauwolfia serpentina on some biochemical parameters of type 1 diabetic mice.

Present work seeks to investigate the biochemical parameters in terms of hypoglycemic and hypolipidemic effects of hydro-methanolic roots extract (HyMREt) of Rauwolfia serpentina in type 1 (alloxan induced) diabetic mice. Animals were divided into seven groups, four control groups, and three were test groups (HyMREt at 50, 100, & 150mg/kg). Each treatment was repeated for 14 days regularly in all seven respective groups and afterwards the body weights, fasting blood glucose (FBG), insulin, and serum lipid levels were determined. Total body weights of diabetic mice treated with HyMREt extract were dose dependently (p<0.05) improved. FBG of test groups were significantly (p<0.0001) reduced in comparison with diabetic controls which displayed elevated fasting blood glucose level. The insulin levels of HyMREt treated groups were significantly (p<0.0001) higher than those of diabetic controls. Lower triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-c) and very low density lipoprotein cholesterol (VLDL-c) whereas elevated level of high density lipoprotein cholesterol (HDL-c) were observed in test dose treated groups. In comparison with diabetic controls, the converse levels of serum lipid were observed. Significant improvement in cardio-protective indices and coronary risk index was also observed. Findings of present study support the hypoglycemic and hypolipidemic potential of HyMREt of R. serpentina.

Homeostasis model assessment of insulin resistance in non-diabetic heart failure patients: A case-control investigation at tertiary care Hospitals of Karachi.

eart failure is a progressive, chronic disorder. Insulin resistance (IR) has been more and more involved as a preliminary metabolic perturbance predisposing to hyperglycemia, hyperlipidemia and atherosclerosis with others heart diseases. To investigate the relation of insulin resistance (IR) in non-diabetic heart failure patients this case-control study was carried out to ascertain the presence of IR with the aid of Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) in non-diabetic heart failure patients (NDHF patients) compared with healthy controls. The sample size was calculated for both, cases (NDHF patients) and control (healthy subjects), which was initially consisted of 113 respondents each. The study consisted of two phase duration. In Phase I, NDHF patients were approached initially; only 80 patients with NDHF completed the study procedure. In Phase II, 80 healthy subjects were targeted and matched. Fasting blood glucose level (FBGL) and serum insulin was estimated. Mathematical model to quantify ß-cell function and insulin resistance was also computed through Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) in both groups. Data was analyzed on SPSS version 16. Mean values with ± standard deviation (SD) of insulin (10.2±4.36) and HOMA-IR (2.52±1.15) were significantly (p<0.05) higher in NDHF patients as compared to control subject (6.4±3.39, 1.45±0.80). Average insulin to glucose ratio was 0.10±0.044 in NDHF patients which was significantly (p<0.0001) lowered in controls i.e., 0.073±0.039. Marginal and matrix plot analysis revealed that a higher patients count have had the HOMA-IR values <1.5 units while opposite scenario was observed in control group. Regression analyses of HOMA-IR with FBGL (as independent indicator) also authenticate the similar pattern. The present study concludes that insulin resistance (decreased insulin sensitivity) is a characteristic finding in Pakistani population of heart failure as compared to matched healthy controls.

Serum Adiponectin Level Association with Breast Cancer Risk: Evidence from a Case-Control Study.

BACKGROUND: Adiponectin is a protein, synthesized by adipose tissue, which has a questionable role in breast cancer pathogenesis. This study focused on serum levels of adiponectin among cases and controls, and its possible association with breast cancer risk. MATERIALS AND METHODS: This case-control study covered 175 diagnosed breast cancer patients with positive histopathology from Breast Clinic, surgical unit-1, Civil Hospital, Karachi and 175 healthy controls from various screening programs. Blood samples were obtained after an overnight fast and later analyzed. RESULTS: Fasting blood glucose was significantly raised, while serum adiponectin was significantly decreased in breast cancer cases when compared to control subjects. To find the precise influence of serum adiponectin on the risk of breast cancer, subjects were divided in to quartiles based on serum levels of adiponectin. It is observed that risk of breast cancer decreased with rising quartile. Q2 (25th-50th) had the highest OR=1.76 (CI: 0.93-3.34), Q3 (50th-75th) has OR=0.89 (CI: 0.48-1.64), while subjects in the highest quartile Q4 (>75th) had significantly (p< 0.05) decreased risk of breast cancer having OR=0.06 (CI: 0.02-0.15), when compared to the lowest quartile. CONCLUSIONS: We demonstrated a statistically significant association of elevated serum adiponectin with decreased risk of breast cancer. This signifies a protective role of adiponectin via an intricate mechanism of masking mitogenic growth factors in breast carcinogenesis.

Frequency and risk of metabolic syndrome in prediabetics versus normal glucose tolerant subjects -- a comparative study.

OBJECTIVE: To compare the frequency and risk of metabolic syndrome in pre-diabetics against normal glucose-tolerant subjects attending diabetes screening camps in an urban centre. METHODS: The cross-sectional study was carried out at the Jinnah Postgraduate Medical Centre, Karachi, from January to August, 2008, and comprised subjects recruited through diabetes screening camps. They were >30 years of age, without prior history of diabetes and were screened through fasting plasma glucose and 2-hour oral glucose tolerance test. Demographic, anthropometric, clinical and biochemical measurements were done. Frequency of different components and their constellation as metabolic syndrome were determined according to the Adult Treatment Panel-III criteria. Relative risk was estimated to find the risk of metabolic syndrome in pre-diabetics versus normal glucose-tolerant subjects. RESULTS: The study sample comprised 80 subjects; 40(50%) normal glucose-tolerant in Group A and 40(50%) pre-diabetics in Group B. In Group A, there were 25(62.5%) men and 15(37.5%) women, while Group B had 22(55%) men and 18(45%) women. The mean age in Group A was 38.08±5.35 years, while in Group B it was 39.09±6.12 years. The frequency of various cardiovascular risk factors was higher in pre-diabetics (p< 0.05). Central obesity was the most prevalent risk factor (85%, CI: 75.74-96.06), followed by low levels of high density lipoprotein (82.5%, CI: 72.64-94.27), raised triglycerides (67.5%, CI: 55.35-82.01), hypertension (57.5%, CI: 44.68-72.82), and fasting plasma glucose >100mg/dl (42.5%, CI: 29.68-57.82). Metabolic syndrome was found in 23(57.5%) in Group B compared to 9(22.5%) Group B, according to Adult Treatment Panel-III criteria. Calculated relative risk indicated that Group B was 1.9 times more prone to be suffering from metabolic syndrome compared to Group A. CONCLUSIONS: Pre-diabetics were more prone to developing cardiovascular disorders than normal glucose-tolerant subjects.

Status of serum adiponectin related to insulin resistance in prediabetics.

UNLABELLED: OBEJCTIVE: To find the status of serum adiponectin in individuals progressing towards Type 2 diabetes mellitus and compare it with normal glucose tolerant subjects to determine the stage where alteration of adiponectin occurred. METHODS: The cross-sectional study was carried out at the Department of Biochemistry, Jinnah Postgraduate Medical Centre, Karachi, during January to August 2008. Subjects were invited through various diabetes screening camps. A total of 608 subjects > or = 30 years of age without prior history of diabetes were screened through fasting plasma glucose and 2-hour oral glucose tolerance test. Forty randomly selected pre-diabetic subjects and 40 age and gender-matched subjects were included in the study. Anthropometric measurements were done. Serum insulin and adiponectin were estimated by enzyme-linked immunosorbent assay. Homeostasis model assessment of insulin resistance (HOMA-IR) was used to calculate insulin resistance mathematically. RESULT: Mean fasting and two-hour plasma glucose, body mass index, waist, hip circumference and blood pressure were significantly raised in pre-diabetics compared to those with normal glucose tolerance. Adiponectin was significantly decreased, while insulin and HOMA-IR were raised significantly in the pre-diabetics. Adiponectin showed significant negative correlation with body mass index (r = -0.31, p = 0.005), fasting plasma glucose (r = -0.24, p = 0.032), 2-hour plasma glucose (r = -0.42, p < 0.0001)), insulin (r = -0.43, p < 0.0001) and HOMA-IR (r = -0.43, p < 0.0001) and remained significant after adjustment of body mass index, gender and insulin level in pre-diabetics. CONCLUSION: Adiponectin estimation may help in earlier identification of impending diabetes. However, casual link between adiponectin and pre-diabetes remained unexplored due to the study design and small sample size that warrants longitudinal large-scale studies.

Exploring teaching style in an undergraduate medical college following traditional curriculum in Pakistan.

OBJECTIVES: To find out the teaching style and its differences among teachers in a medical college setup. METHODS: The observational cross-sectional study was carried out from June to August 2011 at a private medical and dental college in Karachi, using traditional curriculum and comprising 77 faculty members. Self-assessment Staffordshire Evaluation of Teaching Styles questionnaire was used for data collection. SPSS version 15.0 was used for analysis of categorical variables and t-test and analysis of variance were used to compare data variables. RESULTS: Of the total, 28 (36.4%) teachers preferred all round flexible and adaptable style; 19 (24.7%) preferred student-centred, sensitive; 4 (5.2%) official curriculum; 5 (6.5%) straight facts no nonsense; 6 (7.8%) big conference; 1 (1.3%) one-off; and 14 (18.2%) mixed. Percentage of 'all round flexible and adaptable' was higher among females, senior faculty and clinical sciences when compared to males, junior faculty and basic sciences. However, females, junior faculty, and basic science teachers attained significantly higher in official curriculum scores compared to males, senior faculty, and clinical sciences respectively. Similarly, basic sciences teachers attained significantly higher in big conference teaching style score compared to clinical sciences teachers (p < 0.05). CONCLUSION: Traditional curriculum does not support all round flexible and adaptable teaching style. Gender, curriculum, teaching strategies and faculty rank of the teacher influence the teaching style. To train teachers, effective faculty development should be done in medical colleges.

Role of L-carnitine in male infertility.

OBJECTIVE: To test the hypothesis that the free L-camitine helps in maintaining normal fertility. METHODS: The present descriptive study was designed to show comparison of seminal free L-carnitine and sperm quality. Case controlled convenient sampling was used to assess infertile male subjects from fertile. A total of 61 adult males were selected by consent, and were categorized as fertile and infertile on the basis of history and semen analysis. Subjects were selected from Infertility Clinic, Reproductive Health Services Centre of Jinnah Postgraduate Medical Centre, Karachi. Subject's with history of pelvic surgery, or suffering from diabetes mellitus, thyroid diseases or using steroids, antihypertensives and antipsychotics drugs were excluded from the study. Groups were compared using student's t-test and p < 0.05 was considered as statistically significant. RESULTS: The mean values of sperm count, total motility and normal morphology of asthenospermic and oligoasthenoteratospermic were found significantly (p < 0.05) lower when compared with fertile (control). When levels of seminal free L-carnitine were compared among groups, the result showed that infertile subjects had significantly lower (P < 0.001) when compared to fertile subjects with lowest concentration in azoospermic group. CONCLUSION: The results of this study suggested that L-carnitine level in seminal plasma plays an essentialrole in maintaining male fertility. However larger studies on Pakistani population with this approach are warranted.