In silico analysis of the structural and functional implications of SLC19A1 R27H polymorphism

In view of the documented association of solute carrier family 19 member 1 (SLC19A1) G80A (R27H) polymorphism with the risk for different types of cancers and systemic lupus erythematosus (SLE), we have reanalysed the case–control study on breast cancer to ascertain the conditions in which this polymorphic variant exerts the risk of breast cancer. Association statistics have revealed that this polymorphism exerts the risk for breast cancer under the conditions of low folate intake, and in the absence of well-documented protective polymorphism in cytosolic serine hydroxymethyltransferase. To substantiate this observation, we have developed a homology model of SLC19A1 using glycerol-3-phosphate transporter (d1pw4a) as a template where 73% of the residues were modelled at 90% confidence while 162 residues were modelled ab initio. The wild and mutant proteins shared same topology in S3, S5, S6, S7, S11 and S12 transmembrane domains. The topology varied at S1 (28–43 residue vs 28–44 residue), S2 (66–87 residue vs 69–87 residue), S4 (117–140 residue vs 117–139 residue), S8 (305–325 residue vs 305–324 residue), S9 (336–356 residue vs 336–355residue), and S10 (361–386 residue vs 361–385 residue) transmembrane domains between wild versus mutant proteins. S2 domain is shortened by three amino acid residues in themutantwhile in other domains the difference corresponds to one amino acid residue. The 3DLigandSite analysis revealed that the metallic-ligand-binding sites at 273Trp, 277Asn, 379Leu, 439Phe and 442Leu are although unaffected, there is a loss of active sites corresponding to nonmetallic ligand binding. Tetrahydrofolate and methotrexate have lesseraffinity towards the mutant protein than the wild protein. To conclude, the R27H polymorphism affects the secondary and tertiary structures of SLC19A1 with the significant loss in ligand-binding sites.

Cytomorphological alterations of thyroid gland consequent upon fluorosis

Background: Study aimed at assessing the impact of elevated fluoride from drinking water on thyroid gland structure and function in fluorosis prone areas. Iodine is incorporated in the thyroid synthesis by thyroid gland but in the presence of low Iodine levels fluoride is likely to interfere with the concentrating capacity of thyroid of iodine in thyroid production, consequently reflecting changes in thyroid parameters and also cytomorphological features manifesting hypothyroidism in association with different pathological entities.Methods: Prakasam district in Andhra Pradesh is fluorosis prone zone and subjects are picked up from highly vulnerable zone in this district and their specimens are collected to study cytomorphological changes of the thyroid gland and biochemical parameters of blood samples for thyroid function test were considered. Cytological study by way of Fine Needle Aspiration Cytology (FNAC) of thyroid gland, biochemical parameters pertaining to function of thyroid gland namely Free triiodothyronine (FT3), Free Thyroxine (FT4) and Thyroid stimulating hormone (TSH) were assessed in the subjects from fluorosis prone zone.Results: The results were statistically significant with concurrent association of different cytological alterations of thyroid gland in these subjects like Hashimoto’s thyroiditis of hypothyroidism, adenomatous goitre, colloid goitre and few of follicular adenoma/neoplasm. FNAC makes cytological changes evident showing different morphological features that comprise different pathological entities largely with an evidence of hypothyroidism in most of the cases in the given study.Conclusions: The results of the study strongly suggest assessing the magnitude of the problem of fluorosis and also magnitude of its influence on thyroid structure and function that warrants assessment of the thyroid function by biochemical and cytological studies.

A review on application of biomarkers in heart failure

Heart failure (HF) remains the leading cause of death in the elderly population. Since last decade there is an advance in the field of biomarkers in managing these patients. Hence identifying novel and potential biomarkers that help in accessing the risk, predicting the disease and monitoring the prognosis is very crucial in reducing the overall morbidity and mortality. These biomarkers are elevated mainly in response to myocardial stress, dynamic changes in extracellular matrix, myocyte necrosis, oxidative stress, and inflammation. The biomarker that has good clinical correlations may be useful in diagnosis, prognosis, and therapeutic management of HF. Understanding the role of each biomarker and their clinical implication is very crucial. In this review, we summarize the attainments and challenges of using different types of biomarkers in HF.

Association of GSTT1 and GSTM1 polymorphisms in South Indian Epilepsy Patients.

Experimental studies suggest that oxidative stress is one of the contributing factors in the onset of epileptic seizures. Glutathione S-transferases (GSTs) are able to conjugate electrophilic compounds, and thus possess neuroprotective role by removing exogenous and endogenous oxidants, detoxifying therapeutic drugs, environmental toxins through conjugation with glutathione (GSH). Several studies from different ethnic groups showed that polymorphisms of the GST gene have been associated with Epilepsy. In the present study, we investigated the association of GST polymorphism in the South Indian epilepsy patients population. A total 371 samples (110 cases and 261 controls) were genotyped for the GST1 and GSTM1 polymorphism by multiplex PCR method. We observed a significant association of GSTT1 null polymorphism in patients with epilepsy. The frequency of the GSTT1 null genotype was found to be significantly higher in cases (35.45 %) than the controls (18.39 %) (OR: 2.44, 95%CI: 1.4-4.02, P <0.0001). In contrast, the frequency of the GSTM1 null variant was significantly lower in cases (11.81%) than controls (32.95%) (OR: 0.27, 95%CI: 0.14-0.51, P <0.001) indicating a protective role. These results indicated that individuals who have GSTT1 null variant are at higher risk for developing seizure than those of GSTT1 wild genotype. On the other hand, individuals carrying GSTM1 null variant showed protective role against seizure. Further, these two null variants did not show any significant association with antiepileptic drug-induced skin rash.

Association of estrogen receptor 1 (ESR1) haplotypes with risk for systemic lupus erythematosus among South Indians.

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder involving genetic, epigenetic and environmental factors and has higher incidence in women. In this study, we explored the association of estrogen receptor 1 (ESR1) rs2234693 (PvuII) and rs9340799 (XbaI) polymorphisms with susceptibility to SLE. PCR-RFLP and ELISA were used for genetic analysis and determination of specific autoantibodies, respectively. The univariate analysis showed no independent association of rs2234693 (OR: 1.14, 95% CI: 0.87 - 1.49, p = 0.36) and rs9340799 (OR: 0.87, 95% CI: 0.66-1.14, p = 0.34). The haplotype analysis using SHEsis platform revealed strong linkage disequilibrium between these two polymorphisms (D': 0.81, r2: 0.55). Among the four haplotype groups, the C-A haplotype (rs2234693-rs9340799) was strongly associated with the risk for SLE (OR: 2.10, 95% CI: 1.32 - 3.34, p = 0.001). The homozygous variant genotype of rs2234693 exhibited elevated TNF-α and depleted IFN-α, while the effects of rs9340799 were contradictory. The wild genotype of rs2234693 exhibited lower levels of IL-12 with number of rs9340799 variant alleles pronouncing this effect. From this study, it is concluded that the ESR1 haplotypes may influence the Th2 cytokine profile and susceptibility to SLE among the South Indians.

Molecular mechanism of interaction of mitocurcumin-1 with Akt1 and STAT3: An In silico approach.

The bioavailability of curcumin is the limiting factor for its effective use in anti-cancer therapy. Recently, we reported a novel approach to enhance the cellular uptake by conjugating curcumin with triphenyl phosphonium, named mitocurcumin-1. We found that such conjugation significantly increased the uptake of curcumin in various cancer cells and caused cancer cell death by inducing apoptosis by decreasing the phosphorylation of Akt1 (Thr308) and STAT3 (Tyr705). In this study, a molecular mechanistic model deciphering the regulation of phosphorylation of Akt1 and STAT3 by mitocurcumin-1 was investigated and compared with curcumin. The protein structures were obtained from protein data bank data base and protein-ligand interaction studies were performed with mitocurcumin-1 and curcumin. Docking interaction studies of mitocurcumin-1 with Akt1 and STAT3 active sites showed a strong binding affinity of -60.4107 Kcal/mol and -51.1734 Kcal/mol respectively, suggesting mitocurcumin-1 interacted with the residues at the active sites of phosphorylation of these molecules. Further, a Chi rotationary root mean square deviation of 1.468 Å and 3.965 Å at the active sites in Akt1 and STAT3, respectively indicated that changes in the conformation of protein structure at the active site resulted in the inhibition of phosphorylation of these molecules. To conclude, by using molecular modeling approaches for the first time, we demonstrated the inhibition of Akt1 and STAT3 phosphorylation by mitocurcumin-1.

Association of Parkinson's disease with altered serum levels of lead and transition metals among South Indian subjects.

Several epidemiologic studies have suggested an association between the Parkinson’s disease (PD) and exposure to heavy metals, such as lead, iron, copper, manganese, etc. A growing body of evidence suggests that heavy metals stimulate free radical formation in the brain and can lead to neurodegeneration. In the present study, we investigated whether such association exists in PD cases from rural and urban areas in our study population. The plasma levels of copper, iron, manganese and lead in PD cases (n = 150) and controls (n = 170) were determined by inductively coupled plasma mass spectrometry (ICP-MS) and correlated with the oxidative stress markers like malondialdehyde (MDA), protein carbonyl and total glutathione. Results indicated significant increase in the levels of copper (17.73 ± 4.48 vs. 13.0 ± 3.22 ng/ml) and iron (554.4 ± 123.8 vs. 421.7 ± 126.1 ng/ml) in PD cases compared to controls, whereas no significant differences in the levels of manganese and lead were observed. Further, the data based on urban or rural residence showed that plasma copper, iron, manganese levels were comparatively higher in rural subjects, whereas plasma lead levels were significantly higher in urban subjects. Increased plasma iron showed positive correlation with marker of lipid peroxidation (MDA), suggesting that increased iron levels induced oxidative stress in PD. These results substantiated the earlier observations about the role of environmental exposure and metal-induced oxidative stress in the etiology of PD.

Association of genetic variants of xenobiotic metabolic pathway with systemic lupus erythematosus.

In view of documented evidence that catechol estrogen-DNA adducts serve as epitopes for binding of anti-nuclear antibodies, genetic polymorphisms of the xenobiotic metabolic pathway involved in estrogen metabolism might contribute towards pathophysiology of systemic lupus erythematosus (SLE). To test this hypothesis, a case-control study was conducted. Cytochrome P 450 1A1 (CYP1A1) m4 (OR: 4.93, 95% CI: 1.31-18.49), catecholamine-o-methyl transferase (COMT) H108L (OR: 1.39, 95% CI: 1.03-1.88) and glutathione-S-transferase (GST) T1 null (OR: 1.83, 95% CI: 1.11- 3.01) variants showed association with SLE risk. SHEsis web-based platform analysis showed mild to moderate linkage disequilibrium between the CYP1A1 m1, m2 and m4 variants (D’: 0.19-0.37). Among the different haplotypes of CYP1A1, CAC-haplotype harboring CYP1A1 m1 variant showed association with SLE risk (OR: 1.46, 95% CI: 1.11-1.92). Multifactor dimensionality reduction analysis (MDR) showed potential gene-gene interactions between the phase II variants i.e. COMT H108L × GSTT1 null × GSTM1 null (p<0.0001) and also between the phase II and I variants i.e. COMT H108L × GSTT1 null × CYP1A1 m1 × CYP1A1 m2 in inflating the risk of SLE by 3.33-folds (95% CI: 2.30-4.82) and 4.00-folds (95% CI: 2.77-5.78), respectively. To conclude, hyperinducibility of CYP1A1 due to m1 and m4 variants and defective phase-II detoxification due to COMT H108L and GSTT1 null variants increase the susceptibility to SLE.

Impact of COMT H108L, MAOB int 13 A>G and DRD2 haplotype on the susceptibility to Parkinson’s Disease in South Indian subjects.

In view of documented evidence demonstrating the association of dopaminergic metabolism and neurotransmission with Parkinson’s disease (PD), a case-control study was conducted to investigate the impact of particular polymorphisms in the catechol O-methyl transferase (COMT) H108L, monoamine oxidase B (MAOB) int 13 A>G, dopamine transporter 1 (DAT1) A1215G, dopamine receptor D2 (DRD2) Taq1A, DRD2 Taq1B and DRD2 Taq1D genes on the susceptibility to PD. PCR-RFLP method was used for the genetic analysis. The COMT H108L polymorphism increased PD risk by 1.4-fold (95%CI: 1.02-1.98), whereas reduced risk was observed with MAOB int 13 A>G polymorphism (OR: 0.77, 95%CI: 0.51-0.99). Multifactor dimensionality reduction analysis showed gene-gene interactions between these two loci that resulted in loss of the protective role of MAOB G-allele in the presence of COMT L-allele. DAT1A1215G polymorphism in the exon 9 was not associated with PD. Individually, DRD2 polymorphisms showed null association. However, all-variant haplotype of DRD2 locus i.e. T-G-T haplotype showed 29.8-fold risk for PD compared to all-wild haplotype i.e., C-A-C haplotype (95%CI: 6.85-130.4). To conclude, genetic variants of COMT, MAOB and DRD2 loci modulate susceptibility to PD in South Indian subjects.

Association of family history of type 2 diabetes mellitus with markers of endothelial dysfunction in South Indian population.

Studies indicate that risk for type 2 diabetes mellitus (T2D) or cardiovascular disease is detectable in childhood, though these disorders may not emerge until adulthood. This study was aimed to assess the markers of endothelial dysfunction in patients with the family history of T2D from South Indian population. A total of 450 subjects were included in the study comprising Group I (n = 200) of T2D, Group II (n = 200) of age- and sex-matched healthy controls, Group III (n = 25) of children of T2D patients and Group IV (n = 25) of children of healthy controls. Results showed that intimal medial thickening (IMT) was significantly higher in T2D patients, compared with control subjects with no family history of diabetes. The fasting plasma glucose, glycated hemoglobin, serum total cholesterol, triglyceride, LDL-cholesterol, apolipoprotein B (ApoB) and high-sensitive C-reactive protein (hsCRP) levels were significantly increased, whereas HDL-cholesterol and serum nitrite levels were significantly decreased in T2D patients. However, children of T2D patients who were not diabetic did not show significant increase in the IMT, as compared to those of healthy controls. In conclusion, the present study demonstrated that IMT was significantly higher in the T2D patients and increased with age and family history. The increased levels of lipids, hsCRP, IMT and decreased nitrite levels might contribute to the risk of endothelial dysfunction in patients with T2D. However, further studies are warranted with other biomarkers of endothelial dysfunction in T2D patients with increased sample size.

Glutamate carboxypeptidase II (GCPII) genetic variants as determinants of hyperhomocysteinemia: Implications in stroke susceptibility.

The rationale of this case-control study is to ascertain whether glutamate carboxypeptidase II (GCPII) variants serve as determinants of hyperhomocysteinemia and contribute to the etiology of stroke. Hyperhomocysteinemia was observed in stroke cases compared to controls (14.09 ± 7.62 mmol/L vs. 8.71 ± 4.35, P<0.0001). GCPII sequencing revealed two known variants (R190W and H475Y) and six novel variants (V108A, P160S, Y176H, G206R, G245S and D520E). Among the haplotypes of GCPII, all wild-haplotype H0 showed independent association with stroke risk (OR: 9.89, 95% CI: 4.13-23.68), while H2 representing P160S variant showed reduced risk (OR: 0.17, 95% CI: 0.06-0.50). When compared to subjects with H2 haplotype, H0 haplotype showed elevated homocysteine levels (18.26 ± 4.31 mmol/L vs. 13.66 ± 3.72 mmol/L, P = 0.002) and reduced plasma folate levels (7.09 ± 1.19 ng/ml vs. 8.21 ± 1.14 ng/ml, P = 0.007). Using GCPII genetic variants, dietary folate and gender as predictor variables and homocysteine as outcome variable, a multiple linear regression model was developed. This model explained 36% variability in plasma homocysteine levels. To conclude, GCPII haplotypes influenced susceptibility to stroke by influencing homocysteine levels.

Role of CYP1A1 haplotypes in modulating susceptibility to coronary artery disease.

To investigate the role of cytochrome P450 1A1 (CYP1A1) haplotypes in modulating susceptibility to coronary artery disease (CAD), a case-control study was conducted by enrolling 352 CAD cases and 282 healthy controls. PCR-RFLP, multiplex PCR, competitive ELISA techniques were employed for the analysis of CYP1A1 [m1 (T→C), m2 (A→G) and m4 (C→A)] haplotypes, glutathione-S-transferase (GST)T1/GSTM1 null variants and plasma 8-oxo-2’deoxyguanosine (8-oxodG) respectively. Two CYP1A1 haplotypes, i.e. CAC and TGC showed independent association with CAD risk, while all-wild CYP1A1 haplotype i.e. TAC showed reduced risk for CAD. All the three variants showed mild linkage disequilibrium (D’: 0.05 to 0.17). GSTT1 null variant also exerted independent association with CAD risk (OR: 2.53, 95% CI 1.55–4.12). Among the conventional risk factors, smoking showed synergetic interaction with CAC haplotype of CYP1A1 and GSTT1 null genotype in inflating CAD risk. High risk alleles of this pathway showed dose-dependent association with percentage of stenosis and number of vessels affected. Elevated 8-oxodG levels were observed in subjects with CYP1A1 CAC haplotype and GSTT1 null variant. Multiple linear regression model of these xenobiotic variants explained 36% variability in 8-oxodG levels. This study demonstrated the association of CYP1A1 haplotypes and GSTT1 null variant with CAD risk and this association was attributed to increased oxidative DNA damage.

Modulatory effect of plasma folate and polymorphisms in one-carbon metabolism on catecholamine methyltransferase (COMT) H108L associated oxidative DNA damage and breast cancer risk.

The present study was aimed to investigate the modulatory role of plasma folate and eight putatively functional polymorphisms of one-carbon metabolism on catecholamine methyltransferase (COMT)-mediated oxidative DNA damage and breast cancer risk. Plasma folate and 8-oxo-2’-deoxyguanosine (8-oxodG) were estimated by commercially available kits, while polymorphisms were screened by PCR-RFLP and PCR-AFLP methods. COMT H108L polymorphism showed independent association with breast cancer (OR: 1.73, 95% CI: 1.31-2.30). No significant interaction was observed between folate status and COMT genotype. Multifactor dimensionality reduction (MDR) analysis gave evidence for the significant epistatic (gene-gene) interactions (p<0.0001) of COMT H108L with reduced folate carrier 1 (RFC1) G80A, thymidylate synthase (TYMS) 5’-UTR 3R2R, TYMS 3’-UTR ins6/del6. Increased plasma 8-oxodG were observed in cases compared to controls (mean ± SE: 5.59 ± 0.60 vs. 3.50 ± 0.40 ng/ml, p<0.004). Plasma folate deficiency alone was not a significant predictor of 8-oxodG elevation. The genotype combinations namely, RFC1 G80A/methionine synthase reductase (MTRR) A66G, RFC1 G80A/SHMT C1420T/TYMS 3R2R and serine hydroxymethyltransferase (SHMT) C1420T/TYMS 3R2R/methionine synthase (MTR) A2756G/COMT H108L were strong predictors of 8-oxodG elevation in the order of risk. To conclude, the current study provides substantial evidence for a cross talk between one-carbon metabolism and COMT catalysis that might influence oxidative DNA damage and breast cancer risk.

Oxidative Stress in Cardiovascular Disease.

Over the last two decades, it has become increasingly clear that reactive oxygen species (ROS), including free radicals are involved in cardiovascular disease. In recent years, there has been a growing interest in the clinical implications of these oxidants. The ROS are common by-products of many oxidative biochemical and physiological processes. They can be released by xanthine oxidase, NAD(P)H oxidase, lipoxygenases, mitochondria, or the uncoupling of nitric oxide synthase in vascular cells. ROS mediate various signaling pathways that underlie vascular inflammation in atherogenesis. Various animal models of oxidative stress support that ROS have causal role in atherosclerosis and other cardiovascular diseases. They are too reactive to be tolerated in living tissue, and aerobic organisms use sophisticated defense system, both enzymatic and non-enzymatic for prevention of overload of free radicals. In a number of pathophysiological conditions, the delicate equilibrium between free-radical production and antioxidant capability can be altered in favor of the former, thus leading to oxidative stress and increased tissue injury. This review focuses on the biochemical evidences concerning involvement of ROS in several cardiovascular diseases, namely atherosclerosis, heart failure, hypertension and ischemia/reperfusion injury.