Development of pharmacogenomic algorithm to optimize nateglinide dose for the treatment of type 2 diabetes mellitus.

BACKGROUND: Nateglinide is a meglitinide used for the treatment of type 2 diabetes mellitus. Individual studies demonstrated the association of CYP2C9, SLCO1B1, and MTNR1B variants with the safety and efficacy of nateglinide. The current study aimed to develop a pharmacogenomic algorithm to optimize nateglinide therapy. METHODS: Multiple linear regression (MLR) and classification and regression tree (CART) were used to develop a pharmacogenomic algorithm for nateglinide dosing based on the published nateglinide pharmacokinetic data on the area under the curve data (AUC) and Cmax (n = 143). CYP2C9 metabolizer phenotype, SLCO1B1, MTNR1B genotypes, and CYP2C9 inhibitor usage were used as the input variables. The results and associations were further confirmed by meta-analysis and in silico studies. RESULTS: The MLR models of AUC and Cmax explain 87.4% and 59% variability in nateglinide pharmacokinetics. The Bland and Altman analysis of the nateglinide dose predicted by these two MLR models showed a bias of ± 26.28 mg/meal. The CART algorithm was proposed based on these findings. This model is further justified by the meta-analysis showing increased AUCs in CYP2C9 intermediate metabolizers and SLCOB1 TC and CC genotypes compared to the wild genotypes. The increased AUC in SLCO1B1 mutants is due to decreased binding affinity of nateglinide to the mutant affecting the influx of nateglinide into hepatocytes. MTNR1B rs10830963 G-allele-mediated poor response to nateglinide is attributed to increased transcriptional factor binding causing decreased insulin secretion. CONCLUSION: CYP2C9, SLCO1B1, and MTNR1B genotyping help in optimizing nateglinide therapy based on this algorithm and ensuring safety and efficacy.

Pharmacogenetic determinants of warfarin in the Indian population.

BACKGROUND: Several studies optimized the warfarin dose based on CYP2C9*2, CYP2C9*3, VKORC1 -1639 G > A, CYP4F2 V433M. But, the information on the rare variants is lacking. In this study, we have explored the prevalence of common and rare pharmacogenetic determinants of warfarin and determined their damaging nature. METHODS: We have analyzed 2000 healthy adults using the Infinium global screening array (GSA) for 15 pharmacogenetic determinants of warfarin. In addition, we have elucidated the impact of these variants on protein function, stability, dynamics, evolutionary preservation, and ligand binding propensity. RESULTS: The GSA Analysis has revealed that CYP4F2 V433M (MAF: 39.425%), VKORC1 -1639 G > A (MAF: 20.5%), CYP2C9*3 (MAF:9.925%), and CYP2C9*2 (MAF:4.575%) are common, while CYP2C9*14 (MAF: 1.475%), CYP2C9*4 (0.175%), CYP2C9*5 (0.125%), and CYP2C9*11 (0.125%) are rare. Position-specific evolutionary preservation (PSEP) analysis has revealed that CYP2C9*4 is possibly damaging, while CYP2C9*5, CYP2C9*11, and CYP2C9*14 are probably damaging. CYP2C9*4 has high thermolability (-10.14 kcal/mol). Among the rare CYP2C9 variants, CYP2C9*4 and CYP2C9*11 exert destabilizing effects and may have increased molecular flexibility, while CYP2C9*5 and CYP2C9*14 exert stabilizing effects and may have decreased molecular flexibility. DNase I footprint analysis has revealed the loss of the E-box consensus sequence due to VKORC1 -1639 G > A polymorphism. CONCLUSION: CYP2C9*2, CYP2C9*3, VKORC1 -1639 G > A and CYP4F2 V433M are common; CYP2C9*4, CYP2C9*5, CYP2C9*11, and CYP2C9*14 variants are rare in Indian subjects. All the CYP2C9 variants are found to be damaging. DNase I footprint analysis provided the mechanistic rationale for the association of VKORC1 -1639 G > A with warfarin sensitivity.

Influence of RFC1 c.80A>G Polymorphism on Methotrexate-Mediated Toxicity and Therapeutic Efficacy in Rheumatoid Arthritis: A Meta-analysis.

BACKGROUND: Methotrexate (MTX) is an antirheumatic drug, transported by reduced folate carrier-1 (RFC1). The most common RFC1 gene variant, c.80 A>G (rs1051266) is ambiguously linked to adverse effects of MTX therapy in some rheumatoid arthritis (RA) patients. OBJECTIVE: The purpose of meta-analysis was to summarize all major published studies on c.80 A>G SNP to clarify this ambiguity in MTX therapy. METHODS: A total of 18 studies representing 3592 RA patients comprising 699 men and 2893 women were included. Both fixed and random effect models were applied to study the data. RESULTS: The RFC1 80A-allele showed null association with MTX-mediated toxicity in both fixed (odds ratio [OR] = 0.91; 95% CI = 0.80-1.03) and random effects (OR = 0.89; 95% CI: 0.71-1.11) models. Because heterogeneity was observed in this association (P = 0.0006), data were segregated based on use of folate therapy. In 7 studies (n = 1191) where folate was used along with MTX, RFC1 AA patients showed reduced risk for MTX-mediated toxicity (OR = 0.67; 95% CI: 0.50-0.89; P = 0.0006). The RFC1 80A-allele was found to increase the efficacy of MTX therapy by 1.53-fold (95% CI: 1.24-1.88), whereas the 80AA-genotype increased the efficacy by 1.85-fold (95% CI: 1.41-2.42). No publication bias was observed in these associations. CONCLUSION AND RELEVANCE: RFC1 c.80 A>G is an important pharmacogenetic determinant of MTX therapy in RA. The RFC1 80A-allele robustly increased therapeutic efficacy and safety when folate was used along with MTX. Findings are relevant to decision-making in the clinical use of MTX as a treatment for RA patients harboring the RFC1 gene variant.

Pharmacogenetic determinants of thiopurines in an Indian cohort.

BACKGROUND: Several genetic variants of thiopurine metabolic pathway are associated with 6-thiopurine-mediated leucopenia. A population-based evaluation of these variants lays the foundation for Pharmacogenetic-guided thiopurine therapy. METHODS: A total of 2000 subjects were screened for the pharmacogenetic determinants using the infinium global screening array (GSA). The functional relevance of these variants was deduced using SNAP2, SIFT, Provean, Mutalyzer, Mutation Taster, Phyre2, SwissDock, AGGRESCAN, and CUPSAT. RESULTS: The minor allele frequencies of NUDT15*3, NUDT15*5, TPMT*3C, TPMT*3B variant alleles were 6.78%, 0.11%, 1.98% and 0.69%, respectively. TPMT*3A genotype was observed in 0.35% subjects. No gender-based differences were observed in the incidence of these variants. Data from studies of the Indian population showed that 92.86% subjects heterozygous for NUDT15*3 and 60% subjects heterozygous for TPMT*3C exhibit thiopurine-mediated hematological toxicity. NUDT15 variants have no impact on the binding of 'dGTP' to the NUDT protein. NUDT15*3 variant increases aggregation 'hot spot' region and induces unfavourable torsion in the protein. NUDT15*5 destabilizes the protein and impairs Mg/Mn binding. TPMT*3A, TPMT*3B and TPMT*3C variants lower binding affinity to 6-mercaptopurine compared to the wild protein. TPMT*3C variant destabilizes the TPMT protein in the thermal experiment. Compared to the data of European and African/African American populations, NUDT15*3 frequency is higher and TPMT*3C frequency is lower in our population. CONCLUSIONS: TPMT variants were less frequent in Indian population, while NUDT15*3 is more frequent compared to European and African/African American populations. NUDT15*3 increases aggregation 'hot spot' and induces unfavourable torsion in the protein. NUDT15*5 and TPMT*3C destabilize the respective proteins. TPMT*3A, TPMT*3B and TPMT*3C are associated with a lower binding affinity towards 6-mercaptopurine.

Pharmacogenetic profiling of dihydropyrimidine dehydrogenase (DPYD) variants in the Indian population.

BACKGROUND: The present study aimed to delineate the pharmacologically relevant dihydropyrimidine dehydrogenase (DPYD) variants in the Indian population. METHODS: We screened 2000 Indian subjects for DPYD variants using the Infinium Global Screening Array (GSA) (Illumina Inc., San Diego, CA, USA). RESULTS: The GSA analysis identified seven coding, two intronic and three synonymous DPYD variants. Level 1A alleles (rs75017182, rs3918290, P633Qfs*5 and D949V) were found to be rare (minor allele frequency: 1.889%), whereas Level 3 alleles were observed to be predominant (C29R: 24.91%, I543V: 9.047%, M166V: 8.993% and V732I: 8.44%). In silico predictions revealed that all Level 1A alleles were deleterious, whereas three (M166V, S534N and V732I) of seven Level 3 alleles were damaging. CUPSAT analysis revealed that two Level 1A (P633Qfs*, D949V) and three Level 3 (I543V, V732I and S534N) variants were thermolabile. The pooled Indian data showed that V732I, S534N and rs3918290 variants were associated with 5-FU/capecitabine toxicity, whereas C29R, I543V and M166V variants exhibited the null association. A comparison of our data with other population data from the 'Allele Frequency Aggregator' (https://www.ncbi.nlm.nih.gov/snp/docs/gsr/alfa/) database showed similarities with the South Asian data. CONCLUSIONS: We have identified four Level 1A (non-functional/dysfunctional) and seven Level 3 variants in the DPYD gene. The pooled Indian data revealed the association of V732I, S534N and rs3918290 variants with 5-FU/capecitabine toxicity. Clustering analysis revealed the similarities in the DPYD profiles of the Indian and South Asian populations.

The Potential of Pandanus Amaryllifolius Leaves Extract in Fabrication of Dense and Uniform ZnO Microrods.

Zinc oxide (ZnO) micro and nanorods were successfully prepared using Pandanus amaryllifolius and hexamethylenetetramine (HMTA) separately as stabilizers using the solution immersion method. Two types of ZnO seed layer were prepared using the same pre-cursor with the different stabilizers. The fabricated ZnO microrods exhibit absorption at ~375 nm as revealed from the UV-Visible absorption spectrum, and this is comparable with ZnO nanorods synthesized using HMTA. X-ray diffraction (XRD) measurement displayed a sharp peak corresponding to the hexagonal wurtzite structure of ZnO microrods. Field emission scanning electron microscopy (FESEM) of ZnO microrods showed average diameter at approximately 500 nm compared to 70 nm of those synthesized from HMTA. A new finding is the ability of Pandanus amaryllifolius as a green stabilizer to grow a dense ZnO microrod structure with high crystallinity. Results reveal that both samples from different stabilizers during the preparation of the ZnO seed layer greatly improved the morphological and structural properties and optical absorption of ZnO. The main outcomes from this study will benefit optoelectronic application, such as in ultraviolet (UV) sensors.

High Surface Reactivity and Biocompatibility of Y2O3 NPs in Human MCF-7 Epithelial and HT-1080 FibroBlast Cells.

This study aimed to generate a comparative data on biological response of yttrium oxide nanoparticles (Y2O3 NPs) with the antioxidant CeO2 NPs and pro-oxidant ZnO NPs. Sizes of Y2O3 NPs were found to be in the range of 35±10 nm as measured by TEM and were larger from its hydrodynamic sizes in water (1004 ± 134 nm), PBS (3373 ± 249 nm), serum free culture media (1735 ± 305 nm) and complete culture media (542 ± 108 nm). Surface reactivity of Y2O3 NPs with bovine serum albumin (BSA) was found significantly higher than for CeO2 and ZnO NPs. The displacement studies clearly suggested that adsorption to either BSA, filtered serum or serum free media was quite stable, and was dependent on whichever component interacted first with the Y2O3 NPs. Enzyme mimetic activity, like that of CeO2 NPs, was not detected for the NPs of Y2O3 or ZnO. Cell viability measured by MTT and neutral red uptake (NRU) assays suggested Y2O3 NPs were not toxic in human breast carcinoma MCF-7 and fibroblast HT-1080 cells up to the concentration of 200 µg/mL for a 24 h treatment period. Oxidative stress markers suggested Y2O3 NPs to be tolerably non-oxidative and biocompatible. Moreover, mitochondrial potential determined by JC-1 as well as lysosomal activity determined by lysotracker (LTR) remained un-affected and intact due to Y2O3 and CeO2 NPs whereas, as expected, were significantly induced by ZnO NPs. Hoechst-PI dual staining clearly suggested apoptotic potential of only ZnO NPs. With high surface reactivity and biocompatibility, NPs of Y2O3 could be a promising agent in the field of nanomedicine.

Adaptive Neuro-Fuzzy Inference System-Based Exploration of the Interrelationships of 25-Hydroxyvitamin D, Calcium, Phosphorus with Parathyroid Hormone Production.

The rationale of the current study was to assess the prevalence of 25-hydroxyvitamin D (25-OHD) deficiency and hyperparathyroidism in South Indian population and to explore interrelationships of 25-OHD, Ca, P towards parathyroid hormone (PTH) production using adaptive neuro-fuzzy inference system (ANFIS). A total of 407 subjects (228 men 179 women) with the mean age 56.8 ± 14.1 were tested for these parameters. In view of the skewed distribution of biochemical variables, data segregation was performed in tertiles and this data was trained to generate fuzzy interference system based on subclusters. The optimized model had 358 nodes and followed 44 fuzzy rules for prediction. This ANFIS model demonstrates that the deficiency of 25-OHD and Calcium triggers PTH production. PTH elevation is significant when Phosphorus is in the highest tertile. The associations observed by this model were consistent with the Kendall-Tau correlation matrix, which revealed inverse associations of Ca with P; and Ca with PTH and positive associations of P with PTH, and Ca with 25-OHD. Furthermore, the association statistics of the machine learning algorithm were also consistent, which suggested that depletion of Ca below 8.245 mg/dl was shown to elevate PTH levels greater than 167 pg/ml when P > 4.66. Subnormal depletion in 25-OHD (9.3-16.2 ng/ml) is associated with subnormal elevation in PTH (47-73.6 pg/ml). To conclude, ANFIS and machine learning algorithm are in agreement with each other in stating that 25-OHD deficiency triggers lower calcium levels, lower calcium and higher phosphorus trigger PTH production.

Mitochondrial dysfunction, autophagy stimulation and non-apoptotic cell death caused by nitric oxide-inducing Pt-coated Au nanoparticle in human lung carcinoma cells.

BACKGROUND: Reactive oxygen species (ROS)-mediated cancer therapeutic has been at higher appreciation than those mediated by reactive nitrogen species. Cytotoxic mechanism of a novel nitric oxide (NO) inducing-Pt coated Au nanoparticle (NP) has been comparatively studied with the well-established ROS inducing Pt-based anticancer drug cisplatin in human lung A549 carcinoma cells. METHODS: Cytotoxicity was evaluated by MTT assay, lactate dehydrogenase (LDH) release, thiobarbituric acid substances (TBARS) and C11-Boron dipyrromethene (BODIPY). ROS (O2·- and H2O2) was measured with dihydroethidium (DHE) and H2O2-specific sensor. Nitric oxide (NO) and mitochondrial dysfunction were evaluated respectively by NO-specific probe DAR-1 and JC-1. Autophagy was determined by lysotracker (LTR) and monodansylcadaverine (MDC) applied tandemly whereas apoptosis/necrosis by Hoechst/PI and caspase 3 activity. RESULTS: IC50 (concentration that inhibited cell viability by 50%) of Pt coated Au NP came to be 0.413 µM whereas IC50 of cisplatin came out to 86.5 µM in A549 cells treated for 24 h meaning NPs toxicity was over 200 times higher than cisplatin. However, no significant stimulation of intracellular ROS was observed at the IC50 of Pt coated Au NPs in A549 cells. However, markers like LDH release, TBARS, BODIPY and ROS were significantly higher due to cisplatin in comparison to Pt coated Au NP. CONCLUSIONS: Pt coated Au NP caused NO-dependent mitochondrial dysfunction and autophagy. Mode of cell death due to NP was much different from ROS-inducing cisplatin. GENERAL SIGNIFICANCE: Pt coated Au NP offer promising opportunity in cancer therapeutic and warrants advanced study in vivo models of cancer.

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 the mutant while 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 lesser affinity 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.

Recipient ABCB1, donor and recipient CYP3A5 genotypes influence tacrolimus pharmacokinetics in liver transplant cases.

BACKGROUND: Effective immunosuppression through optimization of trough levels tacrolimus reduces post-transplant mortality rate in liver transplant cases. METHODS: Meta-analysis was carried out to evaluate how donor/recipient CYP3A5 (n = 678) and recipient ABCB1 (n = 318) genotypes influence tacrolimus pharmacokinetics till one-month of transplantation. RESULTS: The donor CYP3A5*3/*3 genotype exhibited higher concentration/dose (C/D) ratio of tacrolimus in week 1 (mean difference: 65.04, 95% CI: 15.30-114.79 ng/ml/mg/kg), week 2 (mean difference: 21.7, 95% CI: 12.6-30.9 ng/ml/mg/kg) and week 4 (mean difference: 43.28, 95% CI: 17.09 - 69.49 ng/ml/mg/kg) compared to *1/*1 and *1/*3 genotypes. The recipient CYP3A5 *3/*3 genotype did not showed significant difference in tacrolimus C/D ratio in week 1 compared to other two genotypes. However, week 2 (mean difference: 44.16, 95% CI: 3.68-84.65 ng/ml/mg/kg) and week 4 (mean difference: 43.74, 95% CI: 12.50-75.00 ng/ml/mg/kg) availability was higher in *3/*3 mutant recipients. However, the recipient ABCB1 3435 C > T polymorphism has no significant influence on tacrolimus pharmacokinetics till one month of transplant. CONCLUSIONS: The donor and recipient CYP3A5*3 polymorphism influences tacrolimus pharmacokinetics in the first month post-transplantation, whereas the association with recipient ABCB1 3435 C > T is inconclusive.

Classification and regression tree-based prediction of 6-mercaptopurine-induced leucopenia grades in children with acute lymphoblastic leukemia.

PURPOSE: The rationale of the current study was to develop 6-mercaptopurine (6-MP)-mediated hematological toxicity prediction model for acute lymphoblastic leukemia (ALL) therapeutic management. METHODS: A total of 96 children with ALL undergoing therapy with MCP-841 protocol were screened for all the ten exons of TPMT, exon 2, exon 3 and intron 2 of ITPA using bidirectional sequencing. This dataset was used to construct prediction models of leucopenia grade by constructing classification and regression trees (CART) followed by smart pruning. RESULTS: The developed CART model indicated TPMT*12 and TPMT*3C as the key determinants of toxicity. TPMT int3, int4 and int7 polymorphisms exert toxicity when co-segregated with one mutated allele of TPMT*12 or TPMT*3C or ITPA exon 3. The developed CART model exhibited 93.6% accuracy in predicting the toxicity. The area under the receiver operating characteristic curve was 0.9649. CONCLUSIONS: TPMT *3C and TPMT*12 are the key determinants of 6-MP-mediated hematological toxicity while other variants of TPMT (int3, int4 and int7) and ITPA ex2 interact synergistically with TPMT*3C or TPMT*12 variant alleles to enhance the toxicity. TPMT and ITPA variants cumulatively are excellent predictors of 6-MP-mediated toxicity.

Oxidative stress mediated cytotoxicity and apoptosis response of bismuth oxide (Bi2O3) nanoparticles in human breast cancer (MCF-7) cells.

Bismuth oxide nanoparticles (Bi2O3 NPs) have shown great potential for several applications including cosmetics and biomedicine. However, there is paucity of research on toxicity of Bi2O3 NPs. In this study, we first examined dose-dependent cytotoxicity and apoptosis response of Bi2O3 NPs in human breast cancer (MCF-7) cells. We further explored the potential mechanisms of cytotoxicity of Bi2O3 NPs through oxidative stress. Physicochemical study demonstrated that Bi2O3 NPs have crystalline structure and spherical shape with mean size of 97 nm. Toxicity studies have shown that Bi2O3 NPs reduce cell viability and induce membrane damage dose-dependently in the concentration range of 50-300 µg/ml. Bi2O3 NPs also disturbed cell cycle of MCF-7 cells. Oxidative stress response of Bi2O3 NPs was evident by generation of reactive oxygen species (ROS), higher lipid peroxidation, reduction of glutathione (GSH) and low superoxide dismutase (SOD) enzyme activity. Interestingly, supplementation of external antioxidant N-acetyl-cysteine almost negated the effect of Bi2O3 NPs induced oxidative stress and cell death. We also found that exposure of Bi2O3 NPs induced apoptotic response in MCF-7 cells suggested by impaired regulation of Bcl-2, Bax and caspase-3 genes. Altogether, we found that Bi2O3 NPs induced cytotoxicity in MCF-7 cells through modulating the redox homeostasis via Bax/Bcl-2 pathway. This study warranted further research to delineate the underlying mechanism of Bi2O3 NPs induced toxicity at in vivo level.

MgO nanoparticles cytotoxicity caused primarily by GSH depletion in human lung epithelial cells.

Bio-response of magnesium oxide nanoparticles (MgO NPs) is emerging, obviously, with a conflicting flavor. This study evaluates the underlying mechanism of bio-responses of MgO NPs in human lung epithelial (A549) cell. TEM size of NPs was 40-50 nm and cuboidal in shape. EDS data showed no detectable impurity. Zeta potential of MgO NPs suggested a fair dispersion in complete culture media and in PBS. MgO NPs induced a concentration dependent cytotoxicity when measured by MTT and NRU. MgO NPs induced cytotoxicity strongly correlated with intracellular depletion of antioxidant GSH. MgO NPs did not induce concentration dependent ROS. All live treatment conditions caused autophagy, a survival mechanism when deprived of nutrients and antioxidant. At highest cytotoxic concentration of MgO NPs, there was significant elevation in MMP and caspase-3 activity. GSH depletion mediated autophagy failure lead to MgO NPs induced death at higher concentrations that might have potentiated by induced ROS. This study suggested a mechanism of cytotoxicity caused by MgO NPs that was primarily dependent on GSH depletion, and ROS induction played secondary role in toxicity. Significantly higher toxicity observed for MgO NPs in comparison to Mg salt clearly indicated the involvement of nanoparticulate form in toxicity.

Machine learning algorithm-based risk prediction model of coronary artery disease.

In view of high mortality associated with coronary artery disease (CAD), development of an early predicting tool will be beneficial in reducing the burden of the disease. The database comprising demographic, conventional, folate/xenobiotic genetic risk factors of 648 subjects (364 cases of CAD and 284 healthy controls) was used as the basis to develop CAD risk and percentage stenosis prediction models using ensemble machine learning algorithms (EMLA), multifactor dimensionality reduction (MDR) and recursive partitioning (RP). The EMLA model showed better performance than other models in disease (89.3%) and stenosis prediction (82.5%). This model depicted hypertension and alcohol intake as the key predictors of CAD risk followed by cSHMT C1420T, GCPII C1561T, diabetes, GSTT1, CYP1A1 m2, TYMs 5'-UTR 28 bp tandem repeat and MTRR A66G. MDR and RP models are in agreement in projecting increasing age, hypertension and cSHMTC1420T as the key determinants interacting in modulating CAD risk. Receiver operating characteristic curves exhibited clinical utility of the developed models in the following order: EMLA (C = 0.96) > RP (C = 0.83) > MDR (C = 0.80). The stenosis prediction model showed that xenobiotic pathway genetic variants i.e. CYP1A1 m2 and GSTT1 are the key determinants of percentage of stenosis. Diabetes, diet, alcohol intake, hypertension and MTRR A66G are the other determinants of stenosis. These eleven variables contribute towards 82.5% stenosis. To conclude, the EMLA model exhibited higher predictability both in terms of disease prediction and stenosis prediction. This can be attributed to higher number of iterations in EMLA model that can increase the prediction accuracy.

Application of adaptive neuro-fuzzy inference systems (ANFIS) to delineate estradiol, glutathione and homocysteine interactions.

The rationale of the current study was to elucidate the contributing factors for the gender-based differences in total plasma homocysteine levels. A total of 413 subjects comprising of 293 men and 120 women were enrolled for the study. Chemiluminescence technology for vitamin B12, folate and total plasma homocysteine; ELISA for estradiol and 8-oxo-2-deoxyguanosine; Ellman's method for total glutathione; and PCR-RFLP analysis for the detection of methylene tetrahydrofolate reductase (MTHFR) C677T polymorphism were employed. No statistically significant differences were observed between the men and women in the distribution of age (p = 0.82), vitamin B12 (p = 0.23), folate (p = 0.36) and MTHFR C677T polymorphism (p = 0.35). However, the total plasma homocysteine levels were higher in men compared to women (28.4 ± 17.9 vs. 20.6 ± 13.6 µmol/L, p < 0.0001). In order to explain this gender differences in homocysteine, adaptive neuro-fuzzy inference systems (ANFIS) were developed to understand trivariate interactions among estradiol, glutathione and homocysteine. In the presence of adequate estradiol levels, inverse association was observed between glutathione and homocysteine. This association is lost when estradiol levels were inadequate. Estradiol was found to quench homocysteine mediated oxidative DNA damage. Irrespective of gender, combined deficiency of vitamin B12 and folate showed positive association with hyperhomocysteinemia and vice versa. Homocysteine reduction in response to vitamin status varied according to gender with men responding to folate and women responding to B12. To conclude, gender-differences in homocysteine are attributable estradiol mediated lowering of homocysteine that prevents inactivation of glutathione mediated oxidative defense in women.

Clinical utility of folate pathway genetic polymorphisms in the diagnosis of autism spectrum disorders.

BACKGROUND: The rationale of the current study was to test the clinical utility of the folate pathway genetic polymorphisms in predicting the risk for autism spectrum disorders (ASD) and to address the inconsistencies in the association of MTHFR C677T and hyperhomocysteinemia with ASD. PATIENTS AND METHODS: An artificial neural network (ANN) model was developed from the data of 138 autistic and 138 nonautistic children using GCPII C1561T, SHMT1 C1420T, MTHFR C677T, MTR A2756G, and MTRR A66G as the predictors of autism risk. A neuro fuzzy model was developed to explore the genetic determinants of homocysteine. Meta-analyses were carried out on 1361 ASD children and 6591 nonautistic children to explore the association of MTHFR C677T and homocysteine with the risk for ASD. RESULTS: The ANN model showed 63.8% accuracy in predicting the risk of autism. Hyperhomocysteinemia was observed in autistic children (9.67±4.82 vs. 6.99±3.21 µmol/l). The neuro fuzzy model showed synergistic interactions between MTHFR C677T and MTRR A66G inflating homocysteine levels. The meta-analysis showed MTHFR to be a genetic risk factor for autism in both fixed-effects (odds ratio: 1.47, 95% confidence interval: 1.31-1.65) and random-effects (odds ratio: 1.57, 95% confidence interval: 1.16-2.11) models. The meta-analysis of nine studies showed hyperhomocysteinemia as a significant risk factor for autism in both fixed-effects (P<0.0001) and random-effects (P=0.026) models. CONCLUSION: Genetic polymorphisms of the folate pathway were moderate predictors of autism risk. MTHFR C677T and hyperhomocysteinemia have been identified as risk factors for autism worldwide. Synergistic interactions between MTHFR C677T and MTRR A66G increase homocysteine.

Population-level diversity in the association of genetic polymorphisms of one-carbon metabolism with breast cancer risk.

Aberrations in one-carbon metabolism were reported to increase breast cancer risk by influencing the DNA synthesis and methylation of DNA and catecholamines. However, the results of these association studies remain inconclusive. We have explored the contribution of eight genetic polymorphisms in modulating the susceptibility to breast cancer by performing a meta-analysis of worldwide studies. In total, 62 case-control studies representing 17 different populations involving 18,117 breast cancer cases and 23,573 healthy controls were included in this meta-analysis. Out of the eight polymorphisms analyzed, methylenetetrahydrofolate reductase (MTHFR) C677T exhibited positive association with the breast cancer risk in both fixed effects (OR 1.14, 95 % CI 1.10-1.17) and random effects (OR 1.10, 95 % CI 1.02-1.18) models. Solute carrier family 19 (folate transporter), member 1 (SLC19A1) G80A exhibited positive association (OR 1.16, 95 % CI 1.03-1.31) while MTR A2756G exhibited an inverse association (OR 0.78, 95 % CI 0.75-0.82) with the breast in fixed effect model alone. Significant heterogeneity was observed in the association of MTHFR C677T with breast cancer even between studies from the same geographical area, specifically among Chinese, Indians, and Turks. Subgroup analysis revealed MTHFR C677T-mediated breast cancer risk in post-menopausal women and women with low dietary intake of folate. Geographical area wise segregation of data revealed MTHFR-mediated increased breast cancer risk in populations who consume methionine-rich diet. Altitude-level variations were observed in the association of SHMT1 C1420T with breast cancer. India and Brazil of same altitude showed an inverse association with this polymorphism, while USA and China that share similar altitude showed a null association. MTHFR C677T and SLC19A1 G80A are the two polymorphisms of one-carbon metabolic pathway that increase breast cancer in the worldwide population. Dietary patterns and altitudinal variations are the likely risk modulators that are contributing toward ethnic- and population-level variations in genetic associations.

In silico approaches to identify the potential inhibitors of glutamate carboxypeptidase II (GCPII) for neuroprotection.

To develop a potential inhibitor for glutamate carboxypeptidase II (GCPII) effective against all the eight common genetic variants reported, PyMOL molecular visualization system was used to generate models of variants using the crystal structure of GCPII i.e. 2OOT as a template. High-throughput virtual screening of 29 compounds revealed differential efficacy across the eight genetic variants (pIC50: 4.70 to 10.22). Pharmacophore analysis and quantitative structure-activity relationship (QSAR) studies revealed a urea-based N-acetyl aspartyl glutamate (NAAG) analogue as more potent inhibitor, which was effective across all the genetic variants of GCPII as evidenced by glide scores (-4.32 to -7.08) and protein-ligand interaction plots (13 interactions in wild GCPII). This molecule satisfied Lipinski rule of five and rule of three for drug-likeliness. Being a NAAG-analogue, this molecule might confer neuroprotection by inhibiting glutamatergic neurotransmission mediated by N-acetylated alpha-linked acidic dipeptidase (NAALADase), a splice variant of GCPII.

Deleterious effects of incense smoke exposure on kidney function and architecture in male albino rats.

CONTEXT: Previous studies, including ours, have shown adverse effects of incense smoke on human health. However, the effect of incense smoke on kidney function and structure remains unknown. OBJECTIVE: To evaluate possible adverse effects of incense smoke on kidney function and architecture in albino rats after chronic exposure to Arabian incense. MATERIALS AND METHODS: Emission characteristics including particle size distribution, volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs) were determined by gravimetric and GCMS analyses. Kidney functional markers, oxidative stress and inflammatory markers were measured by standard or ELISA based procedures. Ultrastructural changes in kidney were examined by transmission electron microscope (TEM) and the gene expression of xenobiotic metabolizing enzymes including cytochrome P-450-1A1 (CYP1A1) and CYP1A2 were studied by real time PCR. RESULTS: Rats exposed to incense smoke demonstrated a significant increase in serum creatinine, uric acid, blood urea nitrogen (BUN), tissue malondialdehyde (MDA), tumor necrosis factor-alpha (TNF-α) and interleukin-4 (IL-4) levels and a significant decline in tissue reduced glutathione (GSH) and catalase activity. Incense smoke exposed rats also displayed marked ultrastructural changes in kidney tissue. Further, a significant increase in tissue gene expression of both CYP1A1 and CYP1A2 was noted in exposed rats. DISCUSSION: Changes to kidney functional markers and architecture appear to be mediated through augmented oxidative stress and inflammation. CONCLUSION: Long-term exposure to incense smoke may have deleterious effects on kidney function and architecture. Though, inhalation is the rout of exposure, findings of this study underscore that incense smoke may also have an effect on non-pulmonary tissues.