Genetic polymorphisms in Nrf2 and FoxO1: implications for antioxidant enzyme activity in diabetes.

In diabetes, persistent hyperglycemia generates excess reactive oxygen species (ROS), leading to oxidative stress (OS). In response to OS, transcription factors (TFs) Nrf2 and FoxO1 get activated, which induce the expression of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). It is well documented that the antioxidant response in diabetic individuals is very low. Since Nrf2 and FoxO1 are the major TFs activating these genes, we were interested in determining if single nucleotide polymorphisms (SNPs) in genes for these TFs have any association with lowered antioxidant enzyme activity in diabetic individuals. The activity of CAT and SOD and total antioxidant capacity (TAC) were quantified from the serum samples of diabetic (n = 98) and non-diabetic (n = 90) individuals. Genomic DNA was isolated, and Nrf2 and FoxO1 were amplified and sequenced by Illumina NextSeq500. Data were screened for SNPs in amplified regions. An independent samples t-test to find an association between CAT, SOD, and TAC and allele frequency of SNP with the diabetic condition was carried out. We found decreased CAT and SOD activity and significantly low TAC in diabetic individuals. Thirty-two and thirty-four SNPs and Single-nucleotide variants (SNVs) were observed in Nrf2 and FoxO1, respectively. However, a statistically significant difference in the allele frequency distribution between study groups was observed only in two intronic SNPs, rs17524059:A > C and rs60373589:Indel(A) of Nrf2 and FoxO1, respectively. SNPs, rs17524059 in the Nrf2 and rs60373589 of FoxO1, were not associated with reduced CAT and SOD activity and level of TAC in Indian diabetic individuals.Communicated by Ramaswamy H. Sarma.

Towards standardized and reproducible research in skin microbiomes.

Skin is a complex organ serving a critical role as a barrier and mediator of interactions between the human body and its environment. Recent studies have uncovered how resident microbial communities play a significant role in maintaining the normal healthy function of the skin and the immune system. In turn, numerous host-associated and environmental factors influence these communities' composition and diversity across the cutaneous surface. In addition, specific compositional changes in skin microbiota have also been connected to the development of several chronic diseases. The current era of microbiome research is characterized by its reliance on large data sets of nucleotide sequences produced with high-throughput sequencing of sample-extracted DNA. These approaches have yielded new insights into many previously uncharacterized microbial communities. Application of standardized practices in the study of skin microbial communities could help us understand their complex structures, functional capacities, and health associations and increase the reproducibility of the research. Here, we overview the current research in human skin microbiomes and outline challenges specific to their study. Furthermore, we provide perspectives on recent advances in methods, analytical tools and applications of skin microbiomes in medicine and forensics.

Skin microbiota diversity among genetically unrelated individuals of Indian origin.

Background: Human skin harbors complex transient and resident microbial communities that show intra- & inter-individual variation due to various environmental and host-associated factors such as skin site, diet, age, gender, genetics, or the type and use of cosmetics. This variation remains largely uncharacterized in the Indian population; hence, the present study aims to characterize the variation in skin microbiota among individuals of Indian origin and quantify associations with age, diet, and geography. Methods: Axillary sweat samples from genetically unrelated individuals (N = 58) residing in the three geographical locations of Maharashtra, India, were collected using a sterile cotton swab. Bacterial DNA was extracted using a standard protocol and checked for quality. Variable regions (V3-V4) of the 16S rRNA gene were sequenced using the Illumina platform. We used standard methods from microbiota bioinformatics, including alpha and beta diversity, community typing, and differential abundance, to quantify the association of skin microbiota with age, diet, and geographical location. Results: Our study indicated the prevalence of phyla- Firmicutes, Proteobacteria, and Actinobacteria, consistent with previous reports on skin microbiota composition of the world population level. The alpha diversity (Shannon index) was significantly associated with the age group (Kruskal-Wallis test, p = 0.02), but not with geography (p = 0.62) or diet (p = 0.74). The overall skin microbiota community composition was significantly associated with geographical location based on Community State Types (CST) analysis and PERMANOVA (R2 = 0.07, p = 0.01). Differential abundance analysis at the genus level indicated a distinctively high abundance of Staphylococcus and Corynebacterium among individuals of the Pune district. Pseudomonas and Anaerococcus were abundant in individuals from Ahmednagar whereas, Paenibacillus, Geobacillus, Virgibacillus, Jeotgalicoccus, Pullulanibacillus, Delsulfosporomusa, Citinovibrio, and Calditerricola were abundant in individuals from Nashik district. Conclusion: Our work provides one of the first characterizations of skin microbiota variation in different sub-populations in India. The analysis quantifies the level of individuality, as contrasted to the other factors of age, geography, and diet, thus helping to evaluate the applicability of skin microbiota profiles as a potential biomarker to stratify individuals.

MC1R diversity and its role in skin pigmentation variation in West Maharashtra, India.

OBJECTIVES: MC1R polymorphisms have been reported to be under a selective constraint in populations inhabiting high UVR regions such as Africans; however, these patterns are not consistent. Here we analyze the MC1R gene in West Maharashtra, India to see if sequence diversity corresponds to their diverse pigmentary profiles and if MC1R is constrained in dark skinned tribal as compared to lighter skinned caste populations. METHODS: A 2648 bp region of this gene was sequenced in 102 individuals and the data was compared for π, Ï´ diversity indices. Tajima's D was assessed for signatures of purifying selection and MC1R variants were associated with MI measures using the additive, dominant, and recessive models. Pairwise FST was tested among study populations and between study populations and 1000 Genomes regional samples. RESULTS: MC1R diversity was not uniquely patterned among castes and tribes. Non-synonymous variants rs2228479A, rs1805007_T, and rs885479_A showed low variability in these populations. Selection tests did not indicate any constraint on MC1R and pairwise FST were also low among the study populations (-0.0163 to 0.06112). The SNP rs3212359 was significantly associated with MI measures when tested using different association models. CONCLUSIONS: We do not find evidence of a selective constraint on MC1R. The presence of a large number of unique haplotypes and low FST values at this locus suggests that MC1R polymorphisms may not be influencing pigmentation variation among castes and tribes in this region. Observed associations between rs3212359 and MI measures need to be validated through studies on larger samples and in-vitro functional studies.

Polyalanine polymorphism in the signal peptide of Glutathione peroxidase 1 (GPX1) gene & its association with osteoporosis.

Background & objectives: Osteoporosis is a systemic skeletal disease, characterized by a low bone mass leading to increased bone fragility and hence, a greater susceptibility to the risk of fracture. Since age-related oxidative stress is one of the factors that has been implicated in developing low bone mineral density (BMD), leading to osteoporosis, this study wanted to explore the expression of antioxidant enzymes in individuals with osteoporosis. The present study focused on mapping polymorphism in an important antioxidant enzyme glutathione peroxidase 1 (GPx1) among osteoporosis and healthy Asian Indians. Methods: Dual-energy X-ray absorptiometry was used to assess BMD of individuals and was classified into normal (n=96) and osteoporotic (n=88) groups. Biochemical parameters such as vitamin D, total oxidant status (TOS), and GPx1 enzyme activity were estimated from plasma samples of recruited individuals. Quantitative real-time qRT-PCR was carried out using GAPDH as an endogenous control. Genomic DNA was isolated from whole blood, and polymorphisms were evaluated by sequencing. Results: The BMD was lower in osteoporotic individuals, and further analysis of biochemical parameters indicated significantly low 25-hydroxy vitamin D and GPx1 with higher TOS levels in osteoporotic as compared to healthy individuals. Furthermore, qRT-PCR revealed low expression of GPX1 in osteoporotic individuals. GPX1 sequence analysis of the promoter and two exons revealed the lower frequency of five alanine repeats in the osteoporotic individuals. Interpretation & conclusions: In this study, the in silico analysis revealed the lower frequency of five alanine repeats in exon 1 of GPX1 and high TOS to be associated with osteoporosis. However, no polymorphism was found in exon 2 of GPX1 among the two study groups.

The phytochemical plumbagin reciprocally modulates osteoblasts and osteoclasts.

Bone metabolism is essential for maintaining bone mineral density and bone strength through a balance between bone formation and bone resorption. Bone formation is associated with osteoblast activity whereas bone resorption is linked to osteoclast differentiation. Osteoblast progenitors give rise to the formation of mature osteoblasts whereas monocytes are the precursors for multi-nucleated osteoclasts. Chronic inflammation, auto-inflammation, hormonal changes or adiposity have the potential to disturb the balance between bone formation and bone loss. Several plant-derived components are described to modulate bone metabolism and alleviate osteoporosis by enhancing bone formation and inhibiting bone resorption. The plant-derived naphthoquinone plumbagin is a bioactive compound that can be isolated from the roots of the Plumbago genus. It has been used as traditional medicine for treating infectious diseases, rheumatoid arthritis and dermatological diseases. Reportedly, plumbagin exerts its biological activities primarily through induction of reactive oxygen species and triggers osteoblast-mediated bone formation. It is plausible that plumbagin's reciprocal actions - inhibiting or inducing death in osteoclasts but promoting survival or growth of osteoblasts - are a function of the synergy with bone-metabolizing hormones calcitonin, Parathormone and vitamin D. Herein, we develop a framework for plausible molecular modus operandi of plumbagin in bone metabolism.

Plumbagin, a Biomolecule with (Anti)Osteoclastic Properties.

Plumbagin is a plant-derived naphthoquinone that is widely used in traditional Asian medicine due to its anti-inflammatory and anti-microbial properties. Additionally, plumbagin is cytotoxic for cancer cells due to its ability to trigger reactive oxygen species (ROS) formation and subsequent apoptosis. Since it was reported that plumbagin may inhibit the differentiation of bone resorbing osteoclasts in cancer-related models, we wanted to elucidate whether plumbagin interferes with cytokine-induced osteoclastogenesis. Using C57BL/6 mice, we unexpectedly found that plumbagin treatment enhanced osteoclast formation and that this effect was most pronounced when cells were pre-treated for 24 h with plumbagin before subsequent M-CSF/RANKL stimulation. Plumbagin caused a fast induction of NFATc1 signalling and mTOR-dependent activation of p70S6 kinase which resulted in the initiation of protein translation. In line with this finding, we observed an increase in RANK surface expression after Plumbagin stimulation that enhanced the responsiveness for subsequent RANKL treatment. However, in Balb/c mice and Balb/c-derived RAW264.7 macrophages, these findings could not be corroborated and osteoclastogenesis was inhibited. Our results suggest that the effects of plumbagin depend on the model system used and can therefore either trigger or inhibit osteoclast formation.

A Genome-Wide Association Study of Skin and Iris Pigmentation among Individuals of South Asian Ancestry.

South Asia has a complex history of migrations and is characterized by substantial pigmentary and genetic diversity. For this reason, it is an ideal region to study the genetic architecture of normal pigmentation variation. Here, we present a meta-analysis of two genome-wide association studies (GWASs) of skin pigmentation using skin reflectance (M-index) as a quantitative phenotype. The meta-analysis includes a sample of individuals of South Asian descent living in Canada (N = 348), and a sample of individuals from two caste and four tribal groups from West Maharashtra, India (N = 480). We also present the first GWAS of iris color in South Asian populations. This GWAS was based on quantitative measures of iris color obtained from high-resolution iris pictures. We identified genome-wide significant associations of variants within the well-known gene SLC24A5, including the nonsynonymous rs1426654 polymorphism, with both skin pigmentation and iris color, highlighting the pleiotropic effects of this gene on pigmentation. Variants in the HERC2 gene (e.g., rs12913832) were also associated with iris color and iris heterochromia. Our study emphasizes the usefulness of quantitative methods to study iris color variation. We also identified novel genome-wide significant associations with skin pigmentation and iris color, but we could not replicate these associations due to the lack of independent samples. It will be critical to expand the number of studies in South Asian populations in order to better understand the genetic variation driving the diversity of skin pigmentation and iris color observed in this region.

Epigenetic regulation of BMP2 gene in osteoporosis: a DNA methylation study.

Osteoporosis is a multifactorial disease in which genetic factors and epigenetic modifications play a major role. DNA methylation is known for gene silencing and its effect on BMP2 promoter has been studied here to understand its regulatory activity in osteoporosis pathogenicity. CpG methylation in the BMP2 promoter was analyzed by performing bisulfite specific PCR on the gDNA samples extracted from whole blood of osteoporotic (n = 24) and healthy (n = 24) individuals. Disproportionate allele frequency of CpG sites was calculated statistically. Differential BMP2 expression was estimated using quantitative RT-PCR technique. Luciferase reporter assay was performed to determine and confirm differential transcriptional activity of BMP2 promoter due to methylation. Total of 14 CpG sites were reporter in the BMP2 promoter of which, CpG site at - 267th position upstream to TSS was found to have disproportionate allele frequency among osteoporotic and healthy individuals and was found to be significantly associated with osteoporosis condition. Functional and gene expression analysis of this methylated site using luciferase reporter vector and Real Time PCR approach, suggested reduced transcriptional activity of BMP2 promoter as well as decreased gene expression in disease condition. BMP2 is being a central signaling molecule, aberrant methylation in the promoter region may result into down regulation of osteoblast markers involved in bone formation.

Characterization of Chemical Constituents of Human Sweat: A Study Based on Indian Population.

There is a strong evidence in the literature that human odor is unique to an individual; therefore, the focus of this study was to strengthen this evidence through the testing of sweat samples on unrelated individuals with the same ethnicity. Sweat samples were collected from 42 unrelated Indian males and females residing in the same city to determine the chemical constituents in human sweat. The volatile compounds of sweat were subsequently analyzed and identified using gas chromatography-mass spectrometry, and a National Institute of Standards and Technology library was used for individual profiling. A total of 78 compounds were identified in human sweat tested with 22 compounds found to be unique to the individual (frequency of occurrence one). A scent profile, or "chexmotype," unique to the sweat of each individual was obtained. This is the first extensive study on an Indian population with 36 new compounds detected in human sweat.

Association of genetic variants with skin pigmentation phenotype among populations of west Maharashtra, India.

OBJECTIVES: South Asians exhibit extensive variation in skin melanin index (MI) which is observed across the broader region of South Asia as well as within restricted geographic regions. However, the genetic variants associated with variation in the skin pigmentation phenotype are poorly understood in these populations. The present study examines the association between MI measures and genetic variants from 5 candidate pigmentation genes among 533 individuals representing 6 populations of West Maharashtra. METHODS: Associations between five single nucleotide polymorphisms (SNPs) known to play a role in pigmentation (rs1426654-SLC24A5, rs1042602-TYR, rs16891982-SLC45A2, rs6058017-ASIP, and rs642742-KITLG) and MI measures were tested using standard one-way analysis of variance (ANOVA) within each population. Multiple linear regression was used to test the effects of these SNPs in the full West Maharashtra sample using sex, age, and population or social group as covariates. RESULTS: rs1426654 showed significant association with MI in all six study populations (P < 0.01). Association tests using sex, age, and population as covariates showed rs1426654 and rs1042602 to be significantly (P < 0.01) associated with lighter skin pigmentation in West Maharashtra as a whole. By contrast, when social group was added as a covariate instead of population, rs1426654, rs1042602, and rs16891982 were significantly (P < 0.01) associated with lighter skin pigmentation. CONCLUSIONS: Only rs1426654 is significantly associated with MI in each individual population; however, rs1426654, rs1042602, and rs16891982 are significantly associated with pigmentation in the broader West Maharashtra region after controlling for population and social group, with rs1426654 (SLC24A5) explaining the majority of the observed variation. Am. J. Hum. Biol. 28:610-618, 2016. © 2016 Wiley Periodicals, Inc.

Skin pigmentation variation among populations of West Maharashtra, India.

OBJECTIVES: Global patterns of skin pigmentation have evolved as an adaptation to local ultraviolet radiation (UVR). Indian populations exposed to intense UVR show great variation in skin pigmentation. The UVR-based selection model cannot satisfactorily address the high prevalence of light skin among these populations. Thus, the present study examines pigmentation variation among populations of West Maharashtra and the Indian subcontinent within the context of population structure and social hierarchy. METHODS: Melanin index (MI) was measured from 555 individuals representing six endogamous populations of West Maharashtra. Skin pigmentation was assessed in terms of variation between populations and differences between and among castes and tribes. A linear regression analysis was run to assess the relationship among MI, UVR, and social hierarchy using published MI data from 13 Indian endogamous populations. RESULTS: Skin pigmentation differed significantly among populations of West Maharashtra. Significant pigmentation variation exists between castes and tribes of West Maharashtra as well as across the Indian subcontinent. We observe a significant negative relationship between social hierarchy and skin pigmentation, whereas the relationship between UVR and MI is weak. CONCLUSIONS: Our results suggest that various factors may have contributed to pigmentation diversity across the Indian subcontinent. The lack of correlation between UVR and MI suggests that natural selection may not have played a significant role in shaping pigmentation variation across the subcontinent. We discuss other possible explanations, including metabolic conservation and cultural factors such as traditional social hierarchies and strict endogamy that have led to the development of population structure.

Superoxide Dismutase 2 Polymorphisms and Osteoporosis in Asian Indians: A Genetic Association Analysis.

Oxidative stress plays an important role in the development of osteoporosis. The present cross-sectional study focuses on mapping single nucleotide polymorphisms (SNPs) in the mitochondrial manganese superoxide dismutase (SOD2) gene in Asian Indians. The bone mineral density (BMD) of study subjects was assessed by dual x-ray absorptiometry. Individuals were classified as normal (n = 82) or osteoporotic (n = 98). Biochemical parameters such as vitamin D, total oxidant status (TOS) and SOD2 enzyme activity were estimated from plasma samples. Semi-quantitative PCR was carried out using GAPDH as an endogenous control. Genomic DNA was isolated from whole blood and SNPs were evaluated by PCR sequencing. Thirteen SNPs are reported in the examined region of the SOD2 gene, out of which in our samples SNPs rs5746094 and rs4880 were found to be polymorphic. Allele G of rs5746094 (intronic) and allele C of rs4880 (exonic) are significantly higher in the osteoporotic individuals. Presence of allele C of rs4880 and increased level of TOS among osteoporotic individuals were found to be associated with disease risk.

Genetic epidemiology of osteoporosis across four microsatellite markers near the VDR gene.

The large amount of positive genetic association data in a number of bone diseases suggests functional consequences of Vitamin D receptor (VDR) gene polymorphism. In the present study, four microsatellite markers viz., D12S1633, D12S1635, D12S347, and D12S96, that lie in the vicinity of the VDR gene on chromosome 12 were selected to assess the allele distribution pattern and diversity among three groups of individuals - normal, osteopenia and osteoporosis. Genetic association study was performed using allele frequency data. Total genomic DNA was isolated from the whole blood of 226 individuals, after recording their bone mineral density (BMD) using Dual X-ray absorptiometry (DXA). All DNA samples were subjected to multiplex Polymerase Chain Reaction (PCR) - genotyping. Allele frequencies and genetic diversity parameters like - number of alleles, average variance and average heterozygosity across all the four markers among three groups were computed. Effect of population stratification was excluded by investigating population structure. A trend of decreasing genetic diversity across four loci from normal to pre- and post-disease condition has been observed. Lesser recombination rate (θ) indicates linkage between studied microsatellite markers and VDR gene. Statistically significant linkage disequilibrium was detected for the allele - 22 of locus D12S96 with osteoporosis. A positive association of allele - 22 suggests susceptibility to disease whereas predominance of allele - 27 among non - diseased group implicates its association with normal bone health.

Host microsatellite alleles in malaria predisposition?

BACKGROUND: Malaria is a serious, sometimes fatal, disease caused by Plasmodium infection of human red blood cells. The host-parasite co-evolutionary processes are well understood by the association of coding variations such as G6PD, Duffy blood group receptor, HLA, and beta-globin gene variants with malaria resistance. The profound genetic diversity in host is attributed to polymorphic microsatellites loci. The microsatellite alleles in bacterial species are known to have aided their survival in fatal environmental conditions. The fascinating question is whether microsatellites are genomic cushion in the human genome to combat disease stress and has cause-effect relationships with infections. PRESENTATION OF THE HYPOTHESIS: It is hypothesized that repeat units or alleles of microsatellites TH01 and D5S818, located in close proximity to beta-globin gene and immune regulatory region in human play a role in malaria predisposition. Association of alleles at aforesaid microsatellites with malaria infection was analysed. To overrule the false association in unrecognized population stratification, structure analysis and AMOVA were performed among the sampled groups. TESTING OF HYPOTHESIS: Associations of microsatellite alleles with malaria infection were verified using recombination rate, Chi-square, and powerful likelihood tests. Further investigation of population genetic structure, and AMOVA was done to rule out the confounding effects of population stratification in interpretation of association studies. IMPLICATION OF THE HYPOTHESIS: Lower recombination rate (theta) between microsatellites and genes implicated in host fitness; positive association between alleles-13 (D5S818), 9 (TH01) and strong susceptibility to Plasmodium falciparum; and alleles-12 (D5S818) and 6 (TH01) rendering resistance to human host were evident. The interesting fact emerging from the study was that while predisposition to malaria was a prehistoric attribute, among TH01 alleles; evolution of resistant allele-6 was a recent phenomenon, which could conceivably be driven by infection related selective forces. The host's microsatellite allelic associations with malaria infection were valid in the light of low genetic variance between sampled groups and no population stratification.

Deciphering diversity in populations of various linguistic and ethnic affiliations of different geographical regions of India: analysis based on 15 microsatellite markers.

The extent of genetic polymorphism at fifteen autosomal microsatellite markers in 54 ethnically, linguistically and geographically diverse human populations of India was studied to decipher intrapopulation diversity. The parameters used to quantify intrapopulation diversity were average allele diversity, average heterozygosity, allele range (base pairs), and number of alleles. Multilocus genotype frequencies calculated for selected populations were utilized for testing conformity with the assumption of Hardy-Weinberg equilibrium. The exact test values, after Bonferroni correction, showed significant deviation amongst Gowda (vWA, Penta E); Dhangar, Satnami and Gounder (D8S1179); Hmar (FGA); Kuki and Balti (vWA) groups. Relatively low number of alleles and allelic diversity (base-pairs size) had been observed in populations of central India as compared with southern and northern regions of the country. The communities of Indo-Caucasoid ethnic origin and Indo-European linguistic family (Kshatriya of Uttar Pradesh) showed highest allelic diversity, as well as rare alleles, not reported in any other Indian populations. Analysis based on average heterozygosity was also found to be lowest among the populations of central India (0.729) and highest among the populations from north (0.777) and west (0.784) regions of the country, having Indo-Caucasoid ethnic origin and Austro-Asiatic linguistic affiliation. The maximum power of discrimination (85%-89%) had been observed at loci FGA, Penta E, D18S51 and D21S11, suggested high intrapopulation diversity in India. Genetic diversity revealed by STR markers was consistent with the known demographic histories of populations. Thus, the present study clearly demonstrated that the intrapopulation diversity is not only present at the national level, but also within smaller geographical regions of the country. This is the first attempt to understand the extent of diversity within populations of India at such a large scale at genomic level.