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Protein adopts multitude of flexible and rapidly interconverting conformers, many which are governed by specific protein-interaction domains. ApoA1, a key player involved in high-density lipoprotein (HDL) regulation exists in structurally diverse forms with varying degree of cholesterol association, and each state is associated with different functional properties. While disc-shaped HDL and its oligomeric ApoA1 protein components have been the focus of several investigations, structural properties of monomeric ApoA1 are poorly understood. Here, we undertook large-scale structural analysis of ApoA1 in apo and cholesterol-bound forms using tens of independent simulations with total computing time exceeding 50 μs. Examination of multiple lipid-free trajectories of monomeric ApoA1 revealed a common conformation, with distinct spatial proximity between N- and C-terminal domains. With incorporation of physiologically known cholesterol concentration (~100 cholesterol molecules) in ApoA1 simulations, the monomeric protein spontaneously formed an open circular topology. Remarkably, these drastic structural perturbations are driven by specific binding site at C-terminal and a novel cholesterol binding site at the N-terminal. We proposed a mechanism of stage-wise opening of ApoA1 and demonstrated that less cholesterol concentration around interaction sites or mutation within N-terminal binding sites does not lead to open bell-shaped topology. The kinetic barriers between open- and closed-states also showed an ensemble of loosely packed helix bundle (H1-H7; H4-H7) that posed as a slow-intermediate step. Lastly we performed complementary experiments, including ITC and CD measurements to confirm that structural changes are induced by ligand association and not driven by random hydrophobic effect. Collectively, our study suggests a previously unknown mechanism of cholesterol sequestering by ApoA1 that could directly aid in developing modulators for cholesterol efflux with chronic cardiovascular diseases.

Proteins are the major functional unit that dictates the structure and function of a cell, tissue or organism. The term proteome refers to the pool of proteins coded by the whole genome. Significant efforts towards deciphering the secrets of the human genome in the next-generation genomics era, with high-throughput technologies, failed to provide functional relevance, as gene expressions do not necessarily correlate with protein expression. Therefore, proteomics has emerged as an independent stream of biological research for the large-scale comprehensive systematic study of the proteome of a given cell, organism or condition. Mass spectrometry has proven to be central to proteomics-based studies as it not only helps in the identification of proteins but has also been successfully used for quantification. Initially gel-based (both 1 and 2 dimensions) proteomics played a significant role in the identification and quantification of proteins in a given experimental condition. However gel-based proteomics, has its own limitations and is a cumbersome process. The evolution of liquid chromatography and mass spectrometry has played a pivotal role for the development of proteomics and they are now routinely used for qualitative and quantitative analyses. One of the most important aspects of proteomics is data analysis. A large amount of high-throughput and multidimensional data generated from proteomics experiments needs to be analyzed with profound accuracy. Therefore understanding the principles of the various analysis tools is also essential to making sense of the complex proteomics data. Applications of these advanced sophisticated tools have helped enormously in deciphering critical nodes in biological research. In this chapter, we aim to provide a snapshot of “proteomics”, from sample collection to data analysis, and, most importantly, its application towards solving critical biological questions.

Myocardial infarction (MI) is defined as cardiac cell death due to prolonged ischemia. Although necrotic cell death was considered to be solely responsible for myocyte death during MI, it was recently revealed that apoptosis also plays its part in this death process. Our laboratory has recently shown that endoplasmic reticulum (ER) stress-induced apoptosis is the predominant route for apoptosis during MI and the conventional mitochondrial pathway is bypassed by activation of a small heat shock protein α-crystallin B (CRYAB). Since CRYAB is a direct target of P38 mitogen-activated protein kinase (MAPK) cascade, we were prompted to check the role of P38 MAPK in 20-week-old male Wister rats immediately after infarct formation. Interestingly, parallel activation of mitochondrial apoptotic pathway with an increase in ER stress-induced apoptotic load was observed along with decreased activation of CRYAB and Nrf2 (a pro-survival protein activated in response to ER stress) in MI rats treated with SB203580, a specific inhibitor of P38α and P38β compared to the MI alone. As a cumulative effect, this inhibitor treatment also resulted in significant increase in the levels of caspase3 activity and TUNEL positivity, the end point apoptotic markers. Furthermore, SB203580-treated hypoxic adult cardiomyocytes showed formation of desmin aggregates which were previously associated with impaired cardiac function. Thus, this study shows for the first time the precise mechanism by which P38 MAPK plays a pro-survival role and confers protection of cardiomyocytes, during infarct formation.

Background The alteration in the epigenome forms an interface between the genotype and the environment. Epigenetic alteration is expected to make a significant contribution to the development of cardiovascular disease where environmental interactions play a key role in disease progression. We had previously shown that global DNA hypermethylation per se is associated with coronary artery disease (CAD) and is further accentuated by high levels of homocysteine, a thiol amino acid which is an independent risk factor for cardiovascular disease and is also a key modulator of macromolecular methylation. Results We have identified 72 differentially methylated regions (DMRs) that were hypermethylated in CAD patients in the background of varying homocysteine levels. Following deep bisulfite sequencing of a few of the selected DMRs, we found significantly higher methylation in CAD cases. We get six CpG sites in three DMRs that included the intronic region of C1QL4 gene and upstream region of CCDC47 and TGFBR3 genes. Conclusion To the best of our knowledge, this is the first study to identify hypermethylated regions across the genome in patients with coronary artery disease. Further validation in different populations is necessary for this information to be used for disease risk assessment and management.

Oxygen-compromised environments, such as high altitude, air travel, and sports, and pathological conditions, such as solid tumors, have been suggested to be prothrombotic. Despite the indispensable role of platelets in thrombus formation, the studies linking hypoxia, platelet reactivity, and thrombus formation are limited. In the present study, platelet proteome/reactivity was analyzed to elucidate the acute hypoxia-induced prothrombotic phenotype. Rats exposed to acute simulated hypoxia (282 torr/8% oxygen) demonstrated a decreased bleeding propensity and increased platelet reactivity. Proteomic analysis of hypoxic platelets revealed 27 differentially expressed proteins, including those involved in coagulation. Among these proteins, calpain small subunit 1, a 28-kDa regulatory component for calpain function, was significantly upregulated under hypoxic conditions. Moreover, intraplatelet Ca(2+) level and platelet calpain activity were also found to be in accordance with calpain small subunit 1 expression. The inhibition of calpain activity demonstrated reversal of hypoxia-induced platelet hyperreactivity. The prothrombotic role for calpain was further confirmed by an in vivo model of hypoxia-induced thrombosis. Interestingly, patients who developed thrombosis while at extreme altitude had elevated plasma calpain activities and increased soluble P-selectin level. In summary, this study suggests that augmented calpain activity is associated with increased incidence of thrombosis under hypoxic environments.

Gene environment interactions leading to epigenetic alterations play pivotal role in the pathogenesis of Coronary Artery Disease (CAD). Altered DNA methylation is one such epigenetic factor that could lead to altered disease etiology. In this study, we comprehensively identified methylation sites in several genes that have been previously associated with young CAD patients.The study population consisted of 42 healthy controls and 33 young CAD patients (age group50 years). We performed targeted bisulfite sequencing of promoter as well as gene body regions of several genes in various pathways like cholesterol synthesis and metabolism, endothelial dysfunction, apoptosis, which are implicated in the development of CAD.We observed that the genes like GALNT2, HMGCR were hypermethylated in the promoter whereas LDLR gene promoter was hypomethylated indicating that intracellular LDL uptake was higher in CAD patients. Although APOA1 did not show significant change in methylation but APOC3 and APOA5 showed variation in methylation in promoter and exonic regions. Glucokinase (GCK) and endothelial nitric oxide synthase 3 (NOS3) were hyper methylated in the promoter. Genes involved in apoptosis (BAX/BCL2/AKT2) and inflammation (PHACTR1/LCK) also showed differential methylation between controls and CAD patients. A combined analysis of the methylated CpG sites using machine learning tool revealed 14 CpGs in 11 genes that could discriminate CAD cases from controls with over 93% accuracy.This study is unique because it highlights important gene methylation alterations which might predict the risk of young CAD in Indian population. Large scale studies in different populations would be important for validating our findings and understanding the epigenetic events associated with CAD.

Accumulation of cysteine induces translational defects and metabolic rewiring that are abrogated by leucine in a transfer RNA (tRNA) methyltransferase NCL1-dependent manner in yeast., Nutritional limitation has been vastly studied; however, there is limited knowledge of how cells maintain homeostasis in excess nutrients. In this study, using yeast as a model system, we show that some amino acids are toxic at higher concentrations. With cysteine as a physiologically relevant example, we delineated the pathways/processes that are altered and those that are involved in survival in the presence of elevated levels of this amino acid. Using proteomics and metabolomics approach, we found that cysteine up-regulates proteins involved in amino acid metabolism, alters amino acid levels, and inhibits protein translation—events that are rescued by leucine supplementation. Through a comprehensive genetic screen, we show that leucine-mediated effect depends on a transfer RNA methyltransferase (NCL1), absence of which decouples transcription and translation in the cell, inhibits the conversion of leucine to ketoisocaproate, and leads to tricarboxylic acid cycle block. We therefore propose a role of NCL1 in regulating metabolic homeostasis through translational control.

Isomeric asymmetric and symmetric dimethyl arginine (ADMA and SDMA respectively) residues are excreted in urine and are putative markers of cardiovascular and chronic kidney diseases. In this work, we demonstrate simultaneous and quantitative detection of endogeneous ADMA and SDMA from urine samples of healthy subjects using MALDI-TOF MS without any chromatographic separation. The DMA isomers yielded [M + H]+ ions along with their product ions formed due to MALDI in-source fragmentation. The precursor ions were validated using MALDI-TOF MS/MS as well as direct injection ESI-Q-TOF MS/MS. ADMA and SDMA generated unique product ions at ∼ m/z 46 and ∼ m/z 172 respectively in the MS-mode itself. These were advantageously used for full scan-mode absolute quantification of the isomeric metabolites. The m/z observed for all the ions was within 10 ppm mass accuracy. The calibration method was established by generating internal standard normalized peak area-based concentration response curves using synthetic standards. Good linearities (R2 > 0.95) with acceptable intra-assay, inter-assay variation (within 15% RSD) and excellent recoveries were observed for quality control samples. Finally, endogeneous concentrations of the metabolites were determined in urine from healthy subjects (n = 11). ADMA and SDMA were found to be in the range of 1.6–8 μM and 2.9–9.1 μM in urine and were in agreement with previously reported physiological levels.

Indians undergoing socioeconomic and lifestyle transitions will be maximally affected by epidemic of type 2 diabetes (T2D). We conducted a two-stage genome-wide association study of T2D in 12,535 Indians, a less explored but high-risk group. We identified a new type 2 diabetes–associated locus at 2q21, with the lead signal being rs6723108 (odds ratio 1.31; P = 3.32 × 10−9). Imputation analysis refined the signal to rs998451 (odds ratio 1.56; P = 6.3 × 10−12) within TMEM163 that encodes a probable vesicular transporter in nerve terminals. TMEM163 variants also showed association with decreased fasting plasma insulin and homeostatic model assessment of insulin resistance, indicating a plausible effect through impaired insulin secretion. The 2q21 region also harbors RAB3GAP1 and ACMSD; those are involved in neurologic disorders. Forty-nine of 56 previously reported signals showed consistency in direction with similar effect sizes in Indians and previous studies, and 25 of them were also associated (P < 0.05). Known loci and the newly identified 2q21 locus altogether explained 7.65% variance in the risk of T2D in Indians. Our study suggests that common susceptibility variants for T2D are largely the same across populations, but also reveals a population-specific locus and provides further insights into genetic architecture and etiology of T2D.

Maternal vitamin deficiencies are associated with low birth weight and increased perinatal morbidity and mortality. We hypothesize that maternal folate and/or vitamin B(12) restrictions alter body composition and fat metabolism in the offspring. Female weaning Wistar rats received ad libitum for 12 weeks a control diet (American Institute of Nutrition-76A) or the same with restriction of folate, vitamin B(12) or both (dual deficient) and, after confirming vitamin deficiency, were mated with control males. The pregnant/lactating mothers and their offspring received their respective diets throughout. Biochemical and body composition parameters were determined in mothers before mating and in offspring at 3, 6, 9 and 12 months of age. Vitamin restriction increased body weight and fat and altered lipid profile in female Wistar rats, albeit differences were significant with only B(12) restriction. Offspring born to vitamin-B(12)-restricted dams had lower birth weight, while offspring of all vitamin-restricted dams weighed higher at/from weaning. They had higher body fat (specially visceral fat) from 3 months and were dyslipidemic at 12 months, when they had high circulating and adipose tissue levels of tumor necrosis factor α, leptin and interleukin 6 and low levels of adiponectin and interleukin 1β. Vitamin-restricted offspring had higher activities of hepatic fatty acid synthase and acetyl-CoA-carboxylase and higher plasma cortisol levels. In conclusion, maternal and peri-/postnatal folate and/or vitamin B(12) restriction increased visceral adiposity (due to increased corticosteroid stress), altered lipid metabolism in rat offspring perhaps by modulating adipocyte function and may thus predispose them to high morbidity later.

To demonstrate the efficacy of oral methylcobalamin in treating vitamin B12 (vitB12) deficiency anemia, our prospective observational study enrolled 28 children with both macrocytic anemia and low holotranscobalamin (HoloTC) levels. Their hematological and biochemical parameters pre- and posttreatment at 1 month were compared. Hemoglobin showed mean increase of 2.89 g/dl (P

Coronary artery disease (CAD) has remained as one of the most important causes of mortality and morbidity worldwide. According to WHO, almost seven million deaths occur yearly due to this disease1. It is estimated that global cardiovascular death would increase from 17.1 million (in 2004) to 23.4 million by 2030 with CAD contributing a significant proportion2. Furthermore, the burden of this disease has increased rapidly in the developing countries3. In India alone the mortality due to CAD has increased from 1.17 million to 1.59 million from 1990 to early 20004. Since CAD is a complex disorder where both genetic and lifestyle (including dietary habits) contribute significantly, finding new potential markers holds its own clinical importance specifically in early detection and efficient management of the disease5. In recent years, several studies focussed to identify genetic markers that could be associated with CAD. To this end several Genome Wide Association Studies (GWAS) have been undertaken by various groups to identify single nucleotide polymorphisms (SNPs) that are associated with CAD6,7,8,9. However, other than a few SNPs most of them could not be replicated in different populations. Further, most of the SNPs have low discriminative accuracy and the common variants account for about 10% of predicted genetic heritability of CAD5. Thus, even now, the classical risk factors in blood like total cholesterol, HDL, LDL etc. are routinely determined to assess the risk of CAD1. These traditional risk factors are useful in identifying people at high risk of developing CAD. Wang et al. showed a predictive accuracy of 0.7 area under the curve with classical risk factors for major cardiovascular events in Framingham heart study. However, identification of newer markers are necessary to increase the predictive accuracy especially since various prospective studies using the classical markers of CAD did not show a high predictive significance for the disease10,11. With the advent of mass spectrometry based high throughput proteomic technologies, protein markers have gained attention as it is feasible to compare the proteome of diseased and healthy individuals and identify differentially expressed proteins that could potentially act as disease markers. Proteomics has helped in identifying markers for several diseases like cancer, neurological diseases etc12,13,14. Although several groups have also attempted to identify markers for various cardiovascular diseases including acute coronary syndrome, unstable and stable angina, myocardial infarction etc15,16,17, surprisingly, studies on identifying markers for stable coronary artery disease are limited. A couple of studies have been done based on peptide profiling in urine and a few peptide signatures were identified as potential biomarkers18,19. However the use of peptide signatures has an inherent problem since they can be detected only using mass spectrometer thereby limiting their utility as biomarkers in clinical practice as mass spectrometers are till date not routinely used as a tool for biomarker profiling20,21. Donahue et al. reported a qualitative proteomic analysis using pooled plasma from 53 CAD cases and 53 controls22. However, qualitative proteomic measurement will have limited application in terms of biomarkers. Thus, a panel of defined quantifiable proteins (and not peptide signatures) from an easily accessible biological fluid (like urine or plasma) is necessary for it to be considered as potential biomarkers23. In the present study using high throughput iTRAQ (Isobaric tag for relative and absolute quantitation) based relative quantitation of plasma proteome we have identified proteins that are differentially expressed in stable CAD patients. The differentially expressed proteins in the discovery phase were further validated in two phases in more than 500 samples. After multivariate logistic regression we report four proteins involved in the reverse cholesterol pathway to be significantly associated with CAD.

Cyclosporine A, a potent immunosuppressive agent extensively used to prevent allograft rejections, is under scrutiny due to severe toxic effects. CsA therapy is often continued during pregnancy in conditions such as organ transplantations and autoimmune diseases. Herein, we investigated the effects of CsA on early morphogenesis of zebrafish and identified a spectrum of proteins whose expression was altered in the drug treated embryos. Time-lapse fluorescence imaging of germ-line double transgenic zebrafish embryos treated with CsA revealed severe blood regurgitation in heart chambers, absence of blood circulation in vessels, pericardial and yolk sac edema. We also observed lack of mature blood vessels and down-regulation of endothelial markers in CsA treated embryos. Proteomic analysis using 2D-DIGE followed by mass-spectrometry led to the identification of 37 proteins whose expression was significantly modulated in presence of the drug. These proteins were mostly associated with cytoskeletal/structural assembly, lipid-binding, stress response and metabolism. Furthermore, mRNA expression analysis of eight proteins and Western blotting of actin revealed consistency between the changes observed in protein expression and its corresponding mRNA levels. Our findings demonstrate that CsA administration during early morphogenesis in zebrafish modulates the expression of some proteins which are known to be involved in important physiological processes.

Our current understanding of molecular mechanisms of cardiomyopathies has been elucidated from genetic animal models. Induced pluripotent stem cells (iPSCs) can provide a platform to improve our understanding of familial cardiomyopathies diseases. iPSCs are a type of pluripotent stem cell that can be generated directly from adult cells. Identification of mutations in patients with inherited cardiomyopathies has revealed substantial molecular complexity. In spite of the complexity, the advances of iPSC-based technology may improve our understanding of familial cardiomyopathies.

Transcobalamin (TCII) is a key enzyme involved in intracellular transport of vitamin B12. We had earlier shown that vitamin B12 levels are associated with Coronary Artery Disease (CAD). Herein, we evaluated the association of four nonsynonymous single nucleotide polymorphisms (SNPs) of TCII gene with CAD in 1398 individuals (589 CAD cases and 809 controls). Using logistic regression, we found that three SNPs (G1196A, C776G and C1043T) were significantly associated with CAD and one (G1196A) with vitamin B12 levels even after controlling for confounding factors. Thus, polymorphisms in TCII gene may play an important role in the etiology of CAD.

An elevated level of homocysteine, a thiol amino acid, is associated with various complex disorders. The cellular effects of homocysteine and its precursors S-adenosylhomocysteine (AdoHcy) and S-adenosylmethionine (AdoMet) are, however, poorly understood. We used Saccharomyces cerevisiae as a model to understand the basis of pathogenicity induced by homocysteine and its precursors. Both homocysteine and AdoHcy but not AdoMet inhibited the growth of the str4Δ strain (which lacks the enzyme that converts homocysteine to cystathionine-mimicking vascular cells). Addition of AdoMet abrogated the inhibitory effect of AdoHcy but not that of homocysteine indicating that an increase in the AdoMet/AdoHcy ratio is sufficient to overcome the AdoHcy-mediated growth defect but not that of homocysteine. Also, the transcriptomic profile of AdoHcy and homocysteine showed gross dissimilarity based on gene enrichment analysis. Furthermore, compared with homocysteine, AdoHcy treatment caused a higher level of oxidative stress in the cells. However, unlike a previously reported response in wild type (Kumar, A., John, L., Alam, M. M., Gupta, A., Sharma, G., Pillai, B., and Sengupta, S. (2006) Biochem. J. 396, 61-69), the str4Δ strain did not exhibit an endoplasmic reticulum stress response. This suggests that homocysteine induces varied response depending on the flux of homocysteine metabolism. We also observed altered expression of mitochondrial genes, defective membrane potential, and fragmentation of the mitochondrial network together with the increased expression of fission genes indicating that the imbalance in homocysteine metabolism has a major effect on mitochondrial functions. Furthermore, treatment of cells with homocysteine or AdoHcy resulted in apoptosis as revealed by annexin V staining and TUNEL assay. Cumulatively, our results suggest that elevated levels of homocysteine lead to mitochondrial dysfunction, which could potentially initiate pro-apoptotic pathways, and this could be one of the mechanisms underlying homocysteine-induced pathogenicity.

This paper proposes a methodology to limit the variations in generation cost in a power system under normal and contingent state using Differential Evolution (DE) optimization technique keeping consumer welfare in view. The aim of this proposed methodology is to minimize the deviations of generation cost, during contingency, from a preferred value by rescheduling of the generation with a controlled load curtailment technique and hence relieving the lines from overloading for congestion management. A comparative study between rescheduling with and without load curtailment has also been presented in this paper. Numerical results on test system, namely IEEE 30 Bus System, are presented for illustration purpose and the same has been verified by a well esteemed optimization technique, Particle Swarm Optimization (PSO). The comprehensive simulation results establish that the developed method not only reduces the economic variations of the market but also ensures the voltage stability of the system during contingency.

Cobalamin is an essential micronutrient in mammals. Deficiencies of this micronutrient have been implicated as risk factors for various complex diseases. Cobalamin is transported to the cells by the transport protein transcobalamin II (TCII), and hence genetic variations (like single nucleotide polymorphisms) in TCII could be perceived to affect the binding of cobalamin to TCII, thereby modulating the intracellular concentrations of cobalamin. To understand whether three nonsynonymous mutations in TCII (I5V, P241R, and R381Q) alter the structure of the protein which could potentially affect cobalamin binding, we performed molecular dynamics simulation in silico. Superimposition of active sites of the four simulated models (wild type and three variants) with the human TCII crystal structure revealed that the distance between the Nε nitrogen atom of His-173 and the cobalt ion of cobalamin deviated considerably in the I5V model as compared to wild type and other variants. His-173 directly coordinates with the cobalt ion of cobalamin. Further, from our dynamic cross-correlation and principal component analysis it appears that in the I5V model the β-domain moves apart from the α-domain creating a wide gap between the two domains. This might facilitate the initial binding of cobalamin in the I5V model as cobalamin enters the binding site through the gap between the two domains. These observations were not found in the other variants. We thus speculate that binding of cobalamin will be more facile in the I5V variant.

Altered metabolism of homocysteine in children with idiopathic nephrotic syndrome leads to raised plasma-free homocysteine levels. Elevated free homocysteine causes endothelial cell dysfunction and promotes early atherosclerosis and glomerulosclerosis. In this analytical study with a longitudinal follow-up, 29 children with first episode of nephrotic syndrome (FENS) aged 1–16 years along with 30 age andgender-matched healthy controls were enrolled. Plasma-free homocysteine was measured using high-performance liquid chromatography (HPLC). Other variables were measured using standard biochemical methods. The primary outcome measure was plasma-free homocysteine level in children with FENS and in controls. The secondary outcome measure was to observe the levels of plasma-free homocysteine in children with FENS at 12 weeks in remission and in steroid resistant states. Plasma-free homocysteine levels were significantly elevated in children with FENS at disease onset [Median (IQR) 2.170 (1.54–2.71); N = 29; P < 0.001], at 12 weeks of steroid-induced remission [Median (IQR) 1.946 (1.53–2.71); N = 22; P < 0.001], and in steroid-resistant states [Median (IQR) 2.262 (1.53–2.74); N = 7; P < 0.001] compared to controls. The levels did not decrease significantly at 12 weeks of steroid-induced remission compared to onset of nephrotic syndrome. Plasma-free homocysteine levels correlated positively with serum total cholesterol (P = 0.005; r = 0.362) and negatively with serum albumin (P = 0.032; r = 0.281). Plasma-free homocysteine levels are raised in children with FENS posing a risk of endothelial dysfunction which persists at least in short term. Long-term effects of raised plasma-free homocysteine needs to be studied.

Coronary artery disease (CAD) is one of the leading causes of death worldwide that is influenced by both environmental as well as genetic factors. Several recent genome-wide association studies (GWAS) have reported the association of multiple single nucleotide polymorphisms (SNPs) mainly in the 9p21 region with CAD. However, the association of these SNPs with CAD has not been rigorously tested in Indian population, which accounts for the largest incidences of CAD in the world. Herein, we genotyped six such SNPs (rs10116277, rs10757274, rs1333040, rs2383206, rs2383207 and rs1994016) identified through GWAS, in 754 individuals (311 angiography-confirmed CAD patients and 443 treadmill test controls) recruited mainly from North India to evaluate if these SNPs were associated with CAD. The minor allele frequency of these six SNPs was comparable to that reported in the respective GWAS. We found that three of these SNPs (rs10116277, rs1333040 and rs2383206) present at the locus 9p21 were significantly associated with CAD even after controlling for the confounding factors such as age, sex, body mass index, homocysteine, hypertension, diabetes, smoking, diet, etc. In conclusion, the locus 9p21 found to be significantly associated with cardiovascular diseases in the Caucasian populations seems to be also important in North Indian population.

Cystathionine beta-synthase (CBS) is a key enzyme that plays a critical role in homocysteine metabolism and intracellular redox balance. We have analysed the association of the CBS 844Ins68 polymorphism alone and in combination with methylenetetrahydrofolate reductase (MTHFR C677T) and choline dehydrogenase (CHDH A119C) polymorphisms (the two polymorphisms recently shown to be associated with levels of homocysteine) with homocysteine, cysteine, folate and vitamin B(12) in 817 individuals (397 patients with coronary artery disease and 420 controls). The CBS 844Ins68 polymorphism alone or in combination with MTHFR C677T and CHDH A119C polymorphisms was not significantly associated with any of the biochemical variables studied.

Human papillomavirus is considered to be a major aetiological factor but is not sufficient for the development of cervical cancer. Other host factors, including altered homocysteine levels, a functional marker of folate inadequacy, might contribute to the carcinogenic process. Herein we investigated the potential association of homocysteine levels and MTHFR polymorphisms with cervical cancer in 203 histologically confirmed cases including 39 precancer cases and 231 healthy controls with normal cervical cytology. Both patients and controls were screened for human papillomavirus infection. We found that homocysteine and consequently cysteine levels were significantly higher in cases, both cancer and precancer (p < 0.001) than controls. However, polymorphisms in the MTHFR gene (677C/T and 1298A/C) that are reported to modulate homocysteine levels were not associated with disease. Thus, our study establishes an association of total homocysteine levels with the risk of developing carcinoma of the uterine cervix.

Background— An elevated level of homocysteine (hyperhomocysteinemia) has been implicated as an independent risk factor for cardiovascular diseases. Deficiency of dietary factors like vitamin B 12 , folate, and genetic variations can cause hyperhomocysteinemia. The prevalence of hyperhomocysteinemia in the Indian population is likely to be high because most Indians adhere to a vegetarian diet, deficient in vitamin B 12 . In the background of vitamin B 12 deficiency, variations in genes involved in homocysteine metabolism might have a greater impact on homocysteine levels. Methods and Results— We genotyped 44 nonsynonymous single-nucleotide polymorphisms (nsSNPs) from 11 genes involved in homocysteine metabolism and found only 14 to be polymorphic. These 14 nsSNPs were genotyped in 546 individuals recruited from a tertiary care center in New Delhi, India, and it was found that choline dehydrogenase ( CHDH A119C) and methylenetetrahydrofolate reductase ( MTHFR C677T) were significantly associated with plasma total homocysteine levels ( P =0.009 and P =0.001, respectively). These 2 SNPs were further genotyped in 330 individuals recruited from the same center, and the association remained significant even after increasing the sample size. Furthermore, we found the possibility of a significant interaction between vegetarian diet and the 2 polymorphisms that could explain the variation of homocysteine levels. We also genotyped all the polymorphic nsSNPs in apparently healthy individuals recruited from 24 different subpopulations (based on their linguistic lineage) spread across the country to determine their basal frequencies. The frequencies of these SNPs varied significantly between linguistic groups. Conclusion— Vegetarian diet along with CHDH A119C and MTHFR C677T play an important role in modulating the homocysteine levels in Indian population.

Objective In recent years elevated homocysteine (Hcy) levels have been widely recognized as a risk factor for cardiovascular diseases (CVDs) and a connection between hyperhomocysteinemia and lipid metabolism has been suggested to have a possible role in endothelial vascular damage as lipoprotein fractions contain higher Hcy levels in hypercholesterolemia, compared to normolipidemic individuals. However, the biochemical events underlying the interaction between Hcy and LDL are still poorly understood. Methods and results Herein we have investigated the interaction of LDL with Hcy by measuring thiols S -linked to apoprotein using capillary electrophoresis and have evaluated the effect of S -homocysteinylated LDL on human endothelial cells (HECs). We found that Hcy binds to LDL in a dose dependent manner and the saturation binding is achieved at 100μmol/L Hcy in about 5h. Addition of Hcy resulted in a rapid displacement of other thiols bound to apoprotein and this was dependent on the concentration of Hcy added. For the first time we also demonstrated that treatment of HECs with homocysteine- S -LDL (Hcy- S -LDL) resulted in the induction of significantly higher levels of reactive oxygen species (ROS) compared to N-LDL (native LDL). Furthermore, the Hcy- S -LDL-induced a rise in intracellular ROS production was followed by a marked reduction of HECs proliferation and viability. Conclusions Although the mechanism by which Hcy- S -LDL elicits the current cellular effects needs further investigation, our data suggest that intracellular ROS production induced by Hcy- S -LDL might be responsible for the observed HECs damage and indicate that Hcy- S -LDL may have some role in CVD.

Epigenetic modifications, especially alteration in DNA methylation, are increasingly being recognized as a key factor in the pathogenesis of complex disorders, including atherosclerosis. However, there are limited data on the epigenetic changes in the coronary artery disease (CAD) patients. In the present study we evaluated the methylation status of genomic DNA from peripheral lymphocytes in a cohort of 287 individuals: 137 angiographically confirmed CAD patients and 150 controls. The differential susceptibility of genomic DNA to methylation-sensitive restriction enzymes was utilized to assess the methylation status of the genome. We observed that the genomic DNA methylation in CAD patients is significantly higher than in controls (p0.05). Since elevated homocysteine levels are known to be an independent risk factor for CAD and a key modulator of macromolecular methylation, we investigated the probable correlation between plasma homocysteine levels and global DNA methylation. We observed a significant positive correlation of global DNA methylation with plasma homocysteine levels in CAD patients (p = 0.001). Further, within a higher range of serum homocysteine levels (/=12-50 muM), global DNA methylation was significantly higher in CAD patients than in controls. The alteration in genomic DNA methylation associated with cardiovascular disease per se appears to be further accentuated by higher homocysteine levels.

A 63-year-old lady presented with syncope and atypical chest pain. Conventional two-dimensional (2D) contrast echocardiogram showed a highly vascular mass compressing the right atrium without direct communication. A real-time three-dimensional echocardiogram (RT3DE) revealed its outer spatial relationship with the surrounding structures and its inner heterogeneity as well. A chest computed tomography revealed a solid cardiac mass (6 x 7 cm(2)) arising from the right atrium and multiple pulmonary nodules. Histopathology of the pericardial fluid confirmed angiosarcoma. To our knowledge, this is the first report of histology-confirmed primary cardiac angiosarcoma, which was completely evaluated by conventional 2D, contrast, and RT3DE.

Phosphoproteomics-based platforms have been widely used to identify post translational dynamics of cellular proteins in response to viral infection. The present study was undertaken to assess differential tyrosine phosphorylation during early hours of rotavirus (RV) SA11 infection. Heat shock proteins (Hsp60) were found to be enriched in the data set of RV-SA11 induced differentially tyrosine-phosphorylated proteins at 2 hr post infection (hpi). Hsp60 was further found to be phosphorylated by an activated form of Src kinase on 227th tyrosine residue, and tyrosine phosphorylation of mitochondrial chaperonin Hsp60 correlated with its proteasomal degradation at 2-2.5hpi. Interestingly, mitochondrial Hsp60 positively influenced translocation of the rotaviral nonstructural protein 4 to mitochondria during RV infections. Phosphorylation and subsequent transient degradation of mitochondrial Hsp60 during early hours of RV-SA11 infection resulted in inhibition of premature import of nonstructural protein 4 into mitochondria, thereby delaying early apoptosis. Overall, the study highlighted one of the many strategies rotavirus undertakes to prevent early apoptosis and subsequent reduced viral progeny yield.

The high incidence of obesity has resulted in increased morbidity and mortality worldwide. Obesity, a common lifestyle disorder, is caused by multiple factors with heredity playing a strong causal role. Recently, a genetic variation upstream of insulin-induced gene 2 (INSIG2) (rs7566605) has been reported to be associated with obesity in four separate cohorts. Because the lifestyle and food preferences of a large proportion of Indian population differ from the rest of the world, we studied the impact of this polymorphism with body mass index (BMI). The study consisted of two cohorts - 1577 healthy individuals from three major linguistic lineages in India and 610 coronary artery disease cases and controls. In the two cohorts studied, no significant association was observed between the polymorphism and BMI. However, frequency of homozygous variant genotype was higher in non-obese individuals as compared with obese individuals in both cohorts although the difference was marginally significant only in the case-control cohort under the assumption of a recessive model. Furthermore, regardless of age and sex, mean BMI did not vary with genotype under the assumptions of recessive model. Thus, in contrast to earlier reports, the variant upstream of INSIG2 is not a determinant of BMI in Indian population.

The early diagnosis and successful treatment of breast cancer (BC) is still a challenging task due to the diverse origin and functional heterogeneity of cancer cells. The heterogeneity of BC may likely to explained by molecular BC subtypes, comprises Luminal-A (LA), Luminal-B (LB), Triple-negative (TN) and HER2-positive (HP), which are governed by a variety of cancer associated pathways. To identify protein signatures in different BC subtypes, we performed isobaric tag for absolute and relative quantitation (iTRAQ) of enriched blood plasma samples of BC subtypes (N=32) and healthy subjects (N=8). After analyses of data, 58 proteins were found to be modulated in BC subtypes from healthy subjects (p0.05) and among these; Fibronectin (FN1), Alpha-2-macroglobulin (A2M), and Complement component-4-binding protein-alpha (C4BPA) and Complement factor-B (CFB) were selected for validation in BC subtypes and healthy subjects in the independent set of blood plasma (N=100) and tissue samples (N=25). Statistical analysis showed the significant modulation of FN1 and C4BPA in LB, and A2M in TN patients in both plasma as well as tissues comparatively control (p0.05). Further, FN1 and C4BPA in LB subtype revealed a good diagnostic accuracy in plasma level validation. The receiver operating characteristic (ROC) curve and regression analysis demonstrated that these proteins with associated criterion of expression could act as discriminating signatures among BC subtypes with diagnostic and prognostic relevance.The heterogeneity of breast cancer (BC) has gained many challenges for successful management of BC, thus, the delineating proteomic alterations BC subtypes may provide great clinical values in diagnostic, prognostic and therapeutics of BC. The findings from the present quantitative proteomic study have deciphered the altered proteomic patterns and their possible molecular interactions in each BC subtype. The study showed a strong association of FN1, A2M, C4BPA and CFB in molecular subtypes of BC, in which, C4BPA and A2M demonstrated a potent signature in blood plasma and tissue samples of LB and TN subtypes in BC patients, respectively. The findings also revealed the altered level expressions of these selected proteins could classify BC subtypes through plasma and tissue based expression analysis in patients and control samples. Hence, these proteins could have clinical importance for the diagnosis and prognosis purposes among molecular BC subtypes.

Epigenetic modifications of the nuclear genome have been well studied and it is established that these modifications play a key role in nuclear gene expression. However, the status of mitochondrial epigenetic modifications has not been delved in detail. The recent technological advancements in the genome analyzing tools and techniques, have helped in investigating mitochondrial epigenetic modifications with greater resolution and studies have indicated a regulatory role of the mitochondrial epigenome. Association of mitochondrial DNA methylation with various disease conditions, drug treatment, aging, exposure to environmental pollutants etc. has lent credence to this belief. Herein, we have reviewed studies on mitochondrial epigenetic modifications with a focus to comprehend its regulatory role in gene expression and disease association.

Quantitative proteomics using LC-MS has emerged as an essential tool for addressing different biological questions. Various labelling methods have been effectively employed for quantitative proteomics studies. However, these are fraught with several challenges, including reproducibility and the number of samples that can be analysed at a given time. To this end, unlabelled proteomics is a promising field, and the recently developed sequential window acquisition of all theoretical fragment ion spectra (SWATH-MS) method aims to address these limitations. In this study, we compared SWATH-MS to isobaric tag for relative and absolute quantitation (iTRAQ), a widely used labelled method for relative quantitation. For this, we used yeast, Saccharomyces cerevisiae, since almost all its proteins are identified. More importantly, the abundance of each protein is well documented. We found that although a similar number of proteins could be quantitated using the two techniques, SWATH had the advantage of quantifying a larger percentage of low abundance proteins (below 60 ppm). Thus, based on our analysis, we believe that these two techniques are complementary and can synergistically improve the number of quantifiable proteins. SWATH's ability to quantify low abundant proteins could be an asset in biomarker discovery studies.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Transport of L-homocysteine into and out of the human vascular endothelium is poorly understood. We hypothesized that cultured human aortic endothelial cells (HAEC) would import L-homocysteine on one or more of the L-cysteine transport systems. Inhibitors of the transporters were used to characterize the uptake of [35S]L-homocysteine, [35S]L-homocystine, and [35S]L-cysteine. We found that L-homocysteine uptake is mediated by the sodium-dependent cysteine transport systems X(AG), ASC, and A, and the sodium-independent transport system L. Thus, HAEC utilize multiple cysteine transporters (X(AG) > or = L > ASC > A) to import L-homocysteine. Kinetic analysis supported the uptake results. Michaelis-Menten constants (Km) for the four systems yielded values of 19.0, 27.1, 112, and 1000 microM for systems L, X(AG), ASC, and A, respectively. The binding and uptake of [35S]L-homocystine, the disulfide homodimer of L-homocysteine, was mediated by systems X(AG), L, and ASC but not by system A. In contrast to [35S]L-homocysteine, system x(c) was active for [35S]L-homocystine uptake. A similar pattern was observed for [35S]L-cysteine. Thus, L-homocysteine and L-homocystine found in hyperhomocysteinemic subjects can gain entry into the vascular endothelium by way of multiple L-cysteine transporters.

Genomic rearrangements leading to deletion or duplication of gene(s) resulting in alterations in gene copy number underlie the molecular lesion in several genetic disorders. Methods currently used to determine gene copy number including real time PCR, southern hybridization, fluorescence in situ hybridization, densitometric scanning of PCR product etc. have certain disadvantages and are also expensive and time consuming. Herein, we describe a simple and rapid method to assess gene copy number using denaturing high performance liquid chromatography (dHPLC). We used X chromosome genes as model to compare the gene copy numbers present on this chromosome in males and females. DNA from these samples were amplified by biplex PCR using primer pairs specific for X chromosome genes only (target gene) and for genes present on both X and Y chromosomes (internal control). Amplified products were analyzed using HPLC under non-denaturing conditions. The ratio of peak areas (target gene/internal control) of the amplified products was approximately twice in female samples than male samples (p < 0.001) demonstrating that the differential gene copy number can be easily detected using this method. This method can potentially be used for diagnostic purpose where the need is to distinguish samples based on the differential gene copy numbers.

Cardiovascular disease has remained as the largest cause of morbidity and mortality worldwide. From dissecting the disease aetiology to identifying prognostic markers for better management of the disease is still a challenge for researchers. In the post human genome sequencing era much of the thrust has been focussed towards application of advanced genomic tools along with evaluation of traditional risk factors. With the advancement of next generation proteomics and metabolomics approaches it has now become possible to understand the protein interaction network & metabolic rewiring which lead to the perturbations of the disease phenotype. Further, elucidating different post translational modifications using advanced mass spectrometry based methods have provided an impetus towards in depth understanding of the proteome. The past decade has observed a plethora of studies where proteomics has been applied successfully to identify potential prognostic and diagnostic markers as well as to understand the disease mechanisms for various types of cardiovascular diseases. In this review, we attempted to document relevant proteomics based studies that have been undertaken either to identify potential biomarkers or have elucidated newer mechanistic insights into understanding the patho-physiology of cardiovascular disease, primarily coronary artery disease, cardiomyopathy, and myocardial ischemia. We have also provided a perspective on the potential of proteomics in combating this deadly disease. Biological significance This review has catalogued recent studies on proteomics and metabolomics involved in understanding several cardiovascular diseases (CVDs). A holistic systems biology based approach, of which proteomics and metabolomics are two very important components, would help in delineating various pathways associated with complex disorders like CVD. This would ultimately provide better mechanistic understanding of the disease biology leading to development of prognostic biomarkers. This article is part of a Special Issue entitled: Proteomics in India.

DNA methylation and histone modifications across the nuclear genome have been extensively analyzed, but the epigenetic modifications associated with the mitochondrial genome have not yet been analyzed at high resolutions. In the present work, we analyzed methyl-cytosine profiles from methylated DNA immunoprecipitation datasets from 39 different human cell and tissue types from the NIH Roadmap Epigenomics project and validated the data using an orthologous bisulfite sequencing dataset. We observe a distinct distribution of zmethyl-cytosine in mitochondrial genomes which are conserved across all cell and tissue types. This study thus describes the first comprehensive map of methyl cytosines across the human mitochondrial genome.

Does a differential abundance of high mobility group box 1 (HMGB1) protein in uterine fluid (UF) have a functional significance?In rats, an excess of HMGB1 in UF during the receptive phase is detrimental to pregnancy.The identification of constituents of the human uterine secretome has been a subject of renewed interest, due to the advent of high throughput proteomic technologies. Proteomic-based investigations of human UF have revealed the presence of several proteins such as mucins, host defense proteins S100, heat shock protein 27 and haptoglobin, etc. The present study reports on the presence of HMGB1, a nuclear protein, in human UF. Activated macrophages/monocytes, natural killer cells, mature dendritic cells, pituicytes and erythroleukemic cells are also known to secrete HMGB1. Existing data suggest that extracellular HMGB1 plays a role in inflammation.The human part of this study was cross-sectional in design. UF and endometrial tissues were collected from regularly cycling women in the early secretory (i.e. pre-receptive phase, Day 2 post-ovulation, n = 7) or secretory phase (i.e. receptive phase, Day 6 post-ovulation, n = 7) of their menstrual cycles. Samples were also collected from cycling rats in the proestrous (n = 8) or metestrous (n = 8) phase of their estrous cycles. Uteri were also collected from HMGB1-treated pregnant (n = 7) and untreated pseudo-pregnant (n = 7) rats and from pregnant rats at Day 3-5 post-coitum (p.c.) (n = 18, 3 each for six-time points).In each group of human samples, four samples were used for isobaric tag for relative and absolute quantification (iTRAQ) analysis and three samples were used for immunoblotting experiments to determine the abundance of HMGB1 in pre-receptive and receptive phase UF samples. HMGB1 levels in rat UF and endometrial tissue samples were estimated by ELISA and immunohistochemical studies, respectively. The expression of inflammation-associated molecules, such as nuclear factor kappa B (NFκB), receptor for advanced glycation end products (RAGEs), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), was analyzed by immunohistochemistry in HMGB1-treated and pseudo-pregnant rats.HMGB1 was identified as one of the differentially abundant proteins in the list generated by 8-plex iTRAQ analysis of receptive and pre-receptive phase UF samples. In both humans and rats, secreted and cellular levels of HMGB1 showed a similar pattern, i.e. significantly (P0.05) lower abundance in the receptive phase compared with that in the pre-receptive phase. A significant (P0.05) decline was also observed in the endometrial expression of HMGB1 on the day of implantation in pregnant rats. Exogenous administration of recombinant HMGB1, on Day 3 p.c., led to pregnancy failure, whereas administration of recombinant leukemia inhibitory factor or saline had no effect on pregnant rats. Further investigations revealed morphological changes in the endometrium, an increase in the expression of luminal epithelial NFκB and significantly (P0.05) higher expression levels of endometrial RAGE, TNF-α and IL-6 in HMGB1-treated rats, compared with untreated pseudo-pregnant rats.The mechanisms, contributing to a decline in the cellular and extracellular levels of HMGB1 during the receptive phase, remain to be ascertained.An excess of HMGB1 in the UF may be associated with infertility in women.

Disulfide forms of homocysteine account for >98% of total homocysteine in plasma from healthy individuals. We recently reported that homocysteine reacts with albumin-Cys(34)-S-S-cysteine to form homocysteine-cysteine mixed disulfide and albumin-Cys(34) thiolate anion. The latter then reacts with homocystine or homocysteine-cysteine mixed disulfide to form albumin-bound homocysteine (Sengupta, S., Chen, H., Togawa, T., DiBello, P. M., Majors, A. K., Budy, B., Ketterer, M. E., and Jacobsen, D. W. (2001) J. Biol. Chem. 276, 30111-30117). We now extend these studies to show that human albumin, but not ceruloplasmin, mediates the conversion of homocysteine to its low molecular weight disulfide forms (homocystine and homocysteine-cysteine mixed disulfide) by thiol/disulfide exchange reactions. Only a small fraction of homocystine is formed by an oxidative process in which copper bound to albumin, but not ceruloplasmin, mediates the reaction. When copper is removed from albumin by chelation, the overall conversion of homocysteine to its disulfide forms is reduced by only 20%. Ceruloplasmin was an ineffective catalyst of homocysteine oxidation, and immunoprecipitation of ceruloplasmin from human plasma did not inhibit the capacity of plasma to mediate the conversion of homocysteine to its disulfide forms. In contrast, ceruloplasmin was a highly efficient catalyst for the oxidation of cysteine and cysteinylglycine to cystine and bis(-S-cysteinylglycine), respectively. However, when thiols (cysteine and homocysteine) that are disulfide-bonded to albumin-Cys(34) are removed by treatment with dithiothreitol to form albumin-Cys(34)-SH (mercaptalbumin), the conversion of homocysteine to its disulfide forms is completely blocked. In conclusion, albumin mediates the formation of disulfide forms of homocysteine by thiol/disulfide exchange, whereas ceruloplasmin converts cysteine to cystine by copper-dependent autooxidation.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Greater than 70% of homocysteine in circulation is protein-bound. An in vitro model system using human plasma has been developed to study mechanisms of protein-bound homocysteine formation and establish the equilibrium binding capacities of plasma for homocysteine. Addition of homocysteine to plasma caused an initial rapid displacement of cysteine and a subsequent increase in protein-bound homocysteine. This rapid reaction was followed by a slower oxygen-dependent reaction forming additional protein-bound homocysteine. To determine the equilibrium binding capacity of plasma proteins for homocysteine, plasma was treated with 0.5-10 mM dl-homocysteine for 4 h at 37 degrees C under aerobic conditions. Under these conditions the equilibrium binding capacity was 4.88 +/- 0.51 and 4.74 +/- 0.68 micromol/g protein for male (n = 10) and female (n = 10) donors, respectively. The mechanism of protein-bound homocysteine formation involves both thiol-disulfide exchange and thiol oxidation reactions. We conclude that plasma proteins have a high capacity for binding homocysteine in vitro.

Background Homocysteine metabolism is altered in children with idiopathic nephrotic syndrome. Hyperhomocysteinemia is a risk factor of early atherosclerosis and glomerulosclerosis and may occur at time of first occurrence of idiopathic nephrotic syndrome. Methods Thirty children with first episode of idiopathic nephrotic syndrome (FENS) aged 1–16 years along with 30 age- and sex-matched healthy controls were enrolled in this study. Homocysteine and cysteine were measured with HPLC; vitamin B12 and folic acid were measured with electro-chemilumiscence immunoassay. Primary outcome measure was plasma homocysteine level in children with FENS and in controls. Secondary outcome measures were (1) plasma and urine homocysteine and cysteine levels in children with FENS at 12 weeks and 1 year (remission) and (2) plasma and urine levels of vitamin B12 and folic acid in children with FENS, at 12 weeks and 1 year (remission). Results Plasma homocysteine and cysteine levels were comparable to controls in children with FENS, at 12 weeks and 1-year remission. Plasma levels of vitamin B12 and folic acid were significantly decreased compared to controls in FENS due to increased urinary excretion, which normalize during remission at 12 weeks and 1 year. Urinary homocysteine and cysteine levels were significantly raised in FENS compared to controls and continued to be raised even at 12-week and 1-year remission. Conclusion Homocysteine metabolism is deranged in children with FENS. Renal effects of long-term raised urinary homocysteine levels need to be studied.

Pathogenic bacteria sense the host environment and regulate expression of virulence-related genes. Environmental signals like temperature, bicarbonate/CO2 and glucose induce toxin production in Bacillus anthracis, but the mechanisms by which these signals contribute to virulence and overall physiological adaptation remains elusive. An integrated, systems level investigation using transcriptomics and iTRAQ-based proteomics was done to assess the effect of temperature, bicarbonate/CO2 and glucose on B. anthracis. Significant changes observed in amino acid, carbohydrate, energy and nucleotide metabolism indicates events of metabolic readjustments by environmental factors. Directed induction of genes involved in polyamine biosynthesis and iron metabolism revealed the redirection of cellular metabolite pool towards iron uptake. Protein levels of glycolytic enzymes, ptsH and Ldh along with transcripts involved in immune evasion (mprF, bNOS, Phospholipases and asnA), cell surface remodeling (rfbABCD, antABCD, and cls) and utilization of lactate (lutABC) and inositol showed constant repression under environmental perturbations. Discrepancies observed in mRNA/protein level of genes involved in glycolysis, protein synthesis, stress response and nucleotide metabolism hinted at the existence of additional regulatory layers and illustrated the utility of an integrated approach. The above findings might assist in the identification of novel adaptive strategies of B. anthracis during host associated survival and pathogenesis. Biological Significance In this study, the changes observed at both transcript and protein level were quantified and integrated to understand the effect of host environmental factors (host temperature, bicarbonate and glucose) in shaping the physiology and adaptive strategies of a fully virulent strain of B. anthracis for efficient survival and virulence in its host. Perturbations affecting toxin production were found to concordantly affect vital metabolic pathways and several known as well as novel virulence factors. These changes act as a valuable asset for generating testable hypotheses that can be further verified by detailed molecular and mutant studies to identify novel adaptive strategies of B. anthracis during infection. Adaptation of an integrated transcriptomics and proteomics approach also led to the identification of discrepancies between mRNA/protein levels among genes across major functional categories. Few of these discrepancies have been previously reported in literature for model organisms. However their existence in B. anthracis and that too as a result of growth perturbations have not been reported till date. These findings demonstrate a substantial role of regulatory processes post mRNA synthesis via post transcriptional, translational or protein degradation mechanisms. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Maternal under-nutrition increases the risk of developing metabolic diseases. We studied the effects of chronic maternal dietary vitamin B12 restriction on lean body mass (LBM), fat free mass (FFM), muscle function, glucose tolerance and metabolism in Wistar rat offspring. Prevention/reversibility of changes by rehabilitating restricted mothers from conception or parturition and their offspring from weaning was assessed. Female weaning Wistar rats (n = 30) were fed ad libitum for 12 weeks, a control diet (n = 6) or the same with 40% restriction of vitamin B12 (B12R) (n = 24); after confirming deficiency, were mated with control males. Six each of pregnant B12R dams were rehabilitated from conception and parturition and their offspring weaned to control diet. While offspring of six B12R dams were weaned to control diet, those of the remaining six B12R dams continued on B12R diet. Biochemical parameters and body composition were determined in dams before mating and in male offspring at 3, 6, 9 and 12 months of their age. Dietary vitamin B12 restriction increased body weight but decreased LBM% and FFM% but not the percent of tissue associated fat (TAF%) in dams. Maternal B12R decreased LBM% and FFM% in the male offspring, but their TAF%, basal and insulin stimulated glucose uptake by diaphragm were unaltered. At 12 months age, B12R offspring had higher (than controls) fasting plasma glucose, insulin, HOMA-IR and impaired glucose tolerance. Their hepatic gluconeogenic enzyme activities were increased. B12R offspring had increased oxidative stress and decreased antioxidant status. Changes in body composition, glucose metabolism and stress were reversed by rehabilitating B12R dams from conception, whereas rehabilitation from parturition and weaning corrected them partially, highlighting the importance of vitamin B12 during pregnancy and lactation on growth, muscle development, glucose tolerance and metabolism in the offspring.

Imbalance in protein homeostasis in specific subcellular organelles is alleviated through organelle-specific stress response pathways. As a canonical example of stress activated pathway, accumulation of misfolded proteins in ER activates unfolded protein response (UPR) in almost all eukaryotic organisms. However, very little is known about the involvement of proteins of other organelles that help to maintain the cellular protein homeostasis during ER stress. In this study, using iTRAQ-based LC-MS approach, we identified organelle enriched proteins that are differentially expressed in yeast (Saccharomyces cerevisiae) during ER stress in the absence of UPR sensor Ire1p. We have identified about 750 proteins from enriched organelle fraction in three independent iTRAQ experiments. Induction of ER stress resulted in the differential expression of 93 proteins in WT strains, 40 of which were found to be dependent on IRE1. Our study reveals a cross-talk between ER- and mitochondrial proteostasis exemplified by an Ire1p-dependent induction of Hsp60p, a mitochondrial chaperone. Thus, in this study, we show changes in protein levels in various organelles in response to ER stress. A large fraction of these changes were dependent on canonical UPR signalling through Ire1, highlighting the importance of interorganellar cross-talk during stress.

Myocardial infarction (MI) is defined as cardiac cell death due to prolonged ischemia. Although necrotic cell death was considered to be solely responsible for myocyte death during MI, it was recently revealed that apoptosis also plays its part in this death process. Our laboratory has recently shown that endoplasmic reticulum (ER) stress-induced apoptosis is the predominant route for apoptosis during MI and the conventional mitochondrial pathway is bypassed by activation of a small heat shock protein α-crystallin B (CRYAB). Since CRYAB is a direct target of P38 mitogen-activated protein kinase (MAPK) cascade, we were prompted to check the role of P38 MAPK in 20-week-old male Wister rats immediately after infarct formation. Interestingly, parallel activation of mitochondrial apoptotic pathway with an increase in ER stress-induced apoptotic load was observed along with decreased activation of CRYAB and Nrf2 (a pro-survival protein activated in response to ER stress) in MI rats treated with SB203580, a specific inhibitor of P38α and P38β compared to the MI alone. As a cumulative effect, this inhibitor treatment also resulted in significant increase in the levels of caspase3 activity and TUNEL positivity, the end point apoptotic markers. Furthermore, SB203580-treated hypoxic adult cardiomyocytes showed formation of desmin aggregates which were previously associated with impaired cardiac function. Thus, this study shows for the first time the precise mechanism by which P38 MAPK plays a pro-survival role and confers protection of cardiomyocytes, during infarct formation.

Maternal nutritional deficiency in-utero is known to predict risk of complex disorders like cardiovascular disease, diabetes and many neurological disorders in the offspring and vitamin B12 is one such critical micronutrient. Here we performed 2D-DIGE followed by MALDI TOF/TOF analysis to identify proteins that are differentially expressed in liver of pups born to mothers fed vitamin B12 deficient diet vis-a-vis control diet. To further establish causality, we analyzed the effect of B12 rehabilitation at parturition on the protein levels and the phenotype in pups. We identified 38 differentially expressed proteins that were enriched in pathways involved in the regulation of amino acid, lipid and carbohydrate metabolism. Further, three enzymes in the β-oxidation pathway (hydroxyacyl-coenzyme A dehydrogenase, medium-chain specific acyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase) were down-regulated in pups born to mothers fed vitamin B12 deficient diet. We observed age-dependent differential expression of peroxisome proliferator activated-receptor (PPAR) α and γ in the deficient pups. Interestingly, expression of 27 proteins that were differentially expressed was restored to the control levels after rehabilitation of female rats with vitamin B12 from parturition. Our study thus provides the first evidence that maternal vitamin B12 deficiency influences lipid and other micronutrient metabolism in pups through regulation of PPAR signaling pathway. Biological significance Maternal vitamin B12 deficiency has been shown to predict the onset of complex disorders like atherosclerosis, type II diabetes etc. in the next generation during their adulthood. We have shown earlier that pups born to female rats fed with vitamin B12 deficient diet were obese and developed high levels of other intermediate traits such as triglycerides, cholesterol etc. that are related to the risk of diabetes and cardiovascular disorders. In this piece of work using differential proteomic approach we have identified the altered metabolic processes in the liver of vitamin B12 deficient pups. We have also documented that the proteins involved in β-oxidation pathway are down-regulated. Further, differential expression of PPARα and PPARγ was evidently documented as the master regulator for the alteration of lipid, amino acid and carbohydrate metabolism during maternal vitamin B12 deficiency.

Mycobacterium tuberculosis modulates expression of various metabolism-related genes to adapt in the adverse host environment. The gene coding for M. tuberculosis S-adenosylhomocysteine hydrolase (Mtb-SahH) is essential for optimal growth and the protein product is involved in intermediary metabolism. However, the relevance of SahH in mycobacterial physiology is unknown. In this study, we analyze the role of Mtb-SahH in regulating homocysteine concentration in surrogate host Mycobacterium smegmatis. Mtb-SahH catalyzes reversible hydrolysis of S-adenosylhomocysteine to homocysteine and adenosine and we demonstrate that the conserved His363 residue is critical for bi-directional catalysis. Mtb-SahH is regulated by serine/threonine phosphorylation of multiple residues by M. tuberculosis PknB. Major phosphorylation events occur at contiguous residues Thr219, Thr220 and Thr221, which make pivotal contacts with cofactor NAD+. Consequently, phosphorylation negatively modulates affinity of enzyme towards NAD+ as well as SAH-synthesis. Thr219, Thr220 and Thr221 are essential for enzyme activity, and therefore, responsible for SahH-mediated regulation of homocysteine.

Cardiac hypertrophy and myocardial infarction (MI) are two major causes of heart failure with different etiologies. However, the molecular mechanisms associated with these two diseases are not yet fully understood. So, this study was designed to decipher the process of cardiomyocyte apoptosis during cardiac hypertrophy and MI in vivo. Our study revealed that mitochondrial outer membrane channel protein voltage-dependent anion channel-1 (VDAC1) was upregulated exclusively during cardiac hypertrophy, whereas 78 kDa glucose-regulated protein (GRP78) was exclusively upregulated during MI, which is an important upstream regulator of the endoplasmic reticulum (ER) stress pathway. Further downstream analysis revealed that mitochondrial pathway of apoptosis is instrumental in case of hypertrophy, whereas ER stress-induced apoptosis is predominant during MI, which was confirmed by treatment with either siRNA against VDAC1 or ER stress inhibitor tauroursodeoxycholic acid (TUDCA). Very interestingly, our data also showed that the expression and interaction of small heat-shock protein α-crystallin B (CRYAB) with VDAC1 was much more pronounced during MI compared with either hypertrophy or control. The study demonstrated for the first time that two different organelles--mitochondria and ER have predominant roles in mediating cardiomyocyte death signaling during hypertrophy and MI, respectively, and activation of CRYAB acts as a molecular switch in bypassing mitochondrial pathway of apoptosis during MI.

Accurate assessment of left ventricular (LV) function, LV volume, ejection fraction and segmental wall motion abnormalities is essential for clinical decision-making and follow-up assessment. Currently, two-dimensional echocardiography is the most widely used method to obtain this information. Three-dimensional echocardiography has shown to be an accurate and reproducible method for LV quantitation. In this review, we describe various methods to acquire a 3D-dataset for LV volume and wall motion analysis, including their advantages and limitations. We provide an overview of studies comparing LV volume and function measurement by various gated and realtime 3D echocardiography methods of acquisition compared to magnetic resonance imaging. New technical advances like online assessment and single beat acquisition of data have minimized the errors in assessment of LV function.