The potential of Cystatin C and small dense LDL as biomarkers of coronary artery disease risk in a young Indian population

Coronary artery disease (CAD) affects Indians 5–6 years earlier than in the west, is diffuse and malignant, and poses a heavy burden on India’s developing economy. Traditional risk factors have failed to explain this high incidence of premature CAD and hence this study investigated the association of two novel risk biomarkers, cystatin C and small dense LDL (sdLDL) with the presence and severity of CAD. Cystatin C and sdLDL were estimated in 204 CAD patients ≤45 years of age and compared with 161 age-matched healthy controls. The traditional lipid profile parameters, i.e., cholesterol, LDL, HDL, triglycerides, apolipoproteins A1 and B, and Lp(a) were also measured in both groups. Cystatin C was significantly raised and mean LDL particle size significantly reduced in CAD patients as compared to controls. 62.7 % of CAD patients showed pattern B while 37.3 % patients showed pattern A. Of the traditional lipid tests, only HDL and apolipoprotein A1 showed a significant decrease in the CAD group. sdLDL was significantly associated with the severity of CAD, while cystatin C was not. Both cystatin C and sdLDL emerged as independent risk factors, however, of the two, sdLDL was a more sensitive predictor of CAD events. Cystatin C and mean LDL particle size are significantly and independently associated with the presence of CAD events in patients ≤45 years with normal kidney function. Hence, these novel risk biomarkers can be useful tools in reducing the morbidity and mortality associated with CAD in the productive Indian workforce.     

Matrix-Assisted Refolding,Purification and Activity Assessment Using a ‘Form Invariant’ Assay for Matrix Metalloproteinase 2 (MMP2)

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed ‘form invariant’ assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.     

Genetic characterization of Chikungunya virus from New Delhi reveal emergence of a new molecular signature in Indian isolates

ABSTRACT: BACKGROUND: Chikungunya (CHIK) is currently endemic in South and Central India and exist as co-infections with dengue in Northern India. In 2010, New Delhi witnessed an outbreak of CHIK in the months October-December. This was the first incidence of a dominant CHIK outbreak in Delhi and prompted us to characterize the Delhi virus strains. We have also investigated the evolution of CHIK spread in India. FINDINGS: Clinical samples were subjected to RT-PCR to detect CHIK viral RNA. The PCR amplified products were sequenced and the resulting sequences were genetically analyzed. Phylogenetic analysis based on partial sequences of the structural proteins E1 and E2 revealed that the viruses in the latest outbreak exhibited ECSA lineage. Two novel mutations, E1 K211E and E2 V264A were observed in all Delhi isolates. In addition, CHIKV sequences from eight states in India were analyzed along with Delhi sequences to map the genetic diversity of CHIKV within the country. Estimates of average evolutionary divergence within states showed varying divergence among the sequences both within the states and between the states. We identified distinct molecular signatures of the different genotypes of CHIKV revealing emergence of a new signature in the New Delhi clade. Statistical analyses and construction of evolutionary path of the virus within the country revealed gradual spread of one specific strain all over the country. CONCLUSION: This study has identified unique mutations in the E1 and E2 genes and has revealed the presence of ancestral CHIKV population with maximum diversity circulating in Maharashtra. The study has further revealed the trend of CHIK spread in India since its first report in 1963 and its subsequent reappearance in 2005.     

DJ-1 Protects Pancreatic Beta Cells from Cytokine- and Streptozotocin-Mediated Cell Death

A hallmark feature of type 1 and type 2 diabetes mellitus is the progressive dysfunction and loss of insulin-producing pancreatic beta cells, and inflammatory cytokines are known to trigger beta cell death. Here we asked whether the anti-oxidant protein DJ-1 encoded by the Parkinson’s disease gene PARK7 protects islet cells from cytokine- and streptozotocin-mediated cell death. Wild type and DJ-1 knockout mice (KO) were treated with multiple low doses of streptozotocin (MLDS) to induce inflammatory beta cell stress and cell death. Subsequently, glucose tolerance tests were performed, and plasma insulin as well as fasting and random blood glucose concentrations were monitored. Mitochondrial morphology and number of insulin granules were quantified in beta cells. Moreover, islet cell damage was determined in vitro after streptozotocin and cytokine treatment of isolated wild type and DJ-1 KO islets using calcein AM/ethidium homodimer-1 staining and TUNEL staining. Compared to wild type mice, DJ-1 KO mice became diabetic following MLDS treatment. Insulin concentrations were substantially reduced, and fasting blood glucose concentrations were significantly higher in MLDS-treated DJ-1 KO mice compared to equally treated wild type mice. Rates of beta cell apoptosis upon MLDS treatment were twofold higher in DJ-1 KO mice compared to wild type mice, and in vitro inflammatory cytokines led to twice as much beta cell death in pancreatic islets from DJ-1 KO mice versus those of wild type mice. In conclusion, this study identified the anti-oxidant protein DJ-1 as being capable of protecting pancreatic islet cells from cell death induced by an inflammatory and cytotoxic setting.     

Identification and characterization of a hitherto unknown nucleotide-binding domain and an intricate interdomain regulation in HflX-a ribosome binding GTPase

A role for HflX in 50S-biogenesis was suggested based on its similarity to other GTPases involved in this process. It possesses a G-domain, flanked by uncharacterized N- and C-terminal domains. Intriguingly, Escherichia coli HflX was shown to hydrolyze both GTP and adenosine triphosphate (ATP), and it was unclear whether G-domain alone would explain ATP hydrolysis too. Here, based on structural bioinformatics analysis, we suspected the possible existence of an additional nucleotide-binding domain (ND1) at the N-terminus. Biochemical studies affirm that this domain is capable of hydrolyzing ATP and GTP. Surprisingly, not only ND1 but also the G-domain (ND2) can hydrolyze GTP and ATP too. Further; we recognize that ND1 and ND2 influence each other’s hydrolysis activities via two salt bridges, i.e. E29-R257 and Q28-N207. It appears that the salt bridges are important in clamping the two NTPase domains together; disrupting these unfastens ND1 and ND2 and invokes domain movements. Kinetic studies suggest an important but complex regulation of the hydrolysis activities of ND1 and ND2. Overall, we identify, two separate nucleotide-binding domains possessing both ATP and GTP hydrolysis activities, coupled with an intricate inter-domain regulation for Escherichia coli HflX.     

Integrin-binding Protein Nischarin Interacts with Tumor Suppressor Liver Kinase B1 (LKB1) to Regulate Cell Migration of Breast Epithelial Cells

Biallelic inactivation of LKB1, a serine/threonine kinase, has been detected in 30% of lung adenocarcinomas, and inhibition of breast tumor growth has been demonstrated. We have identified the tumor suppressor, Nischarin, as a novel binding partner of LKB1. Our mapping analysis shows that the N terminus of Nischarin interacts with amino acids 44–436 of LKB1. Time lapse microscopy and Transwell migration data show that the absence of both Nischarin and LKB1 from an invasive breast cancer cell line (MDA-MB-231) enhances migration as measured by increased distance and speed of migrating cells. Our data suggest that this is a result of elevated PAK1 and LIMK1 phosphorylation. Moreover, the absence of Nischarin and LKB1 increased tumor growth in vivo. Consistent with this, the percentage of S phase cells was increased, as demonstrated by flow cytometry and enhanced cyclin D1. The absence of Nischarin and LKB1 also led to a dramatic increase in the formation of lung metastases. Our studies, for the first time, demonstrate functional interaction between LKB1 and Nischarin to inhibit cell migration and breast tumor progression. Mechanistically, we show that these two proteins together regulate PAK-LIMK-Cofilin and cyclin D1/CDK4 pathways.     

Infrastructure for the life sciences: design and implementation of the UniProt website

Background  

The UniProt consortium was formed in 2002 by groups from the Swiss Institute of Bioinformatics (SIB), the European Bioinformatics Institute (EBI) and the Protein Information Resource (PIR) at Georgetown University, and soon afterwards the website was set up as a central entry point to UniProt resources. Requests to this address were redirected to one of the three organisations' websites. While these sites shared a set of static pages with general information about UniProt, their pages for searching and viewing data were different. To provide users with a consistent view and to cut the cost of maintaining three separate sites, the consortium decided to develop a common website for UniProt. Following several years of intense development and a year of public beta testing, the domain was switched to the newly developed site described in this paper in July 2008.     

Microbial production of dihydroxyacetone

Dihydroxyacetone is extensively used in cosmetic industry as an artificial suntan besides having clinical and biological applications. Thus, it is important to meet the commercial demand of dihydroxyacetone at an economical and qualitative level. Microbial route of production is found to be more favorable for dihydroxyacetone as compared to chemical methods. This review gives detailed information about the microbial route of dihydroxyacetone production. Till date the microorganism which is most utilized for dihydroxyacetone production is Gluconobacter oxydans. Some limitations associated with dihydroxyacetone production by G. oxydans like substrate inhibition, product inhibition and oxygen limitation are discussed here. Various fermentation modes and culture conditions have been tried for their ability to overcome these limitations. It has been found that fed-batch mode of fermentation provides a better yield as compared to batch mode for dihydroxyacetone production. Two-stage repeated fed-batch mode of fermentation has been found to be the most optimized mode. Immobilization has also been recognized as a much better alternative for fermentation since it avoids the problem of substrate and product inhibition to a greater extent. Although these methods have increased the dihydroxyacetone production to a prominent level yet the production has not reached the level required to meet the commercial demand. One looks for future prospects of developing recombinant microbial method for dihydoxyacetone production.