Strategies towards understanding the genetics of ischemic stroke

Stroke is the second largest cause of death and disability in the world and 80% of all strokes are ischemic in nature. While most risk factors have been investigated in depth, others such as South Asian ethnicity, long term blood pressure variability and symptomatic carotid stenosis remain largely understudied. In order to understand the genetic etiology underlining ischemic stroke, it is important to study the genetic burden shouldered by these phenotypes as well. This thesis examined the genetics of ischemic stroke in the presence of the above risk factors using three independent lines of investigation: a) literature based meta-analysis, b) candidate gene based approach and c) genome-wide association study. Using a literature based meta-analysis comprising 2529 ischemic stroke cases and 2881 healthy controls, genetic risk variants associated with ischemic stroke in South Asians were investigated. Genes PDE4D SNP 83, ACE I/D and IL10 G1082A were associated with South Asian ischemic stroke risk, with no major differences in strength of association for the same susceptibility genes in other ethnic groups. A candidate gene study was conducted using 8295 ischemic stroke cases and 12722 controls of European ancestry to test the association of GWAS-derived blood pressure variability associated cluster of 17 NLGN1 intronic SNPs. The study did not confirm the risk association of NLGN1 with ischemic stroke. Finally, a genome-wide association study was conducted to identify novel gene variants associated with ≥ 50% carotid stenosis in ischemic stroke, using 1164 cases and 13,703 healthy controls from seven independent cohorts. Three genetic loci at LRIG1, ROBO1 and CAPN7 were found to be associated at genome-wide significance. The findings of this thesis provide new insights into the genetic mechanisms underpinning ischemic stroke, by examining genetic variants associated with unusual stroke related phenotypes. Future directions include replication of sentinel SNPs in larger study populations and a GWAS for South Asian ischemic stroke cases.Open Acces

Phytotoxic Effects of Cinnamic Acid on Cabbage (Brassica oleracea var. capitata)

The present study deals with the effects of exogenous application of cinnamic acid (CA) on growth and metabolism in growing seedlings of Brassica oleracea var. capitata (cabbage) in hydroponic culture. CA was added at 0.5, 1.0 and 1.5 mM concentrations. CA has shown inhibitory effects on shoot and root length, fresh and dry weight of seedlings. CA significantly decreased the photosynthetic pigments, nitrate reductase activity and protein content. Graded concentrations of CA increased lipid peroxidation and sugar content. The increasing concentrations of CA significantly increased the antioxidative enzyme activities viz. superoxide dismutase, catalase, peroxidase against the oxidative stress caused by CA

DEVELOPMENT OF CIPROFLOXACIN LOADED THROAT PAINT FOR THE TREATMENT OF STREP THROAT INFECTION

Objective: This study is to enhance the solubility and sustained release of ciprofloxacin (CPX) drug by amplifying the adhesive capability of formulation by forming throat paint for the Streptococcal pharyngitis, a sore throat infection. Methods: Solid dispersion was prepared by solvent evaporation technique, in which three different ratios of Polyethylene glycol-6000 (PEG-6000) were selected, and the best ratio of solid dispersion was selected after characterization including Scanning electron microscopy (SEM) and Differential scanning calorimetry (DSC) with evaluation parameters including % yield, drug content, and drug solubility. In the case of throat paint, out of six different formulations, the best formulation was selected through viscosity, in vitro mucoadhesion, in situ release study, and spreadability parameters. Results: The DSC and SEM data proved that solid dispersion has a different moiety than its ingredients but it is quite a stable form. Formulation MD-2 was selected as the best formulation which able to increase the solubility of the drug by more than 3.5 folds, at the same time it shows the highest rate of drug dissolution of 13.951 μg/ml with % yield (97.199±0.167%) and drug content (96.425%). Throat paint was formed by fusion and trituration process and out of all six formulations F3 was selected as the best formulation on the basis of Viscosity (11932 Centi poise), Spreadability (17.621), Mucoadhesion (3937.481 dyne/cm2), and drug release (90.336±0.6%). Conclusion: Solid dispersion was successfully prepared with 3.5 times of solubility enhancement capability in comparison with pure CPX drug. The throat paint releases the drug (≥3 h) in a sustained manner with high mucoadhesive force

Comparative transcriptomics of human multipotent stem cells during adipogenesis and osteoblastogenesis

<p>Abstract</p> <p>Background</p> <p>A reciprocal relationship between bone and fat development in osteoporosis is clinically well established. Some of the key molecular regulators involved in this tissue replacement process have been identified. The detailed mechanisms governing the differentiation of mesenchymal stem cells (MSC) – the key cells involved – are however only now beginning to emerge. In an attempt to address the regulation of the adipocyte/osteoblast balance at the level of gene transcription in a comprehensive and unbiased manner, we performed a large-scale gene expression profiling study using a unique cellular model, human multipotent adipose tissue-derived stem cells (hMADS).</p> <p>Results</p> <p>The analysis of 1606 genes that were found to be differentially expressed between adipogenesis and osteoblastogenesis revealed gene repression to be most prevalent prior to commitment in both lineages. Computational analyses suggested that this gene repression is mediated by miRNAs. The transcriptional activation of lineage-specific molecular processes in both cases occurred predominantly after commitment. Analyses of the gene expression data and promoter sequences produced a set of 65 genes that are candidates for genes involved in the process of adipocyte/osteoblast commitment. Four of these genes were studied in more detail: <it>LXRα </it>and phospholipid transfer protein (<it>PLTP</it>) for adipogenesis, the nuclear receptor <it>COUP-TF1 </it>and one uncharacterized gene, <it>TMEM135 </it>for osteoblastogenesis. <it>PLTP </it>was secreted during both early and late time points of hMADS adipocyte differentiation. <it>LXRα</it>, <it>COUP-TF1</it>, and the transmembrane protein <it>TMEM135 </it>were studied in primary cultures of differentiating bone marrow stromal cells from healthy donors and were found to be transcriptionally activated in the corresponding lineages.</p> <p>Conclusion</p> <p>Our results reveal gene repression as a predominant early mechanism before final cell commitment. We were moreover able to identify 65 genes as candidates for genes controlling the adipocyte/osteoblast balance and to further evaluate four of these. Additional studies will explore the precise role of these candidate genes in regulating the adipogenesis/osteoblastogenesis switch.</p

Implications of tolerance to iron toxicity on root system architecture changes in rice (Oryza sativa L.)

IntroductionToxicity due to excess soil iron (Fe) is a significant concern for rice cultivation in lowland areas with acidic soils. Toxic levels of Fe adversely affect plant growth by disrupting the absorption of essential macronutrients, and by causing cellular damage. To understand the responses to excess Fe, particularly on seedling root system, this study evaluated rice genotypes under varying Fe levels.MethodsSixteen diverse rice genotypes were hydroponically screened under induced Fe levels, ranging from normal to excess. Morphological and root system characteristics were observed. The onset of leaf bronzing was monitored to identify the toxic response to the excess Fe. Additionally, agronomic and root characteristics were measured to classify genotypes into tolerant and sensitive categories by computing a response stability index.ResultsOur results revealed that 460 ppm of Fe in the nutrient solution served as a critical threshold for screening genotypes during the seedling stage. Fe toxicity significantly affected root system traits, emphasizing the consequential impact on aerial biomass and nutrient deprivation. To classify genotypes into tolerant and sensitive categories, leaf bronzing score was used as a major indicator of Fe stress. However, the response stability index provided a robust basis for classification for the growth performance. Apart from the established tolerant varieties, we could identify a previously unrecognized tolerant variety, ILS 12–5 in this study. Some of the popular mega varieties, including BPT 5204 and Pusa 44, were found to be highly sensitive.DiscussionOur findings suggest that root system damage, particularly in root length, surface area, and root volume, is the key factor contributing to the sensitivity responses under Fe toxicity. Tolerant genotypes were found to retain more healthy roots than the sensitive ones. Fe exclusion, by reducing Fe2+ uptake, may be a major mechanism for tolerance among these genotypes. Further field evaluations are necessary to confirm the behavior of identified tolerant and sensitive lines under natural conditions. Insights from the study provide potential scope for enhancement of tolerance through breeding programs as well as throw light on the role root system in conferring tolerance

Bio-Repository of DNA in stroke (BRAINS): A study protocol

<p>Abstract</p> <p>Background</p> <p>Stroke is one of the commonest causes of mortality in the world and anticipated to be an increasing burden to the developing world. Stroke has a genetic basis and identifying those genes may not only help us define the mechanisms that cause stroke but also identify novel therapeutic targets. However, large scale highly phenotyped DNA repositories are required in order for this to be achieved.</p> <p>Methods</p> <p>The proposed Bio-Repository of DNA in Stroke (BRAINS) will recruit all subtypes of stroke as well as controls from two different continents, Europe and Asia. Subjects recruited from the UK will include stroke patients of European ancestry as well as British South Asians. Stroke subjects from South Asia will be recruited from India and Sri Lanka. South Asian cases will also have control subjects recruited.</p> <p>Discussion</p> <p>We describe a study protocol to establish a large and highly characterized stroke biobank in those of European and South Asian descent. With different ethnic populations being recruited, BRAINS has the ability to compare and contrast genetic risk factors between those of differing ancestral descent as well as those who migrate into different environments.</p

Runx proteins mediate protective immunity against Leishmania donovani infection by promoting CD40 expression on dendritic cells.

The level of CD40 expression on dendritic cells (DCs) plays a decisive role in disease protection during Leishmania donovani (LD) infection. However, current understanding of the molecular regulation of CD40 expression remains elusive. Using molecular, cellular and functional approaches, we identified a role for Runx1 and Runx3 transcription factors in the regulation of CD40 expression in DCs. In response to lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα) or antileishmanial drug sodium antimony gluconate (SAG), both Runx1 and Runx3 translocated to the nucleus, bound to the CD40 promoter and upregulated CD40 expression on DCs. These activities of Runx proteins were mediated by the upstream phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Notably, LD infection attenuated LPS- or TNFα-induced CD40 expression in DCs by inhibiting PI3K-Akt-Runx axis via protein tyrosine phosphatase SHP-1. In contrast, CD40 expression induced by SAG was unaffected by LD infection, as SAG by blocking LD-induced SHP-1 activation potentiated PI3K-Akt signaling to drive Runx-mediated CD40 upregulation. Adoptive transfer experiments further showed that Runx1 and Runx3 play a pivotal role in eliciting antileishmanial immune response of SAG-treated DCs in vivo by promoting CD40-mediated type-1 T cell responses. Importantly, antimony-resistant LD suppressed SAG-induced CD40 upregulation on DCs by blocking the PI3K-Akt-Runx pathway through sustained SHP-1 activation. These findings unveil an immunoregulatory role for Runx proteins during LD infection

Candidate-gene analysis of white matter hyperintensities on neuroimaging

Background: White matter hyperintensities (WMH) are a common radiographic finding and may be a useful endophenotype for small vessel diseases. Given high heritability of WMH, we hypothesised that certain genotypes may predispose individuals to these lesions and consequently, to an increased risk of stroke, dementia and death. We performed a meta-analysis of studies investigating candidate genes and WMH to elucidate the genetic susceptibility to WMH and tested associated variants in a new independent WMH cohort. We assessed a causal relationship of WMH to methylene tetrahydrofolate reductase (MTHFR). Methods: Database searches through March 2014 were undertaken and studies investigating candidate genes in WMH were assessed. Associated variants were tested in a new independent ischaemic cohort of 1202 WMH patients. Mendelian randomization was undertaken to assess a causal relationship between WMH and MTHFR. Results: We identified 43 case-control studies interrogating eight polymorphisms in seven genes covering 6,314 WMH cases and 15,461 controls. Fixedeffects meta-analysis found that the C-allele containing genotypes of the aldosterone synthase CYP11B2 T(-344)C gene polymorphism were associated with a decreased risk of WMH (OR=0.61; 95% CI, 0.44 to 0.84; p=0.003). Using mendelian randomisation the association among MTHFR C677T, homocysteine levels and WMH, approached, but did not reach, significance (expected OR=1.75; 95% CI, 0.90-3.41; observed OR=1.68; 95% CI, 0.97-2.94). Neither CYP11B2 T(-344)C nor MTHFR C677T were significantly associated when tested in a new independent cohort of 1202 patients with WMH. Conclusions: There is a genetic basis to WMH but anonymous genome wide and exome studies are more likely to provide novel loci of interest

Mendelian Randomization to compare estimated risk with observed risk for gene polymorphisms associated with ischemic stroke.

<p>The mean difference (ΔX) was calculated from a meta-analysis relating MTHFR C677T genotype with homocysteine variation in healthy South Asians. The expected OR was calculated using the following formula: <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057305#pone.0057305-Casas1" target="_blank">[16]</a> where 1.68 was the OR associated with 2.90 µmol/L mean difference in homocysteine levels between stroke cases and controls.</p