Clinical Utility of a Coronary Heart Disease Risk Prediction Gene Score in UK Healthy Middle Aged Men and in the Pakistani Population

BackgroundNumerous risk prediction algorithms based on conventional risk factors for Coronary Heart Disease (CHD) are available but provide only modest discrimination. The inclusion of genetic information may improve clinical utility.MethodsWe tested the use of two gene scores (GS) in the prospective second Northwick Park Heart Study (NPHSII) of 2775 healthy UK men (284 cases), and Pakistani case-control studies from Islamabad/Rawalpindi (321 cases/228 controls) and Lahore (414 cases/219 controls). The 19-SNP GS included SNPs in loci identified by GWAS and candidate gene studies, while the 13-SNP GS only included SNPs in loci identified by the CARDIoGRAMplusC4D consortium.ResultsIn NPHSII, the mean of both gene scores was higher in those who went on to develop CHD over 13.5 years of follow-up (19-SNP p=0.01, 13-SNP p=7x10^-3). In combination with the Framingham algorithm the GSs appeared to show improvement in discrimination (increase in area under the ROC curve, 19-SNP p=0.48, 13-SNP p=0.82) and risk classification (net reclassification improvement (NRI), 19-SNP p=0.28, 13-SNP p=0.42) compared to the Framingham algorithm alone, but these were not statistically significant. When considering only individuals who moved up a risk category with inclusion of the GS, the improvement in risk classification was statistically significant (19-SNP p=0.01, 13-SNP p=0.04). In the Pakistani samples, risk allele frequencies were significantly lower compared to NPHSII for 13/19 SNPs. In the Islamabad study, the mean gene score was higher in cases than controls only for the 13-SNP GS (2.24 v 2.34, p=0.04). There was no association with CHD and either score in the Lahore study.ConclusionThe performance of both GSs showed potential clinical utility in European men but much less utility in subjects from Pakistan, suggesting that a different set of risk loci or SNPs may be required for risk prediction in the South Asian population.     

Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus

Type IV pili are expressed from a wide variety of Gram‐negative bacteria and play a major role in host cell adhesion and bacterial motility. PilC is one of at least a dozen different proteins that are implicated in Type IV pilus assembly in Thermus thermophilus and a member of a conserved family of integral inner membrane proteins which are components of the Type II secretion system (GspF) and the archeal flagellum. PilC/GspF family members contain repeats of a conserved helix‐rich domain of around 100 residues in length. Here, we describe the crystal structure of one of these domains, derived from the N‐terminal domain of Thermus thermophilus PilC. The N‐domain forms a dimer, adopting a six helix bundle structure with an up‐down‐up‐down‐up‐down topology. The monomers are related by a rotation of 170°, followed by a translation along the axis of the final α‐helix of approximately one helical turn. This means that the regions of contact on helices 5 and 6 in each monomer are overlapping, but different. Contact between the two monomers is mediated by a network of hydrophobic residues which are highly conserved in PilC homologs from other Gram‐negative bacteria. Site‐directed mutagenesis of residues at the dimer interface resulted in a change in oligomeric state of PilC from tetramers to dimers, providing evidence that this interface is also found in the intact membrane protein and suggesting that it is important to its function. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Plant growth-promoting Bacillus sp. strain SDA-4 confers Cd tolerance by physio-biochemical improvements,better nutrient acquisition and diminished Cd uptake in Spinacia oleracea L.

Cadmium (Cd) is highly toxic metal for plant metabolic processes even in low concentration due to its longer half-life and non-biodegradable nature. The current study was designed to assess the bioremediation potential of a Cd-tolerant phytobeneficial bacterial strain Bacillus sp. SDA-4, isolated, characterized and identified from Chakera wastewater reservoir, Faisalabad, Pakistan, together with spinach (as a test plant) under different Cd regimes. Spinach plants were grown with and without Bacillus sp. SDA-4 inoculation in pots filled with 0, 5 or 10 mg kg⁻¹ CdCl₂-spiked soil. Without Bacillus sp. SDA-4 inoculation, spinach plants exhibited reduction in biomass accumulation, antioxidative enzymes and nutrient retention. However, plants inoculated with Bacillus sp. SDA-4 revealed significantly augmented growth, biomass accumulation and efficiency of antioxidative machinery with concomitant reduction in proline and MDA contents under Cd stress. Furthermore, application of Bacillus sp. SDA-4 assisted the Cd-stressed plants to sustain optimal levels of essential nutrients (N, P, K, Ca and Mg). It was inferred that the characterized Cd-tolerant PGPR strain, Bacillus sp. SDA-4 has a potential to reduce Cd uptake and lipid peroxidation which in turn maintained the optimum balance of nutrients and augmented the growth of Cd-stressed spinach. Analysis of bioconcentration factor (BCF) and translocation factor (TF) revealed that Bacillus sp. SDA-4 inoculation with spinach sequestered Cd in rhizospheric zone. Research outcomes are important for understanding morpho-physio-biochemical attributes of spinach-Bacillus sp. SDA-4 synergy which might provide efficient strategies to decrease Cd retention in edible plants and/or bioremediation of Cd polluted soil colloids.     

Biodegradation of α- and β-endosulfan by soil bacteria

Extensive applications of persistent organochlorine pesticides like endosulfan on cotton have led to the contamination of soil and water environments at several sites in Pakistan. Microbial degradation offers an effective approach to remove such toxicants from the environment. This study reports the isolation of highly efficient endosulfan degrading bacterial strains from soil. A total of 29 bacterial strains were isolated through enrichment technique from 15 specific sites using endosulfan as sole sulfur source. The strains differed substantially in their potential to degrade endosulfan in vitro ranging from 40 to 93% of the spiked amount (100 mg l⁻¹). During the initial 3 days of incubation, there was very little degradation but it got accelerated as the incubation period proceeded. Biodegradation of endosulfan by these bacteria also resulted in substantial decrease in pH of the broth from 8.2 to 3.7 within 14 days of incubation. The utilization of endosulfan was accompanied by increased optical densities (OD₅₉₅) of the broth ranging from 0.511 to 0.890. High performance liquid chromatography analyses revealed that endosulfan diol and endosulfan ether were among the products of endosulfan metabolism by these bacterial strains while endosulfan sulfate, a persistent and toxic metabolite of endosulfan, was not detected in any case. The presence of endosulfan diol and endosulfan ether in the bacterial metabolites was further confirmed by GC-MS. Abiotic degradation contributed up to 21% of the spiked amount. The three bacterial strains, Pseudomonas spinosa, P. aeruginosa, and Burkholderia cepacia, were the most efficient degraders of both α- and β-endosulfan as they consumed more than 90% of the spiked amount (100 mg l⁻¹) in the broth within 14 days of incubation. Maximum biodegradation by these three selected efficient bacterial strains was observed at an initial pH of 8.0 and at an incubation temperature of 30°C. The results of this study may imply that these bacterial strains could be employed for bioremediation of endosulfan polluted soil and water environments.     

Synthesis and evaluation of modified chalcone based p53 stabilizing agents

Tumor suppressor protein p53 induces cell cycle arrest and apoptotic cell death in response to various cellular stresses thereby preventing cancer development. Activation and stabilization of p53 through small organic molecules is, therefore, an attractive approach for the treatment of cancers retaining wild-type p53. In this context, a series of nineteen chalcones with various substitution patterns of functional groups including chloro, fluoro, methoxy, nitro, benzyloxy, 4-methyl benzyloxy was prepared using Claisen-Schmidt condensation. The compounds were characterized using NMR, HRMS, IR and melting points. Evaluation of synthesized compounds against human colorectal (HCT116) and breast (CAL-51) cancer cell lines revealed potent antiproliferative activities. Nine compounds displayed GI₅₀ values in the low micromolar to submicromolar range; for example (E)-1-phenyl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (SSE14108) showed GI₅₀ of 0.473 ± 0.043 µM against HCT116 cells. Further analysis of these compounds revealed that (E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (SSE14105) and (E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (SSE14106) caused rapid (4 and 8-h post-treatment) accumulation of p53 in HCT116 cells similar to its induction by positive control, Nutlin-3. Such activities were absent in 3-(4-methoxyphenyl)propiophenone (SSE14106H2) demonstrating the importance of conjugated ketone for antiproliferative and p53 stabilizing activity of the chalcones. We further evaluated p53 levels in the presence of cycloheximide (CHX) and the results showed that the p53 stabilization was regulated at post-translational level through blockage of its degradation. These chalcones can, therefore, act as fragment leads for further structure optimization to obtain more potent p53 stabilizing agents with enhanced anti-proliferative activities.     

Activation of adrenergic receptor in H9c2 cardiac myoblasts co-stimulates Nox2 and the derived ROS mediate the downstream responses

Isorhamnetin, a flavonoid compound extracted from the Chinese herb Hippophae rhamnoides L., is well known for its anti-inflammatory, anti-oxidative, anti-adipogenic, anti-proliferative, and anti-tumor activities. However, the role of isorhamnetin in cardiac hypertrophy has not been reported. The aims of the present study were to find whether isorhamnetin could alleviate cardiac hypertrophy and to define the underlying molecular mechanisms. Here, we investigated the effects of isorhamnetin (100 mg/kg/day) on cardiac hypertrophy induced by aortic banding in mice. Cardiac hypertrophy was evaluated by echocardiographic, hemodynamic, pathological, and molecular analyses. Our data demonstrated that isorhamnetin could inhibit cardiac hypertrophy and fibrosis 8 weeks after aortic banding. The results further revealed that the effect of isorhamnetin on cardiac hypertrophy was mediated by blocking the activation of phosphatidylinositol 3-kinase–AKT signaling pathway. In vitro studies performed in neonatal rat cardiomyocytes confirmed that isorhamnetin could attenuate cardiomyocyte hypertrophy induced by angiotensin II, which was associated with phosphatidylinositol 3-kinase–AKT signaling pathway. In conclusion, these data indicate for the first time that isorhamnetin has protective potential for targeting cardiac hypertrophy by blocking the phosphatidylinositol 3-kinase–AKT signaling pathway. Thus, our study suggests that isorhamnetin may represent a potential therapeutic strategy for the treatment of cardiac hypertrophy and heart failure.     

CFD assessment of the effect of convective mass transport on the intracellular clearance of intracellular triglycerides in macrosteatotic hepatocytes

Donor livers available to transplant for patients with end-stage liver disease are in severe shortage. One possible avenue to expand the donor pool is to recondition livers that would be otherwise discarded due to excessive fat content. Severely steatotic livers (also known as fatty livers) are highly susceptible to ischemia-reperfusion injury and as a result, primary liver non-function post-transplantation. Prior studies in isolated perfused rat livers suggest that “defatting” may be possible in a timeframe of a few hours; thus, it is conceivable that fatty liver grafts could be recovered by machine perfusion to clear stored fat from the organ prior to transplantation. However, studies using hepatoma cells and adult hepatocytes made fatty in culture report that defatting may take several days. Because cell culture studies were done in static conditions, we hypothesized that the defatting kinetics are highly sensitive to flow-mediated transport of metabolites. To investigate this question, we experimentally evaluated the effect of increasing flow rate on the defatting kinetics of cultured HepG2 cells and developed an in silico combined reaction-transport model to identify possible rate-limiting steps in the defatting process. We found that in cultured fatty HepG2 cells, the time required to clear stored fat down to lean control cells can be reduced from 48 to 4–6 h by switching from static to flow conditions. The flow required resulted in a fluid shear of .008 Pa, which did not adversely affect hepatic function. The reaction-transport model suggests that the transport of l-carnitine, which is the carrier responsible for taking free fatty acids into the mitochondria, is the key rate-limiting process in defatting that was modulated by flow. Therefore, we can ensure higher levels of l-carnitine uptake by the cells by choosing flow rates that minimize the limiting mass transport while minimizing shear stress.     

The 3MRA Risk Assessment Framework—A Flexible Approach for Performing Multimedia,Multipathway, and Multireceptor Risk Assessments Under Uncertainty

A flexible framework for conducting nationwide multimedia, multipathway and multireceptor risk assessments (3MRA) under uncertainty was developed to estimate protective chemical concentration limits in a source area. The framework consists of two components: risk assessment and uncertainty analysis. The risk component utilizes linked source, fate/transport, exposure and risk assessment models to estimate the risk exposures for the receptors of concern. Both human and ecological receptors are included in the risk assessment framework. The flexibility of the framework is based on its ability to address problems varying in spatial scales from site-specific to regional and even national levels; and its ability to accommodate varying types of source, fate/transport, exposure and risk assessment models. The uncertainty component of the 3MRA framework is based on a two-stage Monte Carlo methodology. It allows the calculation of uncertainty in risk estimates, and the incorporation of the effects of uncertainty on the determination of regulatory concentration limits as a function of variability and uncertainty in input data, as well as potential errors in fate and transport and risk and exposure models. The framework can be adapted to handle a wide range of multimedia risk assessment problems. Two examples are presented to illustrate its use, and to demonstrate how regulatory decisions can be structured to incorporate the uncertainty in risk estimates.     

Evaluation of glucose sensitive affinity binding assay entrapped in fluorescent dissolved‐core alginate microspheres

The feasibility of dissolved‐core alginate‐templated fluorescent microspheres as “smart tattoo” glucose biosensors was investigated in simulated interstitial fluid (SIF). The sensor works on the principle of competitive binding and fluorescence resonance energy transfer. The sensor consists of multilayer thin film coated alginate microspheres incorporating dye‐labeled glucose receptor and competing ligand within the partially dissolved alginate core. In this study, different approaches for the sensing and detection chemistry were studied, and the response of encapsulated reagents was compared with the solution‐phase counterparts. The glucose sensitivity of the encapsulated TRITC‐Con A/FITC‐dextran (500 kDa) assay in DI water was estimated to be 0.26%/mM glucose while that in SIF was observed to be 0.3%/mM glucose. The glucose sensitivity of TRITC‐apo‐GOx/FITC‐dextran (500 kDa) assay was estimated to be 0.33%/mM glucose in DI water and 0.5%/mM glucose in SIF and both demonstrated a response in the range of 0–50 mM glucose. Therefore, it is hypothesized that the calcium ion concentration outside the microsphere (in the SIF) does not interfere with the response sensitivity. The sensor response was observed to exhibit a maximum response time of 120 s. The system further exhibited a sensitivity of 0.94%/mM glucose with a response in range of 0–50 mM glucose, using near‐infrared dyes (Alexa Fluor‐647‐labeled dextran as donor and QSY‐21‐conjugated apo‐GOx as acceptor), thereby making the sensor more amenable to in vivo use, when implanted in scattering tissue. Biotechnol. Bioeng. 2009; 104: 1075–1085. © 2009 Wiley Periodicals, Inc.     

Studies on the production of nigerloxin using agro-industrial residues by solid-state fermentation

Nigerloxin, a new and potent lipoxygenase inhibitor, was discovered in our laboratory through solid-state fermentation of wheat bran by Aspergillus niger V. Teigh (MTCC-5166). The aim of this study is to investigate the possibility of using different agro-industrial residues as nutritional supplements along with wheat bran to enhance the production of nigerloxin. Nigerloxin produced by SSF was quantified spectrophotometrically at 292 nm. The results indicate that the inhibitor production was influenced by the type of solid substrate supplemented, moisture content, pH and size of the inoculum. Individually optimized supplements were tested in different combinations to determine their effects on nigerloxin production. A twofold increase in the production of nigerloxin (4.9 ± 0.3 mg gds⁻¹) was achieved by supplementing wheat bran with 10% w/w sweet lemon peel and 5% v/w methanol at optimized process parameters, that is, an initial moisture content of 65% v/w and incubation period of 6 days with an initial inoculum size of 2 ml (8 × 10⁵ spores gds⁻¹). Nigerloxin production was stable between pH of 4 and 5.