Comparison of expression optimization of new derivative of staphylokinase (SAK-2RGD-TTI) with the rSAK

Staphylokinase (SAK) is a promising thrombolytic agent for the treatment of patients suffering from blood-clotting disorders. To increase the potency of SAK and to minimize vessel reocclusion, a new construct bearing SAK motif fused to tsetse thrombin inhibitor (TTI) via a 20-amino acid linker with 2 RGD (2 × arginine-glycine-aspartic acid inhibiting platelet aggregation via attachment to integrin receptors of platelet) was codon optimized and expressed comparatively in Pichia pastoris GS115 as a Mut⁺ strain and KM71H as a Mut^s strain. Fusion protein was optimized in terms of best expression condition and fibrinolytic activity and compared with the rSAK. Expression level of the designed construct reached up to 175 mg/L of the culture medium after 72-hr stimulation with 2.5% methanol and remained steady for 3–4 days. The highest expression was obtained at the range of 2–3% methanol. The SAK-2RGD-TT (relative activity >82%) was more active at 25–37 °C than rSAK (relative activity of 93%). Further, it showed relative activity >80% at pH ranges of 7–9. Western blot analysis showed two bands of nearly 27 and 24 kDa at ratio of 5 to 3, respectively. The specific fibrinolytic activity of the SAK-2RGD-TTI was measured as 8,269 U/mg, and 19,616 U/mg for the nonpurified and purified proteins, respectively. Deglycosylation by using tunicamycin in culture medium resulted in higher fibrinolytic activity of SAK-2RGD-TTI (2.2 fold). Consequently, compared to the rSAK, at the same equimolar proportion, addition of RGD and TTI fragments could increase fibrinolytic activity. Also, P. pastoris can be considered as an efficient host for overexpression of the soluble SAK-2RGD-TTI with high activity without requiring a complicated purification procedure.     

High-density lipoprotein lipid peroxidation as a molecular signature of the risk for developing cardiovascular disease: Results from MASHAD cohort

High-density lipoprotein (HDL) function rather than level may better predict cardiovascular disease (CVD). However, the contribution of the impaired antioxidant function of HDL that is associated with increased HDL lipid peroxidation (HDLox) to the development of clinical CVD remains unclear. We have investigated the association between serum HDLox with incident CVD outcomes in Mashhad cohort. Three-hundred and thirty individuals who had a median follow-up period of 7 years were recruited as part of the cohort. The primary end point was cardiovascular event, including myocardial infarction, stable angina, unstable angina, or coronary revascularization. In both univariate/multivariate analyses adjusted for traditional CVD risk factors, HDLox was an independent risk factor for CVD (odds ratio, 1.62; 95% confidence interval, 1.41–1.86; p < 0.001). For every increase in HDLox by 0.1 unit, there was an increase in CVD risk by 1.62-fold. In an adjusted analysis, there was a >2.5-fold increase in cardiovascular risk in individuals with HDLox higher than cutoff point of 1.06 compared to those with lower scores, suggesting HDLox > 1.06 is related to the impaired HDL oxidant function and in turn exposed to elevated risk of CVD outcomes (hazard ratio, 2.72; 95% CI, 1.88–3.94). Higher HDLox is a surrogate measure of reduced HDL antioxidant function that positively associated with cardiovascular events in a population-based cohort.     

The effect of carbon source supplementation on the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator

The objective of the present study was to investigate the ability of Cupriavidus necator to produce poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) on various carbon sources in batch cultivation. These results show that C. necator produces poly-3-hydroxybutyrate from single carbon sources. The highest poly-3-hydroxybutyrate (P3HB) content was achieved at growth on fructose in the exponential growth phase. The maximum yield of the P3HV content was obtained when fructose was mixed with acetate. The highest content P3HB-co-3HV was also achieved by C. necator when we supplied C-excess and N- and P-normal conditions. These results indicate that C. necator accumulates high polyhydroxyalkanoates (PHA) content by depleting these elements in the culture medium. Nitrogen and phosphorus limitation has no significant effect on the PHA production, whereas C-excess leads to an increase in PHA formation of up to 92% PHAs of cell dry weight after growth on 5 g/L acetate and 40 g/L fructose.     

Methylenetetrahydrofolate reductase gene polymorphism in diabetes and obesity

Methylenetetrahydrofolate reductase (MTHFR) polymorphism may play an important role in the pathophysiology of obesity and diabetes accompanied by obesity due to its influence on plasma homocysteine levels. There are significant and sometimes very strong relationship between levels of homocysteine and several multi-system diseases including CHD and CVA. To examine the association between MTHFR gene C677T polymorphism in diabetes and obesity with serum homocysteine levels. A total of 682 subjects were recruited in four groups (Normal, obese, diabetic and obese and diabetics). MTHFR gene C677T polymorphism was detected using PCR-RFLP technique. Serum homocysteine levels were measured using HPLC. There was a significant increase in the mean serum homocysteine levels in subjects carrying TT genotype (34.6 ± 26.5) compared to subjects carrying CC (15.1 ± 8) or CT genotype (16.4 ± 7.8) (P < 0.000). We found no significant differences for MTHFR allele and genotype frequencies between different groups. Our data have confirmed the association between serum homocysteine levels and MTHFR C677T genotype reported in other populations.     

Hybrid chitosan–ß‐glycerol phosphate–gelatin nano‐/micro fibrous scaffolds with suitable mechanical and biological properties for tissue engineering

Scaffold‐based tissue engineering is considered as a promising approach in the regenerative medicine. Graft instability of collagen, by causing poor mechanical properties and rapid degradation, and their hard handling remains major challenges to be addressed. In this research, a composite structured nano‐/microfibrous scaffold, made from a mixture of chitosan–ß‐glycerol phosphate–gelatin (chitosan–GP–gelatin) using a standard electrospinning set‐up was developed. Gelatin–acid acetic and chitosan ß‐glycerol phosphate–HCL solutions were prepared at ratios of 30/70, 50/50, 70/30 (w/w) and their mechanical and biological properties were engineered. Furthermore, the pore structure of the fabricated nanofibrous scaffolds was investigated and predicted using a theoretical model. Higher gelatin concentrations in the polymer blend resulted in significant increase in mean pore size and its distribution. Interaction between the scaffold and the contained cells was also monitored and compared in the test and control groups. Scaffolds with higher chitosan concentrations showed higher rate of cell attachment with better proliferation property, compared with gelatin‐only scaffolds. The fabricated scaffolds, unlike many other natural polymers, also exhibit non‐toxic and biodegradable properties in the grafted tissues. In conclusion, the data clearly showed that the fabricated biomaterial is a biologically compatible scaffold with potential to serve as a proper platform for retaining the cultured cells for further application in cell‐based tissue engineering, especially in wound healing practices. These results suggested the potential of using mesoporous composite chitosan–GP–gelatin fibrous scaffolds for engineering three‐dimensional tissues with different inherent cell characteristics. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 163–175, 2016.     

The Association Between rs1748195 and rs11207997 Variants of the ANGPTL3 Gene and Susceptibility to Cardiovascular Disease in the MASHAD Cohort Study

Biochemical Genetics - There is a strong genetic predisposition to cardiovascular disease (CVD). Loss-of-function variants of the angiopoietin-like 3 (ANGPTL3) gene have been reported to be...     

Response of biomass,hydrology and biogeochemistry to alternative approaches of cutting a northern forest: model comparisons

Biogeochemistry - The biogeochemical model, PnET-BGC, was modified and parameterized using field data from an experimental whole-tree harvest of watershed (W5) in 1983–1984 at the Hubbard...