土壤中棉花黄萎病菌SYBR Green Ⅰ荧光RT-PCR定量检测技术研究

为实现田间土壤棉花黄萎病菌的早期检测,建立了土壤中棉花黄萎病菌的SYBR Green I荧光定量PCR检测方法。以含342bp PCR扩增产物的阳性质粒为参考,构建了标准曲线,并对该曲线的特异性、敏感性、可重复性进行了评价。结果表明,该方法具有快速、特异性强、敏感度高等特点。检测范围在3.8×103-3.8×108copies/μL之间有良好的线性关系,相关系数R2为0.996,扩增效率为101.5%,灵敏度比常规PCR方法高102倍。     

Analysis of Non-stationary Data for Heart-rate Fluctuations in Terms of Drift and Diffusion Coefficients

We describe a method for analyzing the stochasticity in non-stationary data for the beat-to-beat fluctuations in the heart rates of healthy subjects, as well as those with congestive heart failure. The method analyzes the return time series of the data as a Markov process, and computes the Markov time scale, i.e., the time scale over which the data are a Markov process. We also construct an effective stochastic continuum equation for the return series. We show that the drift and diffusion coefficients, as well as the amplitude of the return time series for healthy subjects are distinct from those with CHF. Thus, the method may potentially provide a diagnostic tool for distinguishing healthy subjects from those with congestive heart failure, as it can distinguish small differences between the data for the two classes of subjects in terms of well-defined and physically-motivated quantities. PACS: 05.10.Gg 05.40.-a, 05.45.Tp, 87.19.Hh     

Cytoprotective effects of antioxidant supplementation on mesenchymal stem cell therapy

Mesenchymal stem cells (MSCs) are earmarked as perfect candidates for cell therapy and tissue engineering due to their capacity to differentiate into different cell types. However, their potential for application in regenerative medicine declines when the levels of the reactive oxygen and nitrogen species (RONS) increase from the physiological levels, a phenomenon which is at least inevitable in ex vivo cultures and air-exposed damaged tissues. Increased levels of RONS can alter the patterns of osteogenic and adipogenic differentiation and inhibit proliferation, as well. Besides, oxidative stress enhances senescence and cell death, thus lowering the success rates of the MSC engraftment. Hence, in this review, we have selected some representatives of antioxidants and newly emerged nano antioxidants in three main categories, including chemical compounds, biometabolites, and protein precursors/proteins, which are proved to be effective in the treatment of MSCs. We will focus on how antioxidants can be applied to optimize the clinical usage of the MSCs and their associated signaling pathways. We have also reviewed several paralleled properties of some antioxidants and nano antioxidants which can be simultaneously used in real-time imaging, scaffolding techniques, and other applications in addition to their primary antioxidative function.     

Role of mechanosignaling on pathology of varicose vein

Varicose veins are the most common vascular disease in humans. Veins have valves that help the blood return gradually to the heart without leaking blood. When these valves become weak, blood and fluid collect and pool by pressing against the walls of the veins, causing varicose veins. In the cardiovascular system, mechanical forces are important determinants of vascular homeostasis and pathological processes. Blood vessels are constantly exposed to a variety of hemodynamic forces, including shear stress and environmental strains caused by the blood flow. In varicose veins within the leg, venous blood pressure rises in the vein of the lower extremities due to prolonged standing, creating a peripheral tension in the vessel wall thereby causing mechanical stimulation of endothelial cells and vascular smooth muscle. Studies have shown that long-term increased exposure to vascular wall tension is associated with the overexpression of HIF-1α and HIF-2α and increased levels of MMP-2 and MMP-9, thereby reducing venous contraction and progressive venous dilatation, which is involved in the development of varicose veins. Following the expression of metalloproteinase, the expression of type 1 collagen increases, and the amount of type 3 collagen decreases. Therefore, collagen imbalance will cause the varicose veins to not stretch. Loss of structural proteins (type 3 collagen and elastin) in the vessel wall causes the loss of the biophysical properties of the varicose vein wall. This review article tries to elaborate on the effect of mechanical forces and sensors of these forces on the vascular wall in creating the mechanism of mechanosignaling, as well as the role of the onset of molecular signaling cascades in the pathology of varicose veins.     

The prevalence of factor V Leiden,prothrombin G20210A and methylenetetrahydrofolate reductase polymorphism C677T among G6PD deficient individuals from Western Iran

It has been suggested that the allele frequency of thrombophilic mutations is affected by glucose-6-phosphate dehydrogenase (G6PD) deficiency. The prevalence of thrombophilic mutations were studied in sixty G6PD deficient individuals including 57 males and three females with the mean age of 15 ± 3.08 and 110 age and sex matched healthy individuals consisted of 95 males and 15 females with the mean age of 16.19 ± 2.17 from the Kermanshah Province of Iran. Using a combination of PCR-RFLP technique, single strand conformation polymorphism (SSCP) analysis and DNA sequencing polymorphic G6PD mutations were identified. The factor V Leiden, prothrombin G20210A and methylenetetrahydrofolate reductase (MTHFR) C677T were detected by PCR-RFLP method using MnlI, HindIII and HinfI restriction enzymes, respectively. Three mutations, G6PD Mediterranean, G6PD Chatham and G6PD Cosenza were identified in 60 G6PD deficient individuals with highest prevalence of G6PD Mediterranean (91.6%). In G6PD deficient individuals the prevalence of factor V Leiden tended to be higher (5%) compared to healthy individuals (2.7%). The prevalence of prothrombin G20210A mutation in G6PD deficient individuals was 1.7%. However, in normal subjects the prevalence of this mutation was 2.7%. The frequency of T allele in G6PD deficient individuals were insignificantly higher (29.16%) than those in healthy individuals (26.8%). Our finding indicates that the prevalence of factor V Leiden, prothrombin G20210A and MTHFR C677T in G6PD deficient individuals is not statistically different compared to normal subjects and G6PD deficiency is not associated with these thrombophilic mutations in Western Iran.     

IQGAP1-Dependent Signaling Pathway Regulates Endothelial Cell Proliferation and Angiogenesis

Background

Vascular endothelial growth factor receptor-2 (VEGFR-2) signaling is an obligate requirement for normal development and pathological angiogenesis such as cancer and age-related macular degeneration. Although autophosphorylation of tyrosine 1173 (Y1173) of VEGFR-2 is considered a focal point for its angiogenic signal relay, however, the mechanism of phosphorylation of Y1173, signaling proteins that are recruited to this residue and their role in angiogenesis is not fully understood.

Methodology/Principal Findings

In this study we demonstrate that c-Src kinase directly through its Src homology 2 (SH2) domain and indirectly via c-Cbl binds to phospho-Y1057 of VEGFR-2. Activation of c-Src kinase by a positive feedback mechanism phosphorylates VEGFR-2 at multi-docking site, Y1173. c-Src also catalyzes tyrosine phosphorylation of IQGAP1 and acts as an adaptor to bridge IQGAP1 to VEGFR-2. In turn, IQGAP1 activates b-Raf and mediates proliferation of endothelial cells. Silencing expression of IQGAP1 and b-Raf revealed that their activity is essential for VEGF to stimulate angiogenesis in an in vivo angiogenesis model of chicken chorioallantoic membrane (CAM).

Conclusions/Significance

Angiogenesis contributes to the pathology of numerous human diseases ranging from cancer to age-related macular degeneration. Determining molecular mechanism of tyrosine phosphorylation of VEGFR-2 and identification of molecules that are relaying its angiogenic signaling may identify novel targets for therapeutic intervention against angiogenesis-associated diseases. Our study shows that recruitment and activation of c-Src by VEGFR-2 plays a pivotal role in relaying angiogenic signaling of VEGFR-2; it phosphorylates VEGFR-2 at Y1173, facilitates association and activation of IQGAP1 and other signaling proteins to VEGFR-2. IQGAP1-dependent signaling, in part, is critically required for endothelial cell proliferation, a key step in angiogenesis. Thus, Y1057 of VEGFR-2 serves to regulate VEGFR-2 function in a combinatorial manner by supporting both diversity of recruitment of angiogenic signaling proteins to VEGFR-2, and its ability to promote angiogenesis.     

The anabolic action of intermittent parathyroid hormone on cortical bone depends partly on its ability to induce nitric oxide‐mediated vasorelaxation in BALB/c mice

There is strong evidence that vasodilatory nitric oxide (NO) donors have anabolic effects on bone in humans. Parathyroid hormone (PTH), the only osteoanabolic drug currently approved, is also a vasodilator. We investigated whether the NO synthase inhibitor L‐NAME might alter the effect of PTH on bone by blocking its vasodilatory effect. BALB/c mice received 28 daily injections of PTH[1–34] (80 µg/kg/day) or L‐NAME (30 mg/kg/day), alone or in combination. Hindlimb blood perfusion was measured by laser Doppler imaging. Bone architecture, turnover and mechanical properties in the femur were analysed respectively by micro‐CT, histomorphometry and three‐point bending. PTH increased hindlimb blood flow by >30% within 10 min of injection (P < 0.001). Co‐treatment with L‐NAME blocked the action of PTH on blood flow, whereas L‐NAME alone had no effect. PTH treatment increased femoral cortical bone volume and formation rate by 20% and 110%, respectively (P < 0.001). PTH had no effect on trabecular bone volume in the femoral metaphysis although trabecular thickness and number were increased and decreased by 25%, respectively. Co‐treatment with L‐NAME restricted the PTH‐stimulated increase in cortical bone formation but had no clear‐cut effects in trabecular bone. Co‐treatment with L‐NAME did not affect the mechanical strength in femurs induced by iPTH. These results suggest that NO‐mediated vasorelaxation plays partly a role in the anabolic action of PTH on cortical bone. © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.