Rapid Generation of Recombinant Flaviviruses Using Circular Polymerase Extension Reaction.

The genus Flavivirus is a group of arthropod-borne single-stranded RNA viruses, which includes important human and animal pathogens such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), Dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Tick-borne encephalitis virus (TBEV). Reverse genetics has been a useful tool for understanding biological properties and the pathogenesis of flaviviruses. However, the conventional construction of full-length infectious clones for flavivirus is time-consuming and difficult due to the toxicity of the flavivirus genome to E. coli. Herein, we applied a simple, rapid, and bacterium-free circular polymerase extension reaction (CPER) method to synthesize recombinant flaviviruses in vertebrate cells as well as insect cells. We started with the de novo synthesis of the JEV vaccine strain SA-14-14-2 in Vero cells using CPER, and then modified the CPER method to recover insect-specific flaviviruses (ISFs) in mosquito C6/36 cells. Chimeric Zika virus (ChinZIKV) based on the Chaoyang virus (CYV) backbone and the Culex flavivirus reporter virus expressing green fluorescent protein (CxFV-GFP) were subsequently rescued in C6/36 cells. CPER is a simple method for the rapid generation of flaviviruses and other potential RNA viruses. A CPER-based recovery system for flaviviruses of different host ranges was established, which would facilitate the development of countermeasures against flavivirus outbreaks in the future.

TEM8 functions as a receptor for uPA and mediates uPA-stimulated EGFR phosphorylation.

BACKGROUND: TEM8 is a cell membrane protein predominantly expressed in tumor endothelium, which serves as a receptor for the protective antigen (PA) of anthrax toxin. However, the physiological ligands for TEM8 remain unknown. RESULTS: Here we identified uPA as an interacting partner of TEM8. Binding of uPA stimulated the phosphorylation of TEM8 and augmented phosphorylation of EGFR and ERK1/2. Finally, TEM8-Fc, a recombinant fusion protein comprising the extracellular domain of human TEM8 linked to the Fc portion of human IgG1, efficiently abrogated the interaction between uPA and TEM8, blocked uPA-induced migration of HepG2 cells in vitro and inhibited the growth and metastasis of human MCF-7 xenografts in vivo. uPA, TEM8 and EGFR overexpression and ERK1/2 phosphorylation were found co-located on frozen cancer tissue sections. CONCLUSIONS: Taken together, our data provide evidence that TEM8 is a novel receptor for uPA, which may play a significant role in the regulation of tumor growth and metastasis.

Protective effects of choline against hypoxia-induced injuries of vessels and endothelial cells.

The current study aimed to lay a theoretical foundation for further development of choline as an anti-hypoxia damage drug. Wild-type, 3- to 5-month-old male Sprague-Dawley rats, weighing 180-220 g, were used in this study. The rats were randomly divided into a normoxic control group (n=16) and a chronic intermittent hypoxia (CIH) group (n=16). The effects of CIH on acetylcholine (ACh)-mediated endothelium-dependent vasodilatation in the rat cerebral basilar arterioles and mesenteric arterioles, as well as the protective effects of choline on the arterioles damaged by hypoxia were observed. Moreover, the effects of choline on endothelial cell proliferation during hypoxia were observed, and choline's functional mechanism further explored. The ACh-mediated vasodilatation of rat cerebral basilar and mesenteric arterioles significantly reduced during hypoxia (P<0.01). Choline significantly increased dilation in the rat cerebral basilar (P<0.01) and mesenteric arterioles (P<0.05) damaged by CIH compared with those in the control group. In addition, under hypoxic conditions, choline significantly promoted the proliferation of rat aortic endothelial cells (P<0.05) and significantly reduced lactate dehydrogenase activity in the cell culture supernatant in vitro (P<0.05). Furthermore, the effect of choline could be related to its ability to significantly increase the secretion of vascular endothelial growth factor (P<0.01) and activation of α7 non-neuronal nicotinic acetylcholine receptors under hypoxia (P<0.01). This study demonstrated that choline could have protective effects against hypoxic injuries.

Diazoxide protects rat vascular endothelial cells against hypoxia and cold-induced damage.

The present study aimed to examine the effects of hypoxia and cold on vascular endothelial cells (VECs), as well as the protective ability of novel VECs-protective drugs against these injuries. A rat model simulating exposure to hypoxia and cold at high altitude environments was established. Based on these animal experiments, rat aortic VECs were established as injury models and exposed to hypoxia and/or adrenaline (ADR) in vitro. The results revealed that hypoxia significantly altered the levels of nitric oxide and vascular endothelial growth factor, while the cold temperature significantly increased the release of ADR and noradrenaline. Exposure to hypoxia combined with cold temperature significantly affected all these indices. In vitro experiments demonstrated that hypoxia, ADR (which was used to simulate cold in the animal experiments) and the combination of the two factors resulted in damage to the VECs and endothelial dysfunction. In addition, the results also showed that diazoxide, a highly selective mitoKATP opener, protected VECs against these injuries. In conclusion, hypoxia and cold temperature induced endothelial cell dysfunction and endocrine disorders, respectively. Improving endothelial function using diazoxide may be an effective therapeutic strategy in patients with altitude-associated disorders. However, the potential for clinical application requires further study.

Proteomics reveals potential non-neuronal cholinergic receptor-effectors in endothelial cells.

AIM: The non-neuronal acetylcholine system (NNAS) in endothelial cells participates in modulating endothelial function, vascular tone, angiogenesis and inflammation, thus plays a critical role in cardiovascular diseases. In this study, we used a proteomic approach to study potential downstream receptor-effectors of NNAS that were involved in regulating cellular function in endothelial cells. METHODS: Human umbilical vein endothelial cells were incubated in the presence of acetylcholine, oxotremorine, pilocarpine or nicotine at the concentration of 10 µmol/L for 12 h, and the expressed proteins in the cells were separated and identified with two-dimensional electrophoresis (2-DE) and LC-MS. The protein spots with the largest changes were identified by LC-MS. Biowork software was used for database search of the peptide mass fingerprints. RESULTS: Over 1200 polypeptides were reproducibly detected in 2-DE with a pH range of 3-10. Acetylcholine, oxotremorine, pilocarpine and nicotine treatment caused 16, 9, 8 and 9 protein spots, respectively, expressed differentially. Four protein spots were identified as destrin, FK506 binding protein 1A (FKBP1A), macrophage migration inhibitory factor (MIF) and profilin-1. Western blotting analyses showed that treatment of the cells with cholinergic agonists significantly decreased the expression of destrin, FKBP1A and MIF, and increased the expression of profilin-1. CONCLUSION: A set of proteins differentially expressed in endothelial cells in response to cholinergic agonists may have important implications for the downstream biological effects of NNAS.

[Effect of electroacupuncture on cerebral PKC isozyme expression levels in cerebral ischemia-reperfusion rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Dingzhongxian" (MS 5) and "Dingpangxian" (MS 8) on the expression of cerebral protein kinase C (PKC) isozymes in local cerebral ischemia reperfusion injury (CI/RI) rats so as to explore its underlying mechanism in protecting ischemic brain tissue. METHODS: Seventy-two Wistar rats were randomized into normal control (n = 8), CI/RI model (model, n = 32), and EA (n = 32) groups. The later two groups were further divided into 4, 12, 24 and 72 h subgroups, respectively, with 8 rats in each. CI/RI model was established by occlusion of the middle cerebral artery under anesthesia and reperfusion for 4, 12, 24 and 72 h, respectively. EA (1 mA, 2 Hz/15 Hz) was applied to "Dingzhongxian" (MS5) and "Dingpangxian" (MS8) for 10 min every time, and once again every 12 h after modeling. The expression of PKCgamma and PKCdelta in the ischemic cortex tissue was detected using immunohistochemistry. TdT-mediated dUTP Nick-End Labeling was used to detect neuronal apoptosis of the local ischemic cerebral cortex. RESULTS: In comparison with the normal group, the expression levels of cerebral PKCgamma and PKCdelta proteins as well as the number of the apoptotic neurons at time-points of 4, 12, 24 and 72 h after modeling were increased apparently in the model group (P < 0.01); while compared with the model group, cerebral PKCgamma and PKCdelta protein expressions and the apoptotic neuronal number were decreased considerably in the EA group (P < 0.01, P < 0.05). No significant differences were found among the 4 time-points in the expression levels of PKCgamma and PKCdelta and apoptotic neuronal number in the model group and EA group (P > 0.05). CONCLUSION: EA intervention can effectively down-regulate expressions of cerebral PKCgamma, PKCdelta proteins and apoptotic neuronal number in cerebral ischemia rats, which may contribute to its effect in protecting the ischemic cerebral tissue.

[Observation on therapeutic effect of comprehensive therapy for acute large area cerebral infarction].

OBJECTIVE: To search for a method for increasing clinical therapeutic effect of acute large area cerebral infarction. METHODS: Ninety-six cases were randomly divided into a comprehensive treatment group and a western medicine group, 48 cases in each group. The western medicine group were treated with routine western methods, dehydration for decreasing intracranial pressure, brain-protection, anti-platelet agglutination, improving circulation and expectant treatment, etc.; the comprehensive treatment group were treated with the routine treatment of western medicine combined with acupuncture, moxibustion and enema of Chinese drugs. The therapeutic effect was observed after treatment for 28 days. RESULTS: The total effective rate was 68.8% in the comprehensive treatment group and 39.6% in the western medicine group with a very significant difference between the two groups (P < 0.01), and the score of the National Institute of Health's Stroke Scale (NIHSS) and the score of the Glasgow Coma Scale (GCS) in the comprehensive treatment group were better than those in the western medicine group (P < 0.001, P < 0.01). CONCLUSION: Combined treatment of Chinese medicine and western medicine for large area cerebral infarction is better than simple western medicine, and it can significantly improve neurological defect of the patient and decrease death rate.