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Blood-brain barrier (BBB) disruption caused by reperfusion injury after ischemic stroke is an intractable event conducive to further injury. Brain pericytes play a vital role in maintaining BBB integrity by interacting with other components of the BBB. In this study, we found that sphingosine-1-phosphate receptor (S1PR)2 expressed in pericytes was significantly up-regulated after ischemia in vivo and in vitro. By using a S1PR2 antagonist (JTE-013), we showed that S1PR2 plays a critical role in the induction of BBB permeability of transient middle cerebral artery occlusion (tMCAO) rats and the in vitro BBB model. Furthermore, we discovered that S1PR2 may decrease N-cadherin expression and increase pericyte migration via NF-κB p65 signal and found that S1PR2 could be regulated by miR-149-5p negatively, which was decreased in the ischemic boundary zone and cultured pericytes after ischemia. Overexpression of miR-149-5p in cultured pericytes substantially increased N-cadherin expression and decreased pericyte migration, which decreased BBB leakage in the in vitro model. Up-regulating miR-149-5p by intracerebroventricular injection of agomir-149-5p attenuated BBB permeability and improved the outcomes of tMCAO rats significantly. Thus, our data suggest that miR-149-5p may serve as a potential target for treatment of BBB disruption after ischemic stroke.-Wan, Y., Jin, H.-J., Zhu, Y.-Y., Fang, Z., Mao, L., He, Q., Xia, Y.-P., Li, M., Li, Y., Chen, X., Hu, B. MicroRNA-149-5p regulates blood-brain barrier permeability after transient middle cerebral artery occlusion in rats by targeting S1PR2 of pericytes.

Blood-brain barrier (BBB) disruption plays a critical role in brain injury induced by cerebral ischemia, and preserving BBB integrity during ischemia could alleviate cerebral injury. We examined the role of miR-130a in ischemic BBB disruption by using models of rat middle cerebral artery occlusion and cell oxygen-glucose deprivation. We found that ischemia significantly increased microRNA-130a (miR-130a) level and that miR-130a was predominantly from brain microvascular endothelial cells. Antagomir-130a, an antagonist of miR-130a, could attenuate brain edema, lower BBB permeability, reduce infarct volume, and improve neurologic function. MiR-130a overexpression induced by miR-130a mimic increased monolayer permeability, and intercellular inhibition of miR-130a by a miR-130a inhibitor suppressed oxygen-glucose deprivation-induced increase in monolayer permeability. Moreover, dual luciferase reporter system showed that Homeobox A5 was the direct target of miR-130a. MiR-130a, by inhibiting Homeobox A5 expression, could down-regulate occludin, thereby increasing BBB permeability. Our results suggested that miR-130a might be implicated in ischemia-induced BBB dysfunction and serve as a target for the treatment of ischemic stroke.-Wang, Y., Wang, M.-D., Xia, Y.-P., Gao, Y., Zhu, Y.-Y., Chen, S.-C., Mao, L., He, Q.-W., Yue, Z.-Y., Hu, B. MicroRNA-130a regulates cerebral ischemia-induced blood-brain barrier permeability by targeting Homeobox A5.

Background Every year, strokes take millions of lives and leave millions of individuals living with permanent disabilities. Recently more researchers embrace the concept of the neurovascular unit (NVU), which encompasses neurons, endothelial cells (ECs), pericytes, astrocyte, microglia, and the extracellular matrix. It has been well-documented that NVU emerged as a new paradigm for the exploration of mechanisms and therapies in ischemic stroke. To better understand the complex NVU and broaden therapeutic targets, we must probe the roles of multiple cell types in ischemic stroke. The aims of this paper are to introduce the biological characteristics of brain pericytes and the available evidence on the diverse functions and mechanisms involving the pericytes in the context of ischemic stroke. Methods Research and online content related to the biological characteristics and pathophysiological roles of pericytes is review. The new research direction on the Pericytes in ischemic stroke, and the potential therapeutic targets are provided. Results During the different stages of ischemic stroke, pericytes play different roles: 1) On the hyperacute phase of stroke, pericytes constriction and death may be a cause of the no-reflow phenomenon in brain capillaries; 2) During the acute phase, pericytes detach from microvessels and participate in inflammatory-immunological response, resulting in the BBB damage and brain edema. Pericytes also provide benefit for neuroprotection by protecting endothelium, stabilizing BBB and releasing neurotrophins; 3) Similarly, during the later recovery phase of stroke, pericytes also contribute to angiogenesis, neurogenesis, and thereby promote neurological recovery. Conclusion This emphasis on the NVU concept has shifted the focus of ischemic stroke research from neuro-centric views to the complex interactions within NVU. With this new perspective, pericytes that are centrally positioned in the NVU have been widely studied in ischemic stroke. More work is needed to elucidate the beneficial and detrimental roles of brain pericytes in ischemic stroke that may serve as a basis for potential therapeutic targets.

Summary Aims Angiogenesis is a harmonized target for poststroke recovery. Therefore, exploring the mechanisms involved in angiogenesis after stroke is vitally significant. In this study, we are reporting a miR-150-based mechanism underlying cerebral poststroke angiogenesis. Methods Rat models of middle cerebral artery occlusion (MCAO) and cell models of oxygen–glucose deprivation were conducted. Capillary density, tube formation, cell proliferation, and cell migration were measured by FITC-dextran assay, matrigel assay, Ki-67 staining, and wound healing assay, respectively. The expression of miR-150 and vascular endothelial growth factor (VEGF) was, respectively, measured by RT-PCR and Western blotting. Dual-luciferase assay was conducted to confirm the binding sites between miR-150 and VEGF. Results We found that miR-150 expression in the brain and serum of rats subjected to cerebral ischemia, and in oxygen–glucose-deprived brain microvascular endothelial cells (BMVECs) and astrocytes. Upregulation of miR-150 expression could decrease vascular density of infarct border zone in rat after MCAO and decrease tube formation, proliferation, and migration of BMVECs. We also found that miR-150 could negatively regulate the expression of VEGF, and VEGF was confirmed to be a direct target of miR-150. Moreover, VEGF mediated the function of miR-150 on tube formation, proliferation, and migration of BMVECs. Conclusions Our data suggested that miR-150 could regulate cerebral poststroke angiogenesis in rats through VEGF.

The mechanism of blood-brain barrier (BBB) disruption, involved in poststroke edema and hemorrhagic transformation, is important but elusive. We investigated microRNA-150 (miR-150)-mediated mechanism in the disruption of BBB after stroke in rats. We found that up-regulation of miR-150 increased permeability of BBB as detected by MRI after permanent middle cerebral artery occlusion in vivo as well as increased permeability of brain microvascular endothelial cells after oxygen-glucose deprivation in vitro. The expression of claudin-5, a key tight junction protein, was decreased in the ischemic boundary zone after up-regulation of miR-150. We found in brain microvascular endothelial cells that overexpression of miR-150 decreased not only cell survival rate but also the expression levels of claudin-5 after oxygen-glucose deprivation. With dual-luciferase assay, we confirmed that miR-150 could directly regulate the angiopoietin receptor Tie-2. Moreover, silencing Tie-2 with lentivirus-delivered small interfering RNA reversed the effect of miR-150 on endothelial permeability, cell survival, and claudin-5 expression. Furthermore, poststroke treatment with antagomir-150, a specific miR-150 antagonist, contributed to BBB protection, infarct volume reduction, and amelioration of neurologic deficits. Collectively, our findings suggested that miR-150 could regulate claudin-5 expression and endothelial cell survival by targeting Tie-2, thus affecting the permeability of BBB after permanent middle cerebral artery occlusion in rats, and that miR-150 might be a potential alternative target for the treatment of stroke.-Fang, Z., He, Q.-W., Li, Q., Chen, X.-L., Baral, S., Jin, H.-J., Zhu, Y.-Y., Li, M., Xia, Y.-P., Mao, L., Hu, B. MicroRNA-150 regulates blood-brain barrier permeability via Tie-2 after permanent middle cerebral artery occlusion in rats.

With the intensification of the aging process of the world, Alzheimer's disease (AD), which is the main type of senile dementia, has become a primary problem in the present society. Lots of strategies have been used to prevent and treat AD in animal models and clinical trials, but most of them ended in failure. Panax notoginseng saponins (PNS) contain a variety of monomer compositions which have been separated and identified. Among of the monomer compositions, notoginseng saponin Rg1 (Rg1) accounts for 20% of the cultivation of panax notoginseng roots. And now PNS have been reported to be widely used to treat cardio-cerebrovascular diseases and have neuroprotective effects to restrain the β-amyloid peptide (Aβ)25-35-mediated apoptosis. Moreover, it is reported that PNS could accelerate the growth of nerve cells, increase the length of axons and promote synaptic plasticity. Whether Rg1 can ameliorate the cognitive impairment and the underlying mechanism has not been elucidated. To study the preventive effect of Rg1 on cognitive impairment and the possible mechanism, we established the cognitive impairment model in rats through Aβ

Objectives This study examined the neurologists’ perspective toward intravenous thrombolysis for the treatment of acute ischemic stroke and the influencing factors in a Chinese Province. Methods A cross‐sectional study was conducted from 1 October 2014 to 31 January 2015. A total of 719 neurologists from 66 hospitals in Hubei Province were included. A questionnaire was designed, and multivariable logistic regression models were used to identify the factors associated with the neurologists’ perspective toward intravenous thrombolysis. Results Among the responding neurologists, 67.3% reported using intravenous thrombolysis and 32.9% believed the treatment was unsafe. Approximately 51.4% reported deficits in their knowledge of intravenous thrombolysis and 45.8% felt unconfident about their ability to employ the treatment. The majority (90.1%) supported hospitals in performing intravenous thrombolysis for eligible patients. Their safety concern was associated with hospital grade (odds ratio[OR] = 2.3; 95% confidence interval [CI], 1.4–3.7) and previous experiences with thrombolysis (OR = 3.1; 95% CI, 2.1–4.6). Their confidence was associated with their educational background (OR = 2.5; 95% CI, 1.3–4.5), knowledge mastery (OR = 10.4; 95% CI, 6.6–16.3), and previous experiences with thrombolysis (OR = 3.3; 95% CI, 2.1–5.3). Their attitudes were associated with gender (OR = 0.6; 95% CI, 0.3–1.0) and previous experiences with thrombolysis (OR = 4.9; 95% CI, 2.5–9.4). Conclusions Most neurologists in Hubei Province, China, identified with intravenous thrombolysis for the treatment of acute ischemic stroke. However, they were weak in knowledge and lack confidence. Therefore, training, especially practical training, is needed to promote the use of thrombolysis in the region.

The healthspan of mice is enhanced by killing senescent cells using a transgenic suicide gene. Achieving the same using small molecules would have a tremendous impact on quality of life and the burden of age-related chronic diseases. Here, we describe the rationale for identification and validation of a new class of drugs termed senolytics, which selectively kill senescent cells. By transcript analysis, we discovered increased expression of pro-survival networks in senescent cells, consistent with their established resistance to apoptosis. Using siRNA to silence expression of key nodes of this network, including ephrins (EFNB1 or 3), PI3Kδ, p21, BCL-xL, or plasminogen-activated inhibitor-2, killed senescent cells, but not proliferating or quiescent, differentiated cells. Drugs targeting these same factors selectively killed senescent cells. Dasatinib eliminated senescent human fat cell progenitors, while quercetin was more effective against senescent human endothelial cells and mouse BM-MSCs. The combination of dasatinib and quercetin was effective in eliminating senescent MEFs. In vivo, this combination reduced senescent cell burden in chronologically aged, radiation-exposed, and progeroid Ercc1(-/Δ) mice. In old mice, cardiac function and carotid vascular reactivity were improved 5 days after a single dose. Following irradiation of one limb in mice, a single dose led to improved exercise capacity for at least 7 months following drug treatment. Periodic drug administration extended healthspan in Ercc1(-/∆) mice, delaying age-related symptoms and pathology, osteoporosis, and loss of intervertebral disk proteoglycans. These results demonstrate the feasibility of selectively ablating senescent cells and the efficacy of senolytics for alleviating symptoms of frailty and extending healthspan.

To describe the epidemiological characteristics of patients with Creutzfeldt-Jakob disease (CJD) in Shanghai from 2006 to 2012.Clinical and epidemiological information on CJD patients from Shanghai CJD Surveillance Network was analyzed. Cerebral spinal fluid (CSF)and blood specimens from patients were collected and used for detecting the 14-3-3 protein, and polymorphism of 129 amino acid and mutation of PRNP genes. Data was processed by EpiData(V3.0)and analyzed by SPSS(V17.0).In totally, one definite CJD patient together with 56 probable and 17 possible sporadic CJD patients were identified. One E200K genetic CJD case was diagnosed and another one was clinically diagnosed. No period- or geographic-related events were observed for these cases, but the houses of the two genetic CJD cases were close to each other. The mean age of onset of the probable CJD patients was 62 years old which was significantly older than that of those possible CJD patients.Most of the CJD patients identified in Shanghai were sporadic and the number was stable from 2006 to 2012. The mean age of onset of those probable CJD patients was older than that of the possible CJD patients.

The aim of this study was to systemically analyze the epidemiologic, serological and genetic characteristics of hepatitis E virus (HEV) in Shanghai from 1997 to 2012.We analyzed the data related to the epidemics of hepatitis E from China Information System for Disease Control and Prevention. We implemented serological surveillance program, based on community healthy population with enzyme-linked immunosorbent assay method and estimated the standardized sero-prevalence. We also obtained nucleotide sequences of hepatitis E patients using the nested RT-PCT assays, together with prototype sequences in the GenBank to construct a HEV genetic database in Shanghai.In this paper, we found that the distribution of hepatitis E patients was sporadic in the past 16 years in Shanghai. The morbidity kept declining, but with seasonal and periodical fluctuation. Morbidity in males was significantly higher than in females, with the hard hit population between 30 and 65 year-olds. In total, 3979 sera samples were collected through the serological surveillance programs in 2001, 2004, 2007 and 2012. The standardized sero-prevalence rates of the said years were 22.32%, 18.56%, 10.22% and 34.43% which all showing strong relationship with age groups and the regions where the populations were being monitored. 73 nucleotide sequences of hepatitis E patients from hospitals were identified, during 2004 and 2008.from phylogenetic analysis revealed that all HEV isolates belonged to genotype IV and including 4 known subtypes 4a, 4d, 4h and 4i which sharing 83.09% - 97.96%, 85.87% - 97.26% and 83.80% - 95.10% nucleotide sequence identities with the swine HEV genotype IV of GU188851, DQ450072 and EF570133. Meanwhile, 59 HEV isolates from different districts shared 99% nucleotide sequence identities with each other.Hepatitis E would still be a challenge for long time and the zoonotic questions that related to hepatitis E, need to be explored and explained in the future.

Hepatic nuclear factor 4α(HNF4α) modulates the transcriptional activation of numerous metabolic genes in liver. In this study, gene-array analysis revealed that HNF4αoverexpression increased peroxisome proliferator-activated receptorγ(PPARγ) greatly in cultured rat primary hepatocytes. PPAR-response-element-driven reporter gene expression could be elevated by HNF4α. Bioinformatics analysis revealed a high-affinity HNF4αbinding site in the human PPARγ2 promoter andin vitroexperiments showed that this promoter could be transactivated by HNF4α. The presence of HNF4αon the promoter was then confirmed by ChIP assay.In vivo, hepatic overexpression of HNF4αdecreased cholesterol levels both in plasma and liver and several hepatic genes related to cholesterol metabolism, including scavenger receptor BI (SR-BI), were upregulated. The upregulation of SR-BI by HNF4αcould be inhibited by a PPARγantagonistin vitro. In conclusion, HNF4αregulates cholesterol metabolism in rat by modulating the expression of SR-BI in the liver, in which the upregulation of PPARγwas involved.

To understand the epidemiological characteristics of Japanese Encephalitis in Shanghai and to provide evidence for preventing JE.Epidemic characteristics, JEV antibody in healthy population and swine infection rate in Shanghai were analyzed by methods of field survey, serology and molecular biology.JE incident rate in Shanghai was 0.077/100,000 in 2006; and 0.129/100,000 in 2007. Antibody positive rate before JE epidemic fastigium was 60.39%; postive rate after epidemic was 85.44%. JE IgG positive rate was 26.92% in 3-month swine and 14.86% in swine for sale; JE Gene in mosquito was analyzed for type 1.The JE prevalence rate is relatively low in Shanghai. JE antibody positive rate is high in Shanghai population. Swine as a media is infected by JE virus. The JE virus in mosquitoes belongs to genotype 1.

Purpose: We have previously done large-scale cDNA transfection screening on human hepatocellular carcinoma (HCC) cells and have identified 3,806 cDNA genes that possess the ability of either stimulating or inhibiting cell growth. In this study, we characterized one of these growth suppressor genes, carboxypeptidase of glutamate like-B (CPGL-B), in HCC. Experimental Design: Semiquantitative reverse-transcription PCR was used to examine the expression levels of CPGL-B. The cellular localization and functions of CPGL-B were investigated by enforced expression of CPGL-B in HCC cells. Results: From our previous cDNA transfection screening, we identified a gene named CPGL and its isoform, CPGL-B. With computational analysis, CPGL was located at chromosome 18q22.3 and was a homologue of peptidase family M20. CPGL was expressed in all adult and fetal tissues, whereas its isoform, CPGL-B, lacking exons 3 and 4, was expressed in all fetal tissues but only in liver and placenta of adult tissues. In HCC, CPGL-B was frequently underexpressed (35 of 90, 38.9%) in tumorous tissues compared with the corresponding nontumorous livers. Intriguingly, the underexpression was significantly associated with the presence of venous invasion (P = 0.018) and tumor microsatellite formation (P = 0.004). Stable transfection of CPGL-B in SMMC7721 HCC cells showed significant inhibition in cell viability, colony formation, cell invasion, and tumor formation in nude mice. CPGL-B also down-regulated CXCR3, matrix metalloproteinase 11, and CD44s, which are involved in cell growth and cell migration. Conclusions: These findings suggest that the frequent underexpression of CPGL-B may be associated with cell growth and metastasis of HCC.

To study the risk factors of acute sporadic hepatitis E virus (HEV) cases and to analyze its partial sequence in some districts of Shanghai.30 blood samples were collected from the acute sporadic HEV cases in 2003-2004 and the RT-nPCR method was applied to obtain the sequence of HEV in these cases. Meanwhile, a 1:2 case-control study was used to identify risk factors in the process of sporadic HEV infection in these regions of Shanghai.Data from the sequential analysis showed that HEV of the sporadic cases belonged to HEV genotype IV. Finding from the case-control study implicated that the housing condition, outside eating history, especially seafoods (OR = 7.048) played an important role in the infection of HEV. Results from multiple logistic regression showed that eating raw seafoods appeared to be one of the risk factors of HEV infection.HEV sequences isolated from the sporadic cases of HEV in some districts of Shanghai belonged to HEV genotype IV. Foods, especially seafood, were the risk factors in the infection of HEV.

Background MicroRNAs (miRNAs), a ubiquitous class of short RNAs, play vital roles in physiological and biochemical processes in plants by mediating gene silencing at post-transcriptional (PTGS) level. Tomato is a model system to study molecular basis of fleshy fruit ripening and senescence, ethylene biosynthesis and signal transduction owing to its genetic and molecular tractability. To study the functions of miRNAs in tomato fruit ripening and senescence, and their possible roles in ethylene response, the next generation sequencing method was employed to identify miRNAs in tomato fruit. Bioinformatics and molecular biology approaches were combined to profile the miRNAs expression patterns at three different fruit ripening stages and by exogenous ethylene treatment. Results In addition to 7 novel miRNA families, 103 conserved miRNAs belonging to 24 families and 10 non-conserved miRNAs matching 9 families were identified in our libraries. The targets of many these miRNAs were predicted to be transcriptional factors. Other targets are known to play roles in the regulation of metabolic processes. Interestingly, some targets were predicted to be involved in fruit ripening and softening, such as Pectate Lyase, beta-galactosidase, while a few others were predicted to be involved in ethylene biosynthesis and signaling pathway, such as ACS, EIN2 and CTR1. The expression patterns of a number of such miRNAs at three ripening stages were confirmed by stem-loop RT-PCR, which showed a strong negative correlation with that of their targets. The regulation of exogenous ethylene on miRNAs expression profiles were analyzed simultaneously, and 3 down-regulated, 5 up-regulated miRNAs were found in this study. Conclusions A combination of high throughput sequencing and molecular biology approaches was used to explore the involvement of miRNAs during fruit ripening. Several miRNAs showed differential expression profiles during fruit ripening, and a number of miRNAs were influenced by ethylene treatment. The results suggest the importance of miRNAs in fruit ripening and ethylene response.

Background Increasing evidence indicates an important role of transcription factor Yin Yang-1 (YY1) in human tumorigenesis. However, its function in cancer remains controversial and the relevance of YY1 to pancreatic ductal adenocarcinoma (PDAC) remains to be clarified. Methods In this study, we detected YY1 expression in clinical PDAC tissue samples and cell lines using quantitative RT-PCR, immunohistochemistry and western blotting. We also detected MUC4 and MMP10 mRNA levels in 108 PDAC samples using qRT-PCR and analyzed the correlations between YY1 and MUC4 or MMP10 expression. The role of YY1 in the proliferation, invasion and metastatic abilities of PDAC cells in vitro was studied by CCK-8 assay, cell migration and invasion assays. In vivo pancreatic tumor growth and metastasis was studied by a xenogenous subcutaneously implant model and a tail vein metastasis model. The potential mechanisms underlying YY1 mediated tumor progression in PDAC were explored by digital gene expression (DGE) sequencing, signal transduction pathways blockage experiments and luciferase assays. Statistical analysis was performed using the SPSS 15.0 software. Results We found that the expression of YY1 in PDACs was higher compared with their adjacent non-tumorous tissues and normal pancreas tissues. However, PDAC patients with high level overexpression of YY1 had better outcome than those with low level overexpression. YY1 expression levels were statistically negatively correlated with MMP10 expression levels, but not correlated with MUC4 expression levels. YY1 overexpression suppressed, whereas YY1 knockdown enhanced, the proliferation, invasion and metastatic properties of BXPC-3 cells, both in vitro and in vivo. YY1 suppresses invasion and metastasis of pancreatic cancer cells by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism. Conclusions The present study suggested that YY1 plays a negative role, i.e. is a tumor suppressor, in PDAC, and may become a valuable diagnostic and prognostic marker of PDAC.