Glycogen synthase kinase-3beta regulates growth, calcium homeostasis, and diastolic function in the heart

Glycogen synthase kinase (GSK) 3beta is a negative regulator of stress-induced cardiomyocyte hypertrophy. It is not clear, however, if GSK-3beta plays any role in regulating normal cardiac growth and cardiac function. Herein we report that a transgenic mouse expressing wild type GSK-3beta in the heart has a dramatic impairment of normal post-natal cardiomyocyte growth as well as markedly abnormal cardiac contractile function. The most striking phenotype, however, is grossly impaired diastolic relaxation, which leads to increased filling pressures of the left ventricle and massive atrial enlargement. This is due to profoundly abnormal calcium handling, leading to an inability to normalize cytosolic [Ca2+] in diastole. The alterations in calcium handling are due at least in part to direct down-regulation of the sarcoplasmic reticulum calcium ATPase (SERCA2a) by GSK-3beta, acting at the level of the SERCA2 promoter. These studies identify GSK-3beta as a regulator of normal growth of the heart and are the first of which we are aware, to demonstrate regulation of expression of SERCA2a, a critical determinant of diastolic function, by a cytosolic signaling pathway, the activity of which is dynamically modulated. De-regulation of GSK-3beta leads to severe systolic and diastolic dysfunction and progressive heart failure. Because down-regulation of SERCA2a plays a central role in the diastolic and systolic dysfunction of patients with heart failure, these findings have potential implications for the therapy of this disorder.     

Chimeric contribution of human extended pluripotent stem cells to monkey embryos ex vivo

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Minimal sample requirement for highly multiplexed protein quantification in cell lines and tissues by PCT‐SWATH mass spectrometry

The amount of sample available for clinical and biological proteomic research is often limited and thus significantly restricts clinical and translational research. Recently, we have integrated pressure cycling technology (PCT) assisted sample preparation and SWATH‐MS to perform reproducible proteomic quantification of biopsy‐level tissue samples. Here, we further evaluated the minimal sample requirement of the PCT‐SWATH method using various types of samples, including cultured cells (HeLa, K562, and U251, 500 000 to 50 000 cells) and tissue samples (mouse liver, heart, brain, and human kidney, 3–0.2 mg). The data show that as few as 50 000 human cells and 0.2–0.5 mg of wet mouse and human tissues produced peptide samples sufficient for multiple SWATH‐MS analyses at optimal sample load applied to the system. Generally, the reproducibility of the method increased with decreasing tissue sample amounts. The SWATH maps acquired from peptides derived from samples of varying sizes were essentially identical based on the number, type, and quantity of identified peptides. In conclusion, we determined the minimal sample required for optimal PCT‐SWATH analyses, and found smaller sample size achieved higher quantitative accuracy.     

Focal adhesion kinase maintains,but not increases the adhesion of dental pulp cells

Focal adhesion kinase (FAK) functions as a key enzyme in the integrin-mediated adhesion-signalling pathway. Here, we aimed to investigate the effects of FAK on adhesion of human dental pulp (HDP) cells. We transfected lentiviral vectors to silence or overexpress FAK in HDP cells ex vivo. Early cell adhesion, cell survival and focal contacts (FCs)-related proteins (FAK and paxillin) were examined. By using immunofluorescence, the formation of FCs and cytoskeleton was detected, respectively. We found that both adhesion and survival of HDP cells were suppressed by FAK inhibition. However, FAK overexpression slightly inhibited cell adhesion and exhibited no change in cell survival compared with the control. A thick rim of cytoskeleton accumulated and smaller dot-shaped FCs appeared in FAK knockdown cells. Phosphorylation of paxillin (p-paxillin) was inhibited in FAK knockdown cells, verifying that the adhesion was inhibited. Less cytoskeleton and elongated FCs were observed in FAK-overexpressed cells. However, p-paxillin had no significant difference compared with the control. In conclusion, the data suggest that FAK maintains cell adhesion, survival and cytoskeleton formation, but excessive FAK has no positive effects on these aspects.     

广州地区急性散发性HEV毒株基因特征和生物学性状的研究

利用ＲＴ－ＰＣＲ技术对我国广州地区急性性戊型肝炎病毒细胞培养分离株Ｇ９３－１、Ｇ９３－２、Ｇ９３－３和Ｇ９３－４基因组聚合酶区部分核苷序列进行检测,ＰＣＲ阳性产物经纯化、克隆后测定。结果Ｇ９３－１、Ｇ９３－３和Ｇ９３－４株病毒的这段序列完全相同,且与我国新疆暴发流行的ＨＥＶ８７Ａ株及ＨＥＶ代表株的同源性为１００％;而Ｇ９３－２株与它们的差异较大,同源性只有７９．９％;但是,它与１９９７年厦门地区急     

Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71

The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen''s neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.     

绞股蓝种子休眠机理及其破除方法研究

以采自广西金秀县的绞股蓝种子为研究材料,对其休眠原因、休眠类型及其破眠方法进行了研究,为绞股蓝种子繁殖提供理论依据和技术支持。结果表明:(1)绞股蓝新采收成熟种子的生活力达91%,在10℃~35℃恒温和15℃/25℃变温中的发芽率均低于10%,新种子的生活力极显著大于发芽率,具有显著的休眠现象。(2)绞股蓝种皮不限制吸水,胚分化发育完全,离体胚发芽率为(78.0±4.8)%,且能够长成正常幼苗,说明绞股蓝种子的胚在离体条件下无休眠现象。(3)绞股蓝完整种子及其粉碎种子的水提液对白菜种子的萌发率、苗高及根长均有抑制作用,随水提液浓度增加抑制作用均显著增强,且粉碎种子的抑制作用较强;当粉碎种子的水提液浓度为5%时白菜种子萌发率、苗高、根长分别为18.0%、0.1cm、0.1cm,分别显著低于对照77.1%、97.3%、95.8%,说明绞股蓝种子的种皮和胚乳中存在水溶性萌发抑制物质,是绞股蓝种子休眠的主要原因。(4)GA3和6-BA不能促进绞股蓝种子萌发,低温层积对绞股蓝种子休眠的解除具有促进作用;绞股蓝种子的休眠属于生理休眠类型,休眠水平属于中间型。(5)低温干藏能够打破绞股蓝种子休眠,是绞股蓝种子破除休眠及种子保存较为理想的方式。