VEGF-A promotes angiogenesis after acute myocardial infarction through increasing ROS production and enhancing ER stress-mediated autophagy

Proangiogenesis is generally regarded as an effective approach for treating ischemic heart disease. Vascular endothelial growth factor (VEGF)-A is a strong and essential proangiogenic factor. Reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and autophagy are implicated in the process of angiogenesis. This study is designed to clarify the regulatory mechanisms underlying VEGF-A, ROS, ER stress, autophagy, and angiogenesis in acute myocardial infarction (AMI). A mouse model of AMI was successfully established by occluding the left anterior descending coronary artery. Compared with the sham-operated mice, the microvessel density, VEGF-A content, ROS production, expression of vascular endothelial cadherin, positive expression of 78 kDa glucose-regulated protein/binding immunoglobulin protein (GRP78/Bip), and LC3 puncta in CD31-positive endothelial cells of the ischemic myocardium were overtly elevated. Moreover, VEGF-A exposure predominantly increased the expression of beclin-1, autophagy-related gene (ATG) 4, ATG5, inositol-requiring enzyme-1 (IRE-1), GRP78/Bip, and LC3-II/LC3-I as well as ROS production in the human umbilical vein endothelial cells (HUVECs) in a dose and time-dependent manner. Both beclin-1 small interfering RNA and 3-methyladenine treatment predominantly mitigated VEGF-A-induced tube formation and migration of HUVECs, but they failed to elicit any notable effect on VEGF-A-increased expression of GRP78/Bip. Tauroursodeoxycholic acid not only obviously abolished VEGF-A-induced increase of IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I, but also negated VEGF-A-induced tube formation and migration of HUVECs. Furthermore, N-acetyl- l -cysteine markedly abrogated VEGF-A-increased ROS production, IRE-1, GRP78/Bip, beclin-1 expression, and LC3-II/LC3-I in the HUVECs. Taken together, our data demonstrated that increased spontaneous production of VEGF-A may induce angiogenesis after AMI through initiating ROS–ER stress-autophagy axis in the vascular endothelial cells.     

CERNA2: A predictor for clinical progression and poor prognosis in cervical carcinoma

Competing long noncoding RNA 2 (lncRNA 2) for microRNA let-7b (CERNA2) has emerged as an important regulator of tumorigenesis and cancer progression but the clinical value and regulatory function of CERNA2 is yet to be investigated in cervical carcinoma. In our study, we found the CERNA2 expression was obviously increased in cervical carcinoma tissues compared with adjacent normal cervical tissues. In addition, we observed that metastatic lymph nodes exhibited high levels of CERNA2 expression in contrast to primary cervical carcinoma tissues. Furthermore, high CERNA2 expression was associated with advanced clinical stage, lymph node metastasis, distant metastasis poor histological grade, and short overall survival in cervical carcinoma patients. Moreover, high CERNA2 expression acted as an independent unfavorable predictor for overall survival in cervical carcinoma patients. The cell migration and invasion assays in vitro suggested that knockdown of CERNA2 remarkably inhibited cell migration and invasion in cervical carcinoma. In conclusion, CERNA2 functions as an oncogenic lncRNA and may be as a potential therapeutic target in cervical carcinoma.     

An automated process to extract plasmid DNA by alkaline lysis

With advances in the development of DNA vaccines and gene therapy, there is a growing need for plasmid DNA with high quality for fundamental research and clinical trials. In this report, a scalable automated process for large-scale preparation of plasmid is described. This process is based on alkaline lysis and can be easily scaled up to meet demands for larger quantities. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to affect lysis and control alkalinity. The resulting solution is passed through a series of filters to remove contaminants, and ethanol precipitated. System parameters are examined to maximize the quantity and quality of the prepared plasmid. Using this procedure, plasmid can be extracted and purified from 1 l of Escherichia coli cultures at an OD600 nm of 50 in less than 45 min. The plasmid yields are approximately 90 mg/l culture.     

小麦穗发芽基因等位变异及其区域分布研究

为了解中国不同麦区小麦种质资源抗穗发芽基因的等位变异与分布特征,利用小麦抗穗发芽相关的Vp1B3和Dorm-1标记对7个不同麦区的446份小麦种质资源的等位变异和分布差异进行了检测。结果表明:(1)利用标记Vp1B3检测出Vp1的等位基因共有3种类型,分别为Vp1Bc(与抗穗发芽相关)、Vp1Ba(与感穗发芽相关)、Vp1Bb(与抗穗发芽相关),其频率分别为62.8%、32.9%和4.3%。(2)标记Dorm-1在供试材料中共检测出2种特异性条带,分别为468bp(DormB1a,与感穗发芽相关)和606bp(DormB1b,与抗穗发芽相关),所占比例分别为98.6%和1.4%。(3)具有DormB1b基因型的种质主要分布在黄淮冬麦区和长江中下游冬麦区,比例分别为5.1%和1.9%;具有(Vp1Bc+Vp1Bb)基因型(组合)的种质在长江中下游冬麦区和西南冬麦区分布最多,比例分别为75.0%和74.1%。(4)通过标记Vp1B3和Dorm-1的综合鉴定,共筛选出同时携带Vp1与Dorm-B1位点种质6份,分别是‘陕麦611’、‘郑农19’、‘豫49-198’、‘信阳0913’、‘咸阳大穗’与‘徐州8066’,可以作为抗穗发芽育种的参考亲本。     

异源表达古菌TRAM基因增强水稻的耐旱性

干旱会直接影响水稻的生长发育,导致其产量和品质下降。在水稻中异源表达细菌RNA分子伴侣Csp能够显著提高水稻的耐旱能力,并且不影响水稻的正常生长。古菌中也发现具有类似细菌分子伴侣Csp功能的TRAM (TRM2 and MiaB)蛋白,且古菌的DNA复制、转录和翻译等过程与真核生物有着更为相似的调控方式,然而,古菌中RNA分子伴侣蛋白能否调控植物耐旱能力还未见报道。我们选取了嗜冷甲烷古菌Methanolobus psychrophilus R15中两个TRAM蛋白在水稻中进行研究,发现在水稻中过量表达Mpsy_3066和Mpsy_0643两个TRAM蛋白均能显著提高水稻苗期和成株期时对干旱胁迫的耐受能力。同时,我们在水稻原生质体中验证了TRAM蛋白可以发挥其分子伴侣的功能消除RNA的错误折叠对翻译的影响,这可能是TRAM转基因植物发挥其耐旱能力的作用机制。该工作初步展示了异源表达古菌TRAMs可以作为提高水稻耐旱能力的一种有效手段。     

Direct and tunable modulation of protein levels in rice and wheat with a synthetic small molecule

Direct control of protein level enables rapid and efficient analyses of gene functions in crops. Previously, we developed the RDDK‐Shield1 (Shld1) system in the model plant Arabidopsis thaliana for direct modulation of protein stabilization using a synthetic small molecule. However, it was unclear whether this system is applicable to economically important crops. In this study, we show that the RDDK‐Shld1 system enables rapid and tunable control of protein levels in rice and wheat. Accumulation of RDDK fusion proteins can be reversibly and spatio‐temporally controlled by the synthetic small‐molecule Shld1. Moreover, RDDK‐Bar and RDDK‐Pid3 fusions confer herbicide and rice blast resistance, respectively, in a Shld1‐dependent manner. Therefore, the RDDK‐Shld1 system provides a reversible and tunable technique for controlling protein functions and conditional expression of transgenes in crops.     

SALL4 as an Epithelial-Mesenchymal Transition and Drug Resistance Inducer through the Regulation of c-Myc in Endometrial Cancer

SALL4 plays important roles in the development and progression of many cancers. However, the role and molecular mechanism of SALL4 in endometrial cancer remain elusive. In the present research, we have demonstrated that the expression of SALL4 was upregulated in endometrial cancer and correlated positively with tumor stage, metastases and poor survival of patients. The overexpression of SALL4 promoted the invasiveness in endometrial cancer cells, as indicated by the upregulation of mesenchymal cell marker N-cadherin and downregulation of the epithelial marker E-cadherin, and invasion assays in vitro. Additionally, there was also an increase in drug resistance in these cell models due to the upregulation of ATP-binding cassette multidrug transporter ABCB1 expression. Moreover, we also found that ABCB1 was critical for SALL4-induced drug resistance. In contrast, SALL4 knockdown restored drug sensitivity, reversed EMT, diminished cell metastasis and suppressed the downregulation of E-cadherin and the upregulation of N-cadherin and ABCB1. Furthermore, we showed that SALL4 upregulated c-Myc expression and c-Myc was a direct target for SALL4 by ChIP assay, depletion of c-Myc with siRNA abolished the SALL4-induced downregulation of E-cadherin, upregulation of N-cadherin and ABCB1, suggesting that c-Myc was a downstream target for SALL4 and required for SALL4-induced EMT, invasion and drugs resistance in endometrial cancer cells. These results indicated that SALL4 could induce EMT and resistance to antineoplastic drugs through the regulation of c-Myc. SALL4 and c-Myc may be novel therapeutic targets for endometrial cancer.     

Unit Operation Optimization for the Manufacturing of Botanical Injections Using a Design Space Approach: A Case Study of Water Precipitation

Quality by design (QbD) concept is a paradigm for the improvement of botanical injection quality control. In this work, water precipitation process for the manufacturing of Xueshuantong injection, a botanical injection made from Notoginseng Radix et Rhizoma, was optimized using a design space approach as a sample. Saponin recovery and total saponin purity (TSP) in supernatant were identified as the critical quality attributes (CQAs) of water precipitation using a risk assessment for all the processes of Xueshuantong injection. An Ishikawa diagram and experiments of fractional factorial design were applied to determine critical process parameters (CPPs). Dry matter content of concentrated extract (DMCC), amount of water added (AWA), and stirring speed (SS) were identified as CPPs. Box-Behnken designed experiments were carried out to develop models between CPPs and process CQAs. Determination coefficients were higher than 0.86 for all the models. High TSP in supernatant can be obtained when DMCC is low and SS is high. Saponin recoveries decreased as DMCC increased. Incomplete collection of supernatant was the main reason for the loss of saponins. Design space was calculated using a Monte-Carlo simulation method with acceptable probability of 0.90. Recommended normal operation region are located in DMCC of 0.38–0.41 g/g, AWA of 3.7–4.9 g/g, and SS of 280–350 rpm, with a probability more than 0.919 to attain CQA criteria. Verification experiment results showed that operating DMCC, SS, and AWA within design space can attain CQA criteria with high probability.