An In Vitro Anticancer,Antioxidant, and Phytochemical Study on Water Extract of Kalanchoe daigremontiana Raym.-Hamet and H. Perrier

Kalanchoe species are succulents with anti-inflammatory, antioxidant, and analgesic properties, as well as cytotoxic activity. One of the most popular species cultivated in Europe is Kalanchoe daigremontiana Raym.-Hamet and H. Perrier. In our study, we analyzed the phytochemical composition of K. daigremontiana water extract using UHPLC-QTOF-MS and estimated the cytotoxic activity of the extract on human ovarian cancer SKOV-3 cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, flow cytometry, luminometric, and fluorescent microscopy techniques. The expression levels of 92 genes associated with cell death were estimated via real-time PCR. The antioxidant activity was assessed via flow cytometry on human keratinocyte HaCaT cell line. The DPPH (2,2-diphenyl-1-picrylhydrazyl) radical and FRAP (ferric-reducing antioxidant power) assays were also applied. We identified twenty bufadienolide compounds in the water extract and quantified eleven. Bersaldegenin-1,3,5-orthoacetate and bryophyllin A were present in the highest amounts (757.4 ± 18.7 and 573.5 ± 27.2 ng/mg dry weight, respectively). The extract showed significant antiproliferative and cytotoxic activity, induced depolarization of the mitochondrial membrane, and significantly arrested cell cycle in the S and G2/M phases of SKOV-3 cells. Caspases-3, 7, 8, and 9 were not activated during the treatment, which indicated non-apoptotic cell death triggered by the extract. Additionally, the extract increased the level of oxidative stress in the cancer cell line. In keratinocytes treated with menadione, the extract moderately reduced the level of oxidative stress. This antioxidant activity was confirmed by the DPPH and FRAP assays, where the obtained IC₅₀ values were 1750 ± 140 and 1271.82 ± 53.25 μg/mL, respectively. The real-time PCR analysis revealed that the extract may induce cell death via TNF receptor (tumor necrosis factor receptor) superfamily members 6 and 10.     

Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening

Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC–OCT4–EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC–AAVS1–EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC–AAVS1–EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes.     

Effect of Tomato Peel Extract Grown under Drought Stress Condition in a Sarcopenia Model

Tomatoes and their derivates represent an important source of natural biologically active components. The present study aims to investigate the protective effect of tomato peel extracts, grown in normal (RED-Ctr) or in drought stress (RED-Ds) conditions, on an experimental model of sarcopenia. The phenolic profile and total polyphenols content (TPC) of RED-Ctr and RED-Ds were determined by Ultra High-Performance Liquid Chromatography (UHPLC) analyses coupled to electrospray ionization high-resolution mass spectrometry (ESI-HR-MS). Human skeletal muscle myoblasts (HSMM) were differentiated in myotubes, and sarcopenia was induced by dexamethasone (DEXA) treatment. Differentiation and sarcopenia were evaluated by both real-time PCR and immunofluorescent techniques. Data show that myosin heavy chain 2 (MYH2), troponin T (TNNT1), and miogenin (MYOG) were expressed in differentiated myotubes. 5 μg Gallic Acid Equivalent (GAE/mL) of TPC from RED-Ds extract significantly reduced muscle atrophy induced by DEXA. Moreover, Forkhead BoxO1 (FOXO1) expression, involved in cell atrophy, was significantly decreased by RED-Ds extract. The protective effect of tomato peel extracts depended on their qualitative polyphenolic composition, resulting effectively in the in vitro model of sarcopenia.     

人顶体酶三维结构的同源模建及其与KF950的分子对接研究

采用同源模建方法首次构建了人顶体酶的三维结构模型, 模型的可靠性经Ramachandran图和Profile_3D图验证. 采用InsightII/Binding site方法准确定位了人顶体酶的活性位点, 并研究了顶体酶重要功能残基在活性位点的立体分布. 在此基础上, 通过柔性分子对接方法首次阐明了顶体酶高效抑制剂KF950与靶酶活性位点的相互作用模式, 发现特异性的氢键相互作用是KF950产生高抑制活性的重要分子基础. 其研究结果将为合理设计新型顶体酶抑制剂, 寻找男性口服避孕药奠定坚实基础.     

变温下荧光猝灭和加强理论公式合理性的比较

通过荧光和紫外方法研究盐酸左氧氟沙星、氟罗沙星、加替沙星和沃氟沙星四种喹诺酮类药物与人血清白蛋白(HSA)的作用, 在不同温度下对荧光加强和猝灭理论公式的等价性和精细差异进行了比较, 结果表明: 在不同温度下两种理论公式尽管在求取解离常数上所得结果都可视为有效, 但猝灭方程的双倒数图反而不及荧光加强方程的双倒数图与体现动态、静态猝灭机理的猝灭曲线一致. 对于能量转移效率E, 我们首次定义: 按自由荧光体白蛋白的荧光强度F₀和加入药物至荧光不再变化时的荧光强度F值计算F/F₀, 进而将F/F₀代入E＝１－F/F₀计算能量转移效率E. 它标示出每个体系发生能量转移的最大限度, 向体系中再添加药物, 已不能发生进一步的猝灭, 这一新定义的引入, 使得能量转移效率E这一量值有可能推广到估算白蛋白运载药物能力的最大限度.     

稀土金属离子与人血清白蛋白的相互作用

本文用荧光光谱、紫外-可见吸收光谱法和循环伏安法研究了稀土金属离子Eu(Ⅲ)、Pr(Ⅲ)与人血清白蛋白(HSA)的相互作用。实验发现：Eu(Ⅲ)和Pr(Ⅲ)对HSA有较强的荧光猝灭作用。用Stern-Volmer方程分别对实验数据进行处理，结果发现：HSA与Eu(Ⅲ)、Pr(Ⅲ)发生反应生成了新的复合物，发生了分子内的非辐射能量转移。Eu(Ⅲ)、Pr(Ⅲ)对HAS的荧光猝灭作用，属于静态荧光猝灭。荧光猝灭图表明：Eu³⁺和Pr³⁺在HSA分子中至少有两类结合位点，Eu³⁺与HSA形成2.76∶1的复合物，结合常数lgK分别为12.03和9.05；Pr³⁺与HSA形成2.2∶1的复合物，结合常数lgK分别为9.89和6.97。同时用圆二色谱及同步荧光光谱法探讨了 Eu(Ⅲ)和Pr(Ⅲ)对HSA构象的影响。     

石英芯片电泳分离检测尿中蛋白的研究

通过对缓冲体系、缓冲液浓度、酸度、乳酸钙浓度、乙胺浓度、电泳电压和进样时间的优化选择，用石英芯片电泳一紫外检测法分离了纯人白蛋白和人运铁蛋白；在75mmol／L硼酸盐（pH10．55）（含0．8mmol／L乳酸钙、1％（φ）乙胺）运行缓冲液中，上述两组分在3min内完全分离；纯人白蛋白和人运铁蛋白的线性范围分别为1．0～15．0g/L和1．0～10．0g／L；检出限（S／N=3）均为0．5g／L，应用于临床尿蛋白分离测定，并与Helena琼脂糖凝胶电泳仪电泳结果进行比较，获得一致结果。     

镉的人体健康效应

综述了镉暴露对人体健康的危害、人体对镉的需要量和居民膳食日摄入量、镉暴露生物标记物及危险性评估。过量镉暴露可引起肾、肺、肝、骨和生殖效应以及癌症。在中国和其他国家进行的最新研究表明，生活在镉污染区的一般人群可发生以骨矿密度降低和骨折发生率增加为特征的骨效应；出现肾小管功能异常的镉累积量比骨效应剂量更低，也比从前估计的临界剂量更低。附4图11表25篇参考文献。     

Simultaneous quantitation of rosuvastatin and gemfibrozil in human plasma by high-performance liquid chromatography and its application to a pharmacokinetic study

A simple, sensitive and specific high-performance liquid chromatography method is described for simultaneous determination of rosuvastatin (RST) and gemfibrozil (GFZ) in human plasma using celecoxib as an internal standard (IS). The assay procedure involved extraction of RST, GFZ and IS from plasma into acetonitrile. Following separation and evaporation of the organic layer the residue was reconstituted in the mobile phase and injected onto an X-Terra C(18) column (4.6 x 150 mm, 5.0 microm). The chromatographic run time was less than 20 min using flow gradient (0.0-1.60 mL/min) with a mobile phase consisting of 0.01 M ammonium acetate:acetonitrile:methanol (50:40:10, v/v/v) and UV detection at 275 nm. Nominal retention times of RST, GFZ and IS were 6.7, 13.9 and 16.4 min, respectively. Absolute recovery of both analytes and IS was greater than 90%. The lower limit of quantification (LLOQ) of RST and GFZ was 0.03 and 0.30 microg/mL, respectively. Linearity was excellent (r(2) = 0.999) in the 0.03-10 microg/mL and 0.3-100 microg/mL ranges for RST and GFZ, respectively. The inter- and intra-day precisions in the measurement of RST quality control (QC) samples 0.03, 0.09, 2.50 and 8.00 microg/mL were in the range 2.37-9.78% relative standard deviation (RSD) and 0.92-10.08% RSD, respectively. Similarly, the inter- and intra-day precisions in the measurement of GFZ quality control (QC) samples 0.30, 0.90, 25.0 and 80.0 microg/mL were in the ranges 2.79-6.27 and 0.96-9.69% RSD, respectively. Accuracies in the measurement of QC samples for RST and GFZ were in the range 85.43-107.23 and 84.98-102.35% respectively, of the nominal values. RST and GFZ were stable in the array of stability studies viz., bench-top, auto-sampler and freeze-thaw cycles. Stability of RST and GFZ was established for 1 month at -80C. The application of the assay in an oral pharmacokinetic study in rats co-administered with RST and GFZ is described.     

CHROMATOGRAPHIC SEPERATION AND PURIFICATION OF HUMAN CHORIONIC GONADOTROPIN

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