反相高效液相色谱/电喷雾质谱法测定血浆中盐酸舍曲林

采用反相高效液相色谱-电喷雾质谱法(LC-ESI/MS)测定人体血浆中盐酸舍曲林。以盐酸丙咪嗪为内标,按内标法定量。血浆样品经pH 10.00碳酸钠溶液碱化,加入甲基叔丁基醚萃取,离心分离,取上清液吹干,用流动相溶解进样。色谱柱:RP-Extend-C18(150 mm×4.6 mm,5μm),柱温:40℃;流动相:pH 3.00的三氟乙酸溶液-甲醇(40∶60,V/V),流速0.6 mL/m in。质谱采用选择离子监控模式,检测离子的核质比(m/z)分别是281(丙咪嗪)和159(舍曲林)。舍曲林和丙咪嗪的保留时间分别是5.4 m in和3.8 m in;舍曲林标准曲线线性范围为1~80μg/L;检出限为1.0μg/L(S/N>10);日间、日内相对标准差均小于6.0%,相对回收率为90%~106%;提取回收率在75%~93%范围内。此法适合人体药代动力学的血浆样品的分析,结果准确、可靠。     

Design and Activity of Novel Oxadiazole Based Compounds That Target Poly(ADP-ribose) Polymerase

Novel PARP inhibitors with selective mode-of-action have been approved for clinical use. Herein, oxadiazole based ligands that are predicted to target PARP-1 have been synthesized and screened for the loss of cell viability in mammary carcinoma cells, wherein seven compounds were observed to possess significant IC₅₀ values in the range of 1.4 to 25 µM. Furthermore, compound 5u, inhibited the viability of MCF-7 cells with an IC₅₀ value of 1.4µM, when compared to Olaparib (IC₅₀ = 3.2 µM). Compound 5s also decreased cell viability in MCF-7 and MDA-MB-231 cells with IC₅₀ values of 15.3 and 19.2 µM, respectively. Treatment of MCF-7 cells with compounds 5u and 5s produced PARP cleavage, H2AX phosphorylation and CASPASE-3 activation comparable to that observed with Olaparib. Compounds 5u and 5s also decreased foci-formation and 3D Matrigel growth of MCF-7 cells equivalent to or greater than that observed with Olaparib. Finally, in silico analysis demonstrated binding of compound 5s towardsthe catalytic site of PARP-1, indicating that these novel oxadiazoles synthesized herein may serve as exemplars for the development of new therapeutics in cancer.     

Unravelling the Helianthus tuberosus L. (Jerusalem Artichoke,Kiku-Imo) Tuber Proteome by Label-Free Quantitative Proteomics

The present research investigates the tuber proteome of the ‘medicinal’ plant Jerusalem artichoke (abbreviated as JA) (Helianthus tuberosus L.) using a high-throughput proteomics technique. Although JA has been historically known to the Native Americans, it was introduced to Europe in the late 19th century and later spread to Japan (referred to as ‘kiku-imo’) as a folk remedy for diabetes. Genboku Takahashi research group has been working on the cultivation and utilization of kiku-imo tuber as a traditional/alternative medicine in daily life and researched on the lowering of blood sugar level, HbA1c, etc., in human subjects (unpublished data). Understanding the protein components of the tuber may shed light on its healing properties, especially related to diabetes. Using three commercially processed JA tuber products (dried powder and dried chips) we performed total protein extraction on the powdered samples using a label-free quantitate proteomic approach (mass spectrometry) and catalogued for the first time a comprehensive protein list for the JA tuber. A total of 2967 protein groups were identified, statistically analyzed, and further categorized into different protein classes using bioinformatics techniques. We discussed the association of these proteins to health and disease regulatory metabolism. Data are available via ProteomeXchange with identifier PXD030744.     

Are Words the Quanta of Human Language? Extending the Domain of Quantum Cognition

In previous research, we showed that ‘texts that tell a story’ exhibit a statistical structure that is not Maxwell–Boltzmann but Bose–Einstein. Our explanation is that this is due to the presence of ‘indistinguishability’ in human language as a result of the same words in different parts of the story being indistinguishable from one another, in much the same way that ’indistinguishability’ occurs in quantum mechanics, also there leading to the presence of Bose–Einstein rather than Maxwell–Boltzmann as a statistical structure. In the current article, we set out to provide an explanation for this Bose–Einstein statistics in human language. We show that it is the presence of ‘meaning’ in ‘texts that tell a story’ that gives rise to the lack of independence characteristic of Bose–Einstein, and provides conclusive evidence that ‘words can be considered the quanta of human language’, structurally similar to how ‘photons are the quanta of electromagnetic radiation’. Using several studies on entanglement from our Brussels research group, we also show, by introducing the von Neumann entropy for human language, that it is also the presence of ‘meaning’ in texts that makes the entropy of a total text smaller relative to the entropy of the words composing it. We explain how the new insights in this article fit in with the research domain called ‘quantum cognition’, where quantum probability models and quantum vector spaces are used in human cognition, and are also relevant to the use of quantum structures in information retrieval and natural language processing, and how they introduce ‘quantization’ and ‘Bose–Einstein statistics’ as relevant quantum effects there. Inspired by the conceptuality interpretation of quantum mechanics, and relying on the new insights, we put forward hypotheses about the nature of physical reality. In doing so, we note how this new type of decrease in entropy, and its explanation, may be important for the development of quantum thermodynamics. We likewise note how it can also give rise to an original explanatory picture of the nature of physical reality on the surface of planet Earth, in which human culture emerges as a reinforcing continuation of life.     

Micro- and Nanosecond Pulses Used in Doxorubicin Electrochemotherapy in Human Breast and Colon Cancer Cells with Drug Resistance

(1) Background: Pulsed electric field (PEF) techniques are commonly used to support the delivery of various molecules. A PEF seems a promising method for low permeability drugs or when cells demonstrate therapy resistance and the cell membrane becomes an impermeable barrier. (2) Methods: In this study, we have used doxorubicin-resistant and sensitive models of human breast cancer (MCF-7/DX, MCF-7/WT) and colon cancer cells (LoVo, LoVoDX). The study aimed to investigate the susceptibility of the cells to doxorubicin (DOX) and electric fields in the 20–900 ns pulse duration range. The viability assay was utilized to evaluate the PEF protocols’ efficacy. Cell confluency and reduced glutathione were measured after PEF protocols. (3) Results: The obtained results showed that PEFs significantly supported doxorubicin delivery and cytotoxicity after 48 and 72 h. The 60 kV/cm ultrashort pulses × 20 ns × 400 had the most significant cytotoxic anticancer effect. The increase in DOX concentration provokes a decrease in cell viability, affected cell confluency, and reduced GSSH when combined with the ESOPE (European Standard Operating Procedures of Electrochemotherapy) protocol. Additionally, reactive oxygen species after PEF and PEF-DOX were detected. (4) Conclusions: Ultrashort electric pulses with low DOX content or ESOPE with higher DOX content seem the most promising in colon and breast cancer treatment.     

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.