Assessing G4-Binding Ligands In Vitro and in Cellulo Using Dimeric Carbocyanine Dye Displacement Assay

G-quadruplexes (G4) are the most actively studied non-canonical secondary structures formed by contiguous repeats of guanines in DNA or RNA strands. Small molecule mediated targeting of G-quadruplexes has emerged as an attractive tool for visualization and stabilization of these structures inside the cell. Limited number of DNA and RNA G4-selective assays have been reported for primary ligand screening. A combination of fluorescence spectroscopy, AFM, CD, PAGE, and confocal microscopy have been used to assess a dimeric carbocyanine dye B6,5 for screening G4-binding ligands in vitro and in cellulo. The dye B6,5 interacts with physiologically relevant DNA and RNA G4 structures, resulting in fluorescence enhancement of the molecule as an in vitro readout for G4 selectivity. Interaction of the dye with G4 is accompanied by quadruplex stabilization that extends its use in primary screening of G4 specific ligands. The molecule is cell permeable and enables visualization of quadruplex dominated cellular regions of nucleoli using confocal microscopy. The dye is displaced by quarfloxin in live cells. The dye B6,5 shows remarkable duplex to quadruplex selectivity in vitro along with ligand-like stabilization of DNA G4 structures. Cell permeability and response to RNA G4 structures project the dye with interesting theranostic potential. Our results validate that B6,5 can serve the dual purpose of visualization of DNA and RNA G4 structures and screening of G4 specific ligands, and adds to the limited number of probes with such potential.     

Investigation and comparison of biological effects of regioselectively synthesized thiazole derivatives

In this study, anticancer, antibacterial (against hospital-isolated antibiotic-resistant Escherichia coli strains), antifungal, and antioxidant effects of synthesized heterocyclic compounds 5 and 7 containing thiazole core were examined. Cytotoxicity testing was utilized against MCF-7 breast cancer cells via MTT cell viability assay. Antibacterial and antifungal activities were checked out according to Clinical and Laboratory Standards Institute (CLSI) guidelines, and antioxidant properties were evaluated through scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. Results showed the viability of breast cancer cell lines was reliant on concentration of heterocycles and time of incubation. Synthetic compounds exhibited excellent antibacterial and antifungal properties base on their minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) values as well as high antioxidant activities according to their IC₅₀ values. Higher anticancer and antibacterial properties were observed with compound 7; on the contrary, thiazole 5 had better antioxidant effects. They can be introduced as potent antimicrobial, antioxidant, and anticancer agents.     

血液中苯丙氨酸的测定方法研究进展

高效、简便、快速测定血液中苯丙氨酸含量在医学上尤其是对新生儿的苯丙酮尿症的筛查有着重要的意义；本文就血液中苯丙氨酸的测定方法研究进展进行了综述，内容包括：细菌抑制法、荧光法、高效液相色谱法、气相色谱—质谱联用、质谱—质谱联用、毛细管电泳、酶／比色法以及酶电极法等。     

应用毛细管区带电泳测定人血清蛋白

摘要：研究了一种用于临床检测血清蛋白的毛细管区带电泳方法。弹性石英毛细管50μmi.d.×47cm（40cm有效长度）,检测波长200nm,血清用运行缓冲液（含12.5mmol/L四硼酸钠、1mmol/L乳酸钙、0.7mmol/L硫酸镁,pH9.70）稀释40倍,气压进样17.23kPa·s,分析电压23kV。正常血清蛋白分为6种,孕妇的分7种（多一个未知的α0峰）。将正常人、孕妇、多发性骨髓瘤和强直性脊柱炎患者的血清蛋白的毛细管电泳与传统的醋酸纤维膜电泳相比较,前者具有高分辨率、在线数据处理和自动化的特     

酶联免疫吸附分析法测定水样中的阿特拉津

应用阿特拉津半抗原衍生物共价交联于载体蛋白分子上，制成免疫抗原，经免疫得到高质量的兔抗阿特拉津抗血清。经条件优选，建立了测定阿特拉津的酶联吸附分析竞争法，测定性范围为０．０５－５．００μｇ／Ｌ，最低检出限仅为０．０１８－０．０２２μｇ／Ｌ，且精密度高，特异性强。     

偶氮喹啉-镍离子配合物应用于HSO-4识别研究

考察了5-(4'-氰基苯)偶氮-8-羟基喹啉(1)-镍配合物的光谱特性并将其应用于阴离子识别研究中.研究结果表明,1-Ni2+金属配合物可选择性识别不同的阴离子,并对HSO-4具有独特的光谱响应.化合物1在乙腈中为无色溶液,与Ni2+可形成稳定常数为9.2×10 6mol-1·L的1∶1型红色配合物,在此配合物中加入HSO-4后,红色的溶液逐渐褪色,而加入CH3CO-2,F-和H2PO-4却产生增色效应,Cl-和Br-加入后主体溶液无明显颜色和光谱变化,据此建立了选择性识别HSO-4新体系,并探讨其作用机理.     

稀土元素对人肝癌细胞SMMC-7721增殖的影响

用MTT法研究了14种稀土元素(La，Ce，Pr，Nd，Sm，Eu，Gd，Tb，Dy，Ho，Er，Tm，Yb和Lu)对人肝癌细胞SMMC-7721增殖的影响。他们对肝癌细胞的生长作用可分为3类。其中La^3 、Ce^3 和Eu^3 对肝癌细胞的增殖有剂量依赖性正效应，能够在一定浓度范围内刺激细胞生长；Sm^3 ，Gd^3 ，Ho^3 ，Er^3 ，Yb^3 对肝癌细胞生长的刺激作用没有剂量依赖性特征；而Pr^3 ，Nd^3 ，Tb^3 ，Dy^3 ，Tm^3 和Lu^3 则表现出对肝癌细胞的增殖具有不用程度的抑制。推测14种稀土元素作用方式的不同与他们的原子结构有一定的关系，它们对肝癌细胞的相对增殖率随着原子序数的增加呈现出一定的规律性。     

Quantitation of memantine hydrochloride bulk drug and its tablet formulation using proton nuclear magnetic resonance spectrometry

The use of quantitative nuclear magnetic resonance spectrometry for the determination of non‐UV active memantine hydrochloride with relative simplicity and precision has been demonstrated in this study. The method was developed on a 500 MHz NMR instrument and was applied to determination of the drug in a tablet formulation. The analysis was performed by taking caffeine as an internal standard and D₂O as the NMR solvent. The signal of methyl protons of memantine hydrochloride appeared at 0.75 ppm (singlet) relative to the signal of caffeine (internal standard) at 3.13 ppm (singlet). The method was found to be linear (r² = 0.9989) in the drug concentration range of 0.025 to 0.80 mg/ml. The maximum relative standard deviation for accuracy and precision was <2. The limits of detection and quantification were 0.04 and 0.11 mg/ml, respectively. The robustness of the method was revealed by changing nine different parameters. The deviation for each parameter was also within the acceptable limits. The study highlighted possibility of direct determination of memantine hydrochloride in pure form and in its marketed tablet formulation by the use of quantitative NMR, without the need of derivatization, as is the requirement in HPLC studies. Copyright © 2016 John Wiley & Sons, Ltd.     

Development of an on-line automated sample clean-up method and liquid chromatography–tandem mass spectrometry analysis: application in an in vitro proteolytic assay

Fluorescence detection has been a method of choice in industry for screening assays, including identification of enzyme inhibitors, owing to its high-throughput capabilities, excellent reproducibility, and sensitivity. Occasionally, inhibitors are identified that challenge the fluorescence assay limit, necessitating the development of more sensitive detection methods to assess these compounds. For data mining purposes, however, original assay conditions may be required. A direct method transfer to highly sensitive and specific LC-MS-based methods has not always been possible due to the presence of MS-incompatible neutral detergents and non-volatile salts in the assay matrix. Utilizing an in vitro proteolytic screening assay for the serine protease hepatitis C virus (HCV) nonstructural (NS) 3 protease as a test case, we report the development of an automated sample clean-up procedure implemented on-line with liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis to complement fluorescence detection. Ion exchange and peptide microtraps were employed to remove MS-incompatible assay matrix components. Three protease inhibitors were used to validate the MS/MS method. Comparable potencies were achieved for these compounds when assessed by fluorescence and MS/MS detection. Furthermore, four-fold less enzyme could be utilized when employing the MS/MS method compared to fluorescence detection. The longer analysis time, however, resulted in reduced sample capacity. The potency of our designed HCV NS3 protease inhibitors are thus routinely evaluated using a continuous fluorescence-based assay. Only pertinent inhibitors approaching the fluorescence assay sensitivity limit are subsequently analyzed further by LC-MS/MS. This methodology allows us to maintain a database and to compare results independent of the detection method. Despite the relatively slow sample turnaround time of this LC-MS approach, the versatility of the automated on-line clean-up procedure and sample analysis can be applied to assays containing reagents which were historically considered to be MS incompatible.     

一种基于纳米二氧化硅增强凝集反应的压电免疫传感器

本文提出了一种基于抗体包被纳米粒子的简单快速的压电免疫凝集法,用于蛋白质检测。该方法原理是利用羊抗人IgG（G-anti-hIgG）包被的二氧化硅（或金）纳米粒子和人IgG（hIgG）发生免疫凝集反应而使得压电晶体频率发生改变进行测定。当凝集反应发生时,修饰在探针表面的G-anti-hIgG通过hIgG与G-anti-hIgG包被的纳米粒子结合,将质量效应和粘弹性因素叠加作用于压电晶体。结果表明这使得背景值大幅减小而信号明显增强。另外,对修饰后了抗体及结合免疫复合物的探针表面进行了SEM表征,对使用聚乙二醇作为增敏剂和实验最佳离子强度、pH值进行了优化选择。该传感器检测hIgG线性范围是0.26-16.7 mg mL-1,最低检出限为84 ng mL-1。