CO2 fixation by [WIVO(S2C2(CN)2)2]2?: functional model for the tungsten-formate dehydrogenase of Clostridium thermoaceticum

(NEt₄)₂[W^IVO(S₂C₂(CN)₂)₂] (1), isolated by reaction of Na₂ WO₄, Na₂S₂C₂(CN)₂ (Na₂mnt) in acidified (pH5.5) aqueous medium in the presence of excess of sodium dithionite and NEt₄Br, reduces CO₂/HCO ₃ ⁻ (pH 7.5) to yield HCOO⁻ and (NEt₄)₂[W^VIO₂(S₂C₂(CN)₂)₂] (2) mimicking tungsten-formate dehydrogenase (W-FDH) activity. (1) reacts with Na₂MoO₄ in acidic medium to produce [Mo^IvO(S₂C₂(CN)₂)₂]²⁻ implicating the displacement of tungsten by molybdenum from the cofactor complex in W-FDH.     

Development of a fast spectroscopic enzyme assay for on-site measurement of total polyphenol content in grapes and wine

In recent years interest in polyphenols as a nutrient in vegetables and fruits has increased because of polyphenols’ positive effects on human health. The interest focuses on the sensory properties of polyphenols and their influence on the taste of fruits and derived products. This article presents the development of a bioanalytical measurement technique enabling the determination of the total polyphenol content (TPC) of fresh grapes within a few minutes. Furthermore this technique allows the control of TPC during production processes, e. g. fermentation of wine.     

Label-free characterization of cell adhesion using reflectometric interference spectroscopy (RIfS)

Reflectometric interference spectroscopy (RIfS) is a label-free, time-resolved technique for detecting interactions of molecules immobilized on a surface with ligands in solution. Here we show that RIfS also permits the detection of the adhesion of tissue culture cells to a functionalized surface in a flow system. Interactions of T cells with other leukocytes or epithelial cells of blood vessels are crucial steps in the regulating immune response and inflammatory reactions. Jurkat T cell leukemia cells rapidly attached to a transducer functionalized with a monoclonal antibody directed against the T cell receptor (TCR)/CD3 complex, followed by activation-dependent cell spreading. RIfS curves were obtained for the Jurkat derivative JCaM 1.6 (which lacks the key signaling protein Lck), cells preincubated with cytochalasin D (an inhibitor of actin polymerization), and for surfaces functionalized with an antibody directed against the coreceptor CD28. These curves differed with respect to the maximum signal and the initial slope of the increase in optical thickness. The testing of chemical inhibitors, cell surface molecules and gene products relevant to a key event in T cell immunity illustrates the potential of label-free techniques for the analysis of activation-dependent cell-surface contacts. The first two authors contributed equally to this paper     

Preparation of a novel composite particles and its application in the fluorescent detection of proteins

A new fluorescence method for the detection of proteins with novel composite nanoparticles (CdS/PPA) has been developed. The composite nanoparticles have been prepared through an in-situ polymerization method under ultrasonic irradiation. The surface of the composite nanoparticles was covered with functional groups (-COOH). These groups may play a major role in the improving the water solubility and biocompatibility of the nanoparticles. The composite particles is combined with proteins in NaAc-HCl buffer solution (pH=1.99), which can result in strong fluorescence, and the response is linearly proportional to the concentration of proteins. In λem/λex=650 nm/365 nm place (the stoke’ shift is 285 nm), its fluorescent strength reaches the maximum. Under the optimum conditions, the linear range is 0.10–20.0 μg·ml⁻¹ with the detection limit of 41 ng·ml⁻¹ for HSA, and 0.10–15.0 μg·ml⁻¹ with the detection limit of 35 ng·ml⁻¹ for Human γ-IgG . The method has been applied to the determination of the total protein in human serum samples collected from the hospital and the results are satisfactory.     

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.     

初发系统性红斑狼疮患者外周血清IL-17的表达

目的探讨IL-17在初发系统性红斑狼疮（SLE）患者外周血清中的表达水平及其临床意义。方法选择42例初次诊断、未经治疗的SLE患者（SLE组）及26例同期健康志愿者（对照组）,采用双抗体夹心酶联免疫吸附试验（ELISA）方法测定外周血清IL-17水平,同时检测外周血抗ds- DNA等抗体及免疫球蛋白、补体等水平并进行相关分析。结果活动期和缓解期SLE患者血清IL-17水平均较对照组明显升高,差异均有统计学意义（均P＜0.05）。活动期和缓解期SLE患者间血清IL-17水平的差异无统计学意义（P＞0.05）。具有皮肤损害的SLE患者血清IL-17水平较无皮肤损害者高,使用环磷酰胺的SLE患者血清IL-17水平较未使用环磷酰胺者高,差异有统计学意义（P＜0.05）。SLE患者血清IL-17水平与SLEDAI、血沉、CRP、IgG、C3、C4均无相关性（均P＞0.05）。19例SLE活动期患者治疗1个月后血清IL-17水平较治疗前明显下降,差异有统计学意义（P＜0.05）。结论 IL-17与狼疮患者的皮肤损害密切相关;IL-17在SLE的发病中起到重要作用并与疾病严重性有关。     

Fluorometric analysis of borohydrides based on reductive aldehyde-to-alcohol conversion of arylaldehydes

Fluorometric analysis of borohydride (BH₄^–) species by the reduction of arylaldehydes to the corresponding arylmethanols was investigated. 9-Anthracenecarboxaldehyde (9-AA) exhibited pronounced ratiometric fluorescence signaling behavior toward borohydride in alkaline aqueous media. The borohydride-selective signaling of 9-AA was unaffected by the presence of commonly encountered metal ions and anions. 1-Pyrenecarboxaldehyde (1-PA) also showed comparable borohydride signaling behavior. The detection limit was found to be 7.4?μM (0.11?ppm) for 9-AA and 15.7?μM (0.23?ppm) for 1-PA. The utility of the probe with μPAD as a convenient tool for the determination of borohydrides was demonstrated.     

Novel 2,4-disubstituted quinazolines as cytotoxic agents and JAK2 inhibitors: Synthesis,in vitro evaluation and molecular dynamics studies

Recent studies reported the involvement of JAK2/STAT3 pathway in various solid tumours including breast, ovarian, prostate and lung cancers. Clinical literature also reported the lowered burden in breast and ovarian cancers by targeting JAK2 pathway. In this study, a series of novel 2,4-disubstituted quinazolines (2a-2 j and 3a-3 j) were synthesized and were evaluated for their cytotoxicity against human breast cancer (MDA-MB-231) and ovarian cancer (SK-O-V3) cell lines using MTT assay. Moderate to good in vitro cytotoxic potentials of the newly synthesized molecules were reported against selected human cancer cell lines. Among the tested molecules, compound 3b has shown better cytotoxic activity against MD-MB-231 (10.1 ± 0.51 μM). in vitro JAK2 inhibition assay elucidated the mechanistic profile of the derivatives with moderate percentage of inhibition. Compounds 3b and 3d were reported with 35.4% and 34.2% inhibition of JAK2 protein. SAR studies suggest that the larger hydrophobic aromatic nucleus with hydrophilic linkage could probably increase the cytotoxic and JAK2 potentials and hydroxyl or nitro substitution could be more beneficial. Molecular dynamics simulation studies with JAK2-3b, and JAK2-3d complexes elucidated the conformational changes. With the reported bioactivities of these derivatives, further studies on the derivatization could elucidate the broader cytotoxic potentials.     

Design,synthesis, antimicrobial activity and computational studies of novel azo linked substituted benzimidazole,benzoxazole and benzothiazole derivatives

Novel azo linked substituted benzimidazole, benzoxazole, and benzothiazole were synthesized by diazo coupling and characterized by ¹H NMR, elemental analysis, FTIR and UV–vis spectroscopy. The newly synthesized compounds were evaluated for invitro antibacterial activity against Staphylococcus aureus and Escherichia Coli strains by Resazurin microtiter assay method (REMA). The minimum inhibitory concentration (MIC in μg/mL) were used to express the antibacterial activities. The azo linked compounds exhibited good to moderate or high antibacterial activities in vitro. Computational studies were performed to correlate HOMO-LUMO gap with antibacterial activity. The comparative molecular docking studies revealed better insights into binding mechanisms.     

Study of blood collection and sample preparation for analysis of vitamin D and its metabolites by liquid chromatography–tandem mass spectrometry

The analysis of vitamin D status, with special emphasis on 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, is gaining interest in clinical studies due to the classical and non-classical effects attributed to this prohormone. In this research, the influence of the two steps preceding determination (viz. sample collection and preparation) on the quantitative analysis of vitamin D and its more important metabolites has been studied. Two preparation approaches, deproteination and solid-phase extraction (SPE), have been evaluated in terms of sensitivity to delimit their application, thus establishing that detection of 1,25-dihydroxyvitamin D cannot be addressed by protein precipitation. Concerning sample collection, serum and plasma reported high accuracy (above 83.3%) for vitamin D and metabolites, while precision, expressed as relative standard deviation, was below 12.9% for all analytes in both samples. Statistical analysis revealed that serum and plasma provided similar physiological levels for vitamin D₃, 24,25-dihydroxyvitamin D₃ and 25-hydroxyvitamin D₃, while significantly different levels were obtained for 1,25-dihydroxyvitamin D₃, always higher in plasma than in serum. Sample collection and treatment have proved to be significant in the analysis of vitamin D and its relevant metabolites.