Redox active or thiol reactive? Optimization of rapid screens to identify less evident nuisance compounds

Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.     

VP1-Binding Protein Glucose-regulated protein 78 as an important mediator for Enterovirus 71 infecting Human Brain Microvascular Endothelial Cells

Purpose: Enterovirus 71 (EV71) is one of the main pathogens causing hand, foot and mouth disease, which could even induce severe brain damage in some patients. As the underlying mechanism of the invasion and replication process still remains largely unknown, we investigated the role of candidate proteins expressed during EV71 invasion in human brain microvascular endothelial cells (HBMECs) to delineate the pathophysiological mechanism of EV-71 infection. Materials and Methods: Ninety-one candidate EV71-associated proteins which could bind the major capsid protein (viral protein 1 [VP1]) of EV71 on the HBMEC were identified by applying an analysis of glutathione-S-transferase pull-down coupling with liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). Seventy-eight kDa glucose-regulated protein 78 (GRP78) binding to the VP1 protein was further validated by co-immunoprecipitation, immunofluorescence and western blot analysis. To explore the role of GRP78 in EV71 infection, GRP78 was knocked down and overexpressed in HBMEC and was verified by TCID50 assay. Results: LC-ESI-MS/MS-identified 91 proteins were subjected to gene ontology analysis, and on molecular and biological function analysis revealed GRP78 act as an important binding protein in mediating EV71 infection. In addition, immunofluorescence demonstrated the co-localisation of GRP78 and VP1 in cytoplasm of the infected HBMEC. The TCID50 assay showed that knockdown of GRP78 could attenuate the replication capacity of EV71 in HBMEC, and the overexpression could increase the virus titre in HBEMC at 24 h post-infection suggesting that GRP78 was associated with the replication capacity of EV71 in HBMEC. Conclusion: These findings provided evidence that GRP78 plays an important role during the progression of EV71 infection as a mediator in HBMEC.     

前交叉韧带重建失败的影响因素分析

目的探讨性别、年龄、术后运动水平及移植物直径对前交叉韧带（anterior cruciate ligament，ACL）重建术疗效的影响。方法研究纳入 2012 年 2 月—2017 年 6 月，于关节镜下行自体腘绳肌腱单束重建 ACL 2 年以上、二次关节镜手术取出内固定物的 179 例患者。男 111 例，女 68 例；年龄 11～63 岁，平均 30.0 岁。患者均为运动损伤；受伤至手术时间 3 d～26 年，中位时间 120 d。Lachman 试验及轴移试验均为阳性。对性别、年龄、术后运动水平（取内固定物时 Tegner 评分）、移植物直径以及股数进行单因素分析，初步筛选 ACL 重建失败的影响因素；进一步采用 logistic 回归进行多因素分析，筛选危险因素。结果患者术后均获随访，随访时间 24～90 个月，平均 29.1 个月。末次随访时，Lachman 试验阳性 25 例，轴移试验阳性 28 例。KT-2000 检查双侧膝关节胫骨前向位移差值–1～7 mm，平均 1.89 mm。根据 ACL 重建失败评价标准，共 11 例（6.15%）患者重建失败。单因素及多因素分析均显示年龄、性别、术后运动水平、移植物直径及股数不是 ACL 重建失败的影响因素（P>0.05）。 结论ACL 重建术后不同年龄、性别、运动水平患者间重建失败风险无明显差异，而且通过增加股数方法增加移植物直径，对于降低重建失败率无显著意义。     

Interchangeability of two Enterovirus 71 inactivated vaccines in Chinese children: A phase IV,open-label,and randomized controlled trial

BackgroundIn China, three inactivated Enterovirus 71 (EV71) vaccines have been approved. Although the vaccines in an immunization series should be from a single manufacture, children sometimes have to receive EV71 vaccines from more than one manufacturers. The aim of this study was to evaluate the interchangeability and safety of vaccination with EV71 vaccines from two manufacturers among Chinese children.MethodsWe conducted an open label and randomized controlled study among children aged 6–35 months from November 2018 to January 2019. The participants were randomly assigned (1:1:1:1) to receive EV71 vaccines in one of the four different schedules (two using a single vaccine for all doses from one manufacture, and two mixed schedules using vaccines from two manufactures). Blood samples were collected pre-vaccination (Day 0) and one month after the second dose (Day 60) for neutralizing antibody assay. Immunogenicity was assessed in the per-protocol cohort and safety was assessed in the total vaccinated cohort.ResultsA total of 300 children were enrolled and randomized, of whom 89.0% (267/300) were included in the per-protocol cohort for immunogenicity analysis. The seroconversion rates of the EV71 neutralizing antibody in four groups ranged from 98.4% to 100.0%, and were not significantly different among the groups. Compared with other groups, geometric mean titer was higher in group D, in which the participants received Institute of Medical Biology Chinese Academy of Medical Sciences (CAMS) vaccine in the first dose and the Sinovac vaccine in the second dose. Safety profiles were similar among the four groups and no serious adverse events related to the vaccination were reported.ConclusionsInterchangeability of EV71 vaccines from two manufactures to complete an immunization series showed good immunogenicity and safety. The antibody response levels may vary by vaccination sequences of EV71 vaccines from the two manufacturers.Trial registration: ClinicalTrials.govNCT03873740.     

中老年患者后路腰椎椎体间融合术并发症及临床获益的年龄分层随访研究

目的比较不同年龄段中老年患者接受后路腰椎椎体间融合术（posterior lumbar intervertebral fusion，PLIF）的并发症及临床评分改善情况，为医务人员评估不同年龄段中老年患者接受 PLIF 的并发症风险和临床获益提供参考。方法回顾分析 2013 年 6 月—2016 年 6 月符合选择标准的 1 136 例行 PLIF 治疗的 55 岁以上患者临床资料。根据患者接受手术时的年龄分为 55～64 岁组、65～74 岁组和≥75 岁组。比较 3 组患者一般特征、合并症情况及手术资料，统计并比较不同年龄组并发症发生例数及发病率，并按照最小临床显著标准（minimal clinical important difference，MCID），对患者的疼痛视觉模拟评分（VAS）评分和 Oswestry 功能障碍指数（ODI）的改善情况进行比较。采用单因素 logistic 回归比较不同年龄段并发症发病及 VAS 和 ODI 评分改善达到 MCID 的情况，并对并发症的危险因素进行多因素 logistic 回归分析。结果3 组患者手术融合节段数和合并骨质疏松情况比较，差异有统计学意义（P<0.05）；性别、体质量指数、手术时间、术前美国麻醉医师协会（ASA）分级和合并症数量比较，差异无统计学意义（P>0.05）。所有患者均获随访，随访时间 6～62 个月，平均 27.4 个月。术后并发症结果中，3 组术中并发症、系统性并发症、微小并发症总发生率以及术后 ODI 评分改善达到 MCID 比例比较，差异有统计学意义（P<0.05）；远期随访终点并发症总发生率及术后 VAS 评分改善达到 MCID 比例比较，差异无统计学意义（P>0.05）。单因素 logistic 回归分析显示，调整混杂因素后，55～64 岁组和 65～74 岁组间术中并发症发生情况和术后 ODI 评分改善达到 MCID 情况比较，差异有统计学意义（P<0.05）；≥75 岁组系统性并发症、微小并发症、远期随访终点并发症发生情况以及术后 ODI 评分改善达到 MCID 情况与其他两组比较，差异有统计学意义（P<0.05）。多因素 logistic 回归分析显示，年龄增长是系统性并发症、微小并发症和远期随访终点并发症的危险因素。除年龄外，手术时间长是术中并发症的危险因素，融合节段数增加是系统性并发症的危险因素，合并症数量是微小并发症的危险因素，合并骨质疏松是远期随访终点并发症的危险因素。 结论与年龄<75岁的中老年患者相比，年龄≥75 岁的腰椎退行性疾病患者 PLIF 手术并发症风险较高，但术后 VAS 和 ODI 评分改善相似。在严格把握手术适应证前提下行 PLIF，对提高此类患者的生活质量具有积极意义。     

Deficient glucose uptake is linked to impaired Glut1 expression upon CD3/CD28 stimulation in memory T cells from pleural effusions secondary to lung cancer

Glucose and nutrient uptake is essential in supporting T cell activation and is increased upon CD3/CD28 stimulation. As T cells from pleural effusions secondary to lung cancer show impaired function, we hypothesized that these cells might have altered expression of nutrient transporters. Here, we analysed by flow cytometry the expression of the transferrin receptor CD71, amino acid transporter CD98 and glucose transporter Glut1 and glucose uptake in pleural effusion‐derived T cells from lung cancer patients, after stimulation via CD3/CD28 under normoxia or hypoxia (2% O₂). We compared the response of T cells from pleural effusions secondary to lung cancer with that of T cells from nonmalignant effusions. In memory T cells from both groups, anti‐CD3/CD28‐stimulation under normoxia upregulated CD98 and CD71 expression (measured as median fluorescence intensity, MFI) in comparison with anti‐CD3‐stimulation. Costimulation under hypoxia tended to increase CD98 expression compared to CD3‐stimulation in memory T cells from both groups. Remarkably, in the cancer group, memory T cells stimulated via CD3/CD28 under hypoxia failed to increase CD71 and Glut1 expression levels compared to the cells receiving anti‐CD3 stimulation, a phenomenon that contrasted with the behaviour of memory T cells from nonmalignant effusions. Consequently, glucose uptake by memory T cells from the cancer group was not increased after CD3/CD28 stimulation under hypoxia, implying that their glycolytic metabolism is defective. As this process is required for inducing an antitumoural response, our study suggests that memory T cells are rendered dysfunctional and are unable to eliminate lung tumour cells.     

影像学评估在斜外侧腰椎椎间融合术中的指导作用

目的总结近年影像学评估对斜外侧腰椎椎间融合术（oblique lumbar interbody fusion，OLIF）的指导作用。方法广泛查阅近年来国内外于 OLIF 的影像学研究文献，并进行总结分析。结果术前影像学评估对 OLIF 手术入路操作、椎间融合器放置位置、适应证选择、减少并发症发生等方面具有重要指导作用。结论术前详细影像学评估可以更好地把握 OLIF 手术适应证，有效避免神经、血管、肌肉损伤的发生。     

人肠道病毒71型及柯萨奇病毒A组16型VP1蛋白的真核表达

目的：克隆人肠道病毒71型（EV71）及柯萨奇病毒A组16型（CA16）的VP1蛋白编码基因，构建相应真核表达质粒在体外进行重组表达，检测其细胞内定位，为EV71及CA16的疫苗研究提供基础。方法：通过PCR扩增分别获取EV71及CA16的VP1编码序列，插入pcFlag载体，分别构建EV71及CA16 VP1与FLAG标签融合的真核表达质粒；瞬时转染人胚肾293T细胞及横纹肌肉瘤RD细胞，Western blot法检测融合蛋白的表达，免疫荧光检测融合蛋白的细胞内定位情况。结果：测序表明两种来源VP1蛋白的重组表达质粒构建正确；分别转染两种不同的细胞后，均可通过Western blot检测到相应蛋白表达；免疫荧光检测显示两种毒株的VP1均表达在细胞质中。结论：外源融合表达EV71或CA16 VP1蛋白的真核表达质粒构建成功；所构建质粒分别转染细胞后，均可在细胞质内检测到VP1融合蛋白的表达。