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1.
《Drug discovery today》2022,27(6):1733-1742
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. 相似文献
2.
目的:研究Survivin-shRNA对视网膜母细胞瘤HXO-RB44细胞自噬及凋亡的影响。方法:培养视网膜母细胞瘤HXO-RB44细胞,构建Survivin-shRNA载体。按照处理不同分为Survivin-shRNA组、GFP组和CON组。分别采用流式细胞术检测Survivin-shRNA对HXO-RB44细胞凋亡和细胞周期的影响,应用MTT法检测Survivin-shRNA对HXO-RB44细胞活性的影响,Western blot法检测Survivin-shRNA对HXO-RB44 细胞自噬相关蛋白LC3、p62 与mTOR 表达的影响。结果:流式细胞术结果表明,与Control组和GFP组相比,Survivin-shRNA组细胞凋亡率增加,差异有统计学意义(P<0.05);随着作用时间的延长,S期出现阻滞,48 h阻滞最强,显著高于对照组(P<0.05)。MTT结果发现Survivin-shRNA可抑制HXO-RB44细胞增殖活性,与Control组和GFP组相比,差异有统计学意义(P<0.05);Western blot结果发现,与对照组相比,LC3表达量显著增加(P<0.01);而mTOR表达量降低(P<0.01)。结论:Survivin-shRNA可促进视网膜母细胞瘤HXO-RB44细胞的凋亡和自噬水平,进而特异性杀伤肿瘤细胞。 相似文献
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4.
《Clinical neurophysiology》2020,131(4):951-957
ObjectiveTo establish a noninvasive method to measure the neuromagnetic fields of the median nerve at the carpal tunnel after electrical digital nerve stimulation and evaluate peripheral nerve function.MethodsUsing a vector-type biomagnetometer system with a superconducting quantum interference device, neuromagnetic fields at the carpal tunnel were recorded after electrical stimulation of the index or middle digital nerve in five healthy volunteers. A novel technique for removing stimulus-induced artifacts was applied, and current distributions were calculated using a spatial filter algorithm and superimposed on X-ray.ResultsA neuromagnetic field propagating from the palm to the carpal tunnel was observed in all participants. Current distributions estimated from the magnetic fields had five components: leading and trailing components parallel to the conduction pathway, outward current preceding the leading component, inward currents between the leading and trailing components, and outward current following the trailing component. The conduction velocity and peak latency of the inward current agreed well with those of sensory nerve action potentials.ConclusionRemoving stimulus-induced artifacts enabled magnetoneurography to noninvasively visualize with high spatial resolution the electrophysiological neural activity from the palm to the carpal tunnel.SignificanceThis is the first report of using magnetoneurography to visualize electrophysiological nerve activity at the palm and carpal tunnel. 相似文献
5.
目的:获得热休克蛋白90β(HSP90β)基因干扰和过表达慢病毒表达系统,并检测其在人骨肉瘤细胞株Saos-2中的表达水平。方法:设计合成shRNA,以慢病毒表达质粒构建HSP90β干扰和过表达载体,酶切电泳、测序技术鉴定载体构建是否成功。重组病毒转染H1299细胞,以嘌呤霉素筛选稳定转染的Saos-2细胞,通过荧光显微镜观察计数获得转染效率。将感染好的细胞分为干扰NC组(NEG-shRNA)、干扰组(shRNA-HSP90β)、过表达NC对照组(NEG-pEZ)及过表达组(pEZ-HSP90β)。通过qRT-PCR 与Western blotting 分别从mRNA 和蛋白表达水平验证目的基因的干扰和过表达水平。结果:插入慢病毒表达载体的基因片段与目的基因的碱基序列完全一致。病毒包装成功后,嘌呤霉素最小致死浓度1 μg/ml,感染复数200,感染人Saos-2的感染效率达80%,其中shRNA-HSP90β组干扰效率为86.35%,pEZ-HSP90β组的mRNA相对表达量增加2.8倍。进一步研究发现,shRNA-HSP90β组较NEG-shRNA组中HSP90β蛋白表达明显降低,pEZ-HSP90β组较NEG-pEZ组HSP90β蛋白表达增加。结论:HSP90β基因干扰和过表达慢病毒载体构建成功,并能够在Saos-2中稳定表达。 相似文献
6.
Maja Kodani Amanda Poe Jan Drobeniuc Tonya Mixson-Hayden 《Journal of medical virology》2020,92(12):3875-3879
Biotin taken orally can interfere with some diagnostic immunoassays, including those for thyroid hormones, ferritin, and markers of infectious disease. Assays affected are ones that use streptavidin-biotin in their design. The goal of our study was to examine the effect of biotin concentrations of up to 1200 ng/mL on three serological assays performed on VITROS 3600 system, Immunoglobulin M (IgM) antibodies to hepatitis A virus (anti-HAV), total anti-HAV, and IgM antibodies to hepatitis B virus core antigen (anti-HBc), by spiking serum samples with variable amounts of biotin. No false-negative results were generated with either concentration of biotin for total anti-HAV (65/65). Likewise, biotin caused no false-positive IgM anti-HAV results (59/59) with either concentration of biotin; however, 6.7% false negativity was found for IgM anti-HAV when samples were spiked with 1200 ng/mL of biotin. Conversely, 100% false positivity (30/30) was produced by biotin interference in total anti-HAV negative specimens with both concentrations of biotin. False negativity rate was 87.5% in IgM anti-HBc positive samples when biotin levels were at 1200 ng/mL. These data show that individuals taking biotin-containing supplements may test false-positive in some serologic assays using streptavidin-biotin chemistries. Further studies are warranted to determine the extent of biotin interference resulting in false-positive and negative results and their impact, if any, on surveillance and diagnostic settings. 相似文献
7.
目的:利用慢病毒介导的RNA干扰(RNAi)技术,构建稳定敲降NF-YB基因的HeLa细胞系。方法:设计shRNA-NF-YB引物,利用分子克隆技术构建H1/GFP-Puro-shRNA-NF-YB质粒,转染293T后收集病毒感染HeLa细胞;用嘌呤霉素筛选细胞得到稳定敲降细胞株;利用Western blot和实时荧光定量PCR对细胞系进行鉴定。结果:构建的质粒经测序后与设计的引物对比序列一致,Western blot和实时荧光定量PCR结果表明构建细胞的NF-YB蛋白和mRNA表达抑制效果明显。结论:本实验成功构建了NF-YB-shRNA-HeLa细胞系,获得了特异性NF-YB基因敲降的HeLa细胞。 相似文献
8.
Basic fibroblast growth factor‐treated adipose tissue‐derived mesenchymal stem cell infusion to ameliorate liver cirrhosis via paracrine hepatocyte growth factor 下载免费PDF全文
9.
《Annales d'endocrinologie》2019,80(5-6):280-285
Resistance to thyroid hormones syndrome is defined as increased thyroxine (T4) and triiodothyronine (T3) concentrations associated with normal or sometimes increased thyrotropin (TSH) concentration. This is usually due to a pathogenic variant with loss of function of the gene coding for thyroid hormone receptor β (THRB). This discrepancy in thyroid hormones (TH) and TSH concentrations is also frequently observed in the presence of analytical interference, notably alteration of TH transport proteins in serum. During 2017, 58 samples were sent to our laboratory in the Angers University Hospital Rare Thyroid and Hormone Receptor Disease Reference Center in order to identify an etiology for discrepant TSH and TH results. We sequenced the genes involved in TH regulation, action and transport (THRB, THRA, SECISBP2, SLC16A, ALB, TTR, SERPINA7). Free T4 and free T3 assay were performed with a second immunoassay (Siemens ADVIA Centaur). A genetic cause of discrepancy in TH and TSH concentrations, with mutation in THRB, was found in 26% of cases (15/58). Biological interference due to TH serum transport protein variant was found in 24% (14/58) of cases. No pathogenic variants were found in the other genes studied. Biological interference was also suspected in 26% of cases without genetic variant, in which the biological discrepancy was not confirmed by a second analytical technique (15/58). Finally, no etiology for the biological discrepancy could be found in 24% of cases (14/58). Clinically, patients in whom biological discrepancy was due to analytic interference were more often asymptomatic, and patients with no identified etiology tended to be older. To limit diagnostic errors associated with the finding of discrepant TSH and TH, we recommend initially conducting a second thyroid function test (TSH, free T4 and free T3) with a different assay, and then screening for a genetic variant in gene coding for thyroid hormone receptor β (THRB) and the TH serum transport proteins (ALB, TTR, SERPINA7). 相似文献
10.