Discovery of novel elongator protein 2 inhibitors by compound library screening using surface plasmon resonance

Tumour necrosis factor-α (TNF-α) is a pleiotropic cytokine that becomes elevated in chronic inflammatory states, including slowing down osteogenic differentiation, which leads to bone dysplasia in long-term inflammatory microenvironments. The elongator complex plays a role in gene regulation and association with various cellular activities, including the downstream signal transduction of TNF-α in osteogenic cells. To find an inhibitor of Elongator Protein 2 (Elp2), we performed a compound library screen and verified the pharmaceutical effects of candidate compounds on the mouse myoblast cell (C2C12) and mouse osteoblastic cells (MC3T3-E1). The commercial FDA-approved drug (FD) library and the bioactive compound (BC) library were used as candidate libraries. After a label-free, high-throughput affinity measurement with surface plasmon resonance (SPRi), seven kinds of compounds showed binding affinity with mouse Elp2 protein. The seven candidates were then used to perform an inhibition test with TNF-α-induced C2C12 and MC3T3-E1 cell lines. One candidate compound reduced the differentiation suppression caused by TNF-α with resuscitated alkaline phosphatase (ALP) activity, mineralization intensity and expression of osteogenic differentiation marker genes. The results of our study provide a competitive candidate to mitigate the TNF-α-induced osteogenic differentia.

This study employed a label-free high-throughput library screening method and verified a drug candidate to reduce TNF-α induced differentiation inhibition.     

Improved specificity of newborn screening for congenital adrenal hyperplasia by second-tier steroid profiling using tandem mass spectrometry

BACKGROUND: Newborn screening for congenital adrenal hyperplasia (CAH) involves measurement of 17alpha-hydroxyprogesterone (17-OHP), usually by immunoassay. Because this testing has been characterized by high false-positive rates, we developed a steroid profiling method that uses liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure 17-OHP, androstenedione, and cortisol simultaneously in blood spots. METHODS: Whole blood was eluted from a 4.8-mm (3/16-inch) dried-blood spot by an aqueous solution containing the deuterium-labeled internal standard d(8)-17-OHP. 17-OHP, androstenedione, and cortisol were extracted into diethyl ether, which was subsequently evaporated and the residue dissolved in LC mobile phase. This extract was injected into a LC-MS/MS equipped with pneumatically assisted electrospray. The steroids were quantified in the selected-reaction monitoring mode by use of peak areas in reference to the stable-isotope-labeled internal standard. We analyzed 857 newborn blood spots, including 14 blood spots of confirmed CAH cases and 101 of false-positive cases by conventional screening. RESULTS: Intra- and interassay CVs for 17-OHP were 7.2-20% and 3.9-18%, respectively, at concentrations of 2, 30, and 50 microg/L. At a cutoff for 17-OHP of 12.5 microg/L and a cutoff of 3.75 for the sum of peak areas for 17-OHP and androstenedione divided by the peak area for cortisol, 86 of the 101 false-positive samples were within reference values by LC-MS/MS, whereas the 742 normal and 14 true-positive results obtained by conventional screening were correctly classified. CONCLUSION: Steroid profiling in blood spots can identify false-positive results obtained by conventional newborn screening for CAH.     

Improved urine screening using a combination of leukocyte esterase and the Lumac system

Three rapid urine screening tests, leukocyte esterase, nitrite, and the Lumac System for detection of bacterial ATP, were evaluated alone and in combination to determine their utility in screening urine specimens from male patients for bacteruria. The combination of leukocyte esterase and Lumac testing resulted in significant improvement in the sensitivity of urine screening over each test individually and the combination of leukocyte esterase and nitrite. The leukocyte esterase/Lumac combination detected 98% of those specimens with greater than or equal to 10(5) CFU/ml and had a negative predictive value of 99%. The results obtained from this type of testing can be used with confidence to minimize the number of urine specimens cultured and to provide rapid reporting of negative results.     

Complex ciprofloxacin resistome revealed by screening a Pseudomonas aeruginosa mutant library for altered susceptibility

Pseudomonas aeruginosa offers substantial therapeutic challenges due to its high intrinsic resistance to many antibiotics and its propensity to develop mutational and/or adaptive resistance. The PA14 comprehensive mutant library was screened for mutants exhibiting either two- to eightfold increased susceptibilities (revealing genes involved in intrinsic resistance) or decreased susceptibilities (mutational resistance) to the fluoroquinolone ciprofloxacin. Thirty-five and 79 mutants with increased and decreased susceptibilities, respectively, were identified, as confirmed by broth dilution.     

从睾丸cDNA表达文库筛选胰腺癌肿瘤抗原

目的利用胰腺癌患者血清筛选睾丸cDNA噬菌体表达文库，寻找胰腺癌特异性抗原，尤其是肿瘤-睾丸抗原。方法采用血清学筛选重组cDNA表达文库（SEREX）技术筛选睾丸cDNA噬菌体表达文库。对获得的阳性克隆片段进行测序、鉴定和同源性比较。检测40例胰腺导管腺癌患者和40名健康对照血清中抗阳性克隆的抗体。结果筛选共获得107个阳性克隆，代表14个不同抗原基因，其中有13个基因与基因库中已知基因有很高的同源性，这些基因为activin受体AⅡ、LOC92912、KLHL12、IFI16和CAGE等。另有1个阳性克隆HS1在基因库和SEREX数据库中均未发现有同源基因。胰腺癌患者组HS1和HS2克隆抗体阳性率显著高于健康对照组（分别15％与0％比较,Χ^2=4．50，P〈0．05；22．5％与5％比较，Χ^2=5．16，P〈0．05）。结论HS1可能是1个新的肿瘤一睾丸抗原基因。有必要进一步研究这些阳性克隆的生物学功能和临床价值。     

Westgard多规则质量控制法在HPLC筛查珠蛋白生成障碍性贫血中的应用

目的探讨Westgard多规则质量控制法在应用高效液相色谱(HPLC)技术对珠蛋白生成障碍性贫血进行分子检测和筛查中的应用价值。方法应用全自动HPLC分析系统对血红蛋白分子中胎儿血红蛋白(HbF)和血红蛋白A2(HbA_2)两个组分进行定量检测分析,对每一组分的低值和高值质量控制品与每批次样品进行同批检测,绘制Levey-Jennings质量控制图。应用Westgard多规则1_(2s)/1_(3s)2_(2s)R_(4s)对每批次检测结果进行室内质量控制。分析HbF和HbA_2的两个质量控制品在选用的质量控制警戒值1_(2s)出现前后的重测比对结果,验证质量控制规则在临床诊断中的适用性并探讨该规则在珠蛋白生成障碍性贫血筛查中的应用价值。珠蛋白生成障碍性贫血的基因诊断分别采用跨越断裂点聚合酶链反应(Gap-PCR)法检测缺失和反向点杂交技术检测常见的基因突变位点。结果 Westgard质量控制警戒值1_(2s)出现后,重测比对结果差异无统计学意义(P0.05),与该批次的基因诊断符合率无明显差异。研究建立并验证Westgard多规则1_(2s)/1_(3s)2_(2s)R_(4s)在应用HPLC分析技术对HbF和HbA_2定量检测的质量控制作用,证实可以通过质控品检测出误差类型,降低假失控概率,可应用于定量检测珠蛋白生成障碍性贫血的临床诊断和筛查中。结论 Westgard多规则质量控制法在应用全自动HPLC定量分析珠蛋白生成障碍性贫血的筛查诊断中起到重要的室内质量监控作用,可以评价临床检测报告的可靠性,并且对导致结果不可靠的误差实时监控。     

用细胞DNA定量分析方法进行宫颈癌普查的临床研究

目的　探索用细胞DNA定量分析方法进行宫颈癌普查 ,以提高普查的工作效率和准确性。方法　对参与宫颈癌普查的 2 0 0 0 0名妇女用宫颈刷取材 ,进行液基薄层制片 ,分别进行巴氏染色和DNA染色。对巴氏染色片做常规细胞学检查 ,对DNA染色片进行自动扫描诊断。结果　对常规细胞检查结果在LSIL以上病例和全自动DNA倍体分析系统检查可见异倍体细胞或异倍体细胞峰的病例 ,建议进一步做阴道镜检查以及宫颈活检。 10 16名妇女做了病理活检。以病理诊断结果为标准 ,计算出细胞DNA定量分析方法在筛查CIN3以上宫颈病变的敏感性为82 % ,特异性为 71% ,而常规细胞学方法则分别为 5 2 %和 92 %。结论　细胞DNA定量分析方法在有条件的降低特异性的情况下 ,能明显提高宫颈癌普查的阳性检出率。     

Virtual screening of natural products against 5-enolpyruvylshikimate-3-phosphate synthase using the Anagreen herbicide-like natural compound library

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes a reaction involved in the production of amino acids essential for plant growth and survival. EPSPS is the main target of glyphosate, a broad-spectrum herbicide that acts as a competitive inhibitor concerning phosphoenolpyruvate (PEP), which is the natural substrate of EPSPS. In the present study, we introduce a natural compound library, named Anagreen, which is a compendium of herbicide-like compounds obtained from different natural product databases. Herein, we combined the structure- and ligand-based virtual screening strategies to explore Anagreen against EPSPS using the structure of glyphosate complexed with a T102I/P106S mutant of EPSPS from Eleusine indica (EiEPSPS) as a starting point. First, ligand-based pharmacophore screening was performed to select compounds with a similar pharmacophore to glyphosate. Then, structure-based pharmacophore modeling was applied to build a model which represents the molecular features of glyphosate. Then, consensus docking was performed to rank the best poses of the natural compounds against the PEP binding site, and then molecular dynamics simulations were performed to analyze the stability of EPSPS complexed with the selected ligands. Finally, we have investigated the binding affinity of the complexes using free energy calculations. The selected hit compound, namely {"type":"entrez-nucleotide","attrs":{"text":"AG332841","term_id":"47906151"}}AG332841, showed a stable conformation and binding affinity to the EPSPS structure and showed no structural similarity to the already known weed EPSPS inhibitors. Our computational study aims to clarify the inhibition of the mutant EiEPSPS, which is resistant to glyphosate, and identify new potential herbicides from natural products.

Identifying new potential herbicides from natural products and describing their interactions with a double EPSP synthase mutant.     

Improved assessment of plasmodium vivax response to antimalarial drugs by a colorimetric double-site plasmodium lactate dehydrogenase antigen capture enzyme-linked immunosorbent assay

The occurrence of Plasmodium vivax resistance to chloroquine has been reported in several countries of Asia and South America. However, the resistance of P. vivax is insufficiently documented for three reasons: it has received far less attention than P. falciparum; in vivo investigations are handicapped by the existence of hypnozoites, which make it difficult to distinguish between recrudescences due to drug failure and relapses due to dormant forms in the liver; and in vitro studies are greatly limited by the poor growth of P. vivax. We report on the adaptation to P. vivax of a colorimetric double-site Plasmodium lactate dehydrogenase antigen capture enzyme-linked immunosorbent assay previously developed for P. falciparum. The assay proved remarkably sensitive, as under optimal conditions it could detect P. vivax parasitemia levels as low as 10(-8). The technique, which relies on the detection of protein synthesis by the parasite, yielded steep drug-response curves, leading to the precise determination of the 50% inhibitory concentrations for a high proportion of isolates. Chloroquine-resistant parasites were identified in an area where this phenomenon had been documented by in vivo methods. Thus, the results indicate that the in vitro susceptibility of P. vivax can now be monitored easily and efficiently. The data suggest that the threshold of resistance is similar to that of P. falciparum, i.e., in the range of 100 nM for chloroquine and 15 nM for pyronaridine. However, further studies are required to precisely define the cutoff for resistance and the sensitivity to each drug.     

Toxicological screening of drugs by microbore high-performance liquid chromatography with photodiode-array detection and ultraviolet spectral library searches.

We use ultraviolet data, acquired with a photodiode-array detector coupled to a reversed-phase liquid-chromatographic system, to identify unknown drugs in plasma samples of acutely poisoned patients. Both retention time and spectra of the peaks obtained with a microbore Hypersil ODS column under gradient elution are compared with a library of approximately 350 compounds. We present our three-year experience with this system, which identifies drugs in less than 1 h, with a high degree of confidence.     

提高护士利用图书馆医学信息资源实践的能力

21世纪，世界医学科技发展日新月异，各级医院规模建设日益扩大，相应地医院图书馆的建设越来越受到重视。依托网络技术和医学信息学的迅速发展，图书馆医学信息资源日益丰富，能直接为医院的临床、教学、科研提供高质量服务。医院图书馆的读者群体日益扩展，护士正逐渐成为医院图书馆利用文献量最大、借阅频率最高的群体，也是最活跃的信息检索用户。     

提高护士利用图书馆医学信息资源实践的能力

21世纪,世界医学科技发展日新月异,各级医院规模建设日益扩大,相应地医院图书馆的建设越来越受到重视。依托网络技术和医学信息学的迅速发展,图书馆医学信息资源日益丰富,能直接为医院的临床、教学、科研提供高质量服务。医院图书馆的读者群体日益扩展,护士正逐渐成为医院图书馆利用文献量最大、借阅频率最高的群体,也是最活跃的信息检索用户。但是护士由于自身素质和工作特点,业余时间少,读书困难,信息意识差及不懂检索技巧,对图书馆医学信息资源利用不够,长此以往不利于提高她们的自身素质和业务理论水平,影响医院医疗质量发展[1]。因此图…     

Improved green fluorescent protein reporter gene-based microplate screening for antituberculosis compounds by utilizing an acetamidase promoter

The green fluorescent protein (GFP) gene offers many advantages as a viability reporter for high-throughput antimicrobial drug screening. However, screening for antituberculosis compounds by using GFP driven by the heat shock promoter, hsp60, has been of limited utility due to the low signal-to-noise ratio. Therefore, an alternative promoter was evaluated for its enhanced fluorescence during microplate-based culture and its response to 18 established antimicrobial agents by using a green fluorescent protein microplate assay (GFPMA). Mycobacterium tuberculosis strains H37Rv, H37Ra, and Erdman were transformed with pFPCA1, which contains a red-shifted gfp gene driven by the acetamidase promoter of M. smegmatis mc(2)155. The pFPCA1 transformants achieved higher levels of GFP-mediated fluorescence than those carrying the hsp60 construct, with signal-to-noise ratios of 20.6 to 27.8 and 3.8 to 4.5, respectively. The MICs of 18 established antimicrobial agents for all strains carrying pFPCA1 in the GFPMA were within 1 to 2 twofold dilutions of those determined by either the fluorometric or the visual microplate Alamar Blue assay (MABA). No significant differences in MICs were observed between wild-type and pFPCA1 transformants by MABA. The optimized GFPMA is sufficiently simple, robust, and inexpensive (no reagent costs) to be used for routine high-throughput screening for antituberculosis compounds.     

Convenient screening for hemoglobin variants by using the Diamat HPLC system.

Hemolysates from 102 different patients previously assessed for the presence of hemoglobin variants by cellulose acetate electrophoresis were reanalyzed with the Diamat, a microprocessor-controlled step-gradient HPLC technique designed to determine glycohemoglobin (Hgb A1c). This pool contained 81 abnormal specimens, each with one of seven different variant abnormalities. In all cases the HPLC technique correctly detected the presence or absence of a variant. The common S trait gave a large peak immediately after Hgb A, and the SS and SC variants gave clearly abnormal patterns, caused in part by the absence of Hgb A. The pattern from EE variants was distinguished by the appearance of the glycated fraction Hgb E1c, which is eluted at 4.7 instead of 4.2 min, whereas the AE pattern had two glycated peaks. Hemoglobins F, "fast", S, and C were discerned by the presence of a major peak at 3, 5.2, 6.5, and 7.5 min, respectively. The findings suggest that the Diamat is a convenient tool to detect and help diagnose variants in a screening pool.