In vitro study of pesticide hematotoxicity in human and rat progenitors

Some pesticides are hematotoxic and cause aplastic anemia, agranulocytosis, neutropenia, and thrombopenia. In order to evaluate hematopoietic progenitor cultures in the exploration of pesticide hematotoxicity, human and rat colony-forming unit-granulocyte and macrophage (CFU-GM) were cultured in the presence of different concentrations of pesticides, known to be either hematotoxic or innocuous for blood cells. The results were compared to the control culture of the same sample. Four insecticides (lindane, azinphos, mevinphos, parathion methyl), three herbicides, (2,4,5, T, bromacil, MCPA), and two fungicides (fosethyl-aluminum, DNOC) were tested and exhibited the following data: 1) Pesticides, known to be hematotoxic, inhibited the development of progenitors. Different phenomena were observed and suggested different mechanisms: cell destruction, block in mitosis, decrease or delay in mitosis. 2) Difference in sensitivity to molecules between human and rat progenitors was observed. Human progenitors were more sensitive to pesticides, except for 2,4,5 T.     

谷胱苷肽转移酶抑制剂高通量筛选模型的建立和初步应用

谷胱苷肽 - S-转移酶 -π( GST-π)的活性在某些类型的癌症患者中有显著的升高并且在癌症细胞对抗癌药物抗药性的形成过程中起着重要的作用。抑制过量表达的 GST-π酶的活性将有助于克服肿瘤细胞的耐药性 ,因此 ,GST-π被认为是一个潜在的药物作用的靶位点。为了筛选新的抗癌药物的增敏剂 ,我们建立了一个 GST-π酶抑制剂的高通量筛选模型 ,该模型以 CDNB和 GSH为底物 ,在 96 -孔板上通过对 340 nm处吸光度的变化来检测样品对 GST-π酶的抑制活性。经过初筛和复筛 ,从 4 80 0个微生物发酵提取物中筛选到10个阳性样品 ,阳性率为 0 .2 %。     

流式细胞仪高通量药物筛选技术的建立和应用

目的建立流式细胞仪高通量药物筛选技术,并用于筛选逆转肿瘤细胞多药耐药性的药物。方法使用流式细胞仪高通量附件,建立高通量药物筛选技术;以罗丹明123作为荧光指示剂,利用耐药HR-20活细胞筛选逆转耐药性的药物。结果通过阳性药物,确定了逆转耐药性药物筛选模型的Z′因子为0.88。应用此模型筛选8 000多种化合物。结论流式细胞仪高通量药物筛选技术是一种精确度较高、稳定性较好、有效的高通量药物筛选方法。     

重离子束辐照诱变及高通量筛选金霉素高产菌株

应用不同剂量12C+6重离子束对金霉素链霉菌出发菌株B9-34-125进行辐照诱变,并应用96和48孔板高通量筛选金霉素高产菌株。结果表明：当重离子束12C+6离子的辐照剂量为60Gy时,对金霉素链霉菌的诱变效果显著,筛选出5株优势菌株,其中Z-1452菌株摇瓶发酵效价较对照提高14.4%,60m3发酵效价较对照提高5.7%,产量提高了2.6%。     

High throughput methods to characterize protein permeation and release

Spectroscopic methods have been developed to study protein permeation and release kinetics in multi-well plates. The permeation of bovine serum albumin (BSA) through a membrane, which separated a 96-well plate in two compartments, was characterized. A change in fluorescence intensity was measured corresponding to the permeation of BSA from one compartment to another. The permeation of BSA was influenced by the pore size and pore density size of the membrane. The multi-well plates were also used to study the release of a protein drug, hirudin, from an agar hydrogel. A hirudin formulation was mixed at 60 °C with liquid agar and the mixture turned to a gel by cooling at room temperature. The gel entrapping hirudin was formed inside the wells of a 96-well plate. On top of the 100 μl agar–hirudin gel a volume of 200 μl of 10 mM phosphate buffer pH 7.4, 140 mM NaCl was added. The release kinetics of hirudin from the gel were measured following the changes in the hirudin intrinsic tyrosine fluorescence. The release of hirudin over 12 h was measured at three positions: at the bottom of the agar gel, at the interface of the gel with the solution, and in the middle of the receiver solution. The data presented in this paper indicate that high throughput methods can be applied in the characterization of protein drug release from drug delivery systems using small sample volumes.     

极化荧光在高通量药物筛选中的应用

1　前言基于荧光技术的检测分析方法是近年来被应用于药物高通量筛选 (high throughputscreening,HTS)的重要方法之一,荧光检测方法具有灵敏度高、方法简便的优点,目前应用于HTS的荧光技术包括均相时间分辨荧光分析法 (homogeneoustimeresolvedfluorescence,HTRF),荧光共振能量     

A high throughput screening system for predicting chemically-induced reproductive organ deformities

There is a great need for alternative testing methods for reproductive toxicants that are practical, fast, cost-effective and easy to interpret. Previously we followed a pragmatic approach using readily available tests, which was successful in predicting reproductive toxicity of chemicals [13]. This initial battery still contained apical tests and is fairly complex and low in its throughput. The current study aimed to simplify this screening battery using a mechanistic approach and a panel of high throughput CALUX reporter gene assays. A mechanistic approach was taken to validate this high throughput test battery. To this end it was challenged with two preselected sets of chemicals addressing two major apical effect classes relevant in reproductive toxicity. We found selectivity in this battery in that 82% of the compounds inducing reproductive organ deformities were predicted correctly, while for compounds inducing neural tube defects this was the case in 47% only. This is consistent with the mechanisms of toxicity covered in the battery. The most informative assays in the battery were ERalpha CALUX to measure estrogenicity and the AR-anti CALUX assay to measure androgen receptor antagonism.     

药物高通量筛选的设计与实施

本文在简要介绍药物高通量筛选体系的基础上,就组建高效能的高通量筛选体系需要关注的因素,如作用靶点、测试方法、检测方法与作用靶点的匹配、关键参数、效能评价等进行了重点阐述,为高通量筛选体系的设计和实施提供了有益的借鉴。     

以微管蛋白为靶的高通量药物筛选方法的建立与应用

目的：以微管蛋白为靶建立高通量筛选（HTS）模型，以便有效地发现抗肿瘤化合物。方法：细胞培养与免疫荧光技术。结果：以常规的免疫组化（玻片）方法为基础优化实验条件，在96孔板上建立了以微管蛋白为靶点的高通量药物筛选模型；抗肿瘤药物紫杉醇和秋水仙碱作用于人肝癌HepG2细胞后，细胞的免疫荧光强度发生了明显的可检测的变化，间接反映药物对细胞徽管蛋白聚合／解聚作用的影响，与理论预测结果一致。结论：基于人肿瘤细胞的以徽管蛋白为靶点的高通量筛选方法可用于抗肿瘤化合物的筛选。     

Protective effect of thymoquinone against diazinon-induced hematotoxicity,genotoxicity and immunotoxicity in rats

Several studies have shown that oxidative stress and cell damage can occur in the very early stages of diazinon (DZN) exposure. The present study was designed to determine the beneficial effect of thymoquinone (Thy), the main component of Nigella sativa (black seed or black cumin) against DZN immunotoxicity, hematotoxicity and genotoxicity in rats. In the present experimental study, 48 male Wistar rats were randomly divided into six groups, (eight per group) as follows: control (receiving corn oil as the DZN solvent), DZN (20 mg/kg), Thy (10 mg/kg), Thy (2.5 mg/kg) + DZN, Thy (5 mg/kg) + DZN and Thy (10 mg/kg) + DZN. After four weeks of treatment, the hematological parameters of red blood cells (RBCs), white blood cells (WBCs), hemoglobin (Hb), hematocrit (Hct) and platelets (PLTs) were evaluated. The evaluation of genotoxicity was carried out using the micronucleus assay. For measurement of cytokine production, interferon gamma (IFN-γ), interleukin 10 (IL10) and interleukin 4 (IL4) were chosen as immunotoxicity indicators of DZN toxicity. DZN was found to decrease RBCs, WBCs, Hb, Hct, PLTs, butyrl- and acetyl-cholinesterase activity and I FN-γ and increased the micronucleus indices of IL10 and IL4 as compared with the control group. Treatment with Thy reduced DZN hematotoxicity and immunotoxicity, but, significantly, did not prevent genotoxicity. This study showed that Thy (without the significant effect on genotoxicity) decreased the hematological toxicity, immunotoxicity and butyrl and acetyl cholinesterase activity induced by DZN. The success of Thy supplementation against DZN toxicity can be attributed to the antioxidant effects of its constituents.     

紫外分光光度法快速测定庆大霉素 C1a 含量在高通量筛选中的应用

目的 探索庆大霉素 C1a 含量的快速检测方法,实现诱变菌株的高通量筛选。方法 以紫外分光光度法作为检测庆 大霉素 C1a 含量的手段,对庆大霉素 C1a 单组分生产菌株进行 ARTP、LiCl、微波和 ARTP+LiCl 诱变,通过高通量筛选获得高产 菌株,并在 5L 发酵罐中对高产稳定性菌株进行验证。结果 紫外分光光度法和高效液相色谱法对发酵液中庆大霉素 C1a 含量进 行测定,最大相对误差为 3.98%;筛选获得了 10 株高产菌株,其中 AL324 菌株与出发菌株相比,摇瓶效价提高了 52%;通过稳 定性传代实验,获得了 4 株高产稳定性菌株。5L 发酵罐验证实验表明 AL324 与出发菌株相比效价提高了 81.3%。结论 紫外分 光光度法可以快速检测发酵液中庆大霉素 C1a 含量,该方法应用于高通量筛选中,获得了高产菌株,筛选结果表明 ARTP+LiCl 复合诱变方法的正突变率较高,是一种有效的庆大霉素 C1a 高产菌株诱变方式。     

P-gp蛋白功能抑制剂高通量筛选模型的建立与应用

目的建立高通量筛选模型,寻找疗效好、毒副作用小的肿瘤多药耐药逆转剂。方法以罗丹明123荧光染料胞内积累实验为基础,建立基于细胞水平的高通量筛选模型,对480个中药样品进行了高通量筛选,并采用剂量—活性依赖实验和抗性增敏实验验证阳性样品。结果模型Z′因子为0.72,筛选得到2个中药样品。结论建立的P-gp蛋白功能抑制剂高通量筛选模型稳定性好,灵敏度高,筛选得到的2种中药样品具有较好的P-gp蛋白功能抑制活性。     

G-蛋白偶联受体的功能测定和高通量药物筛选

G 蛋白偶联受体家族是药物开发中最大的一类药物靶点 ,高通量药物筛选是开发药物早期阶段的最重要工具之一。根据G 蛋白偶联受体与配体结合及激发的信号通路 ,人们设计了各种可行的功能测试方法 ,用于G 蛋白偶联受体为药靶的高通量药物筛选 ,如 :微体积荧光数字图像测定技术 (Fluorometicmicrovolumeassaytechnology ,FMAT)、荧光偏振 (Fluoresencepolarization ,FP)、竞争性ELISA (Com petitiveenzyme linkedimmunosorbent)、闪烁邻近测定法(Scintillation proximityassay ,SPA)、载黑色素细胞测定法(Melanophoreassay)、报告基因测定法 (Reportergeneassay)和钙离子测定法等测定方法。在这些方法中 ,报告基因测定法和钙离子测定法占了主导地位。非放射性、无需底物和辅助剂的报告基因测定方法和荧光钙离子指示剂的钙离子测定方法可能是将来G 蛋白偶联受体的功能分析和高通量药物筛选的发展方向     

人与不同种属动物骨髓粒系祖细胞对硫芥敏感性的比较

本文用半固体琼脂体外培养技术和琼脂扩散盒体内培养技术测定了人和不同种属动物骨髓粒系祖细胞(CFU—GM),比较了在体内与体外条件下硫芥对不同种属动物骨髓CFU—GM的剂量存活曲线。人、犬、大鼠和小鼠离体骨髓细胞受硫芥作用后CFU—GM的剂量存活曲线在半对数座标图中呈指数性直线,D_(10)(μmol/L)分别为:人0.79,犬0.24,大鼠0.49,小鼠0.84,对硫芥的敏感性以犬最敏感.其次是大鼠.人和小鼠相对最低.硫芥中毒犬、大鼠和小鼠骨髓CFU—GM对硫芥敏感性与体外结果基本一致.     

以大肠埃希氏菌肽脱甲酰基酶为靶点的高通量筛选模型的建立

本文用PCR法从大肠埃希氏菌K-12中克隆出肽脱甲酰基酶(peptide deformylase，PDF)基因，通过测序确证与文献报道的pdf基因序列完全一致。将pdf连接到原核蛋白表达载体pET-30-a( )上，并转化到大肠埃希氏菌BL21(DE3)菌株中，IPTG诱导表达。过量表达的PDF酶用QSepharoseHP离子交换柱和Superdex75纯化得到高纯度Ni—PDF。应用荧光测试仪Polar Fluostar检测PDF对底物For—Met—Ala—Ser的水解活性，PDF酶的已知抑制剂放线酰胺素(actinonin)为阳性对照，通过优化酶反应条件，建立了以PDF为靶点的高通量药物筛选模型。     

人高密度脂蛋白受体CLA-1上调剂9179D的分离、结构鉴定和活性研究

目的从微生物代谢产物中分离能够上调人高密度脂蛋白受体（CLA-1）表达的新型活性化合物,并对其活性进行研究。方法应用已建立的CLA-1表达上调剂的模型,对阳性菌株链霉菌104A-9179发酵产物的活性成分进行分离纯化,获得活性化合物9179D;通过理化性质、质谱、紫外和核磁等波谱学数据进行结构鉴定;利用RT-PCR和Western Blot方法检测9179D对HepG2中CLA-1表达的影响;利用流式细胞仪检测其对小鼠巨噬细胞RAW264.7结合DiI-HDL的影响。结果从链霉菌104A-9179发酵产物中得到活性化合物9179D,并确定了其结构为曲占柳菌素D（Trichostatin D）;9179D在CLA-1上调剂模型上的EC₅₀为46.02μmol/L,表达活性最高值为934%;9179D能增加HepG2中CLA-1的mRNA和蛋白表达;增加RAW264.7对DiI-HDL的结合。结论得到一个微生物来源的具有强的上调CLA-1的表达活性的化合物-9179D（曲古柳菌素D）,属首次报道。     

In-house assessment of a modified in vitro cytotoxicity assay for higher throughput estimation of acute toxicity

The main objective of this study was to assess an in-house 3T3 NRU cytotoxicity assay for compatibility with a prediction model for acute rodent oral toxicity endorsed by an NIEHS–ICCVAM workshop. The aim is to use the NRU assay as one test component of HTS strategies for both acute oral toxicity and acute skin irritation, enabling the rejection of the most toxic materials and prioritisation of other materials for further testing. Groups of model cytotoxins and irritants were tested using the NRU assay and their EC₅₀ values obtained from dose–response curves. These values were compared with those estimated from a limited (three)-dose protocol, deemed more suitable for HTS. A good correlation was observed between the EC₅₀ values from both dose–response curves (R²=0.94). The relationships between EC₅₀ values and acute rodent oral toxicity were compared by application of the prediction model to the model cytotoxins. The results from both full and limited dose–responses fitted within the acceptance limits of the prediction model, with regression lines similar to that of the model. Results indicated that the performance of the currently used 3T3 NRU cytotoxicity assay was similar to that of the assays used to generate the data employed in developing the prediction model. This prediction model can be applied with both the standard and HT assays to estimate acute rodent oral toxicity.     

人高密度脂蛋白受体CLA-1表达上调剂4776B和4776C的分离、结构鉴定及生物活性研究

目的 从微生物代谢产物中筛选和分离能够上调人高密度脂蛋白受体（CLA-1）表达的新型活性化合物。方法 应用本实验室已经建立的CLA-1表达上调剂的高通量筛选模型，从6000多株微生物中筛选出8株能使CLA-1表达上调的阳性菌株。对其中一株阳性菌株放线菌04-4776发酵产物的活性成分进行活性跟踪分离纯化，从中获得CLA-1表达上调的化合物4776B和4776C，并测定了化合物的理化特性和波谱学数据及相关的生物活性。结果通过理化性质、质谱、紫外和核磁等波谱学数据的分析，确定了化合物4776B和4776C的结构；4776B和4776C对CLA-1的上调率分别在2．08和3．26μmol／L时达到最大，与对照相比分别上调了60％和78％。结论 放线菌04-4776产生的两个活性化合物分别与文献报道的红车轴草素和（9R，13S）-7-脱氧-13-二氢道诺霉素酮结构一致；两个化合物均具有强的上调CLA-1的表达活性，属首次报道。     

Recent advances in quantitative high throughput and high content data analysis

ABSTRACT

Introduction: High throughput screening has become a basic technique with which to explore biological systems. Advances in technology, including increased screening capacity, as well as methods that generate multiparametric readouts, are driving the need for improvements in the analysis of data sets derived from such screens.

Areas covered: This article covers the recent advances in the analysis of high throughput screening data sets from arrayed samples, as well as the recent advances in the analysis of cell-by-cell data sets derived from image or flow cytometry application. Screening multiple genomic reagents targeting any given gene creates additional challenges and so methods that prioritize individual gene targets have been developed. The article reviews many of the open source data analysis methods that are now available and which are helping to define a consensus on the best practices to use when analyzing screening data.

Expert opinion: As data sets become larger, and more complex, the need for easily accessible data analysis tools will continue to grow. The presentation of such complex data sets, to facilitate quality control monitoring and interpretation of the results will require the development of novel visualizations. In addition, advanced statistical and machine learning algorithms that can help identify patterns, correlations and the best features in massive data sets will be required. The ease of use for these tools will be important, as they will need to be used iteratively by laboratory scientists to improve the outcomes of complex analyses.     

In silico prediction of SARS protease inhibitors by virtual high throughput screening

A structure-based in silico virtual drug discovery procedure was assessed with severe acute respiratory syndrome coronavirus main protease serving as a case study. First, potential compounds were extracted from protein-ligand complexes selected from Protein Data Bank database based on structural similarity to the target protein. Later, the set of compounds was ranked by docking scores using a Electronic High-Throughput Screening flexible docking procedure to select the most promising molecules. The set of best performing compounds was then used for similarity search over the 1 million entries in the Ligand.Info Meta-Database. Selected molecules having close structural relationship to a 2-methyl-2,4-pentanediol may provide candidate lead compounds toward the development of novel allosteric severe acute respiratory syndrome protease inhibitors.