Identification of Small Molecule Inhibitors of Jumonji AT-rich Interactive Domain 1B (JARID1B) Histone Demethylase by a Sensitive High Throughput Screen

JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC₅₀ of about 3 μm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.     

宏基因组的生物信息分析

基于高通量测序的宏基因组学研究是近年来的研究热点之一。宏基因组的生物信息分析正在逐渐完善成熟．各种分析软件和流程的开发与应用,极大地促进了宏基因组研究的发展,特别是在遗传与进化、基因发现、宏基因组和人类疾病的相关研究等方面取得了显著成果。本文旨在结合宏基因组学的研究内容和研究方向,对宏基因组的生物信息分析方法进行综述,探讨宏基因组的生物信息分析面临的机遇和挑战。     

Drugs, hERG and sudden death

Early recognition of potential QT/TdP liability is now an essential component of the drug discovery/drug development program. The hERG assay is an indispensable step and a high-quality assay must accompany any investigational new drug (IND) application. While it is the gold standard at present, the hERG assay is too labor-intensive and too low throughput to be used as a screen early in the discovery/development process. A variety of indirect high throughput screens have been used.     

Next generation genome-wide association tool: design and coverage of a high-throughput European-optimized SNP array

The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. We describe the development of a next-generation microarray based on the new highly-efficient Affymetrix Axiom genotyping technology that we are using to genotype individuals of European ancestry from the Kaiser Permanente Research Program on Genes, Environment and Health (RPGEH). The array contains 674,517 SNPs, and provides excellent genome-wide as well as gene-based and candidate-SNP coverage. Coverage was calculated using an approach based on imputation and cross validation. Preliminary results for the first 80,301 saliva-derived DNA samples from the RPGEH demonstrate very high quality genotypes, with sample success rates above 94% and over 98% of successful samples having SNP call rates exceeding 98%. At steady state, we have produced 462 million genotypes per week for each Axiom system. The new array provides a valuable addition to the repertoire of tools for large scale genome-wide association studies.     

Enhanced COD and nitrogen removals for the treatment of swine wastewater by combining submerged membrane bioreactor (MBR) and anaerobic upflow bed filter (AUBF) reactor

In order to enhance performances of organics removal and nitrification for the treatment of swine wastewater containing high concentration of organic solids and nitrogen than conventional biological nitrogen removal process, a submerged membrane bioreactor (MBR) was followed by an anaerobic upflow bed filter (AUBF) reactor in this research (AUBF–MBR process). The AUBF reactor is a hybrid reactor, which is the combination of an anoxic filter for denitrification and upflow anaerobic sludge blanket (UASB) for acid fermentation. In the AUBF–MBR process, it showed a considerable enhancement of the effluent quality in terms of COD removal and nitrification. The submerged MBR could maintain more than 14,000 mg VSS/L of the biomass concentration. Total nitrogen (T-N) removal efficiency represented 60% when internal recycle ratio was three times of flow-rate (Q), although the nitrification occurred completely. Although the volatile fatty acids produced in AUBF reactor can enhance denitrification rate, but the AUBF–MBR process showed reduction of overall removal efficiency of the nitrogen due to the reduction of carbon source by methane production in the AUBF reactor compared to that of theoretical nitrogen removal efficiency.

Long-term operation of the submerged MBR showed that the throughputs of the submerged MBR were respectively 74, 63, and 31 days at 10, 15, and 30 L/m² h (LMH) of permeate flux. Resistance to filtration by rejected solid is the primary cause of fouling, however the priority of cake resistance (R_c) and fouling resistance (R_f) with respect to filtration phenomenon was different according to the amount of permeate flux. The submerged MBR, here, achieved a steady-state flux of 15 LMH at 0.4 atm. of trans-membrane pressure (TMP) but the flux can be enhanced in the future because shear force by tangential flow will be greater when multi-layer sheets of membrane were used.     

Capturing ecological complexity: OCI,a novel combination of ecological indices as applied to benthic marine habitats

The novel Overall Complexity Index (OCI) is proposed to measure ecological complexity, incorporating four complexity indices: (1) exergy and (2) throughput as extensive metrics, (3) specific exergy and (4) information as intensive metrics. Exergy and specific exergy estimate structural complexity while throughput and information functional complexity. OCI was applied to benthic habitats in a coastal marine tract encompassing a Marine Protected Area (MPA) in north-western Italy. The four individual indices did not always show homogeneous results in assigning complexity to different habitats. On the contrary, the additive measure provided by OCI showed that seagrass meadows and coralligenous reefs are in all the most complex habitats. Applying OCI provided results consistent with traditional approaches based on expert judgement, which usually attach more interest to seagrass meadows and hard bottoms with respect to soft bottoms, but expressed a synthetic, objective and quantitative approach. OCI can be mapped for management purposes, resolving the discordances evidenced by the individual indices. Ecological complexity in the study area is concentrated in some hot spots, as mapped by OCI, while the greatest part of the seafloor is occupied by low complexity habitats. Only some of these complexity hotspots are included within the Marine Protected Area, while this study suggests that high complexity areas, adjacent to the existing MPA, should be considered for protection possibly reshaping MPA's limits.     

Genome-wide Screen for Modulation of Hepatic Apolipoprotein A-I (ApoA-I) Secretion

Control of plasma cholesterol levels is a major therapeutic strategy for management of coronary artery disease (CAD). Although reducing LDL cholesterol (LDL-c) levels decreases morbidity and mortality, this therapeutic intervention only translates into a 25–40% reduction in cardiovascular events. Epidemiological studies have shown that a high LDL-c level is not the only risk factor for CAD; low HDL cholesterol (HDL-c) is an independent risk factor for CAD. Apolipoprotein A-I (ApoA-I) is the major protein component of HDL-c that mediates reverse cholesterol transport from tissues to the liver for excretion. Therefore, increasing ApoA-I levels is an attractive strategy for HDL-c elevation. Using genome-wide siRNA screening, targets that regulate hepatocyte ApoA-I secretion were identified through transfection of 21,789 siRNAs into hepatocytes whereby cell supernatants were assayed for ApoA-I. Approximately 800 genes were identified and triaged using a convergence of information, including genetic associations with HDL-c levels, tissue-specific gene expression, druggability assessments, and pathway analysis. Fifty-nine genes were selected for reconfirmation; 40 genes were confirmed. Here we describe the siRNA screening strategy, assay implementation and validation, data triaging, and example genes of interest. The genes of interest include known and novel genes encoding secreted enzymes, proteases, G-protein-coupled receptors, metabolic enzymes, ion transporters, and proteins of unknown function. Repression of farnesyltransferase (FNTA) by siRNA and the enzyme inhibitor manumycin A caused elevation of ApoA-I secretion from hepatocytes and from transgenic mice expressing hApoA-I and cholesterol ester transfer protein transgenes. In total, this work underscores the power of functional genetic assessment to identify new therapeutic targets.     

Antimycobacterial evaluation of novel hybrid arylidene thiazolidine-2,4-diones

A series of novel hybrid heterocycles comprising arylidene thiazolidine-2,4-dione and 1-cyclopropyl-2-(2-fluorophenyl)ethanone were synthesized. These compounds were evaluated for their antimycobacterial activity against Mycobacterium tuberculosis H₃₇Rv in High Throughput Screen. Most of the hybrid arylidene thiazolidine-2,4-diones displayed moderate to good activity with MIC of less than 50 μM. Compound 1m exhibited maximum potency being 5.87 fold more active at EC₅₀ and 6.26 fold more active at EC₉₀ than the standard drug pyrimethamine.     

以Neprilysin蛋白酶为靶标的药物筛选模型的建立及应用

目的:建立以蛋白酶Neprilysin(NEP)为靶点的高通量药物筛选模型,应用该模型筛选抑制剂。方法:利用毕赤酵母表达系统。构建重组质粒p PICZα-A-NEP,表达载体通过与酵母菌X-33基因组染色体发生同源重组,将外源基因整合于染色体后实现目的蛋白的表达。应用荧光共振能量转移法(FRET)检测蛋白酶活性,优化反应条件,建立药物筛选体系,筛选抑制剂。结果:成功构建表达载体p PICZα-A-NEP;建立了以NEP为靶标的药物筛选模型,获得模型反应动力学参数Vmax=3.6μM/s,Kcat/Km=4.5×105M-1s-1,测定模型Z-因子为0.89,说明体系稳定可用于以NEP为靶标的药物的高通量筛选;并用该模型对天然产物组分库进行筛选,在0.5mg/ml的药物浓度下,得到抑制率较高的药物为4种,并测得半数抑制浓度IC50值,其中MDCNCL01000242的IC50值最低,为(8.31±0.03)μg/ml。结论:建立的药物筛选模型较为理想,适用于NEP抑制剂的筛选,可促进药物的研发。