Identification of ChIP-seq and RIME grade antibodies for Estrogen Receptor alpha

Estrogen Receptor alpha (ERα) plays a major role in most breast cancers, and it is the target of endocrine therapies used in the clinic as standard of care for women with breast cancer expressing this receptor. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used against the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments

Dynamic tracking of functional gene modules in treated juvenile idiopathic arthritis

Background We have previously shown that childhood-onset rheumatic diseases show aberrant patterns of gene expression that reflect pathology-associated co-expression networks. In this study, we used novel computational approaches to examine how disease-associated networks are altered in one of the most common rheumatic diseases of childhood, juvenile idiopathic arthritis (JIA). Methods Using whole blood gene expression profiles derived from children in a pediatric rheumatology clinical trial, we used a network approach to understanding the impact of therapy and the underlying biology of response/non-response to therapy. Results We demonstrate that therapy for JIA is associated with extensive re-ordering of gene expression networks, even in children who respond inadequately to therapy. Furthermore, we observe distinct differences in the evolution of specific network properties when we compare children who have been treated successfully with those who have inadequate treatment response. Conclusions Despite the inherent noisiness of whole blood gene expression data, our findings demonstrate how therapeutic response might be mapped and understood in pathologically informative cells in a broad range of human inflammatory diseases

Daedalus and Gasz recruit Armitage to mitochondria, bringing piRNA precursors to the biogenesis machinery

The Piwi-interacting RNA (piRNA) pathway is a small RNA-based immune system that silences mobile genetic elements in animal germlines. piRNA biogenesis requires a specialized machinery that converts long singlestranded precursors into small RNAs of ∼25-nucleotides in length. This process involves factors that operate in two different subcellular compartments: the nuage/Yb body and mitochondria. How these two sites communicate to achieve accurate substrate selection and efficient processing remains unclear. Here, we investigate a previously uncharacterized piRNA biogenesis factor, Daedalus (Daed), that is located on the outer mitochondrial membrane. Daed is essential for Zucchini-mediated piRNA production and the correct localization of the indispensable piRNA biogenesis factor Armitage (Armi). We found that Gasz and Daed interact with each other and likely provide a mitochondrial “anchoring platform” to ensure that Armi is held in place, proximal to Zucchini, during piRNA processing. Our data suggest that Armi initially identifies piRNA precursors in nuage/Yb bodies in a manner that depends on Piwi and then moves to mitochondria to present precursors to the mitochondrial biogenesis machinery. These results represent a significant step in understanding a critical aspect of transposon silencing; namely, how RNAs are chosen to instruct the piRNA machinery in the nature of its silencing targets

Association of Liver Injury From Specific Drugs, or Groups of Drugs, With Polymorphisms in HLA and Other Genes in a Genome-Wide Association Study

BACKGROUND & AIMS: We performed a genome-wide association study (GWAS) to identify genetic risk factors for drug-induced liver injury (DILI) from licensed drugs without previously reported genetic risk factors. METHODS: We performed a GWAS of 862 persons with DILI and 10,588 population-matched controls. The first set of cases was recruited before May 2009 in Europe (n = 137) and the United States (n = 274). The second set of cases were identified from May 2009 through May 2013 from international collaborative studies performed in Europe, the United States, and South America. For the GWAS, we included only cases with patients of European ancestry associated with a particular drug (but not flucloxacillin or amoxicillin-clavulanate). We used DNA samples from all subjects to analyze HLA genes and single nucleotide polymorphisms. After the discovery analysis was concluded, we validated our findings using data from 283 European patients with diagnosis of DILI associated with various drugs. RESULTS: We associated DILI with rs114577328 (a proxy for A*33:01 a HLA class I allele; odds ratio [OR], 2.7; 95% confidence interval [CI], 1.9-3.8; P = 2.4 × 10-8) and with rs72631567 on chromosome 2 (OR, 2.0; 95% CI, 1.6-2.5; P = 9.7 × 10-9). The association with A*33:01 was mediated by large effects for terbinafine-, fenofibrate-, and ticlopidine-related DILI. The variant on chromosome 2 was associated with DILI from a variety of drugs. Further phenotypic analysis indicated that the association between DILI and A*33:01 was significant genome wide for cholestatic and mixed DILI, but not for hepatocellular DILI; the polymorphism on chromosome 2 was associated with cholestatic and mixed DILI as well as hepatocellular DILI. We identified an association between rs28521457 (within the lipopolysaccharide-responsive vesicle trafficking, beach and anchor containing gene) and only hepatocellular DILI (OR, 2.1; 95% CI, 1.6-2.7; P = 4.8 × 10-9). We did not associate any specific drug classes with genetic polymorphisms, except for statin-associated DILI, which was associated with rs116561224 on chromosome 18 (OR, 5.4; 95% CI, 3.0-9.5; P = 7.1 × 10-9). We validated the association between A*33:01 terbinafine- and sertraline-induced DILI. We could not validate the association between DILI and rs72631567, rs28521457, or rs116561224. CONCLUSIONS: In a GWAS of persons of European descent with DILI, we associated HLA-A*33:01 with DILI due to terbinafine and possibly fenofibrate and ticlopidine. We identified polymorphisms that appear to be associated with DILI from statins, as well as 2 non-drug-specific risk factors

Association of Liver Injury From Specific Drugs, or Groups of Drugs, With Polymorphisms in HLA and Other Genes in a Genome-Wide Association Study

BACKGROUND & AIMS: We performed a genome-wide association study (GWAS) to identify genetic risk factors for druginduced liver injury (DILI) from licensed drugs without previously reported genetic risk factors. METHODS: We performed a GWAS of 862 persons with DILI and 10,588 population-matched controls. The first set of cases was recruited before May 2009 in Europe (n = 137) and the United States (n = 274). The second set of cases were identified from May 2009 through May 2013 from international collaborative studies performed in Europe, the United States, and South America. For the GWAS, we included only cases with patients of European ancestry associated with a particular drug (but not flucloxacillin or amoxicillin-clavulanate). We used DNA samples from all subjects to analyze HLA genes and single nucleotide polymorphisms. After the discovery analysis was concluded, we validated our findings using data from 283 European patients with diagnosis of DILI associated with various drugs. RESULTS: We associated DILI with rs114577328 (a proxy for A* 33: 01 a HLA class I allele; odds ratio [OR], 2.7; 95% confidence interval [CI], 1.9 - 3.8; P = 2.4 x 10(-8)) and with rs72631567 on chromosome 2 (OR, 2.0; 95% CI, 1.6 - 2.5; P = 9.7 x 10(-9)). The association with A* 33: 01 was mediated by large effects for terbinafine-, fenofibrate-, and ticlopidine-related DILI. The variant on chromosome 2 was associated with DILI from a variety of drugs. Further phenotypic analysis indicated that the association between DILI and A* 33: 01 was significant genome wide for cholestatic and mixed DILI, but not for hepatocellular DILI; the polymorphism on chromosome 2 was associated with cholestatic and mixed DILI as well as hepatocellular DILI. We identified an association between rs28521457 (within the lipopolysaccharide-responsive vesicle trafficking, beach and anchor containing gene) and only hepatocellular DILI (OR, 2.1; 95% CI, 1.6 - 2.7; P = 4.8 x 10(-9)). We did not associate any specific drug classes with genetic polymorphisms, except for statin-associated DILI, which was associated with rs116561224 on chromosome 18 (OR, 5.4; 95% CI, 3.0 - 9.5; P = 7.1 x 10(-9)). We validated the association between A* 33: 01 terbinafine-and sertraline-induced DILI. We could not validate the association between DILI and rs72631567, rs28521457, or rs116561224. CONCLUSIONS: In a GWAS of persons of European descent with DILI, we associated HLA-A* 33: 01 with DILI due to terbinafine and possibly fenofibrate and ticlopidine. We identified polymorphisms that appear to be associated with DILI from statins, as well as 2 non-drug-specific risk factors.Peer reviewe

Gene expression profiles as indicators of ecotoxicological effect in zebrafish (Danio rerio) embryos

The development of DNA microarrays has provided a powerful new tool for investigating the gene expression changes that underlie response to toxicant exposure. The technology promised great improvements in understanding modes of action, the possibility of rapid toxicity screening of novel chemicals, and the potential for developing new methods of environmental monitoring. However, the technology has yet to truly deliver on these initial promises. Largely this can be attributed to the use of suboptimal experimental and statistical approaches. The aim of this study was to develop the application of gene expression profiling using microarrays to toxicity testing in the zebrafish embryo.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Implications of the solvent vehicles dimethylformamide and dimethylsulfoxide for establishing transcriptomic endpoints in the zebrafish embryo toxicity test

Current aquatic chemical testing guidelines recognize that solvents can potentially interfere with the organism or environmental conditions of aquatic ecotoxicity tests and therefore recommend concentration limits for their use. These recommendations are based on evidence of adverse solvent effects in apical level tests. The growing importance of subapical and chronic endpoints in future test strategies, however, suggests that the limits may need reassessment. To address this concern, microarrays were used to determine the effects of organic solvents, dimethylformamide (DMF) and dimethylsulfoxide (DMSO), on the transcriptome of zebrafish (Danio rerio) embryos. Embryos were exposed for 48h to a range of concentrations between 0.025 and 32.0ml/L. Effects on survival and development after 24 and 48h were assessed microscopically, with no effects on mortality or morphology up to 2.0 and 16.0ml/L for DMF and DMSO. However, analysis of 48-h embryonic RNA revealed large number s of differentially expressed genes at concentrations well below the 0.1ml/L solvent limit level. The enrichment of differentially expressed genes was found for metabolic, developmental, and other key biological processes, some of which could be linked to observed morphological effects at higher solvent concentrations. These findings emphasize the need to remove or lower as far as possible the concentrations of solvent carriers in ecotoxicology tests

An Information-Rich Alternative, Chemicals Testing Strategy Using a High Definition Toxicogenomics and Zebrafish (Danio rerio) Embryos

Large-scale toxicogenomic screening approaches offer great promise for generating a bias-free system-wide view of toxicological effects and modes-of-action of chemicals and ecotoxicants. However, early applications of microarray technology have identified relatively small groups of responding genes with which to define new targets for analysis by conventional means. We have trialled a more intensive approach to the design and interpretation of array experiments incorporating a balanced interwoven ANOVA design with higher levels of biological replication, a more thorough analysis of errors and false discovery rates, and an analysis of response patterns using gene network models. Zebrafish embryos were exposed from 1.5 h post-fertilization for 72 h to ecotoxicants representing different classes—2,4-dichlorophenol, 3,4-dichloroaniline, pentachlorophenol, and cadmium chloride—at low concentrations producing a developmental disturbance to 10% of embryos and half of this dose. Extracted whole embryo RNA was then analyzed on microarrays. Analysis revealed responses of 3000–5000 genes, which is 10–1000 times greater than previously reported, with significance at lower levels of fold change. Some gene responses were common to multiple toxicants, and others were restricted to just one or two toxicants. The gene expression profiles for the different toxicants were distinctive, and analysis using network-based models provided a high level of detail of affected processes, some of which were novel. This approach provides a more highly refined view of toxic effects, from which meaningful patterns of response can be discerned and related to functional deficits and from which more reliable indicators of toxicological effect can be predicted.