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Collins B.C.,University College Dublin | Collins B.C.,ETH Zurich | Miller C.A.,Agilent Technologies | Sposny A.,Institute for Toxicology | And 4 more authors.
Molecular and Cellular Proteomics | Year: 2012

There is a pressing and continued need for improved predictive power in preclinical pharmaceutical toxicology assessment as substantial numbers of drugs are still removed from the market, or from late-stage development, because of unanticipated issues of toxicity. In recent years a number of consortia have been formed with a view to integrating -omics molecular profiling strategies to increase the sensitivity and predictive power of preclinical toxicology evaluation. In this study we report on the LC-MS based proteomic analysis of the effects of the hepatotoxic compound EMD 335823 on liver from rats using an integrated discovery to targeted proteomics approach. This compound was one of a larger panel studied by a variety of molecular profiling techniques as part of the InnoMed PredTox Consortium. Label-free LC-MS analysis of hepatotoxicant EMD 335823 treated animals revealed only moderate correlation of individual protein expression with changes in mRNA expression observed by transcriptomic analysis of the same liver samples. Significantly however, analysis of the protein and transcript changes at the pathway level revealed they were in good agreement. This higher level analysis was also consistent with the previously suspected PPARα activity of the compound. Subsequently, a panel of potential biomarkers of liver toxicity was assembled from the label-free LC-MS proteomics discovery data, the previously acquired transcriptomics data and selected candidates identified from the literature. We developed and then deployed optimized selected reaction monitoring assays to undertake multiplexed measurement of 48 putative toxicity biomarkers in liver tissue. The development of the selected reaction monitoring assays was facilitated by the construction of a peptide MS/MS spectral library from pooled control and treated rat liver lysate using peptide fractionation by strong cation exchange and off-gel electrophoresis coupled to LC-MS/MS. After iterative optimization and quality control of the selected reaction monitoring assay panel, quantitative measurements of 48 putative biomarkers in the liver of EMD 335823 treated rats were carried out and this revealed that the panel is highly enriched for proteins modulated significantly on drug treatment/hepatotoxic insult. This proof-of-principle study provides a roadmap for future large scale pre-clinical toxicology biomarker verification studies whereby putative toxicity biomarkers assembled from multiple disparate sources can be evaluated at medium-high throughput by targeted MS. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

News Article
Site: phys.org

Eggs can be 'tricked' into developing into an embryo without fertilisation, but the resulting embryos, called parthenogenotes, die after a few days because key developmental processes requiring input from sperm don't happen. However, scientists from the Department of Biology & Biochemistry at the University of Bath have developed a method of injecting mouse parthenogenotes with sperm that allows them to become healthy baby mice with a success rate of up to 24 per cent. This compares to a rate of zero per cent for parthenogenotes or about two per cent for nuclear transfer cloning. The study is published today (Tuesday, 13 September, 2016) in the journal Nature Communications. Molecular embryologist Dr Tony Perry, senior author of the study, said: "This is first time that full term development has been achieved by injecting sperm into embryos. "It had been thought that only an egg cell was capable of reprogramming sperm to allow embryonic development to take place. "Our work challenges the dogma, held since early embryologists first observed mammalian eggs around 1827 and observed fertilisation 50 years later, that only an egg cell fertilised with a sperm cell can result in a live mammalian birth." The idea was the brain child of Dr Toru Suzuki in Dr Perry's team in the University of Bath's Laboratory of Mammalian Molecular Embryology, who performed the study together with team member Dr Maki Asami and colleagues from the University of Regensburg and the Fraunhofer Institute for Toxicology and Experimental Medicine in Germany. The baby mice born as a result of the technique seem completely healthy, but their DNA started out with different epigenetic marks compared with normal fertilisation. This suggests that different epigenetic pathways can lead to the same developmental destination, something not previously shown. The discovery has ethical implications for recent suggestions that human parthenogenotes could be used as a source of embryonic stem cells because they were considered inviable. It also hints that in the long-term future it could be possible to breed animals using non-egg cells and sperm. Although this is still only an idea, it could have potential future applications in human fertility treatment and for breeding endangered species. Dr Paul Colville-Nash, from the Medical Research Council (MRC) who funded the work, said: "This is an exciting piece of research which may help us to understand more about how human life begins and what controls the viability of embryos, mechanisms which may be important in fertility. It may one day even have implications for how we treat infertility, though that's probably still a long way off." More information: Suzuki, T. et al. Mice produced by mitotic reprogramming of sperm injected into haploid parthenogenotes. Nat. Commun. 7:12676, DOI: 10.1038/ncomms12676 (2016).

Jawerka M.,Institute for Stem Cell Research | Colak D.,Institute for Stem Cell Research | Dimou L.,Ludwig Maximilians University of Munich | Spiller C.,Institute for Toxicology | And 8 more authors.
Neuron Glia Biology | Year: 2010

Gene expression changes during cell differentiation are thought to be coordinated by histone modifications, but still little is known about the role of specific histone deacetylases (HDACs) in cell fate decisions in vivo. Here we demonstrate that the catalytic function of HDAC2 is required in adult, but not embryonic neurogenesis. While brain development and adult stem cell fate were normal upon conditional deletion of HDAC2 or in mice lacking the catalytic activity of HDAC2, neurons derived from both zones of adult neurogenesis die at a specific maturation stage. This phenotype is correlated with an increase in proliferation and the aberrant maintenance of proteins normally expressed only in progenitors, such as Sox2, also into some differentiating neurons, suggesting that HDAC2 is critically required to silence progenitor transcripts during neuronal differentiation of adult generated neurons. This cell-autonomous function of HDAC2 exclusively in adult neurogenesis reveals clear differences in the molecular mechanisms regulating neurogenesis during development and in adulthood. © Cambridge University Press 2010.

Collins B.C.,University College Dublin | Sposny A.,Institute for Toxicology | McCarthy D.,Bio Rad Laboratories Inc. | Brandenburg A.,Genedata AG | And 5 more authors.
Proteomics | Year: 2010

A serious bottleneck in the drug development pipeline is the inability of current pre-clinical toxicology evaluation methods to predict early on, and with good accuracy, that a drug candidate will have to be removed from development due to toxicology/safety issues. The InnoMed PredTox consortium attempted to address this issue by assessing the value of using molecular profiling techniques (proteomics, transcriptomics, and metabonomics), in combination with conventional toxicology measurements, on decision making earlier in preclinical safety evaluation. In this study, we report on the SELDI-TOF-MS proteomics component of the InnoMed PredTox project. In this large scale, multi-site, multi-compound study, tissue and plasma samples from 14-day in vivo rat experiments conducted for 16 hepato- and nephro-toxicants with known toxicology endpoints (including 14 proprietary compounds and 2 reference compounds) were analyzed by SELDI-TOF-MS. We have identified seven plasma proteins and four liver proteins which were shown to be modulated by treatment, and correlated with histopathological evaluations and can be considered potential biomarker candidates for the given toxicology endpoints. In addition, we report on the intraand inter-site variations observed based on measurements from a reference sample, and steps that can be taken to minimize this variation. © 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

Li Y.,Institute for Toxicology | Gao S.,Institute for Toxicology | Jing H.,Institute for Toxicology | Qi L.,Institute for Toxicology | And 10 more authors.
Toxicology Research | Year: 2013

The utility of any non-rodent model system for chemical toxicity screening depends on the level of correlation between its responses and toxic reactions in rodents. Toxicity assays in the nematode Caenorhabditis elegans (C. elegans) can be fast and inexpensive; however few studies have been performed comparing toxic responses in the nematode with data on acute rodent toxicity. We assayed the acute toxicity of 21 types of chemicals in different toxicity categories using C. elegans. The nematodes were exposed to different concentrations of chemicals in 96-well plate for 24 h. The lethality rate was observed at 2, 4, 12 and 24 h, and median lethal concentration (LC50) was calculated by the Probit method. The lethality rate was counted at 1, 8, 16 and 20 h additionally at the concentrations of 10.000, 21.500 and 46.400 mg ml -1 to acquire median lethal times (LT50). The results indicated that the chemical pH could affect the C. elegans LC50 value. The pH toleration range for C. elegans was more than 2.75. Excluding 4 types of acidic chemicals, there were positive correlations between LC 50s of C. elegans and LD50s of mouse/rat (r > 0.72, p < 0.01) after both 12 h and 24 h exposure. As to the LC50 data following a 24 h exposure in C. elegans, the correlation of C. elegans LC 50s vs. rat LD50s (r = 0.885) was greater than the correlation of mouse vs. rat LD50s (r = 0.879), while the correlation of C. elegans LC50s vs. mouse LD50s (r = 0.741) was lower relative to that of mouse vs. rat LD50s. The data were further compared with an in vitro cytotoxicity model utilizing human epidermal keratinocytes (NHK). The data indicate that the correlation of C. elegans LC50s vs. rat LD50s was equal to the correlation of mouse vs. rat LD50s (r = 0.879), and was stronger than the correlation of NHK cell IC50s vs. rat LD50s (r = 0.844). In addition, LT50 was significantly correlative with the LC50 of C. elegans, indicating that both can be utilized as toxic effect index for further study on acute toxicity testing of chemicals. In summary, C. elegans may be a valuable model for predicting chemicals' acute toxicity in rodents. © 2013 The Royal Society of Chemistry.

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