Effect of Lead on Genome-Wide Expression in Developing Mouse Brain during Synaptogenesis
ABSTRACT: Lead exposure causes a variety of health effects, especially in children, that may include cognitive and behavioural problems. This study explores the mechanisms associated with this relationship by assessing alterations in gene expression of C57BL/6J pups treated with 50mg/kg lead compared to controls. In addition this study also analyzed brain gene expression differences in Metallothionein I and II (Mt-I and Mt-II) knockout mice treated with lead. Pups of three genotypes (C57BL/6J, Mt-KO with a C57BL/6J background and Heterozygote Mt-KO) were injected with lead acetate during synaptogenesis and weight-matched controls were injected with saline at the same time points. Whole brains were harvested and RNA extracted and pooled from 5 pups and hybridized to Mouse Genome 430 2.0 Arrays. In total 6 arrays were used, one for each genotype and treatment.
Project description:The blossom of immunotherapy in melanoma highlights the need to delineate mechanisms of immune resistance. Recently, we have demonstrated that the RNA editing protein, adenosine deaminase acting on RNA-1 (ADAR1) is down-regulated during metastatic transition of melanoma, which enhances melanoma cell proliferation and tumorigenicity. Here we investigate the role of ADAR1 in melanoma immune resistance. Importantly, knockdown of ADAR1 in human melanoma cells induces resistance to tumor infiltrating lymphocytes in a cell contact-dependent mechanism. We show that ADAR1, in an editing-independent manner, regulates the biogenesis of miR-222 at the transcription level and thereby Intercellular Adhesion Molecule 1 (ICAM1) expression, which consequently affects melanoma immune resistance. ADAR1 thus has a novel, pivotal, role in cancer immune resistance. Corroborating with these results, the expression of miR-222 in melanoma tissue specimens was significantly higher in patients who had no clinical benefit from treatment with ipilimumab as compared to patients that responded clinically, suggesting that miR-222 could function as a biomarker for the prediction of response to ipilimumab. These results provide not only novel insights on melanoma immune resistance, but also pave the way to the development of innovative personalized tools to enable optimal drug selection and treatment. 13 formalin fixed paraffin embedded (FFPE) melanoma tissues were stained with hematoxilin and eosin for examination by an expert pathologist. Non-tumor tissue was removed. Total RNA was isolated using miRNeasy FFPE kit (Qiagen) according to the manufacture guidelines.
Project description:Transcriptomic analysis of gene expression during the differentiation of cell suspension cultures into tracheary elements using the biological system published by Pesquet et al., Current Biology (2010): tracheary element differentiation was triggered by externally supplying hormone-free habituated cell suspension cultures of Arabidopsis thaliana Col-0 with auxin, cytokinin and epibrassinolides; RNA samples extracted from 3 independent time-courses every 12h from 0h to 4 days were analyzed using ATH1 Arabidopsis Affymetrix micro-array Time-course design during tracheary element differentiation (3 independepent time-course were harvested from initiation 0h to 4 days every 12h) - total of 27 samples corresponding to the 3 replicates of 9 time point time-courses - gene expression was monitored along the time-course compared to 0h
Project description:Nuclear Protein 1 (Nupr1) is a major actor of the cell stress response required for KrasG12D-driven formation of pancreatic intraepithelial neoplastic (PanINs) lesions in mice. We investigated the impact of Nupr1-depletion on the development and biology of murin pancreatic adenocarcinomas (PDAC) in the Pdx1-cre;LSL-KrasG12D;Ink4a/Arffl/fl (KIC) mice. We found that only one half of Nupr1-deficient mice developed PDAC. This is related to increased caspase 3 activity and low IER3 expression in Nupr1-deficient;KIC in the pancreas. Moreover, when Nupr1-deficient;KIC mice do develop PDAC, tumors present with impaired epithelial-to-mesenchymal transition (EMT). Transcriptoma analysis revealed that Nupr1-deficient and Nupr1wt;KIC PDACs presented enrichment of gene signatures of the human classical- and quasi-mesenchymal (QM)-PDAC respectively. Moreover, Nupr1-deficient;KIC PDACs shared with human classical-PDACs overexpression of Kras-activation genes. In addition, cells derived from Nupr1-deficient;KIC PDACs formed fewer microspheres in vitro compared to Nupr1wt;KIC cells, indicative of stemness impairment in the absence of Nupr1. Finally, we found that Nupr1-deficient;KIC cells were more sensitive to some anticancer drugs than their Nupr1wt counterpart. Hence, this study establishes the pivotal role of Nupr1 in PDAC progression after PanIN and in PDAC EMT in vivo, with an impact in PDAC cell stemness. As a consequence, according to absence or presence of Nupr1, KIC mice develop tumors that phenocopy human classical- or QM-PDAC, respectively, thus becoming attractive models for preclinical drug trials. We investigated the impact of the homozygous deletion of the Nupr1 gene on pancreatic adenocarcinoma development and biology in the Pdx1-cre;LSL-KrasG12D;Ink4a/Arffl/fl (KIC) mouse model.
Project description:Uterine NK cells (uNK) play a role in the regulation of placentation but their functions in non-pregnant endometrium are not understood. We have previously reported suppression of endometrial bleeding and alteration of spiral artery morphology in women exposed to asoprisnil, a progesterone receptor modulator (PRM). We now compare global endometrial gene expression in asoprisnil-treated versus control women and demonstrate a statistically significant reduction of genes in the IL-15 pathway, known to play a key role in uNK development and function. Suppression of IL-15 by asoprisnil was also observed at mRNA level (p<0.05), and immunostaining for NK cell marker CD56 revealed a striking reduction of uNK in asoprisnil-treated endometrium (p<0.001). IL-15 levels in normal endometrium are progesterone-responsive. Progesterone receptor (PR) positive stromal cells transcribe both IL-15 and IL-15RA. Thus, the response of stromal cells to progesterone will be to increase IL-15 trans-presentation to uNK, supporting their expansion and differentiation. In asoprisnil-treated endometrium, there is a marked down-regulation of stromal PR expression and virtual absence of uNK. These novel findings indicate that the IL-15 pathway provides a missing link in the complex interplay between endometrial stromal cells, uNK and spiral arteries affecting physiological and pathological endometrial bleeding. 39 Samples
Project description:Two null mutants of MYB103 were found to have decreased expression of FERULATE-5-HYDROXYLASE and reduced syringyl lignin in the basal inflorescence stem. The aim with this microarray experiment was to investigate the transcriptome of these myb103 mutants to better understand the mechanism underlying MYB103 action.
Project description:This experiment was performed in order to assess the specificity of Rad9 binding to S. cerevisiae genome. In another ChIP-chip experiment in SC BCS BPS growth conditions we have found Rad9 present to a significant number of genomic loci with a bias to transcriptionally active regions. In order to see if that pattern was random and depended or not on the activity state of the genes, we conducted a Rad9 ChIP on chip experiment with the strain grown in medium with galactose instead of glucose, where it is known that particular gene groups are transcriptionally activated. We then compared to the results of our previous experiment where the strain was grown in glucose.
Project description:A 3rd generation Wye3aHamster microarray chip was used to carry out a differential expression microarray study of a panel of 30 slow- and fast-growing production cell lines, identifying a priority list of 240 transcripts (110 Up; 130 Down) that passed a statisical filter (ANOVA p-value <0.05, 1.3 fold-change) when the samples were grouped into fast versus slow and pairwise compared and a minimum Pearson Correlation Coefficent (PCC) of >0.7 when growth rate was used as a continuous variable (i.e. included genes have to satisfy BOTH criteria, not just one). This yielded a list of 240 genes. The 240-member genelist yielded 223 Annotated IDs, 203 of which are Unique and all of which are associated with contributing to a high rate of growth in production CHO cell lines. Prospective samples were isolated from 2 distinct groups of clones (slow growers and fast growers), all chosen from the same project (transfection of product transgene into parent line) generated at Pfizer Inc., Bioprocess R&D, Andover, MA, USA. Each group comprised 5 distinct clones, all of which had comparable Qp characteristics. The clones were passaged for 3 or 4 passages over 3-4 days to stabilise the phenotype and freeze stocks. Samples for array hybridization were generated in batch shake flask culture (60ml working volume) with AS1 medium and without feeds or temperature shift. Samples were collected at a single time point (72hrs - mid/late log) and each clone was grown in triplicate flasks, yielding a total of 30 samples for collection and processing. Two bioprocess-relevant variables, viable cell density (VCD) and viability were measured. From the VCD, the growth rate (h-1) was calculated.
Project description:Huntington's disease (HD) is a dominantly inherited genetic disease caused by mutant huntingtin (htt) protein with expanded polyglutamine tracts. A neuropathological hallmark of HD is the presence of neuronal inclusions of mutant htt. p62 is an important regulatory protein in selective autophagy, a process by which aggregated proteins are degraded, and it is associated with several neurodegenerative disorders including HD. Here we investigated the effect of p62 depletion in three HD model mice: R6/2, HD190QG and HD120QG mice. We found that loss of p62 in these models led to longer lifespans and reduced nuclear inclusions, although cytoplasmic inclusions increased with polyglutamine length. In mouse embryonic fibroblasts (MEFs) with or without p62, mutant htt with a nuclear localization signal (NLS) showed no difference in nuclear inclusion between the two MEF types. In the case of mutant htt without NLS, however, p62 depletion increased cytoplasmic inclusions. Furthermore, to examine the effect of impaired autophagy in HD model mice, we crossed R6/2 mice with Atg5 conditional knockout mice. These mice also showed decreased nuclear inclusions and increased cytoplasmic inclusions, similar to HD mice lacking p62. These data suggest that the genetic ablation of p62 in HD model mice enhances cytoplasmic inclusion formation by interrupting autophagic clearance of polyQ inclusions. This reduces polyQ nuclear influx and paradoxically ameliorates disease phenotypes by decreasing toxic nuclear inclusions. Gene expression profiles were analyzed to examine the effects of p62 depletion in mouse with or without mutant huntingtin exon 1 To examine the effect of p62 depletion on the transcriptome of Huntington's disease model mice, we crossed p62 knockout mice with HD model mice. We extracted total RNA from the striatum of these mice at 8 weeks and used for a microaaray analysis. The samples are HD transgenic mice with p62 knockout (HD_p62KO), HD mice with normal p62 (HD_p62WT), non-HD-transgenic mice with p62 knockout (NT_p62KO), and non-HD-transgenic mice with normal p62 (NT_p62WT).
Project description:To investigate whether the transcriptional response to carbon (C) depletion and sucrose re-addition depend on the duration of C-depletion, Arabidopsis thaliana seedlings growing in liquid culture in weak continuous light were harvested 3, 6, 12, 24, 48 and 72 h after removing sucrose from the medium, and 30 min after resupplying sucrose at each of these times. After removing sucrose, soluble sugars fell strongly within 3 h, and starch was gradually depleted over 24 h, and hexose phosphates and ATP declined gradually over 72 h. Expression profiling using ATH arrays pointed to Overall the transcriptional response pointed to early transcriptional remodelling of metabolism to conserve C, followed by induction of photosynthesis and pathways that recycle C, and repression of growth-related processes. The time-dependent transcriptional response to C-depletion differed from that during a light/dark cycle and an extended night. Re-supplying sucrose for 30 min led to near-complete recovery of seedling sucrose levels, partial recovery of reducing sugars and phosphorylated intermediates, but no immediate change of starch or ATP. The rapid transcriptional response to sucrose readdition was conserved across the entire C-depletion time course, became larger with time. , and was highly enriched for regulatory genes. Whilst there was a rapid decrease of many C-depletion-induced transcripts, fewer transcripts increased. The majority of the transcripts that responded rapidly after resupplying sucrose also decreased after treating C-depleted seedlings with the transcriptional inhibitor cordycepin A, pointing to an important role for transcript turnover in the rapid response to sucrose.