Project description:Gene expression of Condine wild type tomato seedling aerial parts grown on MS medium was compared with that of alpha-dioxygenase2 Divaricata (div) mutant
Project description:Leaf growth is a complex developmental process that is continuously fine-tuned by the environment. Various abiotic stresses, including mild drought stress, have been shown to inhibit leaf growth in Arabidopsis thaliana (Arabidopsis), but the underlying mechanisms remain largely unknown. Here we identify the redundant Arabidopsis transcription factors ETHYLENE RESPONSE FACTOR 5 (ERF5) and ERF6 as master regulators which adapt leaf growth to environmental changes. ERF5 and ERF6 gene expression is induced very rapidly and specifically in actively growing leaves after sudden exposure to osmotic stress that mimics mild drought. Subsequently, enhanced ERF6 expression inhibits cell proliferation and leaf growth by a process involving GA and DELLA signaling. Using an ERF6 inducible overexpression line, we demonstrate that the GA-degrading enzyme GA2-OX6 is transcriptionally induced by ERF6 and that consequently DELLA proteins are stabilized. As a result, ERF6 gain-of-function lines are dwarfed and hypersensitive to osmotic stress, while growth of erf5erf6 loss-of-function mutants is less affected by stress. Next to its role in plant growth under stress, ERF6 also activates the expression of a plethora of osmotic stress-responsive genes, including the well-known stress tolerance genes STZ, MYB51 and WRKY33. Interestingly, the activation of the stress tolerance genes by ERF6 occurs independently from the ERF6-mediated growth inhibition. Together, these data fit into a leaf growth regulatory model in which ERF5 and ERF6 form a missing link between the previously observed stress-induced 1-aminocyclopropane-1-carboxylic acid (ACC) accumulation and DELLA-mediated cell cycle exit and execute a dual role by regulating both stress tolerance and growth-inhibition. Samples were obtained from three independent experiments and from multiple plates within the experiment. Whole seedlings were harvested rapidly in an excess of RNAlater® solution (Ambion), and after overnight storage at 4°C, dissected under a binocular microscope on a cooling plate with precision microscissors. Dissected leaves were transferred to a new tube, frozen in liquid nitrogen, and ground with a Retsch machine and 3-mm metal balls. RNA was extracted with TriZol (Invitrogen) and further purified with the RNeasy Mini Kit (Qiagen). DNA digestion was done on columns with RNase-free DNase I (Roche). For the identification of genome-wide expression changes, samples of the strong ERF6-overexpressing line (ERF6IOE-S) and the control line (GFP:IOE) were harvested 4 h after transfer to DEX. Two µg of pure RNA samples were hybridized to AGRONOMICS1 Arabidopsis Tiling Arrays (Rehrauer et al., 2010) at the VIB Microarray Facility (Leuven, Belgium).
Project description:In this study, rats were exposed to three different doses of nanomaterials (SiO2_7, TiO2_NM105) using short-term inhalations. Effects on exposure and recovery groups were compared using untargeted proteomics.
Project description:In this study, rats were exposed to three different doses of nanomaterials (SiO2_15_Amino, SiO2_15_Unmod, SiO2_7, SiO2_40) using instillations. Effects on exposure and recovery groups were compared using untargeted proteomics.
Project description:BRCA1/2-deficient ovarian carcinoma (OC) has been shown to be particularly sensitive to PARP inhibitors (PARPis) and BRCA1/2 mutation status is currently used as a predictive biomarker for PARPi therapy. Despite eliciting major clinical benefit for the majority of patients, a significant proportion of BRCA1/2-deficient OC tumors do not respond to PARPis for reasons that are incompletely understood. Using an integrated chemical, phospho- and ADP-ribosylation proteomics approach we sought to develop additional mechanism-based biomarker candidates for PARPi therapy in OC and identify new targets for combination therapy to overcome primary resistance. Chemical proteomics with PARPi baits in a BRCA1-isogenic OC cell line pair, as well as patient-derived BRCA1-profiecient and deficient tumor samples, and subsequent validation by co-immunoprecipitation showed differential PARP1 and PARP2 protein complex composition with Ku70 and Ku80 in PARPi-sensitive, BRCA1-deficient UWB1.289 (UWB) cells compared to PARPi-insensitive, BRCA1-reconstituted UWB1.289 (UWB+B) cells. Global phosphoproteomics and ADP-ribosylation proteomics furthermore revealed that rucaparib induced the cell cycle pathway and NHEJ pathway in UWB cells, but down-regulated ErbB signaling in UWB+B cells. In addition, we observed AKT PARylation and pro-survival AKT-mTOR signaling in UWB+B cells after PARPi treatment. Consistently, synergy of PARPis with DNAPK or AKT inhibitors was more pronounced in UWB+B cells identifying these pathways as actionable vulnerabilities. In conclusion, the combination of chemical proteomics, phosphoproteomics and ADP-ribosylation proteomics can identify differential PARP1/2 complexes and diverse, but actionable drug compensatory signaling in OC.
Project description:We report the application of next-generation sequencing technology for high-throughput profiling of H3K27ac and transcriptome analysis in pancreatic islets derived from C57Bl/6 mice fed a high-fat diet. We find genomic regions showing change in acetylation of histone H3K27 in response to long-term HFD feeding, which was significantly associated with differential gene expression. Furthermore, increased H3K27ac showed a distinctive genomic distribution surrounding proximal-promoter regions. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cells under various environments.
Project description:Pseudomonas aeruginosa strain PAO1 was grown at 22M-BM-0C and 37M-BM-0C in Lysogeny broth (LB) and RNA was hybridized on the Affymetrix P. aeruginosa chip. PAO1 was grown in triplicate in Lysogeny broth at 22M-BM-0C or 37M-BM-0C. Total RNA from each sample was pooled and amplified then assayed in triplicate resulting in 6 total samples.
Project description:Pseudomonas aeruginosa strains PAHM4 and PAO1 were grown at 37C on LB and RNA was hybridized on the Affymetrix P. aeruginosa chip to compare transcript differences from a BQ isolate to a well characterized wound isolate. Strains PAHM4 and PAO1 were grown in triplicate. Total RNA from each sample was pooled and amplified then assayed in triplicate resulting in 6 total samples. PAO1 is the control strain while PAHM4 is the experimental strain.
Project description:Hypersensitive response-related programmed cell death (PCD) has been extensively analyzed in various plant–virus interactions. However, little is known about changes in gene expression associated with cell death caused by compatible viruses. The synergistic interaction of Potato virus X (PVX) with Plum pox virus (PPV) results in increased symptoms that lead to systemic necrosis (SN) in Nicotiana benthamiana. Here, we performed three transcriptome comparisons in response to i) a PVX recombinant virus expressing the helper component-proteinase (HC-Pro) gene from PPV that leads to SN, ii) a systemic incompatible interaction conferred by the Tobacco mosaic virus (TMV)-resistance gene N (SHR), and iii) the depletion of the PBE subunit of the proteasome that leads to PCD by virus induced gene silencing (VIGS Prot), at early and late stages of infection. Our analysis indicated that the SN response was clustered with SHR by the similarity of their overall gene expression profiles. However, the expression profiles of defence-related and hormone-responsive genes in response to SN were more closely related to the response to VIGS Prot than to that elicited by SHR. This suggests the potential contribution of proteasome dysfunction to the increase in pathogenicity observed in PVX-potyvirus infections We compare the gene expression profiles of Nicotiana benthamiana plants infected with either necrosis-inducing viruses or non-necrosis-inducing viruses, as follow: PVX/HCWT-infected plants versus plants infected with a PVX recombinant virus expressing a PPV HC-Pro mutant (PVX/HCLH) that was unable to induce the SN response (SN comparison), at 7 and 11 days postinoculation (dpi), (ii) TRV:NbPBE-silenced plants versus plants infected with the TRV empty vector (VIGS Prot comparison), at 4 and 8 days after infiltration (dpa), and (iii) TMV-GFP-infected, N-transgenic plants versus wild-type plants infected with TMV-GFP (SHR comparison), at 24 and 72 hours after temperature shift (hts). Per time and treatment, three independent biological replicates were used to monitor differences in gene expression between treatments
Project description:5-methylcytosine (5-mC) can be oxidized to 5-hydroxymethylcytosine (5-hmC). Genome-wide profiling of 5-hmC thus far indicated 5-hmC may not only be an intermediate form of DNA demethylation but could also constitute an epigenetic mark per se. We describe a cost-effective and selective method to detect both the hydroxymethylation and methylation status of cytosines in more than 1.8 million MspI sites in the human genome. This method involves the selective glucosylation of 5-hmC residues, short-sequence tag generation and high-throughput sequencing. We tested this method by screening H9 human embryonic stem cells and their differentiated embroid body cells, and found that differential hydroxymethylation preferentially occur in bivalent genes during cellular differentiation. Especially, our results support hydroxymethylation can regulate key transcription regulators with bivalent marks through demethylation and affect cellular decision on choosing active or inactive state of these genes upon cellular differentiation. We developed a cost-effective and selective method to detect both the hydroxymethylation and methylation status of cytosines in more than 1.8 million MspI sites in the human genome. In this method, we took advantage of the differential enzymatic sensitivities of the isoschizomers MspI and HpaII. HpaII cleaves only a completely unmodified site, any modification at either cytosine blocks the cleavage, while MspI recognizes and cleaves both 5-mC and 5-hmC, but not the newly discovered 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Furthermore, beta-glucosyltransferase (beta-GT) can transfer a glucose to the hydroxyl group of 5-hmC and generate beta-glucosyl-5-hydroxymethylcytosine (5-ghmC) that blocks MspI digestion. Thus, either by combining beta-GT treatment with MspI digestion or simply applying MspI/HpaII digestion, short sequence tags generated can be used for inferring hydroxymethylation or methylation status in around 1.8 million cytosine sites in the human genome. We tested this method by screening H9 human embryonic stem cells and their differentiated embroid body cells.