Project description:To study the early events of priming response leading to changes in gene expression in Arabidopsis thaliana Col0 wild type plant on infiltration with the potent elicitor molecule Diffusible Signal Factor (DSF). In the present experimentation we have employed total RNA microarray expression profiling as a discovery platform to identify differentially expressing genes that are upregulated and down regulated in the various pathways involved in priming the defense responses by the elicitor molecule DSF on infiltration in the host plant Arabidopsis thaliana. This would help understand the possible defense pathways elicited by the elicitor molecule during interaction. The rosette leaves of Arabidopsis thaliana Col0 plants of 4 weeks old were infiltrated with 100uM DSF and the total RNA samples at 4hpi, 8hpi and 16hpi were extracted along with methanol treatment serving as a control in two biological replicates 1A and 2A. Further, these RNA samples were checked for the quality and subjected to microarray analysis. The differentially expressing candidate genes were identified specific to DSF treatment and were quantified by real-time PCR post DSF treatments.
Project description:We examined changes in steady-state transcript level in leaves of Arabidopsis plants subjected to salinity, heat stress and their combination by a transcriptome analysis of leaves.
Project description:Introduction: The application of single-cell RNA sequencing has greatly improved our under-standing of various cellular and molecular mechanisms involved in physiological and pathophysi-ological processes. However, obtaining living cells for this technique can be difficult under certain conditions. To solve this problem, the methanol fixation method appeared as a promising alternative for routine clinical use. Materials and Methods: In this study, we selected two AML samples that had been fixed in methanol for 12–18 months. Once the cells were rehydrated, these samples were subjected to single-cell RNA sequencing. We then compared the results obtained from these samples with those obtained from the same samples cryopreserved in DMSO. Results: We used a previously validated methanol fixation protocol to perform scRNA-seq on DMSO cryopreserved cells and cells fixed in methanol for more than one year. Preliminary results show that methanol fixation induces some genetic and transcriptional modification compared with DMSO cryopreservation but remains a valuable method for single-cell analysis of primary human leukemia cells. Conclusions: The initial findings from this study highlight certain resemblances in methanol fixation over a 12-month period and cryopreservation with DMSO, along with associated transcriptional level modifications. However, we observed genetic degradation in the fixation condition when extending beyond one year. Despite certain study limitations, it is evident that short-term methanol fixation can be effec-tively used for leukemia blast samples. Its ease of implementation holds the potential to simplify the integration of this technique into routine clinical practice.
Project description:Methanol is considered as an interesting carbon source in biobased microbial production processes. As Corynebacterium glutamicum is an important host in industrial biotechnology, in particular for amino acid production, we performed studies on the response of this organism to methanol. C. glutamicum wild type was able to convert 13C-labeled methanol to 13CO2. Analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be up-regulated, indicating that some of the corresponding enzymes are involved in methanol oxidation. Indeed, a mutant lacking the alcohol dehydrogenase gene adhA showed a 62% reduced methanol consumption rate, indicating that AdhA is mainly responsible for methanol oxidation to formaldehyde. Further studies revealed that oxidation of formaldehyde to formate is catalyzed predominantly by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. The deletion mutants aldadhE and aldmshC were severely impaired in their ability to oxidize formaldehyde, but residual methanol oxidation to CO2 was still possible. The oxidation of formate to CO2 is catalyzed by the formate dehydrogenase FdhF recently identified by us. Similar to ethanol, methanol catabolism is subject to carbon catabolite repression in the presence of glucose and is dependent on the transcriptional regulator RamA, which was previously shown to be essential for expression of adhA and ald. In conclusion, we were able to show that C. glutamicum possesses an endogeneous pathway for methanol oxidation to CO2 and to identify the enzymes and a transcriptional regulator involved in this pathway.