Project description:Drought is a stressor for many soil-inhabiting organisms. Although plants have been extensively investigated for drought-adaptive mechanisms, little information is available for fungi. Antioxidants are especially relevant, since desiccation is accompanied by an excessive intracellular production of reactive oxygen species. Riboflavin (vitamin B2) is one antioxidant regulating drought tolerance in plants. A similar function may exist in fungi. Here, we examined the respiratory and transcriptional responses of Agaricus bisporus to drought and the impact of riboflavin. Mesocosm experiments with four groups were established: hyphae were treated with or without 50 µM riboflavin under drought or no drought conditions. Drought increased riboflavin content in hyphae about 5 times with, but also without, addition of riboflavin. Without addition of riboflavin, fungal respiration decreased by more than 50% at water potentials of about -20 MPa. With addition of riboflavin, respiration remained about 2-3 times higher. The transcriptional responses to only drought or only riboflavin strongly overlapped and were mainly based on factors regulating transcription and translation. This was even stronger in combined treatments. Riboflavin induced protective mechanisms in drought-stressed hyphae. Most pronounced was the methylglyoxal (cytotoxic by-product of glycolysis) detoxifying of lactoylglutathione lyase. Thus, our data suggest a stress-priming function and a role of riboflavin in drought responses of A. bisporus.
Project description:Transcriptome analysis of RNA samples from riboflavin-depleted HEK293T cells. Riboflavin is an essential component of the human diet and its derivative cofactors have an established role in oxidative metabolism. Riboflavin deficiency has been linked with various human diseases. In this study, we have modelled riboflavin deficiency in HEK293T cell line, shown remarkable increase of cell proliferation. The aim of the present study is to develop a cell culture model of riboflavin depletion and analyze its molecular mechanism of acceleration cell proliferation. So, we used Affymetrix Human Transcriptome Array 2.0 to identify genes that were differentially expressed upon riboflavin-depleted HEK293T cells.
Project description:Microarray analysis has been applied to the cell proliferation in a human colonic cel line, Caco-2. We have shown previously that a moderate riboflavin depletion around weaning has a profound impact on the structure and function of the small intestine of the rat, which is not reversible following riboflavin repletion. In this study we have modelled riboflavin deficiency in a human cell line, shown irreversible loss of cell viability associated with impaired mitosis and identified candidate effectors of riboflavin depletion in the cell. The aim of the present study is to develop a cell culture model of riboflavin depletion and analyse its behaviour using a using a combination of cell biology approaches including microarray analysis. A human colonic cell line, Caco-2, was grown in culture and treated to riboflavin depletion. Treated and untreated cells were collected at appropriate time points and isolated RNA subjected to microarray analysis.
Project description:Riboflavin-derived molecules such as flavin mononucleotide and flavin adenine dinucleotide comprise the most important redox coenzymes all across life kingdoms. Vibrio cholerae, a pandemic diarrheagenic bacterium, is able to synthesize riboflavin through the riboflavin biosynthetic pathway (RBP) and to internalize exogenous riboflavin using the RibN importer. This bacterium thrives in different environments such as estuarine waters and the human intestinal tract. The presence of two independent riboflavin supply pathways in this species may be related to the variable riboflavin availability and requirements across different niches. In order to gain insights into the role of the riboflavin provision pathways in the physiology of V. cholerae, we analyzed the bacterial transcriptomics response to extracellular riboflavin and to the deletions of ribD (RBP-deficient strain) or ribN. Notably, many genes responding to riboflavin have been previously reported to belong to the iron regulon. Real time PCR analysis confirmed this effect and further documented that reciprocally, iron regulates RBP and ribN genes in a riboflavin-dependent way. A subset of genes were responding to both ribD and ribN deletions. Nonetheless, a group of genes was specifically affected in the ∆ribD strain, on which the functional terms protein folding and oxidation reduction process were enriched. Also, a subset of genes was affected specifically in the ∆ribN strain, on which the cytochrome complex assembly functional term was enriched. Results indicate that iron and riboflavin interrelate to regulate its respective provision genes and suggest that both common and specific effects of biosynthesized and internalized riboflavin exist.