Synphilin-1 is a major component of Parkinson’s disease (PD) inclusion bodies implicated in PD pathogenesis. However, the machinery controlling synphilin-1 inclusion formation remains unclear. Here, we investigated synphilin-1 inclusion formation using a systematic genome-wide, high-content imaging based screening approach (HCI) in the yeast Saccharomyces cerevisiae. By combining with a secondary screening for mutants showing significant changes on fluorescence signal intensity, we filtered out hits that significantly decreased the expression level of synphilin-1. We found 133 yeast genes that didn’t affect synphilin-1 expression but that were required for the formation of synphilin-1 inclusions. Functional enrichment and physical interaction network analysis revealed these genes to encode for functions involved in cytoskeleton organization, histone modification, sister chromatid segregation, glycolipid biosynthetic process, DNA repair and replication. All hits were confirmed by conventional microscopy. Complementation assays were performed with a selected group of mutants, results indicated that the observed phenotypic changes in synphilin-1 inclusion formation were directly caused by the loss of corresponding genes of the deletion mutants. Further growth assays of these mutants showed a significant synthetic sick effect upon synphilin-1 expression, which supports the hypothesis that matured inclusions represent an end stage of several events meant to protect cells against the synphilin-1 cytotoxicity.
Background
For microorganisms on a paper surface, the lack of water is one of the most important stress factors. A strain of Bacillus megaterium FDU301 was isolated from plaques on a paper surface using culture medium with polyethylene glycol 200 (PEG200) to simulate an arid condition. Global transcriptomic analysis of B. megaterium FDU301 grown under normal and simulated arid conditions was performed via RNA-seq technology to identify genes involved in arid stress adaptation.
Results
The transcriptome of B. megaterium FDU301 grown in LB medium under arid (15% PEG200 (w/w)) and normal conditions were compared. A total of 2941 genes were differentially expressed, including 1422 genes upregulated and 1519 genes downregulated under arid conditions. Oxidative stress-responsive regulatory genes perR, fur, and tipA were significantly upregulated, along with DNA protecting protein (dps), and catalase (katE). Genes related to Fe2+ uptake (feoB), sporulation stage II (spoIIB, spoIIE, spoIIGA), small acid-soluble spore protein (sspD), and biosynthesis of compatible solute ectoine (ectB, ectA) were also highly expressed to various degrees. Oxidative phosphorylation-related genes (atpB, atpE, atpF, atpH, atpA, atpG, atpD, atpC) and glycolysis-related genes (pgk, tpiA, frmA) were significantly downregulated.
Conclusion
This is the first report about transcriptomic analysis of a B. megaterium to explore the mechanism of arid resistance. Major changes in transcription were seen in the arid condition simulated by PEG200 (15%), with the most important one being genes related to oxidative stress. The results showed a complex mechanism for the bacteria to adapt to arid stress.
Probiotics are universally recognized for their health benefits, despite the fact that their effects depend on the strain. Identification and enumeration of probiotic strains are required prior to evaluating their effectiveness. Lacticaseibacillus rhamnosus X253 is a potential probiotic strain with antioxidant capacity. Comparative genomics and single nucleotide polymorphisms (SNPs) were used to identify a strain-specific locus within the holA gene for strain X253 that was distinct in 30 different L. rhamnosus strains. Using quantitative PCR, the primers and probe designed for the locus were able to distinguish L. rhamnosus X253 from the other 20 probiotic strains. The chosen locus remained stable over 19 generations. The sensitivity of the assay was 0.2 pg genomic DNA of L. rhamnosus X253, or 103 cfu/mL bacteria of this strain. In terms of repeatability and reproducibility, relative standard deviations (RSD) were less than 1% and 3%, respectively. Additionally, this assay achieved accurate enumerations of L. rhamnosus X253 in spiked milk and complex powder samples. The strain-specific assay could be used for quality control and compliance assessment of dairy products.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.