Project description:Definition of different Tn4001-derived mini-transposons carrying reporters with no translation initiation codons so they can only be expressed when fused to an endogenous protein. We used Chloramphenicol acetyltransferase (Cm) as a positive selection marker while the RNAse barnase (Barn) was used as a negative one. In this case, the mini-transposon has a constitutively expressed chloramphenicol resistance gene downstream to the Barn gene. Also, Erythromycin esterase EreA (Ery) was used to evaluate the effect of oligomerization of protein-fusion resistances, as Ery is a dimeric resistance while Cm forms a tetramer. In the positive selection, for the bacteria to survive in the presence of Chloramphenicol, the transposed reporter needs to be inserted in-frame to a genome protein-coding sequence. In the negative selection, the transposed gene should be out of frame with the genome protein-coding sequence for the bacteria to survive.
Project description:We performed shallow whole genome sequencing (WGS) on circulating free (cf)DNA extracted from plasma or cerebrospinal fluid (CSF), and shallow WGS on the tissue DNA extracted from the biopsy in order to evaluate the correlation between the two biomaterials. After library construction and sequencing (Hiseq3000 or Ion Proton), copy number variations were called with WisecondorX.
Project description:The ability to sense and adapt to a hostile host environment is a crucial element for virulence of pathogenic fungi, including Cryptococcus neoformans. These cellular responses are evoked by diverse signaling cascades, including the stress-activated HOG pathway. Despite previous analysis of central components of the HOG pathway, its downstream signaling network is poorly characterized in C. neoformans. Here we performed comparative transcriptome analysis with HOG signaling mutants to explore stress-regulated genes and their correlation with the HOG pathway in C. neoformans. In this study we not only provide important insights into remodeling patterns of global gene expression for counteracting external stresses, but also elucidated novel characteristics of the HOG pathway in C. neoformans. First, inhibition of the HOG pathway increases expression of ergosterol biosynthesis genes and cellular ergosterol content, conferring a striking synergistic antifungal activity with amphotericin B and providing an excellent opportunity to develop a novel therapeutic method for treatment of cryptococcosis. Second, a number of cadmium-sensitive genes are differentially regulated by the HOG pathway, and their mutation causes resistance to cadmium. Finally, we have discovered novel stress-defense and HOG-dependent genes, which encodes a sodium/potassium efflux pump, protein kinase, multidrug transporter system, and elements of the ubiquitin-dependent system.
Project description:Whole genome sequencing (WGS) of tongue cancer samples and cell line was performed to identify the fusion gene translocation breakpoint. WGS raw data was aligned to human reference genome (GRCh38.p12) using BWA-MEM (v0.7.17). The BAM files generated were further analysed using SvABA (v1.1.3) tool to identify translocation breakpoints. The translocation breakpoints were annotated using custom scripts, using the reference GENCODE GTF (v30). The fusion breakpoints identified in the SvABA analysis were additionally confirmed using MANTA tool (v1.6.0).
Project description:The ability to sense and adapt to a hostile host environment is a crucial element for virulence of pathogenic fungi, including Cryptococcus neoformans. These cellular responses are evoked by diverse signaling cascades, including the stress-activated HOG pathway. Despite previous analysis of central components of the HOG pathway, its downstream signaling network is poorly characterized in C. neoformans. Here we performed comparative transcriptome analysis with HOG signaling mutants to explore stress-regulated genes and their correlation with the HOG pathway in C. neoformans. In this study we not only provide important insights into remodeling patterns of global gene expression for counteracting external stresses, but also elucidated novel characteristics of the HOG pathway in C. neoformans. First, inhibition of the HOG pathway increases expression of ergosterol biosynthesis genes and cellular ergosterol content, conferring a striking synergistic antifungal activity with amphotericin B and providing an excellent opportunity to develop a novel therapeutic method for treatment of cryptococcosis. Second, a number of cadmium-sensitive genes are differentially regulated by the HOG pathway, and their mutation causes resistance to cadmium. Finally, we have discovered novel stress-defense and HOG-dependent genes, which encodes a sodium/potassium efflux pump, protein kinase, multidrug transporter system, and elements of the ubiquitin-dependent system. There is more than 95% genome homology between JEC21 (Cryptococcus neoformans var. neoformans serotype D) and H99 (Cryptococcus neoformans var. grubii serotype A). Therefore, 100 slides of JEC21 70-mer oligo arrays are used in this analysis. 3 biological replicate experiments are performed. Total RNAs are extracted under 3 conditions (1M NaCl, 20ug/ul Fludioxonil, 2.5mM Hydrogen peroxide) at 3 time points (0time, 30min, 60min) with 4 strains from H99 (wild type, hog1Î , ssk1Î , skn7Î). We use a mix of all the total RNAs from this experiment as the control RNA. We use Cy5 as the sample dye and Cy3 as the control dye. Several samples are dye swapped.
Project description:Extracellular vesicles (EVs) are increasingly recognized as key mediators of intercellular communication and immune regulation. However, their proteomic composition in allergic asthma remains poorly understood. In this study, we performed tandem mass tag (TMT)-based quantitative proteomic analyses of the whole bronchoalveolar lavage fluid (BALF) supernatant and EVs enriched from BALF in a murine model of house dust mite (HDM)-induced asthma. Using LC-MS/MS analysis in the data-dependent acquisition (DDA) mode, we identified more than 400 proteins across all samples, revealing distinct enrichment of canonical EV markers in EV fractions. HDM challenge induced significant upregulation of Th2-associated proteins, e.g., CLCA1, FCGBP, CHIL3, CHIL4, and RETNLA, consistent with hallmark features of asthma. Notably, proteins such as EPX and CKM were detected exclusively in EVs, not in whole BALF, underscoring the potential of EVs to selectively carry disease-relevant proteins. Our results provided insights into the distinct proteomic profiles of EV-associated versus BALF proteins, highlighting potential EV-mediated mechanisms in asthma pathogenesis.