Project description:Polymicrobial biofilms are of large medical importance, but little is known about their physiology and the underlying interspecies interactions. Here we studied two human pathogens, the opportunistic fungus Candida albicans and the caries promoting bacterium Streptococcus mutans. Both species formed biofilms in monoculture, with C. albicans growing mainly in the virulence-associated hyphae form, and S. mutans forming a thick layer of extracellular polymeric substances (EPS). Biofilm growth was enhanced in dual-species biofilms, which reached twice the biomass of monospecies biofilms and higher cell numbers of both S. mutans and C. albicans. EPS production by S. mutans was strongly suppressed in dual-species biofilms. Virulence traits of S. mutans, e.g. genetic competence, biofilm formation and bacteriocin synthesis are controlled by quorum sensing through activation of the alternative sigma factor SigX. SigX is induced by the pheromones CSP (competence stimulating factor) or XIP (sigX inducing peptide). Strong induction of sigX was observed in dual species biofilms indicated by fluorescence of a reporter strain for the sigX promoter, S. mutans PcomX-gfp, as well as by qRT-PCR of comX. The peak of sigX expression occurred after 10 h of biofilm growth. Conditioned media from mixed biofilms but not from C. albicans or S. mutans cultivated alone activated sigX in the reporter strain. Deletion mutants for the comC and comS genes encoding the precursors of CSP and XIP, respectively, were constructed. Conditioned media from mixed biofilms with S. mutans DcomS were unable to induce sigX in the reporter strain, while deletion of comC had no effect. These data show that synthesis of XIP was induced in S. mutans by coculture with C. albicans. Transcriptome analysis of S. mutans in single and mixed biofilms confirmed strong induction of comS, sigX, and the downstream late competence genes in dual-species biofilms. Among the late competence genes, fratricins were discovered for the first time. The comCDE operon and bacteriocin related genes were also induced, but much weaker. Genes related to oxidative stress, chaperones and glycosyltransferase genes required for EPS synthesis from sucrose were down-regulated, while glycogen synthesis genes were up-regulated, indicating that S. mutans was protected from oxidative stress and provided with excess sugar for storage polymer synthesis in mixed biofilms. The data show that in dual-species biofilms, C. albicans improves growth of S. mutans, suppresses its EPS formation and induces the complete quorum sensing signalling system, thus fundamentally changing the virulence properties of the caries pathogen, including its potential interactions with other members of the polymicrobial dental plaque community. Dual-species biofilms of S. mutans and C. albicans and single-species biofilms of S. mutans were cultivated in 24-well microtitre plates in YNBB medium. Transcriptional profiles of S. mutans in single- and dual-species biofilms were analysed at early (6 h) and late (10 h) logarithmic phase of the biofilm growth, as well as after 24 h when biofilms entered stationary phase. Transcriptional profiles of S. mutans grown in the dual-species biofilms were compared to profiles obtained for single-species biofilm from the same time point. Three biological and one to two technical replicas were used in the microarray study. RNA samples were labeled with Cy3 or Cy5 using the ULS fluorescent labeling kit (Kreatech, Germany). Seven hundred nanograms of Cy3 or Cy5 labeled RNA after fragmentation were hybridized to the microarray at 65°C for 17 h using the Agilent hybridization chamber according to the manufacturer's instructions. The arrays were scanned using the Agilent DNA microarray scanner and the raw data were extracted using Agilent Feature Extraction software (v. 10.7).
Project description:Functional-assay limitations are an emerging issue in characterizing human pluripotent stem cells (hPSCs). With rodent PSCs, chimera formation, using pre-implantation embryos, is the gold-standard assay of pluripotency. In hPSCs, this can only be monitored via teratoma formation or in vitro differentiation, as ethical concerns preclude generation of human-animal chimera. To circumvent this issue, we established a functional assay utilizing interspecific blastocyst injection and in vitro culture (interspecies in vitro chimera assay). The assay uses mouse pre-implantation embryos and human PSCs to make interspecies chimeras cultured in vitro to the early egg cylinder stage. When hiPSCs, both conventional and naive type, which called “reset cell”, were injected into mouse embryos and cultured. The cells were never integrated into the epiblast of egg cylinder stage-embryo. These results suggest that hPSCs, including naïve type, are unable to form chimera with mouse embryo. Reset cells were converted from conventional human iPSC line PB004, and then compared their gene expression profile with or without transgene overexpression induced by doxycyclin treatment.
Project description:By investigating the germinal center (GC) formation in STAT6ko/WT bone marrow-mixed chimera we found that GC formation in type 2 immune responses is dependent on B cell intrinsic expression of IL-4/IL-13-induced genes. We therefore used microarrays to find Stat6 dependent genes that are important for germinal center formation and/or organization after infection with the nematode Nippostrongylus brasiliensis (N. brasiliensis). Bone marrow of STAT6ko (CD45.2+) and WT (CD45.1+) were mixed and injected in lethaly irradiated WT (CD45.1+) mice. After 8 weeks, 5 Bone marrow-mixed chimera were infected with N. brasiliensis and draining lymph nodes were collected at day 14 after the infection and pooled. RNA was isolated from sort-purified CD45.1+ or CD45.2+ GC B cells (B220+CD38loGL-7hi).
Project description:This is a dataset which comprises the following two different kinds of genomic data in Drosophila species: First, triplicate ChIP-seq data of CTCF (CCCTC binding factor) binding profiles in each of the four closely related Drosophila species : Drosophila melanogaster, Drosophila simulans, Drosophila yakuba and Drosophila pseudoobscura at white pre pupa stage; Second, triplicate RNA-seq data of white pre pupa whole animals of three Drosophila species: Drosophila melanogaster, Drosophila simulans and Drosophila yakub. The binding site/region/peaks are called using a modified method of QuEST( please see details in our related publication). The sequence read counts and RPKM values are calculated following the method in Mortazavi et al 2008 Nature Methods paper. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Examination of CTCF binding in 4 Drosophila species and their correlation with gene expression levels in the same development stages
Project description:Analyses of new genomic, transcriptomic or proteomic data commonly result in trashing many unidentified data escaping the ‘canonical’ DNA-RNA-protein scheme. Testing systematic exchanges of nucleotides over long stretches produces inversed RNA pieces (here named “swinger” RNA) differing from their template DNA. These may explain some trashed data. Here analyses of genomic, transcriptomic and proteomic data of the pathogenic Tropheryma whipplei according to canonical genomic, transcriptomic and translational 'rules' resulted in trashing 58.9% of DNA, 37.7% RNA and about 85% of mass spectra (corresponding to peptides). In the trash, we found numerous DNA/RNA fragments compatible with “swinger” polymerization. Genomic sequences covered by «swinger» DNA and RNA are 3X more frequent than expected by chance and explained 12.4 and 20.8% of the rejected DNA and RNA sequences, respectively. As for peptides, several match with “swinger” RNAs, including some chimera, translated from both regular, and «swinger» transcripts, notably for ribosomal RNAs. Congruence of DNA, RNA and peptides resulting from the same swinging process suggest that systematic nucleotide exchanges increase coding potential, and may add to evolutionary diversification of bacterial populations.
Project description:Design a human-mouse dual-species microarray to provide a technology for investigation of cancer-stromal interaction in xenograft model. The 2 reference RNA were used to evaluate and identify probes with potential the cross-species hybridization, so that we can label these probes and exclude them before analysis of biological samples/data. Reference RNA from human and mouse were labeled using Cy3. Samples were hybridized on customized-commercial array with Agilent probes and user-designed species-specific probes (Human-mouse dual-species array HomoMus_v2). This array is designed and improved from hte first version GPL10714
Project description:Using EWS-FLI and its parental transcription factor, FLI1, we created a unique experimental system to address questions regarding the genomic mechanisms by which chimeric transcription factors cause cancer. We found that in tumor cells, EWS-FLI targets regions of the genome distinct from FLI1, despite identical DNA-binding domains. In primary endothelial cells, however, EWS-FLI and FLI1 demonstrate similar targeting. To understand this mistargeting, we examined chromatin organization. Regions targeted by EWS-FLI are normally repressed and nucleosomal in primary endothelial cells. In tumor cells, however, bound regions are nucleosome-depleted and harbor the chromatin signature of enhancers. We next demonstrated that through chimerism, EWS-FLI acquired the ability to alter chromatin. Expression of EWS-FLI results in nucleosome depletion at targeted sites, whereas silencing of EWS-FLI in tumor cells restored nucleosome occupancy. Thus, the EWS-FLI chimera acquired chromatin-altering activity, leading to mistargeting, chromatin disruption, and ultimately transcriptional dysregulation. Examination of two transcription factors in two different cell types, as well as three histone methylation marks and FAIRE
Project description:Design a human-mouse dual-species microarray to provide a technology for investigation of cancer-stromal interaction in xenograft model. The 2 reference RNA were used to evaluate and identify probes with potential the cross-species hybridization, so that we can label these probes and exclude them before analysis of biological samples/data. Overall design: Reference RNA from human and mouse were labeled using Cy3. Samples were hybridized on customized-commercial array with Agilent probes and user-designed species-specific probes (Human-mouse dual-species array HomoMus_v2). This array is designed and improved from hte first version GPL10714
Project description:We examined the differential gene expression of Staphylococcus epidermidis and Staphylococcus epidermidis in dual species biofilms. Therefore, we performed RNA-Seq on single and dual species biofilms and we compared the gene expression levels in dual species biofilms to those in single species biofilms. Overall design: RNA was extracted from single and dual species biofilms of S. epidermidis ET-024 and S. aureus Mu50, converted to cDNA and RNA-Seq was performed. Three biological replicates were included.
Project description:Somatic cells can be reverted back to a pluripotent state by nuclear transfer, cell fusion, and defined factors, representing three distinct approaches to reprogram cell fate. Unlike cell fusion, reprogramming by nuclear transfer or defined factors gives rise to cells that contribute to the development of a live animal in mouse, thus making a quasi-direct comparison possible Combining optimized culture conditions and modified reprogramming factors, we report here that mouse fibroblasts can be reprogrammed by defined factors into cells capable of contributing to chimera formation and germline transmission in as short as 96 hours, comparable to the time required for the formation of inner cell mass in cloned embryos by nuclear transfer. Gene expression profiling analysis shows that this accelerated reprogramming process in fact corresponds well with previously reported longer ones with similar molecular signatures. Additionally we find a new set of genes activated as the reprogramming cells acquire chimera competency. In a broader sense, this platform may be adopted for applications such as high throughput screenings for reprogramming mediators both chemical and biological, as well as omics-related mechanistic investigations. Overall design: We examined transcriptome transition during somatic cells reprogramming using two reprogramming systems, OSK system and OvSvK system (Ov is Oct4-vp16 and Sv is Sox2-vp16). We can get reprogrammed cells capable of contributing to chimera formation and germline transmission in 4 day using OvSvK system and 7 day in OSK system, so, we got samples at D0, D1, D2, D3, D4 after OvSvK transfection and performed RNA-seq, similarly, we sequenced the samples at D0, D1, D3, D5, D7 in OSK system. Furthermore, we also sequenced several control samples, such as GFP samples at different time points of reprogramming. At the same time, we examined the effect of several elements on transcriptome during reprogramming, including Vitamin C, bGFG, GSK3 inhibitor(CHIR-99021 and LiCl).