Project description:The expression of interferon-related genes was more enhanced in irradiated ATM-deficient mouse embryonic fibroblasts (MEFs) than in irradiated ATM wild-type MEFs.
Project description:Exosome-based cell-free therapeutics has received increasing attention in recent decades. Due to the potential demand for therapeutic exosomes, appropriate methods for preservation and storage of exosomes are essential. Current cryopreservation strategies mostly focus on addition of cryoprotectants. However, due to the high concentration of cryoprotectant required, this approach can lead to unfavorable effects. Thus, other storage methods are urgently needed. In this study, we found that Tetraspanin 4 (TSPAN4) and other Tetraspanin family proteins play an essential role in protecting exosomes from cryo-damage. Moreover, we engineered TSPAN4-loaded exosomes which are resistant to cryo-damage. These engineered exosomes show similar properties to wild-type exosomes in protein composition, uptake by recipient cells, and cargo delivery efficiency. We believe our strategy of exosome cryopreservation, without the need for additional agents, will promote the clinical translation of exosomes as therapeutic agents.
Project description:We found that cardiac fibroblasts produce and secrete exosomes. miRNA profiling and TaqMan qRT-PCR experiments identified miR-21 expression to be higher in cardiac fibroblasts compared to those of miR-21*, whereas in exosomes miR-21* expression was higher compared to miR-21. The purpose of the study was to validate these findings by miRNA sequencing in cardiac fibroblasts and fibroblasts-derived exosomes. Neonatal rat cardiac fibroblasts were cultured in DMEM + 1% exosome-depleted FBS for 48h. Conditioned medium was collected and exosomes were purified by several centrifugation and filtration steps, following ultracentrifugation. Afterwards total RNA from cardiac fibroblasts and exosomes was isolated for miRNA sequencing.
Project description:Microarray analysis of differentially expressed genes in wild-type and NFAT5-deficient hematopoietic stem cells (HSC) and multipotent progenitors (MPP)
Project description:RNAseq differential gene expression profiling of olfactory mucosa in young wild type, ACE2 deficient mice. Purpose: Next-generation gene expression profiling has revolutionized analysis of molecular pathways. The goals of this study were to compare NGS-derived olfactory mucosa transcriptome profiles (RNAseq) of wild-type mice (control) and ACE2 knockout mice. Selected genes and pathways will be analyze further by low-throughput techniques such as real-time RT-PCR. Methods: Olfactory mucosa mRNA profiles of 2 months old wild-type (WT) and ACE2 knockout mice were generated by deep sequencing, in triplicate, using Illumina NovaSeq6000 S4 PE150 XP. For inspecting the quality of RNA-Seq data, the 100 most abundant genes are taken from all the samples and heatmaps were generated to observe the relation between samples/conditions. Results: Using our data analysis workflow, we mapped at least 47 million sequence reads per sample to the mouse genome (build mm10). Conlusion: Our study represent the first detailed analysis of differential gene expression by RNAseq technology in murine olfactory mucosa. It is also the first study examining effects of gene knockout for ACE2 on gene expression profile in murine olfactory mucosa. We conclude that these data would help future studies in olfactory mucosa cells aimed to reveal molecular mechanisms associated with aging and biological function of ACE2 genes. RNAseq differential gene expression profiling of olfactory mucosa in wild type and ACE2 deficient mice.
Project description:The expression of interferon-related genes was more enhanced in irradiated ATM-deficient mouse embryonic fibroblasts (MEFs) than in irradiated ATM wild-type MEFs. Nonirradiated-ATM-WT vs Irradiated-ATM-WT vs Nonirradaited-ATM-KO vs IrradiatedATM-KO
Project description:The daily organisation of most mammalian cellular functions is attributed to circadian regulation of clock-controlled protein expression, driven by daily cycles of CRYPTOCHROME-dependent transcriptional feedback repression. To test this, we used quantitative mass spectrometry to compare wild type and CRY-deficient fibroblasts under constant conditions. In CRY-deficient cells, we found that temporal variation in protein, phosphopeptide, and K+ abundance was at least as great as wild type controls. Most strikingly. the extent of temporal variation within either genotype was much smaller than overall differences in proteome composition between WT and CRY-deficient cells. This proteome imbalance in CRY-deficient cells and tissues was associated with increased susceptibility to proteotoxic stress, which impairs circadian robustness, and may contribute to the wide-ranging phenotypes of CRY-deficient mice. Rather than generating largescale daily variation in proteome composition, we suggest it is plausible that the various transcriptional and post-translational functions of CRY proteins ultimately act to promote protein and osmotic homeostasis against daily perturbation.
Project description:Although long thought to act cell autonomously, mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. We have previously shown that mutant KRAS induces EGFR-ligand trafficking to exosomes and drastically alters exosomal protein contents, leading to activities that contribute to neoplastic growth. We have performed small library RNAseq analysis on cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status to determine whether mutant KRAS regulates the composition of secreted small RNAs. Exosomal small RNA profiles were distinct from cellular profiles, with principle component analysis showing clusters of mutant KRAS cell-derived exosomes distinct from wild type KRAS cell-derived exosomes. Secreted RNA species encompassed several different classes of small RNAs, including ribosomal and tRNA fragments, as well as mature miRNA sequences. miR-10b, was selectively increased in wild type KRAS-derived exosomes, whereas miR-100 was selectively increased in mutant KRAS-derived exosomes. Ceramide inhibition resulted in accumulation of miR-100 in mutant KRAS cells, suggesting KRAS-dependent miRNA export. In Transwell cell culture experiments, mutant, but not wild type, KRAS donor cells conferred miR-100-mediated target repression in wild type KRAS recipient cells
Project description:RNAseq differential gene expression profiling of olfactory mucosa in young wild type, aged wild type, APP deficient, APLP2 deficient and PSEN2 deficient mice. Purpose: Next-generation gene expression profiling has revolutionized analysis of molecular pathways. The goals of this study were to compare NGS-derived olfactory mucosa transcriptome profiles (RNAseq) of aged wild-type mice, APP knockout mice, APLP2 knockout mice and PSEN2 knockout mice with young wild-type controls. Selected genes and pathways will be analyze further by low-throughput techniques such as real-time RT-PCR. Methods: Olfactory mucosa mRNA profiles of 2 months old wild-type (WT), 2 years old wild-type, APP knockout, APLP2 knockout and PSEN2 knockout mice were generated by deep sequencing, in triplicate, using Illumina NovaSeq6000 . For inspecting the quality of RNA-Seq data, the 100 most abundant genes are taken from all the samples and heatmaps were generated to observe the relation between samples/conditions. The sequence reads that passed the quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. Results: Using our data analysis workflow, we mapped at least 30 million sequence reads per sample to the mouse genome (build mm10) and identified approximately 25,867 transcripts in the olfactory epithelium of WT and genetically modified mice with TopHat workflow. RNAseq data confirmed stable expression of 20 known housekeeping genes, and 3 of them were validated with qRT-PCR. Approximately 20% of transcripts showed differential expression between WT and aged samples and 0.1-1.0 % showed differential expression between WT and genetically modified lines (fold change >1.5; p value < 0.05). Altered expression of 20 genes for aged samples and 10 genes for PS-deficient samples was confirmed by qRT-PCR, demonstrating the high degree of sensitivity of RNAseq approach. Conlusion: Our study represent the first detailed analysis of differential gene expression by RNAseq technology in murine olfactory mucosa in aged animals. It is also the first study examining effects of gene knockout for APP, APLP2 and PSEN2 on gene expression profile in murine olfactory mucosa. We conclude that these data would help future studies in olfactory mucosa cells aimed to reveal molecular mechanisms associated with aging and biological function of APP, APLP2 and PSEN2 genes. RNAseq differential gene expression profiling of olfactory mucosa in young wild type, aged wild type, APP deficient, APLP2 deficient and PSEN2 deficient mice. Purpose: Next-generation gene expression profiling has revolutionized analysis of molecular pathways. The goals of this study were to compare NGS-derived olfactory mucosa transcriptome profiles (RNAseq) of aged wild-type mice, APP knockout mice, APLP2 knockout mice and PSEN2 knockout mice with young wild-type controls. Selected genes and pathways will be analyze further by low-throughput techniques such as real-time RT-PCR. Methods: Olfactory mucosa mRNA profiles of 2 months old wild-type (WT), 2 years old wild-type, APP knockout, APLP2 knockout and PSEN2 knockout mice were generated by deep sequencing, in triplicate, using Illumina NovaSeq6000 . For inspecting the quality of RNA-Seq data, the 100 most abundant genes are taken from all the samples and heatmaps were generated to observe the relation between samples/conditions. The sequence reads that passed the quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. Results: Using our data analysis workflow, we mapped at least 30 million sequence reads per sample to the mouse genome (build mm10) and identified approximately 24,000 transcripts in the olfactory epithelium of WT and genetically modified mice with TopHat workflow. RNAseq data confirmed stable expression of 20 known housekeeping genes, and 3 of them were validated with qRT-PCR. Approximately 20% of transcripts showed differential expression between WT and aged samples and 0.1-1.0 % showed differential expression between WT and genetically modified lines (fold change >1.5; p value < 0.05). Altered expression of 20 genes for aged samples and 10 genes for PS-deficient samples was confirmed by qRT-PCR, demonstrating the high degree of sensitivity of RNAseq approach. Conlusion: Our study represent the first detailed analysis of differential gene expression by RNAseq technology in murine olfactory mucosa in aged animals. It is also the first study examining effects of gene knockout for APP, APLP2 and PSEN2 on gene expression profile in murine olfactory mucosa. We conclude that these data would help future studies in olfactory mucosa cells aimed to reveal molecular mechanisms associated with aging and biological function of APP, APLP2 and PSEN2 genes. This RNAseq project has been supported by a grant of Polish National Science Centre (UMO-2013/09/NZ3/02359).