Project description:Focusing on adult skeletal muscle stem cells, we have developed a protocol that circumvents the impact of the isolation procedures and captures the cells in their native quiescent state. We show that current isolation protocols induce major transcriptional changes accompanied by specific histone modifications, while having negligible effects on DNA methylation.
Project description:Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and a broad range of pathological associations. A major technical limitation to understanding the role of EVs in normal and diseased specimens has been the inability to visualize the spatial localization of EVs in tissue microenvironments. Here, we use bovine ocular tissue, the vitreous humor, as a model system to study EV imaging. We show that mammalian tissues crosslinked with conventional formaldehyde solutions result in significant EV loss, with subsequent reduced or negative EV signals; however, EV escape can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) that permanently holds these nano-sized particles and allows for visualization of EVs in normal and cancer tissues in situ.
Project description:We compared transcriptome of real quiescent MuSCs and freshly isolated MuSCs with in-situ fixation,FISC cultured for 24hrs,48hrs and 72hrs by RNA-sequencing.
Project description:Archival formalin-fixed paraffin-embedded (FFPE) tissue samples hold a wealth of transcriptomic information; however, little is known about potential artifacts. Previously, we identified a consistent shift in global RNA-sequencing profiles between matching frozen and FFPE samples. We hypothesized that this shift was from fixing fresh tissue in formalin. To test this idea, RNA-sequencing was performed on liver samples collected from male mice treated with 600 ppm of a reference chemical (phenobarbital, 600 ppm phenobarbital) or vehicle control for 7 days. Samples were divided into: (1) fresh-frozen (FR); (2) directly fixed in 10% buffered formalin for 18 hours followed by paraffin embedding (FFPE); (3) frozen then fixed as FFPE (FR>FFPE); or (4) frozen then fixed in 70% ethanol followed by paraffin embedding (FR>OH) (n=6/group/condition). Direct fixation resulted in 2946 differentially expressed genes (DEGs), 98% of which were down-regulated. Freezing prior to fixation resulted in ≥95% fewer DEGs vs. FR, indicating that most formalin-derived transcriptional effects occurred with fixation. This was supported by follow-up studies, which identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription elongation pathways with formalin fixation. Notably, formalin fixation in the parent study did not significantly impact chemical response profiles, which were consistent with CAR/PXR activation and 600 ppm phenobarbital exposure. Our results demonstrate distinct transcriptional effects of formalin fixation that could impact gene expression studies using FFPE samples.
Project description:Background: Interest in single-cell whole transcriptome analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works, investigators have used live cells which represent several inconveniences and limitations. Some recent cell fixation methods did not work with most primary cells including immune cells. Methods: The methanol-fixation and new processing method was introduced to preserve PBMCs for single-cell RNA sequencing (scRNA-Seq) analysis on 10X Chromium platform. Results: When methanol fixation protocol was broken up into three steps, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to three-month storage steps. Resuspension but not rehydration in 3X saline sodium citrate (SSC) buffer instead of PBS preserved PBMC RNA integrity and prevented RNA leakage. Diluted SSC buffer did not interfere with full-length cDNA synthesis. The methanol-fixed PBMCs resuspended in 3X SSC were successfully implemented into 10X Chromium standard scRNA-seq workflows with no elevated low quality cells and cell doublets. The fixation process did not alter the single-cell transcriptional profiles and gene expression levels. Major subpopulations classified by marker genes could be identified in fixed PBMCs at a similar proportion as in live PBMCs. This new fixation processing protocol was validated in CD8+ T cell and several other cell types. Conclusions: We expect that the methanol-based cell fixation procedure presented here will substantially enable complex experimental design with primary cells at single cell resolution.
Project description:Our study revealed a synergistic effect between biological nitrogen fixation and current generation by G. sulfurreducens, providing a green nitrogen fixation alternative through shifting the nitrogen fixation field from energy consumption to energy production and having implications for N-deficient wastewater treatment.
Project description:Prospective study of accuracy of colonic polyp characterisation in vivo using high resolution white light endoscopy, narrow band imaging and chromoendoscopy.
Project description:A1501 NFI is a genomic island derived from Pseudomonas stutzeri A1501. To study the molecular interactions of the P. stutzeri nif genes with the E. coli genome during nitrogen fixation, the NIF of A1501 was transferred into E. coli and comparative transcriptomics analyses were performed between nitrogen fixation conditions and nitrogen excess conditions.
Project description:Formalin induces inter- and intra-molecular crosslinks within exposed cells. This cross-linking can be exploited to characterise chromatin state as in the FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) and MNase (micrococcal nuclease) assays. Our team aims to optimise these assays for application in museum preserved formalin-exposed specimens. To do so, we first sought to understand the effect of prolonged formalin fixation on the read alignment signatures resulting from FAIRE and MNase treatment. Here we cultured yeast (Saccharomyces cerevisiae) under normal and heat-shock conditions then fixed the cells with formalin for 15 minutes, 1 hour, 6 hours, and 24 hours. We found that heavy formalin fixation modulates rather than eliminates signatures of differential chromatin accessibility and enables semi-quantitative estimates of relative gene expression in this yeast model.