Project description:We report that phosphorylated ribosomes can be immunoprecipitated from mouse brain homogenates, enriching for mRNAs that were selectively expressed in activated neurons
Project description:Exploration of a novel rbcDNA liquid biopsy technique for early detection of colorectal cancer is a promising development in the field of disease diagnosis and screening. This technique has the potential to establish an efficient and sensitive system for the early detection of colorectal cancer, which can provide a new perspective for individual health monitoring.
Project description:We report an approach called physiological optical tagging sequencing (PhOTseq), a technique for tagging cells based on their functional properties and then harvesting them for RNA sequencing. We demonstrate that PhOTseq is capable of selecting physiologically rare ( < 0.2%) cell types and enriching them by nearly one hundred-fold.
Project description:RNA-seq workflows have become progressively more efficient over time however, RNA extraction still remains a significant bottleneck. For small numbers of sample, highly efficient RNA extraction is simple to perform but becomes costly and laborious at the scale of hundreds of samples. Our prior work has demonstrated that qPCR can be accurately performed using bulk cells samples lysate instead of RNA extraction. We combined this method with the recently developed simple method for rapid RNA-seq library prep; Smart-3SEQ (Foley et al., 2019) and hypothesized that bulk RNA-seq can be performed in a multi-well plate format, and at low cost by performing RNA-seq library prep cDNA synthesis using in-lysate RNA followed by Smart-3SEQ. The result shows success of our approach in various levels: 1) all quality control measures were achieved, 2) Gene expression profiles, gene differential expression and the response patterns reported by in-lysate and purified RNA library highly correlate with each other, and 3) in-lysate RNA-seq library prep performed similar to the gold standard used here (Illumina Truseq) for DEG calling.
Project description:Clinical treatment protocols for infertility with in vitro fertilization-embryo transfer (IVF-ET) provide a unique opportunity to assess the human vaginal microbiome in defined hormonal milieu. Herein, we have investigated the association of circulating ovarian-derived estradiol (E2) and progesterone (P4) concentrations to the vaginal microbiome. Thirty IVF-ET patients were enrolled in this study, after informed consent. Blood was drawn at four time points during the IVF-ET procedure. In addition, if a pregnancy resulted, blood was drawn at 4-to-6 weeks of gestation. The serum concentrations of E2 and P4 were measured. Vaginal swabs were obtained in different hormonal milieu. Two independent genome-based technologies (and the second assayed in two different ways) were employed to identify the vaginal microbes. The vaginal microbiome underwent a transition with a decrease in E2 (and/or a decrease in P4). Novel bacteria were found in the vagina of 33% of the women undergoing IVF-ET. Our approach has enabled the discovery of novel, previously unidentified bacterial species in the human vagina in different hormonal milieu. While the relationship of hormone concentration and vaginal microbes was found to be complex, the data support a shift in the microbiome of the human vagina during IVF-ET therapy using standard protocols. The data also set the foundation for further studies examining correlations between IVF-ET outcome and the vaginal microbiome within a larger study population.
Project description:Early detection of spoilage microorganisms and food pathogens is of major importance in preventing food recalls and foodborne outbreaks. Although constant effort is invested in developing sensitive methods for rapid microbial detection, none of the current methods enables the detection of food pathogens within a few hours; therefore, development of innovative early-warning food-testing strategies are needed. Herein, we assessed a novel strategy that harnesses the microbiome signature of a food product to determine deviations in the abundance of particular community members and detect production defects. Employing the production process of barbecued (BarBQ) pastrami as a model, we characterized the microbiome profiles of the product along the production line using next-generation sequencing of the 16S rRNA gene, concentrating on the live microbiota. Following the establishment of a microbiome dataset representing a properly produced product, we were able to identify shifts in the microbiome profile of a defective batch produced under potassium lactate deficiency. With the identification of Vibrio and Lactobacillus as potential indicator bacteria for potassium lactate deficiency, rapid qPCR assays were designed for their quantification. Aligned with the microbiome profiling results, these qPCR assays were effective for rapid identification of a defective production event. This implies the use of rapid quantification targeting microbiome profile-derived indicator bacteria for in-house detection of defective batches and identification of food-safety and quality events with results obtained on the same day. The suggested strategy should pave the way toward safer and more efficient food-production systems.
Project description:Our study identifies TACSTD2 as a novel regulator of two major HCV entry factors, CLDN1 and OCLN, which is strongly downregulated in malignant hepatocytes. These results provide new insights into the complex process of HCV entry into hepatocytes and may assist in the development of more efficient cellular systems for HCV propagation in vitro.
Project description:Our study identifies TACSTD2 as a novel regulator of two major HCV entry factors, CLDN1 and OCLN, which is strongly downregulated in malignant hepatocytes. These results provide new insights into the complex process of HCV entry into hepatocytes and may assist in the development of more efficient cellular systems for HCV propagation in vitro.
Project description:We report that phosphorylated ribosomes can be immunoprecipitated from mouse brain homogenates, enriching for mRNAs that were selectively expressed in activated neurons Mice were given an injection of concentrated salt solution (2M NaCl, 325 uL) or PBS, and then sacrificed 2h later and hypothalami dissected. Tissue homogenates were prepared from hypothalamus and phosphorylated ribosomes immunoprecipitated using pS6 244/247 antibodies. The immunoprecipitated RNA and total RNA from both control and salt treated animals were then purified and analyzed by Illumina Microarray.