Project description:3 samples of R1, R2 and R3 bone marrow monocytes were compared from 3 biological replicates in 3 separate experiments. R1, R2 and R3 were sorted from triplicate experiments from pools of mice
Project description:Copy-back defective viral genomes (cbDVGs) are key inducers of antiviral responses during negative-sense RNA virus infection. Once considered byproducts of in vitro viral replication, cbDVGs have since been detected in clinical specimens and implicated in affecting infection outcomes. The molecular mechanism of cbDVG generation remains unclear, thereby hindering our ability to manipulate cbDVG production during infection for therapeutic gain. Previous work showed that respiratory syncytial virus (RSV) cbDVG re-initiation sites cluster in trailer-end hotspots R1, R2, and R3, and that a poly-U mutation in R1 selectively reduced cbDVG formation at the mutated region. Here, we reported that a 10U mutation in R2 drastically reduced cbDVGs in this region in both minigenome and recombinant virus systems. Furthermore, during high-MOI passaging of the R2-10U virus, we observed delayed detection of cbDVGs with re-initiation sites in R1-R3 (trailer cbDVGs) compared to WT, while no differences in virus titers were observed. Interestingly, we observed the rapid emergence and accumulation of a viral variant bearing a 2-ribonucleotide deletion (R2-8U) within the R2-10U mutation sequence as early as P0. Compared to R2-10U, the R2-8U virus was stable, displayed faster generation and accumulation of trailer cbDVGs, restored cbDVGs with R2 re-initiation sites, and exhibited enhanced genomic replication. Overall, our data identify a sequence in the RSV trailer whose mutation critically modulates both viral replication and the generation/propagation of trailer cbDVGs. Our data also suggest that cbDVG generation, particularly near the trailer, may be an evolutionary tradeoff for more rapid virus genomic replication.
Project description:Investigation of whole genome transcription expression level changes in Drosophila mojavensis wild-type populations (1 Punta Onah: PO, 2 Organ Pipe National Monument: OPNM, 3 Punta Prieta:PP, and 4 San Quintin: SQ). The experiment was designed to investigate functional genomic responses to temperature variation (15, 25, and 35 °C) in adult Drosophila mojavensis wild populations. For each treatment 1-5 replicates were used (R1, R2, R3, R4 & R5). SO and BC represents Sonora deserts and Baja California region respectively.
Project description:We reconstituted arrays of CTCF binding sites (L1, L2, L3, L4, R1, R2 and R3) and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. We inserted STITCH into five different positions of the remaining allele of the locus: \\"STITCH+30kb\\", \\"STITCH+440kb\\", \\"STITCH+1760kb\\" and \\"STITCH+1790kb\\" have the STITCH insertions away from the MYC promoter for the indicated distances to the telomeric side of the p arm of the chromosome. \\"STITCH-30kb\\", at the 30-kb upstream from the MYC. We also made a deletion clone of the enhancer region, termed del(30-440). We made deletion of each CTCF array, L (delL) and R (delR), inversion of R (invR), deletion of the middle five binding sites from L2 to R2 (del(L2-R2)), and deletion of the six sites but for R3 (del(L1-R2)) in STITCH+30kb. We also obtained deletion and inversion of the whole of STITCH (del(L1-R3) and inv(L1-R3)). We integrated a transgene consisting of tetR-KRAB followed by DNA encoding the 2A peptide and the puromycin resistant gene with piggyBac transposition into the genome in the STITCH+30kb clone (STITCH/KRAB). We performed 4C-seq (Circular chromatin conformation capture assay followed by deep-sequencing) from the MYC promoter as a viewpoint to see how STITCH impacts on the chromatin conformation.
Project description:Investigation of whole genome transcription expression level changes in Drosophila mojavensis wild-type populations (1 Punta Onah: PO, 2 Organ Pipe National Monument: OPNM, 3 Punta Prieta:PP, and 4 San Quintin: SQ). The experiment was designed to investigate functional genomic responses to temperature variation (15, 25, and 35 °C) in adult Drosophila mojavensis wild populations. For each treatment 1-5 replicates were used (R1, R2, R3, R4 & R5). SO and BC represents Sonora deserts and Baja California region respectively. A total of 97 hybridizations were performed in this entire experiment. We used 135K 12-plex NimbleGen arrays. Total RNA was recovered from each sample listed below. The experimental design consisted a total of four populations (Punta Onah:PO; Organ Pipe National Manument:OPNM, Punta Prieta:PP and San Quintin:SQ), two host diets (Agria:AG and Organ pipe:OP) and three temperature treatments (15, 25 and 35 °C). Each chip measures the expression level of 14528 transcripts. One to 5 replicates were used for each type (R1, R2, R3, R4 and R5). Fly source details are as follows: Punta Onah 2007:PO07; Organ Pipe National Monument 2008:OPNM08; Punta Prieta 2008:PP08; San Quintin 2008:SQ08.
Project description:GITR and GITRL overexpress in sarcomatoid mesotelioma cells than epithelioid subtype, and cisplatin or radiation results an increase of GITR and GITRL expression on tumor cells. After sorting CRL9546 cells into GITR+(R), GITRL+(L), and GITR-/GITRL- (DN) cell populations. Three cell subsets were labelled as ligand positive L1, L2, L3, receptor positive R1, R2, R3, and double negative DN1, DN2, DN3. In total: 9 samples. RNA was extracted to run Affymetrix microarray.