Project description:Precise control of the innate immune response is required for resistance to microbial infections and maintenance of normal tissue homeostasis. Because this response involves coordinate regulation of hundreds of genes, it provides a powerful biological system to elucidate the molecular strategies that underlie signal- and time-dependent transitions of gene expression. Using a combination of genome-wide and gene-specific approaches, we provide evidence that rather than representing off/on transitions, Toll-like receptor 4 (TLR4)-dependent activation of nearly all immediate/early (I/E) and late response genes results from a sequential process in which signal-independent factors, exemplified by Gabpa, initially establish basal levels of gene expression that are then amplified by signal-dependent transcription factors. Promoters of I/E genes are distinguished from those of late genes by the use of distinct sets of signal-dependent transcription factors, preferential binding of TBP and basal enrichment for RNA Pol II immediately downstream of transcriptional start sites. Global nuclear run-on (GRO) sequencing and total RNA sequencing further indicates that TLR4 signaling markedly increases efficiency of transcriptional elongation of nearly all I/E genes, while RNA splicing is largely unaffected. NCoR/SMRT co-repressor complexes are unexpectedly found to be associated with H3K4me3-positive promoters of TLR4-responsive genes that exhibit a broad range of basal expression levels, implying a dynamic, rather than static role in regulation of gene expression. Collectively, these findings reveal mechanisms underlying temporally distinct patterns of TLR4-dependent gene activation required for homeostasis and effective immune responses. ChIP-Seq, Total RNA-Seq, Gro-Seq, and gene expression profiling was performed in macrophages treated with Kdo2 Lipid A. Control samples for H3K4me3 in Macrophages and B cells, in addition to control microarray data are included in GEO accession# GSE21512. FASTA files are available for older experiments that lack quality read information (i.e. no FASTQ file)
Project description:The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression. Keywords: ordered
Project description:The CMS4 sarcoma, kindly provided by A. DeLeo (University of Pittsburgh, Pittsburgh, PA), is a solid tumor of BALB/c (H-2d) origin, which grows aggressively in naive, syngeneic hosts following a s.c. transplant. Although the parental tumor cell line forms few metastatic foci in the lungs following i.v. administration, a highly metastatic subline, termed CMS4-met, was established from lung digests of those mice as described (Ryan MH, Bristol JA, McDuffie E, Abrams SI. Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope. J Immunol 2001;167:4286-92).<br>In this experiment we compare gene expression kinetics in the CMS4 and CMS4-met cell lines after IFNgamma treatment. Both cells were treated with IFNgamma for 4 and 24 hours respectively. The untreated and treated cells were then used for total RNA isolation, and microarray experiments.
Project description:In this experiment we investigated the effect of HDAC3 inhibition on the transcriptome of IFNg-primed macrophages under different tolerization conditions. Peripheral blood mononuclear cells (PBMCs) were isolated from 3 healthy donors. PBMCs were isolated from whole blood of healthy donors using Ficoll gradient (Invitrogen). Monocytes (CD14+ cells) were positively selected from PBMCs using CD14 Microbeads according to the manufacturer’s instructions (Miltenyi Biotec). Monocytes were subsequently treated with or without 500 nM HDAC3i (ITF3100) for 30 minutes prior to overnight IFNg priming (50 ng/mL). Cells were then kept without LPS (non-LPS; N), treated with 10 ng/mL LPS once (non-tolerized; NT), or treated with LPS twice (tolerized; T; second LPS concentration: 100 ng/mL).
Project description:Florida’s coral reefs are currently experiencing a multi-year disease-related mortality event, that has resulted in massive die-offs in multiple coral species. Coral monitoring data and disease prevention/treatment efforts from recent years have identified individual Orbicella faveolata that possess high, moderate, or low resistance to stony coral tissue loss disease (SCTLD). Ninety samples of high, moderate, or low SCTLD resistance were collected from 3 reefs for bottom-up LC-MS/MS analysis (n=30 for each resistance category).
Project description:Leptospirosis, an emerging zoonotic disease with worldwide distribution, is caused by spirochetes belonging to the genus Leptospira. More than 500,000 cases of severe leptospirosis are reported annually, with .10% of these being fatal. Leptospires can survive for weeks in suitably moist conditions before encountering a new host. Reservoir hosts, typically rodents, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. In humans, leptospires can cause a variety of clinical manifestations, ranging from asymptomatic or mild fever to severe icteric (Weil’s) disease and pulmonary haemorrhage. Currently, little is known about how Leptospira persist within a reservoir host. Prior in vitro studies have suggested that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. However, no study has examined gene expression by leptospires within a mammalian host-adapted state. To obtain a more faithful representation of how leptospires respond to host-derived signals, we used RNA-Seq to compare the transcriptome of L. interrogans cultivated within dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats with that of organisms grown in vitro. In addition to determining the relative expression levels of ‘‘core’’ housekeeping genes under both growth conditions, we identified 166 genes that are differentially-expressed by L. interrogans in vivo. Our analyses highlight physiological aspects of host adaptation by leptospires relating to heme uptake and utilization. We also identified 11 novel non-coding transcripts that are candidate small regulatory RNAs. The DMC model provides a facile system for studying the transcriptional and antigenic changes associated with mammalian host-adaption, selection of targets for mutagenesis, and the identification of previously unrecognized virulence determinants. Transcriptome analysis of L. interrogans Copenhageni FIOCRUZ L1-130 using RNA from 2 different conditions using RNA-seq. Also, the reproducibility and robustness of data is ensured by three biological replicates from each condition.
Project description:Aminoacyl-tRNA synthetases (aaRSs) attach amino acids to their cognate transfer RNAs. In eukaryotes, a subset of cytosolic aaRSs is organized into a multi-synthetase complex (MSC), along with specialized scaffolding proteins referred to as aaRS-interacting multifunctional proteins (AIMPs). In Plasmodium, the causative agent of malaria, the tRNA import protein (tRip), is a membrane protein which participates in tRNA trafficking. Here, we show that tRip is also an AIMP. We identified three aaRSs, namely the glutamyl- (ERS), glutaminyl- (QRS), and methionyl- (MRS) tRNA synthetases, which were specifically co-immunoprecipitated with tRip in Plasmodium berghei blood stage parasites. All four proteins contain an N-terminal GST-like domain involved in MSC assembly. Surprisingly, further dissection of GST-like interactions identified two exclusive heterotrimeric complexes: Q-complex (tRip:ERS:QRS) and M-complex (tRip:ERS:MRS). Gel filtration and light scattering suggest a 2:2:2 stoichiometry for both complexes but with distinct biophysical properties and mutational analysis revealed that the GST-like domains of QRS and MRS use different strategies to bind ERS. Furthermore, the modular organization of the assembled aaRSs and the properties of the specific additional modules supported the proposed localization of these complexes at the parasite membrane.
Project description:To identify unique genes associated with tumor progression by comparing a poorly metastatic tumor cell line with a highly metastatic tumor cell line.
Project description:The CMS4 sarcoma, kindly provided by A. DeLeo (University of Pittsburgh, Pittsburgh, PA), is a solid tumor of BALB/c (H-2d) origin, which grows aggressively in naive, syngeneic hosts following a s.c. transplant. Although the parental tumor cell line forms few metastatic foci in the lungs following i.v. administration, a highly metastatic subline, termed CMS4-met, was established from lung digests of those mice as described (Ryan MH, Bristol JA, McDuffie E, Abrams SI. Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope. J Immunol 2001;167:4286-92).<br>In this experiment we compare the gene expression profiles between a control cell line and a cell line surviving exposure to tumor-specific CTL (cytolytic T lymphocyte). CMS4-met cells were injected iv. to the mouse tails. Three days later, either saline or tumor-specific CTL were injected iv into mouse tails. Single cell suspensions were made from mouse lungs and put in culture. Tumor cells were recovered from cultures. Tumor cells established from mice receiving saline were termed CMS4-met.cntl. Tumor cells established from mice receiving CTL were termed CMS4-met.sel. The gene expression profiles of these two tumor cell lines were then compared to CMS4-met.