Project description:The aim of the present study is to summarize the identification of neuropeptides, neuropeptide-like and protein hormones in Z. atratus
Project description:One of the most thoroughly studied insect species, with respect to locomotion behaviour, is the stick insect Carausius morosus. Although detailed information exists on premotor networks controlling walking, surprisingly little is known about neuropeptides, which are certainly involved in motor activity generation and modulation. So far, only few neuropeptides were identified from C. morosus or related stick insects. We performed a transcriptome analysis of the central nervous system to assemble and identify 65 neuropeptide and protein hormone precursors of C. morosus, including five novel putative neuropeptide precursors without clear homology to known neuropeptide precursors of other insects (Carausius neuropeptide-like precursor 1, HanSolin, PK-like1, PK-like2, RFLamide). Using Q Exactive Orbitrap and MALDI-TOF mass spectrometry, 277 peptides including 153 likely bioactive mature neuropeptides were confirmed. Peptidomics yielded a complete coverage for many of the neuropeptide propeptides and confirmed a surprisingly high number of heterozygous sequences. Few neuropeptide precursors commonly occurring in insects, including those of insect kinins and sulfakinins, could neither be found in the transcriptome data nor did peptidomics support their presence. The results of our study represent one of the most comprehensive peptidomic analyses on insects and provide the necessary input for subsequent experiments revealing neuropeptide function in greater detail.
Project description:Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immuno-precipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. Hit validation by a subsequent RNA interference screen revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae. In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both occurs in the nucleus. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.
Project description:The immune system is unique in its dynamic interplay between numerous cell types. However, a system-wide view of how immune cells communicate to protect against disease has not been established. Here, we applied high-resolution mass spectrometry-based proteomics to generate a publicly accessible protein atlas of 28 primary human immune cell populations in steady and activated states at a depth of > 10,000 proteins. Cell-type-specific protein copy numbers reveal that immune cells are most specialized at the level of ligands and receptors, thereby connecting distinct functions of the immune system. By integrating total and secreted proteomes, we deduce paracrine immune dynamics upon microbial encounter and discover fundamental intercellular communication structures as well as novel connections between cellular senders and receivers of biological information. Our comprehensive cell-type-resolved proteomic resource of human immune cells provides a framework for the orchestration of cellular interplay and a reference for altered communication associated with pathology.
Project description:RORγt is a transcription factor required for T helper 17 (Th17) cell development. We identified three RORγt-specific inhibitors that suppress Th17 cell responses including Th17 cell-mediated autoimmune disease. We systemically characterized RORγt binding data in the presence and absence of drug with corresponding whole-transcriptome sequencing for wild-type and RORγt-deficient cells. RORγt is central in a densely interconnected regulatory network, acting both as a direct activator of genes important for Th17 cell differentiation and as a direct repressor of genes from other T-cell lineages. The three inhibitors identified here reversed both of these modes of action, but to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target-loci, the two more potent inhibitors affected transcription predominantly without removing DNA-binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity. DNA binding of RORγt in WT Th17 cells and under chemical perturbations of RORγt; Additional data is included for epitope-tagged exogenous RORγt in EL4 cells (a murine lymphoma cell line)
Project description:The human iPSC line H19101 was differentiated in vitro into cardiomyocytes using a 20-day differentiation protocol (Burridge et al. 2014 PMID 24930130 and Montefiori et al 2018 PMID 29988018 ). 50,000 cardiomyocytes were used in each ATAC-seq experiment. 8 replicates were pooled to obtain the final peak file.
Project description:The Crown-of-Thorns starfish (COTS), Acanthaster planci, is a highly fecund predator of reef-building corals distributed throughout the Indo-Pacific. COTS population outbreaks cause substantial loss of coral cover, diminishing the integrity and resilience of the reef ecosystems thus increasing their susceptibility to climate change. We sequenced genomes of A. planci from the Great Barrier Reef, Australia (GBR) and Okinawa, Japan (OKI) to guide identification of species-specific peptide communication with potential applications in mitigation strategies. The genome-encoded proteins excreted and secreted into the surrounding seawater by COTS forming aggregations and by those escaping the predatory giant triton snail, Charonia tritonis, were identified LC-MS/MS.
Project description:Here we have used a combination of advanced proteomics and genomics approaches to investigate the extent and mechanisms of transcription factor cross-talk at genomic hotspots. We identify ~12,000 transcription factor hotspots in the early phase of adipogenesis, and we find evidence of both simultaneous and sequential binding of transcription factors at these regions. We demonstrate for the first time that hotspots are highly enriched in large super-enhancer regions and that these drive the early adipogenic reprogramming of gene expression. Our results indicate that cooperativity between transcription factors at the level of hotspots as well as super-enhancers is very important for enhancer activity and transcriptional reprogramming. Thus, hotspots and super-enhancers constitute important regulatory hubs integrating external stimuli on chromatin. Genome-wide profiling of transcription factor and co-factor binding, epigenomic marks, and gene expression in 3T3-L1 pre-adipocytes.
Project description:Liver are frequently declined for transplantation due to the donor age. It is still unclear how donor age affects the graft quality. Normothermic machine perfusion (NMP) has been proposed as a useful assessment tool prior to transplantation. We aimed to compare the performance of young and elderly rat liver grafts in a small animal NMP model. Grafts from 24 rats were procured, either 3 or 12 months old and perfused for 6 hours at 37°C or stored on ice as a reference group (n=6/group). Livers in both NMP groups cleared lactate and produced bile, with similar pressure, bile production, and pH levels. However, older rat livers showed higher peak transaminase and urea levels. Proteomic analysis revealed differences in protein composition, particularly higher levels of proteins related to oxidoreductase and catalytic activity in older livers. Older age appeared to increase susceptibility to oxidative stress in the livers.
Project description:We aimed to address the challenge of accommodating an increasing demand for aged laboratory animals in the context of rising elderly population and research on aging-related diseases. We developed a cost-effective environmental enrichment method and assessed its impact on metabolic changes in Sprague Dawley rats' livers as a proof-of-concept organ. Twenty-four male rats were divided into four groups, with two kept in standard cages and two in modified rabbit cages. Half of each type of cage group received additional enrichment through weekly playtime in a larger cage. Over six months, the rats' weight gain was consistent across all groups, and corticosterone levels did not significantly differ. However, the control group had significantly lower DHEA and Testosterone levels at the study's end. Rats in enriched environments exhibited less distress during inspections and were more resistant to accepting treats. Animals accustomed to playpen time left their cages more easily. Overall, the study demonstrated that refining husbandry for aging rats is both simple and cost-effective, without detrimental effects on stress levels, development, or liver metabolism.