Project description:The RNA binding protein IGF2BP2/IMP2 alters the cargo of cancer cell-derived extracellular vesicles supporting tumor-associated macrophages
Project description:The N-methyl-D-aspartate (NMDA) receptor is a glutamate-activated cation channel critical to many processes in the brain. Genome-wide association studies (GWAS) suggest that glutamatergic neurotransmission and NMDA receptor-mediated synaptic plasticity is important for body weight homeostasis1. Here, we report the engineering and preclinical development of a first-in-class bimodal molecule that integrates NMDA receptor antagonism with glucagon-like peptide-1 (GLP-1) receptor agonism to effectively reverse obesity, hyperglycemia, and dyslipidemia in rodent models of metabolic disease. We demonstrate that GLP-1-directed delivery of the NMDA receptor antagonist MK-801 affects NMDA receptor-mediated synaptic plasticity in the hypothalamus. Importantly, peptide-targeting of MK-801 specifically to GLP-1 receptor-expressing brain regions circumvent adverse physiological and behavioral effects associated with MK-801 monotherapy. In sum, our approach demonstrates the feasibility of cell specific ionotropic receptor-modulation via peptide targeting and highlights the therapeutic potential of unimolecular mixed GLP-1 receptor agonism and NMDA receptor antagonism for obesity treatment.
Project description:Myeloproliferative Neoplasms (MPNs) are a heterogenous group of hematologic cancers characterized by excessive JAK/STAT signaling. Mutations of JAK2 signaling components are among the most common drivers of MPN, but alterations in Suppressors of Cytokine Signaling (SOCS) proteins have been implicated in MPN pathogenesis and progression. Cullin 5 (Cul5) is an E3 ubiquitin ligase known to work with suppressors of cytokine signaling (SOCS) proteins which regulate the JAK/STAT pathway. Here we report that mice lacking Cul5 in hematopoietic stem and progenitor cells (HSPCs) develop an MPN-like disease with characteristic features including splenomegaly, extramedullary hematopoiesis, thrombocytosis, and anemia. Cul5-deficient HSPCs have higher phospho-STAT5 (pSTAT5) levels following stimulation with IL-3 and outcompete WT HSPCs in bone marrow transplants. Immunoprecipitation of Cul5 in cultured HSPCs showed interactions with STAT5 as well as several well-studied substrate receptors including SOCS2, SOCS6, ASB2, ASB3, ASB6 and CIS, as well as lesser-known WSB1 and LRRC41. Proteome analysis of Lin- Sca-1+ c-kit+ (LSK) cells from Cul5Vav-Cre bone marrow shared many upregulated genes and signatures with MPN patient cells. Finally, treatment with ruxolitinib, a JAK1/2 inhibitor, ameliorated MPN symptoms in Cul5-deficient mice. These studies demonstrate a novel function of Cul5 in hematopoiesis, delineating a contributing role in MPN.
Project description:Pancreatic cancer remains as one of the most challenging malignancies to diagnose and treat due to its silent progression and limited early diagnostic options. The intraductal papillary mucinous neoplasm (IPMN) provides an early-stage lesion for studying pancreatic cancer progression. However, proteomic analyses of IPMN and its comparison with pancreatic ductal adenocarcinoma (PDAC) and normal duct (ND) have been limited due to their low cellularity. In this study, we present an optimized spatial tissue proteomics workflow, termed SP-Max (Spatial Proteomics Optimized for Maximum Sensitivity and Reproducibility in Minimal Sample), designed to maximize protein recovery and quantification from limited laser micro dissected (LMD) samples. Our workflow enabled the identification of more than 6,000 proteins and the quantification of over 5,200 protein groups from FFPE tissue contours of actual pancreatic cancer patients. Comparative analyses across ND, IPMN, and PDAC revealed critical molecular differences in protein pathways and potential prognostic markers. SP-Max provides a systematic, reproducible approach that significantly enhances our ability to study precancerous lesions and cancer progression in pancreatic tissues at unprecedented resolution.
Project description:In vitro maturation (IVM) of oocytes retrieved from ovum pick-up (OPU) or ovarian tissue (OT) is a standard approach for patients with specific conditions where prior hormonal stimulation is contraindicated. However, the developmental competence of oocytes matured in vitro is still inferior to that of oocytes matured in vivo. Capacitation-IVM (CAPA-IVM) includes an extra step of pre-maturation culture (PMC) with c-type natriuretic peptide (CNP) as a meiotic arrestor to better synchronize cytoplasmic and nuclear maturity in oocytes. This study aims to evaluate the effect of CAPA-IVM on equine oocyte quality and developmental competence. Immature cumulus-oocyte complexes (COCs) were retrieved from slaughterhouse ovaries and matured in vitro either in CAPA-IVM (short or long) or standard IVM. Matured oocytes from each group were analyzed for calcium-releasing potential and single-oocyte proteomics, and embryo development was assessed after fertilization with Piezo-drilled intracytoplasmic sperm injection (ICSI). Genetic analysis of developed blastocysts was performed to detect chromosomal aberrations. Our findings demonstrate that CAPA-IVM of equine COCs yields significantly higher maturation rates than controls. Moreover, short CAPA-IVM with six hours pre-maturation culture showed substantially higher embryo development potential than the control group. Genetic analysis revealed a high euploidy rate in equine blastocysts regardless of the maturation conditions. Live calcium imaging of the fertilized oocytes demonstrated the majority of oocytes with non-continuous calcium oscillation patterns, irrespective of maturation conditions. Single oocyte proteomics reveals a comparable proteomic landscape between matured oocytes from short CAPA-IVM and standard IVM. However, a trend of differential expression was observed in specific proteins related to cytoskeleton, cell cycle, and hemostasis in the short CAPA-IVM group. Our findings indicate that CAPA-IVM holds the potential to improve oocyte quality and competence in horses. However, further fine-tuning of culture conditions based on omics analysis would benefit the effective use of these IVM systems. Moreover, given that the mare serves as an excellent model for human reproduction, the molecular trends identified in this study could provide valuable insights for advancing human artificial reproductive technologies.
Project description:Co-infection of human papillomavirus (HPV) and human immunodeficiency virus type 1 (HIV-1) in women have a six-fold higher risk of developing cervical cancer compared to those without HIV. To evaluate how paracrine signals from HIV-infected T-cells remodeled the proteome of cervical epithelial cells in culture, primary CD4+ T cells isolated from PBMC-enriched leukapheresis products (leukopaks) from two healthy donors were infected or uninfected with a replication-competent pNL4-3 HIV-1 strain for 72 hours. Secretome from the CD4+ T cell cultures was used to stimulate the human HPV-negative cervical epithelial cell line, C33A, for 72 hours. Then, C33A cells were harvested, and cell lysates were digested and subjected to global quantitative mass spectrometry (MS) based abundance proteomics and phosphoproteomics analyses. Both proteomics and phosphoproteomics outputs were analysed using bioinformatics approaches. These datasets revealed altered expression of proteins in the MAPK, PI3K-AKT, and β-catenin signaling pathways. Additionally, MS phosphoproteomics analysis confirmed PI3K-AKT pathway activation in cervical cells exposed to conditioned media from HIV-1-infected T cells.
Project description:Transcription initiation is a highly regulated process that determines gene expression outcomes1,2, yet the dynamics and efficiency of individual initiation events in living cells remain poorly understood. Here we combine endogenous tagging of human RNA polymerase II (Pol II) and the TFIID subunit TAF1 with dual-color live-cell single-molecule imaging to quantitatively map Pol II behavior during transcription initiation and early elongation. Using GRID (Genuine Rate Identification) analysis, we resolved four kinetic populations of chromatin-bound Pol II and find that more than 94% of promoter-bound molecules dissociate within tens of seconds, indicating globally inefficient initiation. Kinetic partitioning of Pol II dwell times into promoter-proximal pausing and gene body elongation reveals a global pausing index that is selectively increased by CDK9 inhibition. Single-cell analysis uncovers substantial heterogeneity in initiation efficiency and pausing that is not explained by cell-cycle state. Spatial heatmap analysis identifies recurrent Pol II ‘hotspots’, nuclear regions enriched for repeated long-lived Pol II binding events, whose abundance and architecture are selectively perturbed by inhibitors that trap Pol II at promoters or disrupt TFIID. Finally, acute depletion of TAF1 leaves initiation efficiency unchanged but significantly increases the pausing index and reduces elongation efficiency, consistent with impaired pause release. Together, these findings establish a general framework to quantify transcription efficiency in vivo and support a model in which TAF1, and by extension TFIID, promotes pause release and productive elongation.
Project description:Background: Aggressiveness guides treatment in IDH-mutant gliomas. Objective grading of oligodendrogliomas is therefore urgently needed. Material and Methods: 211 primary and recurrent resections from 111 oligodendroglioma patients were collected, complemented with 91 samples for validation. Samples were subjected to Ki-67 staining, proteomics and DNA-methylation profiling. Data were analyzed using various multivariate differential models and algorithms. Results & conclusion: We developed CGCψ, a continuous grading coefficient independent of tumor typing, and demonstrate its prognostic value in oligodendrogliomas. Its prognostic value outperformed WHO grade at tumor recurrence. CGCψ is linked to large scale DNA de-methylation, but increased DNA methylation of polycomb transcription factors, aging, Ki-67, and losses on chr4 and chr9p. DNA de-methylation at tumor recurrence/higher grade is sequence context specific and associated with TET recognition sites. Both oligodendrogliomas and astrocytomas progress along this shared epi-genetic axis. Oligosarcomas are characterized by a high CGCψ and low tumor purity.
Project description:LD3 knock out cells were generated in HEK293T and HMC3 cells. Proteomics was done to test if they have any significant changes in the abundance of proteins that metabolize sugar lipids like gangliosides.