Project description:Jellyfish blooms represent a significant, but largely overlooked, source of labile organic matter (jelly-OM) in the ocean, characterized by high protein content with a low C:N ratio. The bloom-decay cycle of jellyfish in coastal waters are important vehicles for carbon export to the ocean’s interior. To accurately incorporate them into biogeochemical models, the interactions between microbes and jelly-OM have yet to be fully characterized. We conducted jelly-OM enrichment experiments to simulate the scenario experienced by the coastal microbiome after the decay of a jellyfish bloom. We combined metagenomics, endo- and exo-metaproteomic approaches to obtain a mechanistic understanding on the metabolic network operated by the jelly-OM degrading bacterial consortium.
Project description:Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy, characterized by poor clinical outcomes, primarily driven by high rate of locoregional recurrence and metastasis. Extensive heterogeneity among the tumor cells as well as modulation of a highly immunosuppressive tumor microenvironment shape cancer progression. Shedding of small extracellular vesicles (sEVs) derived from tumor cells is a critical mediator of the disease initiating horizontal transfer of tumor components into platelets. HNSCC-derived sEVs isolated from HNSCC cell lines (SAS, UD-SCC 5) using Size exclusion chromatography and characterized via Flow cytometry, Electron microscopy, Nanoparticle tracking analysis and Western blotting, were used to induce platelet activation and aggregation, measured by aggregometry, flow cytometry, as well as the release of chemokines and ATP, which were quantified using Enzyme-linked immunosorbent assays (ELISA). Mechanistic investigations included inhibitor assays, thrombin activity measurements, and proteomic analyses. Unexpectedly we can show that sEVs do not activate PLTs through the FcγRIIa–IgG axis but platelet activation and aggregation is induced in a calcium-dependent manner, primarily mediated by sEV-associated Tissue factor. Proteomic analysis confirmed the presence of Tissue factor in these vesicles, implicating its involvement in initiating the coagulation cascade, that leads to platelet activation and aggregation.
Project description:In biopharmaceutical production, Chinese hamster ovary (CHO) cells derived from Cricetulus griseus remain the most commonly used host cell for recombinant protein production, especially antibodies. Over the last decade in-depth multi-omics characterization of these CHO cells provided data for extensive cell line engineering and corresponding increases in productivity. exosomes, extracellular vesicles containing proteins and nucleic acids, are barely researched at all in CHO cells. Exosomes have been proven to be an ubiquitous mediator of intercellular communication and are proposed as new biopharmaceutical format for drug delivery, indicator reflecting host cell condition and anti-apoptotic factor in spent media. Here we sequenced non-coding RNA of Exosomes (EXO) and whole cell lysate (WCL) isolated from CHO-K1 Cell Cultures at different growth phases (logarithmic/exponential phase (log/exp), stationary phase (stat), as well as death phase at 80 % viability (80 % ) and 60 % viability (60 %)) via Lexogen Small RNA-Seq Library Prep Kit for Illumina on the Illumina MiSeq platform in PE mode 2 x 36nt.
Project description:Despite the continuous emergence of multi-drug resistant pathogens, the number of new antimicrobials reaching the market is critically low. Natural product peptides are a rich source of bioactive compounds, and advances in mass spectrometry have achieved unprecedented capabilities for the discovery and characterization of novel molecular species. However, traditional bioactivity assay formats hinder the discovery and biochemical characterization of natural product antimicrobial peptides (AMPs), necessitating large sample quantities and significant optimization of experimental parameters to achieve accurate/consistent activity measurements. Microfluidic devices offer a promising alternative to bulk assay systems. Herein, a microfluidics-based bioassay was compared to the traditional 96-well plate format in respective commercially-available hardware. Bioactivity in each assay type was compared using a Viola inconspicua peptide library screened against E. coli ATCC 25922. Brightfield microcopy was used to determine bioactivity in microfluidic channels while both common optical and fluorescence-based measurements of cell viability were critically assessed in plate-based assays. Exhibiting some variation in optical density and fluorescence-based measurements, all plate-based assays conferred bioactivity in late eluting V. inconspicua library fractions. However, significant differences in the bioactivity profiles of plate-based and microfluidic assays were found, and may be derived from the materials comprising each assay device or the growth/assay conditions utilized in each format. While new technologies are necessary to overcome the limitations of traditional bioactivity assays, we demonstrate that off-the-shelf implementation of microfluidic devices is non-trivial and significant method development/optimization is required before conventional use can be realized for sensitive and rapid detection of AMPs in natural product matrices.
Project description:The knowledge of the host response to the novel coronavirus SARS-CoV-2 is still limited, slowing the understanding of COVID-19 pathogenesis and the development of therapeutic strategies. During the course of a viral infection, host cells release exosomes and other extracellular vesicles carrying viral and host components, which can modulate the immune response. The present study used a shotgun proteomic approach to mapping the host circulating exosomes response to SARS-CoV-2 infection. We investigated how SARS-CoV-2 modulates extracellular vesicles content, their involvement in disease progression and the potential use of plasma exosomes as biomarkers of disease severity. Proteomic analysis of patients derived exosomes identified several molecules involved in immune response, inflammation, activation of coagulation and complement pathways, the main mechanisms of COVID-19-associated tissue damage and multiple organ dysfunctions. In addition, several potential exosomal biomarkers such as C-reactive protein, Fibrinogen gamma chain, C4b-binding protein alpha chain and Alpha-1-acid glycoprotein 1, presenting an area under the curve (AUC) of 1, were identified. Proteins correlated to the severity of the disease were also detected. These data indicate the existence of a significant contribution of circulating exosomes to inflammation, coagulation, and immunomodulation during SARS-CoV-2 infection.
Project description:Recent advances in mass spectrometry (MS)-based technologies are now set to transform translational cancer proteomics from an idea to a practice. Here, we present a robust proteomic workflow for the analysis of clinically relevant human cancer tissues, which allows quantitation of thousands of tumor proteins in several hours of measuring time and a total turnaround of a few days. We applied it to a chemorefractory metastatic case of the extremely rare urachal carcinoma. Quantitative comparison of lung metastases and surrounding tissue revealed several significantly upregulated proteins, among them lysine specific histone demethylase 1 (LSD1/KDM1A). LSD1 is an epigenetic regulator and is the target of active development efforts in oncology. This demonstrates that clinical cancer proteomics can rapidly and efficiently identify actionable therapeutic options. While currently described for a single case study, we envision that it can be applied broadly to other patients in a similar condition.
Project description:Though the rhesus monkey is one of the most valuable non-human primate animal models for various human diseases because of its manageable size and genetic and proteomic similarities with humans, proteomic research using rhesus monkeys still remains challenging due to the lack of a complete protein sequence database and sufficient proteomic information. In this project, proteomic profiling of multiple organ tissues, 9 male and 11 female were performed in an automated, high-throughput manner employing annotated UniProtKB human database. Based on the success of this alternative interpretation of MS data, the list of proteins identified from total 12 organs of male and female subjects will benefit future rhesus monkey proteome research.
Project description:Diverse classes of regulatory small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Based the conserved biochemical properties intrinsic to all ARGONAUTEs, we have developed a universal, 15-min benchtop extraction procedure allowing simultaneous purification of all classes of RISC-associated sRNAs, without prior knowledge of the sample’s -intrinsic ARGONAUTE repertoires. Optimized as a user-friendly kit, the method –coined “TraPR” for Trans-kingdom, rapid, affordable Purification of RISCs– operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct sRNA deep-sequencing, with TraPR-generated libraries being qualitatively at least on-par with those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel excisions. TraPR considerably improves the quality and consistency of sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots and mammalian plasma, and regardless of RNA contaminants or samples’ RNA-degradation status.
Project description:Phosphoribosyl pyrophosphate synthetase (PRPS) is a highly conserved enzyme that conducts the chokepoint reaction of nucleotide biosynthesis by converting ribose-5-phosphate (R-5-P) to phosphoribosyl pyrophosphate (PRPP). Due to the presence of multiple isoforms and related PRPS-associated proteins in opisthokont species, the precise nature of the PRPS enzyme in cells and tissues remains uncertain. Using proteomics approaches and biochemistry, we demonstrate that these individual components assemble together to form a heterogeneous megadalton complex comprising of PRPS1, PRPS2, and two PRPS associated proteins – PRPSAP1 and PRPSAP2. To validate this and identify other protein/protein complexes that exists in a similar high molecular weight (HMW) range in human (HEK293T) cells, we performed shotgun proteomics on size exclusion chromatography fractions containing HMW proteins/protein complexes. This analysis identified a total of 262 unique proteins, which included ribosomal proteins and CAD confirming enrichment for HMW proteins. This dataset also revealed that among the eight cytosolic enzymes residing in HMW range, two were PRPS isozymes, indicating that the PRPS
Project description:A mouse minigut Organoid model analysed using quantitative proteomics to measure proteins expressed in the crypt and villi –like cellular states, additionally providing novel details of the secreted proteins associated with these conditions.