Kenneth Kastaniegaard

A novel community driven software for functional enrichment analysis of extracellular vesicles data

ABSTRACT Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture “what the community needs in a tool”. Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

Comparing the proteome of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human tissue samples

Graphical abstract

Proteome stability analysis of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human colon mucosal biopsies

Large repositories of well characterized RNAlater preserved samples and formalin-fixed, paraffin-embedded samples have been generated worldwide. However, the impact on the proteome of the preservation methods remain poorly described. Therefore, we analyzed the impact on the proteome of preserving samples in RNAlater, and by formalin-fixation, paraffin-embedding on human soft tissue, using directly frozen samples as a control (“Comparing the proteome of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human tissue samples” [1]). We here report the data from the analysis. The comparative analysis was performed on 24 colon mucosa biopsies, extracted from the sigmoideum of two gastroenterologically healthy participants for the purpose of this study. A set of biopsies were additionally stored for 30 min at room temperature prior to formalin-fixation. The samples were analyzed by high throughput gel free quantitative proteomics. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002029.

Identification of brain antigens recognized by autoantibodies in experimental autoimmune encephalomyelitis-induced animals treated with etomoxir or interferon-β

Multiple sclerosis (MS) is a neurodegenerative autoimmune disease, where chronic inflammation plays an essential role in its pathology. A feature of MS is the production of autoantibodies stimulated by an altered-peptide-ligand response and epitope spreading, resulting in loss of tolerance for self-proteins. The involvement of autoantibodies in MS pathogenesis has been suggested to initiate and drive progression of inflammation; however, the etiology of MS remains unknown. The effect of etomoxir and interferon-β (IFN-β) was examined in an experimental-autoimmune-encephalomyelitis (EAE) model of MS. Moreover, the impact of etomoxir and IFN-β on recognition of brain proteins in serum from EAE rats was examined with the purpose of identifying the autoantibody reactivities involved in MS. Animals treated with etomoxir on day 1 exhibited a statistically significantly lower disease score than animals treated with IFN-β (on day 1 or 5) or placebo. Etomoxir treatment on day 5 resulted in a significantly lower disease score than IFN-β treatment on day 1. After disease induction antibodies was induced to a broad pallet of antigens in the brain. Surprisingly, by blocking CPT1 and therewith lipid metabolism several alterations in the antibody response was observed suggesting that autoantibodies play a role in the EAE animal model.

Proteomic analysis of lipopolysaccharide activated human monocytes

HIGHLIGHTSLC–MS/MS analysis identifies 2746 quantifiable proteins in human monocytes.LPS alters the abundance of 244 proteins.LPS reduces the abundance of several lysosomal proteins.LPS alters the abundance of proteins involved in antigen‐presentation and skews antigen‐presentation towards MHC class I presentation. ABSTRACT Monocytes are key mediators of innate immunity and comprise an important cellular defence against invading pathogens. However, exaggerated or dysregulated monocyte activation can lead to severe immune‐mediated pathology such as sepsis or chronic inflammatory diseases. Thus, detailed insight into the molecular mechanisms of monocyte activation is essential to understand monocyte‐driven inflammatory pathologies. We therefore investigated the global protein changes in human monocytes during lipopolysaccharide (LPS) activation to mimic bacterial activation. Purified human monocytes were stimulated with LPS for 17 h and analyzed by state‐of‐the‐art liquid chromatography tandem mass spectrometry (LC–MS/MS). The label‐free quantitative proteome analysis identified 2746 quantifiable proteins of which 101 had a statistically significantly different abundance between LPS‐stimulated cells and unstimulated controls. Additionally, 143 proteins were exclusively identified in either LPS stimulated cells or unstimulated controls. Functional annotation clustering demonstrated that LPS, most significantly, regulates proteasomal‐ and lysosomal proteins but in opposite directions. Thus, seven proteasome subunits were upregulated by LPS while 11 lysosomal proteins were downregulated. Both systems are critically involved in processing of proteins for antigen‐presentation and together with LPS‐induced regulation of CD74 and tapasin, our data suggest that LPS can skew monocytic antigen‐presentation towards MHC class I rather than MHC class II. In summary, this study provides a sensitive high throughput protein analysis of LPS‐induced monocyte activation and identifies several LPS‐regulated proteins not previously described in the literature which can be used as a source for future studies.

Proteomics-driven design of endothelial stress-based protein array for disease prognostics: applied to plasma and cerebrospinal fluid

Extracellular vesicles (EVs) have a demonstrated involvement in modulating the immune system. It has been proposed that EVs could be used as biomarkers for detection of inflammatory and immunological disorders. Consequently, it is of great interest to investigate EVs in more detail with focus on immunological markers. In this study, five major leukocyte subpopulations and the corresponding leukocyte-derived EVs were phenotyped with focus on selected immunological lineage-specific markers and selected vesicle-related markers. The leukocyte-derived EVs displayed phenotypic differences in the 34 markers investigated. The majority of the lineage-specific markers used for identification of the parent cell types could not be detected on EVs released from monocultures of the associated cell types. In contrast, the vesicular presentation of CD9, CD63, and CD81 correlated to the cell surface expression of these markers, however, with few exceptions. Furthermore, the cellular expression of CD9, CD63, and CD81 varied between leukocytes present inwhole blood and cultured leukocytes. In summary, these data demonstrate that the cellular and vesicular presentation of selected lineage-specific and vesicle-relatedmarkersmay differ, supporting the accumulating observations that sorting of molecular cargo into EVs is tightly controlled.ISEV2016 is organized by The Local Organizing Committee Chair Edit I Buzás (Hungary), Aled Clayton (United Kingdom), Dolores Di Vizio (USA), Juan Manuel Falcon-Perez (Spain), Guido Jenster (The Netherlands), Lorraine O’Driscoll (Ireland), Yong Song Gho (South Korea), Marjolein van Driel (The Netherlands), Hans van Leeuwen (The Netherlands), Guillaume van Niel (France), Marca HM Wauben (The Netherlands), Kenneth W Witwer (USA), María Yáñez-Mó (Spain) Together with the Executive ISEV Board (2014 – 2016) President: Jan Lötvall Secretary General: Clotilde Théry Interim Treasurer: Kenneth W Witwer Executive Chair Science / Meetings: Marca Wauben Executive Chair Education: Yong Song Gho Executive Chair Communication: Andrew Hill Members at Large: Peter Quesenberry, Kenneth W Witwer, Susmita Sahoo, Dolores Di Vizio, Chris Gardiner, Edit I Buzás, Hidetoshi Tahara, Suresh Mathivanan, Igor Kurochkin