bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.01.30.526180

An optimized workflow for analyzing extracellular vesicles as biomarkers in liver diseases.

Paluschinski, M.·Loosen, S.·Kordes, C.·Keitel, V.·Kuebart, A.·Brandenburger, T.·Schoeler, D.·Wammers, M.·Neumann, U.·Luedde, T.·Castoldi, M.
View original source ↗Export metadata ↓

Abstract

Background & AimsExtracellular vesicles (EVs) play an important role in intercellular communication, serving as vehicles for the exchange of biological materials and being involved in the regulation of physiological processes. EVs and their associated cargoes are considered a promising source of disease-associated biomarkers. The purpose of this study was to establish an easy-to-use, reproducible, and scalable workflow to efficiently analyze EVs in the context of liver disease. MethodsAn optimized workflow was established for the pre-analytical processing and isolation of EVs from plasma and serum. Nanoparticle Tracking Analysis (NTA) was used to characterize circulating EVs in the serum of patients with nonalcoholic fatty liver disease (NAFLD), autoimmune liver disease (AIH), and animal models with impaired liver function. EVs were separated from soluble proteins by an optimized, polyethylene glycol (PEG)-based enrichment protocol. Enriched EVs were either labeled and functionally characterized by monitoring cellular uptake or lysed for biomarker identification. ResultsCirculating EVs in the serum of patients with NAFLD or AIH and in different animal models have been characterized by NTA. Here we show that both the quantity and size of EVs in the serum of patients/animal models are significantly different from those of healthy individuals. We show that isolated EVs are functional, and their uptake by acceptor cells can be quantified after fluorescence labelling. Enriched EVs were directly used to analyze RNA biomarkers. Several microRNAs, including miR-15b, -16, -21, -122 and -223, were found to be significantly up-regulated in EVs isolated from the sera of patients with NAFLD and AIH. We show that EVs transport cytokines, and that IL-2, IL-6 and IL-8 were significantly up-regulated in EVs enriched from patients with cholangiocarcinoma (CCA) compared to healthy controls. ConclusionsThe workflow presented here represents an accessible and easy-to-use approach that enables the analysis and enrichment of EVs from complex biological fluids and their preparation for functional characterization or downstream analysis. In this study, the levels of several miRNAs were found to be significantly increased in EVs isolated from AIH and NAFLD patients compared with healthy controls. HighlightsO_LIEVs circulating in crude serum reflect the diseased stage of the donors. C_LIO_LIEnrichment of EVs with the approach presented here efficiently separates soluble proteins from EVs, providing optimal material for further characterization. C_LIO_LIExosomal markers are present in the EVs-enriched fraction. C_LIO_LIEnriched EVs are intact and are functionally taken up by acceptor cells. C_LIO_LIEnriched EVs are suitable, and have been used for, biomarkers identification both at RNA and protein level. C_LI

Source connections

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Paluschinski, M., Loosen, S., Kordes, C., Keitel, V., Kuebart, A., Brandenburger, T., Schoeler, D., Wammers, M., Neumann, U., Luedde, T., Castoldi, M.. 2023-01-30. An optimized workflow for analyzing extracellular vesicles as biomarkers in liver diseases.. https://doi.org/10.1101/2023.01.30.526180

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Trans-branching of polyubiquitin chains orchestrates the DNA replication stress response

Polyubiquitin chain geometry dictates functional consequences of ubiquitylation. Although branched polyubiquitin chains are abundant in cells, little is known about their functions. Here we show that branching on the DNA replication factor PCNA, mediated by the ubiquitin-conjugating enzyme UBE2K and involving lysines 63 and 48 of ubiquitin, orchestrates the sequence of events in response to replication stress. By inducing VCP-dependent extraction of PCNA from chromatin, branching promotes re-priming of stalled forks and necessitates a BRCA1-dependent pathway of daughter-strand gap repair. Our study identifies hyper-accumulation of daughter-strand gaps as the mechanistic basis underlying the toxicity of inhibitors of the PCNA-specific isopeptidase, USP1, in BRCA1-deficient cells. Moreover, an unexpected preference of UBE2K to operate in trans suggests a general timing mechanism to organize hierarchies amongst ubiquitin signals.

molecular biology↗
bioRxiv2026

Impaired proteostasis is an early feature of the diabetic heart in humans and mice

Diabetes and obesity increase cardiac lipid levels leading to cardiomyopathy and heart failure. We hypothesized that intermittent fasting would reduce cardiac lipid levels. Surprisingly, intermittent fasting increased myocardial triglyceride content, but rescued mortality and attenuated cardiomyopathy in mice overexpressing cardiomyocyte acyl-CoA synthetase 1 (MHC-ACSL1). Lipid overload caused cardiomyocyte accumulation of polyubiquitinated protein aggregates containing desmin, a scaffolding intermediate filament protein, which intermittent fasting prevented. Furthermore, intermittent fasting reversed elevated myocardial C16:0 ceramide content, and knockdown of ceramide synthase CerS5 and CerS6 reduced palmitate-induced protein aggregation, highlighting a role for C16:0 ceramides in this pathology. Conversely, impairing aggrephagy with cardiomyocyte-specific p62 ablation induced heart failure in mice fed a high-fat diet, with paradoxically reduced cardiac lipid content. Crucially, non-failing diabetic human hearts also exhibited protein aggregate pathology. Taken together, these results demonstrate that impaired proteostasis characterizes cardiomyopathy from cardiac lipid overload and identify a promising new therapeutic target for this condition.

molecular biology↗
bioRxiv2026

Spatial profiling and neurovascular communication in the developing and adolescent cortex following prenatal alcohol exposure

Fetal alcohol spectrum disorders (FASD) constitute a wide range of developmental, cognitive, and behavioral impairments caused by prenatal alcohol exposure (PAE). Although neuronal and vascular consequences of PAE have been studied, how alcohol affects the cerebrovasculature within the framework of the neurovascular unit (NVU) across development remains poorly understood. At minimum, the NVU comprises neurons, astrocyte endfeet, and endothelial cells (ECs), which coordinate to maintain brain homeostasis. Here, we used the NanoString Digital Spatial Profiling platform to characterize spatial transcriptomic data from neurons, astrocytes, and ECs from PAE and saccharin (SAC) control cortices at embryonic day 18 (E18) and postnatal day 28 (P28). Differentially expressed genes were then used for Ingenuity Pathway Analysis (IPA) to identify altered biological pathways and perform comparison analyses across developmental time points, while CellChat was used to infer cell cell communication networks. We uncovered thousands of differentially expressed genes and numerous altered pathways and biological processes in PAE cortices across development. Both IPA and CellChat analyses implicated dysregulation of vascular and extracellular matrix (ECM) remodeling, cell adhesion, and neuroinflammatory signaling. CellChat further predicted the loss of several key bidirectional relationships and altered ligand-receptor interactions among neurovascular cell types at E18 and P28. Overall, these findings identify PAE associated alterations in neurovascular gene expression and intercellular signaling across development, providing potential mechanisms by which PAE may disrupt neurodevelopment.

molecular biology↗