Biology subjects

Witwer, K.

Publications and source records attributed to Witwer, K..

6 recordsLinked to original sources

bioRxiv2026

Isolation and Characterization of Extracellular Vesicles from Mouse Retina Tissue

This protocol provides a standardized workflow for the isolation of extracellular vesicles (EVs) from mouse retinal tissue, and includes an assessment of EV size and concentration, marker expression, and EV visualization in accordance with the International Society for Extracellular Vesicles Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines. Most retinal EV studies rely on cell culture, which may not fully capture in vivo biology. Our approach more accurately reflects physiological and pathological EV states in vivo by enabling the extraction of EVs from intact retinal tissue. This method addresses a key gap in the field by providing a reproducible and rigorous protocol for studying retinal EVs in a biologically relevant context.

cell biology↗

bioRxiv2025

Simultaneous Isolation and Characterization of Lipoprotein Classes in Plasma, Including HDL Subclasses and the Uncharacterized Dense HDL

Lipoproteins (LPP) and extracellular vesicles are carriers of extracellular small RNA, with potential applications both in the areas of diagnostics and therapeutics. Lipid nanoparticles overlap across a range of densities and sizes in plasma, making them difficult to isolate intact and without contamination from other plasma components. Accurate characterization of their cargo through efficient isolation from other plasma components is required to understand their function. Here we describe the simultaneous separation of LPP classes using sequential flotation ultracentrifugation followed by size exclusion chromatography from 500{micro}L of starting plasma. Using western blot, denaturing and non-denaturing gel electrophoresis, nuclear magnetic resonance, and electron microscopy, we demonstrate separation of the LPP classes with minimal contamination. We also show unique lipidomic, proteomic and small RNA signatures for each LPP class, including, for the first time, very high density high density lipoprotein particles in the density range of 1.21-1.25 g/mL. MotivationIn plasma, lipid nanoparticles overlap in size and density, making it difficult to separate and isolate intact particles. Additionally, currently available methods do not allow for simultaneous characterization and isolation of these particles. This method resolves both challenges. The results presented also analyze and describe the presence of known lipoprotein particles, as well as those that have been previously under-characterized.

molecular biology↗

bioRxiv2025

Tetraspanin positivity as a function of extracellular vesicle size measured by a modified immuno-TEM protocol

The identification of surface markers that correlate with specific subpopulations of extracellular vesicles (EVs) is important for EV identification, classification, purification, sorting, and functional analysis. Tetraspanins such as CD9, CD63 and CD81 were once considered to be universal markers of exosomes: small EVs released into the extracellular space when late endosomes / multivesicular bodies fuse with the plasma membrane. In contrast, plasma membrane-derived ectosomes (also called microvesicles) have a different biogenesis, were often regarded as being larger than exosomes, and display a different surface proteome. However, recent studies have shown that tetraspanins such as CD9 and CD81 are highly enriched on ectosomes derived from various sources. Thus, it is currently unclear how tetraspanin content correlates with specific EV subpopulations. Here, we present a modified immuno-TEM protocol that can be easily applied to heterogeneous EV populations comprising both small and large EVs (and presumably also a collection of exosomes and ectosomes). In EVs purified from U-2 OS cells by size-exclusion chromatography, we show that the percentage of particles positive for CD9 and CD81 is significantly higher in the subpopulation of EVs [≤] 100 nm (i.e., small EVs). These results also explain discrepancies in the size distribution profiles that we obtained using the same EV preparations by alternative single-vesicle characterization platforms such as nano flow cytometry and SP-IRIS / ExoView. The latter, when used to capture tetraspanin-positive particles, returns a population that is relatively small in size.

molecular biology↗

bioRxiv2024

Ribonuclease activity undermines immune sensing of naked extracellular RNA

The plasma membrane and the membrane of endosomal vesicles are considered physical barriers preventing extracellular RNA uptake. While naked RNA can be spontaneously internalized by certain cells types, functional delivery of naked RNA into the cytosol has been rarely observed. Here we show that extracellular ribonucleases, mainly derived from cell culture supplements, have so far hindered the study of extracellular RNA functionality. In the presence of active ribonuclease inhibitors (RI), naked bacterial RNA is pro-inflammatory when spiked in the media of dendritic cells and macrophages. In murine cells, this response mainly depends on the action of endosomal Toll-like receptors. However, we also show that naked RNA can perform endosomal escape and engage with cytosolic RNA sensors and ribosomes. For example, naked mRNAs encoding reporter proteins can be spontaneously internalized and translated by a variety of cell types, in an RI-dependent manner. In vivo, RI co-injection enhances the activation induced by naked extracellular RNA on splenic lymphocytes and myeloid-derived leukocytes. Furthermore, naked extracellular RNA is inherently pro-inflammatory in ribonuclease-poor compartments such as the peritoneal cavity. Overall, these results demonstrate that naked RNA is bioactive and does not need encapsulation inside synthetic or biological lipid vesicles for functional uptake, making a case for nonvesicular extracellular RNA-mediated intercellular communication.

molecular biology↗

bioRxiv2022

Viral and host small RNA transcriptome analysis of SARS-CoV-1 and SARS-CoV-2-infected human cells reveals novel viral short RNAs.

RNA viruses have been shown to express various short RNAs, some of which have regulatory roles during replication, transcription, and translation of viral genomes. However, short viral RNAs (svRNAs) generated by SARS-CoV-1 and SARS-CoV-2 remained largely unexplored, mainly due limitations of the widely used library preparation methods for small RNA deep sequencing and corresponding data processing. By analyzing publicly available small RNA-seq datasets, we observed that human cells infected by SARS-CoV-1 or SARS-CoV-2 produce multiple short viral RNAs (svRNAs), ranging in size from 15 to 26 nt and deriving predominantly from (+) RNA strands. In addition, we verified the presence of the five most abundant SARS-CoV-2 svRNAs in SARS-CoV-2-infected human lung adenocarcinoma cells by qPCR. Interestingly, the copy number of the observed SARS-CoV-2 svRNAs dramatically exceeded the expression of previously reported viral miRNAs in the same cells. We hypothesize that the reported SARS-CoV-2 svRNAs could serve as biomarkers for early infection stages due to their high abundance. Finally, we found that both SARS-CoV-1 and SARS-CoV-2 infection induced up- and down-regulation of multiple endogenous human short RNAs that align predominantly to protein-coding and lncRNA transcripts. Interestingly, a significant proportion of short RNAs derived from full-length viral genomes also aligned to various hg38 sequences, suggesting opportunities to investigate regulatory roles of svRNAs during infection. Further characterization of the small RNA landscape of both viral and host genomes is clearly warranted to improve our understanding of molecular events related to infection and to design more efficient strategies for therapeutic interventions as well as early diagnosis.

molecular biology↗

bioRxiv2022

Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids

Nonvesicular extracellular RNAs (nv-exRNAs) constitute the majority of the extracellular RNAome, but little is known about their stability, function and potential use as disease biomarkers. Herein, we measured the stability of several naked RNAs when incubated in human serum, urine and cerebrospinal fluid (CSF). We identified extracellularly produced tRNA-derived small RNAs (tDRs) with half-lives of up to three hours in CSF. Contrary to widespread assumptions, these intrinsically stable small RNAs are full-length tRNAs containing broken phosphodiester bonds (i.e., nicked tRNAs). Standard molecular biology protocols, including phenol-based RNA extraction and heat, induce the artifactual denaturation of nicked tRNAs and the consequent in vitro production of tDRs. Broken bonds are roadblocks for reverse transcriptases, preventing amplification and/or sequencing of nicked tRNAs in their native state. To solve this, we performed enzymatic repair of nicked tRNAs purified under native conditions, harnessing the intrinsic activity of phage and bacterial tRNA repair systems. Enzymatic repair regenerated an RNase R-resistant tRNA-sized band in northern blot and enabled RT-PCR amplification of full-length tRNAs. We also separated nicked tRNAs from tDRs by chromatographic methods under native conditions, identifying nicked tRNAs inside stressed cells and in vesicle-depleted human biofluids. Dissociation of nicked tRNAs produces single-stranded tDRs that can be spontaneously taken up by human epithelial cells, positioning stable nv-exRNAs as potentially relevant players in intercellular communication pathways.

molecular biology↗