Biology subjects

Tosar, J. P.

Publications and source records attributed to Tosar, J. P..

12 recordsLinked to original sources

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

Substantial hierarchical reductions of genetic and morphological traits in the evolution of rotiferan parasites

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/605096v2_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@469ecborg.highwire.dtl.DTLVardef@6330f4org.highwire.dtl.DTLVardef@22faf6org.highwire.dtl.DTLVardef@8a6704_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG During the last 800 million years of evolution animals radiated into a vast range of diversity of species and disparity of forms and lifestyles. The process involved a near hierarchical increase in complexity from life forms with few cell types to organisms with many hundreds of cell-types. However, neither genome size nor number of protein-coding genes can explain these differences and their biological basis remains elusive. Yet, recent studies have suggested that the evolution of complexity is closely linked to the acquisition of a class of protein coding gene-regulators called microRNAs. In a regressive approach, to investigate the association between loss of organismal complexity and microRNAs, we here studied Syndermata, an invertebrate group including free-living rotifers (Monogononta, Bdelloidea), the epibiotic Seisonidea and the endoparasitic Acanthocephala. Genomic, transcriptomic and morphological characterization and comparisons across 25 syndermatan species revealed a strong correlation between loss of microRNAs, loss of protein-coding genes and decreasing morphological complexity. The near hierarchical loss extends to [~]85% loss of microRNAs and a [~]50% loss of BUSCO genes in the endoparasitic Acanthocephala, the most reduced group we studied. Together, the loss of [~]400 protein-coding genes and [~]10 metazoan core gene losses went along with one microRNA family loss. Furthermore, the loss of [~]4 microRNA families or [~]34 metazoan core genes associated with one lost morphological feature. These are the first quantitative insights into the regulatory impact of microRNAs on organismic complexity as a predictable consequence in regressive evolution of parasites.

evolutionary biology↗

bioRxiv2024

PIWIL1 is recruited to centrosomes during mitosis in Colorectal Cancer Cells and is linked to cell cycle progression.

PIWI proteins, traditionally associated with germline development, have recently gained attention for their expression in various cancers, including colorectal cancer (CRC). However, the molecular mechanisms underlying their reactivation and impact on cancer initiation and progression remain elusive. Here, we found that PIWIL1 is expressed at relatively high levels in CRC-derived samples and cell lines, where it undergoes a dynamic re-localization to the centrosome during mitosis. Knockdown of PIWIL1 induces G2/M arrest associated to disruption of mitotic spindle and aberrant metaphase events, highlighting its role in cell cycle progression. We have also found that expression of PIWIL1 is lost during differentiation of Caco-2 cells into enterocytes and that PIWIL1 is expressed in cells at the base of intestinal crypts in normal human colon tissue, where intestinal stem cells are known to reside. Thus, it is possible that the presence of PIWIL1 in cancer cells reflects a physiological role of this protein in stem cell maintenance, what would argue in favor of the proposed stem cell origin of CRC. Supporting this view, dedifferentiation of human fibroblasts into induced pluripotent stem cells (iPSc) implies reactivation of PIWIL2 expression, another member of the PIWI protein family. Overall, our findings suggest a role of PIWIL1 in mediating cell cycle dynamics, both in colorectal cancer cells and possibly also in intestinal stem cells. In a broader aspect, we provide support for an involvement of PIWI proteins in somatic stem cell maintenance, expanding nongonadal functions for this protein family.

cancer 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↗

bioRxiv2024

Simultaneous protein and RNA analysis in single extracellular vesicles, including viruses: SPIRFISH

Interest in using nanoparticles for delivery of therapeutic RNA has been steadily growing, provoking a need to precisely understand their structure and contents. Single-particle and single-molecule analysis techniques provide snapshots of single biological nanoparticles, including viruses, liposomes, and extracellular vesicles (EVs). While existing methods primarily focus on protein detection, RNA delivery is becoming increasingly prevalent. A method to simultaneously detect protein and internal RNA in the same particle would reveal variability in size, structure, and RNA packaging efficiency, enabling optimization of nanoparticle delivery. Here, we introduce SPIRFISH, a high-throughput method for single-particle protein and RNA analysis, combining single particle interferometric reflectance imaging sensor (SP-IRIS) with single-molecule fluorescence in-situ hybridization (smFISH). Using SPIRFISH, we detect HIV-1 envelope protein and genomic RNA within single infectious virions, allowing resolution against EV background and noninfectious virions. We further show that SPIRFISH can be used to detect specific RNA within EVs. SPIRFISH should enable single particle analysis of a broad class of RNA-containing nanoparticles. Teaser: A new single particle analysis technique simultaneously detects specific RNA and protein in biological nanoparticles.

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↗

bioRxiv2020

Characterization of extracellular vesicles and artificial nanoparticles with four orthogonal single-particle analysis platforms

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

cell biology↗

bioRxiv2020

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

A major proportion of extracellular RNAs (exRNAs) do not co-isolate with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed RI-SEC-seq: a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes can be sensed by dendritic cells. Extracellular ribosomes and/or tRNAs could therefore be decoded as damage-associated molecular patterns.

cell biology↗

bioRxiv2018

Evolutionary implications of the first microRNA- and piRNA complement of Lepidodermella squamata (Gastrotricha)

Gastrotrichs - hairy bellies - are microscopic free-living animals inhabiting marine and freshwater habitats. Based on morphological and early molecular analyses, gastrotrichs were placed close to nematodes, but recent phylogenomic analyses have suggested their close relationship to flatworms (Platyhelminthes) within Spiralia. Small non-coding RNA data on e.g. microRNAs (miRNAs) and PIWI-interacting RNAs (piRNA) may help to resolve this long-standing question. MiRNAs are short post-transcriptional gene regulators that together with piRNAs play key roles in development. In a multi-omics approach we here used small-RNA sequencing, available transcriptome and genomic data to unravel the miRNA-and piRNA complements along with the RNAi protein machinery of Lepidodermella squamata (Gastrotricha, Chaetonotida). We identified 52 miRNA genes representing 35 highly conserved miRNA families specific to Eumetazoa, Bilateria, Protostomia, and Spiralia, respectively, with overall high similarities to platyhelminth miRNA complements. In addition, we found four large piRNA clusters that also resemble flatworm piRNAs but not those earlier described for nematodes. Congruently, transcriptomic annotation revealed that the Lepidodermella protein machinery is highly similar to flatworms, too. Taken together, miRNA, piRNA and protein data support a close relationship of gastrotrichs and flatworms.

evolutionary biology↗

bioRxiv2018

An electrochemical biosensor for rapid detection of anti-dsDNA antibodies in absolute scale.

Autoimmune diseases are chronic inflammatory pathologies that are characterized by the presence of antibodies against own epitopes in serum (autoantibodies). Systemic lupus erythematosus (SLE) is a common autoimmune pathology, characterized by the presence of antinuclear antibodies (ANAs). These include anti-dsDNA (-dsDNA) antibodies, which are widely used for diagnosis and disease monitoring. Their determination is carried out by traditional techniques such as Indirect Immunofluorescence (IFI) or Enzyme Linked Immunosorbent Assay (ELISA), which are time consuming, require qualified technicians, and are not compatible with decentralized analysis outside a laboratory facility. Here, we show a sandwich-format electrochemical biosensor-based method for -dsDNA determination in a rapid and simple manner. Total assay time is only 30 minutes and the sensor is capable of detecting 16 ng (8 g / mL) of -dsDNA antibodies. Using the current derived from the detection limit of the method as a cut-off, we could discriminate positive from negative serum samples with 90% sensitivity and 100% specificity. By using monoclonal antibodies for calibration curves, our results are presented in absolute scale (i.e., concentration instead of serum title) what will help to perform comparisons between methods and further improvements of this protocol. In an effort to render the sensor compatible with automation, we minimized manipulation steps without compromise of the analytical performance, even in complex samples such as serum.

bioengineering↗

bioRxiv2018

miR-21-5p and miR-30a-5p are identical in human and bovine, have similar isomiR distribution, and cannot be used to identify xenomiR uptake from cow milk

microRNAs (miRNAs) are often highly conserved across species, but species-specific sequences are known. In addition, miRNA \"isomiRs\" arise from the same precursor molecule but differ in post-processing length and modification, usually at the 3 end. A recently published feeding study reported the intriguing result that two bovine milk-specific miRNAs were taken up into human circulation after ingestion of bovine milk. Unfortunately, this interpretation is based on annotation errors in a public microRNA database. Reanalysis using databses including the MirGeneDB database reveals that the miRNAs in question, miR-21-5p and miR-30a-5p, arise from 100% identical 5 precursor sequences in human and bovine, and the putative bovine-specific isomiRs appear to be depleted, not enriched, in bovine milk. Thus, enrichment of these isomiRs in human blood is inconsistent with uptake of xenomiRs and likely betrays endogenous miRNA regulation in response to diet or technical artifact.

molecular biology↗