Biology subjects

CARAPITO, C.

Publications and source records attributed to CARAPITO, C..

6 recordsLinked to original sources

bioRxiv2025

Fine-Tuning of Label-Free Single-Cell Proteomics Workflows

Mass spectrometry-based single-cell proteomics emerges as the most promising method for studying cellular heterogeneity at the global proteome level with unprecedented depth and coverage. Its widespread application remains limited due to robustness, reproducibility, and throughput requirements, still difficult to meet as analyzing large cohorts of single cells is necessary to ensure statistical confidence. In this context, we have conducted method optimizations at three levels. First, we benchmarked three distinct workflows compatible with the nanoElute2 platform using different sample collection/preparation plate supports (EVO96 oil-free, LF48 oil-based and LF48 oil-free, a streamlined automated sample resuspension and direct injection protocol). Then, we compared the optimized EVO96 workflow on nanoElute2 with EVOSEP-based separations operating at two analytical throughputs (80 and 120 samples per day). Subsequently, we evaluated digestion efficiency using a range of enzyme/protein ratios (1:1; 10:1; 20:1; 50:1) to maximize peptide recovery. Finally, the chromatographic setup was refined to determine the best compromise between throughput and robustness. Altogether, these optimizations allowed to establish a robust workflow quantifying up to 5,000 proteins in 10min gradient time per single Hela cell at a 55 samples-per-day throughput.

biochemistry↗

bioRxiv2025

Therapeutic poxviruses induce the secretion of immunostimulating and anti-tumoral extracellular vesicles

Poxvirus-based vectors provide a versatile cancer immunotherapy platform, enabling the expression of immunostimulatory molecules and cancer-specific antigens. While infections with pathogenic viruses are well known to modulate extracellular vesicle (EV) biogenesis and function, the extent to which therapeutic poxviral vectors influence EV secretion by immune cells and thereby affect therapeutic efficacy remains underexplored. In this study, we showed that poxviruses, including the clinically relevant Modified Vaccinia Ankara (MVA), stimulate the secretion of small EVs (sEVs) containing viral proteins and immune-related signatures from peripheral blood mononuclear cells (PBMCs). Using an engineered MVA vector, we demonstrated the transfer of virus-encoded therapeutic payloads to sEVs, including the model ovalbumin (OVA)-derived peptide SIINFEKL presented by the class I major histocompatibility complex (MHC I) and the immune activators interleukin-12 (IL-12) and CD40 ligand (CD40L). Depending on the isolation method, these sEVs stimulated SIINFEKL-specific CD8 T cells with varying efficiencies in vitro. Remarkably, intravenous injection of these sEVs into E.G7-OVA lymphoma-bearing mice reduced tumor growth to an extent comparable to the virus itself. Taken together, our findings indicate that EVs released from immune cells infected with engineered therapeutic poxviruses exert potent antitumor activity. These vesicles represent actionable mediators whose secretion and functionalization can be harnessed to improve viral vector-based immunotherapies, as well as being considered as therapeutic vectors in their own. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/677320v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1eae18borg.highwire.dtl.DTLVardef@17d6fb5org.highwire.dtl.DTLVardef@311577org.highwire.dtl.DTLVardef@7839c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Effects of manganese dioxide on macrophages under different exposure schemes

Manganese dioxide is a material that is more and more used in its particular form, for many industrial applications such as chemical catalysis or for batteries. Thus, workers can be exposed to this particulate chemical. It is known that overexposure to manganese leads to a brain disease called manganism. However, manganese is also known to impact the pulmonary function, which is important as pulmonary exposure is of prime importance for workers. We thus investigated the effects of manganese dioxide on macrophages, i.e. the scavenger cells that take particulates in charge in our bodies. To this purpose, we used a combination of proteomic and targeted approaches, in order to obtain a wide view of the cellular responses to manganese dioxide. We also used a repeated exposure mode, in order to better mimic occupational exposure. Our results point out the fact that manganese oxide nanoparticles are rather toxic for macrophages and induce mitochondrial dysfunction, oxidative stress and a pro-inflammatory response. Environmental significanceManganese dioxide is more and more used in batteries, so that workers in batteries factories are exposed to this metallic oxide. As always for particulate materials, macrophages are the first line of defense of the organism. We thus investigated the effects of manganese dioxide on macrophages, using a repeated exposure scheme to mimic occupational exposure, and the effects were documented by a combination of proteomic and targeted approaches. The functional effects include mitochondrial dysfunction, oxidative stress and inflammation.

pharmacology and toxicology↗

bioRxiv2025

Beyond the ink: cellular and molecular effects of iron-based pigments on macrophages

As ochre, iron oxide is among the most ancient pigments used by mankind for different purposes, including tattooing as demonstrated on tattoed mummies. Iron oxides are still used in tattooing nowadays and especially in dermopigmentation, an area of medical tattoing aiming at restoring the color of skin. This ancient use of iron oxide does not mean that it has no effect on cells, and especially on macrophages, the cells that maintain pigments particles on site in tattoos. We thus investigated in vitro the delayed/sustained effects of iron oxide pigments on macrophages, i.e. the effects occurring a few days after the exposure to pigments, on pigments-loaded macrophages but in a pigment-free medium, mimicking the status of tattooed skin after all the pigment particles have been captured. By combining proteomic and targeted approaches, we determined that red iron oxide (but not black iron oxide) induces perturbations in mitochondria, altering the mitochondrial transmembrane potential. Red iron oxide also induces oxidative stress and the secretion of pro-inflammatory cytokines such as interleukin 6 and tumor necrosis factor. Thus, red iron oxide induces adverse effects on macrophages that may persist over time, owing to its low intracellular dissolution.

immunology↗

bioRxiv2025

DeepLC introduces transfer learning for accurate LC retention time prediction and adaptation to substantially different modifications and setups

While LC retention time prediction of peptides and their modifications has proven useful, widespread adoption and optimal performance are hindered by variations in experimental parameters. These variations can render retention time prediction models inaccurate and dramatically reduce the value of predictions for identification, validation, and DIA spectral library generation. To date, mitigation of these issues has been attempted through calibration or by training bespoke models for specific experimental setups, with only partial success. We here demonstrate that transfer learning can successfully overcome these limitations by leveraging pre-trained model parameters. Remarkably, this approach can even fit highly performant models to substantially different peptide modifications and LC conditions than those on which the model was originally trained. This impressive adaptability of transfer learning makes it a highly robust solution for accurate peptide retention time prediction across a very wide variety of imaginable proteomics workflows.

bioinformatics↗

bioRxiv2022

The Holdup Multiplex, an assay for high-throughput measurement of protein-ligand affinity constants using a mass-spectrometry readout

The accurate description and subsequent modeling of protein interactomes requires quantification of their affinities at proteome-wide scale. Here we develop and validate the Holdup Multiplex, a versatile assay for high-throughput measurement of protein-ligand affinity constants that uses mass-spectrometry as readout. The method can quantify thousands of affinities in one single run, with high precision and over several orders of magnitude. We applied this strategy to the seven human 14-3-3 isoforms, quantifying in a few sample-runs their interaction with 1,000 different phosphopeptides. We were able to identify hundreds of new 14-3-3 binding sites. We showed that the seven human 14-3-3 display similar specificities but staggered affinities, 14-3-3g being always the best binder and 14-3-3{varepsilon} and {sigma}, the weakest. Finally, we identified dozens of 14-3-3 bindings sites, some intervening in key signaling pathways, that were either stabilized or destabilized by the phytotoxin Fusicoccin-A. Our approach, which throughput can be pushed up to the sensitivity limit of the mass-spectrometry setup, is applicable to any category of protein-ligand interactions and thus bears a wide potential both for high-throughput interactomics and chemoproteomics.

systems biology↗