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Brucale, M.

Publications and source records attributed to Brucale, M..

10 recordsLinked to original sources
bioRxiv2026

Investigating the effect of ginger-derived nanovesicles on the growth and metabolic activity of Bacteroides thetaiotaomicron: an isothermal microcalorimetric study

Plant-derived extracellular vesicles (EVs) have shown numerous health benefits, including modulation of the human gut microbiota. Hereby, we employed isothermal microcalorimetry (IMC) to explore the effects of ginger-derived nanovesicles (G-NVs) on the growth and metabolic activity of Bacteroides thetaiotaomicron (Bt), a dominant saccharolytic commensal with promising next-generation probiotic potential. Bt was exposed to either G-NVs or the ginger extract separated from G-NVs (G-CTL) in defined media under anaerobic conditions. Both ginger samples enhanced bacterial specific growth rate and maximum metabolic activity, inducing the latter earlier. However, higher biomass production and greater secretion of acetic, succinic, and propionic acids occurred only in response to the G-CTL. Complete sugar depletion and unchanged FAA levels indicated preferential carbohydrate utilization by Bt. Overall, these findings revealed that Bts metabolic state is shaped by both G-NVs and G-CTL, yet through distinct mechanisms, with G-NVs inducing rapid stimulation without increasing total metabolic output and G-CTL providing a sustained, dose-dependent effect. To our knowledge, this is the first study applying IMC to monitor real-time the impact of EVs on microbial growth and metabolism, underscoring IMCs utility for mechanistic studies of EV-microbe interactions. Furthermore, this research sets the ground for innovative strategies in nutraceutical and microbiome-targeted therapy development.

microbiology↗
bioRxiv2024

Single-particle analysis of small extracellular vesicles from the follicular fluid of women undergoing fertility treatments reveals distinct PD-L1+ populations

In some cell systems, small extracellular vesicles bearing PD-L1 (PD-L1+ sEVs) have been shown to be able to suppress T-cell immunity. We have herein investigated whether a distinct profile of PD-L1+ sEVs exists in human follicular fluid (FF). Single-particle interferometric reflectance imaging sensing combined with a single-particle antibody capture and immunofluorescence labelling were used to determine the expression and colocalization of CD63, CD81, CD9, and PD-L1 in sEVs derived from FF of women undergoing fertility treatments (n=10). In addition, the size distribution of sEVs was investigated via atomic force microscopy. Our data indicate that the bulk of tetraspanin-expressing EVs in human FF are less than 50 nm in size. Tetraspanins and PD-L1 exhibit distinct expression and colocalization profiles at sEV level across all cohort samples. A total of 42%, 46%, and 50% of all the particles captured by anti-CD63, anti-CD81, and anti-CD9 antibodies, respectively, were positive for CD81. PD-L1 was expressed at the highest level on CD9+ sEVs, with an average value of 5% within the cohort. The presence of distinct PD-L1+ sEV subpopulations suggests that they may play a role in regulating the immune response in the follicular microenvironment. Further research is needed to fully understand the functional significance of PD-L1+ sEVs in this context and their potential as biomarkers for predicting fertility outcomes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/628903v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@77e67dorg.highwire.dtl.DTLVardef@1bd1ce2org.highwire.dtl.DTLVardef@b376d8org.highwire.dtl.DTLVardef@3f6fad_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗
bioRxiv2024

Platelet as key cells in endometriosis patients: insights from small extracellular vesicles in peritoneal fluid and endometriotic lesions analysis

Endometriosis is a chronic inflammatory condition characterized by the presence of endometrium-like tissue outside the uterus, primarily affecting pelvic organs and tissues. In this study, we explored platelet activation in endometriosis. We utilized the STRING database to analyze the functional interactions among proteins previously identified in small extracellular vesicles (EVs) isolated from the peritoneal fluid of endometriosis patients and controls. The bioinformatic analysis indicated enriched signaling pathways related to platelet activation, hemostasis, and neutrophil degranulation. Double immunohistochemistry analysis for CD61 and MPO revealed a significant presence of neutrophils and platelets infiltrating endometriotic lesions, suggesting potential cell-cell interactions. Subsequently, we isolated small EVs from the peritoneal fluid of women diagnosed with endometriosis and from women without endometriosis who underwent surgery for non-inflammatory benign diseases. We performed single-particle phenotyping analysis based on platelet biomarkers GPIIb/IIIa and PF4 using nanoflow cytometry, as well as single-particle morphological and nanomechanical characterization through atomic force microscopy. The study demonstrated that patients with endometriosis had a notably higher proportion of particles testing positive for platelet biomarkers compared to the total number of EVs. This finding implies a potential role for platelets in the pathogenesis of endometriosis. Further research is necessary to delve into the mechanisms underlying this phenomenon and its implications for disease progression.

cell biology↗
bioRxiv2024

Biogenic nanoparticles from liquid and solid matrices: biochemical and biophysical properties of Extracellular Vesicles-enriched samples from human plasma and skeletal muscle tissue.

AimStudies on extracellular vesicles (EVs) focused on samples enriched from liquid matrices, such as cell culture media and blood. Recent research highlights the roles of EVs derived from the extracellular matrix of solid tissues, and how investigating these specific EVs offers insights into their microenvironment and potential biological influences on surrounding cells. This study presents a shared method to separate and compare EV enriched from solid (human skeletal muscle biopsy) and liquid (human plasma) matrices, addressing technical challenges and minimizing biases in separation techniques. MethodsPlasma and skeletal muscle-EVs were obtained combining serial centrifugation steps and discontinuous sucrose density gradient. EVs characterization employed advanced analytical techniques such as Western Blot, Colorimetric Nanoplasmonic assay, Atomic Force Miscoscopy, Nanoparticle Tracking Analysis and Dynamic Light Scattering to focus on biomolecular composition, nanomechanical properties, particle yield, size distribution, and colloidal stability. ResultsThe analysis revealed distinct differences between skeletal muscle-EVs and plasma-EVs including: molecular composition, physical and nanomechanical properties, as well as particle size distribution. Skeletal muscle-EVs exhibited unique colloidal behavior compared to their plasma counterparts, suggesting tissue-specific features that may influence their biological activity and stability. ConclusionsThe findings demonstrate that EVs from skeletal muscle tissue possess unique biochemical and biophysical characteristics when compared to those derived from plasma. These differences reflect their diverse biological origins and microenvironments. Understanding these distinctions could advance the development of EV-based diagnostic tools, particularly for muscular disorders, and broaden our knowledge of EV roles across various tissue contexts. HIGHLIGHTSO_LIShared protocol to enrich and compare extracellular vesicles (EVs) from both solid (skeletal muscle biopsy) and liquid (plasma) human samples, reducing methodological bias across matrices. C_LIO_LISkeletal muscle-derived EVs differ significantly from plasma EVs in biochemical and biophysical properties such as molecular composition, size distribution, nanomechanical properties, and colloidal stability. C_LIO_LIEV heterogeneity across microenvironments emerged as a key biological feature, highlighting their potential for specific diagnostic applications. C_LI

cell biology↗
bioRxiv2022

Compositional profiling of extracellular vesicles/lipoproteins mixtures by AFM nanomechanical imaging

The widely overlapping physicochemical properties of lipoproteins (LPs) and extracellular vesicles (EVs) represents one of the main obstacles for the isolation and characterization of these pervasive biogenic lipid nanoparticles. We herein present the application of an atomic force microscopy (AFM)-based quantitative morphometry assay to the rapid nanomechanical screening of mixed LPs and EVs samples. The method can determine the diameter and the mechanical stiffness of hundreds of individual nanometric objects within few hours. The obtained diameters are in quantitative accord with those measured via cryo-electron microscopy (cryo-EM); the assignment of a specific nanomechanical readout to each object enables the simultaneous discrimination of co-isolated EVs and LPs even if they have overlapping size distributions. EVs and all classes of LPs are shown to be characterized by specific combinations of diameter and stiffness, thus making it possible to estimate their relative abundance in EV/LP mixed samples in terms of stoichiometric ratio, surface area and volume. As a side finding, we show how the mechanical behaviour of specific LP classes is correlated to distinctive structural features revealed by cryo-EM. To the best of our knowledge, these results represent the first systematic single-particle mechanical investigation of lipoproteins. The described approach is label-free, single-step and relatively quick to perform. Importantly, it can be used to analyze samples which prove very challenging to assess with several established techniques due to ensemble-averaging, low sensibility to small particles, or both, thus providing a very useful tool for quickly assessing the purity of EV/LP isolates including plasma- and serum-derived preparations.

biophysics↗
bioRxiv2021

Stiffness of fluid and gel phase lipid nanovesicles: weighting the contributions of membrane bending modulus and luminal pressurization

The mechanical properties of biogenic membranous compartments are thought to be relevant in numerous biological processes; however, their quantitative measurement remains challenging for most of the already available Force Spectroscopy (FS)-based techniques. In particular, the debate on the mechanics of lipid nanovesicles and on the interpretation of their mechanical response to an applied force is still open. This is mostly due to the current lack of a unified model being able to describe the mechanical response of gel and fluid phase lipid vesicles and to disentangle the contributions of membrane rigidity and luminal pressure. In this framework, we herein propose a simple model in which the contributions of membrane rigidity and luminal pressure to the overall vesicle stiffness are described as a series of springs; this approach allows estimating the two contributions for both gel and fluid phase liposomes. Atomic Force Microscopy-based FS (AFM-FS), performed on both vesicles and Supported Lipid Bilayers (SLBs), is exploited for obtaining all the parameters involved in the model. Moreover, the use of coarse-grained full-scale molecular dynamics simulations allowed for better understanding the differences in the mechanical responses of gel and fluid phase bilayers and supported the experimental findings. Results suggest that the pressure contribution is similar among all the probed vesicle types; however, it plays a dominant role in the mechanical response of lipid nanovesicles presenting a fluid phase membrane, while its contribution becomes comparable to the one of membrane rigidity in nanovesicles with a gel phase lipid membrane. The herein presented results offer a simple way to quantify two of the most important parameters in vesicular nanomechanics, and as such represent a first step towards a currently unavailable, unified model for the mechanical response of gel and fluid phase lipid nanovesicles.

biophysics↗
bioRxiv2020

EVs analysis in the COVID-19 era: insights on serum inactivation protocols towards downstream isolation and analysis

Since the outbreak of COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2 positive individuals demanded the use of inactivation protocols to ensure laboratory operators safety. While not standardized, these practices can be roughly divided in two categories, namely heat inactivation and solvent-detergent treatments. As such, these routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the EVs community, yet deep investigations in this direction havent been reported so far. In the attempt of sparking interest on this highly relevant topic, we here provide preliminary insights on SARS-CoV-2 inactivation practices to be adopted prior serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entailed the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat-treatment, however accompanied by a marked enrichment in EVs-associated contaminants. On the contrary, solvent/detergent treatment is promising for small EVs (< 150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

biochemistry↗
bioRxiv2020

Gold Nanoparticles interacting with Synthetic Lipid Rafts: an AFM investigation

Inorganic nanoparticles (NPs) represent promising examples of engineered nanomaterials, providing interesting biomedical solutions in several fields, like therapeutics and diagnostics. Despite the extensive number of investigations motivated by their remarkable potential for nanomedicinal applications, the interactions of NPs with biological interfaces are still poorly understood. The effect of NPs on living organisms is mediated by biological barriers, such as the cell plasma membrane, whose lateral heterogeneity is thought to play a prominent role in NPs adsorption and uptake pathways. In particular, biological membranes feature the presence of rafts, i.e. segregated lipid micro and/or nano-domains in the so-called liquid ordered phase (Lo), immiscible with the surrounding liquid disordered phase (Ld). Rafts are involved in various biological functions and act as sites for the selective adsorption of materials on the membrane. Indeed, the thickness mismatch present along their boundaries generates energetically favorable conditions for the adsorption of NPs. Despite its clear implications in NPs internalization processes and cytotoxicity, a direct proof of the selective adsorption of NPs along the rafts boundaries is still missing to date. Here we use multicomponent Supported Lipid Bilayers (SLBs) as reliable synthetic models, reproducing the nanometric lateral heterogeneity of cell membranes. After being characterized by Atomic Force Microscopy (AFM) and Neutron Reflectivity (NR), multi-domain SLBs are challenged by prototypical inorganic nanoparticles, i.e. citrated gold nanoparticles (AuNPs), under simplified and highly controlled conditions. By exploiting AFM, we demonstrate that AuNPs preferentially target lipid phase boundaries as adsorption sites. The herein reported study consolidates and extends the fundamental knowledge on NPs-membrane interactions, which constitute a key aspect to consider when designing NPs-related biomedical applications.

biophysics↗
bioRxiv2019

AFM-based High-Throughput Nanomechanical Screening of Single Extracellular Vesicles

We herein describe an Atomic Force Microscopy (AFM)-based experimental procedure which allows the simultaneous mechanical and morphological characterization of several hundred individual nanosized vesicles within the hour timescale. When deposited on a flat rigid surface from aqueous solution, vesicles are deformed by adhesion forces into oblate spheroids whose geometry is a direct consequence of their mechanical stiffness. AFM image analysis can be used to quantitatively measure the contact angle of individual vesicles, which is a size-independent descriptor of their deformation and, consequently, of their stiffness. The same geometrical measurements can be used to infer vesicle diameter in its original, spherical shape. We demonstrate the applicability of the proposed approach to natural vesicles obtained from different sources, recovering their size and stiffness distributions by simple AFM imaging in liquid. We show how the combined EV stiffness/size readout is able to discriminate between subpopulations of vesicular and non-vesicular objects in the same sample, and between populations of vesicles with similar sizes but different mechanical characteristics. We also discuss a force spectroscopy calibration procedure to quantitatively link the stiffness of EVs to their average contact angle. Finally, we discuss expected extensions and applications of the methodology.

biophysics↗