Biology subjects

Zappulli, V.

Publications and source records attributed to Zappulli, V..

5 recordsLinked to original sources

bioRxiv2026

Extracellular vesicles from Manila clam (Ruditapes philippinarum): tailored isolation from hemolymph and insights into water-derived vesicles

Extracellular vesicles (EVs) are evolutionarily conserved mediators of intercellular communication released by cells into biological fluids and the extracellular environment. Despite their growing relevance in biomedical and veterinary research, knowledge on EVs in marine bivalves remains limited. The aim of this study was to optimize tailored protocols for EV isolation from the hemolymph of the Manila clam (Ruditapes philippinarum) based on density gradient ultracentrifugation (dgUC) or size exclusion chromatography (SEC). EV-enriched fractions were identified through nanoparticle tracking analysis, protein quantification, transmission electron microscopy, and cryo-electron microscopy. Both methods successfully isolated small EVs (<200 nm). While dgUC yielded higher-purity preparations, SEC provided a higher recovery rate and compatibility with downstream metabolomic analyses. Metabolomics performed on SEC fractions and on hemolymph, revealed that EV-enriched fractions possessed a distinct metabolic signature including enrichment in metabolites associated with nucleotide metabolism, glycolysis, redox regulation, and energy metabolism. Furthermore, we performed a pilot investigation into the presence of EVs released into conditioned water by Manila clams. Using tangential flow filtration and ultrafiltration, EVs were successfully concentrated from water samples and characterized by nanoparticle tracking analysis, CONAN assay, atomic force microscopy, and electron microscopy. Our findings demonstrate the feasibility of isolating EVs both from Manila clam hemolymph and from conditioned water, providing the first evidence of water-derived EV recovery in aquatic animals. Although further methodological refinement is needed to improve the purity of EVs isolated from water, and additional characterization studies are required to better define the molecular composition of clam-derived EVs, these results establish a foundation for future investigations into the role of EVs in bivalve biology and their potential application as minimally invasive biomarkers for aquaculture, environmental monitoring, and ecosystem health assessment.

systems biology↗

bioRxiv2026

Isolation and preliminary characterization of extracellular vesicles from bottlenose dolphin (Tursiops truncatus) and long-finned pilot whale (Globicephala melas) blow

Cetaceans are key sentinel species for environmental health monitoring. Although sampling from free-ranging animals is challenging, the analysis of cetacean blow offers a minimally invasive approach to assess their health status. Extracellular vesicles (EVs) are cell-derived nanostructures present in biological fluids and widely studied as disease biomarkers in humans. Despite the potential for similar uses, EVs have not been studied in cetacean blow to date . This proof-of-concept study aims to assess the feasibility of the isolation and characterization of EVs from blow samples collected from five bottlenose dolphins (Tursiops truncatus) kept under human care and from a free ranging specimen of long-finned pilot whale (Globicephala melas). EVs were purified from bottlenose dolphin blows by ultracentrifugation (UC) or size exclusion chromatography (SEC) and from the long-finned pilot whale by SEC. Particle concentration and size distribution were assessed by Nanoparticle Tracking Analysis (NTA), morphology by Air-atomic force microscopy (AFM) and protein expression by Western Blotting (WB). NTA revealed a higher mean particle concentration in bottlenose dolphin EVs isolated by UC compared to SEC, while EVs isolated from the long-finned pilot whale presented a lower particle concentration. AFM confirmed the presence of EV-like particles within the typical EV size range in bottlenose dolphins EVs obtained both by SEC or UC. All EV samples were positive for CD9 and integrin-{beta} and negative to Calnexin. SEC was more sensitive to detect OmpA, a membrane protein of Gram-negative bacteria, in EVs from both species. Our pilot study demonstrates that EVs are present in cetacean blow and can be isolated and characterized. Future investigations focused on characterizing and quantifying a wider array of EV associated molecules may further the application of blow EV analysis for cetacean health assessments.

zoology↗

bioRxiv2024

Targeting SCD triggers lipotoxicity of cancer cells and enhances anti-tumor immunity in breast cancer brain metastasis mouse models

Breast cancer brain metastases (BCBM) are a significant cause of mortality and are incurable. Thus, identifying BCBM targets that reduce morbidity and mortality is critical. BCBM upregulate Stearoyl-CoA Desaturase (SCD), an enzyme that catalyzes the synthesis of monounsaturated fatty acids, suggesting a potential metabolic vulnerability of BCBM. In this study, we tested the effect of a brain-penetrant clinical-stage inhibitor of SCD (SCDi), on breast cancer cells and mouse models of BCBM. Lipidomics, qPCR, and western blot were used to study the in vitro effects of SCDi. Single-cell RNA sequencing was used to explore the effects of SCDi on cancer and immune cells in a BCBM mouse model. Pharmacological inhibition of SCD markedly reshaped the lipidome of breast cancer cells and resulted in endoplasmic reticulum stress, DNA damage, loss of DNA damage repair, and cytotoxicity. Importantly, SCDi alone or combined with a PARP inhibitor prolonged the survival of BCBM-bearing mice. When tested in a syngeneic mouse model of BCBM, scRNAseq revealed that pharmacological inhibition of SCD enhanced antigen presentation by dendritic cells, was associated with a higher interferon signaling, increased the infiltration of cytotoxic T cells, and decreased the proportion of exhausted T cells and regulatory T cells in the tumor microenvironment (TME). Additionally, pharmacological inhibition of SCD decreased engagement of immunosuppressive pathways, including the PD-1:PD-L1/PD-L2 and PVR/TIGIT axes. These findings suggest that SCD inhibition could be an effective strategy to intrinsically reduce tumor growth and reprogram anti-tumor immunity in the brain microenvironment to treat BCBM.

cancer biology↗

bioRxiv2023

NON-TOXIC ACID-FREE GLYOXAL FIXATIVE FOR VETERINARY HISTOPATHOLOGY, IMMUNOHISTOCHEMISTRY AND MOLECULAR ANALYSIS

Formaldehyde fixation is worldwide the most used system for histopathological examination. However, its toxicity is well known, and preservation of proteins and nucleic acids is not optimal. Alternative fixatives warranting similar morphological quality of tissues and costs, but lacking toxicity and allowing better preservation of proteins and nucleic acids would therefore increase both safety of operators and quality of molecular analysis in pathology. This multi-institutional study aimed to compare the morphological, histochemical, immunohistochemical (IHC), and molecular analyses outcomes of a newly patented, non-toxic, acid-free Glyoxal (GAF) fixative with neutral buffered formaldehyde (NBF). Tissues from a total of 73 subjects were analyzed, including 13 necropsies. Gross features were preserved after GAF fixation, with no tissue hardening or discoloration. Cellular ultrastructure was also better preserved with GAF and histology and histochemistry on GAF-fixed samples showed good results when compared to NBF-fixed samples, with the exception of loss of tinctorial affinity of erythrocytes and mast cell granules. IHC analyses also showed comparable results with only slight and rare protocol adjustment. DNA and RNA yields were higher from GAF-fixed samples (P<0.05) and the tested genes (p53 and COX1) were better amplified. RNA scope showed positive results for c-KIT expression in GAF-fixed mast cell tumors. Based on these data, the non-toxic GAF fixative allows good macroscopical, histological and immunohistochemical analyses of tissue samples, including on-field application, and better molecular analyses when compared to NBF. This represents a promising possibility for teaching, diagnostic, and research in veterinary pathology.

pathology↗

bioRxiv2018

Mitochondria form cholesterol-rich contact sites with the nucleus during retrograde response

Mitochondria drive cellular adaptation to stress by retro-communicating with the nucleus. This process is known as Mitochondrial Retrograde Response (MRR) and is induced by mitochondrial dysfunctions which perturb cell signalling. MRR results in the nuclear stabilization and activation of pro-survival transcription factors such as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). Here we demonstrate that MRR is facilitated by the formation of contact sites between mitochondria and the nucleus which establish microdomains of communication between the two organelles. The 18kD Translocator Protein (TSPO), which de-ubiquitylates and stabilizes the mitochondrial network preventing its mitophagy-mediated segregation, is required for this interaction. The tethering TSPO enacts is mediated by the complex formed with the Protein Kinase A via the A-kinase anchoring protein Acyl-CoA Binding Domain Containing 3 (ACBD3) and allows the redistribution of cholesterol which sustains the pro-survival response by blocking NF-kB de-acetylation. This work proposes a new paradigm in the mitochondrial retro-communication by revealing the existence of contact sites between mitochondrial and the nucleus and a signalling role for cholesterol.

cell biology↗