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Vesna Spasovski,1,2,* Anna Romolo,1,3,* Urška Zagorc,4 Vesna Arrigler,4 Matic Kisovec,5 Apolonija Bedina Zavec,5 Matevž Arko,1 Adrienn Molnár,6,7 Gitta Schlosser,7 Aleš Iglič,3,8 Ksenija Kogej,4 Veronika Kralj-Iglič1 1University of Ljubljana, Faculty of Health Sciences, Laboratory of Clinical Biophysics, Ljubljana, Slovenia; 2Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia; 3University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Physics, Ljubljana, Slovenia; 4University of Ljubljana, Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia; 5National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia; 6Hevesy György PhD School of Chemistry, ELTE Eötvös Loránd University, Budapest, Hungary; 7MTA-ELTE Lendület Ion Mobility Mass Spectrometry Research Group, Faculty of Science, Institute of Chemistry, ELTE Eötvös Loránd University, Budapest, Hungary; 8University of Ljubljana, Faculty of Medicine, Laboratory of Clinical Biophysics, Ljubljana, Slovenia*These authors contributed equally to this workCorrespondence: Veronika Kralj-Iglič, Email veronika.kralj-iglic@zf.uni-lj.siIntroduction: Lipid nanovesicles associated with bioactive phytochemicals from spruce needle homogenate (here called nano-sized hybridosomes or nanohybridosomes, NSHs) were considered.Methods: We formed NSHs by mixing appropriate amounts of lecithin, glycerol and supernatant of isolation of extracellular vesicles from spruce needle homogenate. We visualized NSHs by light microscopy and cryogenic transmission electron microscopy and assessed them by flow cytometry, dynamic light scattering, ultraviolet–visual spectroscopy, interferometric light microscopy and liquid chromatography–mass spectrometry.Results: We found that the particles consisted of a bilayer membrane and a fluid-like interior. Flow cytometry and interferometric light microscopy measurements showed that the majority of the particles were nano-sized. Dynamic light scattering and interferometric light microscopy measurements agreed well on the average hydrodynamic radius of the particles Rh (between 140 and 180 nm), while the concentrations of the particles were in the range between 1013 and 1014/mL indicating that NSHs present a considerable (more than 25%) of the sample which is much more than the yield of natural extracellular vesicles (EVs) from spruce needle homogenate (estimated less than 1%). Spruce specific lipids and proteins were found in hybridosomes.Discussion: Simple and low-cost preparation method, non-demanding saving process and efficient formation procedure suggest that large-scale production of NSHs from lipids and spruce needle homogenate is feasible.Plain Language Summary: Cells shed into their exterior nanoparticles (here referred to as extracellular vesicles – EVs) that are free to move, reach distant cells and are taken up by them. As they carry bioactive constituents, EVs may have important impact on the recipient cells. The mechanisms of EV formation and mediation can be employed in designing therapeutic, prophylactic and diagnostic methods for various medical issues. EVs can be harvested from biological samples; however, their yield is small,12 and there are potential side effects. Artificial vesicles – liposomes – have high yield; however, in vivo, they can be degraded before reaching the target and their reproducibility is yet insufficient. In order to combine advantages of both types of nanoparticles, we have composed nanohybridosomes (NSHs) from soya lecithin, water and supernatant of isolation of EVs from spruce needle homogenate, visualized them by cryogenic electron microscopy and characterized them with respect to their size, concentration and protein/nucleic acid content. We have applied a recently developed interferometric light microscopy to determine the hydrodynamic radius and the concentration of EVs. We found that the majority of composed particles are nano-sized and that they enclose more than 25% of the incoming volume of liquid, which is considerably more than about 1% that can be harvested by isolation of EVs from spruce needle homogenate by (ultra)centrifugation.Keywords: hybridosomes, liposomes, nanovesicles, extracellular particles, small cellular particles, drug delivery

Abstract Background Respirable mineral particles can induce lower airway inflammation, but the role they play in asthma of horses is unknown. Objectives Respirable mineral particles, particularly respirable silica, are an overlooked determinant of chronic lung inflammation (asthma) in horses. Animals Twenty‐three horses from an equine hospital population: 11 moderately affected (MEA), 7 severely asthmatic (SEA), and 5 control horses free from respiratory clinical signs. Methods Prospective observational study. The quantity and quality of mineral particles found in bronchoalveolar lavage fluid (BALF) were characterized, with particular attention to silica content. Polarized light microscopy performed on cytospin slides identified intracellular birefringent particles as silica. Spectrometry‐based analysis performed on whole BALF determined total mineral and silica percentage and concentration. Group‐related differences in BALF mineral and silica load were investigated as well as associations with BALF cytology. Results Intracellular birefringent particles were increased in SEA vs MEA (median [interquartile range, IQR]), 12 [7] vs 4 [5] particles/30 high power fields [hpf], respectively; P = .01) and vs controls (4 [2] particles/30 hpf; P = .02). Total mineral concentration in BALF was similar between the groups studied, whereas silica concentration and percentage were increased in SEA vs MEA (1758 [887] particles/mL and 20 [10]% vs 867 [662] particles/mL and 8 [6]%; P = .009 and P = .001) and control group (355 [330] particles/mL and 6 [3]%; P = .0003 and P = .002). Silica load in BALF was associated with BALF neutrophilia in MEA and SEA. Conclusions and Clinical Importance Respirable silica is associated with neutrophilic lower airway inflammation in horses and might contribute to asthma development.

Pets have a crucial role in cancer research. Specifically, dogs and cats share the same environment as their owners and thus may serve as sentinels of naturally occurring tumors that are linked to the exposure to environmental hazards. Quantitative comparison of tumor types may reveal unusual cancer frequencies, providing directions for research and generation of hypotheses of cancer causation in a specific area and identification of risk factors. The aim of this study was to describe the data collected by the pathology-based animal cancer registry, managed by Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise (IZSAM), during 10 years of activity (2014–2023) and to assess its potential epidemiological relevance. Frequencies of tumor topography and morphology in dogs and cats were described, analyzed and compared. Proportional morbidity ratios (PMRs) were calculated, taking into consideration some potential risk factors such as species, breed, sex, diet and living environment. The database comprises 5311 tumors (n = 4719 in dogs and n = 592 in cats), with a higher prevalence in females (67.3% in dogs and 61.2% in cats). The mean age at the first diagnosis of tumors was similar between sexes and slightly lower in dogs compared to cats. PMRs highlighted certain risk and “protective” factors for the development of tumors in specific topography. The risk of developing tumors of the blood and hematopoietic system (PMR = 0.44; 95% CI: 0.21–0.94), skin and subcutaneous tissues (PMR = 0.70; 95% CI: 0.61–0.80), oral cavity and pharynx (PMR = 0.60; 95% CI: 0.24–0.89), urinary organs (PMR = 0.33; 95% CI: 0.11–0.99) and bones, joints and cartilage (PMR = 0.72; 95% CI: 0.22–0.98) was lower in non-neutered male dogs than in neutered male dogs. Non-spayed female dogs had a greater risk of developing tumors of the mammary gland (PMR = 1.75; 95% CI: 1.57–1.96), female sexual organs (PMR = 2.12; 95% CI: 1.01–4.36) and respiratory system (PMR = 2.25; 95% CI: 1.55–6.74) but less risk for cutaneous and subcutaneous tissue tumors (PMR = 0.44; 95% CI: 0.38–0.51) and blood/hematopoietic system tumors (PMR = 0.47; 95% CI: 0.26–0.85) compared to spayed female dogs. Compared with mixed breed, purebred dogs had a significantly greater risk of developing mammary gland tumors (PMR = 1.36; 95% CI: 1.20–1.54) and lower risk for respiratory (PMR = 0.15; 95% CI: 0.07–0.32), gastrointestinal (PMR = 0.63; 95% CI: 0.34–0.94) and oral (PMR = 0.59; 95% CI: 0.36–0.96) neoplasia. Non-neutered male cats had a lower risk of developing skin and subcutaneous tumors (PMR = 0.68; 95% CI: 0.50–0.92) compared with neutered cats. The risk of developing skin and subcutaneous tissues tumors was higher for dogs and cats that lived mostly outdoor (PMR dogs = 1.21; 95% CI: 1.10–1.33; PMR cats = 1.18; 95% CI: 1.08–1.47), while dogs that live mainly indoor had a greater risk to develop mammary gland tumors (PMR = 0.78; 95% CI: 0.68–0.89). Results described herein highlight the fundamental role of animal cancer registration initiatives. These efforts would contribute to the possibility of conducting multicentric collaborative studies to deepen the knowledge of the epidemiology of tumors in dogs and cats from a comparative perspective, thus fulfilling the One Health approach.

Background: Respirable mineral particles can induce lower airway inflammation, but the role they play in asthma of horses is unknown. Objectives: Respirable mineral particles, particularly respirable silica, are an overlooked determinant of chronic lung inflammation (asthma) in horses. Animals: Twenty‐three horses from an equine hospital population: 11 moderately affected (MEA), 7 severely asthmatic (SEA), and 5 control horses free from respiratory clinical signs. Methods: Prospective observational study. The quantity and quality of mineral particles found in bronchoalveolar lavage fluid (BALF) were characterized, with particular attention to silica content. Polarized light microscopy performed on cytospin slides identified intracellular birefringent particles as silica. Spectrometry‐based analysis performed on whole BALF determined total mineral and silica percentage and concentration. Group‐related differences in BALF mineral and silica load were investigated as well as associations with BALF cytology. Results: Intracellular birefringent particles were increased in SEA vs MEA (median [interquartile range, IQR]), 12 [7] vs 4 [5] particles/30 high power fields [hpf], respectively; P =.01) and vs controls (4 [2] particles/30 hpf; P =.02). Total mineral concentration in BALF was similar between the groups studied, whereas silica concentration and percentage were increased in SEA vs MEA (1758 [887] particles/mL and 20 [10]% vs 867 [662] particles/mL and 8 [6]%; P =.009 and P =.001) and control group (355 [330] particles/mL and 6 [3]%; P =.0003 and P =.002). Silica load in BALF was associated with BALF neutrophilia in MEA and SEA. Conclusions and Clinical Importance: Respirable silica is associated with neutrophilic lower airway inflammation in horses and might contribute to asthma development. [ABSTRACT FROM AUTHOR]

Simple Summary: Pets share the same environment as humans, making them useful for studying how environmental factors, such as pollution, toxicants and microorganisms, could contribute to cancer onset and development. To better understand tumor frequency, diagnoses, topography and related risk factors, we performed a retrospective study of tumor cases in dogs and cats in Abruzzo and Molise regions (central Italy) over a ten-year period. We analyzed over 5300 tumor cases, with the majority of cases found in dogs. The mean age at the first diagnosis of tumors was similar between sexes and slightly lower in dogs compared to cats. Female animals were the majority, and the most affected sites were the skin, mammary glands and subcutaneous tissues. Non-neutered animals and those living outdoors showed higher risks for certain types of tumors, such as malignant mammary tumors and skin and subcutaneous tumors, respectively. This study also revealed that purebred dogs had a higher risk of developing mammary tumors compared to mixed breeds. Understanding cancer epidemiology in pets could ultimately help protect both animal and human health by implementing prevention strategies that align with the One Health approach. Pets have a crucial role in cancer research. Specifically, dogs and cats share the same environment as their owners and thus may serve as sentinels of naturally occurring tumors that are linked to the exposure to environmental hazards. Quantitative comparison of tumor types may reveal unusual cancer frequencies, providing directions for research and generation of hypotheses of cancer causation in a specific area and identification of risk factors. The aim of this study was to describe the data collected by the pathology-based animal cancer registry, managed by Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise (IZSAM), during 10 years of activity (2014–2023) and to assess its potential epidemiological relevance. Frequencies of tumor topography and morphology in dogs and cats were described, analyzed and compared. Proportional morbidity ratios (PMRs) were calculated, taking into consideration some potential risk factors such as species, breed, sex, diet and living environment. The database comprises 5311 tumors (n = 4719 in dogs and n = 592 in cats), with a higher prevalence in females (67.3% in dogs and 61.2% in cats). The mean age at the first diagnosis of tumors was similar between sexes and slightly lower in dogs compared to cats. PMRs highlighted certain risk and "protective" factors for the development of tumors in specific topography. The risk of developing tumors of the blood and hematopoietic system (PMR = 0.44; 95% CI: 0.21–0.94), skin and subcutaneous tissues (PMR = 0.70; 95% CI: 0.61–0.80), oral cavity and pharynx (PMR = 0.60; 95% CI: 0.24–0.89), urinary organs (PMR = 0.33; 95% CI: 0.11–0.99) and bones, joints and cartilage (PMR = 0.72; 95% CI: 0.22–0.98) was lower in non-neutered male dogs than in neutered male dogs. Non-spayed female dogs had a greater risk of developing tumors of the mammary gland (PMR = 1.75; 95% CI: 1.57–1.96), female sexual organs (PMR = 2.12; 95% CI: 1.01–4.36) and respiratory system (PMR = 2.25; 95% CI: 1.55–6.74) but less risk for cutaneous and subcutaneous tissue tumors (PMR = 0.44; 95% CI: 0.38–0.51) and blood/hematopoietic system tumors (PMR = 0.47; 95% CI: 0.26–0.85) compared to spayed female dogs. Compared with mixed breed, purebred dogs had a significantly greater risk of developing mammary gland tumors (PMR = 1.36; 95% CI: 1.20–1.54) and lower risk for respiratory (PMR = 0.15; 95% CI: 0.07–0.32), gastrointestinal (PMR = 0.63; 95% CI: 0.34–0.94) and oral (PMR = 0.59; 95% CI: 0.36–0.96) neoplasia. Non-neutered male cats had a lower risk of developing skin and subcutaneous tumors (PMR = 0.68; 95% CI: 0.50–0.92) compared with neutered cats. The risk of developing skin and subcutaneous tissues tumors was higher for dogs and cats that lived mostly outdoor (PMR dogs = 1.21; 95% CI: 1.10–1.33; PMR cats = 1.18; 95% CI: 1.08–1.47), while dogs that live mainly indoor had a greater risk to develop mammary gland tumors (PMR = 0.78; 95% CI: 0.68–0.89). Results described herein highlight the fundamental role of animal cancer registration initiatives. These efforts would contribute to the possibility of conducting multicentric collaborative studies to deepen the knowledge of the epidemiology of tumors in dogs and cats from a comparative perspective, thus fulfilling the One Health approach. [ABSTRACT FROM AUTHOR]

Nano- and micro-sized vesicular and colloidal structures mediate cell–cell communication. They are important players in the physiology of plants, animals, and humans, and are a subject of increasing interest. We investigated the effect of three surfactants, N-cetylpyridinium chloride (CPC), sodium dodecyl sulfate (SDS), and Triton X-100 (TX100), and two anionic polyelectrolytes, sodium polystyrene sulfonate (NaPSS) and sodium polymethacrylate (NaPMA), on nanoliposomes. In addition, the effect of SDS and TX100 on selected biological membranes (erythrocytes and microalgae) was investigated. The liposomes were produced by extrusion and evaluated by microcalorimetry and light scattering, based on the total intensity of the scattered light (Itot), hydrodynamic radius (Rh), radius of gyration (Rg), shape parameter p (=Rh/Rg,0), and polydispersity index. The EPs shed from erythrocytes and microalgae Dunaliella tertiolecta and Phaeodactylum tricornutum were visualized by scanning electron microscopy (SEM) and analyzed by flow cytometry (FCM). The Rh and Itot values in POPC liposome suspensions with added CPC, SDS, and TX100 were roughly constant up to the respective critical micelle concentrations (CMCs) of the surfactants. At higher compound concentrations, Itot dropped towards zero, whereas Rh increased to values higher than in pure POPC suspensions (Rh ≈ 60–70 nm), indicating the disintegration of liposomes and formation of larger particles, i.e., various POPC–S aggregates. Nanoliposomes were stable upon the addition of NaPSS and NaPMA, as indicated by the constant Rh and Itot values. The interaction of CPC, SDS, or TX100 with liposomes was exothermic, while there were no measurable heat effects with NaPSS or NaPMA. The SDS and TX100 increased the number density of EPs several-fold in erythrocyte suspensions and up to 30-fold in the conditioned media of Dunaliella tertiolecta at the expense of the number density of cells, which decreased to less than 5% in erythrocytes and several-fold in Dunaliella tertiolecta. The SDS and TX100 did not affect the number density of the microalgae Phaeodactylum tricornutum, while the number density of EPs was lower in the conditioned media than in the control, but increased several-fold in a concentration-dependent manner. Our results indicate that amphiphilic molecules need to be organized in nanosized particles to match the local curvature of the membrane for facilitated uptake. To pursue this hypothesis, other surfactants and biological membranes should be studied in the future for more general conclusions. [ABSTRACT FROM AUTHOR]