Detection and Quantification of Nocardia crassostreae, an Emerging Pathogen, in Mytilus galloprovincialis in the Mediterranean Sea Using Droplet Digital PCR.

Nocardia crassostreae is a novel pathogen responsible for infections in oysters (Crassostrea gigas) and mussels (Mytilus galloprovincialis). N. crassostreae is also responsible for nocardiosis both in immunocompetent and immunocompromised patients. We investigated N. crassostreae DNA in mussels grown in marine sites of the Mediterranean Sea in the Campania Region. We examined 185 mussel pooled samples by droplet digital PCR (ddPCR) and real-time quantitative PCR (qPCR), each pool composed of 10 mussels and 149 individual mussels. ddPCR detected N. crassostreae DNA in 48 mussel pooled samples and in 23 individual mussel samples. qPCR detected N. crassostreae DNA in six pooled samples and six individual mussel samples. The two molecular assays for the detection of N. crassostreae DNA showed significant differences both in the pooled and in individual samples. Our study demonstrated that ddPCR outperformed real-time qPCR for N. crassostreae DNA detection, thus confirming that ddPCR technology can identify the pathogens in many infectious diseases with high sensitivity and specificity. Furthermore, in individual mussels showing histological lesions due to N. crassostreae, the lowest copy number/microliter detected by ddPCR of this pathogen was 0.3, which suggests that this dose could be enough to cause infections of N. crassostreae in mussels.

Multipathogen infections and multifactorial pathogenesis involved in noble pen shell (Pinna nobilis) mass mortality events: Background and current pathologic approaches.

Disease outbreaks in several ecologically or commercially important invertebrate marine species have been reported in recent years all over the world. Mass mortality events (MMEs) have affected the noble pen shell (Pinna nobilis), causing its near extinction. Our knowledge of the dynamics of diseases affecting this species is still unclear. Early studies investigating the causative etiological agent focused on a novel protozoan parasite, Haplosporidium pinnae, although further investigations suggested that concurrent polymicrobial infections could have been pivotal in some MMEs, even in the absence of H. pinnae. Indeed, moribund specimens collected during MMEs in Italy, Greece, and Spain demonstrated the presence of a bacteria from within the Mycobacterium simiae complex and, in some cases, species similar to Vibrio mediterranei. The diagnostic processes used for investigation of MMEs are still not standardized and require the expertise of veterinary and para-veterinary pathologists, who could simultaneously evaluate a variety of factors, from clinical signs to environmental conditions. Here, we review the available literature on mortality events in P. nobilis and discuss approaches to define MMEs in P. nobilis. The proposed consensus approach should form the basis for establishing a foundation for future studies aimed at preserving populations in the wild.

Pathology, epidemiology, and phylogeny of mussel egg disease due to the microsporidian Steinhausia mytilovum (Field, 1924) in the Mediterranean mussel (Mytilus galloprovincialis).

Microsporidia are well known fungal pathogens of aquatic animals. However, the taxonomy of microsporidia is generally poorly resolved, which has consequently constrained our understanding of their pathology and epidemiology in marine animals. To date, microsporidia have been reported in both bivalves and gastropods, and microsporidia from mollusks have been classified in different genera. Despite ongoing work to better describe these genera, including detailed microscopic and ultrastructural images, so far we lack information on microsporidian phylogeny and pathogenicity of species within these genera. Here we investigate the microsporidian parasite Steinhausia mytilovum associated with the mussel, Mytilus galloprovincialis, in natural beds and farms along coast of southern Italy. A survey of M. galloprovincialis was conducted in 13 mussel farms and one natural bed between 2009 and 2020. We found the presence of S. mytilovum in 10 of the investigated farms, with a prevalence ranging between 14 and 100% of female mussels, depending on the population and season in which they were sampled. The parasite developed in the oocytes within a sporophorous vesicle (SV) where it produced 1-3 spores per cell, both in the cytoplasm and in the nucleus. Stenhausia mytilovum elicited an infiltrative (24.8%) or a strong capsular inflammatory response (43.4%) at gonadal follicles and surrounding vesicular connective tissue, in some cases accompanied by gonadal atresia (24.8%), leading to loss of gonadal architecture. In 7% of cases no reaction was observed. Ultrastructural observations revealed a mitochondrial re-organization to interact with all the phases of parasite development; the mitochondria were arranged outside the parasitophorous vesicle (PV) or directly interacting with the spore inside vesicle. There are five taxonomic clades of microsporidians as identified by SSU ribosomal gene sequence data. Maximum likelihood analysis assigned S. mytilovum within the Clade IV, defined as the Class Terresporidia, with closest genetic relationship (83.6% identity) to an undetermined invertebrate ovarian microsporidian. The constant presence, prevalence, and severity of S. mytilovum in coastline populations of M. galloprovincialis populations in southern Italy may indirectly reflect immunocompetence at both individual and population levels.

Investigation of the highly endangered Pinna nobilis' mass mortalities: Seasonal and temperature patterns of health status, antioxidant and heat stress responses.

Recently, P. nobilis populations have suffered a tremendous reduction, with pathogens potentially playing a crucial role. Considering its highly endangered status, mechanisms leading to mass mortalities were examined in one or multiple pathogens infected populations. Thus, seasonal antioxidant enzymatic activities, hsp70 and catalase mRNA levels, were investigated in two different Greek populations, during mass mortality events in summer of 2020. Samples were collected from Fthiotis and Lesvos during February (ToC 14 ± 1.2 and 15 ± 1 respectively), April (ToC 18 ± 1.2 and 17 ± 1.3 respectively), and June (ToC 24.5 ± 1.5 and 21.5 ± 1.5 respectively) 2020. In July of the same year (ToC 26.5 ± 1.7 in Fthiotis and 24.5 ± 1.7 in Lesvos), no live specimens were found. All biochemical parameters and phylogenetic analysis suggest that pathogen infection increases P. nobilis sensitivity to water temperature, subsequently leading to mass mortality. The latter was obvious in Fthiotis individuals, in which Haplosporidium pinnae was also observed with Mycobacterium spp., compared to Lesvos individuals.

Calcium phosphates from fish bones in sunscreen: An LCA and toxicity study of an emerging material for circular economy.

The use of sustainable and natural materials is an ever-increasing trend in cosmetic. Natural calcium phosphate (CaP-N) from food by-products and especially from fisheries (i.e., bones), has been suggested as a sustainable option to chemicals commonly used in cosmetic products, in particular to UV-filters in sunscreens. However, the environmental benefits and impacts of its production and use are still uncertain as they have never been quantified. In this paper, we report on toxicological characterization of CaP-N produced from incineration of fish meal in a pilot scale plant. Furthermore, we quantified the environmental burdens linked to the partial substitution of UV-filters by CaP-N through the life cycle assessment (LCA) comparing CaP-N with zinc oxide nanoparticles (ZnO NPs) as alternative option. CaP-N consists in a biphasic mixture 53:47 of hydroxyapatite:ß-tricalcium phosphate, and is made of round particles with a diameter in the range of a few microns. Toxicity tests on 4 aquatic species (Dunaliella tertiolecta, Tigriopus fulvus, Corophium insidiosum and Gammarus aequicauda) revealed that CaP-N does not produce any adverse effect, all the species showing EC/LC50 values higher than 100 mg L-1. Moreover, during the 96 h acute toxicity test on C. insidiosum, which is a tube-building species, the specimens built their tubes with the available CaP-N, further attesting the non-toxicity of the material. The LCA study showed that the environmental performance of CaP-N is better than that of ZnO NPs for 11 out of 16 impact categories analysed in this study, especially for the categories Ecotoxicity and Eutrophication of freshwaters (an order of magnitude lower), and with the exception of fossil resources for which CaP-N has a significantly higher impact than ZnO NPs (+140 %). Concluding, our study demonstrates that the replacement of ZnO NPs with CaP-N thermally extracted from fish bones in cosmetic products can increase their safety and sustainability.

A widespread picornavirus affects the hemocytes of the noble pen shell (Pinna nobilis), leading to its immunosuppression.

Introduction: The widespread mass mortality of the noble pen shell (Pinna nobilis) has occurred in several Mediterranean countries in the past 7 years. Single-stranded RNA viruses affecting immune cells and leading to immune dysfunction have been widely reported in human and animal species. Here, we present data linking P. nobilis mass mortality events (MMEs) to hemocyte picornavirus (PV) infection. This study was performed on specimens from wild and captive populations. Methods: We sampled P. nobilis from two regions of Spain [Catalonia (24 animals) and Murcia (four animals)] and one region in Italy [Venice (6 animals)]. Each of them were analyzed using transmission electron microscopy (TEM) to describe the morphology and self-assembly of virions. Illumina sequencing coupled to qPCR was performed to describe the identified virus and part of its genome. Results and discussion: In 100% of our samples, ultrastructure revealed the presence of a virus (20 nm diameter) capable of replicating within granulocytes and hyalinocytes, leading to the accumulation of complex vesicles of different dimensions within the cytoplasm. As the PV infection progressed, dead hemocytes, infectious exosomes, and budding of extracellular vesicles were visible, along with endocytic vesicles entering other cells. The THC (total hemocyte count) values observed in both captive (eight animals) (3.5 × 104-1.60 × 105 ml-1 cells) and wild animals (14 samples) (1.90-2.42 × 105 ml-1 cells) were lower than those reported before MMEs. Sequencing of P. nobilis (six animals) hemocyte cDNA libraries revealed the presence of two main sequences of Picornavirales, family Marnaviridae. The highest number of reads belonged to animals that exhibited active replication phases and abundant viral particles from transmission electron microscopy (TEM) observations. These sequences correspond to the genus Sogarnavirus-a picornavirus identified in the marine diatom Chaetoceros tenuissimus (named C. tenuissimus RNA virus type II). Real-time PCR performed on the two most abundant RNA viruses previously identified by in silico analysis revealed positive results only for sequences similar to the C. tenuissimus RNA virus. These results may not conclusively identify picornavirus in noble pen shell hemocytes; therefore, further study is required. Our findings suggest that picornavirus infection likely causes immunosuppression, making individuals prone to opportunistic infections, which is a potential cause for the MMEs observed in the Mediterranean.

Strontium-doped apatitic bone cements with tunable antibacterial and antibiofilm ability.

Injectable calcium phosphate cements (CPCs) represent promising candidates for the regeneration of complex-shape bone defects, thanks to self-hardening ability, bioactive composition and nanostructure offering high specific surface area for cell attachment and conduction. Such features make CPCs also interesting for functionalization with various biomolecules, towards the generation of multifunctional devices with enhanced therapeutic ability. In particular, strontium-doped CPCs have been studied in the last years due to the intrinsic antiosteoporotic character of strontium. In this work, a SrCPC previously reported as osteointegrative and capable to modulate the fate of bone cells was enriched with hydroxyapatite nanoparticles (HA-NPs) functionalized with tetracycline (TC) to provide antibacterial activity. We found that HA-NPs functionalized with TC (NP-TC) can act as modulator of the drug release profile when embedded in SrCPCs, thus providing a sustained and tunable TC release. In vitro microbiological tests on Escherichia coli and Staphylococcus aureus strains proved effective bacteriostatic and bactericidal properties, especially for the NP-TC loaded SrCPC formulations. Overall, our results indicate that the addition of NP-TC on CPC acted as effective modulator towards a tunable drug release control in the treatment of bone infections or cancers.

Calcium Phosphates from Fishery Byproducts as a Booster of the Sun Protection Factor in Sunscreens.

Calcium phosphates (CaPs) have been recently proposed as a bio- and eco-compatible alternative to UV filters in sunscreens, which are in the spotlight for being associated with health risks for both people and the environment. Here, natural CaPs extracted from fish bones have been tested as a booster of the sun protection factor (SPF), that is, as material working in synergy with UV filters to increase sunscreen UV-shielding efficiency, in combination with three of the most used UV filters, namely, octocrylene (OCR), octinoxate, and padimate-O, at different concentrations (10.0 and 20.0 wt %). The material obtained by calcination at 800 °C (CaP-N) was also enriched with Zn (CaP-Zn) or Mn (CaP-Mn) in an attempt to increase its SPF-boosting abilities. CaP-N and CaP-Zn consisted of a biphasic mixture of hydroxyapatite and beta tricalcium phosphate, while CaP-Mn presented a small quantity of Mn oxides. CaP-N was the most effective at increasing the SPF of the final emulsions, doubling the SPF of the formulation containing 20.0 wt % of OCR from 40.6 to 80.8. The results show that these CaPs, produced according to a circular economy approach, can be used as effective SPF boosters to decrease the concentration of UV filters used in sunscreen, while retaining high SPF values.

Chemical, Functional, and Technological Features of Grains, Brans, and Semolina from Purple and Red Durum Wheat Landraces.

A main reason of the increasing interest in cereal landraces is their potential to offer more diversified and functional staple food. For instance, landraces are an underexploited resource of pigmented varieties, appreciated for the high accumulation of phytochemicals with known health benefits. This study characterized the chemical, functional, and technological features of the bran, semolina, and grains of two durum wheat (Triticum turgidum L. subsp. durum, Desf.) landraces, named 'Purple' and 'Red' for their grain color, collected in Ethiopia and grown and sold in southern Italy as a niche product. Specifically, we analyzed the protein content, dry gluten, ash, total polyphenols, anthocyanins, proanthocyanidins, and specific phenolic acids. We also evaluated the antioxidant activity using DPPH- and ABTS-based methods. The two landraces had positive nutritional features, such as a high protein content, a rich and composite range of secondary metabolites (which include specific phenolic acids and anthocyanins), and antioxidant activities in all the fractions analyzed. The germplasm under investigation therefore has a well-justified potential to yield functional products and to diversify durum wheat-based foods.

Vibrio splendidus clade associated with a disease affecting Paracentrotus lividus (Lamarck, 1816) in Sardinia (Western Mediterranean).

This work reports for the first time the so-called bald sea urchin disease of the populations of Paracentrotus lividus in Sardinia (Italy). Following the disease occurrence reported by local fishermen of about 35% on the collected specimens for commercial purpose, the phenomenon was evaluated in two areas. Animals displayed external lesions represented by loss of spines and greenish/brownish skeleton patches. Microscopy of exoskeleton and tube feet showed haemocytosis with associated bacteria; gut and intestine histopathology revealed inflammatory lesions of different type and intensity. Microbiological and molecular analysis revealed the presence of the strains belonging to the Vibrio splendidus clade. Due to the important ecological role of Paracentrotus lividus in the Mediterranean Sea, this report represents a wake-up call for the entire basin worth of further insights.

Biocompatible antimicrobial colistin loaded calcium phosphate nanoparticles for the counteraction of biofilm formation in cystic fibrosis related infections.

The use of inhalable nanoparticles (NPs) for cystic fibrosis (CF) has been advocated as a promising tool to improve the efficacy of antimicrobials taking advantage of their ability to penetrate airway mucus and pathogen biofilm and to release the drug in or in proximity to the enclosed bacteria. Here, inhalable calcium phosphate (CaP) NPs were functionalized with colistin (Col) which is one of the most active antimicrobials against Gram-negative bacteria. The adsorption kinetic and isotherm of Col on CaP-NPs were investigated and fitted according to different mathematical models and revealed an electrostatic interaction between positively charged amine groups of Col and negatively charged surface of CaP-NPs. The maximum Col payload was of about 50 mg g-1 of CaP-NPs. After functionalization, despite an increase of size (213 vs 95 nm), in citrate solution, CaP-NPs maintained a dimension and surface charge considered suitable for crossing mucus barrier. CaP-NPs do not interact with mucin and are able to permeate a layer of artificial mucus. In vitro tests on pulmonary cells demonstrated that CaP-NPs are not cytotoxic up to a concentration of 125 µg mL-1. The antimicrobial and antibiofilm activity of Col loaded CaP-NPs tested on Pseudomonas aeruginosa RP73, a clinical strain isolated from a CF patient, was similar to that of free Col demonstrating that the therapeutic effect of Col adsorbed on CaP-NPs was retained. This work represents the first attempt to use CaP-NPs as delivery system for the CF treatment. The encouraging results open the way to further studies.

Cytotoxicity of an Innovative Pressurised Cyclic Solid-Liquid (PCSL) Extract from Artemisia annua.

Therapeutic treatments with Artemisia annua have a long-established tradition in various diseases due to its antibacterial, antioxidant, antiviral, anti-malaria and anti-cancer effects. However, in relation to the latter, virtually all reports focused on toxic effects of A. annua extracts were obtained mostly through conventional maceration methods. In the present study, an innovative extraction procedure from A. annua, based on pressurised cyclic solid-liquid (PCSL) extraction, resulted in the production of a new phytocomplex with enhanced anti-cancer properties. This extraction procedure generated a pressure gradient due to compressions and following decompressions, allowing to directly perform the extraction without any maceration. The toxic effects of A. annua PCSL extract were tested on different cells, including three cancer cell lines. The results of this study clearly indicate that the exposure of human, murine and canine cancer cells to serial dilutions of PCSL extract resulted in higher toxicity and stronger propensity to induce apoptosis than that detected by subjecting the same cells to Artemisia extracts obtained through canonical extraction by maceration. Collected data suggest that PCSL extract of A. annua could be a promising and economic new therapeutic tool to treat human and animal tumours.

The Use of Calcium Phosphates in Cosmetics, State of the Art and Future Perspectives.

Calcium phosphates (CaPs) belong to a class of biomimetic materials widely employed for medical applications thanks to their excellent properties, such as biodegradability, biocompatibility and osteoinductivity. The recent trend in the cosmetics field of substituting potentially hazardous materials with natural, safe, and sustainable ingredients for the health of consumers and for the environment, as well as the progress in the materials science of academics and chemical industries, has opened new perspectives in the use of CaPs in this field. While several reviews have been focused on the applications of CaP-based materials in medicine, this is the first attempt to catalogue the properties and use of CaPs in cosmetics. In this review a brief introduction on the chemical and physical characteristics of the main CaP phases is given, followed by an up-to-date report of their use in cosmetics through a large literature survey of research papers and patents. The application of CaPs as agents in oral care, skin care, hair care, and odor control has been selected and extensively discussed, highlighting the correlation between the chemical, physical and toxicological properties of the materials with their final applications. Finally, perspectives on the main challenges that should be addressed by the scientific community and cosmetics companies to widen the application of CaPs in cosmetics are given.

Calcium Phosphate Particles Coated with Humic Substances: A Potential Plant Biostimulant from Circular Economy.

Nowadays, the use of biostimulants to reduce agrochemical input is a major trend in agriculture. In this work, we report on calcium phosphate particles (CaP) recovered from the circular economy, combined with natural humic substances (HSs), to produce a plant biostimulant. CaPs were obtained by the thermal treatment of Salmo salar bones and were subsequently functionalized with HSs by soaking in a HS water solution. The obtained materials were characterized, showing that the functionalization with HS did not sort any effect on the bulk physicochemical properties of CaP, with the exception of the surface charge that was found to get more negative. Finally, the effect of the materials on nutrient uptake and translocation in the early stages of development (up to 20 days) of two model species of interest for horticulture, Valerianella locusta and Diplotaxis tenuifolia, was assessed. Both species exhibited a similar tendency to accumulate Ca and P in hypogeal tissues, but showed different reactions to the treatments in terms of translocation to the leaves. CaP and CaP-HS treatments lead to an increase of P accumulation in the leaves of D. tenuifolia, while the treatment with HS was found to increase only the concentration of Ca in V. locusta leaves. A low biostimulating effect on both plants' growth was observed, and was mainly scribed to the low concentration of HS in the tested materials. In the end, the obtained material showed promising results in virtue of its potential to elicit phosphorous uptake and foliar translocation by plants.

Pinna nobilis in suboptimal environments are more tolerant to disease but more vulnerable to severe weather phenomena.

We examined a disease outbreak of the fan mussel, Pinna nobilis (L.), in the Alfacs Bay (South Ebro Delta, Spain) during a period of two years in three zones exposed to a summer salinity gradient resulting from agricultural freshwater discharges and distance to the open sea. Long-term monitoring was also conducted in Fangar Bay (North Ebro Delta), featuring lower salinities and no evidence of disease. Results showed that the salinity gradient of Alfacs Bay (37.4-35.7) was associated to cumulative mortality (100% near the mouth, 43% in middle regions, and 13% in inner regions), thus hindering the spread of pathogens. Young specimens showed to be more tolerant to disease than large adults but become vulnerable over time. In Fangar Bay, lower salinities (30.5-33.5) prevented the disease but individuals were highly vulnerable to Storm Gloria which caused 60% mortality in 3 weeks, and ~100% in 6 weeks.

Cosmopolitan Distribution of Endozoicomonas-Like Organisms and Other Intracellular Microcolonies of Bacteria Causing Infection in Marine Mollusks.

Intracellular microcolonies of bacteria (IMC), in some cases developing large extracellular cysts (bacterial aggregates), infecting primarily gill and digestive gland, have been historically reported in a wide diversity of economically important mollusk species worldwide, sometimes associated with severe lesions and mass mortality events. As an effort to characterize those organisms, traditionally named as Rickettsia or Chlamydia-like organisms, 1950 specimens comprising 22 mollusk species were collected over 10 countries and after histology examination, a selection of 99 samples involving 20 species were subjected to 16S rRNA gene amplicon sequencing. Phylogenetic analysis showed Endozoicomonadaceae sequences in all the mollusk species analyzed. Geographical differences in the distribution of Operational Taxonomic Units (OTUs) and a particular OTU associated with pathology in king scallop (OTU_2) were observed. The presence of Endozoicomonadaceae sequences in the IMC was visually confirmed by in situ hybridization (ISH) in eight selected samples. Sequencing data also indicated other symbiotic bacteria. Subsequent phylogenetic analysis of those OTUs revealed a novel microbial diversity associated with molluskan IMC infection distributed among different taxa, including the phylum Spirochetes, the families Anaplasmataceae and Simkaniaceae, the genera Mycoplasma and Francisella, and sulfur-oxidizing endosymbionts. Sequences like Francisella halioticida/philomiragia and Candidatus Brownia rhizoecola were also obtained, however, in the absence of ISH studies, the association between those organisms and the IMCs were not confirmed. The sequences identified in this study will allow for further molecular characterization of the microbial community associated with IMC infection in marine mollusks and their correlation with severity of the lesions to clarify their role as endosymbionts, commensals or true pathogens.

Epidemiology of Noble Pen Shell (Pinna nobilis L. 1758) Mass Mortality Events in Adriatic Sea Is Characterised with Rapid Spreading and Acute Disease Progression.

From May to October 2019, multiple mass mortality events (MMEs) of Pinna nobilis were observed along Croatian coastline starting from the south-east and rapidly progressing in north-western direction. Time dynamics of the MMEs closely followed general speed and direction patterns of surface sea-currents, advancing approximately 350 km in less than 3 months. Surveillance, clinical evaluation, and sample collection were performed on multiple sites with various degrees of mortality rates. Moribund P. nobilis individuals were collected and subjected to pathological, molecular, and microscopical investigation. Affected animals were positive for Mycobacterium in 70% of the individuals, and Haplosporidium pinnae was present in 58% of the cases. Observed pathological lesions were most severe where concurrent presence of both pathogens was confirmed (in 45.8% of moribund individuals). Moderate to strong lesions were observed in animals positive for Mycobacterium only (25% of cases), and lesions were absent or minor to moderate when only H. pinnae was confirmed (16% of cases). Considering the rapid and severe spread of the MMEs, the areas less exposed to major sea currents appeared to be at lower risk of pathogen transmission. Surveillance activities along the Croatian coastline identified several P. nobilis populations in such "lower risk" areas without apparent mortality or clinical symptoms. Such areas are of particular interest as source of potentially healthy individuals to support active recovery actions.

Mycobacteriosis in Aquatic Invertebrates: A Review of Its Emergence.

Mycobacteriosis is a chronic bacterial disease reported in aquatic and terrestrial animals, including humans. The disease affects a wide range of cultured and wild organisms worldwide. Mycobacteriosis is well-known in aquatic vertebrates (e.g., finfish, marine mammals), while in the last few years, reports of its presence in aquatic invertebrates have been on the rise, for both freshwater and marine species. The number of cases is likely to increase as a result of increased awareness, surveillance and availability of diagnostic methods. Domestication of wild aquatic species and the intensification of modern aquaculture are also leading to an increase in the number of reported cases. Moreover, climate changes are affecting fresh and marine aquatic ecosystems. The increasing reports of mycobacteriosis in aquatic invertebrates may also be influenced by global climate warming, which could contribute to the microbes' development and survival rates, pathogen transmission and host susceptibility. Several species of the genus Mycobacterium have been diagnosed in aquatic invertebrates; a few of them are significant due to their wide host spectrum, economic impact in aquaculture, and zoonotic potential. The impact of mycobacteriosis in aquatic invertebrates is probably underestimated, and there is currently no effective treatment other than facility disinfection. In this review, we provide an overview of the diversity of mycobacterial infections reported in molluscs, crustaceans, cnidarians, echinoderms and sponges. We highlight important issues relating to its pathological manifestation, diagnosis and zoonotic considerations.

Mussel Shell-Derived Macroporous 3D Scaffold: Characterization and Optimization Study of a Bioceramic from the Circular Economy.

Fish industry by-products constitute an interesting platform for the extraction and recovery of valuable compounds in a circular economy approach. Among them, mussel shells could provide a calcium-rich source for the synthesis of hydroxyapatite (HA) bioceramics. In this work, HA nanoparticles have been successfully synthesized starting from mussel shells (Mytilus edulis) with a two steps process based on thermal treatment to convert CaCO3 in CaO and subsequent wet precipitation with a phosphorus source. Several parameters were studied, such as the temperature and gaseous atmosphere of the thermal treatment as well as the use of two different phosphorus-containing reagents in the wet precipitation. Data have revealed that the characteristics of the powders can be tailored, changing the conditions of the process. In particular, the use of (NH4)2HPO4 as the phosphorus source led to HA nanoparticles with a high crystallinity degree, while smaller nanoparticles with a higher surface area were obtained when H3PO4 was employed. Further, a selected HA sample was synthesized at the pilot scale; then, it was employed to fabricate porous 3D scaffolds using the direct foaming method. A highly porous scaffold with open and interconnected porosity associated with good mechanical properties (i.e., porosity in the range 87-89%, pore size in the range 50-300 µm, and a compressive strength σ = 0.51 ± 0.14 MPa) suitable for bone replacement was achieved. These results suggest that mussel shell by-products are effectively usable for the development of compounds of high added value in the biomedical field.

Expression of transferrin receptor-1 (TFR-1) in canine osteosarcomas.

Due to high rates of proliferation and DNA synthesis, neoplastic cells have higher requirements of iron than normal cells. For that reason, neoplastic cells have remodelled iron metabolism pathways, over-expressing genes encoding for iron uptake proteins, among which Transferrin Receptor-1 (TFR-1). Accumulating evidence has proven that overexpression of TFR-1 and high Iron concentration, are both widespread condition of cancer cells, both essential to tumour onset and progression. We studied TFR-1 and PCNA immunohistochemical expression in fifteen (15) Canine osteoblastic osteosarcomas (COS). After immunohistochemical staining, counting of TFR-1 positive cells by two independent observers showed that 85%-95% of neoplastic cells were strongly labelled at cytoplasmic level by anti-TFR-1 antibody in all examined COS. Furthermore, 70%-80% of neoplastic cells were positively labelled at the nuclear level by PCNA. Surprisingly, about 100% of intratumour vascular endothelial cells were also positive, whereas extratumour vascular endothelial cells were negative. The latter is an interesting finding, as TFR-1 is usually not expressed in normal vasculature, with the exception of normal brain vascular endothelium, where it allows transport of transferrin, and thus iron, into tissues, suggesting a similar function here to support cancer growth. The early results presented highlight the relevance of TFR-1 expression in canine OS, suggesting therapies involving both TFR-1 and Iron metabolisms in dogs with osteosarcoma should be developed.