3D printing of PLA:CaP:GO scaffolds for bone tissue applications.

Graphene oxide (GO) has attracted increasing interest for biomedical applications owing to its outstanding properties such as high specific surface area, ability to bind functional molecules for therapeutic purposes and solubility, together with mechanical resistance and good thermal conductivity. The combination of GO with other biomaterials, such as calcium phosphate (CaP) and biodegradable polymers, presents a promising strategy for bone tissue engineering. Presently, the development of these advanced biomaterials benefits from the use of additive manufacturing techniques, such as 3D printing. In this study, we develop a 3D printed PLA:CaP:GO scaffold for bone tissue engineering. First, GO was characterised alone by XPS to determine its main bond contributions and C : O ratio. Secondly, we determined the GO dose which ensures the absence of toxicity, directly exposed in vitro (human osteoblast-like cells MG-63) and in vivo (zebrafish model). In addition, GO was microinjected in the zebrafish to evaluate its effect on immune cells, quantifying the genetic expression of the main markers. Results indicated that the GO tested (C : O of 2.14, 49.50% oxidised, main bonds: C-OH, C-O-C) in a dose ≤0.25 mg mL-1 promoted MG63 cells viability percentages above 70%, and in a dose ≤0.10 mg mL-1 resulted in the absence of toxicity in zebrafish embryos. The immune response evaluation reinforced this result. Finally, the optimised GO dose (0.10 mg mL-1) was combined with polylactic acid (PLA) and CaP to obtain a 3D printed PLA:CaP:GO scaffold. Physicochemical characterisation (SEM/EDS, XRD, FT-Raman, nano-indentation) was performed and in vivo tests confirmed its biocompatibility, enabling a novel approach for bone tissue-related applications.

Potential angiogenic biomarkers in hereditary hemorrhagic telangiectasia and other vascular diseases.

Biomarkers are new tools framed in precision and personalized medicine. Hereditary hemorrhagic telangiectasia (HHT) is a rare genetic vascular disease with disturbances in the angiogenic pathways. Descriptive evidence supports that some angiogenesis-related molecules are differently detected in HHT patients compared to healthy subjects. These molecules are also related to diagnosis, prognosis, complications and therapy monitoring in other common vascular diseases. Despite the need for improving knowledge before applying them in daily clinical practice, there are good candidates to be considered as potential biomarkers in HHT and other vascular diseases. In the present review, the authors aim to summarize and discuss current evidence regarding the main putative angiogenic biomarkers by describing the biological role of each biomarker, the evidence related to HHT and their potential use in this and other common vascular diseases from a clinical point-of-view.

Mytilus galloprovincialis releases immunologically functional haemocytes to the intervalvar space in response to tissue injury and infection.

Haemocytes of Mytilus galloprovincialis represent the main component of the internal self-defence system. Although haemocytes from haemolymph are usually studied to analyse these animals' immune response, the presence of haemocytes in the intervalvar liquid, which is essentially sea water, led us to characterize them. Several functional (ROS production, phagocytosis, gene expression, travel velocity and distance) and morphological (area, size and granularity) assays were performed by applying different stimuli to the mussels (waterborne infection, shell injury and their combination). Our results revealed that intervalvar liquid haemocytes share common characteristics with haemolymph haemocytes (for instance, the cell morphology and the cell population structure divided in three main groups) but also show significant differences in size (usually smaller in the intervalvar liquid), mobility (commonly faster in the intervalvar liquid), ROS production (higher in non-stimulated intervalvar liquid cells) and gene expression (IL17, Myd88 and CathL are over expressed in liquid intervalvar cells compared to haemolymph cells). Moreover, differences were observed when mussels were subjected to the mentioned treatments. These free intervalvar haemocytes could constitute the first line of defence as external sentinels extending the immunological alert system outside of the mussel body.

Comprehensive transcriptome profiling and functional analysis of the meagre (Argyrosomus regius) immune system.

Meagre (Argyrosomus regius) belongs to the family Sciaenidae and is a promising candidate for Mediterranean aquaculture diversification. As a relatively recent species in aquaculture, the physiological consequences of the immune system activation in meagre are understudied. Spleen, as a primary lymphoid organ has an essential role in meagre immune and inflammatory responses. In this study, we have evaluated the in vivo effects of lipopolysaccharide (LPS) on the spleen transcriptome of meagre by RNA-seq analysis at 4 and 24 h after injection.

An overview of the internalization and effects of microplastics and nanoplastics as pollutants of emerging concern in bivalves.

Microplastic and nanoplastic pollution in aquatic environments is a topic of emerging concern due to the internalization, retention time and effects of these particles in aquatic biota. Bivalves are considered bioindicators due to their wide distribution, sessile behaviour, occupation of ecological niches and ability to filter a large water volume. The study of microplastics and nanoplastics in bivalves has revealed the uptake mechanisms, internalization, distribution and depuration of these particles as well as their effects on physiological parameters, morphological alterations, immunotoxicity and changes in gene expression and proteomic profiles. In this review, we examine the primary characteristics of microplastics and nanoplastics (type of material, size, coating, density, additives and shapes) involved in their possible toxicity in bivalves. Furthermore, secondary characteristics such as the suspension media, aggregation stage and adsorption of persistent pollutants were also recorded to assess the impact of these materials on bivalves. Here, we have highlighted the efforts exerted thus far and the remaining gaps in understanding the extent of microplastic and nanoplastic impacts on bivalves on the basis of laboratory experiments and mesocosm bioassays and in the field. Furthermore, further microplastic and nanoplastic toxicological studies are proposed to facilitate the realistic assessment of environmental risk.

Size matters: Zebrafish (Danio rerio) as a model to study toxicity of nanoplastics from cells to the whole organism.

The contamination of the aquatic environment by plastic nanoparticles is becoming a major concern due to their potential adverse effects in aquatic biota. Therefore, in-depth knowledge of their uptake, trafficking and effects at cellular and systemic levels is essential to understand their potential impacts for aquatic species. In this work, zebrafish (Danio rerio) was used as a model and our aims were: i) to determine the distribution, uptake, trafficking, degradation and genotoxicity of polystyrene (PS) NPs of different sizes in a zebrafish cell line; ii) to study PS NPs accumulation, migration of immune cells and genotoxicity in larvae exposed to PS NPs; and iii) to assess how PS NPs condition the survival of zebrafish larvae exposed to a pathogen and/or how they impact the resistance of an immunodeficient zebrafish. Our results revealed that the cellular distribution differed depending on the particle size: the 50 nm PS NPs were more homogeneously distributed in the cytoplasm and the 1 µM PS NPs more agglomerated. The main endocytic mechanisms for the uptake of NPs were dynamin-dependent internalization for the 50 nm NPs and phagocytosis for the 1 µm nanoparticles. In both cases, degradation in lysosomes was the main fate of the PS NPs, which generated alkalinisation and modified cathepsin genes expression. These effects at cellular level agree with the results in vivo, since lysosomal alkalization increases oxidative stress and vice versa. Nanoparticles mainly accumulated in the gut, where they triggered reactive oxygen species, decreased expression of the antioxidant gene catalase and induced migration of immune cells. Finally, although PS NPs did not induce mortality in wild-type larvae, immunodeficient and infected larvae had decreased survival upon exposure to PS NPs. This fact could be explained by the mechanical disruption and/or the oxidative damage caused by these NPs that increase their susceptibility to pathogens.

Patients' contribution to drug safety in Catalonia: the interest of personal feelings on adverse drug reactions.

PURPOSE: There are few studies on the personal view retrieved by patients in the spontaneous reports' free-text section of suspected adverse drug reactions. METHODS: We analysed the suspected adverse drug reactions (ADRs) spontaneous reports sent to the Catalan Centre of Pharmacovigilance between 2013 and 2017. The information provided in the free-text section was classified as (1) temporal sequence, (2) description of symptoms, (3) description of psychological impact, (4) withdrawal effects, (5) alternative causes, and (6) rechallenge. The concordance level between the perceived severity by the reporter and the pharmacovigilance team was assessed by the Kappa index (ƙ). Usual descriptive statistics were used to describe variables. RESULTS: Nationally, 190 spontaneous reports described 383 ADRs, which 28.6% were unknown or poorly known in the literature, and 52.1% were serious. The most frequent ADRs were gastrointestinal (19.3%) and neurological (19.1%), and among the most common 213 suspected medicines, there were those used for nervous system conditions (18.8%). The agreement on the perception of ADRs' severity between citizens and centre's technicians was 'good' (K = 0.62 (0.51-0.72)). An analysis of the free-text section of reports showed that one-quarter of the reports provided useful additional data, like the psychobiosocial impact, which could explain the discrepancy between patients and health professionals in the classification of the severity of some ADRs. CONCLUSIONS: Patients' report free-text section provides relevant information, mainly about symptoms description, psychobiosocial impact and feelings. Therefore, it is a section to be enhanced and analysed. These findings should encourage the strengthening of citizens reporting.

Immune-responsive gene 1 (IRG1) and dimethyl itaconate are involved in the mussel immune response.

Immune-responsive gene 1 (irg1) is a gene that is well-conserved among different taxa and is highly expressed in the mussel Mytilus galloprovincialis at the constitutive level. The expression of this gene increases after a bacterial infection, primarily in haemocytes. irg1 catalyses the production of itaconic acid from cis-aconitic acid in the Krebs cycle. Recently, itaconate has been revealed as an immune metabolite involved in macrophage polarization. In this work, we studied the effects of exogenous dimethyl itaconate (DI) on mussels in vitro and in vivo at relevant previously described endogenous concentrations and in mussels infected with Vibrio splendidus. DI did not have adverse effects on the haemocytes viability, apoptotic cells, proliferation and phagocytic activity; however, haemocyte size, velocity and accumulated distance were decreased. The antibacterial activity of DI in vitro and in vivo was observed with high concentrations of DI, that is, 30 and 50 mM, respectively. Furthermore, DI inhibited total ROS, increased mitochondrial ROS and modulated antioxidant genes, such as SOD and CAT, related to Nrf2 activation. In this research, we have demonstrated some important pathways in haemocytes in which itaconate can be involved after its production in a bacterial infection.

Development and validation of a specific real-time PCR assay for the detection of the parasite Perkinsus olseni.

Perkinsus olseni is a protozoan parasite that infects a wide variety of molluscs worldwide, causing economic losses in the aquaculture sector. In the present study, a quantitative PCR (qPCR) assay was developed for the detection and quantification of P. olseni in clam gill tissue and hemolymph (Ruditapes philippinarum and R. decussatus), and the results were compared with those of the standard diagnostic methods recommended by the O.I.E. (World Organisation for Animal Health): Ray's fluid thioglycollate culture method (RFTM), a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay and histopathology. The efficiency, sensitivity and reproducibility of the newly described qPCR assay were also determined. The highest prevalence was detected using the qPCR assay, and the strongest linear correlation was obtained between the RFTM infection levels and the threshold cycle (Ct) number from the gill tissue. Although better results were obtained from gill than from the hemolymph in the qPCR assays, especially with lower infection levels of the parasite, a significant linear correlation was observed between Ct values from the gill and hemolymph. The qPCR assay that was developed in this study showed high sensitivity, specificity and reproducibility for the detection and quantification of P. olseni.

Nanoplastics: From tissue accumulation to cell translocation into Mytilus galloprovincialis hemocytes. resilience of immune cells exposed to nanoplastics and nanoplastics plus Vibrio splendidus combination.

Plastic litter is an issue of global concern. In this work Mytilus galloprovincialis was used to study the distribution and effects of polystyrene nanoplastics (PS NPs) of different sizes (50 nm, 100 nm and 1 µm) on immune cells. Internalization and translocation of NPs to hemolymph were carried out by in vivo experiments, while endocytic routes and effects of PS NPs on hemocytes were studied in vitro. The smallest PS NPs tested were detected in the digestive gland and muscle. A fast and size-dependent translocation of PS NPs to the hemolymph was recorded after 3 h of exposure. The internalization rate of 50 nm PS NPs was lower when caveolae and clathrin endocytosis pathways were inhibited. On the other hand, the internalization of larger particles decreased when phagocytosis was inhibited. The hemocytes exposed to NPs had changes in motility, apoptosis, ROS and phagocytic capacity. However, they showed resilience when were infected with bacteria after PS NP exposure being able to recover their phagocytic capacity although the expression of the antimicrobial peptide Myticin C was reduced. Our findings show for the first time the translocation of PS NPs into hemocytes and how their effects trigger the loss of its functional parameters.

Turbot (Scophthalmus maximus) Nk-lysin induces protection against the pathogenic parasite Philasterides dicentrarchi via membrane disruption.

P. dicentrarchi is one of the most threatening pathogens for turbot aquaculture. This protozoan ciliate is a causative agent of scuticociliatosis, which is a disease with important economic consequences for the sector. Neither vaccines nor therapeutic treatments are commercially available to combat this infection. Numerous antimicrobial peptides (AMPs) have demonstrated broad-spectrum activity against bacteria, viruses, fungi, parasites and even tumor cells; an example is Nk-lysin (Nkl), which is an AMP belonging to the saposin-like protein (SAPLIP) family with an ability to interact with biological membranes. Following the recent characterization of turbot Nkl, an expression plasmid encoding Nkl was constructed and an anti-Nkl polyclonal antibody was successfully tested. Using these tools, we demonstrated that although infection did not clearly affect nkl mRNA expression, it induced changes at the protein level. Turbot Nkl had the ability to inhibit proliferation of the P. dicentrarchi parasite both in vivo and in vitro. Moreover, a shortened peptide containing the active core of turbot Nkl (Nkl71-100) was synthesized and showed high antiparasitic activity with a direct effect on parasite viability that probably occurred via membrane disruption. Therefore, the nkl gene may be a good candidate for genetic breeding selection of fish, and either the encoded peptide or its shortened analog is a promising antiparasitic treatment in aquaculture.

Conserved gene regulation during acute inflammation between zebrafish and mammals.

Zebrafish (Danio rerio), largely used as a model for studying developmental processes, has also emerged as a valuable system for modelling human inflammatory diseases. However, in a context where even mice have been questioned as a valid model for these analysis, a systematic study evaluating the reproducibility of human and mammalian inflammatory diseases in zebrafish is still lacking. In this report, we characterize the transcriptomic regulation to lipopolysaccharide in adult zebrafish kidney, liver, and muscle tissues using microarrays and demonstrate how the zebrafish genomic responses can effectively reproduce the mammalian inflammatory process induced by acute endotoxin stress. We provide evidence that immune signaling pathways and single gene expression is well conserved throughout evolution and that the zebrafish and mammal acute genomic responses after lipopolysaccharide stimulation are highly correlated despite the differential susceptibility between species to that compound. Therefore, we formally confirm that zebrafish inflammatory models are suited to study the basic mechanisms of inflammation in human inflammatory diseases, with great translational impact potential.

Cell mediated immune response of the Mediterranean sea urchin Paracentrotus lividus after PAMPs stimulation.

The Mediterranean sea urchin (Paracentrotus lividus) is of great ecological and economic importance for the European aquaculture. Yet, most of the studies regarding echinoderm's immunological defense mechanisms reported so far have used the sea urchin Strongylocentrotus purpuratus as a model, and information on the immunological defense mechanisms of Paracentrotus lividus and other sea urchins, is scarce. To remedy this gap in information, in this study, flow cytometry was used to evaluate several cellular immune mechanisms, such as phagocytosis, cell cooperation, and ROS production in P. lividus coelomocytes after PAMP stimulation. Two cell populations were described. Of the two, the amoeboid-phagocytes were responsible for the phagocytosis and ROS production. Cooperation between amoeboid-phagocytes and non-adherent cells resulted in an increased phagocytic response. Stimulation with several PAMPs modified the phagocytic activity and the production of ROS. The premise that the coelomocytes were activated by the bacterial components was confirmed by the expression levels of two cell mediated immune genes: LPS-Induced TNF-alpha Factor (LITAF) and macrophage migration inhibitory factor (MIF). These results have helped us understand the cellular immune mechanisms in P. lividus and their modulation after PAMP stimulation.

The complexity of apoptotic cell death in mollusks: An update.

Apoptosis is a type of programmed cell death that produces changes in cell morphology and in biochemical intracellular processes without inflammatory reactions. The components of the apoptotic pathways are conserved throughout evolution. Caspases are key molecules involved in the transduction of the death signal and are responsible for many of the biochemical and morphological changes associated with apoptosis. Nowadays, It is known that caspases are activated through two major apoptotic pathways (the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway), but there are also evidences of at least other alternative pathway (the perforin/granzyme pathway). Apoptosis in mollusks seems to be similar in complexity to apoptosis in vertebrates but also has unique features maybe related to their recurrent exposure to environmental changes, pollutants, pathogens and also related to the sedentary nature of some stages in the life cycle of mollusks bivalves and gastropods. As in other animals, apoptotic process is involved in the maintenance of tissue homeostasis and also constitutes an important immune response that can be triggered by a variety of stimuli, including cytokines, hormones, toxic insults, viruses, and protozoan parasites. The main goal of this work is to present the current knowledge of the molecular mechanisms of apoptosis in mollusks and to highlight those steps that need further study.

Zebrafish Nk-lysins: First insights about their cellular and functional diversification.

Nk-lysins are antimicrobial proteins produced by cytotoxic T lymphocytes and natural killer cells with a broad antimicrobial spectrum (including bacteria, fungi and parasites). Nevertheless, the implication of these proteins in the protection against viral infections is still poorly understood. In this work, four different Nk-lysin genes (nkla, nklb, nklc and nkld) were identified in the zebrafish genome. That means that zebrafish is the species with the higher repertoire of Nk-lysin genes described so far. The differential expression pattern of the Nk-lysins in several tissues, during ontogeny, among the different kidney cell populations, as well as between Rag1(-/-) and Rag1(+/+) individuals, could suggest a certain specialization of different cell types in the production of different Nk-lysin. Moreover, only two of these genes (nkla and nkld) were significantly up-regulated after viral infection, and this observation could be also a consequence of a functional diversification of the zebrafish Nk-lysins.

Dasatinib worsens the effect of cetuximab in combination with fractionated radiotherapy in FaDu- and A431-derived xenografted tumours.

BACKGROUND: Cetuximab is often combined with radiotherapy in advanced SCCHN. Alternative routes bypassing inhibition of EGFR with cetuximab may overshadow the efficacy of this combination. We undertook this study to investigate a possible role of dasatinib in this scenario. METHODS: The SCC5, SCC25, SCC29, FaDu and A431 cell lines were assessed in vitro for cell proliferation under cetuximab and dasatinib treatments. In FaDu and A431 cells, dasatinib plus cetuximab resulted in higher proliferation than cetuximab alone. Then, FaDu and A431 cells were implanted into subcutaneous tissue of athymic mice that were irradiated with 30 Gy in 10 fractions over 2 weeks, and treated with cetuximab and dasatinib. Tumour growth, DNA synthesis and angiogenesis were determined. The EGFR, RAS-GTP activity, phosphorylated AKT, ERK1/2, SRC protein levels and VEGF secretion were determined in vitro. RESULTS: The addition of dasatinib to cetuximab and radiotherapy increased tumour growth, DNA synthesis and angiogenesis that were associated with RAS, AKT and ERK1/2 activation, and SRC inhibition in FaDu and A431 cells. CONCLUSIONS: In xenografts derived from these two cell lines, dasatinib did not improve the efficacy of cetuximab combined with radiotherapy. On the contrary, it worsened tumour control achieved by the combination of these two treatments.

iRGD tumor-penetrating peptide-modified oncolytic adenovirus shows enhanced tumor transduction, intratumoral dissemination and antitumor efficacy.

Endovenously administered oncolytic viruses extravasate and penetrate poorly into tumors. iRGD is a cyclic peptide that enhances tumor penetration when conjugated or coadministered with different types of molecules such as drugs, nanoparticles or phages. iRGD-mediated tumor penetration occurs in three steps: binding to αv-integrins on tumor vasculature or tumor cells, exposure by proteolysis of a C-terminal motif that binds to neuropilin-1 (NRP-1) and cell internalization. We have genetically inserted the iRGD peptide in the fiber C terminus of ICOVIR15K, an oncolytic tumor-retargeted adenovirus to increase its tumor penetration. In vitro, NRP-1 interaction improved binding and internalization of the virus in different cancer cells overexpressing integrins and NRP-1. However, such NRP-1-mediated internalization did not affect transduction or cytotoxicity. In vivo, iRGD did not change the normal organ transduction pattern, with liver and spleen as main targeted organs. In tumors, however, iRGD enhanced transduction and early adenovirus dissemination through the tumor mass leading to an improved antitumor efficacy.

The warm temperature acclimation protein (Wap65) has an important role in the inflammatory response of turbot (Scophthalmus maximus).

Wap65 is a molecule similar to the mammalian hemopexin that is a serum glycoprotein produced mainly by the liver with high affinity to heme. Its primary role is participating in iron metabolism scavenging heme that is released into the plasma and transporting it to the liver. It has been reported an important role of hemopexin in the inflammation as an acute-phase protein and its production is up-regulated by pro-inflammatory cytokines. There are also some evidences suggesting this immune-induction in fish Wap65 genes. Most teleost species presents two Wap65 genes but their physiological functions have not been completely elucidated; in fact, the transcriptional patterns of Wap65 genes to stimulatory treatments are variable and contradictory. In the present study two Wap65 genes, Wap65-1 and Wap65-2, have been characterized for the first time in turbot (Scophthalmus maximus). Their constitutive expression and differential modulation by thermal treatments, immune challenges (bacterial and viral), as well as iron supplementation, have been investigated. Both genes were mainly expressed in liver, but they were detected in all tested tissues. Whereas Wap65-1 and Wap65-2 were up-regulated by temperature rise and bacterial challenge, VHSV infection inhibited the expression of both genes. Moreover, iron-dextran administration induced only the overexpression of Wap65-1. Interestingly, these induction were observed in head kidney buy not in liver. The effect of Wap65 protein purified from turbot serum by hemin-agarose affinity chromatography was also studied to demonstrate a possible anti-inflammatory role, analyzing its inhibitory effect on leucocytes migration induced by zymosan injection to the peritoneal cavity.

The first characterization of two type I interferons in turbot (Scophthalmus maximus) reveals their differential role, expression pattern and gene induction.

Type I interferons (IFNs) are considered the main cytokines directing the antiviral immune response in vertebrates. These molecules are able to induce the transcription of interferon-stimulated genes (ISGs) which, using different blocking mechanisms, reduce the viral proliferation in the host. In addition, a contradictory role of these IFNs in the protection against bacterial challenges using murine models has been observed, increasing the survival or having a detrimental effect depending on the bacteria species. In teleosts, a variable number of type I IFNs has been described with different expression patterns, protective capabilities or gene induction profiles even for the different IFNs belonging to the same species. In this work, two type I IFNs (ifn1 and ifn2) have been characterized for the first time in turbot (Scophthalmus maximus), showing different properties. Whereas Ifn1 reflected a clear antiviral activity (over-expression of ISGs and protection against viral haemorrhagic septicaemia virus), Ifn2 was not able to induce this response, although both transcripts were up-regulated after viral challenge. On the other hand, turbot IFNs did not show any protective effect against the bacteria Aeromonas salmonicida, although they were induced after bacterial challenge. Both IFNs induced the expression of several immune genes, but the effect of Ifn2 was mainly limited to the site of administration (intramuscular injection). Interestingly, Ifn2 but not Ifn1 induced an increase in the expression level of interleukin-1 beta (il1b). Therefore, the role of Ifn2 could be more related with the immune regulation, being involved mainly in the inflammation process.