Unlocking the potential of self-healing and recyclable ionic elastomers for soft robotics applications.

In the field of soft robotics, current materials face challenges related to their load capacity, durability, and sustainability. Innovative solutions are required to address these problems beyond conventional strategies, which often lack long-term ecological viability. This study aims to overcome these limitations using mechanically robust, self-healing, and recyclable ionic elastomers based on carboxylated nitrile rubber (XNBR). The designed materials exhibited excellent mechanical properties, including tensile strengths (TS) exceeding 19 MPa and remarkable deformability, with maximum elongations (EB) over 650%. Moreover, these materials showed high self-healing capabilities, with 100% recovery efficiency of TS and EB at 110 °C after 3 to 5 h, and full recyclability, preserving their mechanical performance even after three recycling cycles. Furthermore, they were also moldable and readily scalable. Tendon-driven soft robotic grippers were successfully developed out of ionic elastomers, illustrating the potential of self-healing and recyclability in the field of soft robotics to reduce maintenance costs, increase material durability, and improve sustainability.

Epoxy Nanocomposites Filled with Carbon Nanoparticles.

Over the past decades, the development of high performance lightweight polymer nanocomposites and, in particular, of epoxy nanocomposites has become one the greatest challenges in material science. The ultimate goal of epoxy nanocomposites is to extrapolate the exceptional intrinsic properties of the nanoparticles to the bulk matrix. However, in spite of the efforts, this objective is still to be attained at commercially attractive scales. Key aspects to achieve this are ultimately the full understanding of network structure, the dispersion degree of the nanoparticles, the interfacial adhesion at the phase boundaries and the control of the localization and orientation of the nanoparticles in the epoxy system. In this Personal Account, we critically discuss the state of the art and evaluate the strategies to overcome these barriers.

Duplication of Dio3 genes in teleost fish and their divergent expression in skin during flatfish metamorphosis.

Deiodinase 3 (Dio3) plays an essential role during early development in vertebrates by controlling tissue thyroid hormone (TH) availability. The Atlantic halibut (Hippoglossus hippoglossus) possesses duplicate dio3 genes (dio3a and dio3b). Expression analysis indicates that dio3b levels change in abocular skin during metamorphosis and this suggests that this enzyme is associated with the divergent development of larval skin to the juvenile phenotype. In larvae exposed to MMI, a chemical that inhibits TH production, expression of dio3b in ocular skin is significantly up-regulated suggesting that THs normally modulate this genes expression during this developmental event. The molecular basis for divergent dio3a and dio3b expression and responsiveness to MMI treatment is explained by the multiple conserved TREs in the proximal promoter region of teleost dio3b and their absence from the promoter of dio3a. We propose that the divergent expression of dio3 in ocular and abocular skin during halibut metamorphosis contributes to the asymmetric pigment development in response to THs.

Transcriptomic profiles of the upper olfactory rosette in cultured and wild Senegalese sole (Solea senegalensis) males.

The aims of this study were the characterization of the upper olfactory epithelium of cultured and wild Senegalese sole mature males at histological and transcriptomic (using RNA-Seq) level. No significant differences in tissue structure, cell types and cellular distribution pattern (olfactory sensory neurons) were identified between cultured and wild specimens. Deep transcriptomic analysis showed 2387 transcripts were differentially expressed between cultured and wild groups. A detailed analysis identified the differentially expressed transcripts included some olfactory receptors (OR, TAAR and V2R-like) and transcripts related with the control of reproduction such as the brain aromatase cytochrome P450 and tachykinin-3. Also a wide set of genes related with lipid sensing, metabolism and transport were differentially expressed and these transcripts were often down-regulated in cultured fish. Furthermore, cultured males presented a higher expression of genes related with goblet cells and mucin production that modulates innate and adaptive immune responses. All these changes in gene expression could be explained by different nutritional status and diet preference. The different expression of transcripts related to olfaction, reproduction, nutrient sensing and immune system demonstrate distinct differences in functionalities between cultured and wild soles providing new clues about the sexual dysfunction in this species.

Molecular characterization and developmental expression patterns of apolipoprotein A-I in Senegalese sole (Solea senegalensis Kaup).

The apolipoprotein A-I (ApoA-I) is an essential component of the high density lipoproteins (HDL). In this study, the cDNA and genomic sequences of this apolipoprotein were characterized for first time in Solea senegalensis. The predicted polypeptide revealed conserved structural features including ten repeats in the lipid-binding domain and some residues involved in cholesterol interaction and binding. The gene structure analysis identified four exons and three introns. Moreover, the synteny analysis revealed that apoA-I did not localize with other apolipoproteins indicating a divergent evolution with respect to the apoA-IV and apoE cluster. The phylogenetic analyses identified two distinct apoA-I paralogs in Ostariophysi (referred to as Ia and Ib) and only one (Ib) in Acanthopterygii. Whole-mount in situ hybridization located the apoA-I signal mainly in the yolk syncytial layer in lecitotrophic larval stages. Later at mouth opening, the mRNA signals were detected mainly in liver and intestine compatible with its role in the HDL formation. Moreover, a clear signal was detected in some regions of the brain, retina and neural cord suggesting a role in local regulation of cholesterol homeostasis. After metamorphosis, apoA-I was also detected in other tissues such as gills, head kidney and spleen suggesting a putative role in immunity. Expression analyses in larvae fed two diets with different triacylglycerol levels indicated that apoA-I mRNA levels were more associated to larval size and development than dietary lipid levels. Finally, qPCR analyses of immature and mature transcripts revealed distinct expression profiles suggesting a posttranscriptional regulatory mechanism.

Genomic characterization and expression analysis of four apolipoprotein A-IV paralogs in Senegalese sole (Solea senegalensis Kaup).

The apolipoprotein A-IV (ApoA-IV) plays a key role in lipid transport and feed intake regulation. In this work, four cDNA sequences encoding ApoA-IV paralogs were identified. Sequence analysis revealed conserved structural features including the common 33-codon block and nine repeated motifs. Gene structure analysis identified four exons and three introns except for apoA-IVAa1 (with only 3 exons). Synteny analysis showed that the four paralogs were structured into two clusters (cluster A containing apoA-IVAa1 and apoA-IVAa2 and cluster B with apoA-IVBa3 and apoA-IVBa4) linked to an apolipoprotein E. Phylogenetic analysis clearly separated the paralogs according to their cluster organization as well as revealed four subclades highly conserved in Acanthopterygii. Whole-mount analyses (WISH) in early larvae (0 and 1day post-hatch (dph)) showed that the four paralogs were mainly expressed in yolk syncytial layer surrounding the oil globules. Later, at 3 and 5dph, the four paralogs were mainly expressed in liver and intestine although with differences in their relative abundance and temporal expression patterns. Diet supply triggered the intensity of WISH signals in the intestine of the four paralogs. Quantification of mRNA abundance by qPCR using whole larvae only detected the induction by diet at 5dph. Moreover, transcript levels increased progressively with age except for apoA-IVAa2, which appeared as a low-expressed isoform. Expression analysis in juvenile tissues confirmed that the four paralogs were mainly expressed in liver and intestine and secondary in other tissues. The role of these ApoA-IV genes in lipid transport and the possible role of apoA-IVAa2 as a regulatory form are discussed.

Genetic parameters and genotype-environment interactions for skeleton deformities and growth traits at different ages on gilthead seabream (Sparus aurata L.) in four Spanish regions.

One of the most important problems of fish aquaculture is the high incidence of fish deformities, which are mainly skeletal. In this study, genetic parameters on gilthead seabream (Sparus aurata L.) for skeleton deformities at different ages (179, 269, 389, 539 and 689 days) and their correlations with growth traits were estimated, as were as their genotype × environment interactions (G × E) at harvesting age. A total of 4093 offspring from the mass spawning of three industrial broodstocks belonging to the PROGENSA(®) breeding programme were mixed and on-grown by different production systems in four Spanish regions: Canary Islands (tanks and cage), Andalusia (estuary), Catalonia (cage) and Murcia (cage). Parental assignment was inferred using the standardized SMsa1 microsatellite multiplex PCR. From three broodstocks, 139 breeders contributed to the spawn and a total of 297 full-sibling families (52 paternal and 53 maternal half-sibling families) were represented. Heritabilities at different ages were medium for growth traits (0.16-0.48) and vertebral deformities (0.16-0.41), and low for any type of deformity (0.07-0.26), head deformities (0.00-0.05) and lack of operculum (0.06-0.11). The genetic correlations between growth and deformity traits were medium and positive, suggesting that to avoid increasing deformities they should be taken into account in breeding programmes when growth is selected. The G × E interactions among the different facilities were weak for length and deformity and strong for growth rate during this period. These results highlight the potential for the gilthead seabream industry to reduce the prevalence of deformities by genetic improvement tools.

Characterization of the genomic responses in early Senegalese sole larvae fed diets with different dietary triacylglycerol and total lipids levels.

The aim of this work was to evaluate the genomic responses of premetamorphic sole larvae (9 days post-hatching, dph) fed diets with different lipid and triacylglycerol (TAG) content. For this purpose, two diets with high (rotifers enriched with a fish oil-based emulsion; referred to as HTAG) and low (rotifers enriched with a krill oil-based emulsion; LTAG) levels of total lipids and TAG were evaluated. Lipid class and fatty acid (FA) profiles, histological characterization of intestine, liver and pancreas and expression patterns using RNA-seq were determined. Discriminant analysis results showed that larvae could be clearly differentiated on the basis of their FA profile as a function of the diet supplied until 9dph although no difference in growth was observed. RNA-seq analysis showed that larvae fed HTAG activated coordinately the transcription of apolipoproteins (apob, apoa4, apoc2, apoe, and apobec2) and other related transcripts involved in chylomicron formation, likely to facilitate proper lipid absorption and delivery. In contrast, larvae fed LTAG showed higher mRNA levels of several pancreatic enzymes (try1a, try2, cela1, cela3, cela4, chym1, chym2, amy2a and pnlip) and appetite modulators (agrp1) and some intra- and extracellular lipases. Moreover, KEGG analysis also showed that several transcripts related to lipid metabolism and glycolysis were differentially expressed with a higher abundance in larvae fed LTAG diet. All these data suggest that early larvae were able to establish compensatory mechanisms for energy homeostasis regulating key molecules for FA and TAG biosynthesis, FA uptake and intracellular management of TAG and FA to warrant optimal growth rates.

A preliminary genetic map in Solea senegalensis (Pleuronectiformes, Soleidae) using BAC-FISH and next-generation sequencing.

This article presents the first physical mapping carried out in the Senegalese sole (Solea senegalensis), an important marine fish species of Southern Europe. Eight probes were designated to pick up genes of interest in aquaculture (candidate genes) from a bacterial artificial chromosome (BAC) library using a method of rapid screening based on a 4-dimension PCR. Seven known and 3 unknown clones were isolated and labeled. The 10 BAC clones were used as probes to map the karyotype of the species by fluorescence in situ hybridization (FISH). Nine out of the 10 clones were localized in only 1 chromosome pair, whereas the remaining one hybridized on 2 chromosome pairs. The 2-color FISH experiments showed colocation of 4 probes in 2 chromosome pairs. In addition, 2-color FISH was carried out both with 5S rDNA and the BAC containing the lysozyme gene published previously. This first genetic map of the Senegalese sole represents a starting point for future studies of the sole genome. In addition, 7 out of the 10 BAC clones were sequenced using next-generation sequencing, and bioinformatic characterization of the sequences was carried out. Hence the anchoring of the sequences to specific chromosomes or chromosome arms is now possible, leading to an initial scaffold of the Senegalese sole genome.

Development of the first standardised panel of two new microsatellite multiplex PCRs for gilthead seabream (Sparus aurata L.).

The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref-sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit-SMsa1 and kit-SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species.

Two Mx genes identified in European sea bass (Dicentrarchus labrax) respond differently to VNNV infection.

Mx proteins are key components of the antiviral state triggered by interferon type I in response to viral infections. In this study, two different Mx genes have been identified in European sea bass (Dicentrarchus labrax), and their sequences were cloned and characterized. MxA cDNA consists of 1881 bp coding for a putative 626 aminoacids protein, while MxB cDNA has 1920 bp and results in a protein with 639 residues. Their corresponding genomic sequences contain 3538 bp and 5326 bp, respectively, and both present 12 exons and 11 introns. The expression patterns of the two Mx genes after an in vivo challenge with the viral nervous necrosis virus (VNNV), a serious pathogen in farmed European sea bass, have been characterized by real-time PCR. The results showed interesting differences in the transcription profile of both Mx, thus suggesting a differential role for each Mx isoform in the immune response of European sea bass to VNNV, and most likely in the general viral response of this species.

Dexamethasone modulates expression of genes involved in the innate immune system, growth and stress and increases susceptibility to bacterial disease in Senegalese sole (Solea senegalensis Kaup, 1858).

Cortisol, the main glucocorticoid in fish, undertakes pleiotropic biological effects in response to stressors to maintain homeostasis. It can exert several actions on the immune system, growth and cellular metabolism, establishing a fine-tune regulation stress response and cross-talk interactions with other regulatory pathways. In this study, we investigated a causal relationship between high levels of glucocorticoids and susceptibility to pathogens and modification of gene expression profiles in Senegalese sole. For this purpose, we carried out two experiments using post-metamorphic individuals (21 days after hatching) that were exposed to dexamethasone (DXM), a potent glucocorticoid, in order to mimic cortisol effects. We quantified transcript levels of a wide set of genes involved in innate immune system (g-type lysozyme and hepcidin (hamp1)), HPI axis (crf, crfbp, pomcα, pomcß, gr1 and gr2), HPT axis (tgb), cellular stress defense system (hsp70 and hsp90aa), GH/IGF axis (igf-I and igf-Ir) and the neuropeptide trh. Short-term exposure to 0.1, 1 and 10 ppm DXM provoked a reduction of pomcß transcripts and an increase of crfbp mRNAs in a dose-dependent manner at 48 and 72 h after treatment. Moreover, g-type lysozyme transcript levels decreased significantly at 72 h whereas hamp1 mRNA levels increased at 48 h after exposure. Long-term DXM treatment (10 ppm DXM) affected negatively weight of soles (~20% lower than controls). Moreover, reduced mRNA levels were observed for pomcß after 1 week and igf-I and hamp1 after 2 weeks. In contrast, crfbp and crf increased mRNA levels after 2 weeks. hsp70 exhibited a dual response increasing transcript levels at 1 week after treatment and reducing thereafter. No significant changes in gene expression were observed at any time during this study for tgb, trh, hsp90aa, pomcα, gr1 and gr2. Finally, a challenge experiment using the pathogen Photobacterium damselae subsp piscicida confirmed earlier and higher mortalities in DXM-treated animals. Taken together, these data indicate that a prolonged exposure to DXM increases the susceptibility to pathogens and reduces growth. Moreover, DXM can trigger a wide cellular response modulating the expression of genes involved in the innate immune system, HPI and GH/IGF axes as well as cellular stress defense. These results are highly valuable to evaluate responses associated to aquaculture stressful conditions and discriminate specific glucocorticoid-mediated effects.

Three Mx genes with differential response to VNNV infection have been identified in Gilthead seabream (Sparus aurata).

Type I interferons are secreted by infected cells and promote an antiviral state in neighbouring cells by the induction of numerous genes, some of which present antiviral activity, as the Mx proteins. In this study, three different Mx cDNAs (Mx1, Mx2 and Mx3) from gilthead seabream (Sparus aurata), the most important fish species in Southern European aquaculture, have been cloned and characterized. A Southern blot assay revealed the existence of three Mx loci, thus the three Mx isoforms correspond to three different genes that seem to have a common origin. The genomic sequences of Mx1, Mx2 and Mx3 have been completely obtained, and consist on 11 introns and 12 exons in a full length of 5971 bp, 7391 bp and 6938 bp, respectively. As a first approach to the functional meaning of these three genes, their response to the viral nervous necrosis virus (VNNV) infection was tested. Important differences in terms of tissue, time course and level of induction were found between them, thus suggesting a differential functional role for each isoform, which can represent a key point in the natural resistance of this fish species, that has been repeatedly reported as an asymptomatic carrier of VNNV.

Molecular cloning and characterization of European seabass (Dicentrarchus labrax) and Gilthead seabream (Sparus aurata) complement component C3.

We present the complete C3 cDNA sequence of Gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) and its molecular characterization with a descriptive analysis of their structural elements. We obtained one sequence for Gilthead seabream (gsbC3) which encodes a predicted protein of 1656 amino acids, and two sequences for European seabass (esbC3_1 and esbC3_2) which encode two predicted proteins of 1654 and 1587 amino acids respectively. All sequences present the characteristic structural features of C3 but interestingly esbC3_2 lacks the anaphylotoxin domain and the cysteine residue responsible for thiolester bond formation. Moreover, we have detected and quantified (by real-time PCR-based absolute quantification) specific isoform expression in European seabass depending on pathogen and density conditions in vivo. In addition, we have analyzed the tissue distribution pattern of European seabass and Gilthead seabream C3 genes under crowding stress and under pathological challenges in vivo, and we have observed that crowding and infection status provoke changes in expression levels, tissue expression pattern and C3 isoform expression balance.

In vivo genotoxicity and stress defences in three flatfish species exposed to CuSO4.

We have used the comet assay to analyse, after 3h, 24h and 6 days, the genotoxic effect in vivo of applying a single intraperitoneal injection of CuSO4, at a concentration of 2mg/kg, to adult specimens of Solea senegalensis, Dicologlossa cuneata and Scophthalmus rhombus. Metals content (Cu, Zn and Cd) in liver was also measured. The activity of key stress defences was evaluated by analysing antioxidant enzyme activity (catalase (CAT), superoxide dismutase (SOD), total glutathione peroxidase (t-GPX), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH)), metallothionein (MT) and heat shock proteins (HSP70 and HSP60). The results show that CuSO4 intake generates high and cumulative levels of genotoxicity throughout the 6 days in all 3 species. After 6 days, metals content detected in specimens showed significant differences from controls. Inter-species differences were detected in enzyme activity (P<0.05). A clear response to CuSO4 was detected only in S. rhombus, with an increase of MT and a decrease of HSPs. Variations in antioxidant defence levels and their comparative responses to the stress-inducing agent are discussed.

Detection of infectious pancreatic necrosis virus (IPNV) from asymptomatic redbanded seabream, Pagrus auriga Valenciennes, and common seabream, Pagrus pagrus (L.), using a non-destructive procedure.

A non-destructive procedure based on nested RT-PCR and dot-blot hybridization has been developed for the detection of asymptomatic IPNV-carrier fish. The pair of primers designed for RT-PCR amplified a 599-bp fragment of the pVP2 region within the polyprotein gene, resulting in the detection of IPNV genotype III.1. The use of a nested RT-PCR allowed the amplification of IPNV genotypes III.1 and I.2. In addition, a 191-bp probe was designed for hybridization studies used in combination with the nested RT-PCR. The application of the nested RT-PCR to analyse blood samples from asymptomatic redbanded seabream, Pagrus auriga, and common seabream, P. pagrus, specimens showed a 53.1% and 77.8% prevalence of IPNV-carriers, respectively. The combination of nested RT-PCR and dot-blot hybridization increased the detection rates up to 100% for redbanded seabream and 94.4% for common seabream. Therefore, the protocol described in this study is highly sensitive and specific for the detection of IPNV in asymptomatic carrier fish, and, in addition, the results demonstrate the carrier state in two newly cultured sparid species in southern Spain.

Cellular and molecular immune responses of the sea bass (Dicentrarchus labrax) experimentally infected with betanodavirus.

Naïve sea bass juveniles (38.4 + or - 4.5 g) were intramuscularly infected with a sublethal dose of betanodavirus isolate 378/I03, followed after 43 days by a similar boosting. This infection resulted in an overall mortality of 7.6%. At various intervals, sampling of fish tissues was performed to investigate: i) B and T lymphocyte content in organs and tissues; ii), proliferation of leucocytes re-stimulated in vitro with inactivated virus; iii) presence of serum antibody specific for betanodavirus; iv) expression of genes coding for the following immunoregulatory molecules involved in innate and acquired responses: type I IFN, Mx, IL-1, Cox-2; IL-10, TGF-beta, TCRbeta, CD4, CD8alpha, IgM, by using a quantitative PCR array system developed for sea bass. The obtained results showed a detectable increase of T cells and B cells in PBL during betanodavirus infection. Furthermore, leucocytes obtained from blood, head kidney, and gills showed a detectable "in vitro" increase in viability upon addition of inactivated viral particles, as determined by measuring intracellular ATP concentration. ELISA analysis of sera showed that exposure to nodavirus induced a low, but specific antibody titer measured 43 days after infection, despite the presence of measurable levels of natural antibody. Finally, a strong upregulation of genes coding for type I IFN, Mx, and IgM was identified after both infection and boosting. Interestingly, an upregulation of Cox-2 until boosting, and of TGF-beta and IL-10 after boosting was also observed, while the other tested genes did not show any significant variations with respect to mock-treated fish. Overall, our work represents a first comprehensive analysis of cellular and molecular immune parameters in a fish species exposed to a pathogenic virus.

Molecular intraspecific characterization of Photobacterium damselae ssp. damselae strains affecting cultured marine fish.

AIMS: The aim of this study was to analyse the intraspecific variability of Photobacterium damselae ssp. damselae strains isolated from different cultured marine fish species using molecular typing methods. METHODS AND RESULTS: Twenty P. damselae ssp. damselae strains isolated from marine fish species were used in this study. Phenotypic characterization of the strains was carried out using standard microbiological methods. Genetic characterization was conducted using three PCR-based methods [random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) and repetitive extragenic palindromic-PCR (REP-PCR)]. Dice coefficient and the unweighted pair group method with average linkage were used for numerical analyses of banding patterns. At phenotypic level, the strains analysed showed seven different profiles, which could not be related to the host fish species, geographic area or outbreak of disease. Isolates were grouped into nine and eight clusters using the RAPD technique with primers 5 and 4, respectively. In both cases, the main cluster grouped 45% of strains. The techniques ERIC-PCR and REP-PCR were more discriminatory, both resulting in 14 different clusters, which grouped 15-20% of the isolates. CONCLUSIONS: In this study, the techniques tested are confirmed as good tools for molecular typing, because they allow discrimination between P. damselae ssp. damselae strains isolated within the same outbreak. In addition, ERIC-PCR and REP-PCR methods were more adequate for rapid typing of P. damselae ssp. damselae than RAPD, allowing the discrimination at strain level. SIGNIFICANCE AND IMPACT OF THE STUDY: The results, in agreement with previous studies, confirmed the high intraspecific variability among isolated P. damselae ssp. damselae strains at both phenotypic and genetic levels. This suggests the existence of different clonal lineages that coexist in the same geographic area, within a short period of time (2-3 years). The discrimination at strain level can be useful to study the traceability of infections.