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Cathelicidins are a ubiquitous family of host defence antimicrobial peptides in vertebrate animals. Unlike other antimicrobial peptide families, it is defined by a large and relatively well conserved proregion rather than by the mature bioactive peptides themselves, which are highly diverse and conform to at least five different structural types, resulting in distinct modes of action. Cathelicidin-derived host defence peptides have a pleiotropic role in immunity, displaying both a direct antimicrobial activity and the ability to boost other host responses to infection and injury. The presence of a relatively well conserved proregion attached to a vast repertoire of structurally and functionally diverse peptides allows mining the increasing number of vertebrate genomes for lead sequences to potentially useful new anti-infective and/or immunomodulatory agents. This should increase the number of cathelicidin-based peptides entering clinical trials, which has been limited to date, despite considerable efforts in the last 2 decades.

The urochordate Ciona robusta exhibits numerous functional and morphogenetic traits that are shared with vertebrate models. While prior investigations have identified several analogies between the gastrointestinal tract (i.e., gut) of Ciona and mice, the molecular mechanisms responsible for these similarities remain poorly understood. This study seeks to address this knowledge gap by investigating the transcriptional landscape of the adult stage gut. Through comparative genomics analyses, we identified several evolutionarily conserved components of signaling pathways of pivotal importance for gut development (such as WNT, Notch, and TGFβ-BMP) and further evaluated their expression in three distinct sections of the gastrointestinal tract by RNA-seq. Despite the presence of lineage-specific gene gains, losses, and often unclear orthology relationships, the investigated pathways were characterized by well-conserved molecular machinery, with most components being expressed at significant levels throughout the entire intestinal tract of C. robusta. We also showed significant differences in the transcriptional landscape of the stomach and intestinal tract, which were much less pronounced between the proximal and distal portions of the intestine. This study confirms that C. robusta is a reliable model system for comparative studies, supporting the use of ascidians as a model to study gut physiology.

SeviL, a galactoside-binding lectin previously isolated from the mussel Mytilisepta virgata, was demonstrated to trigger apoptosis in HeLa ovarian cancer cells. Here, we show that this lectin can promote the polarization of macrophage cell lines toward an M1 functional phenotype at low concentrations. The administration of SeviL to monocyte and basophil cell lines reduced their growth in a dose-dependent manner. However, low lectin concentrations induced proliferation in the RAW264.7 macrophage cell line, which was supported by the significant up-regulation of TOM22, a component of the mitochondrial outer membrane. Furthermore, the morphology of lectin-treated macrophage cells markedly changed, shifting from a spherical to an elongated shape. The ability of SeviL to induce the polarization of RAW264.7 cells to M1 macrophages at low concentrations is supported by the secretion of proinflammatory cytokines and chemokines, as well as by the enhancement in the expression of IL-6- and TNF-α-encoding mRNAs, both of which encode inflammatory molecular markers. Moreover, we also observed a number of accessory molecular alterations, such as the activation of MAP kinases and the JAK/STAT pathway and the phosphorylation of platelet-derived growth factor receptor-α, which altogether support the functional reprogramming of RAW264.7 following SeviL treatment. These results indicate that this mussel β-trefoil lectin has a concentration-dependent multifunctional role in regulating cell proliferation, phenotype, and death in macrophages, suggesting its possible involvement in regulating hemocyte activity in vivo.

Adamussium colbecki is a scallop endemic of the Antarctic Ocean, the only modern survivor of the Adamussiini tribe and one of the few bivalves living in polar environments. Compared with other Antarctic animals, very little is known concerning the evolutionary adaptations which allow this species to thrive at sub-zero temperatures. Due to its local abundance and sensitivity to environmental changes, A. colbecki is an interesting model for studying the effects of pollution and climate change in the Antarctic Ocean. Here, we report, for the first time, the application of transcriptomic tools to the study of the effects of a short-to-medium term exposure to a +1.5 °C water temperature increase on three tissues. Although this approach did not highlight any significant change in response to thermal stress, we observed slight alterations in energetic metabolism and nutrient adsorption in the digestive gland, most likely linked with stabling in experimental tanks. The results of our study suggest that A. colbecki may be particularly vulnerable to the effects of climate change due to its complete inability to adapt to temperature increase at the transcriptomic level.

Summary: Phosphonates—compounds containing a direct C–P bond—represent an important source of phosphorus in some environments. The most common natural phosphonate is 2-aminoethylphosphonate (AEP). Many bacteria can break AEP down through specialized “hydrolytic” pathways, which start with the conversion of AEP into phosphonoacetaldehyde (PAA), catalyzed by the transaminase PhnW. However, the substrate scope of these pathways is very narrow, as PhnW cannot process other common AEP-related phosphonates, notably N-methyl AEP (M1AEP). Here, we describe a heterogeneous group of FAD-dependent oxidoreductases that efficiently oxidize M1AEP to directly generate PAA, thus expanding the versatility and usefulness of the hydrolytic AEP degradation pathways. Furthermore, some of these enzymes can also efficiently oxidize plain AEP. By doing so, they surrogate the role of PhnW in organisms that do not possess the transaminase and create novel versions of the AEP degradation pathways in which PAA is generated solely by oxidative deamination.

Summary: Presence/absence variation (PAV) is a well-known phenomenon in prokaryotes that was described for the first time in bivalves in 2020 in Mytilus galloprovincialis. The objective of the present study was to further our understanding of the PAV phenomenon in mussel biology. The distribution of PAV was studied in a mussel chromosome-level genome assembly, revealing a widespread distribution but with hotspots of dispensability. Special attention was given to the effect of PAV in gene expression, since dispensable genes were found to be inherently subject to distortions due to their sparse distribution among individuals. Furthermore, the high expression and strong tissue specificity of some dispensable genes, such as myticins, strongly supported their biological relevance. The significant differences in the repertoire of dispensable genes associated with two geographically distinct populations suggest that PAV is involved in local adaptation. Overall, the PAV phenomenon would provide a key selective advantage at the population level.

Thalidomide has emerged as an effective immunomodulator in the treatment of pediatric patients with inflammatory bowel disease (IBD) refractory to standard therapies. Cereblon (CRBN), a component of E3 protein ligase complex that mediates ubiquitination and proteasomal degradation of target proteins, has been identified as the primary target of thalidomide. CRBN plays a crucial role in thalidomide teratogenicity, however it is unclear whether it is also involved in the therapeutic effects in IBD patients. This study aimed at identifying the molecular mechanisms underpinning thalidomide action in pediatric IBD. In this study, ten IBD pediatric patients responsive to thalidomide were prospectively enrolled. RNA-sequencing (RNA-seq) analysis and functional enrichment analysis were carried out on peripheral blood mononuclear cells (PBMC) obtained before and after twelve weeks of treatment with thalidomide. RNA-seq analysis revealed 378 differentially expressed genes before and after treatment with thalidomide. The most deregulated pathways were cytosolic calcium ion concentration, cAMP-mediated signaling, eicosanoid signaling and inhibition of matrix metalloproteinases. Neuronal signaling mechanisms such as CREB signaling in neurons and axonal guidance signaling also emerged. Connectivity Map analysis revealed that thalidomide gene expression changes were similar to those exposed to MLN4924, an inhibitor of NEDD8 activating enzyme, suggesting that thalidomide exerts its immunomodulatory effects by acting on the ubiquitin-proteasome pathway. In vitro experiments on cell lines confirmed the effect of thalidomide on candidate altered pathways observed in patients. These results represent a unique resource for enhanced understanding of thalidomide mechanism in pediatric patients with IBD, providing novel potential targets associated with drug response.

R-type lectins are a widespread group of sugar-binding proteins found in nearly all domains of life, characterized by the presence of a carbohydrate-binding domain that adopts a β-trefoil fold. Mytilectins represent a recently described subgroup of β-trefoil lectins, which have been functionally characterized in a few mussel species (Mollusca, Bivalvia) and display attractive properties, which may fuel the development of artificial lectins with different biotechnological applications. The detection of different paralogous genes in mussels, together with the description of orthologous sequences in brachiopods, supports the formal description of mytilectins as a gene family. However, to date, an investigation of the taxonomic distribution of these lectins and their molecular diversification and evolution was still lacking. Here, we provide a comprehensive overview of the evolutionary history of mytilectins, revealing an ancient monophyletic evolutionary origin and a very broad but highly discontinuous taxonomic distribution, ranging from heteroscleromorphan sponges to ophiuroid and crinoid echinoderms. Moreover, the overwhelming majority of mytilectins display a chimera-like architecture, which combines the β-trefoil carbohydrate recognition domain with a C-terminal pore-forming domain, suggesting that the simpler structure of most functionally characterized mytilectins derives from a secondary domain loss.

Abstract Background Real-time quantitative PCR is a widely used method for gene expression analyses in various organisms. Its accuracy mainly relies on the correct selection of reference genes. Any experimental plan involving real-time PCR needs to evaluate the characteristics of the samples to be examined and the relative stability of reference genes. Most studies in mollusks rely on reference genes commonly used in vertebrates. Results In this study, we focused on the transcriptome of the bivalve mollusk Mytilus galloprovincialis in physiological state to identify suitable reference genes in several adult tissues. Candidate genes with highly stable expression across 51 RNA-seq datasets from multiple tissues were selected through genome-wide bioinformatics analysis. This approach led to the identification of three genes (Rpl14, Rpl32 and Rpl34), whose suitability was evaluated together with 7 other reference genes commonly reported in literature (Act, Cyp-A, Ef1α, Gapdh, 18S, 28S and Rps4). The stability analyses performed with geNorm, NormFinder and Bestkeeper identified specific either single or pairs of genes suitable as references for gene expression analyses in specific tissues and revealed the Act/Cyp-A pair as the most appropriate to analyze gene expression across different tissues. Conclusion Mytilus galloprovincialis is a model system increasingly used in ecotoxicology and molecular studies. Our transcriptome-wide approach represents the first comprehensive investigation aimed at the identification of suitable reference genes for expression studies in this species.

Transglutaminase 2 (TG2) is a multifunctional protein widely distributed in various tissues and involved in many physiological and pathological processes. However, its actual role in biological processes is often controversial as TG2 shows different effects in these processes depending on its localization, cell type, or experimental conditions. We characterized the enzymatic and functional properties of TG2 proteins expressed in Danio rerio (zebrafish) to provide the basis for using this established animal model as a reliable tool to characterize TG2 functions in vivo. We confirmed the existence of three genes orthologous to human TG2 (zTGs2) in the zebrafish genome and their expression and function during embryonic development. We produced and purified the zTGs2s as recombinant proteins and showed that, like the human enzyme, zTGs2 catalyzes a Ca2+ dependent transamidation reaction that can be inhibited with TG2-specific inhibitors. In a cell model of human fibroblasts, we also demonstrated that zTGs2 can mediate RGD-independent cell adhesion in the extracellular environment. Finally, we transfected and selected zTGs2-overexpressing HEK293 cells and demonstrated that intracellular zTGs2 plays a very comparable protective/damaging role in the apoptotic process, as hTG2. Overall, our results suggest that zTGs2 proteins behave very similarly to the human ortholog and pave the way for future in vivo studies of TG2 functions in zebrafish.

Abstract Caudata is an order of amphibians with great variation in genome size, which can reach enormous dimensions in salamanders. In this work, we analysed the activity of transposable elements (TEs) in the transcriptomes obtained from female and male gonads of the Chinese fire-bellied newt, Cynops orientalis, a species with a genome about 12-fold larger than the human genome. We also compared these data with genomes of two basal sarcopterygians, coelacanth and lungfish. In the newt our findings highlighted a major impact of non-LTR retroelements and a greater total TE activity compared to the lungfish Protopterus annectens, an organism also characterized by a giant genome. This difference in TE activity might be due to the presence of young copies in newt in agreement also with the increase in the genome size, an event that occurred independently and later than lungfish. Moreover, the activity of 33 target genes encoding proteins involved in the TE host silencing mechanisms, such as Ago/Piwi and NuRD complex, was evaluated and compared between the three species analysed. These data revealed high transcriptional levels of the target genes in both newt and lungfish and confirmed the activity of NuRD complex genes in adults. Finally, phylogenetic analyses performed on PRDM9 and TRIM28 allowed increasing knowledge about the evolution of these two key genes of the NuRD complex silencing mechanism in vertebrates. Our results confirmed that the gigantism of the newt genomes may be attributed to the activity and accumulation of TEs.

The cave systems of the Neretva River basin in the Dinaric Karst are home to Congeria kusceri, one of the very few known examples of stygobiotic bivalve mollusks, which displays several unique life history traits and adaptations that allowed its adaptation to the subterranean environment. This endemic species is undergoing rapid decline, most likely linked with habitat degradation, which might seriously threaten its survival in the next few decades. Unfortunately, the urgent need for effective conservation efforts is hampered by the lack of effective regulations aimed at preserving remnant populations as well as by our limited knowledge of the biology of this species. Although the precise factors underlying the disappearance of C. kusceri from its type locations are not entirely clear, the alteration of seasonal changes in water temperatures and alkalinity is most likely involved, as these are the main drivers of shell growth, spawning, and the onset and progression of the new gametogenic cycle. While these aspects have so far only been studied using morphological and physiological observations, future conservation efforts would certainly benefit from an integrated multidisciplinary approach. Here, using RNA-sequencing, we provide an overview of the modulation of gene expression recorded in five key tissues (the mantle, gonads, gills, adductor muscle, and digestive gland) between early June and late September. We highlight the presence of markedly tissue-specific responses, with the most dramatic changes affecting the gonads, mantle, and gills. We further identified a drastic switch in the use of energy budgets between the two periods, with evidence of ongoing shell growth and high metabolic activity in the mantle and gills at the early time point, followed by a massive redirection of all available energy to the gonads for the generation of new gamete primordia in early autumn.

Antarctica is the most extreme continent of Earth, with strong winds, freezing temperatures on land, and ocean temperatures constantly below 0 °C. Nonetheless, the Antarctic Ocean is home to an astounding diversity of living organisms that adapted to the multiple challenges posed by this environment via a diverse set of evolutionary traits. Although the recent advancements in sequencing technologies clarified the molecular bases of such adaptations in Antarctic fishes, little information is available for Antarctic invertebrates. In this preliminary study, we address this knowledge gap with a comparative transcriptomic approach to obtain insights into some of the adaptations that allow the Antarctic scallop Adamussium colbecki to survive and thrive in the freezing waters of the Antarctic Ocean. Despite some limitations, our analyses highlighted significant over-expression of genes involved in regulation of mRNA transcription, maturation, and degradation, which might compensate for a reduced efficiency of these processes at low temperatures. Other alterations detected in the Antarctic scallop transcriptome include enhanced expression of genes that regulate degradation of misfolded protein products and allow maintenance of cytoskeletal structure and function at subzero temperatures. Altogether, these observations support the presence of multiple previously unreported molecular adaptive traits in A. colbecki, which have important implications for our understanding of adaptation of this important component of the Antarctic trophic chain to such an extreme, but stable environment.

The blue mussel Mytilus chilensis is an endemic and key socioeconomic species inhabiting the southern coast of Chile. This bivalve species supports a booming aquaculture industry, which entirely relies on artificially collected seeds from natural beds that are translocated to diverse physical–chemical ocean farming conditions. Furthermore, mussel production is threatened by a broad range of microorganisms, pollution, and environmental stressors that eventually impact its survival and growth. Herein, understanding the genomic basis of the local adaption is pivotal to developing sustainable shellfish aquaculture. We present a high-quality reference genome of M. chilensis, which is the first chromosome-level genome for a Mytilidae member in South America. The assembled genome size was 1.93 Gb, with a contig N50 of 134 Mb. Through Hi-C proximity ligation, 11,868 contigs were clustered, ordered, and assembled into 14 chromosomes in congruence with the karyological evidence. The M. chilensis genome comprises 34,530 genes and 4795 non-coding RNAs. A total of 57% of the genome contains repetitive sequences with predominancy of LTR-retrotransposons and unknown elements. Comparative genome analysis of M. chilensis and M. coruscus was conducted, revealing genic rearrangements distributed into the whole genome. Notably, transposable Steamer-like elements associated with horizontal transmissible cancer were explored in reference genomes, suggesting putative relationships at the chromosome level in Bivalvia. Genome expression analysis was also conducted, showing putative genomic differences between two ecologically different mussel populations. The evidence suggests that local genome adaptation and physiological plasticity can be analyzed to develop sustainable mussel production. The genome of M. chilensis provides pivotal molecular knowledge for the Mytilus complex.

Mussels (Mytilus spp.) tolerate infections much better than other species living in the same marine coastal environment thanks to a highly efficient innate immune system, which exploits a remarkable diversification of effector molecules involved in mucosal and humoral responses. Among these, antimicrobial peptides (AMPs) are subjected to massive gene presence/absence variation (PAV), endowing each individual with a potentially unique repertoire of defense molecules. The unavailability of a chromosome-scale assembly has so far prevented a comprehensive evaluation of the genomic arrangement of AMP-encoding loci, preventing an accurate ascertainment of the orthology/paralogy relationships among sequence variants. Here, we characterized the CRP-I gene cluster in the blue mussel Mytilus edulis, which includes about 50 paralogous genes and pseudogenes, mostly packed in a small genomic region within chromosome 5. We further reported the occurrence of widespread PAV within this family in the Mytilus species complex and provided evidence that CRP-I peptides likely adopt a knottin fold. We functionally characterized the synthetic peptide sCRP-I H1, assessing the presence of biological activities consistent with other knottins, revealing that mussel CRP-I peptides are unlikely to act as antimicrobial agents or protease inhibitors, even though they may be used as defense molecules against infections from eukaryotic parasites.

Abstract Background The Mediterranean mussel Mytilus galloprovincialis is an ecologically and economically relevant edible marine bivalve, highly invasive and resilient to biotic and abiotic stressors causing recurrent massive mortalities in other bivalves. Although these traits have been recently linked with the maintenance of a high genetic variation within natural populations, the factors underlying the evolutionary success of this species remain unclear. Results Here, after the assembly of a 1.28-Gb reference genome and the resequencing of 14 individuals from two independent populations, we reveal a complex pan-genomic architecture in M. galloprovincialis, with a core set of 45,000 genes plus a strikingly high number of dispensable genes (20,000) subject to presence-absence variation, which may be entirely missing in several individuals. We show that dispensable genes are associated with hemizygous genomic regions affected by structural variants, which overall account for nearly 580 Mb of DNA sequence not included in the reference genome assembly. As such, this is the first study to report the widespread occurrence of gene presence-absence variation at a whole-genome scale in the animal kingdom. Conclusions Dispensable genes usually belong to young and recently expanded gene families enriched in survival functions, which might be the key to explain the resilience and invasiveness of this species. This unique pan-genome architecture is characterized by dispensable genes in accessory genomic regions that exceed by orders of magnitude those observed in other metazoans, including humans, and closely mirror the open pan-genomes found in prokaryotes and in a few non-metazoan eukaryotes.

The Antarctic continent is one of the most pristine environments on planet Earth, yet one of the most fragile and susceptible to the effects of the ongoing climate change. The overwhelming majority of the components of Antarctic marine trophic chain are stenotherm organisms, highly adapted to the extreme, but extremely stable, freezing temperatures of the Antarctic ocean, which have not changed significantly during the past fifteen million years. Notothenioid fishes are the most abundant representatives of ichthyofauna at these latitudes, being ubiquitously found in coastal areas across the entire continent. While different Antarctic fish species have been previously subjected to studies aimed at defining their range of thermal tolerance, or at studying the response to acute thermal stress, just a handful of authors have investigated the effects of the exposure to a moderate increase of temperature, falling within the expected forecasts for the next few decades in some areas of the Antarctica. Here, the emerald rockcod Trematomus bernacchii was used as a model species to investigate the effects of a 20-day long exposure to a +1.5 °C increase in the brain, gills and skeletal muscle, using a RNA-sequencing approach. In parallel, the experimental design also allowed for assessing the impact of stabling (including acclimation, the handling of fishes and their confinement in tanks during the experimental phase) on gene expression profiling. The results of this study clearly identified the brain as the most susceptible tissue to heat stress, with evidence of a time-dependent response dominated by an alteration of immune response, protein synthesis and folding, and energy metabolism-related genes. While the gills displayed smaller but still significant alterations, the skeletal muscle was completely unaffected by the experimental conditions. The stabling conditions also had an important impact on gene expression profiles in the brain, suggesting the presence of significant alterations of the fish nervous system, possibly due to the confinement to tanks with limited water volume and of the restricted possibility of movement. Besides providing novel insights in the molecular mechanisms underlying thermal stress in notothenioids, these findings suggest that more attention should be dedicated to an improved design of the experiments carried out on Antarctic organism, due to their extreme susceptibility to the slightest environmental alterations.

DNA methylation is an essential epigenetic mechanism influencing gene expression in all organisms. In metazoans, the pattern of DNA methylation changes during embryogenesis and adult life. Consequently, differentiated cells develop a stable and unique DNA methylation pattern that finely regulates mRNA transcription during development and determines tissue-specific gene expression. Currently, DNA methylation remains poorly investigated in mollusks and completely unexplored in Mytilus galloprovincialis. To shed light on this process in this ecologically and economically important bivalve, we screened its genome, detecting sequences homologous to DNA methyltransferases (DNMTs), methyl-CpG-binding domain (MBD) proteins and Ten-eleven translocation methylcytosine dioxygenase (TET) previously described in other organisms. We characterized the gene architecture and protein domains of the mussel sequences and studied their phylogenetic relationships with the ortholog sequences from other bivalve species. We then comparatively investigated their expression levels across different adult tissues in mussel and other bivalves, using previously published transcriptome datasets. This study provides the first insights on DNA methylation regulators in M. galloprovincialis, which may provide fundamental information to better understand the complex role played by this mechanism in regulating genome activity in bivalves.

This Special Issue “Marine Glycomics” (https://www.mdpi.com/journal/marinedrugs/special_issues/Marine_Glycomics, accessed on 12 September 2022) provided new approachesand information on bioactive compounds, such as glycans and lectins from marine animals,seaweeds, and microorganisms for the application of clinical therapy and elucidation of thephysiological functions of marine organisms [...]

Despite a growing number of non-model insect species is being investigated in recent years, a greater understanding of their physiology is prevented by the lack of genomic resources. This is the case of the common European stick insect Bacillus rossius (Rossi, 1788): in this species, some knowledge is available on hemocyte-related defenses, but little is known about the physiological changes occurring in response to natural or experimental challenges. Here, the transcriptional signatures of adult B. rossius hemocytes were investigated after a short-term (2 h) LPS stimulation in vivo: a total of 2191 differentially expressed genes, mostly involved in proteolysis and carbohydrate and lipid metabolic processes, were identified in the de novo assembled transcriptome and in-depth discussed. Overall, the significant modulation of immune signals—such as C-type lectins, ML domain-containing proteins, serpins, as well as Toll signaling-related molecules—provide novel information on the early progression of LPS-induced responses in B. rossius.

Abstract This study reports the presence of two distinct MHC class II β genes in the Antarctic icefish Chionodraco hamatus, belonging to the classical (ChhaDAB) and nonclassical (ChhaDBB) evolutionary lineages. By the application of targeted sequencing approach, a remarkable molecular diversity in the exon 2 sequence of the highly expressed gene ChhaDAB has been observed, resulting in an estimate of 92 different variants translated in 87 different peptides from 54 analysed icefish individuals. A highly conservative estimate, based on a 95% sequence identity threshold clustering, translate this variability in 41 different peptide clusters belonging to four different clades and showing the signature of different kinds of selection. In stark contrast, the poorly expressed ChhaDBB gene displayed a very low level of molecular diversity within exon 2, in agreement with expectations for a nonclassical MHC class II β gene.

The striped venus Chamelea gallina is a bivalve mollusc that represents one of the most important fishery resources of the Adriatic Sea. In this work, we investigated for the first time the ability of this species to modulate the expression of genes encoding proteins involved in biomineralization process in response to biotic and abiotic factors. We provided the first comprehensive transcriptome from the mantle tissue of clams collected in two sampling sites located along the Italian Adriatic coast and characterized by different environmental features. Moreover, the assessment of environmental parameters, scanning electron microscopy (SEM), and X-ray diffraction (XRD) measurements on valves were conducted to better contextualize RNA sequencing (RNA-Seq) data. Functional annotation of differentially expressed genes (DEGs) and SEM observations highlighted a different shell mineralization behaviour in C. gallina clams collected from two selected sites characterized by diverse environmental parameters.

Fish are an interesting taxon comprising species adapted to a wide range of environments. In this work, we analyzed the transcriptional contribution of transposable elements (TEs) in the gill transcriptomes of three fish species exposed to different salinity conditions. We considered the giant marbled eel Anguilla marmorata and the chum salmon Oncorhynchus keta, both diadromous, and the marine medaka Oryzias melastigma, an euryhaline organism sensu stricto. Our analyses revealed an interesting activity of TEs in the case of juvenile eels, commonly adapted to salty water, when exposed to brackish and freshwater conditions. Moreover, the expression assessment of genes involved in TE silencing mechanisms (six in heterochromatin formation, fourteen known to be part of the nucleosome remodeling deacetylase (NuRD) complex, and four of the Argonaute subfamily) unveiled that they are active. Finally, our results evidenced for the first time a krüppel-associated box (KRAB)-like domain specific to actinopterygians that, together with TRIM33, might allow the functioning of NuRD complex also in fish species. The possible interaction between these two proteins was supported by structural prediction analyses.

Ovothiols are histidine-derived thiols produced by a variety of marine invertebrates, protists and bacteria. These compounds, which are among the strongest natural antioxidants, are involved in controlling the cellular redox balance due to their redox exchange with glutathione. Although ovothiols were initially reported as protective agents against environmental stressors, new evidence suggests that they can also act as pheromones and participate in fundamental biological processes such as embryogenesis. To get further insight into the biological roles of ovothiols, we compared ovothiol biosynthesis in the sea urchin Paracentrotus lividus and in the mussel Mytilus galloprovincialis, the two species that represent the richest sources of these compounds among marine invertebrates. Ovothiol content was measured in different tissues and in the immune cells from both species and the expression levels of ovoA, the gene responsible for ovothiol biosynthesis, was inferred from publicly available transcriptomes. A comparative analysis of ovothiol biosynthesis in the two species allowed the identification of the tissues and cells synthesizing the metabolite and highlighted analogies and differences between sea urchins and mussels. By improving our knowledge on the biological roles of ovothiols and pointing out the existence of sustainable natural sources for their isolation, this study provides the basis for future biotechnological investigations on these valuable compounds.

Abstract Background Antimicrobial peptides (AMPs) are multifunctional effector molecules that often combine direct antimicrobial activities with signaling or immunomodulatory functions. The skin secretions of anurans contain a variety of such bioactive peptides. The identification of AMPs from frog species often requires sacrificing several specimens to obtain small quantities of crude peptides, followed by activity based fractionation to identify the active principles. Results We report an efficient alternative approach to selectively amplify AMP-coding transcripts from very small amounts of tissue samples, based on RNA extraction and cDNA synthesis, followed by PCR amplification and high-throughput sequencing of size-selected amplicons. This protocol exploits the highly conserved signal peptide region of the AMP precursors from Ranidae, Hylidae and Bombinatoridae for the design of family-specific, forward degenerate primers, coupled with a reverse primer targeting the mRNA poly-A tail. Conclusions Analysis of the assembled sequencing output allowed to identify more than a hundred full-length mature peptides, mostly from Ranidae species, including several novel potential AMPs for functional characterization. This (i) confirms the effectiveness of the experimental approach and indicates points for protocol optimization to account for particular cases, and (ii) encourages the application of the same methodology to other multigenic AMP families, also from other genera, sharing common features as in anuran AMPs.

Due to their high biodiversity and adaptation to a mutable and challenging environment, aquatic lophotrochozoan animals are regarded as a virtually unlimited source of bioactive molecules. Among these, lectins, i.e., proteins with remarkable carbohydrate-recognition properties involved in immunity, reproduction, self/nonself recognition and several other biological processes, are particularly attractive targets for biotechnological research. To date, lectin research in the Lophotrochozoa has been restricted to the most widespread phyla, which are the usual targets of comparative immunology studies, such as Mollusca and Annelida. Here we provide the first overview of the repertoire of the secretory lectin-like molecules encoded by the genomes of six target rotifer species: Brachionus calyciflorus, Brachionus plicatilis, Proales similis (class Monogononta), Adineta ricciae, Didymodactylos carnosus and Rotaria sordida (class Bdelloidea). Overall, while rotifer secretory lectins display a high molecular diversity and belong to nine different structural classes, their total number is significantly lower than for other groups of lophotrochozoans, with no evidence of lineage-specific expansion events. Considering the high evolutionary divergence between rotifers and the other major sister phyla, their widespread distribution in aquatic environments and the ease of their collection and rearing in laboratory conditions, these organisms may represent interesting targets for glycobiological studies, which may allow the identification of novel carbohydrate-binding proteins with peculiar biological properties.

Big defensins are antimicrobial polypeptides believed to be the ancestors of β-defensins, the most evolutionary conserved family of host defense peptides (HDPs) in vertebrates. Nevertheless, big defensins underwent several independent gene loss events during animal evolution, being only retained in a limited number of phylogenetically distant invertebrates. Here, we explore the evolutionary history of this fascinating HDP family and investigate its patchy distribution in extant metazoans. We highlight the presence of big defensins in various classes of lophotrochozoans, as well as in a few arthropods and basal chordates (amphioxus), mostly adapted to life in marine environments. Bivalve mollusks often display an expanded repertoire of big defensin sequences, which appear to be the product of independent lineage-specific gene tandem duplications, followed by a rapid molecular diversification of newly acquired gene copies. This ongoing evolutionary process could underpin the simultaneous presence of canonical big defensins and non-canonical (β-defensin-like) sequences in some species. The big defensin genes of mussels and oysters, two species target of in-depth studies, are subjected to gene presence/absence variation (PAV), i.e., they can be present or absent in the genomes of different individuals. Moreover, big defensins follow different patterns of gene expression within a given species and respond differently to microbial challenges, suggesting functional divergence. Consistently, current structural data show that big defensin sequence diversity affects the 3D structure and biophysical properties of these polypeptides. We discuss here the role of the N-terminal hydrophobic domain, lost during evolution toward β-defensins, in the big defensin stability to high salt concentrations and its mechanism of action. Finally, we discuss the potential of big defensins as markers for animal health and for the nature-based design of novel therapeutics active at high salt concentrations.

Abstract Background Mytilisepta virgata is a marine mussel commonly found along the coasts of Japan. Although this species has been the subject of occasional studies concerning its ecological role, growth and reproduction, it has been so far almost completely neglected from a genetic and molecular point of view. In the present study we present a high quality de novo assembled transcriptome of the Japanese purplish mussel, which represents the first publicly available collection of expressed sequences for this species. Results The assembled transcriptome comprises almost 50,000 contigs, with a N50 statistics of ~1 kilobase and a high estimated completeness based on the rate of BUSCOs identified, standing as one of the most exhaustive sequence resources available for mytiloid bivalves to date. Overall this data, accompanied by gene expression profiles from gills, digestive gland, mantle rim, foot and posterior adductor muscle, presents an accurate snapshot of the great functional specialization of these five tissues in adult mussels. Conclusions We highlight that one of the most striking features of the M. virgata transcriptome is the high abundance and diversification of lectin-like transcripts, which pertain to different gene families and appear to be expressed in particular in the digestive gland and in the gills. Therefore, these two tissues might be selected as preferential targets for the isolation of molecules with interesting carbohydrate-binding properties. In addition, by molecular phylogenomics, we provide solid evidence in support of the classification of M. virgata within the Brachidontinae subfamily. This result is in agreement with the previously proposed hypothesis that the morphological features traditionally used to group Mytilisepta spp. and Septifer spp. within the same clade are inappropriate due to homoplasy.

Abstract Th2 immunity is a primary host defence against metazoan pathogens and two of the important cytokines involved in this immune response in mammals are IL-4 and IL-13. Recently the origin and evolution of Th2 immune responses have been investigated in fish where a molecule with relatedness to both IL-4 and IL-13 is present, termed IL-4/13. Different IL-4/13 paralogues (IL-4/13 A and IL-4/13B) exist in teleost fish. In this paper, we have focused on the IL-4/13 isoforms found in the European sea bass (Dicentrarchus labrax L.). Two tandem duplicated but divergent IL-4/13 A isoforms and one IL-4/13B are present, a unique situation compared to other teleosts. These genes were studied in terms of their in vitro and in vivo transcript levels after different treatments and their biological activities after production of the recombinant isoforms. The results show that the presence of these three paralogues is associated with different activities, both in terms of their expression profiles and the ability of the proteins to modulate the expression of immune genes in head kidney leukocytes. It is clear that the initiation and control of type-2 responses in seabass is complex due to the presence of multiple IL-4/13 isoforms with overlapping but distinct activities.

Mediterranean mussels (Mytilus galloprovincialis) are sessile filter feeders that live in close contact with numerous marine microorganisms. As is the case in all invertebrates, mussels lack an adaptive immune system, but they respond to pathogens, injuries or environmental stress in a very efficient manner. However, it is not known if they are able to modify their immune response when they reencounter the same pathogen. In this work, we studied the transcriptomic response of mussel hemocytes before and after two consecutive sublethal challenges with Vibrio splendidus. The first exposure significantly regulated genes related to inflammation, migration and response to bacteria. However, after the second exposure, the differentially expressed genes were related to the control and inhibition of ROS production and the resolution of the inflammatory response. Our results also show that the second injection with V. splendidus led to changes at the transcriptional (control of the expression of pro-inflammatory transcripts), cellular (shift in the hemocyte population distribution), and functional levels (inhibition of ROS production). These results suggest that a modified immune response after the second challenge allowed the mussels to tolerate rather than fight the infection, which minimized tissue damage.

Mytimycins are cysteine-rich antimicrobial peptides that show antifungal properties. These peptides are part of the immune network that constitutes the defense system of the Mediterranean mussel (Mytilus galloprovincialis). The immune system of mussels has been increasingly studied in the last decade due to its great efficiency, since these molluscs, particularly resistant to adverse conditions and pathogens, are present all over the world, being considered as an invasive species. The recent sequencing of the mussel genome has greatly simplified the genetic study of some of its immune genes. In the present work, we describe a total of 106 different mytimycin variants in 16 individual mussel genomes. The 13 highly supported mytimycin clusters (A–M) identified with phylogenetic inference were found to be subject to the presence/absence variation, a widespread phenomenon in mussels. We also identified a block of conserved residues evolving under purifying selection, which may indicate the “functional core” of the mature peptide, and a conserved set of 10 invariable plus 6 accessory cysteines which constitute a plastic disulfide array. Finally, we extended the taxonomic range of distribution of mytimycins among Mytilida, identifying novel sequences in M. coruscus, M. californianus, P. viridis, L. fortunei, M. philippinarum, M. modiolus, and P. purpuratus.

Far from being devoid of life, Antarctic waters are home to Cryonotothenioidea, which represent one of the fascinating cases of evolutionary adaptation to extreme environmental conditions in vertebrates. Thanks to a series of unique morphological and physiological peculiarities, which include the paradigmatic case of loss of hemoglobin in the family Channichthyidae, these fish survive and thrive at sub-zero temperatures. While some of the distinctive features of such adaptations have been known for decades, our knowledge of their genetic and molecular bases is still limited. We generated a reference de novo assembly of the icefish Chionodraco hamatus transcriptome and used this resource for a large-scale comparative analysis among five red-blooded Cryonotothenioidea, the sub-Antarctic notothenioid Eleginops maclovinus and seven temperate teleost species. Our investigation targeted the gills, a tissue of primary importance for gaseous exchange, osmoregulation, ammonia excretion, and its role in fish immunity. One hundred and twenty genes were identified as significantly up-regulated in Antarctic species and surprisingly shared by red- and white-blooded notothenioids, unveiling several previously unreported molecular players that might have contributed to the evolutionary success of Cryonotothenioidea in Antarctica. In particular, we detected cobalamin deficiency signatures and discussed the possible biological implications of this condition concerning hematological alterations and the heavy parasitic loads typically observed in all Cryonotothenioidea.

Insects produce a large repertoire of antimicrobial peptides (AMPs) as the first line of defense against bacteria, viruses, fungi or parasites. These peptides are produced from a large precursor that contains a signal domain, which is cleaved in vivo to produce the mature protein with antimicrobial activity. At present, AMPs from insects include several families which can be classified as cecropins, ponericins, defensins, lebocins, drosocin, Metchnikowin, gloverins, diptericins and attacins according to their structure and/or function. This short review is focused on attacins, a class of glycine-rich peptides/proteins that have been first discovered in the cecropia moth (Hyalophora cecropia). They are a rather heterogeneous group of immunity-related proteins that exhibit an antimicrobial effect mainly against Gram-negative bacteria. Here, we discuss different attacin and attacin-like AMPs that have been discovered so far and analyze their structure and phylogeny. Special focus is given to the physiological importance and mechanism of action of attacins against microbial pathogens together with their potential pharmacological applications, emphasizing their roles as antimicrobials.

Transposable elements (TEs) represent a considerable fraction of eukaryotic genomes, thereby contributing to genome size, chromosomal rearrangements, and to the generation of new coding genes or regulatory elements. An increasing number of works have reported a link between the genomic abundance of TEs and the adaptation to specific environmental conditions. Diadromy represents a fascinating feature of fish, protagonists of migratory routes between marine and freshwater for reproduction. In this work, we investigated the genomes of 24 fish species, including 15 teleosts with a migratory behaviour. The expected higher relative abundance of DNA transposons in ray-finned fish compared with the other fish groups was not confirmed by the analysis of the dataset considered. The relative contribution of different TE types in migratory ray-finned species did not show clear differences between oceanodromous and potamodromous fish. On the contrary, a remarkable relationship between migratory behaviour and the quantitative difference reported for short interspersed nuclear (retro)elements (SINEs) emerged from the comparison between anadromous and catadromous species, independently from their phylogenetic position. This aspect is likely due to the substantial environmental changes faced by diadromous species during their migratory routes.

Myticins are cysteine-rich antimicrobial peptides highly expressed in hemocytes of Mytilus galloprovincialis. Along with other antimicrobial peptides (AMPs), myticins are potent effectors in the mussel immune response to pathogenic infections. As intertidal filter-feeders, mussels are constantly exposed to mutable environmental conditions, as well as to the presence of many pathogens, and myticins may be key players in the great ability of these organisms to withstand these conditions. These AMPs are known to be characterized by a remarkable sequence diversity, which was further explored in this work, thanks to the analysis of the recently released genome sequencing data from 16 specimens. Altogether, we collected 120 different sequence variants, evidencing the important impact of presence/absence variation and positive selection in shaping the repertoire of myticin genes of each individual. From a functional point of view, both the isoelectric point (pI) and the predicted charge of the mature peptide show unusually low values compared with other cysteine-rich AMPs, reinforcing previous observations that myticins may have accessory functions not directly linked with microbe killing. Finally, we report the presence of highly conserved regulatory elements in the promoter region of myticin genes, which might explain their strong hemocyte-specific expression.

Antimicrobial peptides (AMPs) are short peptides active against a wide range of pathogens and, therefore, they are considered a useful alternative to conventional antibiotics. We have identified a new AMP in a transcriptome derived from the Antarctic fish Trematomus bernacchii. This peptide, named Trematocine, has been investigated for its expression both at the basal level and after in vivo immunization with an endemic Antarctic bacterium (Psychrobacter sp. TAD1). Results agree with the expected behavior of a fish innate immune component, therefore we decided to synthesize the putative mature sequence of Trematocine to determine the structure, the interaction with biological membranes, and the biological activity. We showed that Trematocine folds into a α-helical structure in the presence of both zwitterionic and anionic charged vesicles. We demonstrated that Trematocine has a highly specific interaction with anionic charged vesicles and that it can kill Gram-negative bacteria, possibly via a carpet like mechanism. Moreover, Trematocine showed minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values against selected Gram-positive and Gram-negative bacteria similar to other AMPs isolated from Antarctic fishes. The peptide is a possible candidate for a new drug as it does not show any haemolytic or cytotoxic activity against mammalian cells at the concentration needed to kill the tested bacteria.

The head kidney is a key organ that plays a fundamental role in the regulation of the fish immune response and in the maintenance of endocrine homeostasis. Previous studies indicate that the supplementation of exogenous dietary components, such as krill meal (KM), soybean meal (SM), Bactocell® (BA), and butyrate (BU), can have a significant effect on the immune function of the head kidney. The aim of this study was to investigate the differential effect of these four dietary ingredients on the transcriptional profiles of the head kidney of the Atlantic salmon. This study revealed that just a small number of genes were responsive to the feeding regime after a long-term (12 weeks) treatment, and evidenced that the most significant alterations, both in terms of the number of affected genes and magnitude of changes in gene expression, were detectable in the BU- and KM-fed groups compared with controls, while the SM diet had a nearly negligible effect, and BA had no significant effects at all. Most of the differentially expressed genes were involved in the immune response and, in line with data previously obtained from pyloric caeca, major components of the complement system were significantly affected. These alterations were accompanied by an increase in the density of melanomacrophage centers in the KM- and SM-fed group and their reduction in the BU-fed group. While three types of dietary supplements (BU, KM, and SM) were able to produce a significant modulation of some molecular players of the immune system, the butyrate-rich diet was revealed as the one with the most relevant immune-stimulating properties in the head kidney. These preliminary results suggest that further investigations should be aimed towards the elucidation of the potential beneficial effects of butyrate and krill meal supplementation on farmed salmon health and growth performance.

The CS-αβ architecture is a structural scaffold shared by a high number of small, cationic, cysteine-rich defense peptides, found in nearly all the major branches of the tree of life. Although several CS-αβ peptides involved in innate immune response have been described so far in bivalve mollusks, a clear-cut definition of their molecular diversity is still lacking, leaving the evolutionary relationship among defensins, mytilins, myticins and other structurally similar antimicrobial peptides still unclear. In this study, we performed a comprehensive bioinformatic screening of the genomes and transcriptomes available for marine mussels (Mytilida), redefining the distribution of mytilin-like CS-αβ peptides, which in spite of limited primary sequence similarity maintain in all cases a well-conserved backbone, stabilized by four disulfide bonds. Variations in the size of the alpha-helix and the two antiparallel beta strand region, as well as the positioning of the cysteine residues involved in the formation of the C1−C5 disulfide bond might allow a certain degree of structural flexibility, whose functional implications remain to be investigated. The identification of mytilins in Trichomya and Perna spp. revealed that many additional CS-αβ AMPs remain to be formally described and functionally characterized in Mytilidae, and suggest that a more robust scheme should be used for the future classification of such peptides with respect with their evolutionary origin.

Lysozyme is one of the most important anti-bacterial effectors in the innate immune system of animals. Besides its direct antibacterial enzymatic activity, lysozyme displays other biological properties, pointing toward a significant anti-inflammatory effect, many aspects of which are still elusive. Here we investigate the perturbation of gene expression profiles induced by lysozyme in a monocyte cell line in vitro considering a perspective as broad as the whole transcriptome profiling. The results of the RNA-seq experiment show that lysozyme induces transcriptional modulation of the TNF-α/IL-1β pathway genes in U937 monocytes. The analysis of transcriptomic profiles with IPA® identified a simple but robust molecular network of genes, in which the regulation trends are fully consistent with the anti-inflammatory activity of lysozyme. This study provides the first evidence in support of the anti-inflammatory action of lysozyme on the basis of transcriptomic regulation data resulting from the broad perspective of a whole-transcriptome profiling. Such important effects can be achieved with the supplementation of relatively low concentrations of lysozyme, for a short time of exposure. These new insights allow the potential of lysozyme in pharmacological applications to be better exploited.

C1q-domain-containing (C1qDC) proteins are rapidly emerging as key players in the innate immune response of bivalve mollusks. Growing experimental evidence suggests that these highly abundant secretory proteins are involved in the recognition of microbe-associated molecular patterns, serving as lectin-like molecules in the bivalve proto-complement system. While a large amount of functional data concerning the binding specificity of the globular head C1q domain and on the regulation of these molecules in response to infection are quickly accumulating, the genetic mechanisms that have led to the extraordinary lineage-specific expansion of the C1qDC gene family in bivalves are still largely unknown. The analysis of the chromosome-scale genome assembly of the Eastern oyster Crassostrea virginica revealed that the 476 oyster C1qDC genes, far from being uniformly distributed along the genome, are located in large clusters of tandemly duplicated paralogs, mostly found on chromosomes 7 and 8. Our observations point out that the evolutionary process behind the development of a large arsenal of C1qDC lectin-like molecules in marine bivalves is still ongoing and likely based on an unequal crossing over.

We identified a lectin (carbohydrate-binding protein) belonging to the complement 1q(C1q) family in the feather star Anneissia japonica (a crinoid pertaining to the phylum Echinodermata). The combination of Edman degradation and bioinformatics sequence analysis characterized the primary structure of this novel lectin, named OXYL, as a secreted 158 amino acid-long globular head (sgh)C1q domain containing (C1qDC) protein. Comparative genomics analyses revealed that OXYL pertains to a family of intronless genes found with several paralogous copies in different crinoid species. Immunohistochemistry assays identified the tissues surrounding coelomic cavities and the arms as the main sites of production of OXYL. Glycan array confirmed that this lectin could quantitatively bind to type-2 N-acetyllactosamine (LacNAc: Galβ1-4GlcNAc), but not to type-1 LacNAc (Galβ1-3GlcNAc). Although OXYL displayed agglutinating activity towards Pseudomonas aeruginosa, it had no effect on bacterial growth. On the other hand, it showed a significant anti-biofilm activity. We provide evidence that OXYL can adhere to the surface of human cancer cell lines BT-474, MCF-7, and T47D, with no cytotoxic effect. In BT-474 cells, OXYL led to a moderate activation of the p38 kinase in the MAPK signaling pathway, without affecting the activity of caspase-3. Bacterial agglutination, anti-biofilm activity, cell adhesion, and p38 activation were all suppressed by co-presence of LacNAc. This is the first report on a type-2 LacNAc-specific lectin characterized by a C1q structural fold.

Proteins of the ShK superfamily are characterized by a small conserved domain (ShKT), first discovered in small venom peptides produced by sea anemones, and acting as specific inhibitors of voltage-dependent and calcium-activated K+ channels. The ShK superfamily includes both small toxic peptides and larger multifunctional proteins with various functions. ShK toxins are often important components of animal venoms, where they perform different biological functions including neurotoxic and immunosuppressive effects. Given their high specificity and effectiveness, they are currently regarded as promising pharmacological lead compounds for the treatment of autoimmune diseases. Here, we report on the molecular analysis of ShKT domain containing proteins produced by the Mediterranean vampire snail Cumia reticulata, an ectoparasitic gastropod that feeds on benthic fishes. The high specificity of expression of most ShK transcripts in salivary glands identifies them as relevant components of C. reticulata venom. These ShK proteins display various structural architectures, being produced either as single-domain secretory peptides, or as larger proteins combining the ShKT with M12 or CAP domains. Both ShKT-containing genes and their internal ShKT domains undergo frequent duplication events in C. reticulata, ensuring a high level of variability that is likely to play a role in increasing the range of their potential molecular targets.

The extraordinary progress experienced by sequencing technologies and bioinformatics has made the development of omic studies virtually ubiquitous in all fields of life sciences nowadays. However, scientific attention has been quite unevenly distributed throughout the different branches of the tree of life, leaving molluscs, one of the most diverse animal groups, relatively unexplored and without representation within the narrow collection of well established model organisms. Within this Phylum, bivalve molluscs play a fundamental role in the functioning of the marine ecosystem, constitute very valuable commercial resources in aquaculture, and have been widely used as sentinel organisms in the biomonitoring of marine pollution. Yet, it has only been very recently that this complex group of organisms became a preferential subject for omic studies, posing new challenges for their integrative characterization. The present contribution aims to give a detailed insight into the state of the art of the omic studies and functional information analysis of bivalve molluscs, providing a timely perspective on the available data resources and on the current and prospective applications for the biomonitoring of harmful marine compounds.

Synonymous codon usage bias (CUB) is a defined as the non-random usage of codons encoding the same amino acid across different genomes. This phenomenon is common to all organisms and the real weight of the many factors involved in its shaping still remains to be fully determined. So far, relatively little attention has been put in the analysis of CUB in bivalve mollusks due to the limited genomic data available. Taking advantage of the massive sequence data generated from next generation sequencing projects, we explored codon preferences in 64 different species pertaining to the six major evolutionary lineages in Bivalvia. We detected remarkable differences across species, which are only partially dependent on phylogeny. While the intensity of CUB is mild in most organisms, a heterogeneous group of species (including Arcida and Mytilida, among the others) display higher bias and a strong preference for AT-ending codons. We show that the relative strength and direction of mutational bias, selection for translational efficiency and for translational accuracy contribute to the establishment of synonymous codon usage in bivalves. Although many aspects underlying bivalve CUB still remain obscure, we provide for the first time an overview of this phenomenon in this large, commercially and environmentally important, class of marine invertebrates.

The aim of this research was the identification of novel pharmacogenomic biomarkers for better understanding the complex gene regulation mechanisms underpinning glucocorticoid (GC) action in paediatric inflammatory bowel disease (IBD). This goal was achieved by evaluating high-throughput microRNA (miRNA) profiles during GC treatment, integrated with the assessment of expression changes in GC receptor (GR) heterocomplex genes. Furthermore, we tested the hypothesis that differentially expressed miRNAs could be directly regulated by GCs through investigating the presence of GC responsive elements (GREs) in their gene promoters. Ten IBD paediatric patients responding to GCs were enrolled. Peripheral blood was obtained at diagnosis (T0) and after four weeks of steroid treatment (T4). MicroRNA profiles were analyzed using next generation sequencing, and selected significantly differentially expressed miRNAs were validated by quantitative reverse transcription-polymerase chain reaction. In detail, 18 miRNAs were differentially expressed from T0 to T4, 16 of which were upregulated and 2 of which were downregulated. Out of these, three miRNAs (miR-144, miR-142, and miR-96) could putatively recognize the 3’UTR of the GR gene and three miRNAs (miR-363, miR-96, miR-142) contained GREs sequences, thereby potentially enabling direct regulation by the GR. In conclusion, we identified miRNAs differently expressed during GC treatment and miRNAs which could be directly regulated by GCs in blood cells of young IBD patients. These results could represent a first step towards their translation as pharmacogenomic biomarkers.

The application of high-throughput sequencing technologies to non-model organisms has brought new opportunities for the identification of bioactive peptides from genomes and transcriptomes. From this point of view, marine invertebrates represent a potentially rich, yet largely unexplored resource for de novo discovery due to their adaptation to diverse challenging habitats. Bioinformatics analyses of available genomic and transcriptomic data allowed us to identify myticalins, a novel family of antimicrobial peptides (AMPs) from the mussel Mytilus galloprovincialis, and a similar family of AMPs from Modiolus spp., named modiocalins. Their coding sequence encompasses two conserved N-terminal (signal peptide) and C-terminal (propeptide) regions and a hypervariable central cationic region corresponding to the mature peptide. Myticalins are taxonomically restricted to Mytiloida and they can be classified into four subfamilies. These AMPs are subject to considerable interindividual sequence variability and possibly to presence/absence variation. Functional assays performed on selected members of this family indicate a remarkable tissue-specific expression (in gills) and broad spectrum of activity against both Gram-positive and Gram-negative bacteria. Overall, we present the first linear AMPs ever described in marine mussels and confirm the great potential of bioinformatics tools for the de novo discovery of bioactive peptides in non-model organisms.

Genes involved in sex determination and differentiation have been identified in mice, humans, chickens, reptiles, amphibians and teleost fishes. However, little is known of their functional conservation, and it is unclear whether there is a common set of genes shared by all vertebrates. Coelacanths, basal Sarcopterygians and unique "living fossils", could help establish an inventory of the ancestral genes involved in these important developmental processes and provide insights into their components. In this study 33 genes from the genome of Latimeria chalumnae and from the liver and testis transcriptomes of Latimeria menadoensis, implicated in sex determination and differentiation, were identified and characterized and their expression levels measured. Interesting findings were obtained for GSDF, previously identified only in teleosts and now characterized for the first time in the sarcopterygian lineage; FGF9, which is not found in teleosts; and DMRT1, whose expression in adult gonads has recently been related to maintenance of sexual identity. The gene repertoire and testis-specific gene expression documented in coelacanths demonstrate a greater similarity to modern fishes and point to unexpected changes in the gene regulatory network governing sexual development.

The crustacean Hyperglycemic Hormone (cHH) is a neuropeptide present in many decapods. Two different chiral isomers are simultaneously present in Astacid crayfish and their specific biological functions are still poorly understood. The present study is aimed at better understanding the potentially different effect of each of the isomers on the hepatopancreatic gene expression profile in the crayfish Pontastacus leptodactylus, in the context of short term hyperglycemia. Hence, two different chemically synthesized cHH enantiomers, containing either L- or D-Phe(3), were injected to the circulation of intermolt females following removal of their X organ-Sinus gland complex. The effects triggered by the injection of the two alternate isomers were detected after one hour through measurement of circulating glucose levels. Triggered changes of the transcriptome expression profile in the hepatopancreas were analyzed by RNA-seq. A whole transcriptome shotgun sequence assembly provided the assumedly complete transcriptome of P. leptodactylus hepatopancreas, followed by RNA-seq analysis of changes in the expression level of many genes caused by the application of each of the hormone isomers. Circulating glucose levels were much higher in response to the D-isoform than to the L-isoform injection, one hour from injection. Similarly, the RNA-seq analysis confirmed a stronger effect on gene expression following the administration of D-cHH, while just limited alterations were caused by the L-isomer. These findings demonstrated a more prominent short term effect of the D-cHH on the transcription profile and shed light on the effect of the D-isomer on specific functional gene groups. Another contribution of the study is the construction of a de novo assembly of the hepatopancreas transcriptome, consisting of 39,935 contigs, that dramatically increases the molecular information available for this species and for crustaceans in general, providing an efficient tool for studying gene expression patterns in this organ.

MytiLec is an α-d-galactose-binding lectin with a unique primary structure isolated from the Mediterranean mussel (Mytilus galloprovincialis). The lectin adopts a β-trefoil fold that is also found in the B-sub-unit of ricin and other ricin-type (R-type) lectins. We are introducing MytiLec(-1) and its two variants (MytiLec-2 and -3), which both possess an additional pore-forming aerolysin-like domain, as members of a novel multi-genic “mytilectin family” in bivalve mollusks. Based on the full length mRNA sequence (911 bps), it was possible to elucidate the coding sequence of MytiLec-1, which displays an extended open reading frame (ORF) at the 5′ end of the sequence, confirmed both at the mRNA and at the genomic DNA sequence level. While this extension could potentially produce a polypeptide significantly longer than previously reported, this has not been confirmed yet at the protein level. MytiLec-1 was revealed to be encoded by a gene consisting of two exons and a single intron. The first exon comprised the 5′UTR and the initial ATG codon and it was possible to detect a putative promoter region immediately ahead of the transcription start site in the MytiLec-1 genomic locus. The remaining part of the MytiLec-1 coding sequence (including the three sub-domains, the 3′UTR and the poly-A signal) was included in the second exon. The bacteriostatic activity of MytiLec-1 was determined by the agglutination of both Gram-positive and Gram-negative bacteria, which was reversed by the co-presence of α-galactoside. Altogether, these data support the classification of MytiLec-1 as a member of the novel mytilectin family and suggest that this lectin may play an important role as a pattern recognition receptor in the innate immunity of mussels.