Modulation of Glucose Consumption and Uptake in HepG2 Cells by Aqueous Extracts from the Coelomic Fluid of the Edible Holothuria tubulosa Sea Cucumber.

The cell-free aqueous extract from the coelomic fluid of Holothuria tubulosa was prepared and examined for its glucose-lowering effect on HepG2 cells in vitro. In particular, employing a combination of cytochemical, flow cytometric, PCR, and protein blot techniques, we evaluated its role on glucose internalization and storage and on the upregulation and surface translocation of the two glucose transporters GLUT-2 and -4. The changes in expression, synthesis, and/or activation of the GLUT2-related transcription factor hepatocyte nuclear factor-1 alpha (HNF1α) and the GLUT-4-translocation regulatory factors insulin receptor substrate-1 (IRS-1) and AKT were also studied. Our results showed the improved glucose response by HepG2 cells, leading to an evident increase in glucose consumption/uptake and glycogen storage upon exposure. Moreover, the extract induced molecular reprogramming involving the upregulation of (i) IRS1 gene expression, (ii) the transcription and translation levels of HNF1α, AKT, and GLUT-4, (iii) the phosphorylation level of AKT, (iv) the synthesis of GLUT-2 protein, and (v) the translocation of GLUT-2 and -4 transporters onto the plasma membrane. Cumulatively, our results suggest that the coelomic fluid extract from H. tubulosa can be taken into consideration for the development of novel treatment agents against diabetes mellitus.

Environmental DNA: The First Snapshot of the Vertebrate Biodiversity in Three Sicilian Lakes.

Freshwater ecosystems play a key role in global diversity and are subject to a series of anthropic impacts, often leading to biodiversity loss. The organisms inhabiting these sites continuously release DNA into the environment through cells, excrement, gametes and/or decomposing matter; thus, evaluation of this eDNA could revolutionize the monitoring of biodiversity. In this study, environmental DNA metabarcoding was used for the first time in three Sicilian lakes: Lake Poma, Piana degli Albanesi Lake and Lake Scanzano. Results obtained provide the first snapshot of vertebrate biodiversity in these three lakes, where little is known, to provide valuable information useful for creating a baseline of knowledge regarding the biodiversity in these three lakes. Another important result was the detection of marine species, most likely due to some kind of anthropogenic contamination. Environmental DNA is a useful tool to evaluate both biodiversity and the ecological status of the environment; it has the potential to complement traditional methods, and the use of both approaches may offer a more comprehensive understanding of the ecosystem.

Ciona robusta macrophage migration inhibitory factor (Mif1 and Mif2) genes are differentially regulated in the lipopolysaccharide-challenged pharynx.

The effects of lipopolysaccharide (LPS) on Mif (macrophage migration inhibitory factor) gene expression in the pharynx (haemapoetic tissue) of Ciona robusta were investigated using quantitative reverse-transcription PCR (qRT-PCR) and in situ hybridisation (ISH). To verify the induction of an inflammatory response in the pharynx, a qRT-PCR analysis was performed to evaluate the change in the expression of proinflammatory marker genes such as Mbl, Ptx-like, Tnf-α and Nf-kb, which were shown to be upregulated 1 h post LPS challenge. The change in the expression of the two Mif paralogs in the pharynx was assessed before and after stimulation, and qRT-PCR and ISH results showed that, although Mif2 and Mif2 were expressed in clusters of haemocytes in pharynx vessels, only Mif1 expression increased after LPS stimulation. This indicates that the Mif genes are differently regulated and respond to different ambient inputs that need further analysis.

In Vitro Cytotoxic Effect of Aqueous Extracts from Leaves and Rhizomes of the Seagrass Posidonia oceanica (L.) Delile on HepG2 Liver Cancer Cells: Focus on Autophagy and Apoptosis.

Aqueous extracts from Posidonia oceanica's green and brown (beached) leaves and rhizomes were prepared, submitted to phenolic compound and proteomic analysis, and examined for their potential cytotoxic effect on HepG2 liver cancer cells in culture. The chosen endpoints related to survival and death were cell viability and locomotory behavior, cell-cycle analysis, apoptosis and autophagy, mitochondrial membrane polarization, and cell redox state. Here, we show that 24 h exposure to both green-leaf- and rhizome-derived extracts decreased tumor cell number in a dose-response manner, with a mean half maximal inhibitory concentration (IC50) estimated at 83 and 11.5 µg of dry extract/mL, respectively. Exposure to the IC50 of the extracts appeared to inhibit cell motility and long-term cell replicating capacity, with a more pronounced effect exerted by the rhizome-derived preparation. The underlying death-promoting mechanisms identified involved the down-regulation of autophagy, the onset of apoptosis, the decrease in the generation of reactive oxygen species, and the dissipation of mitochondrial transmembrane potential, although, at the molecular level, the two extracts appeared to elicit partially differentiating effects, conceivably due to their diverse composition. In conclusion, P. oceanica extracts merit further investigation to develop novel promising prevention and/or treatment agents, as well as beneficial supplements for the formulation of functional foods and food-packaging material with antioxidant and anticancer properties.

Transcriptomic and Bioinformatic Analyses Identifying a Central Mif-Cop9-Nf-kB Signaling Network in Innate Immunity Response of Ciona robusta.

The Ascidian C. robusta is a powerful model for studying innate immunity. LPS induction activates inflammatory-like reactions in the pharynx and the expression of several innate immune genes in granulocyte hemocytes such as cytokines, for instance, macrophage migration inhibitory factors (CrMifs). This leads to intracellular signaling involving the Nf-kB signaling cascade that triggers downstream pro-inflammatory gene expression. In mammals, the COP9 (Constitutive photomorphogenesis 9) signalosome (CSN) complex also results in the activation of the NF-kB pathway. It is a highly conserved complex in vertebrates, mainly engaged in proteasome degradation which is essential for maintaining processes such as cell cycle, DNA repair, and differentiation. In the present study, we used bioinformatics and in-silico analyses combined with an in-vivo LPS exposure strategy, next-generation sequencing (NGS), and qRT-PCR to elucidate molecules and the temporal dynamics of Mif cytokines, Csn signaling components, and the Nf-κB signaling pathway in C. robusta. A qRT-PCR analysis of immune genes selected from transcriptome data revealed a biphasic activation of the inflammatory response. A phylogenetic and STRING analysis indicated an evolutionarily conserved functional link between the Mif-Csn-Nf-kB axis in ascidian C. robusta during LPS-mediated inflammation response, finely regulated by non-coding molecules such as microRNAs (miRNAs).

Bioactive Molecules from the Innate Immunity of Ascidians and Innovative Methods of Drug Discovery: A Computational Approach Based on Artificial Intelligence.

The study of bioactive molecules of marine origin has created an important bridge between biological knowledge and its applications in biotechnology and biomedicine. Current studies in different research fields, such as biomedicine, aim to discover marine molecules characterized by biological activities that can be used to produce potential drugs for human use. In recent decades, increasing attention has been paid to a particular group of marine invertebrates, the Ascidians, as they are a source of bioactive products. We describe omics data and computational methods relevant to identifying the mechanisms and processes of innate immunity underlying the biosynthesis of bioactive molecules, focusing on innovative computational approaches based on Artificial Intelligence. Since there is increasing attention on finding new solutions for a sustainable supply of bioactive compounds, we propose that a possible improvement in the biodiscovery pipeline might also come from the study and utilization of marine invertebrates' innate immunity.

Effects of Sulfamethoxazole on Fertilization and Embryo Development in the Arbacia lixula Sea Urchin.

To date, drugs released into the aquatic environment are a real problem, and among antibiotics, sulfamethoxazole is the one most widely found in wastewater; thus, the evaluation of its toxicity on marine organisms is very important. This study, for the first time, investigates the in vitro effects of 4 concentrations of sulfamethoxazole (0.05 mg/L, 0.5 mg/L, 5 mg/L, 50 mg/L) on the fertilization and development of the sea urchin Arbacia lixula. The gametes were exposed to drugs in three different stages: simultaneously with, prior to, and post-fertilization. The results show a significant reduction in the percentage of fertilized oocytes at the highest drug concentrations. Moreover, an increase in anomalies and delays in embryo development following the treatment with the drug was demonstrated. Therefore, the data suggest that this antibiotic can alter the development of marine organisms, making it urgent to act to reduce their release and to determine the concentration range with the greatest impact.

Transcriptomic Analyses Reveal 2 and 4 Family Members of Cytochromes P450 (CYP) Involved in LPS Inflammatory Response in Pharynx of Ciona robusta.

Cytochromes P450 (CYP) are enzymes responsible for the biotransformation of most endogenous and exogenous agents. The expression of each CYP is influenced by a unique combination of mechanisms and factors including genetic polymorphisms, induction by xenobiotics, and regulation by cytokines and hormones. In recent years, Ciona robusta, one of the closest living relatives of vertebrates, has become a model in various fields of biology, in particular for studying inflammatory response. Using an in vivo LPS exposure strategy, next-generation sequencing (NGS) and qRT-PCR combined with bioinformatics and in silico analyses, compared whole pharynx transcripts from naïve and LPS-exposed C. robusta, and we provide the first view of cytochrome genes expression and miRNA regulation in the inflammatory response induced by LPS in a hematopoietic organ. In C. robusta, cytochromes belonging to 2B,2C, 2J, 2U, 4B and 4F subfamilies were deregulated and miRNA network interactions suggest that different conserved and species-specific miRNAs are involved in post-transcriptional regulation of cytochrome genes and that there could be an interplay between specific miRNAs regulating both inflammation and cytochrome molecules in the inflammatory response in C. robusta.

ceRNA Network Regulation of TGF-ß, WNT, FOXO, Hedgehog Pathways in the Pharynx of Ciona robusta.

The transforming growth factor-ß (TGF-ß) family of cytokines performs a multifunctional signaling, which is integrated and coordinated in a signaling network that involves other pathways, such as Wintless, Forkhead box-O (FOXO) and Hedgehog and regulates pivotal functions related to cell fate in all tissues. In the hematopoietic system, TGF-ß signaling controls a wide spectrum of biological processes, from immune system homeostasis to the quiescence and self-renewal of hematopoietic stem cells (HSCs). Recently an important role in post-transcription regulation has been attributed to two type of ncRNAs: microRNAs and pseudogenes. Ciona robusta, due to its philogenetic position close to vertebrates, is an excellent model to investigate mechanisms of post-transcriptional regulation evolutionarily highly conserved in immune homeostasis. The combined use of NGS and bioinformatic analyses suggests that in the pharynx, the hematopoietic organ of Ciona robusta, the Tgf-ß, Wnt, Hedgehog and FoxO pathways are involved in tissue homeostasis, as they are in human. Furthermore, ceRNA network interactions and 3'UTR elements analyses of Tgf-ß, Wnt, Hedgehog and FoxO pathways genes suggest that different miRNAs conserved (cin-let-7d, cin-mir-92c, cin-mir-153), species-specific (cin-mir-4187, cin-mir-4011a, cin-mir-4056, cin-mir-4150, cin-mir-4189, cin-mir-4053, cin-mir-4016, cin-mir-4075), pseudogenes (ENSCING00000011392, ENSCING00000018651, ENSCING00000007698) and mRNA 3'UTR elements are involved in post-transcriptional regulation in an integrated way in C. robusta.

Evolutionary and transcriptional analyses of a pentraxin-like component family involved in the LPS inflammatory response of Ciona robusta.

Pentraxins (PTXs) are a superfamily of conserved proteins which are components of the humoral arm of innate immunity. They are considered to be functional ancestors of antibodies and are classified into short and long types. In this study, we show that a pentraxin-like component (Ptx-like) with a C-terminal PTX domain, highly homologous to the short PTX of H. sapiens CRP, and a long N-terminal domain typical of long PTXs, is involved in the inflammatory response of Ciona robusta under LPS exposure in vivo. Analyses of protein domains as well as 3D modelling and phylogenetic tree supported the close relationship of Ptx-like with mammalian CRP, suggesting that C. robusta Ptx-like shares a common ancestor in the chordate lineages. qRT-PCR analysis showed that Ptx-like was transcriptionally upregulated during the inflammatory process induced by LPS inoculation and that it is involved in the initial phase as well as the secondary phase of the inflammatory response in which matrix remodelling and the achievement of homeostasis occur. In situ hybridisation assays revealed that gene transcription was upregulated in the pharynx post-LPS challenge in vivo, and that Ptx-like was expressed by clusters of haemocytes, mainly granulocytes, inside the pharynx vessels. We also found transcript-expressing granulocytes flowing in the musculature and in the lacunae of the circulatory system. These data supported that Ptx-like is a potential molecule of the acute-phase response in C. robusta immune defence systems against bacterial infection.

Transcriptional and in silico analyses of MIF cytokine and TLR signalling interplay in the LPS inflammatory response of Ciona robusta.

The close phylogenetic relationship between Ciona robusta and vertebrates makes it a powerful model for studying innate immunity and the evolution of immune genes. To elucidate the nature and dynamics of the immune response, the molecular mechanisms by which bacterial infection is detected and translated into inflammation and how potential pattern recognition receptors (PRRs) are involved in pathogen recognition in tunicate C. robusta (formerly known as Ciona intestinalis), we applied an approach combining bacterial infections, next-generation sequencing, qRT-PCR, bioinformatics and in silico analyses (criteria of a p-value < 0.05 and FDR < 0.05). A STRING analysis indicated a functional link between components of the Tlr/MyD88-dependent signalling pathway (Tlr2, MyD88, and Irak4) and components of the Nf-κB signalling pathway (Nf-κB, IκBα, and Ikkα) (p-value < 0.05, FDR < 0.05). A qRT-PCR analysis of immune genes selected from transcriptome data revealed Mif as more frequently expressed in the inflammatory response than inflammation mediator or effector molecules (e.g., Il-17s, Tnf-α, Tgf-ß, Mmp9, Tlrs, MyD88, Irak4, Nf-κB, and galectins), suggesting close interplay between Mif cytokines and Nf-κB signalling pathway components in the biphasic activation of the inflammatory response. An in silico analyses of the 3'-UTR of Tlr2, MyD88, IκBα, Ikk, and Nf-κB transcripts showed the presence of GAIT elements, which are known to play key roles in the regulation of immune gene-specific translation in humans. These findings provide a new level of understanding of the mechanisms involved in the regulation of the C. robusta inflammatory response induced by LPS and suggest that in C. robusta, as in humans, a complex transcriptional and post-transcriptional control mechanism is involved in the regulation of several inflammatory genes.

5' and 3' RACE Method to Obtain Full-Length 5' and 3' Ends of Ciona robusta Macrophage Migration Inhibitory Factors Mif1 and Mif2 cDNA.

The 5' and 3' RACE is a method to obtain full-length 5' and 3' ends of cDNA using known cDNA sequences from expressed sequence tags (ESTs), subtracted cDNA, differential display, or library screening. Here is described the identification of full-length 5' and 3' ends of Ciona robusta Mif1 and Mif2 cDNA by using 5' and 3' RACE method.

The gelatinase MMP-9like is involved in regulation of LPS inflammatory response in Ciona robusta.

Matrix metalloproteinases (MMPs) are a family of endopeptidases collectively able to degrade the components of the extracellular matrix (ECM), with important roles in many biological processes, such as embryogenesis, normal tissue remodelling, angiogenesis and wound healing. New views on the function of MMPs reveal that they regulate inflammatory response and therefore might represent an early step in the evolution of the immune system. MMPs can affect the activity of cytokines involved in inflammation including TGF-ß and TNF-α. MMPs are widely distributed in all kingdoms of life and have likely evolved from a single-domain protein which underwent successive rounds of duplications. In this study, we focused on the Ciona robusta (formerly known as Ciona intestinalis) MMP gelatinase homologue. Gene organization, phylogenetic analysis and 3D modeling supported the closest correlation of C. robusta gelatinase with the human MMP-9. Real-time PCR analysis and zymographic assay showed a prompt expression induced by LPS inoculation and an upregulation of enzymatic activity. Furthermore, we showed that before of the well-known increase of TGF-ß and TNF-α levels, a MMP-9like boost occurred, suggesting a possible involvement of MMP-9like in regulating inflammatory response in C. robusta.

Identification of CPE and GAIT elements in 3'UTR of macrophage migration inhibitory factor (MIF) involved in inflammatory response induced by LPS in Ciona robusta.

Innate immune responses face infectious microorganisms by inducing inflammatory responses. Multiple genes within distinct functional categories are coordinately and temporally regulated by transcriptional 'on' and 'off' switches that account for the specificity of gene expression in response to external stimuli. Mechanisms that control transcriptional and post-transcriptional regulation are important in coordinating the initiation and resolution of inflammation. Macrophage migration inhibitory factor (MIF) is an important cytokine that, in Ciona robusta, is related to inflammatory response. It is well known that in C. robusta, formerly known as Ciona intestinalis, the pharynx is involved in the inflammatory reaction induced by lipopolysaccharide (LPS) injection in the body wall. Using this biological system, we describe the identification of two C. robusta MIFs (CrMIF1 and CrMIF2). The phylogenetic tree and modeling support a close relationship with vertebrate MIF family members. CrMIF1 and CrMIF2 possess two evolutionally conserved catalytic sites: a tautomerase and an oxidoreductase site with a conserved CXXC motif. Real-time PCR analysis shows a prompt expression induced by LPS inoculation in CrMIF1 and a late upregulation of CrMIF2 and in silico analyses of 3'UTR show a cis-acting GAIT element and a CPE element in 3'-UTR, which are not present in the 3'-UTR of CrMIF1, suggesting that different transcriptional and post-transcriptional control mechanisms are involved in the regulation of gene expression of MIF during inflammatory response in C. robusta.

Ci8 short, a novel LPS-induced peptide from the ascidian Ciona intestinalis, modulates responses of the human immune system.

The selective modulation of immunity is an emerging concept driven by the vast advances in our understanding of this crucial host defense system. Invertebrates have raised researchers' interest as potential sources of new bioactive molecules owing to their antibacterial, anticancer and immunomodulatory activities. A LipoPolySaccharide (LPS) challenge in the ascidian Ciona intestinalis generates the transcript, Ci8 short, with cis-regulatory elements in the 3' UTR region that are essential for shaping innate immune responses. The derived amino acidic sequence in silico analysis showed specific binding to human Major Histocompatibility Complex (MHC) Class I and Class II alleles. The role of Ci8 short peptide was investigated in a more evolved immune system using human Peripheral Blood Mononuclear Cells (PBMCs) as in vitro model. The biological activities of this molecule include the activation of 70kDa TCR ζ chain Associated Protein kinase (ZAP-70) and T Cell Receptor (TCR) Vß oligo clonal selection on CD4+ T lymphocytes as well as increased proliferation and IFN-γ secretion. Furthermore Ci8 short affects CD4+/CD25high induced regulatory T cells (iTreg) subset selection which co-expressed the functional markers TGF-ß1/Latency Associated Protein (LAP) and CD39/CD73. This paper describes a new molecule that modulates important responses of the human adaptive immune system.

In the ovary of Ciona intestinalis (Type A), immune-related galectin and phenoloxidase genes are differentially expressed by the follicle accessory cells.

Riboprobes (in situ hybridization) and antibodies (immunohistochemistry), previously used to show the upregulation of Ciona intestinalis (Type A) galectins (CiLgals-a, CiLgals-b) and phenoloxidase (CinPO2) immune-related genes, were tested on histological sections of the ovary. The ovarian follicles are composed of oocytes encased by follicular cells (FCs) and test cells (TCs). Results show the transcription upregulation of both CiLgals and CinPO2 genes in the vitellogenic FCs, conversely distinct cytolocalization of the proteins are shown. At vitellogenic stage, the CiLgals are localized in the FCs, in the oocyte cytoplasm, and close to the germinal vesicle (GV), whereas the CinPO2 was never identified in the FCs. In a presumptive advanced phase and at the post-vitellogenic stage the TCs appear to be labelled by the CinPO2 riboprobe, and the protein identified by the antibody suggesting an mRNA transcytosis process from FCs. At post-vitellogenic stage the CiLgals mainly enrich the GV nucleoplasm, whereas the CinPO2 is contained in TCs and in the ooplasm but never found in the GV. This finding sheds new light on a former paper in which TCs were reported to be the only CinPO2-producing cells in the ovarian follicle. Finally, CiLgals and CinPO2 genes transcription and proteins production seem to be associated with accessory cells during their differentiation from vitellogenic to post-vitellogenic stage. The present findings promote further research on the early upregulation of immune-related genes, and the potential multifunctional role of the produced proteins. In addition further insight on the accessory cells involvement in ascidian oogenesis are reported.

The Ciona intestinalis immune-related galectin genes (CiLgals-a and CiLgals-b) are expressed by the gastric epithelium.

The transcription of two Ciona intestinalis galectin genes (CiLgals-a and CiLgals-b) is uparegulated by LPS in the pharynxis (hemocytes, vessel epithelium, endostilar zones) which is retained the main organ of the immunity. In this ascidian, for the first time we show, by immunohistochemistry and in situ hybridization methods, that these two immune-related genes are expressed in the gastric epithelium of naïve ascidians, whereas the galectins appear to be only contained in the intestine columnar epithelium. In addition, according to previous results on the pharynx, the genes are also expressed and galectins produced by hemocytes scattered in the connective tissue surrounding the gut. The genes expression and galectin localization in several tissues, including the previous findings on the transcription upregulation, the constitutive expression of these genes by endostylar zones and by the gastric epithelium suggest a potential multifunctional role of these galectins. In this respect, it is of interest to define where the CiLgals are normally found as related to the tissue functions. Such an approach should be a starting point for further investigations.

Molecular characterisation, evolution and expression analysis of g-type lysozymes in Ciona intestinalis.

Lysozyme is an important defense molecule of the innate immune system. Known for its bactericidal properties, lysozyme catalyzes the hydrolysis of b-(1,4)-glycosidic bonds between the N-acetyl glucosamine and N-acetyl muramic acid in the peptidoglycan layer of bacterial cell walls. In this study, the complete coding sequence of four g-type lysozymes were identified in Ciona intestinalis. Phylogenetic analysis and modelling supported the hypothesis of a close relationship with the vertebrate g-type lysozymes suggesting that the C. intestinalis g-type lysozyme genes (CiLys-g1, Cilys-g2, CiLys-g3, CiLys-g4) share a common ancestor in the chordate lineage. Protein motif searches indicated that C. intestinalis g-type lysozymes contain a GEWL domain with a GXXQ signature, typical of goose lysozymes. Quantitative Real-Time PCR analysis results showed that transcripts are expressed in various tissues from C. intestinalis. In order to determine the involvement of C. intestinalis g-type lysozymes in immunity, their expression was analyzed in the pharynx, showing that transcripts were significantly up-regulated in response to a challenge with lipopolysaccharide (LPS). These data support the view that CiLys g-type are molecules with potential for immune defense system against bacterial infection.

Evolution of Ciona intestinalis Tumor necrosis factor alpha (CiTNFα): Polymorphism, tissues expression, and 3D modeling.

Although the Tumor necrosis factor gene superfamily seems to be very conserved in vertebrates, phylogeny, tissue expression, genomic and gene organization, protein domains and polymorphism analyses showed that a strong change has happened mostly in invertebrates in which protochordates were a constraint during the immune-molecules history and evolution. RT PCR was used to investigate differential gene expression in different tissues. The expression shown was greater in the pharynx. Single-nucleotide polymorphism has been investigated in Ciona intestinalis Tumor necrosis factor alpha (CiTNFα) mRNA isolated from the pharynx of 30 ascidians collected from Licata, Sicily (Italy), by denaturing gradient gel electrophoresis (DGGE). For this analysis, CiTNFα nucleotide sequence was separated into two fragments, TNF-1 and -2, respectively, of 630 and 540 bp. We defined 23 individual DGGE patterns (named 1 to 10 for TNF-1 and 1 to 13 for TNF-2). Five patterns for TNF-1 accounted for <10% of the individuals, whereas the pattern 13 of TNF-2 accounted for >20% of the individuals. All the patterns were verified by direct sequencing. Single base-pair mutations were observed mainly within COOH-terminus, leading to 30 nucleotide sequence variants and 30 different coding sequences segregating in two main different clusters. Although most of the base mutations were silent, four propeptide variants were detected and six amino acid replacements occurred within COOH-terminus. Statistical tests for neutrality indicated negative selection pressure on signal and mature peptide domains, but possible positive selection pressure on COOH-terminus domain. Lastly we displayed the in silico 3D structure analysis including the CiTNFα variable region.

LPS injection reprograms the expression and the 3' UTR of a CAP gene by alternative polyadenylation and the formation of a GAIT element in Ciona intestinalis.

The diversification of cellular functions is one of the major characteristics of multicellular organisms which allow cells to modulate their gene expression, leading to the formation of transcripts and proteins with different functions and concentrations in response to different stimuli. CAP genes represent a widespread family of proteins belonging to the cysteine-rich secretory protein, antigen 5 and pathogenesis-related 1 superfamily which, it has been proposed, play key roles in the infection process and the modulation of immune responses in host animals. The ascidian Ciona intestinalis represents a group of proto-chordates with an exclusively innate immune system that has been widely studied in the field of comparative and developmental immunology. Using this biological system, we describe the identification of a novel APA mechanism by which an intronic polyadenylation signal is activated by LPS injection, leading to the formation of a shorter CAP mRNA capable of expressing the first CAP exon plus 19 amino acid residues whose sequence is contained within the first intron of the annotated gene. Furthermore, such an APA event causes the expression of a translational controlling cis-acting GAIT element which is not present in the previously isolated CAP isoform and identified in the 3'-UTR of other immune-related genes, suggesting an intriguing scenario in which both transcriptional and post-transcriptional control mechanisms are involved in the activation of the CAP gene during inflammatory response in C. intestinalis.