More than a simple epithelial layer: multifunctional role of echinoderm coelomic epithelium.

In echinoderms, the coelomic epithelium (CE) is reportedly the source of new circulating cells (coelomocytes) as well as the provider of molecular factors such as immunity-related molecules. However, its overall functions have been scarcely studied in detail. In this work, we used an integrated approach based on both microscopy (light and electron) and proteomic analyses to investigate the arm CE in the starfish Marthasterias glacialis during different physiological conditions (i.e., non-regenerating and/or regenerating). Our results show that CE cells share both ultrastructural and proteomic features with circulating coelomocytes (echinoderm immune cells). Additionally, microscopy and proteomic analyses indicate that CE cells are actively involved in protein synthesis and processing, and membrane trafficking processes such as phagocytosis (particularly of myocytes) and massive secretion phenomena. The latter might provide molecules (e.g., immune factors) and fluids for proper arm growth/regrowth. No stem cell marker was identified and no pre-existing stem cell was observed within the CE. Rather, during regeneration, CE cells undergo dedifferentiation and epithelial-mesenchymal transition to deliver progenitor cells for tissue replacement. Overall, our work underlines that echinoderm CE is not a "simple epithelial lining" and that instead it plays multiple functions which span from immunity-related roles as well as being a source of regeneration-competent cells for arm growth/regrowth.

Biomaterials and Bioactive Natural Products from Marine Invertebrates: From Basic Research to Innovative Applications.

Aquatic invertebrates are a major source of biomaterials and bioactive natural products that can find applications as pharmaceutics, nutraceutics, cosmetics, antibiotics, antifouling products and biomaterials. Symbiotic microorganisms are often the real producers of many secondary metabolites initially isolated from marine invertebrates; however, a certain number of them are actually synthesized by the macro-organisms. In this review, we analysed the literature of the years 2010-2019 on natural products (bioactive molecules and biomaterials) from the main phyla of marine invertebrates explored so far, including sponges, cnidarians, molluscs, echinoderms and ascidians, and present relevant examples of natural products of interest to public and private stakeholders. We also describe omics tools that have been more relevant in identifying and understanding mechanisms and processes underlying the biosynthesis of secondary metabolites in marine invertebrates. Since there is increasing attention on finding new solutions for a sustainable large-scale supply of bioactive compounds, we propose that a possible improvement in the biodiscovery pipeline might also come from the study and utilization of aquatic invertebrate stem cells.

An integrated view of asteroid regeneration: tissues, cells and molecules.

The potential for repairing and replacing cells, tissues, organs and body parts is considered a primitive attribute of life shared by all the organisms, even though it may be expressed to a different extent and which is essential for the survival of both individual and whole species. The ability to regenerate is particularly evident and widespread within invertebrates. In spite of the wide availability of experimental models, regeneration has been comprehensively explored in only a few animal systems (i.e., hydrozoans, planarians, urodeles) leaving many other animal groups unexplored. The regenerative potential finds its maximum expression in echinoderms. Among echinoderm classes, asteroids offer an impressive range of experimental models in which to study arm regeneration at different levels. Many studies have been recently carried out in order to understand the regenerative mechanisms in asteroids and the overall morphological processes have been well documented in different starfish species, such as Asterias rubens, Leptasterias hexactis and Echinaster sepositus. In contrast, very little is known about the molecular mechanisms that control regeneration development and patterning in these models. The origin and the fate of cells involved in the regenerative process remain a matter of debate and clear insights will require the use of complementary molecular and proteomic approaches to study this problem. Here, we review the current knowledge regarding the cellular, proteomic and molecular aspects of asteroid regeneration.

Changes in the intestinal mucosal proteome of turkeys (Meleagris gallopavo) infected with haemorrhagic enteritis virus.

Haemorrhagic enteritis (HE) is a viral disease affecting intestinal integrity and barrier function in turkey (Meleagris gallopavo) and resulting in a significant economic loss. Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH-MS) was applied to identify crucial proteins involved in HE infection. A total of 938 proteins were identified and used to generate a reference library for SWATH-MS analysis. In total, 523 proteins were reliably quantified, and 64 proteins were found to be differentially expressed, including 49 up-regulated and 15 down-regulated proteins between healthy and HE-affected intestinal mucosa. Functional analysis suggested that these proteins were involved in the following categories of cellular pathways and metabolisms: 1) energy pathways; 2) intestine lipid and amino acid metabolism; 3) oxidative stress; 4) intestinal immune response. Major findings of this study demonstrated that natural HE infection is related to the changes in abundance of several proteins involved in cell-intrinsic immune defense against viral invasion, systemic inflammation, modulation of excessive inflammation, B and T cell development and function and antigen presentation. mRNA quantitative expression demonstrated that most of the proteins involved in innate immunity that were found to be differentially abundant were produced by intestinal mucosa, suggesting its direct involvement in immune defences against HE infection.

Comparative proteomic analysis of saliva from dogs with and without obesity-related metabolic dysfuntion.

Dogs develop only some of the components of the human metabolic syndrome (MetS). Thus, in order to study possible MetS-related alterations in dogs, human MetS criteria were adapted to define canine MetS or so-called obesity-related metabolic dysfunction (ORMD). The main objective of this study was to identify changes in the salivary proteome of obese dogs with ORMD in comparison with obese dogs without ORMD which may constitute potential salivary biomarkers for assessing ORMD. In a first phase, 12 adult obese dogs with ORMD (N = 6) and without ORMD (N = 6) were included in the study. Subsequently, and with the aim of validating and strengthening the results, additional 12 obese dogs (6 with and 6 without ORMD) were tested in an independent experiment following the same protocol. Saliva samples were subjected to a quantitative proteomics analysis and the levels of nine salivary proteins were found to be significantly different between groups, among them those which had greatest fold-change were proteins involved in glycolysis and oxidative stress. In conclusion, despite metabolic syndrome to include different combinations of diseases, the observation of differences in salivary proteome suggests a potential of this fluid to understand the pathophysiology of the disease. SIGNIFICANCE: This is the first study evaluating proteomes of saliva in dogs, as a non invasive sample, in order to increase knowledge about the metabolic/physiopathological changes related to obesity-related metabolic dysfunction (ORMD) together with the identification of potential biomarkers for its diagnosis. As approximately 20% of dogs with naturally occurring obesity were described to suffer ORMD associated with insulin resistance and hypoadiponectinemia, the fact that indicate possible links between ORMD and associated diseases.

Identification of vaccine candidate antigens of Staphylococcus pseudintermedius by whole proteome characterization and serological proteomic analyses.

The recent emergence of methicillin-resistant Staphylococcus pseudintermedius (MRSP) has complicated considerably the treatment of infections caused by these bacteria. Therefore new treatment strategies are urgently needed, namely through the development of vaccines towards the control of bacterial infections. Our study describes an extensive characterization of the proteome of S. pseudintermedius through a 2-DE MALDI-TOF/TOF approach, followed by SERological Proteome Analysis (SERPA) to identify potential vaccine candidate antigens. We were able to identify 361 unique proteins, of which 39 are surface proteins. In order to assess the immunogenic potential of S. pseudintermedius proteins, a Western blot analysis of two-dimensional gels was carried out with serum from healthy dogs, dogs with atopic dermatitis infected and not infected with S. pseudintermedius. Only immunogenic areas detected by ≥ 50% of the dogs with atopic dermatitis infected with S. pseudintermedius sera and by <50% of the healthy dogs sera were excised and identified from Coomassie-colloidal stained gels. The areas identified by IgE were not considered as vaccine targets, because those proteins could induce hypersensitivity. We were able to identify 13 unique proteins after in-gel digestion of selected protein gel spots, with 4 antigenic proteins showing promising features for vaccine development. No specific antibodies were identified in the dogs with atopic dermatitis not infected with S. pseudintermedius sera that could contribute to prevention of infection. The SERPA approach employed in this study revealed novel candidate therapeutic targets for the control of S. pseudintermedius infections.

Chronic sustained hypoxia-induced redox remodeling causes contractile dysfunction in mouse sternohyoid muscle.

Chronic sustained hypoxia (CH) induces structural and functional adaptations in respiratory muscles of animal models, however the underlying molecular mechanisms are unclear. This study explores the putative role of CH-induced redox remodeling in a translational mouse model, with a focus on the sternohyoid-a representative upper airway dilator muscle involved in the control of pharyngeal airway caliber. We hypothesized that exposure to CH induces redox disturbance in mouse sternohyoid muscle in a time-dependent manner affecting metabolic capacity and contractile performance. C57Bl6/J mice were exposed to normoxia or normobaric CH (FiO2 = 0.1) for 1, 3, or 6 weeks. A second cohort of animals was exposed to CH for 6 weeks with and without antioxidant supplementation (tempol or N-acetyl cysteine in the drinking water). Following CH exposure, we performed 2D redox proteomics with mass spectrometry, metabolic enzyme activity assays, and cell-signaling assays. Additionally, we assessed isotonic contractile and endurance properties ex vivo. Temporal changes in protein oxidation and glycolytic enzyme activities were observed. Redox modulation of sternohyoid muscle proteins key to contraction, metabolism and cellular homeostasis was identified. There was no change in redox-sensitive proteasome activity or HIF-1α content, but CH decreased phospho-JNK content independent of antioxidant supplementation. CH was detrimental to sternohyoid force- and power-generating capacity and this was prevented by chronic antioxidant supplementation. We conclude that CH causes upper airway dilator muscle dysfunction due to redox modulation of proteins key to function and homeostasis. Such changes could serve to further disrupt respiratory homeostasis in diseases characterized by CH such as chronic obstructive pulmonary disease. Antioxidants may have potential use as an adjunctive therapy in hypoxic respiratory disease.

Proteolytic events are relevant cellular responses during nervous system regeneration of the starfish Marthasterias glacialis.

The molecular pathways that trigger the amazing intrinsic regenerative ability of echinoderm nervous system are still unknown. In order to approach this subject, a 2D-DIGE proteomic strategy was used, to screen proteome changes during neuronal regeneration in vivo, using starfish (Asteroidea, Echinodermata) as a model. A total of 528 proteins showed significant variations during radial nerve cord regeneration in both soluble and membrane protein-enriched fractions. Several functional classes of proteins known to be involved in axon regeneration events in other model organisms, such as chordates, were identified for the first time in the regenerating echinoderm nervous system. Unexpectedly, most of the identified proteins presented a molecular mass either higher or lower than expected. Such results suggest a functional modulation through protein post-translational modifications, such as proteolysis. Among these are proteins involved in cytoskeleton and microtubule regulators, axon guidance molecules and growth cone modulators, protein de novo synthesis machinery, RNA binding and transport, transcription factors, kinases, lipid signaling effectors and proteins with neuroprotective functions. In summary, the impact of proteolysis during regeneration events is here shown, although requiring further studies to detail on the mechanisms involving this post-transcriptional event on nervous system regeneration. BIOLOGICAL SIGNIFICANCE: The nervous systems of some organisms present a complete inability of neurons to regrow across a lesion site, which is the case of the adult mammalian central nervous system (CNS). Expanding our knowledge on how other animals regenerate their nervous system offers great potential for groundbreaking biomedical applications towards the enhancement of mammalian CNS regeneration. In order to approach this subject, a 2D-DIGE proteomic strategy was used for the first time, to screen the proteome changes during neuronal regeneration in vivo, using starfish (Asteroidea, Echinodermata) as a model. We strongly believe in the relevance of our results and have clear evidences that this work constitutes a solid basis for new research on starfish regenerating nerve cord. We also believe this work will have a significant impact not only on the general scientific community as we present here an alternative animal model to neurobiology, but also on the scientific community that works with echinoderms or closely related marine invertebrates, which are constantly searching for specific protein markers of several tissues, thus constituting an important advance towards the improvement of large scale protein information of unsequenced, but yet not less important organisms.

Adhesive proteins of stalked and acorn barnacles display homology with low sequence similarities.

Barnacle adhesion underwater is an important phenomenon to understand for the prevention of biofouling and potential biotechnological innovations, yet so far, identifying what makes barnacle glue proteins 'sticky' has proved elusive. Examination of a broad range of species within the barnacles may be instructive to identify conserved adhesive domains. We add to extensive information from the acorn barnacles (order Sessilia) by providing the first protein analysis of a stalked barnacle adhesive, Lepas anatifera (order Lepadiformes). It was possible to separate the L. anatifera adhesive into at least 10 protein bands using SDS-PAGE. Intense bands were present at approximately 30, 70, 90 and 110 kilodaltons (kDa). Mass spectrometry for protein identification was followed by de novo sequencing which detected 52 peptides of 7-16 amino acids in length. None of the peptides matched published or unpublished transcriptome sequences, but some amino acid sequence similarity was apparent between L. anatifera and closely-related Dosima fascicularis. Antibodies against two acorn barnacle proteins (ab-cp-52k and ab-cp-68k) showed cross-reactivity in the adhesive glands of L. anatifera. We also analysed the similarity of adhesive proteins across several barnacle taxa, including Pollicipes pollicipes (a stalked barnacle in the order Scalpelliformes). Sequence alignment of published expressed sequence tags clearly indicated that P. pollicipes possesses homologues for the 19 kDa and 100 kDa proteins in acorn barnacles. Homology aside, sequence similarity in amino acid and gene sequences tended to decline as taxonomic distance increased, with minimum similarities of 18-26%, depending on the gene. The results indicate that some adhesive proteins (e.g. 100 kDa) are more conserved within barnacles than others (20 kDa).

Mass spectrometry and animal science: protein identification strategies and particularities of farm animal species.

Proteomic approaches are gaining increasing importance in the context of all fields of animal and veterinary sciences, including physiology, productive characterization, and disease/parasite tolerance, among others. Proteomic studies mainly aim the proteome characterization of a certain organ, tissue, cell type or organism, either in a specific condition or comparing protein differential expression within two or more selected situations. Due to the high complexity of samples, usually total protein extracts, proteomics relies heavily on separation procedures, being 2D-electrophoresis and HPLC the most common, as well as on protein identification using mass spectrometry (MS) based methodologies. Despite the increasing importance of MS in the context of animal and veterinary science studies, the usefulness of such tools is still poorly perceived by the animal science community. This is primarily due to the limited knowledge on mass spectrometry by animal scientists. Additionally, confidence and success in protein identification is hindered by the lack of information in public databases for most of farm animal species and their pathogens, with the exception of cattle (Bos taurus), pig (Sus scrofa) and chicken (Gallus gallus). In this article, we will briefly summarize the main methodologies available for protein identification using mass spectrometry providing a case study of specific applications in the field of animal science. We will also address the difficulties inherent to protein identification using MS, with particular reference to experiments using animal species poorly described in public databases. Additionally, we will suggest strategies to increase the rate of successful identifications when working with farm animal species.