Dynamics of the Pacific oyster pathobiota during mortality episodes in Europe assessed by 16S rRNA gene profiling and a new target enrichment next-generation sequencing strategy.

Infectious agents such as the bacteria Vibrio aestuarianus or Ostreid herpesvirus 1 have been repeatedly associated with dramatic disease outbreaks of Crassostrea gigas beds in Europe. Beside roles played by these pathogens, microbial infections in C. gigas may derive from the contribution of a larger number of microorganisms than previously thought, according to an emerging view supporting the polymicrobial nature of bivalve diseases. In this study, the microbial communities associated with a large number of C. gigas samples collected during recurrent mortality episodes at different European sites were investigated by real-time PCR and 16SrRNA gene-based microbial profiling. A new target enrichment next-generation sequencing protocol for selective capturing of 884 phylogenetic and virulence markers of the potential microbial pathogenic community in oyster tissue was developed allowing high taxonomic resolution analysis of the bivalve pathobiota. Comparative analysis of contrasting C. gigas samples conducted using these methods revealed that oyster experiencing mortality outbreaks displayed signs of microbiota disruption associated with the presence of previously undetected potential pathogenic microbial species mostly belonging to genus Vibrio and Arcobacter. The role of these species and their consortia should be targeted by future studies aiming to shed light on mechanisms underlying polymicrobial infections in C. gigas.

Gene expression profiling in white blood cells reveals new insights into the molecular mechanisms of thalidomide in children with inflammatory bowel disease.

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.

HMGA Interactome: new insights from phage display technology.

High mobility group A proteins (HMGA1 and HMGA2) are architectural factors involved in chromatin remodelling and regulation of gene expression. HMGA are highly expressed during embryogenesis and in cancer cells and are involved in development and cell differentiation as well as cancer formation and progression. These factors, by binding to DNA and interacting with other nuclear proteins, can organize macromolecular complexes involved in transcription, chromatin dynamics, RNA processing, and DNA repair. The identification of protein partners for HMGA has greatly contributed to our understanding of their multiple functions. He we report the identification of HMGA molecular partners using a gene fragment library in a phage display screening. Using an ORF-enriched cDNA library, we have isolated several HMGA1 interacting clones and for two of them, TBP associated factor 3 (TAF3) and chromatin assembly factor 1 p150/CAF-1, have demonstrated an in vivo association with HMGA1. The identification of these new partners suggests that HMGA can also influence general aspects of transcription and once more underlines their involvement in chromatin remodelling and dynamics.

Suitability of a dual COI marker for marine zooplankton DNA metabarcoding.

As DNA metabarcoding has become an emerging tool for surveying biodiversity, including its application in legally binding assessments, reliable and efficient barcodes are requested, especially for the highly diverse group of zooplankton. This study focuses on comparing the efficiency of two mitochondrial COI barcodes based on the internal primers mlCOIintF and mlCOIintR utilizing mesozooplankton samples collected in a Mediterranean lagoon. Our results indicate that after a slight adjustment, the mlCOIintR primer performs in combination with jdgLCO1490 (herein) very comparably to the much more widely used primer system mlCOIintF/jgHCO2198+dgHCO2198, in terms of level of taxonomic resolution, species detection and their relative abundance in terms of numbers of reads. As for some groups, like Ctenophora, this barcode is not suitable; a combination of them may be the best option to rely on the Folmer region in its entirety without the risk of losing information for a limited primer match.

Molecular Diversity of Mytilin-Like Defense Peptides in Mytilidae (Mollusca, Bivalvia).

The CS-αß 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-αß 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-αß 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-αß 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.

Defining the Helicobacter pylori Disease-Specific Antigenic Repertoire.

The analysis of the interaction between Helicobacter pylori (HP) and the host in vivo is an extremely informative way to enlighten the molecular mechanisms behind the persistency/latency of the bacterium as well as in the progression of the infection. An important source of information is represented by circulating antibodies targeting the bacteria that define a specific "disease signature" with prospective diagnostic implications. The diagnosis of some of the HP induced diseases such as gastric cancer (GC), MALT lymphoma (MALT), and autoimmune gastritis (AIG) is not easy because patients do not show symptoms of illness in early-onset stages, at the same time they progress rapidly. The possibility of identifying markers able to provide an early diagnosis would be extremely beneficial since a late diagnosis results in a delay in undergoing active therapy and reduces the survival rate of patients. With the aim to identify the HP antigens recognized during the host immune-response to the infection and possibly disease progression, we applied a discovery-driven approach, that combines "phage display" and deep sequencing. The procedure is based on the selection of ORF phage libraries, specifically generated from the pathogen's genome, with sera antibodies from patients with different HP-related diseases. To this end two phage display libraries have been constructed starting from genomic DNA from the reference HP 26695 and the pathogenic HP B128 strains; libraries were filtered for ORFs by using an ORF selection vector developed by our group (Di Niro et al., 2005; Soluri et al., 2018), selected with antibodies from patients affected by GC, MALT, and AIG and putative HP antigens/epitopes were identified after Sequencing and ranking. The results show that individual selection significantly reduced the library diversity and comparison of individual ranks for each condition allowed us to highlight a pattern of putative antigens specific for the different pathological outcomes or common for all of them. Within the putative antigens enriched after selection, we have validated protein CagY/Cag7 by ELISA assay as a marker of HP infection and progression. Overall, we have defined HP antigenic repertoire and identified a panel of putative specific antigens/epitopes for three different HP infection pathological outcomes that could be validated in the next future.

Identification of novel non-myelin biomarkers in multiple sclerosis using an improved phage-display approach.

Although the etiology of multiple sclerosis is not yet understood, it is accepted that its pathogenesis involves both autoimmune and neurodegenerative processes, in which the role of autoreactive T-cells has been elucidated. Instead, the contribution of humoral response is still unclear, even if the presence of intrathecal antibodies and B-cells follicle-like structures in meninges of patients has been demonstrated. Several myelin and non-myelin antigens have been identified, but none has been validated as humoral biomarker. In particular autoantibodies against myelin proteins have been found also in healthy individuals, whereas non-myelin antigens have been implicated in neurodegenerative phase of the disease. To provide further putative autoantigens of multiple sclerosis, we investigated the antigen specificity of immunoglobulins present both in sera and in cerebrospinal fluid of patients using phage display technology in a new improved format. A human brain cDNA phage display library was constructed and enriched for open-read-frame fragments. This library was selected against pooled and purified immunoglobulins from cerebrospinal fluid and sera of multiple sclerosis patients. The antigen library was also screened against an antibody scFv library obtained from RNA of B cells purified from the cerebrospinal fluid of two relapsing remitting patients. From all biopanning a complex of 14 antigens were identified; in particular, one of these antigens, corresponding to DDX24 protein, was present in all selections. The ability of more frequently isolated antigens to discriminate between sera from patients with multiple sclerosis or other neurological diseases was investigated. The more promising novel candidate autoantigens were DDX24 and TCERG1. Both are implicated in RNA modification and regulation which can be altered in neurodegenerative processes. Therefore, we propose that they could be a marker of a particular disease activity state.

Functional aspects of cHH C-terminal amidation in crayfish species.

The crustacean hyperglycemic hormone is the most abundant neuropeptide present in the eyestalk of Crustacea and its main role is to control the glucose level in the hemolymph. Our study was aimed at assessing the importance of C-terminal amidation for its biological activity. Two recombinant peptides were produced, Asl-rcHH-Gly with a free carboxyl terminus and Asl-rcHH-amide with an amidated C-terminus. Homologous bioassays performed on the astacid crayfish Astacus leptodactylus showed that the amidated peptide had a stronger hyperglycemic effect compared to the non-amidated peptide. To assess the relevance of amidation also in other decapods and how much the differences in the cHH amino acid sequence can affect the functionality of the peptides, we carried out heterologous bioassays on the cambarid Procambarus clarkii and palaemonid Palaemon elegans. The Asl-rcHH-amide elicited a good response in P. clarkii and in P. elegans. The injection of Asl-rcHH-Gly evoked a weak response in both species. These results prove the importance of C-terminal amidation for the biological activity of cHH in crayfish as well as the role of the peptide primary sequence for the species-specificity hormone-receptor recognition.

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing.

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 ß-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called "domainome", will be well expressed and soluble, making them suitable for structural/functional studies. By cloning and displaying the "domainome" directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification. The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

Diversity and evolution of TIR-domain-containing proteins in bivalves and Metazoa: New insights from comparative genomics.

The Toll/interleukin-1 receptor (TIR) domain has a fundamental role in the innate defence response of plants, vertebrate and invertebrate animals. Mostly found in the cytosolic side of membrane-bound receptor proteins, it mediates the intracellular signalling upon pathogen recognition via heterotypic interactions. Although a number of TIR-domain-containing (TIR-DC) proteins have been characterized in vertebrates, their evolutionary relationships and functional role in protostomes are still largely unknown. Due to the high abundance and diversity of TIR-DC proteins in bivalve molluscs, we investigated this class of marine invertebrates as a case study. The analysis of the available genomic and transcriptomic data allowed the identification of over 400 full-length sequences and their classification in protein families based on sequence homology and domain organization. In addition to TLRs and MyD88 adaptors, bivalves possess a surprisingly large repertoire of intracellular TIR-DC proteins, which are conserved across a broad range of metazoan taxa. Overall, we report the expansion and diversification of TIR-DC proteins in several invertebrate lineages and the identification of many novel protein families possibly involved in both immune-related signalling and embryonic development.

Managing of Procambarus clarkii by X-ray sterilisation of males: Cytological damage to gonads.

Procambarus clarkii is an invasive alien species spreading worldwide. It is therefore mandatory to find new methods to manage this species since traditional techniques are not sufficient for this purpose. The present study investigates gonad damage induced by different doses of ionising irradiation: 20, 40 and 60 Gy. Testis were analysed after 10 and 30 days by means of light, scanning and transmission electron microscopy. Control unirradiated testes present an acinar structure with a well-defined germinative cells maturation from the distal proliferative zone to the proximal stalk of the lobes whilst, in irradiated testes, induced apoptosis of germinative and accessory cells and a high level of vacuolisation inside the acini were identified, progressively increasing in accordance to Gy dosage and time after exposure. We determined the dose of 40 Gy as the best compromise: it causes an extensive damage to germinative tissues without affecting crayfish vitality, differing from 60 Gy. From an applicative point of view, this dose reduces the efforts, in terms of cost and time, for the application of SMRT.

Functional analysis of crustacean Hyperglycemic Hormone by in vivo assay with wild-type and mutant recombinant proteins.

The neuro-endocrine X-organ sinus-gland complex regulates important crustacean physiological processes, such as growth, reproduction and molting. Its major products are the neuropeptides of the cHH/MIH/GIH family. Until now the structure-function relationships of these neuropeptides were established by sequence comparison. To study the functional relevance of conserved amino acid residues or peptide motifs, we generated point and deletion mutants of the Norway lobster Nephrops norvegicus cHH. The wild type mature neuropeptide cHH and its mutant forms were expressed in bacteria as fusion proteins and assayed in vivo to assess their hyperglycemic activity. The wild type cHH had a hyperglycemic activity similar to that of cHH present in an eyestalk extract, and it was blocked by an anti-recombinant cHH antibody. Bioassays of cHHs, obtained by a progressive deletion of five highly conserved motifs, showed that the only deleted cHH, which conserves a hyperglycemic activity, is the one lacking the C-terminal motif, but still retaining all the motifs reported to be important for functional specificity and three-dimensional structure. All the cHH point mutants lacked a hyperglycemic activity. These results identify amino acid residues that are required for the hyperglycemic activity of cHH.

Anti-α-enolase Antibodies in Serum from Pediatric Patients Affected by Inflammatory Diseases: Diagnostic and Pathogenetic Insights.

Human glycolytic enzyme α-enolase was associated with human diseases and with inflammation. An ELISA test was developed to measure anti-α-enolase AAE IgG and AAE IgA in the serum from patients affected by inflammatory diseases with the purpose to evaluate it as a novel diagnostic marker. 80 healthy blood donors and 194 paediatric patients affected by Juvenile idiopathic arthritis (JIA), celiac disease (CD), Crohn's Disease (CrD), hereditary periodic fever (HPF), and PFAPA syndrome were included in the study. HPF patients showed high levels of AAE antibodies, whereas JIA, CD, and CrD presented only partial results. Benign fevers such as PFAPA were almost negative for AAE Abs. These findings suggested that the genetic dysfunction of inflammasome associated with HPF could lead to the formation of AAE Abs that could be used for an early and easy diagnosis.

Role of biogenic amines and cHH in the crustacean hyperglycemic stress response.

In this study, we investigated (using bioassays and ELISA) the variation of cHH (crustacean hyperglycemic hormone) level in the eyestalks and hemolymph of Palaemon elegans (Rathke) (Decapoda, Caridea) following injection of serotonin (5-HT) and dopamine (DA) and correlated cHH profile with the variation in amount and time course of glycemia. 5-HT induced in P. elegans a rapid and massive release of cHH from the eyestalk into the hemolymph followed by hyperglycemia. On the contrary, DA did not significantly affect cHH release and hyperglycemia. In addition, we measured the level and variation of 5-HT in the eyestalk and hemolymph of P. elegans following copper contamination. The release of 5-HT from the eyestalk is very rapid and dose dependent. In the hemolymph, a peak of 5-HT occurs after 30 min, and again the circulating concentration of 5-HT is dose dependent on copper exposure. After 1 h, the level of 5-HT slowly decreases to basal level. The release of 5-HT from the eyestalk into the hemolymph after copper exposure precedes the release of cHH, confirming its role as a neurotransmitter acting on cHH neuroendocrine cells. The fact that copper induced a rapid and massive release of 5-HT from the eyestalk can explain its demonstrated role in inducing the release of cHH and the consequent hyperglycemia in intact but not eyestalkless animals.

Variation of crustacean hyperglycemic hormone (cHH) level in the eyestalk and haemolymph of the shrimp Palaemon elegans following stress.

This study investigates (by means of bioassays and ELISA using an antibody against recombinant cHH) the variation of cHH levels in the eyestalks and haemolymph of Palaemon elegans (Decapoda, Caridea) following exposure to various stresses (heavy metals and lipopolysaccharide), and correlates them with the variation in amount and time course of blood glucose. The dose-relationship between exposure to copper and quick release of cHH from the eyestalk into haemolymph was confirmed by variation of blood glucose with a dose-related hyperglycaemia, that peaked 2 h after immersion in contaminated seawater. Animals exposed to a sublethal concentration of mercury showed the same dose relation between toxicant, release of cHH from the eyestalk, increment of circulating hormone level and subsequent hyperglycaemia as observed for copper contamination. It is of note that although the highest lethal mercury concentration induced the release of cHH from the eyestalk into the haemolymph, it was not followed by a significant variation of blood glucose. Step doses of a bacterial contaminant [such as lipopolysaccharide (LPS) from E. coli injected into shrimps] confirmed the dose-relationship and convergent chain of events that bring about hyperglycaemia. These are the first data that relate the release of cHH from the eyestalk, the circulating hormone level and the consequent glycaemic response to stress. Moreover, they confirm the dose-related pathway that leads to variation of blood glucose as a quantitative biomarker of environmental quality, even at sublethal toxicant concentrations.

Alterations in the Arf6-regulated plasma membrane endosomal recycling pathway in cells overexpressing the tetraspan protein Gas3/PMP22.

Growth arrest specific 3 (Gas3)/peripheral myelin protein 22 (PMP22) is a component of the compact peripheral nerve myelin, and mutations affecting gas3/PMP22 gene are responsible for a group of peripheral neuropathies in humans. We have performed in vivo imaging in order to investigate in detail the phenotype induced by Gas3/PMP22 overexpression in cultured cells. Here we show that Gas3/PMP22 triggers the accumulation of vacuoles, before the induction of cell death or of changes in cell spreading. Overexpressed Gas3/PMP22 accumulates into two distinct types of intracellular membrane compartments. Gas3/PMP2 accumulates within late endosomes close to the juxtanuclear region, whereas in the proximity of the cell periphery, it induces the formation of actin/phosphatidylinositol (4,5)-bisphosphate (PIP(2))-positive large vacuoles. Gas3/PMP22-induced vacuoles do not contain transferrin receptor, but instead they trap membrane proteins that normally traffic through the ADP-ribosylation factor 6 (Arf6) endosomal compartment. Arf6 and Arf6-Q67L co-localize with Gas3/PMP22 in these vacuoles, and the dominant negative mutant of Arf6, T27N, blocks the appearance of vacuoles in response to Gas3/PMP22, but not its accumulation in the late endosomes. Finally a point mutant of Gas3/PMP22 responsible for the Charcot-Marie-Tooth 1A disease is unable to trigger the accumulation of PIP(2)-positive vacuoles. Altogether these results suggest that increased Gas3/PMP22 levels can alter membrane traffic of the Arf6 plasma-membrane-endosomal recycling pathway and show that, similarly to other tetraspan proteins, Gas3/PMP22 can accumulate in the late endosomes.