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

The CS-\u3b1\u3b2 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-\u3b1\u3b2 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-\u3b1\u3b2 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\u2013C5 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-\u3b1\u3b2 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 origi

Gene expression analysis of Coffea arabica seeds processed under different post-harvest processing methods

The mode of coffee processing, either the wet or dry method, determines the characteristic flavour and establishes the differences in quality of the final green coffee produced. The present study focused mainly on identifying the differential gene expression in green coffee seeds of Brazilian arabica coffee (Coffea arabica L.) among samples prepared under three different post-harvest treatments (natural, washed and semi washed method) and grown in two different locations. Expression levels of 16 genes of interest were measured. These genes are involved in various cellular, metabolic and biochemical activities influencing levels of certain compounds, such as lipids, carbohydrates, caffeine and chlorogenic acid, associated with quality characteristics of the beverage. Microarray experiments were designed with cDNA probe sequences. Microarray data was analyzed to identify the differences in gene expression between two altitudes and between two variables: location and post-harvest treatment. Cluster analysis was carried out with samples showing similar patterns, which are characteristic to the group. With this approach, it was possible to identify the important genes in C. arabica seeds that have differential (increased or decreased) expression levels. It was also seen that between the location and treatments, location profoundly impacts the levels of gene expression in samples

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

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 \u3b2-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

Gene Expression Profiling of Trematomus bernacchii in Response to Thermal and Stabling Stress

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

Exposure at the cell surface is required for gas3/PMP22 To regulate both cell death and cell spreading: implication for the Charcot-Marie-Tooth type 1A and Dejerine-Sottas diseases

4nonenoneBRANCOLINI C; EDOMI P; MARZINOTTO S; SCHNEIDER CBrancolini, Claudio; Edomi, P; Marzinotto, S; Schneider, Claudi

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

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

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