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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.

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.

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.

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.

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.

Marchigian Sardinian alcohol-preferring (msP) rats exhibit innate preference for alcohol, are highly sensitive to stress and stress-induced alcohol seeking. Genetic analysis showed that over-expression of the corticotropin-releasing factor (CRF) system of msP rats is correlated with the presence of two single nucleotide polymorphisms (SNPs) occurring in the promoter region (position -1836 and -2097) of the CRF1 receptor (CRF1-R) gene. Here we examined whether these point mutations were associated to the innate alcohol preference, stress-induced drinking and seeking.We have recently re-derived the msP rats to obtain two distinct lines carrying the wild type (GG) and the point mutations (AA), respectively. The phenotypic characteristics of these two lines were compared with those of unselected Wistar rats. Both AA and GG rats showed similar patterns of voluntary alcohol intake and preference. Similarly, the pharmacological stressor yohimbine (0.0, 0.625, 1.25 and 2.5 mg/kg) elicited increased operant alcohol self-administration under fixed and progressive ratio reinforcement schedules in all three lines. Following extinction, yohimbine (0.0, 0.625, 1.25 and 2.5 mg/kg) significantly reinstated alcohol seeking in the three groups. However, at the highest dose this effect was no longer evident in AA rats. Treatment with the CRF1-R antagonist antalarmin (0, 5, 10 and 20 mg/kg) significantly reduced alcohol-reinforced lever pressing in the AA line (10 and 20 mg/kg) while a weaker or no effect was observed in the Wistar and the GG group, respectively. Finally, antalarmin significantly reduced yohimbine-induced increase in alcohol drinking in all three groups.In conclusion, these specific SNPs in the CRF1-R gene do not seem to play a primary role in the expression of the msP excessive-drinking phenotype or stress-induced drinking but may be associated with a decreased threshold for stress-induced alcohol seeking and an increased sensitivity to the effects of pharmacolo

Animal models provide an important tool to investigate the pathogenesis of neuromuscular disorders. In the present study, we analyze fiber composition of the brachial plexus branches to the pectoral muscles: the medial anterior thoracic nerve and the lateral anterior thoracic nerve. The morphological and morphometric characteristics and the percentage of motor fibers within each nerve are here reported, adding information to microscopic anatomy knowledge of the rat brachial plexus. As control, we employed the quadriceps nerve, commonly used for the evaluation of motor fibers at hindlimbs. We demonstrated that the medial anterior thoracic nerve and the lateral anterior thoracic nerve are predominantly composed of large motor fibers and therefore could be employed to evaluate the peripheral nervous system involvement at forelimbs in neurological diseases models, predominantly affecting the motor fiber compartment.

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.

An analysis of a possible function for the murine growth-arrest-specific gene gas 1 in the origin of tumors involved the structural and functional study of the human homolog of gas1, h-gas 1. Inactivation of h-gas 1 in tumor cells is the result of its possible role as a target for genetic alterations. Proliferation in the A549 lung carcinoma cell lines can be inhibited by h-gas 1 overexpression.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2020 Soluri, Puccio, Caredda, Edomi, D’Elios, Cianchi, Troilo, Santoro, Sblattero and Peano.)

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., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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., Competing Interests: Andrea Cortini, Sara Bembich and Paolo Edomi are listed as inventors on PCT patent applications titled Biomarkers for the diagnosis of multiple sclerosis (PCT/EP20 11/072440). Other authors declare that they do not have competing interests related to this study. There are no additional patents, products in development or marketed products to declare. This does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

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., (© 2019 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

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 β-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.

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.