Multifunctional Cell Regulation Activities of the Mussel Lectin SeviL: Induction of Macrophage Polarization toward the M1 Functional Phenotype.

SeviL, a galactoside-binding lectin previously isolated from the mussel Mytilisepta virgata, was demonstrated to trigger apoptosis in HeLa ovarian cancer cells. Here, we show that this lectin can promote the polarization of macrophage cell lines toward an M1 functional phenotype at low concentrations. The administration of SeviL to monocyte and basophil cell lines reduced their growth in a dose-dependent manner. However, low lectin concentrations induced proliferation in the RAW264.7 macrophage cell line, which was supported by the significant up-regulation of TOM22, a component of the mitochondrial outer membrane. Furthermore, the morphology of lectin-treated macrophage cells markedly changed, shifting from a spherical to an elongated shape. The ability of SeviL to induce the polarization of RAW264.7 cells to M1 macrophages at low concentrations is supported by the secretion of proinflammatory cytokines and chemokines, as well as by the enhancement in the expression of IL-6- and TNF-α-encoding mRNAs, both of which encode inflammatory molecular markers. Moreover, we also observed a number of accessory molecular alterations, such as the activation of MAP kinases and the JAK/STAT pathway and the phosphorylation of platelet-derived growth factor receptor-α, which altogether support the functional reprogramming of RAW264.7 following SeviL treatment. These results indicate that this mussel ß-trefoil lectin has a concentration-dependent multifunctional role in regulating cell proliferation, phenotype, and death in macrophages, suggesting its possible involvement in regulating hemocyte activity in vivo.

Taxonomic Distribution and Molecular Evolution of Mytilectins.

R-type lectins are a widespread group of sugar-binding proteins found in nearly all domains of life, characterized by the presence of a carbohydrate-binding domain that adopts a ß-trefoil fold. Mytilectins represent a recently described subgroup of ß-trefoil lectins, which have been functionally characterized in a few mussel species (Mollusca, Bivalvia) and display attractive properties, which may fuel the development of artificial lectins with different biotechnological applications. The detection of different paralogous genes in mussels, together with the description of orthologous sequences in brachiopods, supports the formal description of mytilectins as a gene family. However, to date, an investigation of the taxonomic distribution of these lectins and their molecular diversification and evolution was still lacking. Here, we provide a comprehensive overview of the evolutionary history of mytilectins, revealing an ancient monophyletic evolutionary origin and a very broad but highly discontinuous taxonomic distribution, ranging from heteroscleromorphan sponges to ophiuroid and crinoid echinoderms. Moreover, the overwhelming majority of mytilectins display a chimera-like architecture, which combines the ß-trefoil carbohydrate recognition domain with a C-terminal pore-forming domain, suggesting that the simpler structure of most functionally characterized mytilectins derives from a secondary domain loss.

Comparative analysis of novel and common reference genes in adult tissues of the mussel Mytilus galloprovincialis.

BACKGROUND: Real-time quantitative PCR is a widely used method for gene expression analyses in various organisms. Its accuracy mainly relies on the correct selection of reference genes. Any experimental plan involving real-time PCR needs to evaluate the characteristics of the samples to be examined and the relative stability of reference genes. Most studies in mollusks rely on reference genes commonly used in vertebrates. RESULTS: In this study, we focused on the transcriptome of the bivalve mollusk Mytilus galloprovincialis in physiological state to identify suitable reference genes in several adult tissues. Candidate genes with highly stable expression across 51 RNA-seq datasets from multiple tissues were selected through genome-wide bioinformatics analysis. This approach led to the identification of three genes (Rpl14, Rpl32 and Rpl34), whose suitability was evaluated together with 7 other reference genes commonly reported in literature (Act, Cyp-A, Ef1α, Gapdh, 18S, 28S and Rps4). The stability analyses performed with geNorm, NormFinder and Bestkeeper identified specific either single or pairs of genes suitable as references for gene expression analyses in specific tissues and revealed the Act/Cyp-A pair as the most appropriate to analyze gene expression across different tissues. CONCLUSION: Mytilus galloprovincialis is a model system increasingly used in ecotoxicology and molecular studies. Our transcriptome-wide approach represents the first comprehensive investigation aimed at the identification of suitable reference genes for expression studies in this species.

Spectroscopic investigation of faeces with surface-enhanced Raman scattering: a case study with coeliac patients on gluten-free diet.

Surface-enhanced Raman scattering (SERS) spectra of faecal samples can be obtained by adding AuNP to their methanol extracts according to the reported protocol, and display bands that are due to bilirubin-like species but also to xanthine and hypoxanthine, two metabolic products secreted by gut bacteria. A total of 27 faecal samples from three different groups, i.e. coeliac patients (n = 9), coeliac patients on gluten-free diet (n = 10) and a control group (n = 8), were characterized with both SERS spectroscopy and 16S rRNA sequencing analysis. Significant differences are present between SERS spectra of coeliac patients and those on gluten-free diet, with a marked increase in the relative intensity of both xanthine and hypoxanthine for the latter. Interestingly, these differences do not correlate with bacterial composition as derived from 16S rRNA sequencing.

The role of outdoor and indoor air quality in the spread of SARS-CoV-2: Overview and recommendations by the research group on COVID-19 and particulate matter (RESCOP commission).

There are important questions surrounding the potential contribution of outdoor and indoor air quality in the transmission of SARS-CoV-2 and perpetuation of COVID-19 epidemic waves. Environmental health may be a critical component of COVID-19 prevention. The public health community and health agencies should consider the evolving evidence in their recommendations and statements, and work to issue occupational guidelines. Evidence coming from the current epidemiological and experimental research is expected to add knowledge about virus diffusion, COVID-19 severity in most polluted areas, inter-personal distance requirements and need for wearing face masks in indoor or outdoor environments. The COVID-19 pandemic has highlighted the need for maintaining particulate matter concentrations at low levels for multiple health-related reasons, which may also include the spread of SARS-CoV-2. Indoor environments represent even a more crucial challenge to cope with, as it is easier for the SARS-COV2 to spread, remain vital and infect other subjects in closed spaces in the presence of already infected asymptomatic or mildly symptomatic people. The potential merits of preventive measures, such as CO2 monitoring associated with natural or controlled mechanical ventilation and air purification, for schools, indoor public places (restaurants, offices, hotels, museums, theatres/cinemas etc.) and transportations need to be carefully considered. Hospital settings and nursing/retirement homes as well as emergency rooms, infectious diseases divisions and ambulances represent higher risk indoor environments and may require additional monitoring and specific decontamination strategies based on mechanical ventilation or air purification.

Environmental DNA reveals diversity and abundance of Alternaria species in neighbouring heterogeneous landscapes in Worcester, UK.

Alternaria is a pathogenic and allergenic fungus affecting 400 plant species and 334 million people globally. This study aimed at assessing the diversity of Alternaria species in airborne samples collected from closely located (7 km apart) and heterogeneous sites (rural, urban and unmanaged grassland) in Worcester and Lakeside, the UK. A secondary objective was to examine how the ITS1 subregion varies from ITS2 in Alternaria species diversity and composition. Airborne spores were collected using Burkard 7-day and multi-vial Cyclone samplers for the period 5 July 2016-9 October 2019. Air samples from the Cyclone were amplified using the ITS1and ITS2 subregions and sequenced using Illumina MiSeq platform whereas those from the Burkard sampler were identified and quantified using optical microscopy. Optical microscopy and eDNA revealed a high abundance of Alternaria in the rural, urban and unmanaged sites. ITS1 and ITS2 detected five and seven different Alternaria species at the three sampling sites, respectively. A. dactylidicola, A. metachromatica and A. infectoria were the most abundant. The rural, urban and unmanaged grassland sites had similar diversity (PERMANOVA) of the species due to similarity in land use and proximity of the sites. Overall, the study showed that heterogeneous and neighbouring sites with similar land uses can have similar Alternaria species. It also demonstrated that an eDNA approach can complement the classical optical microscopy method in providing more precise information on fungal species diversity in an environment for targeted management. Similar studies can be replicated for other allergenic and pathogenic fungi. Supplementary Information: The online version contains supplementary material available at 10.1007/s10453-022-09760-9.

Molecular detection of SARS-CoV-2 from indoor air samples in environmental monitoring needs adequate temporal coverage and infectivity assessment.

The relevance of airborne exposure to SARS-CoV-2 in indoor environments is a matter of research and debate, with special importance for healthcare low-risk settings. Experimental approaches to the bioaerosol sampling are neither standardized nor optimized yet, leading in some cases to limited representativity of the temporal and spatial variability of viral presence in aerosols. Airborne viral viability moreover needs to be assessed. A study has been conducted collecting five 24-h PM10 samples in a COVID-19 geriatric ward in late June 2020, and detecting E and RdRp genes by RT-qPCR with a Ct between 36 and 39. The viral RNA detection at Ct = 36 was related to the maximal numerosity of infected patients hosted in the ward. Lacking a direct infectivity assessment for the collected samples an experimental model has been defined, by seeding twelve nasopharyngeal swab extracts from COVID-19 positive patients on Vero E6 cells; only the four extracts with a viral load above E+10 viral copies (approximately Ct<24) have been able to establish a persistent infection in vitro. Therefore, the cytopathic effect, a key feature of residual infectivity, could be considered unlikely for the environmental PM10 samples showing amplification of viral RNA at Ct = 36 or higher. A standardization of airborne SARS-CoV-2 long-term monitoring and of environmental infectivity assessment is urgently needed.

Comparative Genomics Reveals 13 Different Isoforms of Mytimycins (A-M) in Mytilus galloprovincialis.

Mytimycins are cysteine-rich antimicrobial peptides that show antifungal properties. These peptides are part of the immune network that constitutes the defense system of the Mediterranean mussel (Mytilus galloprovincialis). The immune system of mussels has been increasingly studied in the last decade due to its great efficiency, since these molluscs, particularly resistant to adverse conditions and pathogens, are present all over the world, being considered as an invasive species. The recent sequencing of the mussel genome has greatly simplified the genetic study of some of its immune genes. In the present work, we describe a total of 106 different mytimycin variants in 16 individual mussel genomes. The 13 highly supported mytimycin clusters (A-M) identified with phylogenetic inference were found to be subject to the presence/absence variation, a widespread phenomenon in mussels. We also identified a block of conserved residues evolving under purifying selection, which may indicate the "functional core" of the mature peptide, and a conserved set of 10 invariable plus 6 accessory cysteines which constitute a plastic disulfide array. Finally, we extended the taxonomic range of distribution of mytimycins among Mytilida, identifying novel sequences in M. coruscus, M. californianus, P. viridis, L. fortunei, M. philippinarum, M. modiolus, and P. purpuratus.

Cold Adaptation in Antarctic Notothenioids: Comparative Transcriptomics Reveals Novel Insights in the Peculiar Role of Gills and Highlights Signatures of Cobalamin Deficiency.

Far from being devoid of life, Antarctic waters are home to Cryonotothenioidea, which represent one of the fascinating cases of evolutionary adaptation to extreme environmental conditions in vertebrates. Thanks to a series of unique morphological and physiological peculiarities, which include the paradigmatic case of loss of hemoglobin in the family Channichthyidae, these fish survive and thrive at sub-zero temperatures. While some of the distinctive features of such adaptations have been known for decades, our knowledge of their genetic and molecular bases is still limited. We generated a reference de novo assembly of the icefish Chionodraco hamatus transcriptome and used this resource for a large-scale comparative analysis among five red-blooded Cryonotothenioidea, the sub-Antarctic notothenioid Eleginops maclovinus and seven temperate teleost species. Our investigation targeted the gills, a tissue of primary importance for gaseous exchange, osmoregulation, ammonia excretion, and its role in fish immunity. One hundred and twenty genes were identified as significantly up-regulated in Antarctic species and surprisingly shared by red- and white-blooded notothenioids, unveiling several previously unreported molecular players that might have contributed to the evolutionary success of Cryonotothenioidea in Antarctica. In particular, we detected cobalamin deficiency signatures and discussed the possible biological implications of this condition concerning hematological alterations and the heavy parasitic loads typically observed in all Cryonotothenioidea.

Identification, molecular characterization and functional analysis of interleukin (IL)-2 and IL-2like (IL-2L) cytokines in sea bass (Dicentrarchus labrax L.).

In mammals, interleukin (IL)-2, initially known as a T-cell grow factor, is an immunomodulatory cytokine involved in the proliferation of T cells upon antigen activation. In bony fish, some IL-2 orthologs have been identified, but, recently, an additional IL-2like (IL-2L) gene has been found. In this paper, we report the presence of these two divergent IL-2 isoforms in sea bass (Dicentrarchus labrax L.). Genomic analyses revealed that they originated from a gene duplication event, as happened in most percomorphs. These two IL-2 paralogs show differences in the amino acid sequence and in the exon 4 size, and these features could be an indication that they bind preferentially to different specific IL-2 receptors. Sea bass IL-2 paralogs are highly expressed in gut and spleen, which are tissues and organs involved in fish T cell immune functions, and the two cytokines could be up-regulated by both PHA stimulation and vaccination with a bacterial vaccine, with IL-2L being more inducible. To investigate the functional activities of sea bass IL-2 and IL-2L we produced the corresponding recombinant molecules in E. coli and used them to in vitro stimulate HK and spleen leukocytes. IL-2L is able to up-regulate the expression of markers related to different T cell subsets (Th1, Th2 and Th17) and to Treg cells in HK, whereas it has little effect in spleen. IL-2 is not active on these markers in HK, but shows an effect on Th1 markers in spleen. Finally, the stimulation with recombinant IL-2 and IL-2L is also able to induce in vitro proliferation of HK- and spleen-derived leukocytes. In conclusion, we have demonstrated that sea bass possess two IL-2 paralogs that likely have an important role in regulating T cell development in this species and that show distinct bioactivities.

Identification of an IgD/IgT chimera in the European sea bass (Dicentrarchus labrax L.).

Three classes of immunoglobulins have been identified in Teleosts: IgM, IgT/Z and IgD. They are fundamental for fish immune responses and, therefore, their functional activities are heavily investigated. In this paper, we describe the identification of a new IgD/IgT chimera in sea bass (Dicentrarchus labrax) from a gills transcriptome. This transcript joined the first six constant domains of the IgD chain with the two terminal constant domains of IgT, generating a long in-frame coding sequence with a junction between the canonical δ6 exon splicing donor site and the τ3 exon splicing acceptor site. Studies performed on genomic DNA confirmed the presence of the sequence and identifies and intronic region of 656 bp within this joining region. The basal expression of the IgD/IgT chimera was investigated both in silico and in vivo: high level of expression was found in gills, gut and head kidney. Moreover, IgD/IgT transcripts were up-regulated after in vitro stimulation of sea bass HK leukocytes with LPS. The IgD/IgT chimera was found also in two congener species, Morone saxatilis and Morone chrysops. It is not possible to have a precise idea on the evolutionary scenario that lead to the appearance of this sequence due to the lack of genomic information, but we could speculate that an ancestral duplication of the entire IgH locus was followed by the chimerization of Cδ/Cτ in one of the two loci. Finally, the IgD/IgT high basal expression in tissues and organs fundamental for sea bass immune response and its modulation after LPS stimulation provide a very preliminary indication that this unusual Ig variant could have a functional activity.

SARS-Cov-2RNA found on particulate matter of Bergamo in Northern Italy: First evidence.

BACKGROUND: The burden of COVID-19 was extremely severe in Northern Italy, an area characterized by high concentrations of particulate matter (PM), which is known to negatively affect human health. Consistently with evidence already available for other viruses, we initially hypothesized the possibility of SARS-CoV-2 presence on PM, and we performed a first experiment specifically aimed at confirming or excluding this research hyphotesys. METHODS: We have collected 34 PM10 samples in Bergamo area (the epicenter of the Italian COVID-19 epidemic) by using two air samplers over a continuous 3-weeks period. Filters were properly stored and underwent RNA extraction and amplification according to WHO protocols in two parallel blind analyses performed by two different authorized laboratories. Up to three highly specific molecular marker genes (E, N, and RdRP) were used to test the presence of SARS-CoV-2 RNA on particulate matter. RESULTS: The first test showed positive results for gene E in 15 out of 16 samples, simultaneously displaying positivity also for RdRP gene in 4 samples. The second blind test got 5 additional positive results for at least one of the three marker genes. Overall, we tested 34 RNA extractions for the E, N and RdRP genes, reporting 20 positive results for at least one of the three marker genes, with positivity separately confirmed for all the three markers. Control tests to exclude false positivities were successfully accomplished. CONCLUSION: This is the first evidence that SARS-CoV-2 RNA can be present on PM, thus suggesting a possible use as indicator of epidemic recurrence.

Differential Effects of Dietary Supplementation of Krill Meal, Soybean Meal, Butyrate, and Bactocell® on the Gene Expression of Atlantic Salmon Head Kidney.

The head kidney is a key organ that plays a fundamental role in the regulation of the fish immune response and in the maintenance of endocrine homeostasis. Previous studies indicate that the supplementation of exogenous dietary components, such as krill meal (KM), soybean meal (SM), Bactocell® (BA), and butyrate (BU), can have a significant effect on the immune function of the head kidney. The aim of this study was to investigate the differential effect of these four dietary ingredients on the transcriptional profiles of the head kidney of the Atlantic salmon. This study revealed that just a small number of genes were responsive to the feeding regime after a long-term (12 weeks) treatment, and evidenced that the most significant alterations, both in terms of the number of affected genes and magnitude of changes in gene expression, were detectable in the BU- and KM-fed groups compared with controls, while the SM diet had a nearly negligible effect, and BA had no significant effects at all. Most of the differentially expressed genes were involved in the immune response and, in line with data previously obtained from pyloric caeca, major components of the complement system were significantly affected. These alterations were accompanied by an increase in the density of melanomacrophage centers in the KM- and SM-fed group and their reduction in the BU-fed group. While three types of dietary supplements (BU, KM, and SM) were able to produce a significant modulation of some molecular players of the immune system, the butyrate-rich diet was revealed as the one with the most relevant immune-stimulating properties in the head kidney. These preliminary results suggest that further investigations should be aimed towards the elucidation of the potential beneficial effects of butyrate and krill meal supplementation on farmed salmon health and growth performance.

De novo gonad transcriptome analysis of the common littoral shrimp Palaemon serratus: novel insights into sex-related genes.

BACKGROUND: The common littoral shrimp Palaemon serratus is an economically important decapod resource in some European communities. Aquaculture practices prevent the genetic deterioration of wild stocks caused by overfishing and at the same time enhance the production. The biotechnological manipulation of sex-related genes has the proved potential to improve the aquaculture production but the scarcity of genomic data about P. serratus hinders these applications. RNA-Seq analysis has been performed on ovary and testis samples to generate a reference gonadal transcriptome. Differential expression analyses were conducted between three ovary and three testis samples sequenced by Illumina HiSeq 4000 PE100 to reveal sex-related genes with sex-biased or sex-specific expression patterns. RESULTS: A total of 224.5 and 281.1 million paired-end reads were produced from ovary and testis samples, respectively. De novo assembly of ovary and testis trimmed reads yielded a transcriptome with 39,186 transcripts. The 29.57% of the transcriptome retrieved at least one annotation and 11,087 differentially expressed genes (DEGs) were detected between ovary and testis replicates. Six thousand two hundred seven genes were up-regulated in ovaries meanwhile 4880 genes were up-regulated in testes. Candidate genes to be involved in sexual development and gonadal development processes were retrieved from the transcriptome. These sex-related genes were discussed taking into account whether they were up-regulated in ovary, up-regulated in testis or not differentially expressed between gonads and in the framework of previous findings in other crustacean species. CONCLUSIONS: This is the first transcriptome analysis of P. serratus gonads using RNA-Seq technology. Interesting findings about sex-related genes from an evolutionary perspective (such as Dmrt1) and for putative future aquaculture applications (Iag or vitellogenesis genes) are reported here. We provide a valuable dataset that will facilitate further research into the reproductive biology of this shrimp.

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.

The complex evolutionary history of sulfoxide synthase in ovothiol biosynthesis.

Sulfoxide synthases are enzymes involved in the biosynthesis of small sulfur-containing natural products. Their enzymatic activity represents a unique sulfur transfer strategy in nature that is the insertion of a sulfur atom on the imidazole ring of histidine. To date, only two enzymes are known to carry out this function: the sulfoxide synthase EgtB, involved in the biosynthesis of ergothioneine in fungi and bacteria, and the 5-histidylcysteine sulfoxide synthase OvoA, involved in the biosynthesis of ovothiols, found in the eggs and biological fluids of marine invertebrates, some proteobacteria and protists. In particular, ovothiols, thanks to their unique redox properties, are probably the most intriguing marine sulfur-containing molecules. Although they have long been considered as cellular protective molecules, new evidence suggest that their biological activities and ecological role might be more complex than originally thought. Here, we investigate the evolutionary history of OvoA in Metazoa, reporting its monophyletic ancient origins, which could be traced back to the latest common ancestor of Choanozoa. Nevertheless, we show that OvoA is missing in several major extant taxa and we discuss this patchy distribution in the light of the massive genome reduction events documented in Metazoa. We also highlight two interesting cases of secondary acquisition through horizontal gene transfer, which occurred in hydrozoans and bdelloid rotifers. The evolutionary success of this metabolic pathway is probably ascribable to its role in the maintenance of cellular redox homeostasis, which enables organisms to survive in different environmental niches.

Expansion and loss events characterized the occurrence of MIF-like genes in bivalves.

Macrophage migration inhibitory factor (MIF) dynamically connects innate and adaptive immune systems in vertebrate animals, allowing highly orchestrated systemic responses to various insults. The occurrence of MIF-like genes in non-vertebrate organisms suggests its origin from an ancestral metazoan gene, whose function is still a matter of debate. In the present work, by analyzing available genomic and transcriptomic data from bivalve mollusks, we identified 137 MIF-like sequences, which were classified into three types, based on phylogeny and conservation of key residues: MIF, D-DT, and the lineage-specific type MDL. Comparative genomics revealed syntenic conservation of homologous genes at the family level, the loss of D-DT in the Ostreidae family as well as the expansion of MIF-like genes in the Mytilidae family, possibly underpinning the neofunctionalization of duplicated gene copies. In M. galloprovincialis, MIF and one D-DT were mostly expressed in haemocytes and mantle rim of untreated animals, while D-DT paralogs often showed very limited expression, suggesting an accessory role or their persistence as relict genes.

Extensive Tandem Duplication Events Drive the Expansion of the C1q-Domain-Containing Gene Family in Bivalves.

C1q-domain-containing (C1qDC) proteins are rapidly emerging as key players in the innate immune response of bivalve mollusks. Growing experimental evidence suggests that these highly abundant secretory proteins are involved in the recognition of microbe-associated molecular patterns, serving as lectin-like molecules in the bivalve proto-complement system. While a large amount of functional data concerning the binding specificity of the globular head C1q domain and on the regulation of these molecules in response to infection are quickly accumulating, the genetic mechanisms that have led to the extraordinary lineage-specific expansion of the C1qDC gene family in bivalves are still largely unknown. The analysis of the chromosome-scale genome assembly of the Eastern oyster Crassostrea virginica revealed that the 476 oyster C1qDC genes, far from being uniformly distributed along the genome, are located in large clusters of tandemly duplicated paralogs, mostly found on chromosomes 7 and 8. Our observations point out that the evolutionary process behind the development of a large arsenal of C1qDC lectin-like molecules in marine bivalves is still ongoing and likely based on an unequal crossing over.

Lysozyme-Induced Transcriptional Regulation of TNF-α Pathway Genes in Cells of the Monocyte Lineage.

Lysozyme is one of the most important anti-bacterial effectors in the innate immune system of animals. Besides its direct antibacterial enzymatic activity, lysozyme displays other biological properties, pointing toward a significant anti-inflammatory effect, many aspects of which are still elusive. Here we investigate the perturbation of gene expression profiles induced by lysozyme in a monocyte cell line in vitro considering a perspective as broad as the whole transcriptome profiling. The results of the RNA-seq experiment show that lysozyme induces transcriptional modulation of the TNF-α/IL-1ß pathway genes in U937 monocytes. The analysis of transcriptomic profiles with IPA® identified a simple but robust molecular network of genes, in which the regulation trends are fully consistent with the anti-inflammatory activity of lysozyme. This study provides the first evidence in support of the anti-inflammatory action of lysozyme on the basis of transcriptomic regulation data resulting from the broad perspective of a whole-transcriptome profiling. Such important effects can be achieved with the supplementation of relatively low concentrations of lysozyme, for a short time of exposure. These new insights allow the potential of lysozyme in pharmacological applications to be better exploited.

Parallel identification of novel antimicrobial peptide sequences from multiple anuran species by targeted DNA sequencing.

BACKGROUND: Antimicrobial peptides (AMPs) are multifunctional effector molecules that often combine direct antimicrobial activities with signaling or immunomodulatory functions. The skin secretions of anurans contain a variety of such bioactive peptides. The identification of AMPs from frog species often requires sacrificing several specimens to obtain small quantities of crude peptides, followed by activity based fractionation to identify the active principles. RESULTS: We report an efficient alternative approach to selectively amplify AMP-coding transcripts from very small amounts of tissue samples, based on RNA extraction and cDNA synthesis, followed by PCR amplification and high-throughput sequencing of size-selected amplicons. This protocol exploits the highly conserved signal peptide region of the AMP precursors from Ranidae, Hylidae and Bombinatoridae for the design of family-specific, forward degenerate primers, coupled with a reverse primer targeting the mRNA poly-A tail. CONCLUSIONS: Analysis of the assembled sequencing output allowed to identify more than a hundred full-length mature peptides, mostly from Ranidae species, including several novel potential AMPs for functional characterization. This (i) confirms the effectiveness of the experimental approach and indicates points for protocol optimization to account for particular cases, and (ii) encourages the application of the same methodology to other multigenic AMP families, also from other genera, sharing common features as in anuran AMPs.