Targeting Virulence Genes Expression in Vibrio vulnificus by Alternative Carbon Sources.

Vibrio vulnificus is an opportunistic human pathogen causing self-limiting gastroenteritis, life-threatening necrotizing soft tissue infection, and fulminating septicaemia. An increasing rate of infections has been reported worldwide, characterized by sudden onset of sepsis and/or rapid progression to irreversible tissue damage or death. Timely intervention is essential to control the infection, and it is based on antibiotic therapy, which does not always result in the effective and rapid blocking of virulence. Inhibitors of essential virulence regulators have been reported in the last years, but none of them has been further developed, so far. We aimed to investigate whether exposure to some carbon compounds, mostly easily metabolizable, could result in transcriptional down-regulation of virulence genes. We screened various carbon sources already available for human use (thus potentially easy to be repurposed), finding some of them (including mannitol and glycerol) highly effective in down-regulating, in vitro and ex-vivo, the mRNA levels of several relevant -even essential- virulence factors (hlyU, lrp, rtxA, vvpE, vvhA, plpA, among others). This paves the way for further investigations aiming at their development as virulence inhibitors and to unveil mechanisms explaining such observed effects. Moreover, data suggesting the existence of additional regulatory networks of some virulence genes are reported.

The gut microbiota of larvae of Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae).

BACKGROUND: The red palm weevil (RPW) Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae) is one of the major pests of palms. The larvae bore into the palm trunk and feed on the palm tender tissues and sap, leading the host tree to death. The gut microbiota of insects plays a remarkable role in the host life and understanding the relationship dynamics between insects and their microbiota may improve the biological control of insect pests. The purpose of this study was to analyse the diversity of the gut microbiota of field-caught RPW larvae sampled in Sicily (Italy). RESULTS: The 16S rRNA gene-based Temporal Thermal Gradient Gel Electrophoresis (TTGE) of the gut microbiota of RPW field-trapped larvae revealed low bacterial diversity and stability of the community over seasons and among pools of larvae from different host trees. Pyrosequencing of the 16S rRNA gene V3 region confirmed low complexity and assigned 98% of the 75,564 reads to only three phyla: Proteobacteria (64.7%) Bacteroidetes (23.6%) and Firmicutes (9.6%) and three main families [Enterobacteriaceae (61.5%), Porphyromonadaceae (22.1%) and Streptococcaceae (8.9%)]. More than half of the reads could be classified at the genus level and eight bacterial genera were detected in the larval RPW gut at an abundance ≥1%: Dysgonomonas (21.8%), Lactococcus (8.9%), Salmonella (6.8%), Enterobacter (3.8%), Budvicia (2.8%), Entomoplasma (1.4%), Bacteroides (1.3%) and Comamonas (1%). High abundance of Enterobacteriaceae was also detected by culturing under aerobic conditions. Unexpectedly, acetic acid bacteria (AAB), that are known to establish symbiotic associations with insects relying on sugar-based diets, were not detected. CONCLUSIONS: The RPW gut microbiota is composed mainly of facultative and obligate anaerobic bacteria with a fermentative metabolism. These bacteria are supposedly responsible for palm tissue fermentation in the tunnels where RPW larvae thrive and might have a key role in the insect nutrition, and other functions that need to be investigated.

High-Performance PCR for Alleles Discrimination of Chromo-Helicase-DNA Binding Protein (CHD1) Gene in Bird Sexing.

Genetic analyses aiming at assessing the presence of specific sequences or alleles are often carried out by PCR. Sexing of most birds is nowadays based on PCR with "universal" primers and relies on the assessment of the presence of the sex-linked CHD1-Z and -W alleles. The entire workflow is relatively time-consuming, especially for batch analyses, whereas methods that allow carrying out the entire procedure in a short time are highly desirable. The only method for outdoor analyses reported so far relies on LAMP; however; it fails to work properly in Procellariiformes. Besides improving the LAMP test; we have developed a PCR-based DNA amplification procedure (named high-performance PCR); whose unique features allow it to outperform standard PCR; making possible the direct, in-tube visual reading of results. We tested it with specifically designed Procellariiformes-targeted primer sets for rapid sexing of the birds using fluorimetric detection. The protocol, combined with rapid DNA extraction, allows for fast reading of results without electrophoresis within less than 1 h from sampling. The technique could be extended to other species, as well as to many other applications.

A taxonomic revision of the Palaearctic genus Roeseliana (Orthoptera: Tettigoniidae: Tettigoniinae: Platycleidini): a case of ongoing Mediterranean speciation.

The genus Roeseliana presently includes 10 specific or subspecific taxa, but following different authors some of them are considered synonyms. However, the authors who have treated these taxa often did not agree with the synonymies, in particular, concerning some taxa, such as R. fedtschenkoi (Saussure, 1874) and R. roeselii (Hagenbach, 1822). The present authors examined hundreds of specimens of different taxa, for the first time were able to obtain the translation from the Russian of the description of R. fedtschenkoi, compared the main morphological characters used to discriminate different taxa, biometrics, bioacoustics and genetics of some taxa. This allowed them to conclude that it is possible to recognize the following taxa: 1) Roeseliana roeselii (Hagenbach, 1822) widespread in the Palaearctic Region and imported in North America; 2) Roeseliana fedtschenkoi (Saussure, 1874) in Uzbekistan and Turkmenistan; 3) Roeseliana pylnovi (Uvarov, 1924) in the Caucasian region; 4) Roeseliana bispina (Bolívar, 1899) in Turkey; 5) Roeseliana azami (Finot, 1892) from the Mediterranean France through Italian peninsula (formerly R. azami minor Nadig, 1961); 6) R. ambitiosa (Uvarov, 1924) on the Balkan peninsula; 7) Roeseliana n. sp. Lemonnier-Darcemont & Darcemont, (in press) on Epirus (Greece and Albania); 8) Roeseliana brunneri Ramme 1951 in north east Italy (Veneto, Friuli and Po Valley); 9) Roeseliana oporina (Bolívar, 1887) in Spain.

Temporal dynamic of biofilms enhances the settlement of the central-Mediterranean reef-builder Dendropoma cristatum (Biondi, 1859).

Research on marine invertebrate settlement provides baseline knowledge for restoration technique implementation, especially for biogenic engineers with limited dispersion ability. Previously, we determined that the maturity of a biofilm strongly enhances the settlement of the vermetid reef-builder Dendropoma cristatum. To elucidate settlement-related biofilm features, here we analyse the structure and composition of marine biofilms over time, through microscopic observations, eukaryotic and prokaryotic fingerprinting analyses and 16S rDNA Illumina sequencing. The vermetid settlement temporal increase matched with the higher biofilm coverage on the substratum and the reduction of the eukaryotic abundance and diversity. The prokaryotic assemblage become, over time, more similar to that found on the reef-associated biofilm. Vermetids may detect these differences and selectively settle on those biofilms which show an advantageous structure and composition. These outcomes may support the production of ideal substrates for vermetid colonization and their further translocation to repopulate degraded reefs.

Gene Expression Changes after Parental Exposure to Metals in the Sea Urchin Affect Timing of Genetic Programme of Embryo Development.

It is widely accepted that phenotypic traits can be modulated at the epigenetic level so that some conditions can affect the progeny of exposed individuals. To assess if the exposure of adult animals could result in effects on the offspring, the Mediterranean sea urchin and its well-characterized gene regulatory networks (GRNs) was chosen as a model. Adult animals were exposed to known concentrations of zinc and cadmium (both individually and in combination) for 10 days, and the resulting embryos were followed during the development. The oxidative stress occurring in parental gonads, embryo phenotypes and mortality, and the expression level of a set of selected genes, including members of the skeletogenic and endodermal GRNs, were evaluated. Increased oxidative stress at F0, high rates of developmental aberration with impaired gastrulation, in association to deregulation of genes involved in skeletogenesis (dri, hex, sm50, p16, p19, msp130), endodermal specification (foxa, hox11/13b, wnt8) and epigenetic regulation (kat2A, hdac1, ehmt2, phf8 and UBE2a) occurred either at 24 or 48 hpf. Results strongly indicate that exposure to environmental pollutants can affect not only directly challenged animals but also their progeny (at least F1), influencing optimal timing of genetic programme of embryo development, resulting in an overall impairment of developmental success.

Signals from the deep-sea: Genetic structure, morphometric analysis, and ecological implications of Cyclothone braueri (Pisces, Gonostomatidae) early life stages in the Central Mediterranean Sea.

Cyclothone braueri (Stomiiformes, Gonostomatidae) is a widely distributed fish inhabiting the mesopelagic zone of marine tropical and temperate waters. Constituting one of the largest biomasses of the ocean, C. braueri is a key element in most of the ecological processes occurring in the twilight layer. We focused on the ecological processes linked to early life stages in relation to marine pelagic environmental drivers (temperature, salinity, food availability and geostrophic currents) considering different regions of the Central Mediterranean Sea. A multivariate morphometric analysis was carried out using six parameters with the aim of discerning different larval morphotypes, while a fragment of 367 bp representing the 12S ribosomal RNA gene was used to perform molecular analyses aimed at determining the intraspecific genetic variability. Analysis highlighted two geographically distinct morphotypes not genetically discernible and related to the different nutritional conditions due to spatial heterogeneities in terms of temperature and food availability. The body depth (BD) emerged as an appropriate morphometric parameter to detect the larval condition in this species. Molecular analysis highlighted a moderate genetic divergence in the fish population, showing the recurrence of two phylogroups not geographically separated.

Optimized RNA extraction and northern hybridization in streptomycetes.

Northern blot hybridization is a useful tool for analyzing transcript patterns. To get a picture of what really occurs in vivo, it is necessary to use a protocol allowing full protection of the RNA integrity and recovery and unbiased transfer of the entire transcripts population. Many protocols suffer from severe limitations including only partial protection of the RNA integrity and/or loss of small sized molecules. Moreover, some of them do not allow an efficient and even transfer in the entire sizes range. These difficulties become more prominent in streptomycetes, where an initial quick lysis step is difficult to obtain. We present here an optimized northern hybridization protocol to purify, fractionate, blot, and hybridize Streptomyces RNA. It is based on grinding by a high-performance laboratory ball mill, followed by prompt lysis with acid phenol-guanidinium, alkaline transfer, and hybridization to riboprobes. Use of this protocol resulted in sharp and intense hybridization signals relative to long mRNAs previously difficult to detect.

Regeneration of total RNA purification silica-based columns.

Silica-based columns are largely used in RNA purification, allowing fast extractions and good yields of high quality nucleic acid, but their major limitation is the high cost. The reuse of such columns, although desirable, is not recommended because of residual amounts of material from the previous sample trapped in the column matrix, which might be released during further purification. Thus, recycling does need previous complete removal of any detectable RNA trace, but to date no protocol which allows decontamination and reuse is available.We report a very rapid decontamination procedure, based on treatment with warm alkaline solution containing Triton X-100, which ensures no RNA carry-over, and allows the recycling of columns without impairment of their efficiency in high-quality RNA purification even after several regeneration rounds.

Neural Crest-Derived Chondrocytes Isolation for Tissue Engineering in Regenerative Medicine.

Chondrocyte transplantation has been successfully tested and proposed as a clinical procedure aiming to repair articular cartilage defects. However, the isolation of chondrocytes and the optimization of the enzymatic digestion process, as well as their successful in vitro expansion, remain the main challenges in cartilage tissue engineering. In order to address these issues, we investigated the performance of recombinant collagenases in tissue dissociation assays with the aim of isolating chondrocytes from bovine nasal cartilage in order to establish the optimal enzyme blend to ensure the best outcomes of the overall procedure. We show, for the first time, that collagenase H activity alone is required for effective cartilage digestion, resulting in an improvement in the yield of viable cells. The extracted chondrocytes proved able to grow and activate differentiation/dedifferentiation programs, as assessed by morphological and gene expression analyses.

Complete decontamination and regeneration of DNA purification silica columns.

Silica columns are among the most used DNA purification systems, allowing a good yield of high-quality nucleic acids without organic extractions. Silica column regeneration protocols reported up to now to remove DNA traces are time-consuming, and their effectiveness on genomic DNA has not been demonstrated. Here we report a very rapid regeneration procedure that ensures no DNA carryover, independent of its size, without impairing column efficiency. The method takes advantage of the improved DNA removal by low concentrations of Triton X-100.

Advanced Strategies for Food-Grade Protein Production: A New E. coli/Lactic Acid Bacteria Shuttle Vector for Improved Cloning and Food-Grade Expression.

Food-grade production of recombinant proteins in Gram-positive bacteria, especially in LAB (i.e., Lactococcus, Lactobacillus, and Streptococcus), is of great interest in the areas of recombinant enzyme production, industrial food fermentation, gene and metabolic engineering, as well as antigen delivery for oral vaccination. Food-grade expression relies on hosts generally considered as safe organisms and on clone selection not dependent on antibiotic markers, which limit the overall DNA manipulation workflow, as it can be carried out only in the expression host and not in E. coli. Moreover, many commercial expression vectors lack useful elements for protein purification. We constructed a "shuttle" vector containing a removable selective marker, which allows feasible cloning steps in E. coli and subsequent protein expression in LAB. In fact, the cassette can be easily excised from the selected recombinant plasmid, and the resulting marker-free vector transformed into the final LAB host. Further useful elements, as improved MCS, 6xHis-Tag, and thrombin cleavage site sequences were introduced. The resulting vector allows easy cloning in E. coli, can be quickly converted in a food-grade expression vector and harbors additional elements for improved recombinant protein purification. Overall, such features make the new vector an improved tool for food-grade expression.

On the identity of Tettigonia krugeri Massa from Libya with some remarks on variability of Tettigonia species (Orthoptera: Tettigoniidae; Tettigoniinae).

Morphological and genetic data allowed the authors to resurrect the name Tettigonia krugeri Massa, 1998 as a valid species. It is currently known only from two specimens (one male and one female) collected in Cirenaica (Libya).

Vibrio Proteases for Biomedical Applications: Modulating the Proteolytic Secretome of V. alginolyticus and V. parahaemolyticus for Improved Enzymes Production.

Proteolytic enzymes are of great interest for biotechnological purposes, and their large-scale production, as well as the discovery of strains producing new molecules, is a relevant issue. Collagenases are employed for biomedical and pharmaceutical purposes. The high specificity of collagenase-based preparations toward the substrate strongly relies on the enzyme purity. However, the overall activity may depend on the cooperation with other proteases, the presence of which may be essential for the overall enzymatic activity, but potentially harmful for cells and tissues. Vibrios produce some of the most promising bacterial proteases (including collagenases), and their exo-proteome includes several enzymes with different substrate specificities, the production and relative abundances of which strongly depend on growth conditions. We evaluated the effects of different media compositions on the proteolytic exo-proteome of Vibrio alginolyticus and its closely relative Vibrio parahaemolyticus, in order to improve the overall proteases production, as well as the yield of the desired enzymes subset. Substantial biological responses were achieved with all media, which allowed defining culture conditions for targeted improvement of selected enzyme classes, besides giving insights in possible regulatory mechanisms. In particular, we focused our efforts on collagenases production, because of the growing biotechnological interest due to their pharmaceutical/biomedical applications.

A modified culture medium for improved isolation of marine vibrios.

Marine Vibrio members are of great interest for both ecological and biotechnological research, which often relies on their isolation. Whereas many efforts have been made for the detection of food-borne pathogenic species, much less is known about the performances of standard culture media toward environmental vibrios. We show that the isolation/enumeration of marine vibrios using thiosulfate-citrate-bile salts-sucrose agar (TCBS) as selective medium may be hampered by the variable adaptability of different taxa to the medium, which may result even in isolation failure and/or in substantial total count underestimation. We propose a modified TCBS as isolation medium, adjusted for marine vibrios requirements, which greatly improved their recovery in dilution plate counts, compared with the standard medium. The modified medium offers substantial advantages over TCBS, providing more accurate and likely estimations of the actual presence of vibrios. Modified TCBS allowed the recovery of otherwise undetected vibrios, some of which producing biotechnologically valuable enzymes, thus expanding the isolation power toward potentially new enzyme-producers Vibrio taxa. Moreover, we report a newly designed Vibrio-specific PCR primers pair, targeting a unique rpoD sequence, useful for rapid confirmation of isolates as Vibrio members and subsequent genetic analyses.

Aberrant gene expression profiles in Mediterranean sea urchin reproductive tissues after metal exposures.

Marine organisms are simultaneously exposed to numerous pollutants, among which metals probably represent the most abundant in marine environments. In order to evaluate the effects of metal exposure at molecular level in reproductive tissues, we profiled the sea urchin transcriptional response after non-lethal exposures using pathway-focused mRNA expression analyses. Herein, we show that exposures to relatively high concentrations of both essential and toxic metals hugely affected the gonadic expression of several genes involved in stress-response, detoxification, transcriptional and post-transcriptional regulation, without significant changes in gonadosomatic indices. Even though treatments did not result in reproductive tissues visible alterations, metal exposures negatively affected the main mechanisms of stress-response, detoxification and survival of adult P. lividus. Additionally, transcriptional changes observed in P. lividus gonads may cause altered gametogenesis and maintenance of heritable aberrant epigenetic effects. This study leads to the conclusion that exposures to metals, as usually occurs in polluted coastal areas, may affect sea urchin gametogenesis, thus supporting the hypothesis that parental exposure to environmental stressors affects the phenotype of the offspring.

Two distinct amplification events of the c-myc locus in a colorectal tumour.

Southern hybridisation of genomic DNA extracted from a human primary colorectal carcinoma revealed amplification of a fragment containing the wild-type c-myc locus. Two additional rearranged DNA fragments, lying upstream of c-myc, fused to distant non-contiguous sequences from the same chromosome, with an opposite configuration (head to head vs. head to tail), were also found to be amplified. Sequences analysis suggested that these rearrangements resulted from illegitimate recombination at two distinct points within the DNA sequence just upstream of the c-myc ORF and further that these events triggered two different amplification mechanisms, only one of which, involving a strand invasion event following DNA double strand breaks, increased the copy number of the c-myc ORF.

Characterization of Translationally Controlled Tumour Protein from the Sea Anemone Anemonia viridis and Transcriptome Wide Identification of Cnidarian Homologues.

Gene family encoding translationally controlled tumour protein (TCTP) is defined as highly conserved among organisms; however, there is limited knowledge of non-bilateria. In this study, the first TCTP homologue from anthozoan was characterised in the Mediterranean Sea anemone, Anemonia viridis. The release of the genome sequence of Acropora digitifera, Exaiptasia pallida, Nematostella vectensis and Hydra vulgaris enabled a comprehensive study of the molecular evolution of TCTP family among cnidarians. A comparison among TCTP members from Cnidaria and Bilateria showed conserved intron exon organization, evolutionary conserved TCTP signatures and 3D protein structure. The pattern of mRNA expression profile was also defined in A. viridis. These analyses revealed a constitutive mRNA expression especially in tissues with active proliferation. Additionally, the transcriptional profile of A. viridis TCTP (AvTCTP) after challenges with different abiotic/biotic stresses showed induction by extreme temperatures, heavy metals exposure and immune stimulation. These results suggest the involvement of AvTCTP in the sea anemone defensome taking part in environmental stress and immune responses.

Exploiting translational coupling for the selection of cells producing toxic recombinant proteins from expression vectors.

High rates of plasmid instability are associated with the use of some expression vectors in Escherichia coli, resulting in the loss of recombinant protein expression. This is due to sequence alterations in vector promoter elements caused by the background expression of the cloned gene, which leads to the selection of fast-growing, plasmid-containing cells that do not express the target protein. This phenomenon, which is worsened when expressing toxic proteins, results in preparations containing very little or no recombinant protein, or even in clone loss; however, no methods to prevent loss of recombinant protein expression are currently available. We have exploited the phenomenon of translational coupling, a mechanism of prokaryotic gene expression regulation, in order to select cells containing plasmids still able to express recombinant proteins. Here we designed an expression vector in which the cloned gene and selection marker are co-expressed. Our approach allowed for the selection of the recombinant protein-expressing cells and proved effective even for clones encoding toxic proteins.

Maintenance of a Protein Structure in the Dynamic Evolution of TIMPs over 600 Million Years.

Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria phylum, coeval with the origins of connective tissue, to great apes and humans. Despite dramatic sequence differences compared with highest metazoans, the ancestral proteins displayed the canonical TIMP fold. Only small structural changes, represented by an α-helix located in the N-domain, have occurred over the evolution. Both the occurrence of such secondary structure elements and the relative solvent accessibility of the corresponding residues in the three-dimensional structures raises the possibility that these sites represent unconserved element prone to accept variations.