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

Analysis of hollow inclusion–matrix debonding in particulate composites

AbstractThis work aims at understanding the effect of particle–matrix interfacial debonding on the tensile response of syntactic foams. The problem of a single hollow inclusion with spherical-cap cracks embedded in a dissimilar matrix material is studied. Degradation of elastic modulus, cavity formation in the proximity of debonded regions, stress localization phenomena in the inclusion, debonding energetics, and crack kinking are studied for a broad range of inclusion wall thickness and debonding extent. A series solution based on the Galerkin method is proposed and validated through comparison with findings from boundary element and finite element methods. Results are specialized to glass particle-vinyl ester matrix systems widely used in marine structural applications. The insight gained into the role of particle–matrix debonding extent and inclusion wall thickness is useful in understanding the possible failure mechanisms of syntactic foams under tensile and flexural loading conditions and in tailoring their parameters for specific applications

Elastic interaction of interfacial spherical-cap cracks in hollow particle filled composites

AbstractThis work analyzes the elastic interaction between two spherical-cap cracks present along the outer surface of a hollow particle embedded in a dissimilar medium under remote uniaxial tensile loading. A semi-analytical approach based on an enriched Galerkin method is adopted to determine stress and deformation fields as functions of particle wall thickness and cracks’ configuration. The present analysis is limited to multiple interfacial spherical-cap cracks; that is, crack propagation is restrained to the particle-matrix interface and possibility of crack kinking in the matrix is not considered. Interfacial crack growth characteristics, conditions for stable crack propagation, equal crack growth, and shielding are established through energy release rate analysis. The study is relevant to the analysis of tensile and flexural failure of syntactic foams used in marine and aerospace applications. Results specialized to glass-vinyl ester syntactic foams demonstrate that particle wall thickness can be used to control crack stability and growth characteristics as well as tailoring the magnitude of the shielding phenomenon. Predictions are compared to finite element findings for validation and to results for penny-shaped cracks to elucidate the role of crack curvature

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

Methanotrophic activity and diversity of methanotrophs in volcanic-geothermal soils at Pantelleria island (Italy)

Volcanic and geothermal systems emit endogenous gases by widespread degassing from soils, including CH4, a greenhouse gas twenty-five times as potent as CO2. Recently, it has been demonstrated that volcanic/geothermal soils are not only a source of methane, but also sites of methanotrophic activity. Methanotrophs are able to consume 10-40 Tg of CH4 a-1 and to trap more than 50% of the methane degassing through the soils. We report on methane microbial oxidation in the geothermally most active site of Pantelleria island (Italy), Favara Grande, whose total methane emission was previously estimated in about 2.5 Mg a-1 (t a-1). Laboratory incubation experiments with three top-soil samples from Favara Grande indicated methane consumption values up to 59.2 nmol g-1 soil d.w. h-1. One of the three sites, FAV2, where the highest oxidation rate was detected, was further analysed on a vertical soil profile and the maximum methane consumption was measured in the top-soil layer and values >6.23 nmol g-1 h-1 were still detected up to a depth of 13 cm. The highest consumption rate was measured at 37°C, but a still detectable consumption at 80°C (>1.25 nmol g -1 h-1) was recorded. The soil total DNAs extracted from the three samples were probed by PCR using standard proteobacterial primers and newly designed verrucomicrobial primers, targeting the unique methane monooxygenase gene pmoA; the presence of methanotrophs was detected in sites FAV2 and FAV3, but not in FAV1, where harsher chemical-physical conditions and negligible methane oxidation were detected. The pmoA gene libraries from the most active site FAV2 pointed out a high diversity of gammaproteobacterial methanotrophs, distantly related to Methylococcus/Methylothermus genera and the presence of the newly discovered acido-thermophilic methanotrophs Verrucomicrobia. Alphaproteobacteria of the genus Methylocystis were isolated from enrichment cultures, under a methane containing atmosphere at 37°C. The isolates grow at a pH range from 3.5 to 8, temperatures of 18 – 45 °C and consume 160 nmol of CH 4 h-1 ml-1 of culture. Soils from Favara Grande showed the largest diversity of methanotrophic bacteria until now detected in a geothermal soil. While methanotrophic Verrucomicrobia are reported to dominate highly acidic geothermal sites, our results suggest that slightly acidic soils, in high enthalpy geothermal systems, host a more diverse group of both culturable and uncultivated methanotrophs

Methanotrophic activity and diversity of methanotrophs in volcanic geothermal soils at Pantelleria (Italy)

Volcanic and geothermal systems emit endogenous gases by widespread degassing from soils, including CH4, a greenhouse gas twenty-five times as potent as CO2. Recently, it has been demonstrated that volcanic or geothermal soils are not only a source of methane, but are also sites of methanotrophic activity. Methanotrophs are able to consume 10–40 Tg of CH4 a−1 and to trap more than 50% of the methane degassing through the soils. We report on methane microbial oxidation in the geothermally most active site of Pantelleria (Italy), Favara Grande, whose total methane emission was previously estimated at about 2.5Mga−1 (t a−1). Laboratory incubation experiments with three top-soil samples from Favara Grande indicated methane consumption values of up to 59.2 nmol g−1 soil d.w. h−1. One of the three sites, FAV2, where the highest oxidation rate was detected, was further analysed on a vertical soil profile, the maximum methane consumption was measured in the topsoil layer, and values greater than 6.23 nmol g−1 h−1 were still detected up to a depth of 13 cm. The highest consumption rate was measured at 37 C, but a still detectable consumption at 80 C (>1.25 nmol g−1 h−1) was recorded. The soil total DNA extracted from the three samples was probed by Polymerase Chain Reaction (PCR) using standard proteobacterial primers and newly designed verrucomicrobial primers, targeting the unique methane monooxygenase gene pmoA; the presence of methanotrophs was detected at sites FAV2 and FAV3, but not at FAV1, where harsher chemical–physical conditions and negligible methane oxidation were detected. The pmoA gene libraries from the most active site (FAV2) pointed to a high diversity of gammaproteobacterial methanotrophs, distantly related to Methylocaldum-Metylococcus genera, and the presence of the newly discovered acido-thermophilic Verrucomicrobia methanotrophs. Alphaproteobacteria of the genus Methylocystis were isolated from enrichment cultures under a methane-containing atmosphere at 37 C. The isolates grow at a pH range of 3.5 to 8 and temperatures of 18–45 C, and consume 160 nmol of CH4 h−1 mL−1 of culture. Soils from Favara Grande showed the largest diversity of methanotrophic bacteria detected until now in a geothermal soil. While methanotrophic Verrucomicrobia are reported as dominating highly acidic geothermal sites, our results suggest that slightly acidic soils, in high-enthalpy geothermal systems, host a more diverse group of both culturable and uncultivated methanotrophs

Effect of chronic heat stress on gastrointestinal histology and expression of feed intake-regulatory hormones in broiler chickens

Heat stress (HS) dramatically impairs the growth performance of broiler chickens, mainly as a consequence of reduced feed intake due to the loss of appetite. This study was aimed at evaluating the alterations induced by chronic HS conditions on the morphological and morphometric features of the gastrointestinal (GI) tract and on the expression of some enteroendocrine cells (EECs) involved in the regulation of feed intake in chickens. Three hundred male chickens (Ross 308) were divided into two experimental groups and raised either in thermoneutral environment for the whole fattening period (0–41 days) (TNT group) or subjected to chronic HS conditions (30 °C for 24 h/day) from 35 to 41 days (HS group). Samples of proventriculus, duodenum, jejunum and cecum were collected from 24 broilers (12/group). Haematoxylin-eosin was used for the morphometric evaluations, while immunohistochemistry was applied for the evaluation of EECs expressing ghrelin (GHR), cholecystokinin (CCK), neuropeptide Y (NPY), glucagon-like peptide-1 (GLP-1), and serotonin (5-HT). In the proventriculus, HS reduced total wall thickness and mucous layer height (P ≤ 0.01) as well as mean diameter, circumference, and area of the compound tubular glands (P ≤ 0.001) with respect to TNT. The small intestine of HS birds was characterised by decreased villous height and total thickness (duodenum, P ≤ 0.01; jejunum, P ≤ 0.001), whereas crypt depth and width were reduced only in the jejunum (P ≤ 0.01). HS had negligible effects on the morphological aspects of the cecum. In the proventriculus, an increase in GHR and NPY EECs was observed in response to HS (P ≤ 0.001). Similarly, the small intestine villi of the HS group showed greater GLP-1 (P ≤ 0.05), 5-HT (P ≤ 0.001) and CCK (P ≤ 0.01) EECs. Moreover, the expression of 5-HT EECs was higher in the duodenal (P ≤ 0.01) and jejunal (P ≤ 0.01) crypts of HS birds, whereas GLP-1 and CCK EECs increased only in jejunal crypts (P ≤ 0.05). Finally, 5-HT EEC expression was increased in the cecum of HS group (P ≤ 0.01). In conclusion, these outcomes demonstrate that chronic HS induces morphometric alterations not only in the small intestine but also in a key organ such as the proventriculus. Furthermore, HS conditions affect the presence and distribution of EECs, suggesting that some GI peptides and biogenic amine may be implicated in the regulation of appetite and voluntary feed intake in heat-stressed broiler chickens

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

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