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

Videotaped Trial Court Proceedings: The Potential Effect on Appellate Review of Credibility Determinations

Appellate courts have traditionally afforded great deference to a trial court\u27s factual findings, both in jury and non-jury trials

Quaterpyridine Ligands for Panchromatic Ru(II) Dye Sensitizers

A new general synthetic access to carboxylated quaterpyridines (qpy), of interest as ligands for panchromatic dyesensitized solar cell organometallic sensitizers, is presented. The strategic step is a Suzuki−Miyaura cross-coupling reaction, which has allowed the preparation of a number of representative unsubstituted and alkyl and (hetero)aromatic substituted qpys. To bypass the poor inherent stability of 2-pyridylboronic acid derivatives, we successfully applied N-methyliminodiacetic acid (MIDA) boronates as key reagents, obtaining the qpy ligands in good yields up to (quasi)gram quantities. The structural, spectroscopic (NMR and UV−vis), electrochemical, and electronic characteristics of the qpy have been experimentally and computationally (DFT) investigated. The easy access to the bis-thiocyanato Ru(II) complex of the parent species of the qpy series, through an efficient route which bypasses the use of Sephadex column chromatography, is shown. The bis-thiocyanato Ru(II) complex has been spectroscopically (NMR and UV−vis), electrochemically, and computationally investigated, relating its properties to those of previously reported Ru(II)−qpy complexes.“This document is the Accepted Manuscript version of a Published Work that appeared in final form in [The Journal of Organic Chemistry], copyright © American Chemical Society after peer review and technical editing by the publisher

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

a mathematical description accounting for the superfluous hydrogen evolution and the inductive behaviour observed during electrochemical measurements on magnesium

Abstract When electrochemical techniques are used to probe the surface of corroding magnesium with the aim of obtaining quantitative information on the corrosion process, two peculiarities are generally observed: i) with anodic polarization, the rate of hydrogen evolution increases instead of decreasing and ii) during electrochemical impedance spectroscopy measurements, an inductive contribution is often observed at the low-frequency end of the spectra. The presence of these two phenomena clearly has an impact on the methodology that should be applied to correctly estimate corrosion rates from electrochemical data. The aim of this work is to provide a general mathematical description of the corroding magnesium surface that, under minimal a priori assumptions regarding the reaction kinetics, can account simultaneously for both superfluous hydrogen evolution and inductive response. The mathematical results are consistent with the suggestion that the superfluous hydrogen evolution is mainly related to the increase of the surface of the active corrosion front during anodic polarization. Further, the obtained results show that the inductive response is expected when, at the corrosion front, oxidation of magnesium proceeds faster than hydrogen evolution

Evaluation of an Internet Document Delivery Service

An Internet-based Document Delivery Service (DDS) has been developed within the framework of the CNR ( the Italian Research National Council) Project BiblioMIME, in order to take advantage of new Internet technologies and promote cooperation among CNR and Italian university libraries. Adopting such technologies changes the traditional organisation of DDS and may drastically reduce costs and delivery times. An information system managing DDS requests and monitoring the temporal evolution of the service has been implemented, running on the local-area network of a test-site library. It aims to track number and types of documents requested and received, user distribution, delivery times and types (surface mail, fax, Internet), to automate repetitive manual procedures and to deal with the various accounting methods used by other libraries. Transmission of documents is carried out by means of an e-mail/Web gateway system supporting document exchange via Internet, which assists receiving libraries in retrieving requested documents. This paper describes the architecture and main design features of the e-mail/Web gateway server (the BiblioMime server). This approach permits librarians to continue using e-mail service to send large documents, while resolving problems that users may encounter when downloading large size files with e-mail agents. The library operator sends the document as an attachment to the destination address; on fly the e-mail server extracts and saves the attachments in a web-server disk file and substitutes them with a new message part that includes an URL pointing to the saved document. The receiver can download these large objects by means of a user-friendly browser. We further discuss the data gathered during the triennium 1998-2000; this consists of about 5,000 DDS transactions per annum with 300 other Italian scientific and bio-medical libraries and commercial document suppliers. Use of the instruments described above allowed us to evaluate the performance of service "before" and "after" the use of Internet Document Delivery and to extract some critical data regarding DDS. Those include: a) libraries with which we have greater numbers of exchanges and their turnaround times; b) extraordinary reduction in costs and delivery times; c) the most frequently requested serial titles (allowing cost-effective decisions on new subscriptions); d) impact on DDS of library participation in consortia which allow user access to greater numbers of online serials

Validation of an electrogoniometry system as a measure of knee kinematics during activities of daily living

Purpose: The increasing use of electrogoniometry (ELG) in clinical research requires the validation of different instrumentation. The purpose of this investigation was to examine the concurrent validity of an ELG system during activities of daily living. Methods: Ten asymptomatic participants gave informed consent to participate. A Biometrics SG150 electrogoniometer was directly compared to a 12 camera three dimensional motion analysis system during walking, stair ascent, stair descent, sit to stand, and stand to sit activities for the measurement of the right knee angle. Analysis of validity was undertaken by linear regression. Standard error of estimate (SEE), standardised SEE (SSEE), and Pearson’s correlation coefficient r were computed for paired trials between systems for each functional activity. Results: The 95% confidence interval of SEE was reasonable between systems across walking (LCI = 2.43 °; UCI = 2.91 °), stair ascent (LCI = 2.09 °; UCI = 2.42 °), stair descent (LCI = 1.79 °; UCI = 2.10 °), sit to stand (LCI = 1.22 °; UCI = 1.41 °), and stand to sit (LCI = 1.17 °; UCI = 1.34 °). Pearson’s correlation coefficient r across walking (LCI = 0.983; UCI = 0.990), stair ascent (LCI = 0.995; UCI = 0.997), stair descent (LCI = 0.995; UCI = 0.997), sit to stand (LCI = 0.998; UCI = 0.999), and stand to sit (LCI = 0.996; UCI = 0.997) was indicative of a strong linear relationship between systems. Conclusion: ELG is a valid method of measuring the knee angle during activities representative of daily living. The range is within that suggested to be acceptable for the clinical evaluation of patients with musculoskeletal conditions

3d collagen hydrogel promotes in vitro langerhans islets vascularization through ad-mvfs angiogenic activity

Adipose derived microvascular fragments (ad-MVFs) consist of effective vascularization units able to reassemble into efficient microvascular networks. Because of their content in stem cells and related angiogenic activity, ad-MVFs represent an interesting tool for applications in regenerative medicine. Here we show that gentle dissociation of rat adipose tissue provides a mixture of ad-MVFs with a length distribution ranging from 33–955 µm that are able to maintain their original morphology. The isolated units of ad-MVFs that resulted were able to activate transcriptional switching toward angiogenesis, forming tubes, branches, and entire capillary networks when cultured in 3D collagen type-I hydrogel. The proper involvement of metalloproteases (MMP2/MMP9) and serine proteases in basal lamina and extracellular matrix ECM degradation during the angiogenesis were concurrently assessed by the evaluation of alpha-smooth muscle actin (αSMA) expression. These results suggest that collagen type-I hydrogel provides an adequate 3D environment supporting the activation of the vascularization process. As a proof of concept, we exploited 3D collagen hydrogel for the setting of ad-MVF–islet of Langerhans coculture to improve the islets vascularization. Our results suggest potential employment of the proposed in vitro system for regenerative medicine applications, such as the improving of the islet of Langerhans engraftment before transplantation