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

Growing Up with the Rochester Red Wings in the Forties and Fifties

I grew up an avid fan of baseball, football, and basketball in Rochester, NY, in the postwar years. The Rochester Royals were the only major league team but for me basketball was a stopgap until baseball began. The Royals and their players were fine, and I filled scrapbooks with the doings of Bobbie Davies and others. However, the Red Wings were the real deal for me. True, they were a minor league team but they were my minor league team. The games were easy to get to, and I could easily take a bus or even walk to them. The Knothole Gang card let kids into 9 or 10 free games, and my Dad often came with me on weekends for double-headers. He, too, was a baseball nut and socialized me into becoming a Yankee fan. The Italian ballplayers were our heroes, and the Red Wings had a few for us to follow and support. The fact that their parent club was in St. Louis at the time was fine since it was a National League team and good players would not go to Yankee rivals. By the time Baltimore became the parent club I was losing interest in the Wings and had moved on to other cities. However, the Wings kept a hold on me, as tenuous as it was. This paper examines that significant tie between a boy and his home team in a minor league city and its impact on his maturing young years

« Oh! Vous voilà ! » L'anthropologue hétérosexuel et le sexe

* Oh ! Vous voilà ! »L'anthropologue hétérosexuel et le sexeJ'ai réalisé sept enquêtes sur le terrain au Nigeria, une au Kenya, cinq en Angleterre, d'autres aux États-Unis, avec des musiciens de jazz, des Asiatiques ougandais et des Italo-Américains, ainsi qu'au Canada, parmi les immigrants asiatiques ougandais. À chaque voyage je me trouvais à une étape différente de ma vie et de ma carrière. De plus, chaque voyage fut différent quant à sa «signification sexuelle». Par exemple, j'ai cherché des prostituées lors d'un voyage, une autre fois j'étais accompagné par une étudiante adulte, puis par ma fiancée de quelques semaines, enfin ma femme et mes enfants étaient avec moi lors de mon dernier séjour au Nigeria. Je mets à profit mes propres expériences pour discuter plusieurs variables qui affectent les pratiques hétérosexuelles sur le terrain et influencent l'enquête en cours : notamment l'âge, le statut professionnel, le lieu de l'enquête, les personnes qui accompagnent le chercheur, etc. À cette étape de notre connaissance de l'enquête sur le terrain en lien avec la sexualité, une bonne analyse passe d'abord par une bonne description.« Oh ! There You Are ! »Sex and the Heterosexual AnthropologistI have conducted fieldwork in Nigeria on seven separate occasions, in Kenya once, in England on five separate occasions, in the United States for many years with jazz musicians, resettled Ugandan Asians, and Italian-Americans, and in Canada on resettled Ugandan Asians. For each of these trips, I have been at a différent stage of professional and life-span development In addition, each trip has been différent in ils « sexual meaning ». For example, on one trip I sought out prostitutes, on another I had an adult student with me, my bride of a few weeks on yet another, and my wife and children on my last trip to Nigeria. I am using my expéeiences to discuss a number of variables that affect heterosexual practices in the field and influence the fieldwork that is conducted; specifically, age, professional status, place of fieldwork, persons accompanying the field worker, and so on. At this stage of our knowledge of fieldwork and sexuality, good description is essential to good analysis

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

Silibinin modulates lipid homeostasis and inhibits nuclear factor kappa B activation in experimental nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is associated with increased liver-related mortality. Disturbances in hepatic lipid homeostasis trigger oxidative stress and inflammation (ie, lipotoxicity), leading to the progression of NASH. This study aimed at identifying whether silibinin may influence the molecular events of lipotoxicity in a mouse model of NASH. Eight-week-old db/db mice were fed a methionine-choline deficient (MCD) diet for 4 weeks and treated daily with silibinin (20 mg/kg intraperitoneally) or vehicle. Liver expression and enzyme activity of stearoyl-CoA desaturase-1 and acyl-CoA oxidase, and expression of liver fatty acid-binding protein were assessed. Hepatic levels of reactive oxygen species, thiobarbituric acid-reactive substances (TBARS), 3-nitrotyrosine (3-NT), inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NFkB) activities were also determined. Silibinin administration decreased serum alanine aminotransferase and improved liver steatosis, hepatocyte ballooning, and lobular inflammation in db/db mice fed an MCD diet. Gene expression and activity of stearoyl-CoA desaturase-1 were reduced in db/db mice fed an MCD diet compared with lean controls and were increased by silibinin; moreover, silibinin treatment induced the expression and activity of acyl-CoA oxidase and the expression of liver fatty acid-binding protein. Vehicle-treated animals displayed increased hepatic levels of reactive oxygen species and TBARS, 3-NT staining, and iNOS expression; silibinin treatment markedly decreased reactive oxygen species and TBARS and restored 3-NT and iNOS to the levels of control mice. db/db mice fed an MCD diet consistently had increased NFkB p65 and p50 binding activity; silibinin administration significantly decreased the activity of both subunits. Silibinin treatment counteracts the progression of liver injury by modulating lipid homeostasis and suppressing oxidative stress-mediated lipotoxicity and NFkB activation in experimental NASH

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

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

Efficiency of sperm-mediated gene transfer in the ovine by laparoscopic insemination, in vitro fertilization and ICSI

188-196Transgenesis constitutes an important tool for pharmacological protein production and livestock improvement. We evaluated the potential of laparoscopic insemination (LI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) to produce egfp-expressing ovine embryos, using spermatozoa previously exposed to pCX-EGFP plasmid in two different sperm/DNA incubation treatments: "Long Incubation" (2 h at 17 C) and "Short Incubation" (5 min at 5 C). For LI, Merino sheep were superovulated and inseminated with treated fresh semen from Merino rams. The embryos were recovered by flushing the uterine horns. For IVF and ICSI, slaughterhouse oocytes were fertilized with DNA-treated frozen/thawed sperm. All recovered embryos were exposed to blue light (488 nm) to determine green fluorescent morulae and blastocysts rates. High cleavage and morulae/blastocysts rates accompanied the LI and IVF procedures, but no egfp-expressing embryos resulted. In contrast, regardless of the sperm/ plasmid incubation treatment, egfp-expressing morulae and blastocysts were always obtained by ICSI, and the highest transgenesis rate (91.6 percent) was achieved with Short Incubation. In addition, following the incubation of labeled plasmid DNA, after Long or Short exposure treatments, with fresh or frozen/thawed spermatozoa, only non-motile fresh spermatozoa could maintain an attached plasmid after washing procedures. No amplification product could be detected following PCR treatment of LI embryos whose zonae pellucidae (ZP) had been removed. In order to establish conditions for transgenic ICSI in the ovine, we compared three different activation treatments, and over 60 percent of the obtained blastocysts expressed the transgene. For ICSI embryos, FISH analysis found possible signals compatible with integration events. In conclusion, our results show that in the ovine, under the conditions studied, ICSI is the only method capable of producing exogenous gene-expressing embryos using spermatozoa as vectors