Culture Medium Factorial Design Optimization for Fibrinolytic Enzymes Production by Bionectria sp.

Thrombotic diseases can be clinically treated with fibrinolytic enzymes and many attempts have been made at laboratory level to increase fibrinolytic enzymes production from microbial sources and to reduce the process cost, including culture medium design, optimization of environmental conditions, and over expression with genetically modified strains. In this contribution we present the optimization of culture medium composition and incubation temperature for fibrinolytic enzyme production by Bionectria sp., a selected fungal strain from Las Yungas (Tucumán). Optimization was carried out at Erlenmeyer scale (100-mL working volume) via factorial design methodology. All trials included a common mineral base (%, w/v: NaCl 0.2, KH2PO4 0.05, MgSO4·7H2O 0.05). According to four factorial designs it could be demonstrated the convenience of using soy peptone as N-source, glucose as C-source, and the possibility to eliminate starch, meat peptone and meat extract from original medium composition, whilst 25°C was selected as the optimal incubation temperature. Results showed that culture medium could be successfully optimized by factorial design, achieving a reduction in the production process costs by means of a decrease in culture medium components, the improvement in culture broth rheology, mycelial morphology and mass/energy transfer, and the subsequent two-fold enhancement in productivity.Fil: Arnau, Victor Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Rovati, Jose Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Figueroa, L. I. C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Fariña, Julia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaXLVI Reunión Anual Sociedad Argentina de Investigaciones Bioquímicas y MolecularesPuerto Madryn, Chubut, ArgentinaArgentinaSociedad Argentina de Investigaciones Bioquímicas y Moleculare

Potentialities of the combined use of underwater fluorescence imagery and photogrammetry for the detection of fine-scale changes in marine bioconstructors

Marine communities are facing both natural disturbances and anthropogenic stressors. Bioconstructor species are endangered by multiple large-scale and local pressures and the early identification of impacts and damages is a primary goal for preserving coral reefs. Taking advantage of the recent development in underwater photogrammetry, the use of photogrammetry and fluorimetry was coupled to design, test and validate in laboratory a multi-sensor measuring system that could be potentially exploited in open water by SCUBA divers for assessing the health status of corals and detecting relevant biometric parameters with high accuracy and resolution. The approach was tested with fragments of the endemic coral Cladocora caespitosa, the sole zooxanthellate scleractinian reef-builder in the Mediterranean. The most significant results contributing to the scientific advancement of knowledge were: 1) the development of a cost-effective, flexible and easy-to-use approach based on emerging technologies; 2) the achievement of a sub-centimetric resolution for measuring relevant biometric parameters (polyp counting, colony surface areas and volumes); 3) set up of a reliable and repeatable strategy for multi-temporal analyses capable of quantifying changes in coral morphology with sub-centimeter accuracy; 4) detect changes in coral health status at a fine scale and under natural lighting through autofluorescence analysis. The novelty of the present research lies in the coupling of emerging techniques that could be applied to a wide range of 3D morphometrics, different habitats and species, thus paving the way to innovative opportunities in ecological research and more effective results than traditional in-situ measurements. Moreover, the possibility to easily modify the developed system to be installed on an underwater remotely operated vehicle further highlights the possible concrete impact of the research for ecological monitoring and protection purposes

A comparison between in vivo and ex vivo HR-MAS H-1 MR spectra of a pediatric posterior fossa lesion

The present case report was aimed at identifying the molecular profile characteristic of a primitive neuroectodermal tumor (PNET) in a 3-year-old child affected by a lesion localized in the cerebellar region. The histological diagnosis was medulloblastoma. In vivo single voxel H-1 magnetic resonance spectroscopy (MRS) shows high specificity in detecting the main metabolic alterations in the primitive cerebellar lesion; a very high amount of the choline-containing compounds and very low level of creatine derivatives and N-acetylaspartate. Ex vivo high resolution magic angle spinning (HR-MAS) H-1 magnetic resonance spectroscopy, performed at 9.4 Tesla on the neoplastic specimen collected during surgery, allows for the unambiguous identification of several metabolites giving a more in-depth evaluation of the metabolic pattern of the lesion. The ex vivo HR-MAS MR spectra show that the spectral detail is much higher than that obtained in vivo and that, for example, myo-inositol, taurine and phosphorylethanolamine contribute to the in vivo signal at 3.2 ppm, usually attributed to choline-containing compounds. In addition, the spectroscopic data appear to correlate with some morphological features of the medulloblastoma. Consequently, the present study shows that ex vivo HR-MAS H-1 MRS is able to strongly improve the clinical possibility of in vivo MRS and can be used in conjunction with in vivo spectroscopy for clinical purposes

Role of cholecystokinin in dietary fat-promoted azaserine-induced pancreatic carcinogenesis in rats.

The role of cholecystokinin in dietary fat-promoted pancreatic carcinogenesis was investigated in azaserine-treated rats, using lorglumide, a highly specific cholecystokinin-receptor antagonist. The animals were killed 8 months after the start of treatment. Cholecystokinin, but not dietary unsaturated fat, increased pancreatic weight. Rats treated with cholecystokinin developed more acidophilic atypical acinar cell nodules, adenomas and adenocarcinomas than control animals. Rats maintained on the high-fat diet developed significantly more adenomas and adenocarcinomas than controls given a diet low in unsaturated fat. Lorglumide largely inhibited the enhancing effect of cholecystokinin, but not of dietary fat, on pancreatic carcinogenesis indicating that it is unlikely that the promoting effect of dietary unsaturated fat on pancreatic carcinogenesis is mediated via cholecystokinin

ER alpha-independent NRF2-mediated immunoregulatory activity of tamoxifen

Sex differences in immune-mediated diseases are linked to the activity of estrogens on innate immunity cells, including macrophages. Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM) used in estrogen receptor-alpha (ERα)-dependent breast cancers and off-target indications such as infections, although the immune activity of TAM and its active metabolite, 4-OH tamoxifen (4HT), is poorly characterized. Here, we aimed at investigating the endocrine and immune activity of these SERMs in macrophages. Using primary cultures of female mouse macrophages, we analyzed the expression of immune mediators and activation of effector functions in competition experiments with SERMs and 17β-estradiol (E2) or the bacterial endotoxin LPS. We observed that 4HT and TAM induce estrogen antagonist effects when used at nanomolar concentrations, while pharmacological concentrations that are reached by TAM in clinical settings regulate the expression of VEGFα and other immune activation genes by ERα- and G protein-coupled receptor 1 (GPER1)-independent mechanisms that involve NRF2 through PI3K/Akt-dependent mechanisms. Importantly, we observed that SERMs potentiate cell phagocytosis and modify the effects of LPS on the expression of inflammatory cytokines, such as TNFα and IL1β, with an overall increase in cell inflammatory phenotype, further sustained by potentiation of IL1β secretion through caspase-1 activation

Novel bioprinted 3D model to human fibrosis investigation

Fibrosis is shared in multiple diseases with progressive tissue stiffening, organ failure and limited therapeutic options. This unmet need is also due to the lack of adequate pre-clinical models to mimic fibrosis and to be challenged novel by anti-fibrotic therapeutic venues. Here using bioprinting, we designed a novel 3D model where normal human healthy fibroblasts have been encapsulated in type I collagen. After stimulation by Transforming Growth factor beta (TGFβ), embedded cells differentiated into myofibroblasts and enhanced the contractile activity, as confirmed by the high level of α − smooth muscle actin (αSMA) and F-actin expression. As functional assays, SEM analysis revealed that after TGFβ stimulus the 3D microarchitecture of the scaffold was dramatically remolded with an increased fibronectin deposition with an abnormal collagen fibrillar pattern. Picrius Sirius Red staining additionally revealed that TGFβ stimulation enhanced of two logarithm the collagen fibrils neoformation in comparison with control. These data indicate that by bioprinting technology, it is possible to generate a reproducible and functional 3D platform to mimic fibrosis as key tool for drug discovery and impacting on animal experimentation and reducing costs and time in addressing fibrosis

AB0360 EFFICACY AND SAFETY OF THE PROSTAGLANDIN EP4 RECEPTOR ANTAGONIST CR6086 ADDED TO METHOTREXATE IN DMARD-NAÏVE EARLY RA PATIENTS: A PHASE 2 RANDOMIZED CONTROLLED TRIAL

Background:MTX is the first line treatment in early RA. There is robust evidence from cohort studies, but less from RCTs, that a "window of opportunity" exists over 12-16 weeks symptom duration. CR6086 is a selective prostaglandin EP4 receptor antagonist, with an immunomodulatory profile.Objectives:To test efficacy and safety of CR6086 added to MTX in early RA, DMARD-naïve patients.Methods:Patients with RA (ACR/EULAR 2010 criteria), 6 months disease duration. This generated the hypothesis that addition of CR6086 90mg bid may benefit in RA patients initiating MTX after the window of opportunity, to be tested in further studies.Table 1.Patient characteristics & pregnancy outcomesSymptom onset <12 months(principal analysis)Symptom onset 6-12 months(post-hoc analysis)*Placebo+MTX(N=62)CR6086 90mg+MTX(N=60)Placebo+MTX(N=37)CR6086 90mg+MTX(N=38)ACR20, %59.7%70.0%48.6%68.4%ACR50, %33.9%38.3%29.7%39.5%ACR70, %17.7%23.3%10.8%28.9%DAS28 (CRP) <2.6, %12.9%20.0%8.1%18.4%CDAI ≤2.8, %8.1%11.7%5.4%15.8%SDAI ≤3.3, %6.5%10.0%2.7%15.8%Boolean-based remission, %6.5%6.7%2.7%10.5%*In patients with symptom onset <6 months, MTX monotherapy exerted a large 76% ACR20 response, and correspondingly high secondary efficacy parameters, precluding potentiation in this subsetFigure 1.Change in MRI (DEMRIQ-ME#) after 13 weeksDisclosure of Interests:Karel Pavelka Consultant of: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen, Speakers bureau: Abbvie, MSD, BMS, Egis, Roche, UCB, Medac, Pfizer, Biogen, Ivanova Delina2 Delina: None declared, Minodora Mazur: None declared, Massimo D'Amato Employee of: Rottapharm Biotech, GIAMPAOLO GIACOVELLI Employee of: Rottapharm Biotech, Federica Girolami Employee of: Rottapharm Biotech, Marek Krogulec: None declared, René Østgård: None declared, Asger Reinstrup Bihlet Shareholder of: Nordic Bioscience A/S., Olga Kubassova Shareholder of: IAG, Image Analysis Group, Consultant of: Novartis, Takeda, Lilly, Employee of: IAG, Image Analysis Group, Lucio Rovati Shareholder of: Rottapharm Biotech, Employee of: Rottapharm Biotech, Peter C. Taylor Grant/research support from: Celgene, Eli Lilly and Company, Galapagos, and Gilead, Consultant of: AbbVie, Biogen, Eli Lilly and Company, Fresenius, Galapagos, Gilead, GlaxoSmithKline, Janssen, Nordic Pharma, Pfizer Roche, and UC

Nuclear receptor ligands induce TREM-1 expression on dendritic cells: analysis of their role in tumors

Dendritic cells (DCs) initiate adaptive immune responses after their migration to secondary lymphoid organs. The LXR ligands/oxysterols and the RXR ligand 9-cis Retinoic Acid (9-cis RA) were shown to dampen DC migration to lymphoid organs through the inhibition of CCR7 expression. We performed transcriptomics of DCs undergoing maturation in the presence of the LXR ligand 22R-Hydroxycholesterol (22R-HC). The analysis highlighted more than 1500 genes modulated by 22R-HC treatment, including the triggering receptor expressed on myeloid cells (TREM)-1, which was found markedly up-regulated. We tested the effect of other nuclear receptor ligands (NRL) and we reported the induction of TREM-1 following RXR, RAR and VDR activation. From a functional point of view, triggering of TREM-1 induced by retinoids increased TNF\u3b1 and IL-1\u3b2 release, suggesting an active role of NRL-activated TREM-1+ DCs in inflammation-driven diseases, including cancer. Consistently with this hypothesis we detected DCs expressing TREM-1 in pleural effusions and ascites of cancer patients, an observation validated by the induction of TREM-1, LXR and RAR target genes when monocyte-DCs were activated in the presence of tumor-conditioned fluids. Finally, we observed a better control of LLC tumor growth in Trem-1 12/- bone marrow chimera mice as compared to wild type chimera mice. Future studies will be necessary to shed light on the mechanism of TREM-1 induction by distinct NRL, and to characterize the role of TREM-1+ DCs in tumor growth

Boosting Anti-Inflammatory Potency of Zafirlukast by Designed Polypharmacology

Multitarget design offers access to bioactive small molecules with potentially superior efficacy and safety. Particularly multifactorial chronic inflammatory diseases demand multiple pharmacological interventions for stable treatment. By minor structural changes, we have developed a close analogue of the cysteinyl-leukotriene receptor antagonist zafirlukast that simultaneously inhibits soluble epoxide hydrolase and activates peroxisome proliferator-activated receptor \u3b3. The triple modulator exhibits robust anti-inflammatory activity in vivo and highlights the therapeutic potential of designed multitarget agents