Validation of precision-cut liver slices to study drug-induced cholestasis:A transcriptomics approach

Hepatotoxicity is one of the major reasons for withdrawal of drugs from the market. Therefore, there is a need to screen new drugs for hepatotoxicity in humans at an earlier stage. The aim of this study was to validate human precision-cut liver slices (PCLS) as an ex vivo model to predict drug-induced cholestasis and identify the possible mechanisms of cholestasis-induced toxicity using gene expression profiles. Five hepatotoxicants, which are known to induce cholestasis (alpha-naphthyl isothiocyanate, chlorpromazine, cyclosporine, ethinyl estradiol and methyl testosterone) were used at concentrations inducing low (<30 %) and medium (30-50 %) toxicity, based on ATP content. Human PCLS were incubated with the drugs in the presence of a non-toxic concentration (60 µM) of a bile acid mixture (portal vein concentration and composition) as model for bile acid-induced cholestasis. Regulated genes include bile acid transporters and cholesterol transporters. Pathway analysis revealed that hepatic cholestasis was among the top ten regulated pathways, and signaling pathways such as farnesoid X receptor- and liver X receptor-mediated responses, which are known to play a role in cholestasis, were significantly affected by all cholestatic compounds. Other significantly affected pathways include unfolded protein response and protein ubiquitination implicating the role of endoplasmic reticulum stress. This study shows that human PCLS incubated in the presence of a physiological bile acid mixture correctly reflect the pathways affected in drug-induced cholestasis in the human liver. In the future, this human PCLS model can be used to identify cholestatic adverse drug reactions of new chemical entities

Development of a best practice manual in wine tourism in Portugal

Portugal has become a popular tourist destination in the recent years, encouraging the development of wine tourism. With increased demand and greater competition among wineries, managers must enhance wine tourism experiences to attract high-value customers in order to make wine tourism profitable and sustainable. One option is to have an appropriate set of standards to implement best practices in service, hospitality and overall experience. This research paper focuses on the development of a knowledge tool, in the form of a best practice manual. With the support of ViniPortugal, an inter-professional organization dedicated to promoting Portuguese wines, 63 wineries were selected to participate in this project, of which 47 confirmed their participation and provided the basis for the project findings. This paper will show the development behind a comprehensive best practice manual, based on literary theory, research, and real life evidence in wine tourism experiences in Portugal. The framework used for the best practice manual was an adaptation of the Knowledge-To-Action framework [1], which is divided into two sections, Knowledge Creation and Action Cycle. The project methodology is based on the Knowledge Creation format as follows: knowledge inquiry, knowledge synthesis and finally knowledge tool. By accumulating knowledge from different sources, the finished manual will be a powerful tool that provides management with practices, standards, and protocols in all areas of the wine tourism offer. The application, limitations and future use of the best practice manual are also discussed

Evolution de la composition en polyamines des baies de raisin au cours du processus d'infection par Botrytis cinerea

Evolution of polyamine composition in grape berries during infection with Botrytis cinereaPolyamines are growth regulators occurring naturally in grapevine(Vitis vinifera L.) and pathogenic fungi, e.g. Botrytis cinerea. Investigation of polyamines of in vitro-grown Botrytis cinerea mycelium and infected berries has shown modifications in the metabolism of the berries which are directly related to the development of the fungi in the berries. The abnormal polyamine concentrations in infected berries appear to be of fungal origin for free polyamines and of plant origin for conjugated polyamines. The specific role of each type of polyamine is discussed with regard to the host-parasite relation.

Real-time navigation by fluorescence-based enhanced reality for precise estimation of future anastomotic site in digestive surgery.

Fluorescence-based enhanced reality (FLER) is a technique to evaluate intestinal perfusion based on the elaboration of the Indocyanine Green fluorescence signal. The aim of the study was to assess FLER's performances in evaluating perfusion in an animal model of long-lasting intestinal ischemia. An ischemic segment was created in 18 small bowel loops in 6 pigs. After 2 h (n = 6), 4 h (n = 6), and 6 h (n = 6), loops were evaluated clinically and by FLER to delineate five regions of interest (ROIs): ischemic zone (ROI 1), presumed viable margins (ROI 2a-2b), and vascularized areas (3a-3b). Capillary lactates were measured to compare clinical vs. FLER assessment. Basal (V 0 ) and maximal (V max) mitochondrial respiration rates were determined according to FLER. Lactates (mmol/L) at clinically identified resection lines were significantly higher when compared to those identified by FLER (2.43 ± 0.95 vs. 1.55 ± 0.33 p = 0.02) after 4 h of ischemia. Lactates at 2 h at ROI 1 were 5.45 ± 2.44 vs. 1.9 ± 0.6 (2a-2b; p &lt; 0.0001) vs. 1.2 ± 0.3 (3a-3b; p &lt; 0.0001). At 4 h, lactates were 4.36 ± 1.32 (ROI 1) vs. 1.83 ± 0.81 (2a-2b; p &lt; 0.0001) vs. 1.35 ± 0.67 (3a-3b; p &lt; 0.0001). At 6 h, lactates were 4.16 ± 2.55 vs. 1.8 ± 1.2 vs. 1.45 ± 0.83 at ROI 1 vs. 2a--2b (p = 0.013) vs. 3a-3b (p = 0.0035). Mean V 0 and V max (pmolO2/second/mg of tissue) were significantly impaired after 4 and 6 h at ROI 1 (V 0 (4h) = 34.83 ± 10.39; V max (4h) = 76.6 ± 29.09; V 0 (6h) = 44.1 ± 12.37 and V max (6h) = 116.1 ± 40.1) when compared to 2a--2b (V 0 (4h) = 67.1 ± 17.47 p = 0.00039; V max (4h) = 146.8 ± 55.47 p = 0.0054; V 0 (6h) = 63.9 ± 28.99 p = 0.03; V max (6h) = 167.2 ± 56.96 p = 0.01). V 0 and V max were significantly higher at 3a-3b. FLER may identify the future anastomotic site even after repetitive assessments and long-standing bowel ischemia

Synthesis of new N,N′-bis[1-aryl-3-(piperidine-1-yl)propylidene] hydrazine dihydrochlorides and evaluation of their cytotoxicity against human hepatoma and breast cancer cells

N,N0-Bis[1-aryl-3-(piperidine-1-yl)propylidene]hydrazine dihydrochlorides were synthesized by the reaction of 2 mols of 1-aryl-3-(piperidine-1-yl)-1- propanone hydrochlorides with 1 mol of hydrazine hydrate. Aryl part was C 6H5 (P1), 4-CH3C6H4 (P2), 4-CH3OC6H4 (P3), 4-HOC6H 4 (P4), 4-ClC6H4 (P5), 3-CH3OC 6H4 (P6), 4-FC6H4 (P7) and 4-BrC6H4 (P8). Except P1, all compounds were reported for the first time. The chemical structures were confirmed by UV, 1H NMR, 13C NMR and HRMS spectra. P1, P2, P7 and P8 against human hepatoma (Huh7) cells and P1, P2, P4, P5, P6, P7 and P8 against breast cancer (T47D) cells have shown cytotoxicity. P1, P2 and P7 had more potent cytotoxicity against Huh7 cells than the reference compound 5-FU, whereas only P2 was more potent than the 5-FU against T47D cells. Representative compound P7 inhibited the mitochondrial respiration at 144, 264 and 424 mM concentrations dose-dependantly in liver homogenates. The results suggest that P1, P2, P7 and P8 may serve as model compounds for further synthetic studies. © 2014 Informa UK Ltd

Structural insights into Clostridium perfringens delta toxin pore formation

Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins

Developmental control of hypoxia during bud burst in grapevine

Dormant or quiescent buds of woody perennials are often dense, and in the case of grapevine (Vitis vinifera L.) have a low tissue oxygen status. The precise timing of the decision to resume growth is difficult to predict, but once committed the increase in tissue oxygen status is rapid and developmentally regulated. Here we show that more than a third of the grapevine homologues of widely conserved hypoxia-responsive genes, and nearly a fifth of all grapevine genes possessing a plant hypoxia-responsive promoter element were differentially regulated during bud burst, in apparent harmony with resumption of meristem identity and cell-cycle gene regulation. We then investigated the molecular and biochemical properties of the grapevine ERF-VII homologues, which in other species are oxygen labile and function in transcriptional regulation of hypoxia-responsive genes. Each of the three VvERF-VIIs were substrates for oxygen-dependent proteolysis in vitro, as a function of the N-terminal cysteine. Collectively these data support an important developmental function of oxygen-dependent signalling in determining the timing and effective coordination bud burst in grapevine. In addition, novel regulators, including GASA-, TCP-, MYB3R-, PLT- and WUS-like transcription factors, were identified as hallmarks of the orderly and functional resumption of growth following quiescence in buds

Proteomic analysis of the effects of ABA treatments on ripening Vitis vinifera berries

The control of ripening of the non-climacteric grapevine fruit is still a matter of debate, but several lines of evidence point to an important role for the hormone abscisic acid (ABA). The effects of ABA treatments on Cabernet Sauvignon berries before and at véraison were studied using a 2-DE proteomic approach. Proteins from whole deseeded berries (before véraison) and berry flesh and skin (at véraison) treated with 0.76 mM ABA and collected 24 h after treatment were separated and analysed. A total of 60 protein spots showed significant variations between treated and control berries, and 40 proteins, mainly related to general metabolism and cell defence, were identified by LC MS/MS. Our results show that ABA acts mainly through the regulation of mostly the same proteins which are involved in the ripening process, and that several of these changes share common elements with the ABA-induced responses in vegetative tissues

Diabetes Worsens Skeletal Muscle Mitochondrial Function, Oxidative Stress, and Apoptosis After Lower-Limb Ischemia-Reperfusion: Implication of the RISK and SAFE Pathways?

Objectives: Diabetic patients respond poorly to revascularization for peripheral arterial disease (PAD) but the underlying mechanisms are not well understood. We aimed to determine whether diabetes worsens ischemia-reperfusion (IR)-induced muscle dysfunction and the involvement of endogenous protective kinases in this process. Materials and Methods: Streptozotocin-induced diabetic and non-diabetic rats were randomized to control or to IR injury (3 h of aortic cross-clamping and 2 h of reperfusion). Mitochondrial respiration, reactive oxygen species (ROS) production, protein levels of superoxide dismutase (SOD2) and endogenous protective kinases (RISK and SAFE pathways) were investigated in rat gastrocnemius, together with upstream (GSK-3β) and downstream (cleaved caspase-3) effectors of apoptosis. Results: Although already impaired when compared to non-diabetic controls at baseline, the decline in mitochondrial respiration after IR was more severe in diabetic rats. In diabetic animals, IR-triggered oxidative stress (increased ROS production and reduced SOD2 levels) and effectors of apoptosis (reduced GSK-3β inactivation and higher cleaved caspase-3 levels) were increased to a higher level than in the non-diabetics. IR had no effect on the RISK pathway in non-diabetics and diabetic rats, but increased STAT 3 only in the latter. Conclusion: Type 1 diabetes worsens IR-induced skeletal muscle injury, endogenous protective pathways not being efficiently stimulated