Identification of new correctors for traffic-defective ABCB4 variants by a high-content screening approach.

ABCB4 is located at the canalicular membrane of hepatocytes and is responsible for the secretion of phosphatidylcholine into bile. Genetic variations of this transporter are correlated with rare cholestatic liver diseases, the most severe being progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 patients most often require liver transplantation. In this context of unmet medical need, we developed a high-content screening approach to identify small molecules able to correct ABCB4 molecular defects. Intracellularly-retained variants of ABCB4 were expressed in cell models and their maturation, cellular localization and function were analyzed after treatment with the molecules identified by high-content screening. In total, six hits were identified by high-content screening. Three of them were able to correct the maturation and canalicular localization of two distinct intracellularly-retained ABCB4 variants; one molecule was able to significantly restore the function of two ABCB4 variants. In addition, in silico molecular docking calculations suggest that the identified hits may interact with wild type ABCB4 residues involved in ATP binding/hydrolysis. Our results pave the way for their optimization in order to provide new drug candidates as potential alternative to liver transplantation for patients with severe forms of ABCB4-related diseases, including PFIC3.

In Vitro Rescue of the Bile Acid Transport Function of ABCB11 Variants by CFTR Potentiators.

ABCB11 is responsible for biliary bile acid secretion at the canalicular membrane of hepatocytes. Variations in the ABCB11 gene cause a spectrum of rare liver diseases. The most severe form is progressive familial intrahepatic cholestasis type 2 (PFIC2). Current medical treatments have limited efficacy. Here, we report the in vitro study of Abcb11 missense variants identified in PFIC2 patients and their functional rescue using cystic fibrosis transmembrane conductance regulator potentiators. Three ABCB11 disease-causing variations identified in PFIC2 patients (i.e., A257V, T463I and G562D) were reproduced in a plasmid encoding an Abcb11-green fluorescent protein. After transfection, the expression and localization of the variants were studied in HepG2 cells. Taurocholate transport activity and the effect of potentiators were studied in Madin-Darby canine kidney (MDCK) clones coexpressing Abcb11 and the sodium taurocholate cotransporting polypeptide (Ntcp/Slc10A1). As predicted using three-dimensional structure analysis, the three variants were expressed at the canalicular membrane but showed a defective function. Ivacaftor, GLP1837, SBC040 and SBC219 potentiators increased the bile acid transport of A257V and T463I and to a lesser extent, of G562D Abcb11 missense variants. In addition, a synergic effect was observed when ivacaftor was combined with SBC040 or SBC219. Such potentiators could represent new pharmacological approaches for improving the condition of patients with ABCB11 deficiency due to missense variations affecting the function of the transporter.

Out of hospital cardiac arrest: when to resuscitate.

INTRODUCTION: This study explores why resuscitation is withheld when mobile emergency medical team arrive at the scene of a cardiac arrest. METHODS: We conducted a prospective, observational study in pre hospital emergency services. We included adults' patients, with a suspicion of non-traumatic cardiac arrest (CA) in an out of hospital environment, who received or not cardiopulmonary resuscitation (CPR) by our mobile emergency medical service teams. An analytic study was conducted in order to identify independent factors that could influence the decision to resuscitate OHCA. RESULTS: During study, 228 patients were enrolled, the mean age was 64 +/- 14 years and 59% were men. Eighteen patients (8%) received bystander CPR by witnesses. The median time elapsed to arrive at the scene was 13 [8-25] min. The median "noflow" was 22 [10-34] min. The resuscitation decision was taken by the mobile EMS staff for 106 patients (46.5%). For other patients, the decision not to resuscitate was motivated solely by the finding of a confirmed state of death in an elderly patient (p = 0.045). The predictive decision factor for resuscitation was the no flow time less than 18.5 min, Odds Ratio adjusted with 95% confidence interval to: 1.38 (1.24 - 3.55) (p <0.001). Overall out of hospital survival rate was 17% of resuscitated patients. CONCLUSION: The decision to resuscitate a cardiac arrest outside of the hospital depends more on the "no flow" time than on the presumed etiologies.

Screening and characterization of thermo-active enzymes of biotechnological interest produced by thermophilic Bacillus isolated from hot springs in Tunisia.

As part of the contribution to the global efforts in research of thermostable enzymes being of industrial interest, we focus on the isolation of thermophilic bacteria from Tunisian hot springs. Among the collection of 161 strains of thermophilic Bacillus isolated from different samples of thermal water in Tunisia, 20% are capable of growing at 100°C and the rest grow at 70°C or above. Preliminary activity tests on media supplemented with enzyme-substrates confirmed that 35 strains produced amylases, 37 - proteases, 43 - cellulases, 31 - xylanases and 37 - mannanases. The study of the effect of temperature on enzyme activity led to determination of the optimal temperatures of activities that vary between 60 and 100°C. Several enzymes were active at high temperatures (80, 90 and 100°C) and kept their activity even at 110°C. Several isolated strains producing enzymes with high optimal temperatures of activity were described for the first time in this study. Both strains B62 and B120 are producers of amylase, protease, cellulase, xylanase, and mannanase. The sequencing of 16S DNA identified isolated strains as Geobacillus kaustophillus, Aeribacillus pallidus, Geobacillus galactosidasus and Geobacillus toebii.

Purification and Characterization of a New Thermostable, Haloalkaline, Solvent Stable, and Detergent Compatible Serine Protease from Geobacillus toebii Strain LBT 77.

A new thermostable, haloalkaline, solvent stable SDS-induced serine protease was purified and characterized from a thermophilic Geobacillus toebii LBT 77 newly isolated from a Tunisian hot spring. This study reveals the potential of the protease from Geobacillus toebii LBT 77 as an additive to detergent with spectacular proprieties described for the first time. The protease was purified to homogeneity by ammonium sulfate precipitation followed by Sephadex G-75 and DEAE-Cellulose chromatography. It was a monomeric enzyme with molecular weight of 30 kDa. The optimum pH, temperature, and NaCl for maximum protease activity were 13.0, 95°C, and 30%, respectively. Activity was stimulated by Ca(2+), Mg(2+), DTNB, ß-mercaptoethanol, and SDS. The protease was extremely stable even at pH 13.25, 90°C, and 30% NaCl and in the presence of hydrophilic, hydrophobic solvents at high concentrations. The high compatibility with ionic, nonionic, and commercial detergents confirms the utility as an additive to cleaning products. Kinetic and thermodynamic characterization of protease revealed K m = 1 mg mL(-1), V max = 217.5 U mL(-1), K cat/K m = 99 mg mL(-1) S(-1), E a = 51.5 kJ mol(-1), and ΔG (⁎) = 56.5 kJ mol(-1).

[Systemic BCG reactions after intravesical BCG therapy. A report of four cases]. / Réactions systémiques après traitement endovésical par le BCG: à propos de 4 cas.

Local Bacillus Calmette-Guerin (BCG) immunotherapy is an effective and widely used treatment for superficial bladder carcinoma. Local side effects are frequent, whereas systemic side effects are rare, but more serious. We report four cases of systemic BCG reaction. Although uncommon, this infectious complication of BCG therapy should always be considered in the appropriate clinical setting. The best approach to minimize this complication is a strict compliance with precautions and a close and rigorous surveillance of this drug.

[Not Available]. / Réactions systémiques après traitement endovésical par le BCG : à propos de 4 cas.

Local Bacillus Calmette-Guerin (BCG) immunotherapy is an effective and widely used treatment for superficial bladder carcinoma. Local side effects are frequent, whereas systemic side effects are rare, but more serious. We report four cases of systemic BCG reaction. Although uncommon, this infectious complication of BCG therapy should always be considered in the appropriate clinical setting. The best approach to minimize this complication is a strict compliance with precautions and a close and rigorous surveillance of this drug.