Strategy for the identification of micro-organisms producing food and feed products : bacteria producing food enzymes as study case

Recent European regulations require safety assessments of food enzymes (FE) before their commercialization. FE are mainly produced by micro-organisms, whose viable strains nor associated DNA can be present in the final products. Currently, no strategy targeting such impurities exists in enforcement laboratories. Therefore, a generic strategy of first line screening was developed to detect and identify, through PCR amplification and sequencing of the 16S-rRNA gene, the potential presence of FE producing bacteria in FE preparations. First, the specificity was verified using all microbial species reported to produce FE. Second, an in-house database, with 16S reference sequences from bacteria producing FE, was constructed for their fast identification through blast analysis. Third, the sensitivity was assessed on a spiked FE preparation. Finally, the applicability was verified using commercial FE preparations. Using straightforward PCR amplifications, Sanger sequencing and blast analysis, the proposed strategy was demonstrated to be convenient for implementation in enforcement laboratories

COA6 facilitates cytochrome c oxidase biogenesis as thiol-reductase for copper metallochaperones in mitochondria.

The mitochondrial cytochrome c oxidase, the terminal enzyme of the respiratory chain, contains heme and copper centers for electron transfer. The conserved COX2 subunit contains the CuA site, a binuclear copper center. The copper chaperones SCO1, SCO2, and COA6 are required for CuA center formation. Loss of function of these chaperones and the concomitant cytochrome c oxidase deficiency cause severe human disorders. Here we analyzed the molecular function of COA6 and the consequences of COA6 deficiency for mitochondria. Our analyses show that loss of COA6 causes combined complex I and complex IV deficiency and impacts membrane potential driven protein transport across the inner membrane. We demonstrate that COA6 acts as a thiol-reductase to reduce disulphide bridges of critical cysteine residues in SCO1 and SCO2. Cysteines within the CX3CXNH domain of SCO2 mediate its interaction with COA6 but are dispensable for SCO2-SCO1 interaction. Our analyses define COA6 as thiol-reductase, which is essential for CuA biogenesis

Vanadium (β-(Dimethylamino)ethyl)cyclopentadienyl Complexes with Diphenylacetylene Ligands

Reduction of the V(III) (β-(dimethylamino)ethyl)cyclopentadienyl dichloride complex [η5:η1-C5H4(CH2)2NMe2]VCl2(PMe3) with 1 equiv of Na/Hg yielded the V(II) dimer {[η5:η1-C5H4(CH2)2NMe2]V(µ-Cl)}2 (2). This compound reacted with diphenylacetylene in THF to give the V(II) alkyne adduct [η5:η1-C5H4(CH2)2NMe2]VCl(η2-PhC≡CPh). Further reduction of 2 with Mg in the presence of diphenylacetylene resulted in oxidative coupling of two diphenylacetylene groups to yield the diamagnetic, formally V(V), bent metallacyclopentatriene complex [η5:η1-C5H4(CH2)2NMe2]V(C4Ph4).

Ultrasound and microbubbles for the treatment of ocular diseases : From preclinical research towards clinical application

The unique anatomy of the eye and the presence of various biological barriers make efficacious ocular drug delivery challenging, particularly in the treatment of posterior eye diseases. This review focuses on the combination of ultrasound and microbubbles (USMB) as a minimally invasive method to improve the efficacy and targeting of ocular drug delivery. An extensive overview is given of the in vitro and in vivo studies investigating the mechanical effects of ultrasound-driven microbubbles aiming to: (i) temporarily disrupt the blood–retina barrier in order to enhance the delivery of systemically administered drugs into the eye, (ii) induce intracellular uptake of anticancer drugs and macromolecules and (iii) achieve targeted delivery of genes, for the treatment of ocular malignancies and degenerative diseases. Finally, the safety and tolerability aspects of USMB, essential for the translation of USMB to the clinic, are discussed.Peer reviewe

Positive exercise test?

markdownabstractA 46-year-old male was referred for exercise testing after he was evaluated in the emergency department because of progressive fatigue, dyspnoea on exertion and chest pain with tingling of the left arm. He was an otherwise healthy man who was a professional climber. He had no cardiovascular risk factors and was not using any medication. On physical examination he was normotensive and the cardiovascular examination was normal. His troponin level was normal. A bicycle exercise test was performed to rule out ischaemia. He exercised for 13 min and 40 sec, reaching 242 Watt (reference value 208 Watt) with a maximum heart rate of 167 beats/min (96% of predicted maximal heart rate) and a blood pressure of 187/92 mmHg. The test was stopped because of fatigue. He had no chest pain. Two recordings of the exercise test are shown here (Fig. 1a, b). __Questions__ Is this a positive test for ischaemia? What is the prognosis

Dynamic fluorescence microscopy of cellular uptake of intercalating model drugs by ultrasound-activated microbubbles

The combination of ultrasound and microbubbles can facilitate cellular uptake of (model) drugs via transient permeabilization of the cell membrane. By using fluorescent molecules, this process can be studied conveniently with confocal fluorescence microscopy. This study aimed to investigate the relation between cellular uptake and fluorescence intensity increase of intercalating model drugs. SYTOX Green, an intercalating fluorescent dye that displays > 500-fold fluorescence enhancement upon binding to nucleic acids, was used as a model drug for ultrasound-induced cellular uptake. SYTOX Green uptake was monitored in high spatiotemporal resolution to qualitatively assess the relation between uptake and fluorescence intensity in individual cells. In addition, the kinetics of fluorescence enhancement were studied as a function of experimental parameters, in particular, laser duty cycle (DC), SYTOX Green concentration and cell line. Ultrasound-induced intracellular SYTOX Green uptake resulted in local fluorescence enhancement, spreading throughout the cell and ultimately accumulating in the nucleus during the 9-min acquisition. The temporal evolution of SYTOX Green fluorescence was substantially influenced by laser duty cycle: continuous laser (100 % DC) induced a 6.4-fold higher photobleaching compared to pulsed laser (3.3 % DC), thus overestimating the fluorescence kinetics. A positive correlation of fluorescence kinetics and SYTOX Green concentration was found, increasing from 0.6 x 10(-3) to 2.2 x 10(-3) s(-1) for 1 and 20 mu M, respectively. Finally, C6 cells displayed a 2.4-fold higher fluorescence rate constant than FaDu cells. These data show that the temporal behavior of intracellular SYTOX Green fluorescence enhancement depends substantially on nuclear accumulation and not just on cellular uptake. In addition, it is strongly influenced by the experimental conditions, such as the laser duty cycle, SYTOX Green concentration, and cell line