Characterization of two bacterial multi-flavinylated proteins harboring multiple covalent flavin cofactors

In recent years, studies have shown that a large number of bacteria secrete multi-flavinylated proteins. The exact roles and properties, of these extracellular flavoproteins that contain multiple covalently anchored FMN cofactors, are still largely unknown. Herein, we describe the biochemical and structural characterization of two multi-FMN-containing covalent flavoproteins, SaFMN3 from Streptomyces azureus and CbFMN4 from Clostridiaceae bacterium. Based on their primary structure, these proteins were predicted to contain three and four covalently tethered FMN cofactors, respectively. The genes encoding SaFMN3 and CbFMN4 were heterologously coexpressed with a flavin transferase (ApbE) in Escherichia coli, and could be purified by affinity chromatography in good yields. Both proteins were found to be soluble and to contain covalently bound FMN molecules. The SaFMN3 protein was studied in more detail and found to display a single redox potential (-184 mV) while harboring three covalently attached flavins. This is in line with the high sequence similarity when the domains of each flavoprotein are compared. The fully reduced form of SaFMN3 is able to use dioxygen as electron acceptor. Single domains from both proteins were expressed, purified and crystallized. The crystal structures were elucidated, which confirmed that the flavin cofactor is covalently attached to a threonine. Comparison of both crystal structures revealed a high similarity, even in the flavin binding pocket. Based on the crystal structure, mutants of the SaFMN3-D2 domain were designed to improve its fluorescence quantum yield by changing the microenvironment of the isoalloxazine moiety of the flavin cofactor. Residues that quench the flavin fluorescence were successfully identified. Our study reveals biochemical details of multi-FMN-containing proteins, contributing to a better understanding of their role in bacteria and providing leads to future utilization of these flavoprotein in biotechnology.</p

X-ray structure of bovine pancreatic phospholipase A(2) at atomic resolution

Using synchrotron radiation and a CCD camera, X-ray data have been collected from wild-type bovine pancreatic phospholipase A(2) at 100 K to 0.97 Angstrom resolution allowing full anisotropic refinement. The final model has a conventional R factor of 9.44% for all reflections, with a mean standard uncertainty for the positional parameters of 0.031 Angstrom as calculated from inversion of the full positional least-squares matrix. At 0.97 Angstrom resolution, bovine pancreatic phospholipase A(2) reveals for the first time that its rigid scaffolding does not preclude flexibility, which probably plays an important role in the catalytic process. Functionally important regions (the interfacial binding site and calcium-binding loop) are located at the molecular surface, where conformational variability is more pronounced. A cluster of 2-methyl-2,4-pentanediol molecules is present at the entrance of the hydrophobic channel that leads to the catalytic site and mimics the fatty-acid chains of a substrate analogue. Bovine pancreatic phospholipase A(2) at atomic resolution is compared with previous crystallographic structures and with models derived from nuclear magnetic resonance studies. Given the high structural similarity among extracellular phospholipases A(2) observed so far at lower resolution, the results arising from this structural analysis are expected to be of general validity for this class of enzymes

Feasibility of joystick guided colonoscopy

The flexible endoscope is increasingly used to perform minimal invasive interventions. A novel add-on platform allows single-person control of both endoscope and instrument at the site of intervention. The setup changes the current routine of handling the endoscope. This study aims to determine if the platform allows effective and efficient manipulation to position the endoscope at potential intervention sites throughout the bowel. Five experts in flexible endoscopy first performed three colonoscopies on a computer simulator using the conventional angulation wheels. Next they trained with the joystick interface to achieve their personal level of intubation time with low pain score. 14 PhD students (novices) without hands-on experience performed the same colonoscopy case using either the conventional angulation wheels or joystick interface. Both novice groups trained to gain the average expert level. The cecal intubation time, pain score and visualization performance (% of bowel wall) were recorded. All experts reached their personal intubation time in 6 ± 6 sessions. Three experts completed their learning curve with low pain score in 8 ± 6 sessions. The novices required 11 ± 6 sessions using conventional angulation wheels, and 12 ± 6 sessions using the joystick interface. There was no difference in the visualization performance between the novice and between the expert groups. This study shows that the add-on platform enables endoscope manipulation required to perform colonoscopy. Experts need only a relatively short training period. Novices are as effective and as efficient in endoscope manipulation when comparing the add-on platform with conventional endoscope contro

Characterization of Two VAO-Type Flavoprotein Oxidases from Myceliophthora thermophila

The VAO flavoprotein family consists mostly of oxidoreductases harboring a covalently linked flavin cofactor. The linkage can be either monocovalent at position 8 with a histidine or tyrosine or bicovalent at position 8 with a histidine and at position 6 with a cysteine. Bicovalently bound flavoproteins show a preference for bulkier substrates such as oligosaccharides or secondary metabolites. The genome of the thermophilic fungus Myceliophthora thermophila C1 was found to be rich in genes encoding putative covalent VAO-type flavoproteins. Enzymes from this fungus have the advantage of being rather thermostable and homologous overexpression in M. thermophila C1 is feasible. Recently we discovered a new and VAO-type carbohydrate oxidase from this fungus: xylooligosaccharide oxidase. In this study, two other putative VAO-type oxidases, protein sequence XP_003663615 (MtVAO615) and XP_003665713 (MtVAO713), were expressed in M. thermophila C1, purified and characterized. Enzyme MtVAO615 was found to contain a bicovalently bound FAD, while enzyme MtVAO713 contained a monocovalent histidyl-bound FAD. The crystal structures of both proteins were obtained which revealed atypical active site architectures. It could be experimentally verified that both proteins, when reduced, rapidly react with molecular oxygen, a hallmark of flavoprotein oxidases. A large panel of alcohols, including carbohydrates, steroids and secondary alcohols were tested as potential substrates. For enzyme MtVAO713 low oxidase activity was discovered towards ricinoleic acid

Damaging real lives through obstinacy: re-emphasising why significance testing is wrong

This paper reminds readers of the absurdity of statistical significance testing, despite its continued widespread use as a supposed method for analysing numeric data. There have been complaints about the poor quality of research employing significance tests for a hundred years, and repeated calls for researchers to stop using and reporting them. There have even been attempted bans. Many thousands of papers have now been written, in all areas of research, explaining why significance tests do not work. There are too many for all to be cited here. This paper summarises the logical problems as described in over 100 of these prior pieces. It then presents a series of demonstrations showing that significance tests do not work in practice. In fact, they are more likely to produce the wrong answer than a right one. The confused use of significance testing has practical and damaging consequences for people's lives. Ending the use of significance tests is a pressing ethical issue for research. Anyone knowing the problems, as described over one hundred years, who continues to teach, use or publish significance tests is acting unethically, and knowingly risking the damage that ensues

ApoE4 disrupts interaction of sortilin with fatty acid-binding protein 7 essential to promote lipid signaling

Sortilin is a neuronal receptor for apolipoprotein E. Sortilin-dependent uptake of lipidated apoE promotes conversion of polyunsaturated fatty acids (PUFA) into neuromodulators that induce anti-inflammatory gene expression in the brain. This neuroprotective pathway works with apoE3 but is lost with apoE4, the main risk factor for Alzheimer's disease (AD). Here, we elucidated steps in cellular handling of lipids through sortilin, and why they are disrupted by apoE4. Combining unbiased proteome screens with analyses in mouse models, we uncover interaction of sortilin with fatty acid-binding protein (FABP) 7, the intracellular carrier for PUFA in the brain. In the presence of apoE3, sortilin promotes functional expression of FABP7 and its ability to elicit lipid-dependent gene transcription. By contrast, apoE4 binding blocks sortilin sorting, causing catabolism of FABP7 and impairing lipid signaling. Reduced FABP7 levels in the brain of AD patients expressing apoE4 substantiate the relevance of these interactions for neuronal lipid homeostasis. Taken together, we document interaction of sortilin with mediators of extracellular and intracellular lipid transport that provides a mechanistic explanation for loss of a neuroprotective lipid metabolism in AD

Transmission of West Nile Virus by Culex quinquefasciatus Say Infected with Culex Flavivirus Izabal

Unlike most known flaviviruses (Family, Flaviviridae: Genus, Flavivirus), insect-only flaviviruses are a unique group of flaviviruses that only infect invertebrates. The study of insect-only flaviviruses has increased in recent years due to the discovery and characterization of numerous novel flaviviruses from a diversity of mosquito species around the world. The widespread discovery of these viruses has prompted questions regarding flavivirus evolution and the potential impact of these viruses on the transmission of flaviviruses of public health importance such as WNV. Therefore, we tested the effect of Culex flavivirus Izabal (CxFV Izabal), an insect-only flavivirus isolated from Culex quinquefasciatus mosquitoes in Guatemala, on the growth and transmission of a strain of WNV isolated concurrently from the same mosquito species and location. Prior infection of C6/36 (Aedes albopictus mosquito) cells or Cx. quinquefasciatus with CxFV Izabal did not alter the replication kinetics of WNV, nor did it significantly affect WNV infection, dissemination, or transmission rates in two different colonies of mosquitoes that were fed blood meals containing varying concentrations of WNV. These data demonstrate that CxFV probably does not have a significant effect on WNV transmission efficiency in nature