High-Frequency-Induced Cathodic Breakdown during Plasma Electrolytic Oxidation

The present communication shows the possibility of observing microdischarges under cathodic polarization during plasma electrolytic oxidation at high frequency. Cathodic microdischarges can ignite beyond a threshold frequency found close to 2 kHz. The presence (respectively, absence) of an electrical double layer is put forward to explain how the applied voltage can be screened, which therefore prevents (respectively, promotes) the ignition of a discharge. Interestingly, in the conditions of the present study, the electrical double layer requires between 175 and 260 μs to form. This situates the expected threshold frequency between 1.92 and 2.86 kHz, which is in good agreement with the value obtained experimentally

High speed video evidence for localised discharge cascades during plasma electrolytic oxidation

Information is presented from high speed video imaging of the free surface of coatings being grown on aluminium substrates by PEO processing. The exposure time during image capture ranged down to 5.5 μs, while the linear spatial resolution of the images ranged upwards from about 12 μm. The area being viewed was about 2.4 mm2, which was taken to be representative of the substrate area as a whole (~ 129 mm2). PEO processing was carried out at 50 Hz AC. The periods over which image sequences were captured was about 100 ms, covering several cycles of variation of the applied potential. This operation was repeated periodically while the coating thickness increased from a few microns to several tens of microns. During the imaging periods, it was typically observed that tens or hundreds of individual discharges were occurring, all of them readily distinguishable from the background light levels. Their duration was of the order of several tens of microseconds. It was noticeable that they tended to occur in “cascades” at particular locations, each sequence comprising tens or hundreds of individual discharges, with an “incubation” period between them of the order of several hundreds of microseconds. It seems likely that they all occurred during the positive (anodic) half-cycle, while the applied voltage was sufficiently high. An individual cascade tended to persist (at the same location) over several voltage cycles. As the coating became thicker, these characteristics broadly persisted, although individual discharges became longer-lived and more energetic. An attempt is made to relate these observations to the overall picture of how coating growth takes place during PEO processing, and also to the overall energy consumption.This work has been supported by EPSRC (grant number EP/I001174/1), by a Sims Scholarship (for SCT) in Cambridge University and by Keronite plc. The research also forms part of the activities of the COST TD 1208 Network.This is the final published version. It first appeared at http://www.sciencedirect.com/science/article/pii/S0257897215000778#

Synchronised electrical monitoring and high speed video of bubble growth associated with individual discharges during plasma electrolytic oxidation

Synchronised electrical current and high speed video information are presented from individual discharges on Al substrates during PEO processing. Exposure time was 8 μs and linear spatial resolution 9 μm. Image sequences were captured for periods of 2 s, during which the sample surface was illuminated with short duration flashes (revealing bubbles formed where the discharge reached the surface of the coating). Correlations were thus established between discharge current, light emission from the discharge channel and (externally-illuminated) dimensions of the bubble as it expanded and contracted. Bubbles reached radii of 500 μm, within periods of 100 μs, with peak growth velocity about 10 m/s. It is deduced that bubble growth occurs as a consequence of the progressive volatilisation of water (electrolyte), without substantial increases in either pressure or temperature within the bubble. Current continues to flow through the discharge as the bubble expands, and this growth (and the related increase in electrical resistance) is thought to be responsible for the current being cut off (soon after the point of maximum radius). A semi-quantitative audit is presented of the transformations between different forms of energy that take place during the lifetime of a discharge.This work has been supported by EPSRC (grant number EP/I001174/1), by a Sims Scholarship (for SCT) in Cambridge University and by Keronite plc. The research also forms part of the activities of the COST TD 1208 Network. Thanks are due to Steve Hutchins and Suman Shrestha, of Keronite, for many helpful discussions. The technical assistance of Fréderic Brochard (Nancy) and Kevin Roberts (Cambridge) is also gratefully acknowledged.This is the author accepted manuscript. The final version is available from Elsevier via http://dx.doi.org/10.1016/j.apsusc.2015.10.124 In compliance with EPSRC requirements, raw data in the form of selected video and discharge current files are available at www.ccg.msm.cam.ac.uk/publications/resources, and are also accessible via the University repository at http://www.data.cam.ac.uk/repository

A review of recent work on discharge characteristics during plasma electrolytic oxidation of various metals

The review describes recent progress on understanding and quantification of the various phenomena that take place during plasma electrolytic oxidation, which is in increasing industrial use for production of protective coatings and other surface treatment purposes. A general overview of the process, and some information about usage of these coatings, are provided in the first part of the review. The focus is then on the dielectric breakdown that repeatedly occurs over the surface of the work-piece. These discharges are central to the process, since it is largely via the associated plasmas that oxidation of the substrate takes place and the coating is created. The details are complex, since the discharge characteristics are affected by a number of processing variables. The inter-relationships between electrical conditions, electrolyte composition, coating microstructure and rates of growth, which are linked via the characteristics of the discharges, have become clearer over recent years and these improvements in understanding are summarized here. There is considerable scope for more effective process control, with specific objectives in terms of coating performance and energy efficiency, and an attempt is made to identify key points that are likely to assist this

FFAT motif phosphorylation controls formation and lipid transfer function of inter‐organelle contacts

Organelles are physically connected in membrane contact sites. The endoplasmic reticulum possesses three major receptors, VAP‐A, VAP‐B, and MOSPD2, which interact with proteins at the surface of other organelles to build contacts. VAP‐A, VAP‐B, and MOSPD2 contain an MSP domain, which binds a motif named FFAT (two phenylalanines in an acidic tract). In this study, we identified a non‐conventional FFAT motif where a conserved acidic residue is replaced by a serine/threonine. We show that phosphorylation of this serine/threonine is critical for non‐conventional FFAT motifs (named Phospho‐FFAT) to be recognized by the MSP domain. Moreover, structural analyses of the MSP domain alone or in complex with conventional and Phospho‐FFAT peptides revealed new mechanisms of interaction. Based on these new insights, we produced a novel prediction algorithm, which expands the repertoire of candidate proteins with a Phospho‐FFAT that are able to create membrane contact sites. Using a prototypical tethering complex made by STARD3 and VAP, we showed that phosphorylation is instrumental for the formation of ER‐endosome contacts, and their sterol transfer function. This study reveals that phosphorylation acts as a general switch for inter‐organelle contacts

Protein Expr Purif

E6 is a small oncoprotein involved in tumorigenesis induced by papillomaviruses (PVs). E6 often recognizes its cellular targets by binding to short motifs presenting the consensus LXXLL. E6 proteins have long resisted structural analysis. We found that bovine papillomavirus type 1 (BPV1) E6 binds the N-terminal LXXLL motif of the cellular protein paxillin with significantly higher affinity as compared to other E6/peptide interactions. Although recombinant BPV1 E6 was poorly soluble in the free state, provision of the paxillin LXXLL peptide during BPV1 E6 biosynthesis greatly enhanced the protein's solubility. Expression of BPV1 E6/LXXLL peptide complexes was carried out in bacteria in the form of triple fusion constructs comprising, from N- to C-terminus, the soluble carrier protein maltose binding protein (MBP), the LXXLL motif and the E6 protein. A TEV protease cleavage site was placed either between MBP and LXXLL motif or between LXXLL motif and E6. These constructs allowed us to produce highly concentrated samples of BPV1 E6, either covalently fused to the C-terminus of the LXXLL motif (intra-molecular complex) or non-covalently bound to it (inter-molecular complex). Heteronuclear NMR measurements were performed and showed that the E6 protein was folded with similar conformations in both covalent and non-covalent complexes. These data open the way to novel structural and functional studies of the BPV1 E6 in complex with its preferential target motif

TRAF4 is a novel phosphoinositide-binding protein modulating tight junctions and favoring cell migration

Tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4) is frequently overexpressed in carcinomas, suggesting a specific role in cancer. Although TRAF4 protein is predominantly found at tight junctions (TJs) in normal mammary epithelial cells (MECs), it accumulates in the cytoplasm of malignant MECs. How TRAF4 is recruited and functions at TJs is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)-binding domain crucial for its recruitment to TJs. Of interest, this property is shared by the other members of the TRAF protein family. Indeed, the TRAF domain of all TRAF proteins (TRAF1 to TRAF6) is a bona fide PIP-binding domain. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer that binds up to three lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts as a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJs and favoring cell migration