A contact area function for Berkovich nanoindentation : Application to hardness determination of a TiHfCN thin film

In nanoindentation, especially at very low indenter displacements, the indenter/material contact area must be defined in the best possible way in order to accurately determine the mechanical properties of the material. One of the best methodologies for the computation of the contact area has been proposed by Oliver and Pharr [W.C.Oliver, G.M.Pharr, J.Mater. Res. 7 (1992) 1564], which involves a complex phenomenological area function. Unfortunately, this formulation is only valid when the continuous stiffness measurement mode is employed. For other conditions of indentation, different contact area functions, which take into account the effective truncation length or the radius of the rounded indentertip, as well as some fitting parameters, have been proposed. However, most of these functions require a calibration procedure due to the presence of such parameters. To avoid such a calibration, in the present communication a contact area function only related to the truncation length representative of the indenter tip defect, which can be previously estimated with high resolution microscopy, has been proposed. This model allows the determination of consistent indentation data from indenter displacements of only few nanometers indepth. When this proposed contact area function is applied to the mechanical characterization of a TiHfCN film of 2.6 μm in thickness deposited onto a tool steel substrate, the direct determination of the hardness and elastic modulus of the film leads to values of 35.5±2 GPa and 490±50 GPa, respectively

Hardness Evaluation of Porous Hydroxyapatite Coating

The extensive use of appropriate coatings to improve wear resistance, friction coefficient, electrical properties, corrosion resistance and biomedical application has stimulated a growing interest in their mechanical properties and especially hardness testing that is routinely used for coating evaluation. In this study Jönsson and Hogmark model is applied for the porous hydroxyapatite produced by plasma spraying on Ti6A14V substrate. Firstly, the effect of indentation load on hardness values of coating and substrate are studied. The modified Jönsson and Hogmark model is used to explain the composite hardness behavior and the effect of coating porosity

Optimisation of Over-Expression in E. coli and Biophysical Characterisation of Human Membrane Protein Synaptogyrin 1

Progress in functional and structural studies of integral membrane proteins (IMPs) is lacking behind their soluble counterparts due to the great challenge in producing stable and homogeneous IMPs. Low natural abundance, toxicity when over-expressed and potential lipid requirements of IMPs are only a few reasons for the limited progress. Here, we describe an optimised workflow for the recombinant over-expression of the human tetraspan vesicle protein (TVP) synaptogyrin in Escherichia coli and its biophysical characterisation. TVPs are ubiquitous and abundant components of vesicles. They are believed to be involved in various aspects of the synaptic vesicle cycle, including vesicle biogenesis, exocytosis and endocytotic recycling. Even though TVPs are found in most cell types, high-resolution structural information for this class of membrane proteins is still missing. The optimisation of the N-terminal sequence of the gene together with the usage of the recently developed Lemo21(DE3) strain which allows the balancing of the translation with the membrane insertion rate led to a 50-fold increased expression rate compared to the classical BL21(DE3) strain. The protein was soluble and stable in a variety of mild detergents and multiple biophysical methods confirmed the folded state of the protein. Crosslinking experiments suggest an oligomeric architecture of at least four subunits. The protein stability is significantly improved in the presence of cholesteryl hemisuccinate as judged by differential light scattering. The approach described here can easily be adapted to other eukaryotic IMPs

Human DDX3 functions in translation and interacts with the translation initiation factor eIF3

The conserved RNA helicase DDX3 is of major medical importance due to its involvement in numerous cancers, human hepatitis C virus (HCV) and HIV. Although DDX3 has been reported to have a wide variety of cellular functions, its precise role remains obscure. Here, we raised a new antibody to DDX3 and used it to show that DDX3 is evenly distributed throughout the cytoplasm at steady state. Consistent with this observation, HA-tagged DDX3 also localizes to the cytoplasm. RNAi of DDX3 in both human and Drosophila cells shows that DDX3 is required for cell viability. Moreover, using RNAi, we show that DDX3 is required for expression of protein from reporter constructs. In contrast, we did not detect a role for DDX3 in nuclear steps in gene expression. Further insight into the function of DDX3 came from the observation that its major interaction partner is the multi-component translation initiation factor eIF3. We conclude that a primary function for DDX3 is in protein translation, via an interaction with eIF3

A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit

Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg2+ exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change