Polymer Hydrogel Sheets with Perpendicular Cross-Linking Gradient: Non-Monotonic Actuation and Ion-Specific Effects on the Actuation Kinetics.

Non-monotonous actuation, that is, different kinds of motion in response to a single stimulus, is observed in some natural materials but difficult to implement in synthetic systems. Herein, polymer hydrogel sheets made from polyacrylamide (PAAm) or poly(dimethylacrylamide) (PDMAA) with a cross-linking gradient along the sheet thickness are reported. These are obtained by thermally initiated free radical polymerization using a specially designed Teflon mold with a glass lid. The resulting PAAm hydrogels undergo non-monotonous actuation (rolling into a tube and re-opening) when exposed to aqueous media as a single external stimulus. Their actuation kinetics is tuned with anions that have specific ion effects in their interaction with the surrounding solvent and the polymer itself: structure-breaking chloride enhances the hydration of the polymer backbone, structure-making sulfate decreases it, and is thus slowing down the actuation kinetics of the PAAm hydrogels. The PDMAA gel rolls up instantaneously in aqueous NaCl and only re-opens after 24 h. PDMAA actuation in aqueous Na2 SO4 is only moderate as the gel did not swell in that solvent. Bilayer hydrogels made from PAAm and PDMAA (without gradient) show monotonic actuation, closing in NaCl solution and re-opening in Na2 SO4 .

Ni/Al-Hybrid Cellular Foams: An Interface Study by Combination of 3D-Phase Morphology Imaging, Microbeam Fracture Mechanics and In Situ Synchrotron Stress Analysis.

Nickel(Ni)/aluminium(Al) hybrid foams are Al base foams coated with Ni by electrodeposition. Hybrid foams offer an enhanced energy absorption capacity. To ensure a good adhering Ni coating, necessary for a shear resistant interface, the influence of a chemical pre-treatment of the base foam was investigated by a combination of an interface morphology analysis by focused ion beam (FIB) tomography and in situ mechanical testing. The critical energy for interfacial decohesion from these microbending fracture tests in the scanning electron microscope (SEM) were contrasted to and the results validated by depth-resolved measurements of the evolving stresses in the Ni coating during three-point bending tests at the energy-dispersive diffraction (EDDI) beamline at the synchrotron BESSY II. Such a multi-method assessment of the interface decohesion resistance with respect to the interface morphology provides a reliable investigation strategy for further improvement of the interface morphology.

Strategies to Achieve High Strength and Ductility of Pulsed Electrodeposited Nanocrystalline Co-Cu by Tuning the Deposition Parameters.

Strategies to improve tensile strength and ductility of pulsed electrodeposited nanocrystalline Co-Cu were investigated. Parameters of deposition, which are pulse current density, duty cycle, and pulse-on time were adjusted to produce nanocrystalline Co-Cu deposits with different microstructures and morphologies. The most significant improvement of strength and ductility was observed at nanocrystalline Co-Cu deposited, at a low duty cycle (10%) and a low pulse-on time (0.3 ms), with a high pulse current density (1000 A/m2). Enhancement of ductility of nanocrystalline Co-Cu was also obtained through annealing at 200 °C, while annealing at 300 °C leads to strengthening of materials with reduction of ductility. In the as deposited state, tensile strength and ductility of nanocrystalline Co-Cu is strongly influenced by several factors such as concentration of Cu, grain size, and processing flaws (e.g., crystal growth border, porosity, and internal stresses), which can be controlled by adjusting the parameters of deposition. In addition, the presence of various microstructural features (e.g., spinodal and phase decomposition), as well as recovery processes induced by annealing treatments, also have a significant contribution to the tensile strength and ductility.

Microstructure Evolution and Mechanical Stability of Supersaturated Solid Solution Co-Rich Nanocrystalline Co-Cu Produced by Pulsed Electrodeposition.

Thick films of supersaturated solid solution nanocrystalline Co-Cu (28 at.% Cu) were synthesized through the pulsed electrodeposition technique. Microstructural changes of nanocrystalline Co-Cu were intensively studied at various annealing temperatures. Annealing at 300 °C results in a spinodal decomposition within the individual grains, with no grain coarsening. On the other hand, distinct phase separation of Co-Cu is detected at annealing temperatures beyond 400 °C. Static micro-bending tests show that the nanocrystalline Co-Cu alloy exhibits a very high yield strength and ductile behavior, with no crack formation. Static micro-bending tests also reported that a large plastic deformation is observed, but no microstructure change is detected. On the other hand, observation on the fatigue resistance of nanocrystalline Co-Cu shows that grain coarsening is observed after conducting the cyclic micro-bending test.

Effect of Sample Size and Crystal Orientation on the Fatigue Behaviour of Single Crystalline Microbeams.

Beam deflection experiments were used to systematically examine size effects on the low cyclic fatigue (LCF) deformation behaviour of micro-sized bending beams of copper (Cu) single crystals oriented for single slip, critical and coplanar double slip. We present cyclic hardening curves and fatigue surface roughness, as well as dislocations structures of the micro-sized beams with sizes between 1 and 15 µm. A clear crystal orientation and size effect on the cyclic hardening curves, surface roughness, and the dislocation microstructures were observed. Based on the experimental results, the fatigue damage in single slip orientations clearly decreased with decreasing the sample size, however, below a critical size regime, the surface damage suddenly increases. Additionally, samples with sizes larger than 5 µm clearly revealed, besides PSBs-like structures, the emergence of kink bands leading to larger surface roughness in comparison to the smaller ones. Fatigue surface damages in microcrystals oriented for critical double slip became more prevalent compared to single slip orientations. Quantitatively, the correlation of the fatigue surface damage was also demonstrated with the formation of PSBs-like structures.

The Impact of Hydrogen on Mechanical Properties; A New In Situ Nanoindentation Testing Method.

We have designed a new method for electrochemical hydrogen charging which allows us to charge very thin coarse-grained specimens from the bottom and perform nanomechanical testing on the top. As the average grain diameter is larger than the thickness of the sample, this setup allows us to efficiently evaluate the mechanical properties of multiple single crystals with similar electrochemical conditions. Another important advantage is that the top surface is not affected by corrosion by the electrolyte. The nanoindentation results show that hydrogen reduces the activation energy for homogenous dislocation nucleation by approximately 15⁻20% in a (001) grain. The elastic modulus also was observed to be reduced by the same amount. The hardness increased by approximately 4%, as determined by load-displacement curves and residual imprint analysis.

Helicobacter pylori VacA toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane.

The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% beta-strands, similar to pore-forming beta-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.

Role of gamma-subunit N- and C-termini in assembly of the mitochondrial ATP synthase in yeast.

The gamma-subunit is required for the assembly of ATP synthases and plays a crucial role in their catalytic activity. We stepwise shortened the N-terminus and the C-terminus of the gamma-subunit in the mitochondrial ATP synthase of yeast and investigated the relevance of these segments in the assembly of the enzyme and in the growth of the cells. We found that a deletion of 9 residues at the N-terminus or 20 residues at the C-terminus still allowed efficient import of the subunit into mitochondria; however, the assembly of both monomeric and dimeric holoenzymes was partially impaired. gamma-Subunits lacking 13 N-terminal residues or 30 C-terminal residues were not assembled. Yeast strains expressing either of the truncated gamma-subunits did not grow on non-fermentable carbon sources, indicating that non-assembled parts of the ATP synthase accumulated and impaired essential mitochondrial functions.

Dislocation avalanches, strain bursts, and the problem of plastic forming at the micrometer scale.

Under stress, many crystalline materials exhibit irreversible plastic deformation caused by the motion of lattice dislocations. In plastically deformed microcrystals, internal dislocation avalanches lead to jumps in the stress-strain curves (strain bursts), whereas in macroscopic samples plasticity appears as a smooth process. By combining three-dimensional simulations of the dynamics of interacting dislocations with statistical analysis of the corresponding deformation behavior, we determined the distribution of strain changes during dislocation avalanches and established its dependence on microcrystal size. Our results suggest that for sample dimensions on the micrometer and submicrometer scale, large strain fluctuations may make it difficult to control the resulting shape in a plastic-forming process.

Separation of proteins by blue native electrophoresis.

Blue native gel electrophoresis is a native electrophoresis method that can be used for molecular weight determination for most soluble protein complexes as well as for most membrane proteins. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) can be used in a second dimension to resolve the complexes into their subunits. The method has been extensively used for the analysis of the respiratory chain complexes, for the determination of intermediates of mitochondrial protein import, and for the identification of the composition of the protein import machinery for mitochondria and chloroplasts. Here we describe the basic method and some applications in the research of mitochondrial protein import.

Conserved mechanism of Oxa1 insertion into the mitochondrial inner membrane.

Oxa1 is the mitochondrial representative of a family of related proteins that mediate the insertion of substrate proteins into the membranes of bacteria, chloroplasts, and mitochondria. Several studies have demonstrated that the bacterial homologue YidC participates both in the direct uptake of proteins from the bacterial cytosol, and in the uptake of nascent proteins from the Sec translocase. Studies on the biogenesis of membrane proteins in mitochondria established that Oxa1 has the capability to receive substrates at the inner surface of the inner membrane. In this study, we asked if Oxa1 may similarly cooperate with a protein translocase within the membrane. Since Oxa1 is involved in its own biogenesis, we used the precursor of Oxa1 as a model protein and investigated its import pathway. We found that immediately after import into mitochondria, Oxa1 initially accumulates at Tim23 that forms the inner membrane protein translocase. Cleavage of the Oxa1 presequence is dependent on mtHsp70, a heat shock protein of the mitochondrial matrix. However, mutant mtHsp70 showing a defect in the release of bound substrate proteins does not interfere with subsequent membrane insertion, indicating that membrane insertion of the mature protein is essentially mtHsp70-independent. We conclude that Oxa1 has the ability to accept preproteins within the membrane.

The subunit b dimer of the FOF1-ATP synthase: interaction with F1-ATPase as deduced by site-specific spin-labeling.

We have used site-specific spin-labeling of single cysteine mutations within a water-soluble mutant of subunit b of the ATP synthase and employed electron spin resonance (ESR) spectroscopy to obtain information about the binding interactions of the b dimer with F1-ATPase. Interaction of b2 with a delta-depleted F1 (F1-delta) was also studied. The cysteine mutations used for spin-labeling were distributed throughout the cytosolic domain of the b subunit. In addition, each position between residues 101 and 114 of b was individually mutated to cysteine. All mutants were modified with a cysteine-reactive spin label. The room temperature ESR spectra of spin-labeled b2 in the presence of F1 or F1-delta when compared with the spectra of free b2 indicate a tight binding interaction between b2 and F1. The data suggest that b2 packs tightly to F1 between residues 80 and the C terminus but that there are segments of b2 within that region where packing interactions are quite loose. Two-dimensional gel electrophoresis confirmed binding of the modified b mutants to F1-ATPase as well as to F1-delta. Subsequent addition of delta to F1-delta.b2 complex resulted in changes in the ESR spectra, indicating different binding interactions of b to F1 in the presence or absence of delta. The data also suggest that the reconstitution of the ATP synthase is not ordered with respect to these subunits. Additional spectral components observed in b preparations that were spin-labeled between amino acid position 101 and 114 are indicative of either two populations of b subunits with different packing interactions or to helical bending within this region.

Bcl-2 and porin follow different pathways of TOM-dependent insertion into the mitochondrial outer membrane.

The bcl-2 gene encodes a 26kDa protein which functions as a central regulator of apoptosis. Here we investigated the pathway of Bcl-2alpha into the mitochondrial outer membrane using the yeast Saccharomyces cerevisiae as a model organism. We found that interactions of Bcl-2alpha with the mitochondrial import receptor Tom20 are dependent on two positively charged lysine residues in the immediate vicinity of the carboxy-terminal hydrophobic membrane anchor. The targeting function of these residues is independent of Tom22. Subsequent insertion of Bcl-2alpha into the mitochondrial outer membrane does not require Tom5 or Tom40, indicating that Bcl-2alpha bypasses the general import pore (GIP). Bcl-2alpha shows a unique pattern of interactions with the components of the mitochondrial TOM complex, demonstrating that at least two different pathways lead from the import receptor Tom20 into the mitochondrial outer membrane.