We have developed a single-layer UV-nanoimprint process, which was utilized to fabricate 34 × 34 crossbar circuits with a half-pitch of 50 nm (equivalent to a bit density of 10 Gbit/cm 2 ). This process contains two innovative ideas to overcome challenges in the nanoimprint at shrinking dimensions. First, our new liquid resist formulation allowed us to minimize the residual resist layer thickness after curing and requires the relatively low imprint pressure of 20 psi. Second, by engineering the surface energy of the substrate we also eliminated the problem of trapped air during contact with the mold such that it spreads the resist and expels trapped air uniformly. Our overall process required fewer processing steps than any bilayer process and yielded high quality results at 50 nm half-pitch.

30Obligate aerobic organisms rely on a functional electron transport chain for energy 31 generation and NADH oxidation. Because of this essential requirement, the genes of 32 this pathway are likely constitutively and highly expressed to avoid a cofactor imbalance 33 and energy shortage under fluctuating environmental conditions. 34We here investigated the essentiality of the three NADH dehydrogenases of the 35 respiratory chain of the obligate aerobe Pseudomonas taiwanensis VLB120 and the 36 impact of the knockouts of corresponding genes on its physiology and metabolism. 37While a mutant lacking all three NADH dehydrogenases seemed to be nonviable, the 38 generated single or double knockout strains displayed none or only a marginal 39 phenotype. Only the mutant deficient in both type 2 dehydrogenases showed a clear 40 phenotype with biphasic growth behavior and strongly reduced growth rate in the 41 second phase. In-depth analyses of the metabolism of the generated mutants including 42 quantitative physiological experiments, transcript analysis, proteomics and enzyme 43 activity assays revealed distinct responses to type II and type I dehydrogenase 44deletions. An overall high metabolic flexibility enables P. taiwanensis to cope with the 45 introduced genetic perturbations and maintain stable phenotypes by rerouting of 46 metabolic fluxes. 47 This metabolic adaptability has implications for biotechnological applications. While the 48 phenotypic robustness is favorable in large-scale applications with inhomogeneous 49 conditions, versatile redirecting of carbon fluxes upon genetic interventions can frustrate 50 metabolic engineering efforts. 51 52 Importance 53While Pseudomonas has the capability for high metabolic activity and the provision of 54 reduced redox cofactors important for biocatalytic applications, exploitation of this 55 3 characteristic might be hindered by high, constitutive activity of and consequently 56 competition with the NADH dehydrogenases of the respiratory chain. The in-depth 57 analysis of NADH dehydrogenase mutants of Pseudomonas taiwanensis VLB120 58 presented here, provides insight into the phenotypic and metabolic response of this 59 strain to these redox metabolism perturbations. The observed great metabolic flexibility 60 needs to be taken into account for rational engineering of this promising 61 biotechnological workhorse towards a host with controlled and efficient supply of redox 62 cofactors for product synthesis. 63 64 65Many industrially relevant molecules, e. g., ethanol, butanediol or isoprene, are more 66 reduced than the industrially-used sugars glucose and sucrose or alternative, upcoming 67 carbon sources such as xylose or glycerol (1-3). The microbial production of those 68 favored compounds hence is inherently redox limited, i.e. by the supply of reduced 69 redox cofactors, generally NADH or NADPH. This bottleneck has been overcome in 70 some cases, e.g., 1,4 butanediol and 1,3-propanediol production in Escherichia coli (4, 71 5) or L-lysine synthesis in Corynebacter...

Affinity maturation of U33, a recombinant Fab inhibitor of uPA, was used to improve the affinity and the inhibitory effect compared to the parental Fab. Arginine scanning of the six CDR loops of U33 was done to identify initial binding determinants since uPA prefers arginine in its primary substrate binding pocket. Two CDR loops were selected to create an engineered affinity maturation library of U33 that was diversified around ArgL91 (CDR L3) and ArgH52 (CDR H2). Biopanning of the randomized U33 library under stringent conditions resulted in eight Fabs with improved binding properties. One of the most potent inhibitors, AB2, exhibited a 13fold decrease in IC50 when compared to U33 largely due to a decrease in its off rate. To identify contributions of interfacial residues that might undergo structural rearrangement upon interface formation we used X-ray footprinting and mass spectrometry (XFMS). Four residues showed a pronounced decrease in solvent accessibility, and their clustering suggests that AB2 targets the active site and also engages residues in an adjacent pocket unique to human uPA. The 2.9 Å resolution crystal structure of AB2-bound to uPA shows a binding mode in which the CDR L1 loop inserts into the active site cleft and acts as a determinant of inhibition. The selectivity determinant of this binding mode is unlike previously identified inhibitory Fabs against uPA related serine proteases, MTSP-1, HGFA and FXIa. CDRs H2 and L3 loops aid in interface formation and provide critical saltbridges to remodel loops surrounding the active site of uPA providing specificity and further evidence that antibodies can be potent and selective inhibitors of proteolytic enzymes.

The configurations of misfit dislocations in In0.2Ga0.8As/GaAs(001) hetero structures grown on slightly misoriented substrates was investigated by transmission electron microscopy (TEM). Layers 6 nm, 20 nm and 40 nm thick were grown by MBE. The substrate was tilted in [110], [110], [120], [210] and [010] directions at angles between 0° and 10°. Only in the 40 nm thick layers networks of 60° and 90° dislocations were formed. Misfit dislocations were found at the interface in <110> directions. In the substrate tilting range between 0° and 4° the changes in dislocation density can be explained by the differentcharacter of α and β dislocations. For a substrate tilting above 6° the different dislocation sets show an increased anisotropy. The misfit dislocations at the interface were decorated by In atoms. The influence of three-dimensional crystal growth on increasing surface roughness is discussed.