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Repulsive guidance molecule A (RGMa) is a potent inhibitor of axonal growth and a regulator of neuronal cell death. It is up-regulated following neuronal injury and accumulates in chronic neurodegenerative diseases. Neutralizing RGMa has the potential to promote neuroregeneration and neuroprotection. Previously we reported that a rat anti-N terminal RGMa (N-RGMa) antibody r5F9 and its humanized version h5F9 (ABT-207) promote neuroprotection and neuroregeneration in preclinical neurodegenerative disease models. However, due to its cross-reactivity to RGMc/hemojuvelin, ABT-207 causes iron accumulation in vivo, which could present a safety liability. Here we report the generation and characterization of a novel RGMa-selective anti-N-RGMa antibody elezanumab, which is currently under Phase 2 clinical evaluation in multiple disease indications. Elezanumab, a human monoclonal antibody generated by in vitro PROfusion mRNA display technology, competes with ABT-207 in binding to N-RGMa but lacks RGMc cross-reactivity with no impact on iron metabolism. It neutralizes repulsive activity of soluble RGMa in vitro and blocks membrane RGMa mediated BMP signaling. In the optic nerve crush and optic neuritis models, elezanumab promotes axonal regeneration and prevents retinal nerve fiber layer degeneration. In the spinal targeted experimental autoimmune encephalomyelitis (EAE) model, elezanumab promotes axonal regeneration and remyelination, decreases inflammatory lesion area and improves functional recovery. Finally, in the mouse cuprizone model, elezanumab reduces demyelination, which is consistent with its inhibitory effect on BMP signaling. Taken together, these preclinical data demonstrate that elezanumab has neuroregenerative and neuroprotective activities without impact on iron metabolism, thus providing a compelling rationale for its clinical development in neurodegenerative diseases.

$\beta $ -Ga2O3 thin-channel MOSFETs were evaluated using both dc and pulsed ${I}$ – ${V}$ measurements. The reported pulsed ${I}$ – ${V}$ technique was used to study self-heating effects in the MOSFET channel. The device was analyzed over a large temperature range of 23 °C–200 °C. A relationship between dissipated power and channel temperature was established, and it was found that the MOSFET channel was heating up to 208 °C when dissipating 2.5 W/mm of power. The thermal resistance of the channel was found to be 73 °C-mm/W. The results are supported with the experimental Raman nanothermography and thermal simulations and are in reasonable agreement with pulsed ${I}$ – ${V}$ findings. The high thermal resistance underpins the importance of optimizing thermal management in future Ga2O3 devices.

In this work, the fabrication of high-performance metal-oxide-semiconductor field-effect transistors (MOSFETs) on Si-doped homoepitaxial layers on (100) Mg-doped semi-insulating β-Ga2O3 substrates is demonstrated. Furthermore, the influence of additional gate and source field-plates is analyzed in regards to the breakdown properties of the MOSFET devices. The comparative study reveals comparable transistor performances of devices with and without field-plates showing maximum drain currents, on/off-current ratios and ON-resistances of ~75 mA/mm, 109 and 110 Ω∙mm, respectively. Moreover, both MOSFET structures show comparable breakdown voltages at around 1200 V which equals an average breakdown field strength of 2 MV/cm emphasizing no influence of the field-plates on the breakdown properties. Analysis of the thin film composition by secondary ion mass spectrometry on the processed β-Ga2O3 wafers indicates an increased concentration of silicon dopants at the interface between epitaxial layer and substrate. Our findings suggest that this might be the current main limitation factor in reaching higher breakdown voltages using field-plated β-Ga2O3 MOSFET devices.

The influence of chamber pressure and Si-doping on the growth rate, surface morphology and Hall mobility was investigated forβ-Ga2O3thin films homoepitaxially grown by metalorganic vapor phase epitaxy on Mg-dopedβ-Ga2O3(100) substrates with 4° miscut. Transitions from step-bunching to step-flow to 2D island growth modes were achieved by varying the chamber pressure from 10 mbar to 40 mbar and/or by varying the O2/Ga ratio. High-qualityβ-Ga2O3homoepitaxial thin films with a high electron mobility of 153 cm2Vs−1have been obtained at a chamber pressure of 25 mbar and a growth rate of 3.6 nm min−1. The Si-doped films show electron concentrations in the range of 1 × 1017to 2 × 1019cm−3. When increasing the chamber pressure to 40 mbar step-flow growth mode and high charge carrier mobility can only be preserved by adjusting the O2/Ga ratio and increasing the Ar push gas flow. Secondary ion mass spectrometry and Hall measurements for Si and electron concentration, respectively, revealed Si compensation at higher tetraethyl orthosilicate flux.

Homoepitaxial β-(In,Ga)2O3 thin films were grown on (100) β-Ga2O3 substrates by metal organic vapor phase epitaxy using triethylgallium (TEGa) and triethylindium (TEIn). Deposition temperatures from 650 to 825 °C and pressures from 5 to 20 mbar have been explored. The growth rate decreased linearly with increasing deposition temperature and decreased exponentially with increasing pressure. The resulting films were characterized by atomic force microscopy (AFM), high resolution x-ray diffraction (HR-XRD), and transmission electron microscopy (TEM). As the flow rate of TEIn varied from 0 to 0.13 μmol/min during the growth, AFM showed the surface roughness of about 1 nm, while HR-XRD measurements revealed an increase of the vertical lattice spacing. The maximum atomic concentration of indium incorporated in monoclinic β-(In,Ga)2O3 is about 3.5% and shifts the optical absorption edge to lower energy by ∼0.18 eV. Further increase of the indium flow rate leads to an increase of the surface roughness and a decrease in the vertical lattice spacing due to the formation of a separate cubic In2O3 phase that was confirmed by HR-TEM images. X-ray reciprocal space maps showed that the β-(In,Ga)2O3 thin films were grown coherently on β-Ga2O3.

CuGaSe 2 with varying Cu/Ga ratios were grown epitaxial on GaAs (001) substrates by molecular beam epitaxy (MBE). A Cu/Ga stoichiometry range from Cu-poor to Cu-rich (0, 51–1, 65) was prepared. Depending on the stoichiometry a single domain (4×1) reconstruction of the zinc blende surface periodicity was determined by low energy electron diffraction (LEED) for Cu-poor and near stoichiometric samples. For Cu-rich samples instead, a (4×2) reconstruction was found. In addition, for Cu-rich stochiometries a secondary phase of Cu 2−x Se appeared. The electronic structure with respect to the Cu/Ga ratio was investigated by XPS and UPS. Supplementary an investigation of the film morphology was done ex-situ by SEM. In addition to conventional MBE, high quality films could be prepared by using the GaAs (001) substrate at elevated temperature as gallium source instead of an external gallium source.

This article describes the design of HuCAL (human combinatorial antibody library) PLATINUM, an optimized, second-generation, synthetic human Fab antibody library with six trinucleotide-randomized complementarity-determining regions (CDRs). Major improvements regarding the optimized antibody library sequence space were implemented. Sequence space optimization is considered a multistep process that includes the analysis of unproductive antibody sequences in order to, for example, avoid motifs such as potential N-glycosylation sites, which are undesirable in antibody production. Gene optimization has been used to improve expression of the antibody master genes in the library context. As a result, full-length IgGs derived from the library show both significant improvements in expression levels and less undesirable glycosylation sites when compared to the previous HuCAL GOLD library. Additionally, in-depth analysis of sequences from public databases revealed that diversity of CDR-H3 is a function of loop length. Based upon this analysis, the relatively uniform diversification strategy used in the CDR-H3s of the previous HuCAL libraries was changed to a length-dependent design, which replicates the natural amino acid distribution of CDR-H3 in the human repertoire. In a side-by-side comparison of HuCAL GOLD and HuCAL PLATINUM, the new library concept led to isolation of about fourfold more unique sequences and to a higher number of high-affinity antibodies. In the majority of HuCAL PLATINUM projects, 100-300 antibodies each having different CDR-H3s are obtained against each antigen. This increased diversity pool has been shown to significantly benefit functional antibody profiling and screening for superior biophysical properties.

Endotoxins are frequent contaminants of recombinant proteins produced in Escherichia coli. Due to their adverse effects, endotoxins have to be removed from recombinant proteins prior their use in cell-based assays or parenteral application. Reduction of endotoxin to less than 10 EU mg(-1) is, however, one of the most problematic steps during protein purification from E. coli and often associated with substantial loss of biological materials. The present paper describes the use of a single step procedure enabling metal chelate affinity purification and endotoxin clearance from antibody fragments produced in E. coli using a non-ionic detergent. Endotoxin content was as low as 5 to 9 EU mg(-1) with a recovery of antibody fragments of over 90%. Non-ionic detergent treatment did not compromise integrity and functionality of these multimeric molecules. Furthermore, recombinant antibody fragments did not stimulate endotoxin-sensitive cell lines confirming the low endotoxin content. In conclusion, this one-step protocol is a rapid, cost effective and automation-compatible procedure suitable for recombinant antibody fragments.

High levels of hepcidin, the main regulator of systemic iron metabolism, lead to various diseases. Targeting hepcidin and lowering its concentration is a possible form of intervention in order to treat these diseases. High turnover rate of hepcidin is a major drawback of therapies directly targeting this peptide. We developed two monoclonal antibodies ABT-207 and h5F9-AM8 which inhibit hemojuvelin/repulsive guidance molecule C (RGMc) and downregulate hepcidin. We conducted single-application and dose response studies to understand the antibodies’ mechanism and subchronic toxicology studies to exclude safety-related concerns. Investigation was carried out at different biological levels through qPCR, Affymetrix, liquid chromatography coupled with mass spectrometry (LC-MS/MS), histopathology, serum iron, unsaturated iron binding capacity (UIBC), and drug concentration measurements. After a single application of these antibodies, hepcidin expression in liver and its serum protein levels were reduced. Serum iron increased for several weeks. The RGMc antibodies show a pronounced dose response relationship in rats with h5F9-AM8 having an IC50 (UIBC) of approximately 80-fold higher than ABT-207. When hepcidin levels were downregulated, iron deposition in the liver was visible histologically 1 week post application. These antibody-mediated iron depositions were not associated with any adverse toxicologically relevant effect at the doses and time points evaluated. Iron depositions seen after 14 weekly treatments with ABT-207 were reversible in rats and in cynomolgus monkeys. Due to their long-lasting effects and excellent safety profile, both RGMc-blocking antibodies ABT-207 and h5F9-AM8 are favorable clinical candidates for diseases characterized by high serum hepcidin levels like anemia of chronic disease. Electronic supplementary material The online version of this article (doi:10.1208/s12248-015-9770-4) contains supplementary material, which is available to authorized users.

3-Hydroxy-2,2-dimethyl- N -[4({[5-(dimethylamino)-1-naphthyl]sulfonyl}amino)-phenyl]propanamide (BAY 38-4766) is a novel selective nonnucleoside inhibitor of cytomegalovirus (CMV) replication with an excellent safety profile. This compound and structural analogues inhibit neither viral DNA synthesis nor viral transcription and translation. Accumulation of dense bodies and noninfectious enveloped particles coincides with inhibition of both concatemer processing and functional cleavage at intergenomic transitions, pointing to interference with viral DNA maturation and packaging of monomeric genome lengths. Resistant virus populations, including a murine CMV (MCMV) isolate with 566-fold-decreased drug sensitivity, were selected in vitro. Sequencing of the six open reading frames (ORFs) known to be essentially involved in viral DNA cleavage and packaging identified mutations in ORFs UL56, UL89, and UL104. Construction of MCMV recombinants expressing different combinations of murine homologues of mutant UL56, UL89, and UL104 and analysis of drug susceptibilities clearly demonstrated that mutant ORFs UL89 exon II (M360I) and M56 (P202A I208N) individually confer resistance to BAY 38-4766. A combination of both mutant proteins exhibited a strong synergistic effect on resistance, reconstituting the high-resistance phenotype of the in vitro mutant. These findings are consistent with genetic mapping of resistance to TCRB (2,5,6-trichloro-1-β- d -ribofuranosyl benzimidazole) (P. M. Krosky et al., J. Virol. 72:4721–4728, 1998) and provide further indirect evidence that proteins encoded by UL89 and UL56 function as two subunits of the CMV terminase. While these studies also suggest that the molecular mechanism of BAY 38-4766 is distinct from that of benzimidazole ribonucleosides, they also offer an explanation for the excellent specificity and tolerability of BAY 38-4766, since mammalian DNA does not undergo comparable maturation steps.

Bacteriorhodopsin (BR) folms made from purple membranes (PM) containing the wildtype form of BR or one of its variants with single amino acids exchanges Asp96→Asn or Asp85→Glu were compared with respect to their holographic properties. The dependence of the diffraction efficiency of BR films on the parameters pH, temperature, intensity, initial optical density, and spatial frequency was measured. Increasing the initial optical density of a BR material with a long lifetime of the M intermediate and a low steady-state concentration of all other intermediates

Summary 170 Yersinia strains belonging to various species were investigated for the presence of temperate bacteriophages. By induction with mitomycin C seven phages were isolated from Y. enterocolitica strains and one phage from a Y. frederiksenii strain. The phages were characterized on the basis of their morphology, host range, genome size, DNA homology, and protein composition. They belong to different phage families and reveal narrow to moderate wide host ranges. Some of the isolated phages were able to infect pathogenic as well as nonpathogenic strains of Y. enter ocolitica . The genomes of all isolated phages were found to be composed of double stranded DNA ranging from about 40 to 60 kb. In addition to the analysed phages, a number of putative phages were induced in strains of Y. frederiksenii, Y. kristensenii, Y. intermedia , and Y. mollaretii . The putative phages were identified by isolation of phage DNA from cell free lysates but could not be propagated on indicator strains. Southern hybridization experiments revealed relationships between phages belonging to different families. Moreover, DNA homologies were observed between phages isolated from nonpathogenic Yersinia strains and a phage which was isolated from a pathogenic Y. enterocolitica serogroup O:3 strain.

The carcinoembryonic antigen (CEA) gene family members, CEACAM1, CEACAM3, CEACAM5 and CEACAM6, are bound by the Opa outer membrane proteins of pathogenic Neisseria spp., whereas CEACAM8 is not. In this study, we demonstrate that the closely related CEACAM4 and CEACAM7, which are also members of the CEA family, are not Opa receptors. We exploited the high conservation between CEACAM6 and CEACAM8 to generate an extensive set of chimeric receptors in order to delineate the sequences necessary for Opa binding. Using a transfection-based infection system, we showed that binding of Opa52 involves residues 27-42, which are predicted to form beta-strand C and short loops adjacent to it, and residues lying between amino acids 60 and 108 in the amino-terminal domain. The replacement of residues 27-29 in CEACAM6 with the CEACAM1 or CEACAM5 sequences generated recombinant CEACAM6 receptors that are bound by CEACAM1/CEACAM5-specific Opa variants. Together, our data demonstrate that Opa proteins bind to residues exposed on the GFCC' face of the N-terminal domain of CEACAM receptors, and identify an amino acid triplet sequence that is responsible for the differential binding of Opa proteins to CEACAM1, CEACAM5 and CEACAM6.

The photochemical activity of the O-state was investigated in bacteriorhodopsin (BR) films containing wildtype BR at pH 6.5 in the presence of glycerol. The formation of a photoproduct of O with an absorption maximum at 490 nm and 9-cis-retinal configuration was found. This 490-nm product was named P and shows a slow thermal reaction into a compound with a maximal absorption at 380 nm which was named Q and contains free 9-cis-retinal in the proteins binding site. The photoproducts of O, i.e., P and Q, are very similar, or even identical, to those previously observed in blue membranes. Common to the O-state and blue membrane forms of bacteriorhodopsin is a protonated aspartic acid 85, and we suggest that it is the reduced negative charge around the Schiff base which is responsible for the 9-cis photoisomerization. The release of a proton from aspartic acid 85 is linked to the conversion of the O-state back to the initial state of BR. Therefore the conditions of low proton mobility in BR films containing glycerol favor the accumulation of the O-state. For optical and holographic applications such BR films are very attractive. It is possible to create photoproducts with red light which are thermally stable at room temperature and that can be photochemically erased. Dependent on the light composition both properties can be realized in the same sample material. This feature may bridge the gap between information processing and short-term and long-term storage of information with BR.