Development of Bifunctional Electrodes for Closed-loop Fuel Cell Applications

Unitized regenerative fuel cells (URFC) in combination with photovoltaic modules are attractive for space missions because they enable extended operation times and low weight. During the planetary day, electrical energy is stored which can be converted into electricity by the fuel cell during the night. All air-independent applications such as spacecraft or space stations would profit signify¬cantly from such energy conversion devices. A unitized regenerative fuel cell is a combined energy con¬version and storage system based on H2 and O2 which combines the advantages of fuel cells and secondary batteries. Substantial advantages of the specific energy density can be expected from the use of a URFC (400-1000 Wh/kg) in comparison to secondary batteries (220-250 Wh/kg for future advanced Li-polymer batteries). An important topic is the function of so-called bifunc¬tional oxygen electrodes which generally require the combination of favourable properties for both operating modes. In particular, different catalysts for oxygen reduction and for oxygen evolution are needed. This contribution investigates various electrode designs with Pt and IrO2 catalysts. For that purpose, the DLR dry spraying technique for the manufacturing of electrodes is applied for by mixing the two different catalysts together (Pt and IrO2) or applying the catalyst on different areas of electrode or even realising different layers with both catalysts. The different options are explained in Fig. 1. Of interest was to compare the simple mixing of the catalysts (option 1), the layered electrode with two compositions (option 2) and the segmented approach with dedicated areas with just one catalyst (option 3)

Seed-produced anti-globulin VHH-Fc antibodies retrieve globulin precursors in the insoluble fraction and modulate the Arabidopsis thaliana seed subcellular morphology

Key message Nanobody-heavy chain (VHH-Fc) antibody formats have the potential to immunomodulate even highly accumulating proteins and provide a valuable tool to experimentally modulate the subcellular distribution of seed storage proteins. Recombinant antibodies often obtain high accumulation levels in plants, and thus, besides being the actual end-product, antibodies targeting endogenous host proteins can be used to interfere with the localization and functioning of their corresponding antigens. Here, we compared the effect of a seed-expressed nanobody-heavy chain (VHH-Fc) antibody against the highly abundant Arabidopsis thaliana globulin seed storage protein cruciferin with that of a VHH-Fc antibody without endogenous target. Both antibodies reached high accumulation levels of around 10% of total soluble protein, but strikingly, another significant part was present in the insoluble protein fraction and was recovered only after extraction under denaturing conditions. In seeds containing the anti-cruciferin antibodies but not the antibody without endogenous target, the amount of soluble, processed globulin subunits was severely reduced and a major part of the cruciferin molecules was found as precursor in the insoluble fraction. Moreover, in these seeds, aberrant vacuolar phenotypes were observed that were different from the effects caused by the depletion of globulins in knock-out seeds. Remarkably, the seeds with strongly reduced globulin amounts are fully viable and germinate with frequencies similar to wild type, illustrating how flexible seeds can retrieve amino acids from the stored proteins to start germination

Protein Glycosylation in Bryophytes Differs Subtly from That in Vascular Plants

Glycosylation substantially contributes to the physicochemical properties of proteins, and hence also cell walls. Moreover, they are key factors for the recognition of free or cell-bound glycoproteins by internal and external interaction partners. Green plants get by with a highly conserved, limited number of modifications of the pan-eukaryotic basic N-glycan structure. In detail, these are fucosylation of the innermost N-acetylglucosamine residue in 3-position, which renders plant glycoproteins immunogenic to mammals; xylosylation of the branching mannose; frequent occurrence of small N-glycans terminating with mannose or decoration of the antennae with Lewis A determinants. Bryophytes share all these features, but some mosses additionally display two peculiarities not seen in vascular plants. Many mosses exhibit 2,6-di-O-methylated mannose on the 6-arm and some mosses contain modified Lewis A termini with an as yet unspecified methyl pentose. Neither the responsible enzymes nor the function of these novel glycan features is currently known. Targeted glycoengineering of the moss Physcomitrella patens (Hedw.) Bruch & Schimp can allow the production of biopharmaceutical glycoproteins that are difficult to express in more established systems

Lewis A glycans are present on proteins involved in cell wall biosynthesis and appear evolutionarily conserved amongnatural Arabidopsis thaliana accessions

N-glycosylation is a highly abundant protein modification present in all domains of life. Terminal sugar residues on complex-type N-glycans mediate various crucial biological processes in mammals such as cell-cell recognition or protein-ligand interactions. In plants, the Lewis A trisaccharide constitutes the only known outer-chain elongation of complex N-glycans. Lewis A containing complex N-glycans appear evolutionary conserved, having been identified in all plant species analyzed so far. Despite their ubiquitous occurrence, the biological function of this complex N-glycan modification is currently unknown. Here, we report the identification of Lewis A bearing glycoproteins from three different plant species: Arabidopsis thaliana, Nicotiana benthamiana, and Oryza sativa. Affinity purification via the JIM84 antibody, directed against Lewis A structures on complex plant N-glycans, was used to enrich Lewis A bearing glycoproteins, which were subsequently identified via nano-LC-MS. Selected identified proteins were recombinantly expressed and the presence of Lewis A confirmed via immunoblotting and site-specific N-glycan analysis. While the proteins identified in O. sativa are associated with diverse functions, proteins from A. thaliana and N. benthamiana are mainly involved in cell wall biosynthesis. However, a Lewis A-deficient mutant line of A. thaliana showed no change in abundance of cell wall constituents such as cellulose or lignin. Furthermore, we investigated the presence of Lewis A structures in selected accessions from the 1001 genome database containing amino acid variations in the enzymes required for Lewis A biosynthesis. Besides one relict line showing no detectable levels of Lewis A, the modification was present in all other tested accessions. The data provided here comprises the so far first attempt at identifying Lewis A bearing glycoproteins across different species and will help to shed more light on the role of Lewis A structures in plants

Prevalence of marginally unstable periodic orbits in chaotic billiards

The dynamics of chaotic billiards is significantly influenced by coexisting regions of regular motion. Here we investigate the prevalence of a different fundamental structure, which is formed by marginally unstable periodic orbits and stands apart from the regular regions. We show that these structures both {\it exist} and {\it strongly influence} the dynamics of locally perturbed billiards, which include a large class of widely studied systems. We demonstrate the impact of these structures in the quantum regime using microwave experiments in annular billiards.Comment: 6 pages, 5 figure

Engineering vacuolar sorting pathways for efficient secretion of recombinant proteins

Recombinant protein production is an expanding branch of biotechnology with increasing economic importance. Currently, 20% of biopharmaceutical proteins and approximately half of the industrial enzymes are produced in yeasts. Many proteins are efficiently secreted by yeast systems, reaching product titers in the g L-1 range. The expression of more complex proteins, however, may overwhelm the folding and secretion capacity of the host cells. This triggers the unfolded protein response (UPR), which aims at restoring endoplasmic reticulum (ER) homeostasis. The UPR, in turn, is thought to activate ER-associated protein degradation (ERAD). Alternatively, trafficking of correctly folded proteins can be hampered on their way to the cell exterior leading e.g. to missorting and subsequent degradation in the vacuole. The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Vacuolar protein sorting has not been investigated in detail so far in P. pastoris, although there were a few indications that vacuolar mistargeting of recombinant products might occur also in this yeast. Thus we engineered the vacuolar sorting pathways in P. pastoris and investigated their impact on extracellular product titers as well as intracellular localization of the recombinant secretory product. Thereby, differences between vps (vacuolar protein sorting) mutant strains disrupted in genes involved either in the CORVET or the HOPS tethering complexes became obvious. Moreover, we were able to show that engineering of the vacuolar sorting pathways has a positive impact on heterologous protein secretion, however, in some cases simultaneous inactivation of specific vacuolar proteases was necessary. Taken together, these studies allowed us to gain deeper insight into the pathways leading to intracellular degradation of recombinant secretory proteins. Based on these findings, approaches how to efficiently adapt the host cell’s secretion capacity will be presented, which confirm that impairment of vacuolar protein sorting is an effective means of enhancing secretion of heterologous proteins

Silencing β1,2-xylosyltransferase in Transgenic Tomato Fruits Reveals xylose as Constitutive Component of Ige-Binding Epitopes

Complex plant N-glycans containing β1,2-xylose and core α1,3-fucose are regarded as the major class of the so-called “carbohydrate cross-reactive determinants” reactive with IgE antibodies in sera of many allergic patients, but their clinical relevance is still under debate. Plant glycosyltransferases, β1,2-xylosyltransferase (XylT), and core α1,3-fucosyltransferase (FucT) are responsible for the transfer of β1,2-linked xylose and core α1,3-linked fucose residues to N-glycans of glycoproteins, respectively. To test the clinical relevance of β1,2-xylose-containing epitopes, expression of the tomato β1,2-xylosyltransferase was down-regulated by RNA interference (RNAi) in transgenic plants. Fruits harvested from these transgenic plants were analyzed for accumulation of XylT mRNA, abundance of β1,2-xylose epitopes and their allergenic potential. Based on quantitative real-time PCR analysis XylT mRNA levels were reduced up to 10-fold in independent transgenic lines as compared to untransformed control, whereas no xylosylated N-glycans could be revealed by MS analysis. Immunoblotting using anti-xylose-specific IgG antibodies revealed a strong reduction of β1,2-xylose-containing epitopes. Incubating protein extracts from untransformed controls and XylT_RNAi plants with sera from tomato allergic patients showed a patient-specific reduction in IgE-binding, indicating a reduced allergenic potential of XylT_RNAi tomato fruits, in vitro. To elucidate the clinical relevance of β1,2-xylose-containing complex N-glycans skin prick tests were performed demonstrating a reduced responsiveness of tomato allergic patients, in vivo. This study provides strong evidence for the clinical relevance of β1,2-xylose-containing epitopes in vivo

Processing of the Terminal Alpha-1,2-Linked Mannose Residues From Oligomannosidic N-Glycans Is Critical for Proper Root Growth

N-glycosylation is an essential protein modification that plays roles in many diverse biological processes including protein folding, quality control and protein interactions. Despite recent advances in characterization of the N-glycosylation and N-glycan processing machinery our understanding of N-glycosylation related processes in plant development is limited. In Arabidopsis thaliana, failure of mannose trimming from oligomannosidic N-glycans in the endoplasmic reticulum (ER) and cis/medial-Golgi leads to a defect in root development in the mns123 triple mutant. Here, we show that the severe root phenotype of mns123 is restored in asparagine-linked glycosylation (ALG)-deficient plants with distinct defects in the biosynthesis of the lipid-linked oligosaccharide precursor. The root growth of these ALG-deficient plants is not affected by the α-mannosidase inhibitor kifunensine. Genetic evidence shows that the defect is uncoupled from the glycan-dependent ER-associated degradation (ERAD) pathway that removes misfolded glycoproteins with oligomannosidic N-glycans from the ER. Restoration of mannose trimming using a trans-Golgi targeted α-mannosidase suppresses the defect of mns123 roots. These data suggest that processing of terminal mannose residues from oligomannosidic N-glycans is important for an unknown late-Golgi or post-Golgi process that is implicated in proper root formation