[Mortality after high-risk surgery in Jehovah's Witness patients]. / Letalität nach operativen Risikoeingriffen bei Zeugen Jehovas.

BACKGROUND: Jehovah's Witness (JW) patients strictly refuse allogeneic blood transfusion for religious reasons. Nevertheless, JW also wish to benefit from modern therapeutic concepts including major surgical procedures without facing an excessive risk of death. The Northwest Hospital in Frankfurt am Main Germany is a confidential clinic of JW and performs approximately 100 surgical interventions per year on this patient group. MATERIAL AND METHODS: A retrospective analysis of closed medical cases performed in the years 2008-2018 at the Northwest Hospital aimed to clarify (1) the frequency of surgical procedures in JW patients associated with a statistical allogeneic transfusion risk (presence of preoperative anemia and/or in-house transfusion probability >10%) during this time period, (2) the degree of acceptance of strategies avoiding blood transfusion by JW and (3) the anemia-related postoperative mortality rate in JW patients. RESULTS: In the 11- year observation period 123 surgical procedures with a relevant allogeneic transfusion risk were performed in 105 JW patients. Anemia according to World Health Organization (WHO) criteria was present in 44% of cases on the day of surgery. Synthetic and recombinant drugs (tranexamic acid, desmopressin, erythropoetin, rFVIIa) were generally accepted, acute normovolemic hemodilution (ANH) in 92% and cell salvage in 96%. Coagulation factor concentrates extracted from human plasma and therefore generally refused by JW so far, were accepted by 83% of patients following detailed elucidation. Out of 105 JW patients 7 (6.6%) died during the postoperative hospital stay. In 4 of the 7 fatal cases the cause of death could be traced back to severe postoperative anemia. CONCLUSION: Given optimal management JW patients can undergo major surgery without an excessive risk of death. The 6.6% in-hospital mortality observed in this institution was in the range of the 4% generally observed after surgery in Europe. The majority of JW patients accepted a variety of blood conservation strategies following appropriate elucidation. This also included coagulation factor concentrates extracted from human plasma enabling an effective treatment of even severe bleeding complications. In this analysis postoperative hemoglobin concentrations below 6 g/dl in older JW patients were associated with a high mortality risk due to anemia.

Covalent incorporation of tobacco mosaic virus increases the stiffness of poly(ethylene glycol) diacrylate hydrogels.

Hydrogels are versatile materials, finding applications as adsorbers, supports for biosensors and biocatalysts or as scaffolds for tissue engineering. A frequently used building block for chemically cross-linked hydrogels is poly(ethylene glycol) diacrylate (PEG-DA). However, after curing, PEG-DA hydrogels cannot be functionalized easily. In this contribution, the stiff, rod-like tobacco mosaic virus (TMV) is investigated as a functional additive to PEG-DA hydrogels. TMV consists of more than 2000 identical coat proteins and can therefore present more than 2000 functional sites per TMV available for coupling, and thus has been used as a template or building block for nano-scaled hybrid materials for many years. Here, PEG-DA (M n = 700 g mol-1) hydrogels are combined with a thiol-group presenting TMV mutant (TMVCys). By covalent coupling of TMVCys into the hydrogel matrix via the thiol-Michael reaction, the storage modulus of the hydrogels is increased compared to pure PEG-DA hydrogels and to hydrogels containing wildtype TMV (wt-TMV) which is not coupled covalently into the hydrogel matrix. In contrast, the swelling behaviour of the hydrogels is not altered by TMVCys or wt-TMV. Transmission electron microscopy reveals that the TMV particles are well dispersed in the hydrogels without any large aggregates. These findings give rise to the conclusion that well-defined hydrogels were obtained which offer the possibility to use the incorporated TMV as multivalent carrier templates e.g. for enzymes in future studies.

Virus-directed formation of electrocatalytically active nanoparticle-based Co3O4 tubes.

Spinel-type Co3O4 finds applications in a wide range of fields, including clean energy conversion, where nanostructured Co3O4 may provide a cost-efficient alternative to platinum- and iridium-based catalysts for electrocatalytic water-splitting. We here describe a novel strategy in which basic cobalt carbonate - a precursor to Co3O4 - is precipitated as sheet-like structures and microspheres covered with fine surface protrusions, via ammonium carbonate decomposition at room temperature. Importantly, these mild reaction conditions enable us to employ bio-inspired templating approaches to further control the mineral structure. Rod-like tobacco mosaic viruses (TMV) were used as biotemplates for mineral deposition, where we profit from the ability of Co(ii) ions to mediate the ordered assembly of the virus nanorods to create complex tubular superstructures of TMV/ basic cobalt carbonate. Calcination of these tubules is then achieved with retention of the gross morphology, and generates a hierarchically-structured solid comprising interconnected Co3O4 nanoparticles. Evaluation of these Co3O4 materials as electrocatalysts for the oxygen evolution reaction (OER) demonstrates that the activity of Co3O4 prepared by calcination of ammonia diffusion-grown precursors in both, the absence or presence of TMV exceeds that of a commercial nanopowder.

Application perspectives of localization microscopy in virology.

Localization microscopy approaches allowing an optical resolution down to the single-molecule level in fluorescence-labeled biostructures have already found a variety of applications in cell biology, as well as in virology. Here, we focus on some perspectives of a special localization microscopy embodiment, spectral precision distance/position determination microscopy (SPDM). SPDM permits the use of conventional fluorophores or fluorescent proteins together with standard sample preparation conditions employing an aqueous buffered milieu and typically monochromatic excitation. This allowed superresolution imaging and studies on the aggregation state of modified tobacco mosaic virus particles on the nanoscale with a single-molecule localization accuracy of better than 8 nm, using standard fluorescent dyes in the visible spectrum. To gain a better understanding of cell entry mechanisms during influenza A virus infection, SPDM was used in conjunction with algorithms for distance and cluster analyses to study changes in the distribution of virus particles themselves or in the distribution of infection-related proteins, the hepatocyte growth factor receptors, in the cell membrane on the single-molecule level. Not requiring TIRF (total internal reflection) illumination, SPDM was also applied to study the molecular arrangement of gp36.5/m164 glycoprotein (essentially associated with murine cytomegalovirus infection) in the endoplasmic reticulum and the nuclear membrane inside cells with single-molecule resolution. On the basis of the experimental evidence so far obtained, we finally discuss additional application perspectives of localization microscopy approaches for the fast detection and identification of viruses by multi-color SPDM and combinatorial oligonucleotide fluorescence in situ hybridization, as well as SPDM techniques for optimization of virus-based nanotools and biodetection devices.

The movement protein BC1 promotes redirection of the nuclear shuttle protein BV1 of Abutilon mosaic geminivirus to the plasma membrane in fission yeast.

In order to monitor their interaction and cellular localisation, the movement protein (MP; syn. BC1) and the nuclear shuttle protein (NSP; syn. BV1) of the geminivirus Abutilon mosaic virus (AbMV) were ectopically expressed in Schizosaccharomyces pombe cells, either alone or together under the control of an inducible promoter. For highest resolution, electron microscopy using freeze-fracture immunolabelling served to detect these proteins in situ. As expected from previous in planta and yeast experiments, NSP accumulated within the nuclei, whereas MP was targetted to the protoplasmic face of plasma membranes when expressed alone. Upon coexpression, NSP was localised at the plasma membranes, where it was strongly attached. These results support a model in which NSP transports viral DNA to the cell periphery to facilitate cell-to-cell movement of viral DNA within plants. In contrast to AbMV MP, no plant-specific protein seems to be necessary for the translocation of NSP to the plasma membrane.

Yeast two-hybrid systems confirm the membrane- association and oligomerization of BC1 but do not detect an interaction of the movement proteins BC1 and BV1 of Abutilon mosaic geminivirus.

Most of the plant begomoviruses use two proteins to transport their DNA from cell to cell, BV1 to shuttle it between nucleus and cytoplasm and BC1 to facilitate movement across plasmodesmata. In order to analyse their interaction for Abutilon mosaic geminivirus (AbMV) in yeast ( Saccharomyces cerevisiae), BC1 and BV1 genes were cloned into various plasmid vectors suitable for two-hybrid analysis. BC1 was fused to the binding domain (GBD) and BV1 to the activation domain (GAD) of the GAL4 transcription factor to check for interactions in the nucleus. Additionally, BC1 as well as BV1 were integrated into pMyr or pSos vectors to analyze protein binding at the plasma membrane using the CytoTraptrade mark system. Using freeze-fracture immuno-labelling (FreeFI), singly-expressed GBD:BC1 was localized at the plasma membrane although it was fused to a nuclear localization signal provided by the construct. GAD:BV1 was found in the nucleus of transformed cells as expected. Upon co-transformation of both constructs, cells grew poorly and exhibited symptoms of autolysis without any detectable level of GBD:BC1 or GAD:BV1, as revealed by FreeFI. In conclusion, both fusion proteins did not meet in the same compartment and appeared to be harmful to yeast if constitutively co-expressed. When expressed from pSos vector, BC1 induced the CytoTrap detection signal in the absence of pMyr indicating that BC1 protein alone is able to target the effector protein to the inner face of the plasma membrane. A mutated form of BC1 (DeltaBC1) lacking the previously identified membrane-binding domain was no longer able to auto-induce the CytoTrap signal cascade. Using DeltaBC1, an N-terminal, or a C-terminal third of BC1 revealed a homo-oligomerization of the C-terminal region of BC1 in two-hybrid analysis, but no interaction of BC1 with BV1.

Binding the tobacco mosaic virus to inorganic surfaces.

We studied the adsorption behavior and surface chemistry of the tobacco mosaic virus (TMV) on well-defined metal and insulator surfaces. TMV serves as a tubular supramolecular model system with precisely known surface termination. We show that if the surface chemistry of the substrate and the pH-dependent chemistry of the molecular surface match, for example, by hydrogen bonding, a strong adsorption occurs, and lateral movement is impeded. Due to the immobilization, the virion can be imaged by atomic force microscopy (AFM) in contact mode. We also used self-assembled monolayers with an acyl chloride group to induce covalent bonding via ester formation. Noncontact AFM proved that TMV keeps its cylindrical cross section only under weak adsorption conditions, that is, on hydrophobic surfaces, while on hydrophilic substrates a deformation occurs to maximize the number of interacting chemical groups.

Localizing the movement proteins of Abutilon mosaic geminivirus in yeast by subcellular fractionation and freeze-fracture immuno-labelling.

The movement proteins BC1 and BV1 of Abutilon mosaic geminivirus fused to glutathion-S-transferase (GST) and Flag-peptide were expressed in fission yeast (Schizosaccharomyces pombe) cells to analyse the fundamental intracellular distribution of these proteins in an eukaryotic cell in the absence of plant-specific factors. Most of BC1 protein sedimented rapidly after cell lysis and differential centrifugation. Using freeze-fracture immuno-labelling, the protein was detected in situ predominantly at plasma membranes and to a lower extent at cytoplasmic vesicles but not in the cytoplasm, the nuclei, or the mitochondria. Anti-BC1, anti-GST, and anti-Flag antibodies tagged smooth flecks only at the protoplasmic faces of the plasma membrane. The consequences of the BC1 behaviour for its use in two-hybrid analysis in yeast are discussed. In contrast, BV1 was detected mainly in the nucleus and partially in the cytoplasm but never associated with membranes.

Movement proteins (BC1 and BV1) of Abutilon mosaic geminivirus are cotransported in and between cells of sink but not of source leaves as detected by green fluorescent protein tagging.

Two movement proteins (BV1 and BC1) facilitate the intra- and intercellular transport of begomoviruses in plants. In contrast to other geminiviruses the movement protein BC1 of Abutilon mosaic virus (AbMV) remained in the supernatant after centrifuging plant extracts at 20,000 g. To test whether this unusual behavior results from a distinct intracellular distribution of the protein, the BC1 gene has been fused to the gene of green fluorescent protein (GFP). The resulting plasmids were delivered into nonhost plants (Allium cepa) as well as into mature and immature cells of host plants (Nicotiana tabacum, N. benthamiana) by biolistic bombardment for transient expression in planta. BC1 directed GFP to two different cellular sites. In the majority of nonhost cells as well as in mature cells of host leaves, BC1 was mainly localized in small punctate flecks at the cell periphery or, to a lesser extent, around the nucleus. In sink leaves of host plants, GFP:BC1 additionally developed disc-like structures in the cell periphery. Cobombardment of GFP:BC1 with its cognate infectious DNA A and B did not change their subcellular distribution patterns in source leaves but led to the formation of peculiar needle-like structures in sink leaves. The nuclear shuttle protein (BV1) of AbMV accumulated mainly inside the nuclei as shown by immunohistochemical staining and GFP tagging. In sink cells of host plants it was mobilized to the plasma membrane and to the nucleus of the neighboring cell by coexpressed BC1, GFP:BC1, BC1:GFP, or after cobombardment with the cognate viral DNA. Only under these conditions were GFP:BC1 and BC1:GFP also found in the recipient cell.

Fulfilling Koch's postulates for Abutilon mosaic virus.

Cloned Abutilon mosaic geminivirus (AbMV) has been re-transmitted to Abutilon sellovianum (syn. striatum) plants by a two-step combined agroinfection/grafting method. The symptoms induced were indistinguishable from the characteristic mosaic of ornamental Abutilon plants. Therefore, we can exclude that a mixture of different AbMV variants is responsible for the striking variety of Abutilon leaf pattern elements. Analysis of the symptoms on consecutively infected leaves suggests that mosaic formation depends on routing and timing of super-imposed virus waves.