Biochemische und molekularbiologische Charakterisierung des neuartigen Influenza-A-Virus Proteins PB1-F2

This study is focused on the functional and biochemical characterization of the novel 11th influenza A virus (IAV) protein PB1-F2. Recently, it was shown that PB1-F2 contributes to the increased virulence of highly pathogenic IAV strains and enhances the effects of secondary bacterial coinfection in mice. This results in severe pneumonia which is the major cause of influenza related mortality. PB1-F2 shares functional similarities to the pro-apoptotic Vpr protein of HIV-1. Initial experiments in this work are based on the hypothesis that extracellular synthetic (s)PB1-F2 might be able to induce apoptosis in cell culture, similar to the membrane transducing Vpr protein. PB1-F2 is predominantely located in the mitochondria of transfected or infected cells and mediates pro-apoptotic functions by a so far unknown mechanism. No cytotoxic effects were detected after incubation of different cell lines with extracellular PB1-F2. Also the plasmid mediated expression of PB1-F2 after transfection of 293T cells showed no influence on apoptosis induction. Additional experiments revealed that PB1-F2 is an enhancer of virus induced apoptosis after infection of primary human monocytes, measured by caspase 3 acitivity. Therefore it was concluded that the pro-apoptotic effects of PB1-F2 are predominantly mediated after virus infection and in a cell-type-specific manner. Further studies shown in this work describe the discovery of PB1-F2 phopshorylation, which is a totally new finding in the field. PB1-F2 is phopshorylated in vitro by an extract containing the conventional isoforms of the protein kinase C (PKC) and by cellular S10 extract of different cell lines. 32P radiolabelling of transfected 293T cells and infected MDCK cells revealed that PB1-F2 is phosphorylated after expression in cell culture. The in vitro and in vivo phosphorylation of PB1-F2 is sensitive to inhibitors of PKC and could be increased by the PKC activator PMA. Several studies have shown that activation of the PKC occurs subsequent to influenza virus infection. However, the function of the PKC activation in the context of virus replication is unknown. The threonine in position 27 and the serine in position 35 have been identified as the relevant PKC phopshorylation sites of PB1-F2 with the latter being conserved in different influenza isolates which is a prerequisite for a general function of the phosphorylation phenomenon. A recombinant virus which is mutated for the PB1-F2 phopshorylation sites was impaired in its ability to induce apoptosis, similar to a mutant virus which is knocked out for PB1-F2 expression. If PB1-F2 phopshorylation is also important for the recently described immunomodulatory effects and pathogenicity remains to be investigated. PB1-F2 displays the inherent ability to undergo oligomerization. This phenomenon was characterized by the use of sPB1-F2 with chemical crosslinkers and western blot analysis. In silico analysis revealed that the protein displays two oligomerization domains in the C-terminus and a much weaker domain in the N-terminus, which is consistent with the finding that both, a N-terminal and a C-terminal fragment of PB1-F2 are able to form multimers, while the C-terminal fragment has a much higher propensity for oligomerization. Under chemically reducing conditions it was shown that the cystein in position 42 mediates the dimerization of PB1-F2 by disulfid-bridges whereas the protein multimerization depends mainly on the integrity of the oligomerization sites. There has been much speculation about the functional relevance of the oligomerization phenomenon. The most prominent hypothesis suggests that PB1-F2 oligomerization results in the formation of membrane-pores and therefore contributes to the destabilization of the mitochondrial membrane potential, suggesting that PB1-F2 mediates a viroporin-like function.Diese Arbeit beschäftigt sich mit der funktionellen und biochemischen Charakterisierung des im Jahre 2001 entdeckten 11. Influenza-A-Virus-(IAV)-Proteins PB1-F2. Vor kurzem wurde im Mausmodell gezeigt, dass PB1-F2 die Virulenz hochpathogener IAV-Isolate verstärkt. Weiterhin besitzt PB1-F2 funktionelle Ähnlichkeit zum pro-apoptotischen Vpr-Protein von HIV-1, weshalb zunächst untersucht wurde, ob extrazelluläres PB1-F2, ähnlich dem transduzierenden Vpr-Protein in der Lage ist, apoptotische Prozesse einzuleiten. Grundlage für diese Arbeitshypothese war die bekannte Tatsache, dass PB1-F2 in den Mitochondrien lokalisiert und pro-apoptotische Funktionen in einem noch nicht verstandenen Mechanismus vermittelt. In dieser Arbeit wurde gezeigt, dass weder extrazelluläres synthetisches (s)PB1-F2 noch intrazelluläres, durch Plasmid exprimiertes PB1-F2 ausreichend ist, um in verschiedenen Zelllinien Apoptose zu induzieren. Des Weiteren konnte hier gezeigt werden, dass nach der Infektion von primären humanen Monozyten ein apoptoseverstärkender Effekt in Gegenwart von PB1-F2 gemessen werden kann. Wurden dieselben Zellen mit einem PB1-F2-defizienten Virus infiziert, war die Apoptoseinduktion deutlich reduziert. Diese Befunde legen nahe, dass PB1-F2 nur im Kontext einer Virusinfektion pro-apoptotisch wirkt und dieser Effekt zellspezifisch ist, was in späteren Publikationen anderer Labore bestätigt wurde. Ein weiterer Schwerpunkt war die Untersuchung einer möglichen posttranslationalen Modifikation von PB1-F2. Es konnte hier erstmals gezeigt werden, dass PB1-F2 ein Phosphoprotein ist und direkt mit der Proteinkinase C (PKC) interagiert. Das virale Protein wurde von einem Extrakt aus verschiedenen konventionellen PKC-Isoformen sowie den Zellextrakten verschiedener Zelllinien effizient in vitro phosphoryliert. Die radioaktive 32P-Markierung von transfizierten 293-T-Zellen und infizierten MDCK-Zellen ergab, dass PB1-F2 in vivo phopshoryliert wird. Die in vitro und in vivo Phosphorylierung konnte in Gegenwart PKC-spezifischer Inhibitioren dosisabhängig inhibiert und durch den Aktivator PMA verstärkt werden. Es wurde mehrfach berichtet, dass die PKC nach einer IAV-Infektion aktiviert vorliegt, jedoch ist nur wenig über die Funktion dieser Aktivierung im Kontext der Virusreplikation bekannt. Als PB1-F2-relevante Phosphorylierungsstellen wurden in dieser Arbeit die Aminosäuren Threonin in Poistion 27 und Serin in Position 35 ermittelt, wobei die letztere in verschiedenen Isolaten konserviert ist, was eine Grundvoraussetzung für eine mögliche funktionelle Bedeutung der PB1-F2-Phosphorylierung wäre. Ein rekombinantes Virus, das für die Phosphorylierungsstellen in PB1-F2 mutiert wurde, zeigte nach der Infektion von primären Monozyten einen vergleichbaren Phänotyp wie eine Mutante, die durch gezielte Austauschmutationen nicht mehr in der Lage ist, PB1-F2 zu exprimieren. Beide Virusmutanten waren im Vergleich zum Wildtyp-Virus in ihrer Fähigkeit eingeschränkt, effektiv Apoptose zu induzierten. Ob die Phosphorylierung von PB1-F2 auch für die kürzlich beschriebenen immunmodulatorischen Effekte des Proteins im Tiermodell von Bedeutung ist und in welchem Zusammenhang sie zur IAV-aktivierten, zellulären Signaltransduktion steht, bleibt noch Gegenstand zukünftiger Untersuchungen. PB1-F2 besitzt die inhärente Fähigkeit zu oligomerisieren. Dieses Phänomen wurde durch die Behandlung von sPB1-F2 mit chemischen Crosslinkern und anschließenden Westernblot-Analysen untersucht. Es zeigte sich, dass sowohl ein N-terminales als auch C-terminales Fragment von PB1-F2 Oligomere ausbildet, was mit dem in silico Befund übereinstimmt, dass das Protein je eine Oligomerisierungsdomäne im N- und C-terminalen Bereich besitzt, wobei die C-terminale Domäne ein deutlich größeres Potential zur Multimerisierung aufweist. Unter chemisch reduzierenden Bedingungen konnte gezeigt werden, dass das Cystein in Position 42 maßgeblich an der Ausbildung der dimeren Spezies von PB1-F2 durch Disulfidbindung beteiligt ist, wohingegen die PB1-F2-Multimerisierung ausschließlich auf die Integrität der Oligomerisierungsdomänen zurückgeführt werden kann. Über die funktionelle Relevanz der Oligomerisierung von PB1-F2 wurde bisher viel spekuliert. Die prominenteste Hypothese ist, dass PB1-F2 Membranporen ausbildet und möglicherweise zur Destabilisierung des mitochondrialen Membranpotentials beiträgt und damit funktionell der Gruppe der Viroporine zugeordnet werden kann

Effect of Parenchymal Stiffness on Canine Airway Size with Lung Inflation

Although airway patency is partially maintained by parenchymal tethering, this structural support is often ignored in many discussions of asthma. However, agonists that induce smooth muscle contraction also stiffen the parenchyma, so such parenchymal stiffening may serve as a defense mechanism to prevent airway narrowing or closure. To quantify this effect, specifically how changes in parenchymal stiffness alter airway size at different levels of lung inflation, in the present study, we devised a method to separate the effect of parenchymal stiffening from that of direct airway narrowing. Six anesthetized dogs were studied under four conditions: baseline, after whole lung aerosol histamine challenge, after local airway histamine challenge, and after complete relaxation of the airways. In each of these conditions, we used High resolution Computed Tomography to measure airway size and lung volume at five different airway pressures (0, 12, 25, 32, and 45 cm H2O). Parenchymal stiffening had a protective effect on airway narrowing, a fact that may be important in the airway response to deep inspiration in asthma. When the parenchyma was stiffened by whole lung aerosol histamine challenge, at every lung volume above FRC, the airways were larger than when they were directly challenged with histamine to the same initial constriction. These results show for the first time that a stiff parenchyma per se minimizes the airway narrowing that occurs with histamine challenge at any lung volume. Thus in clinical asthma, it is not simply increased airway smooth muscle contraction, but perhaps a lack of homogeneous parenchymal stiffening that contributes to the symptomatic airway hyperresponsiveness

Individual Canine Airway Response Variability to a Deep Inspiration

In healthy individuals, a DI can reverse (bronchodilation) or prevent (bronchoprotection) induced airway constriction. For individuals with asthma or COPD, these effects may be attenuated or absent. Previous work showed that the size and duration of a DI affected the subsequent response of the airways. Also, increased airway tone lead to increased airway size variability. The present study examined how a DI affected the temporal variability in individual airway baseline size and after methacholine challenge in dogs using High-Resolution Computed Tomography. Dogs were anesthetized and ventilated, and on 4 separate days, HRCT scans were acquired before and after a DI at baseline and during a continuous intravenous infusion of methacholine (Mch) at 3 dose rates (17, 67, and 200 μg/min). The Coefficient of Variation was used as an index of temporal variability in airway size

How a Diverse Research Ecosystem Has Generated New Rehabilitation Technologies: Review of NIDILRR’s Rehabilitation Engineering Research Centers

Over 50 million United States citizens (1 in 6 people in the US) have a developmental, acquired, or degenerative disability. The average US citizen can expect to live 20% of his or her life with a disability. Rehabilitation technologies play a major role in improving the quality of life for people with a disability, yet widespread and highly challenging needs remain. Within the US, a major effort aimed at the creation and evaluation of rehabilitation technology has been the Rehabilitation Engineering Research Centers (RERCs) sponsored by the National Institute on Disability, Independent Living, and Rehabilitation Research. As envisioned at their conception by a panel of the National Academy of Science in 1970, these centers were intended to take a “total approach to rehabilitation”, combining medicine, engineering, and related science, to improve the quality of life of individuals with a disability. Here, we review the scope, achievements, and ongoing projects of an unbiased sample of 19 currently active or recently terminated RERCs. Specifically, for each center, we briefly explain the needs it targets, summarize key historical advances, identify emerging innovations, and consider future directions. Our assessment from this review is that the RERC program indeed involves a multidisciplinary approach, with 36 professional fields involved, although 70% of research and development staff are in engineering fields, 23% in clinical fields, and only 7% in basic science fields; significantly, 11% of the professional staff have a disability related to their research. We observe that the RERC program has substantially diversified the scope of its work since the 1970’s, addressing more types of disabilities using more technologies, and, in particular, often now focusing on information technologies. RERC work also now often views users as integrated into an interdependent society through technologies that both people with and without disabilities co-use (such as the internet, wireless communication, and architecture). In addition, RERC research has evolved to view users as able at improving outcomes through learning, exercise, and plasticity (rather than being static), which can be optimally timed. We provide examples of rehabilitation technology innovation produced by the RERCs that illustrate this increasingly diversifying scope and evolving perspective. We conclude by discussing growth opportunities and possible future directions of the RERC program

The intriguing Cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding

<p>Abstract</p> <p>Background</p> <p>Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur <it>in vitro </it>and <it>in vivo</it>. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.</p> <p>Results</p> <p>Characterization of the interactions of human CypA with N-terminal peptides of HIV-1 Vpr has been achieved using a combination of nuclear magnetic resonace (NMR) exchange spectroscopy and surface plasmon resonance spectroscopy (SPR). NMR data at atomic resolution indicate prolyl <it>cis</it>/<it>trans </it>isomerisation of the highly conserved proline residues Pro-5, -10, -14 and -35 of Vpr are catalyzed by human CypA and require only very low concentrations of the isomerase relative to that of the peptide substrates. Of the N-terminal peptides of Vpr only those containing Pro-35 bind to CypA in a biosensor assay. SPR studies of specific N-terminal peptides with decreasing numbers of residues revealed that a seven-residue motif centred at Pro-35 consisting of RHFPRIW, which under membrane-like solution conditions comprises the loop region connecting helix 1 and 2 of Vpr and the two terminal residues of helix 1, is sufficient to maintain strong specific binding.</p> <p>Conclusions</p> <p>Only N-terminal peptides of Vpr containing Pro-35, which appears to be vital for manifold functions of Vpr, bind to CypA in a biosensor assay. This indicates that Pro-35 is essential for a specific CypA-Vpr binding interaction, in contrast to the general prolyl <it>cis</it>/<it>trans </it>isomerisation observed for all proline residues of Vpr, which only involve transient enzyme-substrate interactions. Previously suggested models depicting CypA as a chaperone that plays a role in HIV-1 virulence are now supported by our data. In detail the SPR data of this interaction were compatible with a two-state binding interaction model that involves a conformational change during binding. This is in accord with the structural changes observed by NMR suggesting CypA catalyzes the prolyl <it>cis/trans </it>interconversion during binding to the RHFP³⁵RIW motif of N-terminal Vpr.</p

Impact of intraoperative cytokine adsorption on outcome of patients undergoing orthotopic heart transplantation - an observational study

AIM: The aim of this study was to assess the influence of intraoperative cytokine adsorption on the perioperative vasoplegia, inflammatory response and outcome during orthotopic heart transplantation (OHT). METHODS: 84 OHT patients were separated into the cytokine adsorption (CA) treated group or controls. Vasopressor demand, inflammatory response described by procalcitonin and C-reactive protein and postoperative outcome were assessed performing propensity score matching. RESULTS: In the 16 matched pairs, the median noradrenaline requirement was significantly less in the CA-treated patients than in the controls on the first and second postoperative days (0.14 vs 0.3mug*kg(-1) *min(-1) , P=0.039 and 0.06 vs 0.32mug*kg(-1) *min(-1) , P=0.047). The inflammatory responses were similar in the two groups. There was a trend towards shorter length of mechanical ventilation and intensive care unit (ICU) stay in the CA-treated group compared to the controls. No difference in adverse events was observed between the two groups. However, the frequency of renal replacement therapy was significantly less in the CA-treated than in controls (P=0.031). CONCLUSIONS: Intraoperative CA treatment was associated with reduced vasopressor demand and less frequent renal replacement therapy with a favorable tendency in length of mechanical ventilation and ICU stay. CA treatment was not linked to higher rates of adverse events. This article is protected by copyright. All rights reserved

Inclusion of service robots in the daily lives of frail older users: a step-by-step definition procedure on users' requirements

The implications for the inclusion of robots in the daily lives of frail older adults, especially in relation to these population needs, have not been extensively studied. The “Multi-Role Shadow Robotic System for Independent Living” (SRS) project has developed a remotelycontrolled, semi-autonomous robotic system to be used in domestic environments. The objective of this paper is to document the iterative procedure used to identify, select and prioritize user requirements. Seventy-four requirements were identified by means of focus groups, individual interviews and scenario-based interviews. The list of user requirements, ordered according to impact, number and transnational criteria, revealed a high number of requirements related to basic and instrumental activities of daily living, cognitive and social support and monitorization, and also involving privacy, safety and adaptation issues. Analysing and understanding older users’ perceptions and needs when interacting with technological devices adds value to assistive technology and ensures that the systems address currently unmet needs

Understanding the Use of Crisis Informatics Technology among Older Adults

Mass emergencies increasingly pose significant threats to human life, with a disproportionate burden being incurred by older adults. Research has explored how mobile technology can mitigate the effects of mass emergencies. However, less work has examined how mobile technologies support older adults during emergencies, considering their unique needs. To address this research gap, we interviewed 16 older adults who had recent experience with an emergency evacuation to understand the perceived value of using mobile technology during emergencies. We found that there was a lack of awareness and engagement with existing crisis apps. Our findings characterize the ways in which our participants did and did not feel crisis informatics tools address human values, including basic needs and esteem needs. We contribute an understanding of how older adults used mobile technology during emergencies and their perspectives on how well such tools address human values.Comment: 10 page

The proapoptotic influenza A virus protein PB1-F2 forms a nonselective ion channel

Background: PB1-F2 is a proapoptotic influenza A virus protein of approximately 90 amino acids in length that is located in the nucleus, cytosol and in the mitochondria membrane of infected cells. Previous studies indicated that the molecule destabilizes planar lipid bilayers and has a strong inherent tendency for multimerization. This may be correlate with its capacity to induce mitochondrial membrane depolarization. Methodology/Principal Findings: Here, we investigated whether PB1-F2 is able to form ion channels within planar lipid bilayers and microsomes. For that purpose, a set of biologically active synthetic versions of PB1-F2 (sPB1-F2) derived from the IAV isolates A/Puerto Rico/8/34(H1N1)( IAV(PR8)), from A/Brevig Mission/1/1918( H1N1) (IAV(SF2)) or the H5N1 consensus sequence (IAV(BF2)) were used. Electrical and fluorimetric measurements show that all three peptides generate in planar lipid bilayers or in liposomes, respectively, a barely selective conductance that is associated with stochastic channel type fluctuations between a closed state and at least two defined open states. Unitary channel fluctuations were also generated when a truncated protein comprising only the 37 c-terminal amino acids of sPB1-F2 was reconstituted in bilayers. Experiments were complemented by extensive molecular dynamics simulations of the truncated fragment in a lipid bilayer. The results indicate that the c-terminal region exhibits a slightly bent helical fold, which is stable and remains embedded in the bilayer for over 180 ns. Conclusion/Significance: The data support the idea that PB1-F2 is able to form protein channel pores with no appreciable selectivity in membranes and that the c-terminus is important for this function. This information could be important for drug development