Activation and inhibition of the type I interferon pathway in tick-borne encephalitis virus infection

Das Frühsommer-Meningoenzephalitis (FSME) Virus gehört zur Gattung der Flaviviren, Familie Flaviviridae und besteht aus einem einsträngigem RNA Genom mit positiver Polarität. Angeborene Immunität ist wichtig, um Virusstrukturen und Virusreplikation von RNA Viren zu erkennen. Säugetierzellen verfügen über pathogene Erkennungsrezeptoren (PRR) um Produkte der Virusreplikation zu erkennen und Signalkaskaden zu aktivieren, die zur Etablierung eines antiviralen Zustandes durch die Induktion von Typ I Interferonen (IFN) führen. In dieser Studie untersuchten wir die Interaktion des FSME Virus mit dem angeborenen Wirtsimmunsystem. Von besonderem Interesse war die Rolle von Typ I Interferonen in FSME Virus Infektionen in vitro und die Mechanismen die zur Interferon Aktivierung führen. Wir zeigten, dass die Infektion von Zellen mit replizierendem Virus oder die Transfektion von selbst-replizierender viraler RNA zu der Aktivierung von Typ I IFN mRNA Transkription führte. Zusätzlich war nur ″Ganzlängen″ RNA fähig ein Reportergen mit einem davor geschalteten IFNβ Promoter zu aktivieren. Unsere Ergebnisse bewiesen, dass IFNα/β Signalisierung von RNA Replikation abhängt. Weiters ist die virale RNA Replikation ein zentraler Mechanismus in der Aktivierung von Typ I IFN Induktion. Außerdem zeigten wir dass IRF3 (interferon regulatory factor 3) eine wichtige Rolle in der Kontrolle von viraler Replikation spielt. Mauszellen, die über kein IRF3 mehr verfügten und die mit TBEV infiziert wurden, wiesen eine stark erhöhte Virusreplikation auf. Diese Zellen aktivierten kein IFNα/β. Im Gegensatz induzierten Wildtyp Zellen sehr stark die IFN Produktion, was zu einer Verminderung der Virusreplikation führte und zeigt, dass IRF3 ein Hauptregulator in der Typ I IFN Signalisierung ist. Der Transkriptionsfaktor ist wichtig, um Virusreplikation durch die Induktion von IFNα/β zu bekämpfen. Überaschenderweise führte die Infektion durch das FSME Virus nur zu geringer IFNα/β Aktivierung und aus diesem Grund testeten wir, ob es die Entstehung von IFN hemmt. Schließlich identifizierten wir das FSME Virus als einen Inhibitor des IFNα/β Systems. Das Virus interagiert mit Komponenten der Typ I IFN Signalkaskade in frühen Stadien durch die Inhibierung von IFN Induktion und in später Phase in der Etablierung eines antiviralen Zustandes durch die Inhibierung von STAT-1 Phosphorylierung.Tick-borne encephalitis virus (TBEV) is a member of the genus Flaviviruses, family Flaviviridae and contains a single stranded (ss) RNA genome with positive polarity. Innate immunity is important to recognize viral structures and control viral replication of RNA viruses. Mammalian cells provide pathogen recognition receptors (PRRs) to detect products of viral replication and to trigger signal cascades, which result in the establishment of an antiviral state by the induction of type I interferons (IFNs). Here, we investigated the interaction of TBEV with the host innate immune system. In particular, we were interested in the role of type I interferons on TBEV infections in vitro and on mechanisms leading to IFN activation. We showed that infection of cells with replicating virus or transfection of self replicating viral RNAs lead to the activation of type I IFN mRNA transcription. In addition, only full length RNA of TBEV was able to activate an IFNβ promoter driven reporter gene. Our results indicated that IFNα/β signalling is RNA replication dependent. Moreover, virus RNA replication is a central mechanism in the activation of type I IFN induction. Furthermore, we showed that the interferon regulatory factor 3 plays (IRF3) an important role in the control of viral replication. IRF3 deficient cells infected with TBEV exhibited a strong enhancement of virus replication. These cells did not activate IFNα/β. On the contrary, wild-type cells strongly induced IFN production, which lead to the suppression of virus replication, indicating that IRF3 is a main regulator in type I IFN signalling and essential to combat viral replication by inducing IFNα/β. Surprisingly, TBEV infection only lead to low IFNα/β activation and therefore we analysed if TBEV inhibits IFN expression. Finally, we identified TBEV as an inhibitor of the IFNα/β system. The virus interferes with components of the type I IFN signalling cascade at early stages by inhibiting IFN induction and on later periods in the establishment of an anti viral state by inhibition of STAT-1 phosphorylation

Entwicklung funktionalisierter Zinkpartikel als Anodenmaterial für wiederaufladbare Zink-Luft-Batterien

In dieser Arbeit werden vergleichende Untersuchungen zu partikulären Zinkanodenmaterialien für wiederaufladbare Zink-Luft-Batterien vorgestellt. Dies umfasst die Entwicklung funktionalisierter Anodenmaterialien durch Variation der Zinkmorphologie und Beschichtung der Zinkpartikel mit Silikaten sowie bismutoxidbasierten Oxiden. Dadurch sollen die Degradationen, welche die Lebensdauer von partikulären Zinkanoden begrenzen, verringert werden. Ziel ist es, einerseits eine hohe Aktivmaterialausnutzung und andererseits eine höhere Zyklenzahl zu erzielen, als es mit den Zinkpartikeln aus dem gegenwärtigen Stand der Technik bisher möglich ist. Mit Hilfe verschiedener elektrochemischer und materialwissenschaftlicher Methoden werden die Einflüsse der funktionalisierten Zinkpulver auf die Degradationsmechanismen dargelegt und mit Zinkpartikeln, welche in primären Zink-Luft-Zellen generell Einsatz finden, verglichen. Die Anodenmaterialien werden mit vollständigen Ent- und Beladungen beaufschlagt, um aufzuzeigen, ob Wiederaufladungen nach maximaler Ausnutzung erreicht werden können. Anhand der Ergebnisse werden die Funktionalitäten der unterschiedlichen Zinkmorphologien und Beschichtungen abgeleitet und Schlussfolgerungen für Zinkanoden mit verbesserten elektrochemischen Eigenschaften gezogen

Toxoplasma and Eimeria co-opt the host cFos expression for intracellular development in mammalian cells

Successful asexual reproduction of intracellular pathogens depends on their potential to exploit host resources and subvert antimicrobial defense. In this work, we deployed two prevalent apicomplexan parasites of mammalian cells, namely Toxoplasma gondii and Eimeria falciformis, to identify potential host determinants of infection. Expression analyses of the young adult mouse colonic (YAMC) epithelial cells upon infection by either parasite showed regulation of several distinct transcripts, indicating that these two pathogens program their intracellular niches in a tailored manner. Conversely, parasitized mouse embryonic fibroblasts (MEFs) displayed a divergent transcriptome compared to corresponding YAMC epithelial cells, suggesting that individual host cells mount a fairly discrete response when encountering a particular pathogen. Among several host transcripts similarly altered by T. gondii and E. falciformis, we identified cFos, a master transcription factor, that was consistently induced throughout the infection. Indeed, asexual growth of both parasites was strongly impaired in MEF host cells lacking cFos expression. Last but not the least, our differential transcriptomics of the infected MEFs (parental and cFos-/- mutant) and YAMC epithelial cells disclosed a cFos-centered network, underlying signal cascades, as well as a repertoire of nucleotides- and ion-binding proteins, which presumably act in consort to acclimatize the mammalian cell and thereby facilitate the parasite development.Peer Reviewe

Validation of the newly developed Advanced Practice Nurse Task Questionnaire: A national survey

AIM: To describe psychometric validation of the newly developed Advanced Practice Nurse Task Questionnaire. DESIGN: Cross-sectional quantitative study. METHODS: The development of the questionnaire followed an adapted version of the seven steps described in the guide by the Association for Medical Education in Europe. A nationwide online survey tested the construct and structural validity and internal consistency using an exploratory factor analysis, Cronbach's alpha coefficient and a Kruskal-Wallis test to compare the hypotheses. RESULTS: We received 222 questionnaires between January and September 2020. The factor analysis produced a seven-factor solution as suggested in Hamric's model. However, not all item loadings aligned with the framework's competencies. Cronbach's alpha of factors ranged between .795 and .879. The analysis confirmed the construct validity of the Advanced Practice Nurse Task Questionnaire. The tool was able to discriminate the competencies of guidance and coaching, direct clinical practice and leadership across the three advanced practice nurse roles clinical nurse specialist, nurse practitioner or blended role. CONCLUSION: A precise assessment of advanced practice nurse tasks is crucial in clinical practice and in research as it may be a basis for further refinement, implementation and evaluation of roles. IMPACT: The Advanced Practice Nurse Task Questionnaire is the first valid tool to assess tasks according to Hamric's model of competencies independently of the role or the setting. Additionally, it distinguishes the most common advanced practice nurse roles according to the degree of tasks in direct clinical practice and leadership. The tool may be applied in various countries, independent of the degree of implementation and understanding of advanced nursing practice. REPORTING METHOD: The STARD 2015 guideline was used to report the study. PATIENT OR PUBLIC CONTRIBUTION: No patient or public contribution

Mutation (677C to T) in the methylenetetrahydrofolate reductase gene aggravates hyperhomocysteinemia in hemodialysis patients

Mutation 677C to T in the methylenetetrahydrofolate reductase gene aggravates hyperhomocysteinemia in hemodialysis patients. Hyperhomocysteinemia is frequent in hemodialysis patients and represents an independent risk factor for vascular disease in these patients. Elevated total homocysteine (tHcy) plasma levels can result from defective remethyla-tion of Hcy to methionine due to decreased activity of the enzyme methylenetetrahydrofolate reductase (MTHFR). A genetic aberration in the MTHFR gene (677C to T substitution) has been shown to result in reduced MTHFR activity. We tested the hypothesis that elevation of tHcy plasma levels in hemodialysis patients is influenced by the 677C to T mutation of the MTHFR gene and examined the relation of the genotype with tHcy, folate and vitamin B12 plasma levels in these patients. The allelic frequency of the MTHFR mutation was evaluated in 203 patients maintained on chronic hemodialysis treatment. Total Hcy, folate, vitamin B12 levels and the MTHFR mutation were analyzed in 69 of the 203 patients and in 69 age- and sex-matched healthy control subjects. The allelic frequency of the 677C to T transition in the MTHFR gene in hemodialysis patients was 34.7% versus 35.5% in healthy controls. Of 203 patients 26 (12.8%) were homozygous for the mutation (+/+) versus 10.2% in healthy subjects. The heterozygous (+/−) genotype was identified in 43.8% of patients versus 50.7% in controls. The mean tHcy level in hemodialysis patients was 28.7 ± 11.0 µuunol/liter versus 10.0 ± 3.0 µmol/liter in control subjects. The mean tHcy levels were 36.4 ± 13.4 µmol/liter in (+/+) patients and 12.2 ± 4.5 /mol/liter in (+/+) controls, 28.7 ± 10.8 µmol/liter in (+/−) patients and 9.9 ± 2.7 µmol/liter in (+/−) controls and 25.4 ± 8.5 µmol/liter in (−/−) hemodialysis patients versus 9.7 ± 2.8 µmol/liter in (−/−) controls. There was no significant difference of folate and vitamin B12 concentrations in patients and controls with different MTHFR genotypes. Analysis of covariance including age, gender, folate concentrations, vitamin B12 levels, albumin and creatinine as covariables revealed a significant influence of the (+/+) genotype, albumin and folate status on tHcy levels in hemodialysis patients. Together, our data demonstrate that the extent of hyperhomocysteinemia in hemodialysis patients is not only the result of uremia or folate status, but is also genetically determined by the (+/+) MTHFR genotype. The presence of the 677C to T mutation in the MTHFR gene does not appear to represent a risk factor for development of end-stage renal disease

Validation of the newly developed Advanced Practice Nurse Task Questionnaire: A national survey.

AIM To describe psychometric validation of the newly developed Advanced Practice Nurse Task Questionnaire. DESIGN Cross-sectional quantitative study. METHODS The development of the questionnaire followed an adapted version of the seven steps described in the guide by the Association for Medical Education in Europe. A nationwide online survey tested the construct and structural validity and internal consistency using an exploratory factor analysis, Cronbach's alpha coefficient and a Kruskal-Wallis test to compare the hypotheses. RESULTS We received 222 questionnaires between January and September 2020. The factor analysis produced a seven-factor solution as suggested in Hamric's model. However, not all item loadings aligned with the framework's competencies. Cronbach's alpha of factors ranged between .795 and .879. The analysis confirmed the construct validity of the Advanced Practice Nurse Task Questionnaire. The tool was able to discriminate the competencies of guidance and coaching, direct clinical practice and leadership across the three advanced practice nurse roles clinical nurse specialist, nurse practitioner or blended role. CONCLUSION A precise assessment of advanced practice nurse tasks is crucial in clinical practice and in research as it may be a basis for further refinement, implementation and evaluation of roles. IMPACT The Advanced Practice Nurse Task Questionnaire is the first valid tool to assess tasks according to Hamric's model of competencies independently of the role or the setting. Additionally, it distinguishes the most common advanced practice nurse roles according to the degree of tasks in direct clinical practice and leadership. The tool may be applied in various countries, independent of the degree of implementation and understanding of advanced nursing practice. REPORTING METHOD The STARD 2015 guideline was used to report the study. PATIENT OR PUBLIC CONTRIBUTION No patient or public contribution

Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells

Airway ciliary beat frequency regulation is complex but in part influenced by cyclic adenosine monophosphate (cAMP)-mediated changes in cAMP-dependent kinase activity, yet the cAMP concentration required for increases in ciliary beat frequency and the temporal relationship between ciliary beat frequency and cAMP changes are unknown. A lentiviral gene transfer system was developed to express a fluorescence resonance energy transfer (FRET)-based cAMP sensor in ciliated cells. Expression of fluorescently tagged cAMP-dependent kinase subunits from the ciliated-cell-specifi

NCBP3 positively impacts mRNA biogenesis

The nuclear Cap-Binding Complex (CBC), consisting of Nuclear Cap-Binding Protein 1 (NCBP1) and 2 (NCBP2), associates with the nascent 5' cap of RNA polymerase II transcripts and impacts RNA fate decisions. Recently, the C17orf85 protein, also called NCBP3, was suggested to form an alternative CBC by replacing NCBP2. However, applying protein-protein interaction screening of NCBP1, 2 and 3, we find that the interaction profile of NCBP3 is distinct. Whereas NCBP1 and 2 identify known CBC interactors, NCBP3 primarily interacts with components of the Exon Junction Complex (EJC) and the TRanscription and EXport (TREX) complex. NCBP3-EJC association in vitro and in vivo requires EJC core integrity and the in vivo RNA binding profiles of EJC and NCBP3 overlap. We further show that NCBP3 competes with the RNA degradation factor ZC3H18 for binding CBC-bound transcripts, and that NCBP3 positively impacts the nuclear export of polyadenylated RNAs and the expression of large multi-exonic transcripts. Collectively, our results place NCBP3 with the EJC and TREX complexes in supporting mRNA expression

Influence of natural killer cells and perforin-mediated cytolysis on the development of chemically induced lung cancer in A/J mice

One alternative approach for the treatment of lung cancer might be the activation of the immune system using vaccination strategies. However, most of clinical vaccination trials for lung cancer did not reach their primary end points, suggesting that lung cancer is of low immunogenicity. To provide additional experimental information about this important issue, we investigated which type of immune cells contributes to the protection from lung cancer development. Therefore, A/J mice induced for lung adenomas/adenocarcinomas by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were depleted of CD4+ or CD8+ T cells, CD11b+ macrophages, Gr-1+ neutrophils and asialo GM1+ natural killer (NK) cells. Subsequent analysis of tumour growth showed an increase in tumour number only in mice depleted of NK cells. Further asking by which mechanism NK cells suppressed tumour development, we neutralized several death ligands of the tumour necrosis factor (TNF) family known to be involved in NK cell-mediated cytotoxicity. However, neither depletion of TNF-α, TNF-related apoptosis-inducing ligand, TNF-like weak inducer of apoptosis or FasL alone nor in combination induced an augmentation of tumour burden. To show whether an alternative cell death pathway is involved, we next generated A/J mice deficient for perforin. After challenging with NNK, mice deficient for perforin showed an increase in tumour number and volume compared to wild-type A/J mice. In summary, our data suggest that NK cells and perforin-mediated cytolysis are critically involved in the protection from lung cancer giving promise for further immunotherapeutic strategies for this disease