Epsilonproteobakterielle Hydroxylamin-Oxidoreduktase (εHao): Charakterisierung eines “missing links“ innerhalb der Multihäm Cytochrom c Familie

Die Familie der prokaryotischen Multihäm Cytochrome c (MCC) besitzt eine zentrale Rolle im biogeochemischen Stickstoffkreislauf. Zwei bedeutende Multihäm Cytochrome c, die am Umsatz von Stickstoffverbindungen beteiligt sind, sind die Pentahäm Cytochrom c Nitritreduktase (NrfA) und die Oktahäm Hydroxylamin-Oxidoreduktase (Hao). Die Nitritreduktase ist dabei in den anaeroben Stoffwechselprozess der Ammonifikation involviert wohingegen die Hydroxylamin-Oxidoreduktase an dem aeroben Prozess der Nitrifikation beteiligt ist. Vertreter der MCC-Familie weisen zumeist einen vielseitigen enzymatischen Charakter auf. Ein prominentes Beispiel hierfür ist die Nitritreduktase (NrfA), welche neben der Nitrit- und Hydroxylamin-Reduktion zu Ammonium ebenfalls die Detoxifikation reaktiver Stickstoff-Spezies katalysiert. Die Oktahäm Cytochrom c Hydroxylamin-Oxidoreduktasen (fakultativ) anaerober Nitrat-Ammonifizierer (εHao) sind ebenfalls der MCC-Familie zugehörig. Strukturell weisen εHao-Proteine Identitäten von bis zu 22 % zur Hydroxylamin-Oxidoreduktase der nitrifizierenden Bakterien auf. Epsilonproteobakterielle Hydroxylamin-Oxidoreduktasen (εHao), die in den Genomen diverser Nitrat/Nitrit-Ammonifizierer identifiziert wurden, wurden jedoch bislang weder biochemisch noch enzymatisch charakterisiert. Interessant ist jedoch, dass diese Organismen bis auf wenige Ausnahmen kein Homolog der periplasmatischen Nitritreduktase NrfA besitzen. Aufgrund dessen wurde vermutet, dass die entsprechenden Bakterien über einen bisher nicht untersuchten Weg der Nitritverwertung verfügen. Weiterhin wurde angenommen, dass εHao-Proteine maßgeblich an diesem neuen Reaktionsweg beteiligt sein könnten. Die Ziele dieser Arbeit waren daher die heterologe Produktion diverser epsilonproteobakterieller Hydroxylamin-Oxidoreduktasen im Wirtsorganismus Wolinella succinogenes, die Reinigung der Proteine mittels Affinitätschromatographie und die ortsspezifische Mutagenese εHao-kodierender haoA-Gene. Weiterhin sollten die enzymatische Charakterisierung der Proteine unter Verwendung verschiedener Substrate sowie die Analyse des Substrat- und Produktspektrums erfolgen, um Rückschlüsse auf die biologische Funktion der epsilonproteobakteriellen Hydroxylamin-Oxidoreduktasen zu ziehen. Zusätzlich wurde das Vorkommen, die Lage der entsprechenden Gene im Genom sowie die strukturelle Diversität der Hydroxylamin-Oxidoreduktasen (εHao) mit Hilfe von bioinformatischen Analysen untersucht. Innerhalb dieser Arbeit wurden affinitätsmarkierte epsilonproteobakterielle Hydroxylamin-Oxidoreduktasen der wirtsassoziierten Epsilonproteobakterien Campylobacter fetus (CfHao) und Campylobacter curvus (CcuHao) sowie der Tiefsee-Bakterien Caminibacter mediatlanticus (CmHao) und Nautilia profundicola (NpHao) unter Verwendung des Expressionsplasmids pTMH heterolog als εHao-MBP (Maltose-Bindeprotein) Fusionsproteine in W. succinogenes produziert. Durch doppelt homologe Rekombination erfolgte der Austausch des nrfA Gens von W. succinogenesgegen die haoA Gene der oben aufgeführten Epsilonproteobakterien. Nachfolgend wurden die εHao-Proteine gereinigt und hinsichtlich ihrer Enzymaktivitäten im Vergleich zu bereits charakterisierten Enzymen (W. succinogenes NrfA; Nitrosomonas europaea Hao) untersucht. Innerhalb der Enzymaktivitätsmessungen wurden Nitrit, Hydroxylamin, Sulfit und Hydrazin als potenzielle Substrate verwendet. Bei allen vier charakterisierten Enzymen war die für Nitritreduktasen des NrfA-Typs charakteristische Katalyse von Nitrit und Hydroxylamin nachweisbar, wobei bei der Nitritreduktion Ammonium als Produkt gebildet wurde. Hinsichtlich der Hydroxylamin-Oxidation sowie Hydrazin- und Sulfit-Reduktion konnten hingegen keine signifikanten spezifischen Aktivitäten ermittelt werden. Ein weiteres Ziel dieser Arbeit war die ortsspezifische Mutagenese von Genen, die für epsilonproteobakterielle Hydroxylamin-Oxidoreduktasen kodieren. εHao-Proteine besitzen im Gegensatz zur N. europaea Hao (NeHao) keinen Tyrosinrest im aktiven Zentrum, welcher für die Ausbildung eines Häm P460 essentiell ist. Dieses Häm P460 weist zwei kovalente Bindungen zwischen einem konservierten Tyrosin-Rest und der Häm c-Gruppe des aktiven Zentrums eines benachbarten Hao-Monomers auf. Es bedingt daher vermutlich die oxidative Funktion der NeHao und ist ursächlich für die Ausbildung einer homotrimeren Struktur, in der die einzelnen Untereinheiten kovalent miteinander verknüpft sind. Mittels Mutagenese der entsprechenden haoA-Gene wurde die Aminosäure Tyrosin an den zur NeHao äquivalenten Positionen in die Proteine CcuHao, CfHao und CmHao eingebracht. Nachfolgend wurden die Proteinvarianten hinsichtlich der Ausbildung eines Häm P460, einer Trimerisierung und der Enzymaktivität geprüft. Bei keiner der generierten εHao-Varianten war jedoch eine erhöhte Hydroxylamin-Oxidase-Aktivität oder ein für das Häm P460 charakteristisches Absorptionsmaximum bei 460 nm nachweisbar. Zusätzlich wurde eine bioinformatische Analyse von εHao-kodierenden haoA-Gensequenzen durchgeführt, die Aufschluss über das Vorkommen der haoA-Gene, deren Lage im Genom sowie mögliche Interaktionspartner geben sollte. Mit Hilfe von Sequenzvergleichen wurde ermittelt, dass 18 der insgesamt 40 identifizierten HaoA-Proteine der Klasse der Epsilonproteobakterien zugeordnet werden können. Allerdings wiesen auch die Genome diverser Gamma- und Deltaproteobakterien sowie Angehörige der Phyla Aquificae und Thermodesulfobacteria vorhergesagte HaoA-Proteine auf. Mit Hilfe der vorhandenen Genomdaten war es möglich, in einem Großteil der Organismen das Gen haoB nachzuweisen, welches für ein Tetrahäm Cytochrom c der NapC/NrfH-Familie kodiert und in vielen Fällen stromaufwärts des haoA-Gens gelegen ist. Dieses Resultat lässt die Ausbildung eines membrangebundenen HaoBA-Komplexes vermuten, der eine funktionelle Ähnlichkeit zu dem gut charakterisierten Nitritreduktase-Komplex NrfHA aufweisen sollte. Bedeutend ist hierbei, dass die beiden Tiefseebakterien C. mediatlanticus und N. profundicola als Nitrat-ammonifizierende Organismen beschrieben wurden, obwohl in deren Genomen keine Gene für die periplasmatische Nitritreduktase NrfA vorhanden sind. Somit ist der ammonifizierende Metabolismus dieser Bakterien durch das Vorhandensein von haoA- (und haoB-) Genen erklärbar. Aufgrund dessen besitzt die εHao in diesen Organismen vermutlich eine physiologische Rolle innerhalb der Nitrit-Respiration und/oder Nitrit-Assimilation. Weiterhin ist unter Einbeziehung der biochemischen Ergebnisse eine Funktion der Proteine im Rahmen der Hydroxylamin- und/oder NO-Detoxifizierung denkbar, was den vielseitigen metabolischen Charakter von Vertretern der MCC-Familie unterstreicht. Die konservierte Anordnung der Häm-Gruppen, die Sequenzhomologie zur Hao sowie die mögliche funktionelle Ähnlichkeit zu NrfA-Proteinen lassen die Hypothese zu, dass εHao-Proteine möglicherweise eine bedeutende Rolle als “missing link“ innerhalb der Evolution von NrfA- und Hao-Enzymen einnehmen

Are We Ready for the Use of Foxp3+ Regulatory T Cells for Immunodiagnosis and Immunotherapy in Kidney Transplantation?

The existence of T-cell subsets naturally committed to perform immunoregulation has led to enthusiastic efforts to investigate their role in the immunopathogenesis of transplantation. Being able to modulate alloresponses, regulatory T cells could be used as an immunodiagnostic tool in clinical kidney transplantation. Thus, the measurement of Foxp3 transcripts, the presence of regulatory T cells in kidney biopsies, and the phenotypic characterisation of the T-cell infiltrate could aid in the diagnosis of rejection and the immune monitoring and prediction of outcomes in kidney transplantation. Interestingly, the adoptive transfer of regulatory T cells in animal models has been proven to downmodulate powerful alloresponses, igniting translational research on their potential use as an immunomodulatory therapy. For busy transplant clinicians, the vast amount of information in the literature on regulatory T cells can be overwhelming. This paper aims to highlight the most applicable research findings on the use of regulatory T cells in the immune diagnosis and potential immunomodulatory therapy of kidney transplant patients. However, can we yet rely on differential regulatory T-cell profiles for the identification of rejection or to tailor patient's immunosuppression? Are we ready to administer regulatory T cells as inductive or adjunctive therapy for kidney transplantation

A Missense Mutation in the SERPINH1 Gene in Dachshunds with Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a hereditary disease occurring in humans and dogs. It is characterized by extremely fragile bones and teeth. Most human and some canine OI cases are caused by mutations in the COL1A1 and COL1A2 genes encoding the subunits of collagen I. Recently, mutations in the CRTAP and LEPRE1 genes were found to cause some rare forms of human OI. Many OI cases exist where the causative mutation has not yet been found. We investigated Dachshunds with an autosomal recessive form of OI. Genotyping only five affected dogs on the 50 k canine SNP chip allowed us to localize the causative mutation to a 5.82 Mb interval on chromosome 21 by homozygosity mapping. Haplotype analysis of five additional carriers narrowed the interval further down to 4.74 Mb. The SERPINH1 gene is located within this interval and encodes an essential chaperone involved in the correct folding of the collagen triple helix. Therefore, we considered SERPINH1 a positional and functional candidate gene and performed mutation analysis in affected and control Dachshunds. A missense mutation (c.977C>T, p.L326P) located in an evolutionary conserved domain was perfectly associated with the OI phenotype. We thus have identified a candidate causative mutation for OI in Dachshunds and identified a fifth OI gene

Quantification of silver nanoparticle uptake and distribution within individual human macrophages by FIB/SEM slice and view

Background: Quantification of nanoparticle (NP) uptake in cells or tissues is very important for safety assessment. Often, electron microscopy based approaches are used for this purpose, which allow imaging at very high resolution. However, precise quantification of NP numbers in cells and tissues remains challenging. The aim of this study was to present a novel approach, that combines precise quantification of NPs in individual cells together with high resolution imaging of their intracellular distribution based on focused ion beam/ scanning electron microscopy (FIB/SEM) slice and view approaches. Results: We quantified cellular uptake of 75 nm diameter citrate stabilized silver NPs (Ag 75 Cit) into an individual human macrophage derived from monocytic THP-1 cells using a FIB/SEM slice and view approach. Cells were treated with 10 μg/ml for 24 h. We investigated a single cell and found in total 3138 ± 722 silver NPs inside this cell. Most of the silver NPs were located in large agglomerates, only a few were found in clusters of fewer than five NPs. Furthermore, we cross-checked our results by using inductively coupled plasma mass spectrometry and could confirm the FIB/SEM results. Conclusions: Our approach based on FIB/SEM slice and view is currently the only one that allows the quantification of the absolute dose of silver NPs in individual cells and at the same time to assess their intracellular distribution at high resolution. We therefore propose to use FIB/SEM slice and view to systematically analyse the cellular uptake of various NPs as a function of size, concentration and incubation time

Development and external validation of predictive models for prevalent and recurrent atrial fibrillation: a protocol for the analysis of the CATCH ME combined dataset

Background: Atrial fibrillation (AF) is caused by different mechanisms but current treatment strategies do not target these mechanisms. Stratified therapy based on mechanistic drivers and biomarkers of AF have the potential to improve AF prevention and management outcomes. We will integrate mechanistic insights with known pathophysiological drivers of AF in models predicting recurrent AF and prevalent AF to test hypotheses related to AF mechanisms and response to rhythm control therapy. Methods: We will harmonise and combine baseline and outcome data from 12 studies collected by six centres from the United Kingdom, Germany, France, Spain, and the Netherlands which assess prevalent AF or recurrent AF. A Delphi process and statistical selection will be used to identify candidate clinical predictors. Prediction models will be developed in patients with AF for AF recurrence and AF-related outcomes, and in patients with or without AF at baseline for prevalent AF. Models will be used to test mechanistic hypotheses and investigate the predictive value of plasma biomarkers. Discussion: This retrospective, harmonised, individual patient data analysis will use information from 12 datasets collected in five European countries. It is envisioned that the outcome of this analysis would provide a greater understanding of the factors associated with recurrent and prevalent AF, potentially allowing development of stratified approaches to prevention and therapy management

Analysis of T Cell Subsets in Adult Primary/Idiopathic Minimal Change Disease: A Pilot Study

Aim. To characterise infiltrating T cells in kidneys and circulating lymphocyte subsets of adult patients with primary/idiopathic minimal change disease. Methods. In a cohort of 9 adult patients with primary/idiopathic minimal change recruited consecutively at disease onset, we characterized (1) infiltrating immune cells in the kidneys using immunohistochemistry and (2) circulating lymphocyte subsets using flow cytometry. As an exploratory analysis, association of the numbers and percentages of both kidney-infiltrating immune cells and the circulating lymphocyte subsets with kidney outcomes including deterioration of kidney function and proteinuria, as well as time to complete clinical remission up to 48 months of follow-up, was investigated. Results. In the recruited patients with primary/idiopathic minimal change disease, we observed (a) a dominance of infiltrating T helper 17 cells and cytotoxic cells, comprising cytotoxic T cells and natural killer cells, over Foxp3+ Treg cells in the renal interstitium; (b) an increase in the circulating total CD8+ T cells in peripheral blood; and (c) an association of some of these parameters with kidney function and proteinuria. Conclusions. In primary/idiopathic minimal change disease, a relative numerical dominance of effector over regulatory T cells can be observed in kidney tissue and peripheral blood. However, larger confirmatory studies are necessary

Development and external validation of predictive models for prevalent and recurrent atrial fibrillation:a protocol for the analysis of the CATCH ME combined dataset

Background: Atrial fibrillation (AF) is caused by different mechanisms but current treatment strategies do not target these mechanisms. Stratified therapy based on mechanistic drivers and biomarkers of AF have the potential to improve AF prevention and management outcomes. We will integrate mechanistic insights with known pathophysiological drivers of AF in models predicting recurrent AF and prevalent AF to test hypotheses related to AF mechanisms and response to rhythm control therapy. Methods: We will harmonise and combine baseline and outcome data from 12 studies collected by six centres from the United Kingdom, Germany, France, Spain, and the Netherlands which assess prevalent AF or recurrent AF. A Delphi process and statistical selection will be used to identify candidate clinical predictors. Prediction models will be developed in patients with AF for AF recurrence and AF-related outcomes, and in patients with or without AF at baseline for prevalent AF. Models will be used to test mechanistic hypotheses and investigate the predictive value of plasma biomarkers. Discussion: This retrospective, harmonised, individual patient data analysis will use information from 12 datasets collected in five European countries. It is envisioned that the outcome of this analysis would provide a greater understanding of the factors associated with recurrent and prevalent AF, potentially allowing development of stratified approaches to prevention and therapy management

Understanding metric-related pitfalls in image analysis validation

Validation metrics are key for the reliable tracking of scientific progress and for bridging the current chasm between artificial intelligence (AI) research and its translation into practice. However, increasing evidence shows that particularly in image analysis, metrics are often chosen inadequately in relation to the underlying research problem. This could be attributed to a lack of accessibility of metric-related knowledge: While taking into account the individual strengths, weaknesses, and limitations of validation metrics is a critical prerequisite to making educated choices, the relevant knowledge is currently scattered and poorly accessible to individual researchers. Based on a multi-stage Delphi process conducted by a multidisciplinary expert consortium as well as extensive community feedback, the present work provides the first reliable and comprehensive common point of access to information on pitfalls related to validation metrics in image analysis. Focusing on biomedical image analysis but with the potential of transfer to other fields, the addressed pitfalls generalize across application domains and are categorized according to a newly created, domain-agnostic taxonomy. To facilitate comprehension, illustrations and specific examples accompany each pitfall. As a structured body of information accessible to researchers of all levels of expertise, this work enhances global comprehension of a key topic in image analysis validation.Comment: Shared first authors: Annika Reinke, Minu D. Tizabi; shared senior authors: Paul F. J\"ager, Lena Maier-Hei

Integrating new approaches to atrial fibrillation management: the 6th AFNET/EHRA Consensus Conference.

There are major challenges ahead for clinicians treating patients with atrial fibrillation (AF). The population with AF is expected to expand considerably and yet, apart from anticoagulation, therapies used in AF have not been shown to consistently impact on mortality or reduce adverse cardiovascular events. New approaches to AF management, including the use of novel technologies and structured, integrated care, have the potential to enhance clinical phenotyping or result in better treatment selection and stratified therapy. Here, we report the outcomes of the 6th Consensus Conference of the Atrial Fibrillation Network (AFNET) and the European Heart Rhythm Association (EHRA), held at the European Society of Cardiology Heart House in Sophia Antipolis, France, 17-19 January 2017. Sixty-two global specialists in AF and 13 industry partners met to develop innovative solutions based on new approaches to screening and diagnosis, enhancing integration of AF care, developing clinical pathways for treating complex patients, improving stroke prevention strategies, and better patient selection for heart rate and rhythm control. Ultimately, these approaches can lead to better outcomes for patients with AF