Neue Perspektiven auf post-transkriptionale Regulationsmechanismen in Pseudomonas aeruginosa

Regulation of gene expression plays a key role in bacterial adaptability to changes in the environment. Quorum sensing (QS) systems constitute an integral part of this gene regulatory network and coordinate bacterial responses under high cellular densities. In the nosocomial pathogen Pseudomonas aeruginosa, a sophisticated QS network exists controlling virulence factor synthesis important for pathogenesis. The overall aim of this thesis was to gain insight into novel regulation mechanisms of QS-based gene expression at the post-transcriptional level. In this context, we characterized the S-adenosyl-L-methionine-dependent methyltransferase (PrmC), specifically methylating a conserved GGQ motif of peptide chain release factors. Mutation of prmC was shown to predominantly affect the expression of major virulence-associated factors and caused attenuation of virulence in a Galleria mellonella larvae infection model. Furthermore, a previously unrecognized post-transcriptional regulation mechanism in bacteria was characterized. This involved an RhlR-mediated transcriptional induction of a secondary structure in the 5' untranslated region of the pqs operon transcript. The resulting hairpin-like structure sterically hinders ribosomal access to the ribosome binding site thus repressing pqs signaling. Another important focus of the work was to establish ribosome profiling (Ribo-Seq). The technique Ribo-Seq is based on deep sequencing of ribosome-protected mRNA fragments and provides a global snapshot of translation in vivo. This powerful tool opens up wide-ranging prospects to the developing picture of post-transcriptional regulation in P. aeruginosa. Finally, the present thesis focuses on the role of PqsE as part of the pqs QS system in regulating QS-dependent virulence factor production. We provide evidence that PqsE is an enzyme that interferes with carbon flow at the chorismate branching point in the complex metabolic network of P. aeruginosa. By using a multi-methodological approach, we suggest that PqsE is involved in the biosynthesis of phenylpyruvate, being a possible signal molecule for the rhl QS system.Regulation der Genexpression spielt eine zentrale Rolle in der bakteriellen Anpassungsfähigkeit an sich verändernde Umweltbedingungen. Quorum sensing (QS) Systeme sind ein wesentlicher Bestandteil dieses regulatorischen Netzwerkes und koordinieren bakterielle Reaktionen bei hoher Zelldichte. In dem nosokomialen Pathogen Pseudomonas aeruginosa existiert ein komplexes QS Netzwerk, welches die Synthese von Virulenzfaktoren kontrolliert die wichtig für die Pathogenese sind. Allgemeines Ziel dieser Dissertation war es, Einblicke in neuartige Regulationsmechanismen auf QS-basierender Genexpression auf dem post-transkriptionellen Level zu gewinnen. In diesem Zusammenhang haben wir die S-Adenosyl-L-Methionin-abhängige Methyltransferase (PrmC) charakterisiert, die spezifisch ein konserviertes GGQ Motif der Terminationsfaktoren methyliert. Es wurde gezeigt, dass prmC Mutation vorwiegend die Expression wichtiger Virulenz-assoziierter Faktoren betraf und eine Abschwächung der Virulenz in einem Galleria mellonella Larven Infektionsmodel zur Folge hatte. Zudem wurde ein zuvor unerkannter post-transkriptioneller Regulationsmechanismus charakterisiert. Dieser umfasst eine RhlR-vermittelte transkriptionelle Induktion einer Sekundärstruktur in der 5‘ nicht-codierenden Region des pqs Operon Transkripts. Die daraus resultierende Haarnadel-ähnliche Struktur verhindert sterisch den Zugang des Ribosoms an die ribosomale Bindungsstelle und unterdrückt auf diese Weise das pqs Signalsystem. Ein weiterer wichtiger Fokus dieser Arbeit bestand darin, ribosome profiling (Ribo-Seq) zu etablieren. Die Ribo-Seq Technik basiert auf dem deep sequencing von Ribosom geschützten mRNA Fragmenten, und verschafft eine globale Momentaufnahme der Translation in vivo. Dieses leistungsfähige methodische Werkzeug eröffnet umfangreiche Perspektiven im Hinblick auf das sich entwickelnde Bild der post-transkriptionellen Regulation in P. aeruginosa. Schließlich liegt ein Fokus dieser Dissertation auf der Rolle von PqsE als wichtiger Bestandteil des pqs QS Systems in der Regulation QS-abhängiger Virulenzfaktorproduktion. Wir konnten zeigen, dass PqsE ein Enzym ist, welches in den Kohlenstoffstrom an dem Chorisminsäure Verzweigungspunkt des komplexen metabolischen Netzwerkes von P. aeruginosa eingreift. Durch den Einsatz eines multi-methodischen Ansatzes schlagen wir vor, dass PqsE in der Phenylpyruvat Biosynthese involviert ist, welches ein mögliches Signalmolekül für das rhl QS System darstellt

Rate equations for Coulomb blockade with ferromagnetic leads

We present a density-matrix rate-equation approach to sequential tunneling through a metal particle weakly coupled to ferromagnetic leads. The density-matrix description is able to deal with correlations between degenerate many-electron states that the standard rate equation formalism in terms of occupation probabilities cannot describe. Our formalism is valid for an arbitrary number of electrons on the dot, for an arbitrary angle between the polarization directions of the leads, and with or without spin-orbit scattering on the metal particle. Interestingly, we find that the density-matrix description may be necessary even for metal particles with unpolarized leads if three or more single-electron levels contribute to the transport current and electron-electron interactions in the metal particle are described by the `universal interaction Hamiltonian'.Comment: 10 pages, 4 figures, REVTeX

Conductance Peak Height Correlations for a Coulomb-Blockaded Quantum Dot in a Weak Magnetic Field

We consider statistical correlations between the heights of conductance peaks corresponding to two different levels in a Coulomb-blockaded quantum dot. Correlations exist for two peaks at the same magnetic field if the field does not fully break time-reversal symmetry as well as for peaks at different values of a magnetic field that fully breaks time-reversal symmetry. Our results are also relevant to Coulomb-blockade conductance peak height statistics in the presence of weak spin-orbit coupling in a chaotic quantum dot.Comment: 5 pages, 3 figures, REVTeX 4, accepted for publication in Phys. Rev.

Excitation Spectra and Thermodynamic Response of Segmented Heisenberg Spin Chains

The spectral and thermodynamic response of segmented quantum spin chains is analyzed using a combination of numerical techniques and finite-size scaling arguments. Various distributions of segment lengths are considered, including the two extreme cases of quenched and annealed averages. As the impurity concentration is increased, it is found that (i) the integrated spectral weight is rapidly reduced, (ii) a pseudo-gap feature opens up at small frequencies, and (iii) at larger frequencies a discrete peak structure emerges, dominated by the contributions of the smallest cluster segments. The corresponding low-temperature thermodynamic response has a divergent contribution due to the odd-site clusters and a sub-dominant exponentially activated component due to the even-site segments whose finite-size gap is responsible for the spectral weight suppression at small frequencies. Based on simple scaling arguments, approximate low-temperature expressions are derived for the uniform susceptibility and the heat capacity. These are shown to be in good agreement with numerical solutions of the Bethe ansatz equations for ensembles of open-end chains.Comment: RevTex, 9 pages with 6 figure

Employment and ability to work after kidney transplantation in the Netherlands:The impact of preemptive versus non-preemptive kidney transplantation

Background: Work can have a major positive impact on health and wellbeing. Employment of kidney transplant recipients (KTR) of working age is much lower than in the general population. The first aim of this study was to examine the impact of a preemptive kidney transplantation (PKT) on employment, in addition to other possible influencing factors. The second aim was to explore differences in work ability, absenteeism and work performance among employed KTR with different types of transplantations. Methods: A cross-sectional survey study was conducted between 2018 and 2019 in nine Dutch hospitals. PKT as potential predictor of employment was examined. Furthermore, work ability, absenteeism and loss of work performance were compared between employed preemptive recipients with a living donor (L-PKT) and non-preemptive recipients with a living donor (L-nPKT) and with a deceased donor (D-nPKT). Results: Two hundred and twenty four KTR participated; 71% reported having paid work. Paid work was more common among PKT recipients (82% vs. 65% in L-nPKT and 55% in D-nPKT) and recipients who were younger (OR .950, 95%CI .913-.989), had no comorbidities (1 comorbidity: OR .397, 95%CI .167-.942; 2 comorbidities: OR .347, 95%CI .142-.844), had less fatigue (OR .974, 95%CI .962-.987) and had mentally demanding work tasks (only in comparison with physically demanding tasks, OR .342, 95%CI .145-.806). If recipients were employed, D-nPKT recipients worked fewer hours (mean 24.6 +/- 11.3 vs. PKT 31.1 +/- 9.6, L-nPKT 30.1 +/- 9.5) and D-nPKT and L-nPKT recipients received more often supplemental disability benefits (32 and 33.3%, respectively) compared to PKT recipients (9.9%). No differences were found for self-reported ability to work, sick leave (absenteeism) and loss of work performance with the exception of limitations in functioning at work. Conclusions: Preemptive kidney transplantation recipients with a kidney from a living donor are employed more often, work more hours per week (only in comparison with D-nPKT) and have a partial disability benefit less often than nPKT recipients. More knowledge regarding treatments supporting sustainable participation in the labor force is needed as work has a positive impact on recipients' health and wellbeing and is also beneficial for society as a whole

Barriers to and Facilitators of Sustained Employment:A Qualitative Study of Experiences in Dutch Patients With CKD

Rationale & Objective: Although patients with chronic kidney disease (CKD) are at risk for work disability and loss of employment, not all experience work disruption. We aimed to describe the barriers to and facilitators of sustained employment experienced by Dutch patients with CKD. Study Design: Qualitative study using semi-structured interviews. Setting & Participants: 27 patients with CKD glomerular filtration rate categories 3b-5 (G3b-G5) from 4 nephrology outpatient clinics in The Netherlands. Analytical Approach: Content analyses with constant comparison of interview data based on the International Classification of Functioning, Disability and Health framework. Results: Participants were 6 patients with CKD G3b-G4, 8 patients receiving maintenance dialysis, and 13 patients with functioning kidney transplants. We identified health-related barriers (symptoms, physical toll of dialysis/transplantation, limited work capacity) and facilitators (few physical symptoms, successful posttransplantation recovery, absence of comorbidities, good physical condition), personal barriers (psychological impact, limited work experience) and facilitators (positive disposition, job satisfaction, work attitude, person-job fit), and environmental barriers and facilitators. Environmental barriers were related to nephrology care (waiting time, use of a hemodialysis catheter) and work context (reorganization, temporary contract, working hours, physical demands); environmental facilitators were related to nephrology care (personalized dialysis, preemptive transplant), work context (large employer, social climate, job requiring mental rather than physical labor, flexible working hours, adjustment of work tasks, reduced hours, remote working, support at work, peritoneal dialysis exchange facility), and support at home. Occupational health services and social security could be barriers or facilitators. Limitations: The study sample of Dutch patients may limit the transferability of these findings to other countries. Conclusions: The wide range of barriers and facilitators in all International Classification of Functioning, Disability and Health components suggests great diversity among patients and their circumstances. These findings underline the importance of personalized nephrology and occupational health care as well as the importance of individually tailored workplace accommodations to promote sustained employment for patients with CKD

Detection of Epidemic Scarlet Fever Group A Streptococcus in Australia.

Sentinel hospital surveillance was instituted in Australia to detect the presence of pandemic group A Streptococcus strains causing scarlet fever. Genomic and phylogenetic analyses indicated the presence of an Australian GAS emm12 scarlet fever isolate related to United Kingdom outbreak strains. National surveillance to monitor this pandemic is recommended

Subclinical giant cell arteritis in new onset polymyalgia rheumatica:A systematic review and meta-analysis of individual patient data

Objectives: To determine the prevalence and predictors of subclinical giant cell arteritis (GCA) in patients with newly diagnosed polymyalgia rheumatica (PMR). Methods: PubMed, Embase, and Web of Science Core Collection were systematically searched (date of last search July 14, 2021) for any published information on any consecutively recruited cohort reporting the prevalence of GCA in steroid-naïve patients with PMR without cranial or ischemic symptoms. We combined prevalences across populations in a random-effect meta-analysis. Potential predictors of subclinical GCA were identified by mixed-effect logistic regression using individual patient data (IPD) from cohorts screened with PET/(CT). Results: We included 13 cohorts with 566 patients from studies published between 1965 to 2020. Subclinical GCA was diagnosed by temporal artery biopsy in three studies, ultrasound in three studies, and PET/(CT) in seven studies. The pooled prevalence of subclinical GCA across all studies was 23% (95% CI 14%-36%, I2=84%) for any screening method and 29% in the studies using PET/(CT) (95% CI 13%-53%, I2=85%) (n=266 patients). For seven cohorts we obtained IPD for 243 patients screened with PET/(CT). Inflammatory back pain (OR 2.73, 1.32-5.64), absence of lower limb pain (OR 2.35, 1.05-5.26), female sex (OR 2.31, 1.17-4.58), temperature >37° (OR 1.83, 0.90-3.71), weight loss (OR 1.83, 0.96-3.51), thrombocyte count (OR 1.51, 1.05-2.18), and haemoglobin level (OR 0.80, 0.64-1.00) were most strongly associated with subclinical GCA in the univariable analysis but not C-reactive protein (OR 1.00, 1.00-1.01) or erythrocyte sedimentation rate (OR 1.01, 1.00-1.02). A prediction model calculated from these variables had an area under the curve of 0.66 (95% CI 0.55-0.75). Conclusion: More than a quarter of patients with PMR may have subclinical GCA. The prediction model from the most extensive IPD set has only modest diagnostic accuracy. Hence, a paradigm shift in the assessment of PMR patients in favour of implementing imaging studies should be discussed

Cohesin complex-associated holoprosencephaly

Marked by incomplete division of the embryonic forebrain, holoprosencephaly is one of the most common human developmental disorders. Despite decades of phenotype-driven research, 80–90% of aneuploidy-negative holoprosencephaly individuals with a probable genetic aetiology do not have a genetic diagnosis. Here we report holoprosencephaly associated with variants in the two X-linked cohesin complex genes, STAG2 and SMC1A, with loss-of-function variants in 10 individuals and a missense variant in one. Additionally, we report four individuals with variants in the cohesin complex genes that are not X-linked, SMC3 and RAD21. Using whole mount in situ hybridization, we show that STAG2 and SMC1A are expressed in the prosencephalic neural folds during primary neurulation in the mouse, consistent with forebrain morphogenesis and holoprosencephaly pathogenesis. Finally, we found that shRNA knockdown of STAG2 and SMC1A causes aberrant expression of HPE-associated genes ZIC2, GLI2, SMAD3 and FGFR1 in human neural stem cells. These findings show the cohesin complex as an important regulator of median forebrain development and X-linked inheritance patterns in holoprosencephaly

Combining traditional dietary assessment methods with novel metabolomics techniques: present efforts by the Food Biomarker Alliance

FFQ, food diaries and 24 h recall methods represent the most commonly used dietary assessment tools in human studies on nutrition and health, but food intake biomarkers are assumed to provide a more objective reflection of intake. Unfortunately, very few of these biomarkers are sufficiently validated. This review provides an overview of food intake biomarker research and highlights present research efforts of the Joint Programming Initiative 'A Healthy Diet for a Healthy Life' (JPI-HDHL) Food Biomarkers Alliance (FoodBAll). In order to identify novel food intake biomarkers, the focus is on new food metabolomics techniques that allow the quantification of up to thousands of metabolites simultaneously, which may be applied in intervention and observational studies. As biomarkers are often influenced by various other factors than the food under investigation, FoodBAll developed a food intake biomarker quality and validity score aiming to assist the systematic evaluation of novel biomarkers. Moreover, to evaluate the applicability of nutritional biomarkers, studies are presently also focusing on associations between food intake biomarkers and diet-related disease risk. In order to be successful in these metabolomics studies, knowledge about available electronic metabolomics resources is necessary and further developments of these resources are essential. Ultimately, present efforts in this research area aim to advance quality control of traditional dietary assessment methods, advance compliance evaluation in nutritional intervention studies, and increase the significance of observational studies by investigating associations between nutrition and health. Keywords: Dietary assessment; Food intake biomarkers; Food metabolome; Metabolomics