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Molecular and quantitative genetic variation within and between populations of the declining grassland species Saxifraga granulata
No full textFormerly common plant species are expected to be particularly susceptible to recent habitat fragmentation. We studied the population genetics of 19 recently fragmented Saxifraga granulata populations (max. distance 61 km) in Luxembourg and neighboring Germany using RAPD markers and a common garden experiment. We assessed (1) the relationships between plant fitness, quantitative genetic variation, molecular geneticvariation, and population size; and (2) the relative importance of genetic drift and selection in shaping genetic variation. Molecular genetic diversity was high but did not correlate with population size, habitat conditions, or plant performance. Genetic differentiation was low (FST= 0.079 ± 0.135), and there was no isolation by distance. Longevity, clonality, and the long- lived seed bank of S. granulata may have prevented strong genetic erosion and genetic differentiation among populations. However, ge -netic distinctness increased with decreasing genetic diversity indicating that randomgenetic drift occurred in the studied populations. Quantitative and molecular geneticvariations were correlated, and their differentiation (QST vs. FST) among S. granulatapopulations was similar, suggesting that mainly random processes have shaped the quantitative genetic differentiation among populations. However, pairwise quantita-tive genetic distances increased with geographic and climatic distances, even when adjusted for molecular genetic distances, indicating diversifying selection. Our results indicate that long- lived clonal species may be buffered at least temporarily against the negative effects of fragmentation. The relationship between quantitative geneticand geographic distance may be a more sensitive indicator of selection than QST– FSTdifferences.Gefördert durch den Open-Access-Publikationsfonds der UB Marburg
Comparison of Two Symptom Checkers (Ada and Symptoma) in the Emergency Department: Randomized, Crossover, Head-to-Head, Double-Blinded Study
No full textBackground: Emergency departments (EDs) are frequently overcrowded and increasingly used by nonurgent patients. Symptom checkers (SCs) offer on-demand access to disease suggestions and recommended actions, potentially improving overall patient flow. Contrary to the increasing use of SCs, there is a lack of supporting evidence based on direct patient use.
Objective: This study aimed to compare the diagnostic accuracy, safety, usability, and acceptance of 2 SCs, Ada and Symptoma.
Methods: A randomized, crossover, head-to-head, double-blinded study including consecutive adult patients presenting to the ED at University Hospital Erlangen. Patients completed both SCs, Ada and Symptoma. The primary outcome was the diagnostic accuracy of SCs. In total, 6 blinded independent expert raters classified diagnostic concordance of SC suggestions with the final discharge diagnosis as (1) identical, (2) plausible, or (3) diagnostically different. SC suggestions per patient were additionally classified as safe or potentially life-threatening, and the concordance of Ada's and physician-based triage category was assessed. Secondary outcomes were SC usability (5-point Likert-scale: 1=very easy to use to 5=very difficult to use) and SC acceptance net promoter score (NPS).
Results: A total of 450 patients completed the study between April and November 2021. The most common chief complaint was chest pain (160/437, 37%). The identical diagnosis was ranked first (or within the top 5 diagnoses) by Ada and Symptoma in 14% (59/437; 27%, 117/437) and 4% (16/437; 13%, 55/437) of patients, respectively. An identical or plausible diagnosis was ranked first (or within the top 5 diagnoses) by Ada and Symptoma in 58% (253/437; 75%, 329/437) and 38% (164/437; 64%, 281/437) of patients, respectively. Ada and Symptoma did not suggest potentially life-threatening diagnoses in 13% (56/437) and 14% (61/437) of patients, respectively. Ada correctly triaged, undertriaged, and overtriaged 34% (149/437), 13% (58/437), and 53% (230/437) of patients, respectively. A total of 88% (385/437) and 78% (342/437) of participants rated Ada and Symptoma as very easy or easy to use, respectively. Ada's NPS was -34 (55% [239/437] detractors; 21% [93/437] promoters) and Symptoma's NPS was -47 (63% [275/437] detractors and 16% [70/437]) promoters.
Conclusions: Ada demonstrated a higher diagnostic accuracy than Symptoma, and substantially more patients would recommend Ada and assessed Ada as easy to use. The high number of unrecognized potentially life-threatening diagnoses by both SCs and inappropriate triage advice by Ada was alarming. Overall, the trustworthiness of SC recommendations appears questionable. SC authorization should necessitate rigorous clinical evaluation studies to prevent misdiagnoses, fatal triage advice, and misuse of scarce medical resources.
Trial registration: German Register of Clinical Trials DRKS00024830; https://drks.de/search/en/trial/DRKS00024830.Gefördert durch den Open-Access-Publikationsfonds der UB Marburg
Multiple Orientia clusters and Th1-skewed chemokine profile: a cross-sectional study in patients with scrub typhus from Nepal
No full textObjectives: Scrub typhus is an emerging infectious disease in Asia caused by Orientia tsutsugamushi (Ot). From Nepal, only scant data on the genetic epidemiology of this agent is available, and determinants of immunoregulation are poorly understood.
Methods: Patients (n = 238) referred to the National Public Health Laboratory (Kathmandu, Nepal) from all over Nepal for suspected scrub typhus were enrolled upon positive immunoglobulin (Ig)M testing between July and October 2015. From Ot 16S and 47 kD polymerase chain reaction (PCR)-positive samples, the variable domain I of the 56 kD gene was sequenced and phylogenetically analyzed. T helper (Th) cell-associated cytokines (n = 13) and chemokines (n = 12) were quantified by multiplex bead arrays.
Results: In 93/238 (39.1%) IgM-positive samples, Ot DNA was detected by quantitative PCR. Phylogenetic analysis of 56 kD sequences revealed seven distinct clusters, six of them with high homologies to strains detected in other countries. The Th1-related cytokines interferon-γ and C-X-C motif chemokine ligand 10 were strongly upregulated and correlated with bacteremia, while levels of Th2-associated chemokines were reduced. Bacteremia also correlated with concentrations of interleukin (IL)-6 and IL-10 but not tumor necrosis factor-α.
Conclusion: We identified a considerable genetic heterogeneity of human-pathogenic Ot strains circulating in Nepal. Acute Nepalese scrub typhus patients showed strong Th1 but impaired Th2 responses, especially on the chemokine level.Gefördert durch den Open-Access-Publikationsfonds der UB Marburg
We analyze the impact of psychological pressure on individual performance with handball penalties thrown in the decisive stage vs. the rest of the game. Contrary to the phenomenon of choking under pressure, we observe that most of the analyzed players perform best when it matters the most. The positive effect of pressure on performance is especially pronounced when the score is level or when the thrower’s team is lagging. We control for gender and psychological traits assessed with a survey. Female players score with a higher probability than male players in our sample. The positive impact of pressure is not significantly higher for female players.
No full textThis article investigates the efficiency of the roll-out of Covid-19 vaccinations
when vaccines are scarce. Using Germany as an example, we find considerable differences across federal states in terms of efficiency, defined as the ability to get most vaccinations out of a given number of available doses. Back-of-theenvelope calculations for the past five months show that vaccinations would have been 3.7–6.6% higher if all federal states had adopted a similar ratio between vaccinations given and vaccines stored as the most efficient ones. We also find evidence that the integration of doctor’s offices into the vaccination campaign significantly increased the share of vaccinations out of a given stocks of vaccine doses
Does the method adopted for distribution of services by amalgamating municipalities affect expenditure after amalgamation? Evidence from Japan
No full textMunicipal boundary reform (municipal amalgamation) has been done in many countries in recent years as the result of a push to enlarge the size and coverage of local government units, which in turn is driven mainly by the prospect of economies of scale. However, in a notable body of previous literature, the enlargement of local government has not led to reduction of public expenditures. Decision-making before amalgamation might affect to public expenditure after amalgamation. This study uses Japanese municipal-level data and argues for a relation between the choice of public administration distribution method and expenditure after amalgamation. The results show that a plan for distributed or decentralized facility method is more likely to be adopted in a larger administrative jurisdiction and in one with large differences in finances or political structures between amalgamated sub-regions. In turn, a plan for distributed facilities has the effect of pushing up administrative expenditure
Globalization and Outbreak of COVID-19: An Empirical Analysis
No full textThe purpose of this study is to examine the relationship between globalization, Coronavirus Disease 2019 (COVID-19) cases, and associated deaths in more than 100 countries. Our ordinary least squares multivariate regressions show that countries with higher levels of socio-economic globalization are exposed more to COVID-19 outbreak. Nevertheless, globalization cannot explain cross-country differences in COVID-19 confirmed deaths. The fatalities of coronavirus are mostly explained by cross-country variation in health infrastructures (e.g., share of out of pocket spending on health per capita and the number of hospital beds) and demographic structure (e.g., share of population beyond 65 years old in total population) of countries. Our least squares results are robust to controlling outliers and regional dummies. This finding provides the first empirical insight on the robust determinants of COVID-19 outbreak and its human costs across countries
Calcium Handling Remodeling Underlies Impaired Sympathetic Stress Response in Ventricular Myocardium from Cacna1c Haploinsufficient Rats
No full textCACNA1C encodes the pore-forming α1C subunit of the L-type Ca2+ channel, Cav1.2. Mutations and polymorphisms of the gene are associated with neuropsychiatric and cardiac disease. Haploinsufficient Cacna1c+/− rats represent a recently developed model with a behavioral phenotype, but its cardiac phenotype is unknown. Here, we unraveled the cardiac phenotype of Cacna1c+/− rats with a main focus on cellular Ca2+ handling mechanisms. Under basal conditions, isolated ventricular Cacna1c+/− myocytes exhibited unaltered L-type Ca2+ current, Ca2+ transients (CaTs), sarcoplasmic reticulum (SR) Ca2+ load, fractional release, and sarcomere shortenings. However, immunoblotting of left ventricular (LV) tissue revealed reduced expression of Cav1.2, increased expression of SERCA2a and NCX, and augmented phosphorylation of RyR2 (at S2808) in Cacna1c+/− rats. The β-adrenergic agonist isoprenaline increased amplitude and accelerated decay of CaTs and sarcomere shortenings in both Cacna1c+/− and WT myocytes. However, the isoprenaline effect on CaT amplitude and fractional shortening (but not CaT decay) was impaired in Cacna1c+/− myocytes exhibiting both reduced potency and efficacy. Moreover, sarcolemmal Ca2+ influx and fractional SR Ca2+ release after treatment with isoprenaline were smaller in Cacna1c+/− than in WT myocytes. In Langendorff-perfused hearts, the isoprenaline-induced increase in RyR2 phosphorylation at S2808 and S2814 was attenuated in Cacna1c+/− compared to WT hearts. Despite unaltered CaTs and sarcomere shortenings, Cacna1c+/− myocytes display remodeling of Ca2+ handling proteins under basal conditions. Mimicking sympathetic stress with isoprenaline unmasks an impaired ability to stimulate Ca2+ influx, SR Ca2+ release, and CaTs caused, in part, by reduced phosphorylation reserve of RyR2 in Cacna1c+/− cardiomyocytes.Gefördert durch den Open-Access-Publikationsfonds der UB Marburg
Ein mechanistisches Verständnis der bakteriellen Chromosomenorganisation
No full textChromosomes serve as repositories of genetic information, crucial to the functionality of all living organisms. Bacteria, in their simplicity, offer an ideal model to investigate the intricacies of chromosomal dynamics. Despite their lack of compartmentalisation, bacterial chromosomes display spatiotemporal organization like eukaryotic cells. However, understanding the mechanisms of bacterial chromosome organization and segregation, particularly in the extensively studied model organism, Escherichia coli, remains incomplete.
In E. coli, like most bacteria, the origin of replication is the first part of the chromosome to be replicated and segregated. While centromere-like sequences aiding origin segregation have been identified in other bacteria, none have been discovered in E. coli. The SMC complex, which the origin is attracted to and co-localizes with throughout the cell cycle, has been identified as important for positioning and segregation. However, a sequence identity facilitating this interaction has yet to be determined.
Our study began with comprehensive genomic screening to identify sequences that might aid origin positioning in E. coli. Our hypothesis was motivated by the observation that specific chromosomal regions, when present within unstable low-copy plasmids, lead to their active positioning and maintenance within the population. We found that two loci in the E. coli genome, near the spoT and seqA genes, confer a moderate degree of stability to an unstable plasmid. The stability effect of the spoT locus is strain-specific, while the seqA locus demonstrates stability that is consistent across different strains. Due to time constraints, the precise mechanism for the latter remains unidentified. Our findings at this stage support the established view of the absence of a centromere-like sequence for origin positioning in the E. coli genome.
While the origin of replication has been the focus of many studies, the terminus region—the last chromosomal segment to undergo replication and segregation—also plays a vital role in successful completion of cell cycle, ensuring the equal distribution of genetic material between daughter cells. In slow-growing E. coli cells, the terminus region transitions from the new pole at birth to the midcell during the cell cycle. Unlike other chromosomal regions, it maintains the midcell positioning for the majority of the cell cycle, even after its duplication. Understanding the behavior of the terminus region can provide insights into the broader aspects of bacterial chromosome organization and segregation.
Using a high-throughput single-cell approach, we tracked tens of thousands of cell cycles to quantitatively analyse the transition of the terminus region from the new pole to midcell and investigated its relationship to various cell cycle events. We found that terminus centralisation, a rapid discrete event, is closely associated with the completion of origin segregation, even in the absence of its linkage to the divisome. This revealed a previously unexplored relationship between origin and terminus. Additionally, we found that E. coli exhibits a longitudinal-like chromosome organization even under slow growth conditions.
In conclusion, our research significantly advances our understanding of chromosome organization in E. coli and paves the way for the application of our methodologies to studying chromosomal organization in other bacterial species.Chromosomen dienen als Speicher für genetische Informationen, die für das Überleben aller lebenden Organismen entscheidend sind. Bakterien, in ihrer Einfachheit, bieten ein ideales Modell zur Untersuchung der Komplexität der Chromosomendynamik. Trotz ihres Mangels an Kompartimentierung zeigen bakterielle Chromosomen eine räumlich-zeitliche Organisation, ähnlich wie eukaryotische Zellen. Allerdings ist das Verständnis der Mechanismen der bakteriellen Chromosomenorganisation und -segregation, insbesondere bei dem ausgiebig erforschten Modellorganismus Escherichia coli, noch unvollständig.
Bei E. coli, wie bei den meisten Bakterien, ist der Origin of replication der erste Teil des Chromosoms, der repliziert und segregiert wird. Während bei anderen Bakterien zentromerähnliche Sequenzen zur Segregation des Chromosoms identifiziert wurden, wurden bei E. coli noch keine entdeckt. Der SMC-Komplex, der DNA bindet, zu dem Origin hingezogen wird und mit dem Origin während des gesamten Zellzyklus kolokalisiert, wurde als wichtiger Bestandteil für Positionierung und Segregation identifiziert. Allerdings wurde bisher noch keine DNA-Sequenzen bestimmt, welche diese Interaktionen begünstigen.
Unsere Studie begann mit einem umfassenden genomischen Screening, um zentromerähnliche DNA-Sequenzen zu identifizieren, die die Origin Positionierung in E. coli begünstigen könnten. Unsere Hypothese wurde durch die Beobachtung motiviert, dass spezifische Chromosomenregionen, wenn sie in instabilen Plasmiden mit geringer Kopienzahl vorhanden sind, zu deren aktiver Positionierung und Aufrechterhaltung führen. Wir fanden heraus, dass zwei Loci im E. coli-Genom, in der Nähe der spoT- und seqA-Gene, einem instabilen Plasmid ein moderates Maß an Stabilität verleihen. Der Stabilitätseffekt des spoT-Locus ist stammspezifisch, während der seqA-Locus eine Stabilität aufweist, die über verschiedene Stämme hinweg konsistent ist. Aufgrund von Zeitbeschränkungen bleibt der genaue Mechanismus für letzteres unidentifiziert. Unsere Ergebnisse in diesem Stadium stützen die etablierte Ansicht der Nichtexistenz einer zentromerähnlichen DNA-Sequenz für die Origin Positionierung im E. coli-Genom.
Obwohl Origin oft im Fokus vieler Studien ist, spielt auch die Terminus region - das letzte Chromosomensegment, das eine Replikation und Segregation durchläuft - eine entscheidende Rolle bei der Zellteilung, indem sie die gleichmäßige Verteilung des genetischen Materials zwischen Tochterzellen gewährleistet. In langsam wachsenden E. coli-Zellen wandert Terminsregion während des Zellzyklus vom neuen Pol zur Mitte der Zelle. Im Gegensatz zu anderen Chromosomensegmenten ist Terminus region für den größten Teil des Zellzyklus in der Zellmitte positioniert, selbst nach ihrer Verdoppelung. Das Verständnis des Verhaltens von Ter bietet umfassende Einblicke in verschiedenste Aspekte der bakteriellen Chromosomenorganisation und -segregation.
Mit einem Hochdurchsatz-Ansatz auf Einzelzellenebene verfolgten wir Zehntausende von Zellzyklen, um den Übergang der Terminsregion vom neuen Pol zur Zellmitte quantitativ zu analysieren und ihre Beziehung zu verschiedenen Zellzyklusereignissen zu untersuchen. Wir fanden heraus, dass die Zentralisierung des Termins, ein schnelles diskretes Ereignis, eng mit dem Abschluss der Ori-Segregation verbunden ist, selbst in Abwesenheit seiner Verknüpfung mit dem Divisom. Dies enthüllte eine zuvor unerforschte Beziehung zwischen Origin und Terminus regionen des Chromsoms. Darüber hinaus entdeckten wir, dass E. coli unter Bedingungen langsamen Wachstums eine längsorientierte Chromosomenorganisation aufweist.
Zusammenfassend hat unsere Forschung unser Verständnis der Chromosomen organisation in E. coli erheblich erweitert und den Weg für die Anwendung unserer Methoden auf die Untersuchung der Chromosomenorganisation in anderen Bakterienarten geebnet
Aggregation Kinetics of Therapeutic Monoclonal Antibodies under the Influence of a Chemical Denaturant
No full textTherapeutische monoklonale Antikörper (mAbs) haben das Feld der Arzneimittel revolutioniert und stellen eine wichtige Behandlungsoption für eine Reihe von Krankheiten dar, einschließlich Krebs und Autoimmunerkrankungen. Trotzdem stellt die Neigung von mAbs zur Aggregation ein erhebliches Hindernis dar, das ihre Wirksamkeit und Stabilität beeinträchtigen kann. Im Weiteren kann es durch Aggregation zur Bildung von Partikeln kommen, die potenziell unerwünschte immunologische Reaktionen beim Patienten hervorrufen können.
Die vorliegende Arbeit beabsichtigt daher, das Verständnis für die mAb-Aggregation in einer Lösung zu verbessern, indem ein umfassenderes Wissen für ihren Aggregationsprozess in Gegenwart eines chemischen Denaturierungsmittels gewonnen wird. Zu diesem Zweck wurden zunächst kinetische Profile von mAbs aufgezeichnet und mathematisch beschrieben. Darauf aufbauend wurden neue kinetische Vorhersageparameter, einschließlich der Monomer-Fraktion bei 50 % (Mf50), definiert, die dann für Stabilitätsbewertungen von mAbs in verschiedenen pharmazeutisch relevanten Pufferbedingungen verwendet wurden. Ein Vergleich erfolgte mit einem häufig verwendeten Stabilitätsparameter (Halbwerts-Konzentration des Denaturierungsmittels C50), um deren Fähigkeit zur Verbesserung der Protein-Stabilitätsvorhersage zu bewerten. Darüber hinaus wurde das gewonnene Wissen über den Aggregationsprozess genutzt, um Proteinkeime zu generieren und deren Einfluss auf die Stabilität von nativen Proteinen zu bestimmen.
Die Ergebnisse haben einen vierstufigen Aggregationsprozess für beide mAbs aufgezeigt, bestehend aus Monomer, Dimer, Trimer/Tetramer und Oligomer. Die abgeleiteten kinetischen Parameter zeigten eine hohe Korrelation untereinander sowie mit dem C50. Diese Parameter haben das Potenzial, die Zeit- und Kostenbelastung im Zusammenhang mit der Entwicklung von Biopharmazeutika signifikant zu reduzieren, indem sie genaueres und effizienteres Pufferscreening ermöglichen. Darüber hinaus zeigte die Seeding-Studie, dass die Anwesenheit von Proteinkeimen in einer gestressten nativen Proteinlösung einen signifikanten Einfluss sowohl auf die Partikelkonzentration im Nanometer- bis Mikrometerbereich als auch auf die Morphologieform hat. Diese Erkenntnis könnte potenziell zur Entwicklung neuer Methoden führen, die darauf abzielen, dieses Phänomen zu verhindern oder zumindest zu reduzieren. Insgesamt trägt diese Studie zu einem fundierten Verständnis des Aggregationsprozesses von mAb unter dem Einfluss eines chemischen Denaturierungsmittels bei und stellt neue Vorhersageparameter vor, die erhebliche Auswirkungen auf die Verbesserung der Qualität, Sicherheit und Wirksamkeit von therapeutischen mAbs haben können.Therapeutic monoclonal antibodies (mAbs) have revolutionized the field of pharmaceuticals and represent a crucial treatment option for a range of diseases, including cancer and autoimmune disorders. Nonetheless, the propensity of mAbs to aggregate poses a substantial obstacle, potentially jeopardizing their efficacy and structural stability. Moreover, the emergence of particulate matter as a consequence of aggregation may provoke inadvertent immunological responses within the patient.
Hence, the current thesis seeks to enhance the comprehension of mAb aggregation in solution by gaining a deeper insight into the aggregation process in the presence of a chemical denaturant. To this end, the kinetic profiles of mAbs were recorded and mathematically delineated. Subsequently, novel predictive parameters, including the monomer fraction at 50 % (Mf50), were defined, which were then employed for the stability assessment of mAbs in various pharmaceutically relevant buffer conditions. An evaluation of these parameters was executed by comparing them with a commonly employed stability parameter (midpoint denaturant concentration C50), in order to appraise their capacity to enhance protein stability prediction. Furthermore, the knowledge gleaned from the aggregation process was harnessed to generate protein seeds to assess their influence on the stability of native proteins within the micro- and nanometer size ranges.
The findings of this thesis unveiled a four-stage aggregation process consisting of monomer, dimer, trimer/tetramer, and oligomer formation for both mAbs. The derived kinetic parameters exhibited a high correlation among themselves as well as with the C50. The derived parameters hold the potential to markedly reduce the time and costs associated with biopharmaceutical development by allowing for more accurate and efficient buffer screening. Additionally, the seeding investigation demonstrated that the presence of protein seeds in a stressed native protein solution exerts a substantial influence on both particle formation and morphological shape. This information may potentially guide the formulation of novel strategies to circumvent or mitigate this phenomenon. Overall, this study cultivates a more informed viewpoint regarding the aggregation process of mAb under the influence of a chemical denaturant while introducing novel predictive parameters that have significant implications for improving the quality, safety, and efficacy of therapeutic mAbs
The Emergence of Inter-Municipal cooperation – A Hazard Model Approach
No full textWe use a hazard model to identify the factors that drive the emergence of inter-municipal cooperation (IMC). We focus on IMC in tasks of internal administration in West-Germany be-tween 2001 and 2014 – tasks where IMC may generate economies of scale while regional spill-overs are negligible. Our results support the homophily-hypothesis and the relevance of politi-cal transaction costs. Municipalities situated in clusters of small and shrinking municipalities are more likely to start IMC. At odds with the previous literature, IMC is more likely to emerge among municipalities in fiscally strong clusters. We find no evidence that IMC is more likely in certain phases of the election cycle while state subsidies for IMC are an important driving force behind IMC