Do Aspirin and Other Antiplatelet Drugs Reduce the Mortality in Critically Ill Patients?

Platelet activation has been implicated in microvascular thrombosis and organ failure in critically ill patients. In the first part the present paper summarises important data on the role of platelets in systemic inflammation and sepsis as well as on the beneficial effects of antiplatelet drugs in animal models of sepsis. In the second part the data of retrospective and prospective observational clinical studies on the effect of aspirin and other antiplatelet drugs in critically ill patients are reviewed. All of these studies have shown that aspirin and other antiplatelet drugs may reduce organ failure and mortality in these patients, even in case of high bleeding risk. From the data reviewed here interventional prospective trials are needed to test whether aspirin and other antiplatelet drugs might offer a novel therapeutic option to prevent organ failure in critically ill patients

Key insights for the future of urban ecosystem services research

Understanding the dynamics of urban ecosystem services is a necessary requirement for adequate planning, management, and governance of urban green infrastructure. Through the three-year Urban Biodiversity and Ecosystem Services (URBES) research project, we conducted case study and comparative research on urban biodiversity and ecosystem services across seven cities in Europe and the United States. Reviewing > 50 peer-reviewed publications from the project, we present and discuss seven key insights that reflect cumulative findings from the project as well as the state-of-the-art knowledge in urban ecosystem services research. The insights from our review indicate that cross-sectoral, multiscale, interdisciplinary research is beginning to provide a solid scientific foundation for applying the ecosystem services framework in urban areas and land management. Our review offers a foundation for seeking novel, nature-based solutions to emerging urban challenges such as wicked environmental change issues

Early inhaled budesonide for the prevention of bronchopulmonary dysplasia

BACKGROUND Systemic glucocorticoids reduce the incidence of bronchopulmonary dysplasia among extremely preterm infants, but they may compromise brain development. The effects of inhaled glucocorticoids on outcomes in these infants are unclear. METHODS We randomly assigned 863 infants (gestational age, 23 weeks 0 days to 27 weeks 6 days) to early (within 24 hours after birth) inhaled budesonide or placebo until they no longer required oxygen and positive-pressure support or until they reached a postmenstrual age of 32 weeks 0 days. The primary outcome was death or bronchopulmonary dysplasia, confirmed by means of standardized oxygen-saturation monitoring, at a postmenstrual age of 36 weeks. RESULTS A total of 175 of 437 infants assigned to budesonide for whom adequate data were available (40.0%), as compared with 194 of 419 infants assigned to placebo for whom adequate data were available (46.3%), died or had bronchopulmonary dysplasia (relative risk, stratified according to gestational age, 0.86; 95% confidence interval [CI], 0.75 to 1.00; P = 0.05). The incidence of bronchopulmonary dysplasia was 27.8% in the budesonide group versus 38.0% in the placebo group (relative risk, stratified according to gestational age, 0.74; 95% CI, 0.60 to 0.91; P = 0.004); death occurred in 16.9% and 13.6% of the patients, respectively (relative risk, stratified according to gestational age, 1.24; 95% CI, 0.91 to 1.69; P = 0.17). The proportion of infants who required surgical closure of a patent ductus arteriosus was lower in the budesonide group than in the placebo group (relative risk, stratified according to gestational age, 0.55; 95% CI, 0.36 to 0.83; P = 0.004), as was the proportion of infants who required reintubation (relative risk, stratified according to gestational age, 0.58; 95% CI, 0.35 to 0.96; P = 0.03). Rates of other neonatal illnesses and adverse events were similar in the two groups. CONCLUSIONS Among extremely preterm infants, the incidence of bronchopulmonary dysplasia was lower among those who received early inhaled budesonide than among those who received placebo, but the advantage may have been gained at the expense of increased mortality

Optimization of Bacillus subtilis as an expression system

Bacteria are an integral part of modern biotechnology. They are used to make a variety of products, such as foods, drugs, as well as a multitude of chemicals. In order to increase their production rates molecular biotechnology offers many tuning points, starting from the selection of an applicable host, over its geno- and phenotypical characterization, followed by genetic manipulations for an optimized metabolism and stabilisation of production processes. This work comprises the optimization of Bacillus subtilis as an expression system. It describes the steps taken for selection and genomic characterization of the B. subtilis wild type strain ATCC 6051, the subsequent optimizations of the strain in respect to growth and productivity, as well as the characterization of its behaviour in a variety of cultivation conditions. The B. subtilis strain most commonly found in laboratories around the world is the first sequenced Gram-positive organism B. subtilis 168. Zeigler et al. showed that strain 168 is not a real wild type. Instead it was created through random mutagenesis with X-rays and selected for transformability. This strain has been used as the basis for popular B. subtilis strains in heterologous gene expression such as the extracellular protease deficient WB strains. Growth experiments showed the real wild type strain ATCC 6051 to be superior to its mutated ancestor 168, making it a solid basis for the construction of an optimized B. subtilis expression system. In order to gain a full understanding of the genomic and corresponding physiological differences between the two systems, B. subtilis ATCC 6051 was sequenced and compared to the genome of B. Subtilis 168. Several variations on geno- and phenotypic level could be revealed, that resulted in particular from genes involved in natural competency, the metabolism of amino acids and chemotaxis. This genomically well characterized B. subtilis ATCC 6051 was improved in respect to its application as an expression host. Improvements were achieved through the inactivation of both sporulation and reduction of autolysis, leading to a more robust behaviour during the overproduction and secretion of a reporter enzyme. A positive effect on the activity of an acetoin induced promoter by the addition of second copies for its transcription factors SigmaL and AcoR could be observed. Anaerobic zones and areas with excess glucose caused by insufficient mixing are common conditions in large scale bioprocesses and lead to oscillating conditions for the cells. In turn, this oscillation provokes an excretion of so called overflow metabolites, which can negatively affect the bacterial productivity. Detailed scientific characterizations of industrial scale processes under such oscillating conditions are scarce due to the high costs and logistics involved. A B. Subtilis sporulation mutant was thus examined in respect to its extra- and intracellular metabolites in a scale-down, two-compartment reactor giving hints about conditions the host is exposed to and how it reacts. To improve tolerance thresholds and utilization capacity for such metabolites in B. subtilis, the glyoxylate cycle was transferred from its close relative Bacillus licheniformis into the genome of B. subtilis. This feature enabled our B. subtilis ACE mutant to grow on acetate. The improved strain showed higher tolerance towards excess glucose in a fed-batch as well as higher productivity during the expression of a reporter enzyme in comparison to the wild type. The ACE strain and B. licheniformis showed an increased formation of glycolate during growth with the glyoxylate cycle. This with regard to bacteria undescribed metabolite seems to play a role as a by-product of the glyoxylate cycle. Summarizing, this thesis deals with the characterization and optimization of B. subtilis for growth on overflow metabolites, enhancements of the acoA-expression system and the influence of sporulation and lysis mutants on its activity. Complementary, the host was begun to be characterized in respect to its behaviour in industrial scale processes.Bakterien sind ein integraler Bestandteil der modernen Biotechnologie. Sie werden zur Herstellung verschiedenster Produkte verwendet, von Lebensmitteln über Medikamente sowie einer Vielzahl von Chemikalien. Zur Erhöhung ihrer Produktionsraten bietet die molekulare Biotechnologie viele Stellschrauben. Diese bestehen in der Auswahl eines möglichen Wirts bis hin zur geno- und phänotypischen Charakterisierung gefolgt von genetischen Manipulationen zur Optimierung des Stoffwechsels und der Stabilität in Produktionsprozessen. Diese Arbeit umfaßt die Optimierung von Bacillus subtilis als Expressionssystem. Es werden die Schritte zur Auswahl und genomischen Charakterisierung des B. subtilis Wildtyp-Stamm ATCC 6051, die nachfolgenden Optimierungen in Bezug auf Wachstum und Produktivität sowie eine Charakterisierung des Wirts unter Fed-Batch-Bedingungen im industriellen Maßstab beschrieben. Der in den meisten Laboren der Welt verwendete B. subtilis Stamm ist der erste sequenzierte Gram-positive Organismus B. subtilis 168. Zeigler et al. zeigten, dass Stamm 168 kein echter Wildtyp ist, sondern ein durch Zufalls-Mutagenese mit Röntgenstrahlen verändertes Bakterium, welches auf eine erhöhte Transformierbarkeit hin selektiert wurde. Dieser Stamm bildet auch die Grundlage für populäre B. subtilis Stämme, wie zum Beispiel die protease defizienten WB-Stämme , welche in der heterologen Genexpression und Produktion Verwendung finden. Wachstumsexperimente zeigten ein besseres Wachstum des Wildtyp Stamms ATCC 6051 gegenüber seinem mutierten Nachfahren B. subtilis 168, so dass der Stamm ATCC 6051 eine gute Alternative für die Konstruktion eines optimierten B. subtilis Expressionssystems darstellt. Um ein möglichst lückenloses Verständnis und eine solide Basis für die Konstruktion eines Expressions Stamms zu erhalten, wurde B. subtilis ATCC 6051 sequenziert und mit dem Genom des bereits sehr gut charakterisierten B. subtilis 168 hinsichtlich der Unterschiede auf genotypischer und phänotypischer Ebene untersucht. Es konnten eine Reihe von Abweichungen in Bezug auf die natürliche Kompetenz, auf den Stoffwechsel der Aminosäuren und die Chemotaxis festgestellt werden. Mit dem B. subtilis ATCC 6051 als Ausgangs Stamm wurden Optimierungen durch die Inaktivierung der Sporulation sowie einer Reduktion der Autolyse durchgeführt. Die Inaktivierung der entsprechenden Gene führte zu einem robusteren Verhalten während der Überproduktion und Sekretion eines Reporterenzyms. Eine positive Wirkung auf die Aktivität eines Acetoin induzierten Promotors konnte durch die Integration zusätzlicher Kopien seiner Transkriptionsfaktoren SigmaL und AcoR beobachtet werden. Sauerstoffmangel und Zonen mit einem Überschuss an Glu kose aufgrund unzureichender Durchmischung führen zu oszillierenden Bedingungen in industriellen Bioprozessen. Diese Bedingungen führen zur Exkretion von so genannten Überlauf Metaboliten (overflow metabolites), die sich negativ auf die Produktivität auswirken können. Auf Grund der hohen Kosten und dem Umfang der involvierten Logistik gibt es bislang kaum wissenschaftliche Untersuchungen von Bioprozessen im industriellen Maßstab. Daher wurde eine B. subtilis sporulations-Mutante auf ihre extra-und intrazellulären Metabolite während einer Kultivierung in einem maßstabsverkleinerten Bio-Reaktor untersucht. Damit entstehende Überlauf-Metabolite von B. subtilis besser toleriert und verwendet werden können, wurde der Glyoxylatzyklus aus seinem nahen Verwandten Bacillus licheniformis in das Genom von B. subtilis transferiert. Dadurch konnte diese B. subtilis ACE Mutante mit Azetat als C-Quelle wachsen. Der verbesserte Stamm zeigt im Vergleich zum Wildtyp eine höhere Toleranz gegenüber überschüssiger Glukose in einem Fed-Batch und eine erhöhte Produktivität bei der Expression eines Reporter-Enzyms. Für den ACE-Stamm und B. licheniformis konnte ein Anstieg von Glykolat während des Wachstums mit dem Glyoxylatzyklus festgestellt werden. Dieser für Bakterien bisher kaum beschriebene Metabolit könnte eine wichtige Rolle als Zwischenprodukt des Glyoxylatzyklus darstellen. Zusammenfassend behandelt diese Arbeit die Charakterisierung und Optimierung von B. subtilis für das Wachstum auf Überlauf-Metaboliten, die Erhöhung der Expression durch das acoA-Expressionssystem und den Einfluss einer Sporulations- und Lysemutante auf dessen Aktivität. Komplementiert wird dieses optimierte Expressionssystem durch die Untersuchung des Effekts von Kultivierungen im industriellen Maßstab auf den Stoffwechsel von B. subtilis anhand eines „scale-down“ Experiments

Quantification and isolation of Bacillus subtilis spores using cell sorting and automated gating

The Gram-positive bacterium Bacillus subtilis is able to form endospores which have a variety of biotechnological applications. Due to this ability, B. subtilis is as well a model organism for cellular differentiation processes. Sporulating cultures of B. subtilis form sub-populations which include vegetative cells, sporulating cells and spores. In order to readily and rapidly quantify spore formation we employed flow cytometric and fluorescence activated cell sorting techniques in combination with nucleic acid fluorescent staining in order to investigate the distribution of sporulating cultures on a single cell level. Automated gating procedures using Gaussian mixture modeling (GMM) were employed to avoid subjective gating and allow for the simultaneous measurement of controls. We utilized the presented method for monitoring sporulation over time in germination deficient strains harboring different genome modifications. A decrease in the sporulation efficiency of strain Bs02018, utilized for the display of sfGFP on the spores surface was observed. On the contrary, a double knock-out mutant of the phosphatase gene encoding Spo0E and of the spore killing factor SkfA (Bs02025) exhibited the highest sporulation efficiency, as within 24 h of cultivation in sporulation medium, cultures of BS02025 already consisted of 80% spores as opposed to 18% for the control strain. We confirmed the identity of the different subpopulations formed during sporulation by employing sorting and microscopy

CopySwitch—in vivo Optimization of Gene Copy Numbers for Heterologous Gene Expression in Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis has long been used as a host for production and secretion of industrially relevant enzymes like amylases and proteases. It is imperative for optimal efficiency, to balance protein yield and correct folding. While there are numerous ways of doing so on protein or mRNA level, our approach aims for the underlying number of coding sequences. Gene copy numbers are an important tuning valve for the optimization of heterologous gene expression. While some genes are best expressed from many gene copies, for other genes, medium or even single copy numbers are the only way to avoid formation of inclusion bodies, toxic gene dosage effects or achieve desired levels for metabolic engineering. In order to provide a simple and robust method to address above-mentioned issues in the Gram-positive bacterium Bacillus subtilis, we have developed an automatable system for the tuning of heterologous gene expression based on the host’s intrinsic natural competence and homologous recombination capabilities. Strains are transformed with a linearized, low copy number plasmid containing an antibiotic resistance marker and homology regions up- and downstream of the gene of interest. Said gene is copied onto the vector, rendering it circular and replicative and thus selectable. We could show an up to 3.6-fold higher gfp (green fluorescent protein) expression and up to 1.3-fold higher mPLC (mature phospholipase C) expression after successful transformation. Furthermore, the plasmid-borne gfp expression seems to be more stable, since over the whole cultivation period the share of fluorescent cells compared to all measured cells is consistently higher. A major benefit of this method is the ability to work with very large regions of interest, since all relevant steps are carried out in vivo and are thus far less prone to mechanical DNA damage

Implementation of Spore Display in <i>Paenibacillus polymyxa</i> with Different Hydrolytic Enzymes

Biotechnological processes are essential for producing climate-friendly high-value chemicals or pharmaceutical compounds, which can include steps catalyzed by enzymes. Therefore, establishing new, robust, and cheap enzyme production processes is desirable. One possible way to enhance processes is through the use of the spore display method. Spore display can present heterologous proteins on the surface of bacterial spores, offering numerous advantages in a range of biotechnological applications. This study demonstrates the implementation of the spore display method in Paenibacillus polymyxa, achieved by modifying the spore surface, incorporating an anchoring protein, and attaching green fluorescent protein to it, allowing the visualization of fluorescent spores. Following the initial experiment, a native lipase (Lip3), a heterologous lipase (LipA) from Bacillus subtilis, a native esterase (PnbA) from P. polymyxa, and a lipoyl synthase were expressed during sporulation and displayed on the spore surface. The activity profiles were determined in the temperature range from 4 °C to 70 °C. The PnbA reached its optimum at 4 °C, whereas the LipA from B. subtilis showed 4.4-fold higher activity at 42 °C compared to the control. Furthermore, we explored a possible new technique for the purification of enzymes with the TEV cleavage site between the anchor and the protein of interest. Finally, we showed a not-yet-described side activity of the lipoyl synthase over a wide temperature range

Editorial to the special issue "Advancing urban ecosystem service implementation and assessment considering different dimensions of environmental justice"

Unidad de excelencia María de Maeztu CEX2019-000940-MFins gener del 202

Editorial to the special issue "Advancing urban ecosystem service implementation and assessment considering different dimensions of environmental justice"

Unidad de excelencia María de Maeztu CEX2019-000940-MFins gener del 202