Label-free optical detection of single enzyme-reactant reactions and associated conformational changes

Monitoring the kinetics and conformational dynamics of single enzymes is crucial in order to better understand their biological functions as these motions and structural dynamics are usually unsynchronized among the molecules. Detecting the enzyme-reactant interactions and associated conformational changes of the enzyme on a single molecule basis, however, remain as a challenge with established optical techniques due to the commonly required labeling of the reactants or the enzyme itself. The labeling process is usually non-trivial and the labels themselves might skew the physical properties of the enzyme. Here we demonstrate an optical, label-free method capable of observing enzymatic interactions and the associated conformational changes on the single molecule level. We monitor polymerase/DNA interactions via the strong near-field enhancement provided by plasmonic nanorods resonantly coupled to whispering gallery modes in microcavities. Specifically, we employ two different recognition schemes: one in which the kinetics of polymerase/DNA interactions are probed in the vicinity of DNA-functionalized nanorods, and the other in which these interactions are probed via the magnitude of conformational changes in the polymerase molecules immobilized on nanorods. In both approaches we find that low and high polymerase activities can be clearly discerned via their characteristic signal amplitude and signal length distributions. Furthermore, the thermodynamic study of the monitored interactions suggests the occurrence of DNA polymerization. This work constitutes a proof-of-concept study of enzymatic activities via plasmonically enhanced microcavities and establishes an alternative and label-free method capable of investigating structural changes in single molecules

Zivilgesellschaftliche Akteure in der Außenpolitik: Chancen und Perspektiven von Public Diplomacy

Der Fokus dieser Studie liegt auf dem Handeln deutscher zivilgesellschaftlicher Akteure als außenpolitische Akteure. Entsprechend muss mit einer Vielzahl an Begrifflichkeiten gearbeitet werden: Welches Zivilgesellschaftsverständnis herrscht vor und inwiefern können zivilgesellschaftliche Akteure Außenpolitik betreiben? Mithilfe eines netzwerkbasierten Ansatzes von Public Diplomacy wird auch zivilgesellschaftlichen Akteuren eine relevante Funktion in der Außenpolitik eines Staates zugesprochen. Doch mit welchen Herausforderungen werden die Akteure konfrontiert? Die zentralen Fragestellungen der Arbeit sind daher: Inwiefern können deutsche zivilgesellschaftliche Akteure als außenpolitisch handelnde Akteure eingestuft werden? Welche Chancen und welche Hindernisse erfahren zivilgesellschaftliche Akteure, wenn sie als außenpolitische Akteure auftreten? Mit welchen Unterschieden in der Arbeitsweise und dem Begriffsverständnis von Zivilgesellschaft werden die Akteure in ihrer täglichen Arbeit konfrontiert und welche Rückschlüsse lassen sich hieraus ziehen? Als zentrales Ergebnis steht, dass zivilgesellschaftliche Akteure Außenpolitik betreiben, aber die Anerkennung dieser Arbeit von staatlichen Akteuren als zu niedrig angesehen wird. Das Selbstbild und die Fremdwahrnehmung unterscheiden sich hier. In der täglichen Arbeit müssen die Akteure darüber hinaus vielfältige Hindernisse umgehen, die aber nicht aus unterschiedlichen Verständnissen von Zivilgesellschaftskonzeption stammen, sondern praktischer Natur sind, wie der Partnerakquise im Ausland

Medienkompetenz - Tipps zum sicheren Umgang mit digitalen Medien : für Eltern und alle, die mit Kids zu tun haben

Jugend und Medien. Nationales Programm zur Förderung von Medienkompetenzen. http://www.jugendundmedien.c

Recognition of Peptidoglycan Fragments by the Transpeptidase PBP4 From Staphylococcus aureus

Peptidoglycan (PG) is an essential component of the cell envelope, maintaining bacterial cell shape and protecting it from bursting due to turgor pressure. The monoderm bacterium Staphylococcus aureus has a highly cross-linked PG, with ~90% of peptide stems participating in DD-cross-links and up to 15 peptide stems connected with each other. These cross-links are formed in transpeptidation reactions catalyzed by penicillin-binding proteins (PBPs) of classes A and B. Most S. aureus strains have three housekeeping PBPs with this function (PBP1, PBP2, and PBP3) but MRSA strains have acquired a third class B PBP, PBP2a, which is encoded by the mecA gene and required for the expression of high-level resistance to β-lactams. Another housekeeping PBP of S. aureus is PBP4, which belongs to the class C PBPs, and hence would be expected to have PG hydrolase (DD-carboxypeptidase or DD-endopeptidase) activity. However, previous works showed that, unexpectedly, PBP4 has transpeptidase activity that significantly contributes to both the high level of cross-linking in the PG of S. aureus and to the low level of β-lactam resistance in the absence of PBP2a. To gain insights into this unusual activity of PBP4, we studied by NMR spectroscopy its interaction in vitro with different substrates, including intact peptidoglycan, synthetic peptide stems, muropeptides, and long glycan chains with uncross-linked peptide stems. PBP4 showed no affinity for the complex, intact peptidoglycan or the smallest isolated peptide stems. Transpeptidase activity of PBP4 was verified with the disaccharide peptide subunits (muropeptides) in vitro, producing cyclic dimer and multimer products; these assays also showed a designed PBP4(S75C) nucleophile mutant to be inactive. Using this inactive but structurally highly similar variant, liquid-state NMR identified two interaction surfaces in close proximity to the central nucleophile position that can accommodate the potential donor and acceptor stems for the transpeptidation reaction. A PBP4:muropeptide model structure was built from these experimental restraints, which provides new mechanistic insights into mecA independent resistance to β-lactams in S. aureus

Prevalence and prognostic value of neurological affections in hospitalized patients with moderate to severe COVID-19 based on objective assessments.

Neurological manifestations of coronavirus disease 2019 (COVID-19) have been frequently described. In this prospective study of hospitalized COVID-19 patients without a history of neurological conditions, we aimed to analyze their prevalence and prognostic value based on established, standardized and objective methods. Patients were investigated using a multimodal electrophysiological approach, accompanied by neuropsychological and neurological examinations. Prevalence rates of central (CNS) and peripheral (PNS) nervous system affections were calculated and the relationship between neurological affections and mortality was analyzed using Firth logistic regression models. 184 patients without a history of neurological diseases could be enrolled. High rates of PNS affections were observed (66% of 138 patients receiving electrophysiological PNS examination). CNS affections were less common but still highly prevalent (33% of 139 examined patients). 63% of patients who underwent neuropsychological testing (n = 155) presented cognitive impairment. Logistic regression models revealed pathology in somatosensory evoked potentials as an independent risk factor of mortality (Odds Ratio: 6.10 [1.01-65.13], p = 0.049). We conclude that hospitalized patients with moderate to severe COVID-19 display high rates of PNS and CNS affection, which can be objectively assessed by electrophysiological examination. Electrophysiological assessment may have a prognostic value and could thus be helpful to identify patients at risk for deterioration

Mapping local patterns of childhood overweight and wasting in low- and middle-income countries between 2000 and 2017

A double burden of malnutrition occurs when individuals, household members or communities experience both undernutrition and overweight. Here, we show geospatial estimates of overweight and wasting prevalence among children under 5 years of age in 105 low- and middle-income countries (LMICs) from 2000 to 2017 and aggregate these to policy-relevant administrative units. Wasting decreased overall across LMICs between 2000 and 2017, from 8.4% (62.3 (55.1–70.8) million) to 6.4% (58.3 (47.6–70.7) million), but is predicted to remain above the World Health Organization’s Global Nutrition Target of <5% in over half of LMICs by 2025. Prevalence of overweight increased from 5.2% (30 (22.8–38.5) million) in 2000 to 6.0% (55.5 (44.8–67.9) million) children aged under 5 years in 2017. Areas most affected by double burden of malnutrition were located in Indonesia, Thailand, southeastern China, Botswana, Cameroon and central Nigeria. Our estimates provide a new perspective to researchers, policy makers and public health agencies in their efforts to address this global childhood syndemic

One Size Fits All? The Validity of a Composite Poverty Index Across Urban and Rural Households in South Africa

Composite indices have been prominently used in poverty research. However, validity of these indices remains subject to debate. This paper examines the validity of a common type of composite poverty indices using data from a cross-sectional survey of 2477 households in urban and rural KwaZulu-Natal, South Africa. Multiple-group comparisons in structural equation modelling were employed for testing differences in the measurement model across urban and rural groups. The analysis revealed substantial variations between urban and rural respondents both in the conceptualisation of poverty as well as in the weights and importance assigned to individual poverty indicators. The validity of a ‘one size fits all’ measurement model can therefore not be confirmed. In consequence, it becomes virtually impossible to determine a household’s poverty level relative to the full sample. Findings from our analysis have important practical implications in nuancing how we can sensitively use composite poverty indices to identify poor people