[Diagnostic stewardship in outpatient and hospital medicine with focus on microbiological urinary and bloodstream diagnostics]. / Diagnostic Stewardship in Klinik und Praxis mit Fokus auf die mikrobiologische Harnwegs- und Blutstromdiagnostik.

Targeted infection diagnosis supports decision-making in the rational use of antibiotics usually encompassed as Antibiotic Stewardship (ABS). Similar to ABS, the term "Diagnostic Stewardship" (DGS) is suggested, whereas DGS includes, beneath general, predominantly microbiological infection diagnostics - with specific pathogen detection, conventional via culture or immunology, increasingly also using molecular biological methods. Especially in microbiology, pre-analytics, analytics and post-analytics play an essential role. Pathogen characterization is accompanied by an antimicrobial susceptibility test (with S-I-R classification), which deserves special attention, especially in the context of ABS. All of these aspects are dealt with in this work and represented using two practical examples of urinary and bloodstream diagnostics that are relevant for outpatients and inpatients.

Phase separation in evaporating all-aqueous sessile drops.

The phase transition and phase distribution in an all-aqueous sessile drop containing polyethylene glycol (PEG) and dextran is studied. Evaporation of water triggers the formation of dextran-rich droplets close to the contact line of the drop that subsequently migrate towards the drop center. The likely reason for the droplet migration is Marangoni convection due to stresses at the interface between the dextran-rich droplets and the surrounding liquid.

[Strategies for the rational use of antibiotics in the outpatient sector-results of a workshop with key stakeholders in the healthcare system]. / Strategien zum rationalen Antibiotikaeinsatz im ambulanten Sektor ­ Ergebnisse eines Workshops mit wichtigen Akteuren des Gesundheitswesens.

In November 2021, the Federal Ministry of Health (BMG) organized the one-day virtual workshop "Rational antibiotic use in the outpatient sector - potential and opportunities for change" with scientific support from the Robert Koch Institute (RKI). The aim was to collect strategies for promoting the appropriate use of antibiotics in the outpatient sector. With 114 participants, important stakeholders of the healthcare system were represented. In the run-up to the event, the invited participants had already been asked to take part in an online survey on perspectives, experiences, and ideas for the rational use of antibiotics in the outpatient sector. The answers were analyzed and presented at the workshop.The workshop was introduced with plenary lectures on the German Antibiotic Resistance Strategy (DART) and the antibiotic resistance situation in Germany. All experts participated in 10 working group discussions; the resulting findings were presented in the concluding plenary session. In this conference report, selected aspects of these discussions are presented. The insights gained are to be incorporated into the "DART 2030" strategy.

Manipulation and control of droplets on surfaces in a homogeneous electric field.

A method to manipulate and control droplets on a surface is presented. The method is based on inducing electric dipoles inside the droplets using a homogeneous external electric field. It is shown that the repulsive dipole force efficiently suppresses the coalescence of droplets moving on a liquid-infused surface (LIS). Using a combination of experiments, numerical computations and semi-analytical models, the dependence of the repulsion force on the droplet volumes, the distance between the droplets and the electric field strength is revealed. The method allows to suppress coalescence in complex multi-droplet flows and is real-time adaptive. When the electric field strength exceeds a critical value, tip streaming from the droplets sets in. Based on that, it becomes possible to withdraw minute samples from an array of droplets in a parallel process.

Interaction of proteins with phase boundaries in aqueous two-phase systems under electric fields.

The electric-field driven transport of proteins across the liquid-liquid interface in an aqueous two-phase system (ATPS) is studied in a microfluidic device using fluorescence microscopy. An ATPS containing polyethylene glycol (PEG) and dextran is employed, and bovine serum albumin (BSA) and bovine γ-globulins (BγG) are considered as model proteins. It is shown that both proteins, initially in the dextran-rich phase, accumulate at the liquid-liquid interface, preferably close to the three-phase contact line between the two liquid phases and the microchannel wall. It is in these regions where the proteins penetrate into the PEG-rich phase. The transport resistance of the liquid-liquid interface is higher for BγG than for BSA, such that a much larger molar flux of BSA into the PEG phase is observed. This opens up the opportunity of separating different protein species by utilizing differences in the transport resistance at the interface. A mathematical model is developed, accounting for adsorption and desorption processes at the liquid-liquid interface. The underlying theoretical concept is that of an electrostatic potential minimum formed by superposing the applied electric field and the field due to the Donnan potential at the interface. A fit of the model parameters to the experimental data results in good agreement between theory and experiments, thereby corroborating the underlying picture.

Electric-Field-Induced Pattern Formation in Layers of DNA Molecules at the Interface between Two Immiscible Liquids.

The concentration patterns of DNA molecules attached to the interface between two immiscible aqueous phases forming under an electric field are studied. The pattern formation is driven by hydrodynamic interactions between the molecules originating from the electro-osmotic flow due to the Debye layer around a molecule. A nonlinear integrodifferential equation is derived describing the time evolution of the concentration field at the liquid-liquid interface. A linear stability analysis of this equation shows that a mode of given wavelength is initially stable, but destabilizes after a critical time which is inversely proportional to the wavelength. The scaling behavior of the critical time with electric field strength and viscosity found in the experiments agrees with the predictions by the theoretical model.

Relaxation of surface-tethered polymers under moderate confinement.

We study the relaxation of surface-tethered polymers in microchannels under moderate confinement (i.e. h ∼ Rg, where h is the channel height and Rg is the radius of gyration of the polymer) by experiments with fluorescence-marked DNA molecules and coupled lattice-Boltzmann/molecular dynamics simulations. The determined scaling exponent suggests that the relaxation is dominated by Zimm-dynamics with significant intra-chain hydrodynamic interactions. The relaxation of the DNA molecules is slower in shallower channels, indicating a pronounced effect of confinement on the longest relaxation time. An experimental correlation is obtained for the longest relaxation time as a function of the molecular contour length and the channel height. Good agreement between the experimental and the simulation results is found.

A single N1-methyladenosine on the large ribosomal subunit rRNA impacts locally its structure and the translation of key metabolic enzymes.

The entire chemical modification repertoire of yeast ribosomal RNAs and the enzymes responsible for it have recently been identified. Nonetheless, in most cases the precise roles played by these chemical modifications in ribosome structure, function and regulation remain totally unclear. Previously, we demonstrated that yeast Rrp8 methylates m1A645 of 25S rRNA in yeast. Here, using mung bean nuclease protection assays in combination with quantitative RP-HPLC and primer extension, we report that 25S/28S rRNA of S. pombe, C. albicans and humans also contain a single m1A methylation in the helix 25.1. We characterized nucleomethylin (NML) as a human homolog of yeast Rrp8 and demonstrate that NML catalyzes the m1A1322 methylation of 28S rRNA in humans. Our in vivo structural probing of 25S rRNA, using both DMS and SHAPE, revealed that the loss of the Rrp8-catalyzed m1A modification alters the conformation of domain I of yeast 25S rRNA causing translation initiation defects detectable as halfmers formation, likely because of incompetent loading of 60S on the 43S-preinitiation complex. Quantitative proteomic analysis of the yeast Δrrp8 mutant strain using 2D-DIGE, revealed that loss of m1A645 impacts production of specific set of proteins involved in carbohydrate metabolism, translation and ribosome synthesis. In mouse, NML has been characterized as a metabolic disease-associated gene linked to obesity. Our findings in yeast also point to a role of Rrp8 in primary metabolism. In conclusion, the m1A modification is crucial for maintaining an optimal 60S conformation, which in turn is important for regulating the production of key metabolic enzymes.

Design and validation of a clinical scale for prehospital stroke recognition, severity grading and prediction of large vessel occlusion: the shortened NIH Stroke Scale for emergency medical services.

OBJECTIVE: To develop an NIH Stroke Scale (NIHSS)-compatible, all-in-one scale for rapid and comprehensive prehospital stroke assessment including stroke recognition, severity grading and progression monitoring as well as prediction of large vessel occlusion (LVO). METHODS: Emergency medical services (EMS) personnel and stroke physicians (n=326) rated each item of the NIHSS regarding suitability for prehospital use; best rated items were included. Stroke recognition was evaluated retrospectively in 689 consecutive patients with acute stroke or stroke mimics, prediction of LVO in 741 consecutive patients with ischaemic stroke with acute vessel imaging independent of admission NIHSS score. RESULTS: Nine of the NIHSS items were rated as 'suitable for prehospital use.' After excluding two items in order to increase specificity, the final scale (termed shortened NIHSS for EMS, sNIHSS-EMS) consists of 'level of consciousness', 'facial palsy', 'motor arm/leg', 'sensory', 'language' and 'dysarthria'. Sensitivity for stroke recognition of the sNIHSS-EMS is 91% (95% CI 86 to 94), specificity 52% (95% CI 47 to 56). Receiver operating curve analysis revealed an optimal cut-off point for LVO prediction of ≥6 (sensitivity 70% (95% CI 65 to 76), specificity 81% (95% CI 76 to 84), positive predictive value 70 (95% CI 65 to 75), area under the curve 0.81 (95% CI 0.78 to 0.84)). Test characteristics were non-inferior to non-comprehensive scales. CONCLUSIONS: The sNIHSS-EMS may overcome the sequential use of multiple emergency stroke scales by permitting parallel stroke recognition, severity grading and LVO prediction. Full NIHSS-item compatibility allows for evaluation of stroke progression starting at the prehospital phase.

Correction: Mapping of Complete Set of Ribose and Base Modifications of Yeast rRNA by RP-HPLC and Mung Bean Nuclease Assay.

[This corrects the article DOI: 10.1371/journal.pone.0168873.].

Mapping of Complete Set of Ribose and Base Modifications of Yeast rRNA by RP-HPLC and Mung Bean Nuclease Assay.

Ribosomes are large ribonucleoprotein complexes that are fundamental for protein synthesis. Ribosomes are ribozymes because their catalytic functions such as peptidyl transferase and peptidyl-tRNA hydrolysis depend on the rRNA. rRNA is a heterogeneous biopolymer comprising of at least 112 chemically modified residues that are believed to expand its topological potential. In the present study, we established a comprehensive modification profile of Saccharomyces cerevisiae's 18S and 25S rRNA using a high resolution Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). A combination of mung bean nuclease assay, rDNA point mutants and snoRNA deletions allowed us to systematically map all ribose and base modifications on both rRNAs to a single nucleotide resolution. We also calculated approximate molar levels for each modification using their UV (254nm) molar response factors, showing sub-stoichiometric amount of modifications at certain residues. The chemical nature, their precise location and identification of partial modification will facilitate understanding the precise role of these chemical modifications, and provide further evidence for ribosome heterogeneity in eukaryotes.

Melatonin: Free Radicals and Metabolites Resulting by Emission and Consumption of Solvated Electrons (eaq-): Reaction Mechanisms.

BACKGROUND/AIM: Melatonin not only regulates circadian rhythm, but also induces apoptosis in tumor cells. Hence, elucidation of the basic reaction mechanisms of melatonin and its metabolites is a matter of interest. MATERIAL AND METHODS: Melatonin dissolved in a mixture of water/ethanol=40/60 form associates (unstable complexes). For simulation of biological processes, melatonin was excited by UV light into the singlet state. RESULTS: By using monochromatic UV light (λ=254 nm) melatonin ejects solvated electrons (eaq (-)), a part of which is scavenged by melatonin in ground state contained in the associates. Consequently, with increase of melatonin concentration a decrease of the determined quantum yield of emitted eaq (-), Q(eaq (-)), is obtained. The complex molecular structure of melatonin contains functional groups which can emit eaq (-), as well such consuming eaq (-). As a succession of these processes various types of metabolites are generated, as well as degradation products, with lower molecular weight, are formed. CONCLUSION: Not melatonin per se, but the ejected eaq (-) and thereby resulting various metabolites are responsible for different biological properties of melatonin.

Diastolic heart murmur, nocturnal back pain, and lumbar rigidity in a 7-year girl: an unusual manifestation of lyme disease in childhood.

A 7-year-old girl presented with nocturnal pain in her back and legs. The physical examination revealed a loud opening sound of the mitral valve and lumbar rigidity. With the exception of significantly increased anti-nuclear antibody (ANA) levels, the immunological findings did not show any other abnormal parameters, also spinal magnetic resonance imaging (MRI) and ultrasound examination of the abdomen and pelvis yield no pathological findings. The lumbar puncture showed a lymphocytic pleocytosis as well as an intrathecal synthesis of Borrelia-specific antibodies. Echocardiography showed a thickened mitral valve with mild regurgitation. No other signs of florid endocarditis or myocarditis could be detected. Due to these findings, the diagnosis Lyme neuroborreliosis was made and an intravenous antibiotic therapy was given. The clinical symptoms subsided. Six months later, she had an almost normal mitral valve with only trivial mitral insufficiency. The association between the lumbar rigidity and the thickened mitral valve remains unclear. The case of our patient with nocturnal back and leg pain may be considered a rare case of Lyme neuroborreliosis with meningoradiculitis in children, and to our knowledge these symptoms together with cardiac involvement, such as a significantly thickened mitral valve, have not yet been described in the literature.

Photo-induced regeneration of hormones by electron transfer processes: Potential biological and medical consequences.

Based on the previous results concerning electron transfer processes in biological substances, it was of interest to investigate if hormone transients resulting by e.g. electron emission can be regenerated.The presented results prove for the first time that the hormone transients originating by the electron emission process can be successfully regenerated by the transfer of electrons from a potent electron donor, such as vitamin C (VitC). Investigations were performed using progesterone (PRG), testosterone (TES) and estrone (E1) as representatives of hormones. By irradiation with monochromatic UV light (λ=254 nm) in a media of 40% water and 60% ethanol, the degradation as well as the regeneration of the hormones was studied with each hormone individually and in the mixture with VitC as a function of the absorbed UV dose, using HPLC. Calculated from the obtained initial yields, the determined regeneration of PRG amounted to 52.7%, for TES to 58.6% and for E1 to 90.9%. The consumption of VitC was determined in the same way.The reported results concerning the regeneration of hormones by the transfer of electrons from an electron donor offer a new, promising method for the therapy with hormones. As a consequence of the regeneration of hormones, a decreased formation of carcinogenic metabolites is expected.

The effect of progesterone on the electron emission and degradation of testosterone.

Based on recent findings that hormones can emit electrons () from their excited singlet state in polar media, it was of importance to study a possible mutual interaction of progesterone (PRG) and testosterone (TES) in this respect. Hormones of highest purity were dissolved in an air-free mixture of 40% triply distilled water and 60% ethanol, because the hormones are unsoluble in water. As energy source for substrate excitation in singlet state served a monochromatic UV-light (254 nm), the emitted electrons were scavenged by chloroethanol, whereby the quantum yield of produced Cl⁻ ions, Q (Cl⁻), is equal to Q(e⁻(aq)). Hormone degradation initiated by the electron emission was studied by HPLC method, using a Zorbax Eclipse XDB-C18 column (150 mm x 4.6 mm, 5 µm). The quantum yield of emitted e⁻(aq), Q(e⁻(aq)), from TES was 3.6 times higher than that from PRG, which is explained by the different molecular structures of the hormones. Observed 2nd and 3rd maxima of electron emission indicate the ability of TES and PRG products to also eject e⁻(aq), but with lower yield. It can be stated that a part of the emitted electrons from TES are consumed by PRG⁺ leading to a partial regeneration of hormone. The present results offer a deeper insight in the biological behavior of hormones.

Electron emission and product analysis of estrone: progesterone interactions studied by experiments in vitro.

Recent studies showed that hormones like progesterone, testosterone, etc. can eject [Formula: see text] (solvated electrons). By means of electron transfer processes via the brain, the hormones communicate with other biological systems in the organism. The present study proves that also estrone is able to emit electrons. Their yield strongly depends on the concentration of the hormone, temperature and on the absorbed energy. The metabolites resulting from this process are likewise able to generate electrons, however with much smaller yields. The formation of the estrone metabolites is studied by HPLC-analyses. In vitro experiments with MCF-7 cells demonstrate the distinct effect of progesterone on the carcinogenity of estrone metabolites. Probable reaction mechanisms for explanation of the observed effects are postulated.

Method for regeneration of hormones: 17ß-estradiol, 21α-hydroxyprogesterone and corticosterone. A pathway for a possible medical application.

The hormones 17ß-estradiol (17ßE2), 21α-hydroxyprogesterone (21α-HOPRG) and corticosterone (CORT) were used as representative models for the study. As a source for hormone excitation in singlet state serviced monochromatic UV-light (λ=254 nm), it was stated that the transients resulting by e-aq emission in air-free mixture water/ethanol 40/60, as long as they are in "status nascendi", can be regenerated by electron transfer from a potent electron donor, e.g., vitamin C. The hormone regeneration (%) strongly depends, after all, on specific hormone molecular structure, concentration, temperature, etc. Because of the large heterogenic molecular structures, the substrates dissolved in the solvent mixture form "associates" (unstable complexes) in concentrations >109 mol/L hormone. The hormones eject, but they also consume e-aq with a rather high reaction rate constant (k≈109 up to 2×1010 L/mol.s), therefore, they act as "electron mediators". It was also observed that the hormones by dissolution in aerated solvent mixture are sensitive towards oxygen. For an explanation of the results, probable reaction mechanisms are presented. The described method offers a new pathway and possibilities for application in medicine.

Metabolite formation of 17alpha-hydroxyprogesterone as a consequence of e⁻(aq)-emission and progesterone effect regarding cancer.

Based on previous investigations on several hormones, 17α-hydroxyprogesterone (17α-HOPRG) was studied in respect to cancer initiation by its metabolites resulting from electron emission. The emission of electrons (e⁻(aq)) from its singlet excited state of 17α-HOPRG and HPLC-analysis of products were studied. Possible carcinogenicity of metabolites originating from 17α-HOPRG and the effect of progesterone (PRG) in this respect were studied in vitro. The results showed that 17α-HOPRG is very sensitive towards oxygen. The highest Q(e⁻(aq)) values were obtained by dissolution and UV-irradiation of substrate in airfree media. 17α-HOPRG metabolites showed a strong anticancer activity, which is, however, lower compared to that of PRG-metabolites. Mixture of both hormones, 17α-HOPRG and PRG, in respect to carcinogenicity showed a synergistic effect of PRG on 17α-HOPRG. Reaction mechanisms are presented.

Mutual interaction of 17beta-estradiol and progesterone: electron emission. Free radical effect studied by experiments in vitro.

BACKGROUND: Based on the different behaviour of 17beta-estradiol (17betaE(2)) and progesterone (PRG), it was of interest to investigate the interaction of both hormones in respect of their electron emission and cytotoxicity by experiments in vitro. MATERIALS AND METHODS: The studies include determination of emitted electrons (e(-)(aq)) by the individual hormones as well as by their mixtures, all complexed with cyclodextrin (HBC). Experiments in vitro (Escherichia coli bacteria) were performed for a better understanding of the mechanisms involved. Survival ratios, DeltaD(37)(Gy), were calculated. RESULTS: Aqueous HBC as well as 17betaE(2) and PRG, individually as well as in mixtures, are able to emit e(-)(aq). The resulting transients can lead to the formation of metabolites, some of which can initiate cancer. It was established that both hormones, 17betaE(2) and PRG, interact in respect to their electron emission property. In the frame of experiments in vitro, it was found that oxidizing radicals (OH, O(2)(-)) lead to negative DeltaD(37)(Gy) values, indicating cytostatic properties. On the other hand, the primary reducing radicals (e(-)(aq), H) lead to positive DeltaD(37)(Gy) values, indicating a radical-scavenging effect. CONCLUSION: The main outcome of this work is that PRG in combination with 17betaE(2) can strongly reduce the number of carcinogenic 17betaE(2)-metabolites. This fact offers a new pathway for application of hormones in medical treatment of patients.

17beta-estradiol acting as an electron mediator: experiments in vitro.

BACKGROUND: The present work reports on the effect of oxidizing (OH, O(2)(*-)) and reducing free radicals (e(-)(aq), H) on 17beta-estradiol (17betaE2) in respect to breast cancer initiation. The objectives of the study were based on the following premise: the ability of 17betaE2 to emit electrons (e(-)(aq)) as well as to transfer them to other biological systems. Thereby, the resulting transient hormone products are leading to the formation of metabolites, some of which may initiate the neoplastic process. The present work considers the effect of the simultaneously generated oxidizing and reducing free radicals on the carcinogenic properties of the 17betaE2 metabolites. MATERIALS AND METHODS: Water-soluble 17betaE2 with incorporated 2-hydroxypropyl-beta-cyclodextrin (HBC) in various aqueous media (pH ~7.4), saturated with air, N(2)O or argon, as well as HBC alone, were exposed to the action of free radicals produced by gamma-ray. Escherichia coli bacteria (AB 1157) were used as a model for living systems. RESULTS: From the survival curves obtained under different conditions, the derived DeltaD(37) values (representing the radiation dose at which N/N(0)=0.37; N/N(0) ratio: N(0)=starting number of colonies, N=number after irradiation treatment) illustrate that 17betaE2 as well as HBC act as very powerful scavengers of OH and O(2)(*-) radicals. On the other hand, 17betaE2 and HBC intermediates resulting from attack of the reducing species (e(-)(aq), H) have strong anticancer properties. CONCLUSION: It is stated that DeltaD(37) values strongly depend on the reactivity of the individual free radicals. Oxidizing free radicals lead to positive DeltaD(37) values, illustrating the strongly pronounced radiation protecting ability of the systems. On the contrary, the primary reducing free radicals result in negative DeltaD(37) values, indicating anticancer effect.