In VitroSelection for Different Mutational Patterns in the HIV-1 Reverse Transcriptase Using High and Low Selective Pressure of the Nonnucleoside Reverse Transcriptase Inhibitor HBY 097

AbstractIn vitroresistance of HIV-1 against high levels of HBY 097 ((S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4-dihydro-quinoxaline-2(1H)-thione) and other quinoxaline nonnucleoside reverse transcriptase inhibitors (NNRTIs) is characterized by a specific amino acid substitution in the reverse transcriptase (RT), Gly190Glu. This change results in decreased RT polymerase activity and in reduced growth properties of the corresponding viral variant. Here we show that the appearance of the crippling mutation at codon 190 can be prevented by lowering the selective pressure exerted by HBY 097. Under low selective pressure an accumulation of other NNRTI-specific mutations is observed. Up to five NNRTI-specific substitutions were detected in some of these virus lineages. In addition, we report novel RT amino acid changes which were not observed previously, including Val106Ile, Val106Leu, and Gly190Thr. HBY 097 selects for different mutational patterns under high and low selective pressure conditions, respectively. Thus, the type of mutations which appear in HIV-infected patients undergoing therapy may be determined by the levels of the selecting drug

3D stochastic bicontinuous microstructures: Generation, topology and elasticity

Motivated by recent experimental investigations of the mechanical behavior of nanoporous metal we explore an efficient and robust method for generating 3D representative volume elements (RVEs) with strikingly similar behavior. Our approach adopts Cahn's method of generating a Gaussian random field by taking a superposition of standing sinusoidal waves of fixed wavelength but random in direction and phase. In its theory part, our study describes closed-form expressions for how the solid volume fraction affects the binarization level, mean structure size, specific surface area, averages of mean and Gaussian curvature, and the scaled topological genus. Based on numerical studies we report on criteria for achieving representative realizations of the structure by proper choice of the number of waves and element size. We also show that periodic structures are readily created. We analyze the mechanical properties considering linear and infinitesimal elasticity and evaluate the residual anisotropy (which can be made small) and the effective values of the Young's modulus and Poisson's ratio. The numerical results are in excellent agreement with experimental findings for the variation of stiffness with solid fraction of nanoporous gold made by dealloying. We propose scaling relations that achieve naturally a perfect agreement with the numerical and experimental data. The scaling relation for the stiffness accounts for a percolation-to-cluster transition in the random field microstructure at a finite solid fraction. We propose that this transition is the origin of the previously reported anomalous compliance of nanoporous gold

Single shot time-resolved magnetic x-ray absorption at a Free Electron Laser

Ultrafast dynamics are generally investigated using stroboscopic pump-probe measurements, which characterize the sample properties for a single, specific time delay. These measurements are then repeated for a series of discrete time delays to reconstruct the overall time trace of the process. As a consequence, this approach is limited to the investigation of fully reversible phenomena. We recently introduced an off-axis zone plate based X-ray streaking technique, which overcomes this limitation by sampling the relaxation dynamics with a single femtosecond X-ray pulse streaked over a picosecond long time window. In this article we show that the X-ray absorption cross section can be employed as the contrast mechanism in this novel technique. We show that changes of the absorption cross section on the percent level can be resolved with this method. To this end we measure the ultrafast magnetization dynamics in CoDy alloy films. Investigating different chemical compositions and infrared pump fluences, we demonstrate the routine applicability of this technique. Probing in transmission the average magnetization dynamics of the entire film, our experimental findings indicate that the demagnetization time is independent of the specific infrared laser pump fluence. These results pave the way for the investigation of irreversible phenomena in a wide variety of scientific areas.Comment: 9 pages, 5 figure

Improved methods for microfluorometry recordings with emphasis in the FAD and NAD(P)H autoflourescence as a readout of energy demand in hippocampal neurons

Abstrakt Die „Light Emitting Diode“ (LED) als Beleuchtungseinheit in der Mikroskopie hat in den letzten Jahren einen festen Platz eingenommen. Dies betrifft nicht nur die Durchlichtmikroskopie sondern auch weite Bereiche in der Fluoreszenzmessung und Optogenetik. Ihre Vorteile liegen u.a. in der Lebensdauer, stufenlosen Regulierbarkeit der Intensität und einer periodischen Ansteuerbarkeit im μs Bereich. Gleichzeitig gab es auf dem Gebiet der Fotodetektion mit dem Einzug der „Avalanche Photodiode“ (APD) und weiterentwickelten Hybriddetektoren eine Verbesserung in der Quanteneffizienz (QE) im Vergleich zum „Photomultiplier Tube“ (PMT). Begründet aus dieser Entwicklung ergab sich die Frage, ob eine Verbesserung in Bezug auf Fototobleichen in biologischen Experimenten bei einer kombinierten Anwendung von LED und APD erreicht werden kann. Im Mittelpunkt der Arbeit stand die Entwicklung und Erprobung von optischen, elektronischen und mechanischen Komponenten an einem Mikroskopsystem. Mit den neuen Aufbauten wurde eine Reihe von biologischen Experimenten durchgeführt. So konnten die intrinsischen Fluoreszenzen von Nicotinsäureamid-Adenin-Dinukleotid-Phosphat und Flavin- Adenine-Dinucleotid (NAD(P)H und FAD) parallel untersucht und kombiniert werden mit einem Vergleich von Fotodetektoren bezüglich Sensitivität und Signal-Rausch Verhältnis in Human Embryonic Kidney (HEK) 293 Zellen und akuten Hirnschnitten der Ratte. Zu Beginn wurde die Erholung nach dem Bleichen von NAD(P)H in HEK293 Zellen in Abhängigkeit des Substrates (Glukose, 2-deoxy-D-Glukose, Laktat) aufgezeichnet. Um die Beteiligung astrozytärer Laktatzufuhr im Energiemetabolismus der Nervenzellen zu testen, haben wir Stimulus induzierte Veränderungen der NAD(P)H/FAD Fluoreszenz in hippokampalen Gehirnschnitten mit und ohne Blockade der Laktattransporter aufgezeichnet. Wir konnten zeigen, dass eine mit Pulsen angesteuerte LED im Vergleich zur kontinuierlichen Beleuchtung mit einer LED zu weniger Fotobleichen ohne Verluste an Fluoreszenzintensität bei der Aufzeichnung von FAD führte. Die empirisch ermittelten Parameter an Belichtungszeit und Frequenz sind 5 Hz und 5 ms. Bei allen Experimenten konnte die Überlegenheit der LED gegenüber Quecksilberdampflampen aufgezeigt und genutzt werden. Insbesondere bei der parallelen Aufzeichnung von NAD(P)H und FAD bei Untersuchungen zum Zellmetabolismus kamen die Vorteile der LED zum Tragen.Abstract In recent years Light Emitting Diodes (LEDs) became a preferred component for use in microscopy applications. Not only as light sources for transmitted light microscopy but also as excitation light sources for fluorescent microscopy and optogenetics. The advantages of LEDs are the enhanced life-span, the stepless intensity regulation and their switching capability in the μs range. At the same time, semiconductor-based photodetectors gained a foothold by the introduction of avalanche photodiodes (APDs) and different forms of ´hybrid´ detectors with improved quantum efficiency (QE) as compared to conventional photomultiplier tubes (PMTs). Due to these developments it was plausible to ask whether a replacement of the fluorescent light source and detector by an LED and APD combination would decrease the photo-bleaching in experiments with sensitive biological preparations. The central aim of the study was the design and technological development of the optical, electrical and mechanical components for such a replacement on a commercially available microscope system. By using the novel system we made parallel recordings of the intrinsic fluorescence of Nicotinicacid-Adenine-Dinucleotide-Phosphate und Flavin-Adenine-Dinucleotide (NAD(P)H and FAD), in combination with the comparison of different photo- detectors with respect to their sensitivity and signal-to-noise ratio in Human Embryonic Kidney (HEK) 293 cells and in rat brain slices. First, we monitored photo-bleaching and metabolism dependent recovery of NAD(P)H fluorescence in HEK293 cells in dependence of available substrates (glucose, 2-deoxy-glucose and lactate). In order to identify the contribution of astrocytic-neuronal lactate shuttle to energy metabolism, we measured stimulus induced changes in NAD(P)H / FAD fluorescence in hippocampal slices in the presence and absence of lactate transport inhibitors. We have shown that a pulsed LED illumination protocol achieved reduced photobleaching with the same fluorescence yield of FAD as compared to continuous illumination. The empirical optimum of the illumination parameters with our system were 5 Hz and 5 ms with respect to pulse duration and frequency. In all of these experiments LEDs demonstrated a clear benefit in comparison with conventional Mercury arc lamps. LED excitation was of particular advantage for parallel recordings of NAD(P)H and FAD fluorescence in studies of energy metabolism

Der „verlorenen“ Energie auf der Spur. Einsatzmöglichkeiten von Wärmebildkameras Im Physikunterricht

Stinken-Rösner L. Der „verlorenen“ Energie auf der Spur. Einsatzmöglichkeiten von Wärmebildkameras Im Physikunterricht. In: Maxton-Küchenmeister J, Meßinger-Koppelt J, eds. Naturwissenschaften digital: Toolbox für den Unterricht - Band 2. Hamburg: Joachim Hertz Stiftung ; 2021: 24-27

Contribution of Intrinsic Lactate to Maintenance of Seizure Activity in Neocortical Slices from Patients with Temporal Lobe Epilepsy and in Rat Entorhinal Cortex

Neuronal lactate uptake supports energy metabolism associated with synaptic signaling and recovery of extracellular ion gradients following neuronal activation. Altered expression of the monocarboxylate transporters (MCT) in temporal lobe epilepsy (TLE) hampers lactate removal into the bloodstream. The resulting increase in parenchymal lactate levels might exert both, anti- and pro-ictogen effects, by causing acidosis and by supplementing energy metabolism, respectively. Hence, we assessed the contribution of lactate to the maintenance of transmembrane potassium gradients, synaptic signaling and pathological network activity in chronic epileptic human tissue. Stimulus induced and spontaneous field potentials and extracellular potassium concentration changes (∆[K+]O) were recorded in parallel with tissue pO2 and pH in slices from TLE patients while blocking MCTs by α-cyano-4-hydroxycinnamic acid (4-CIN) or d-lactate. Intrinsic lactate contributed to the oxidative energy metabolism in chronic epileptic tissue as revealed by the changes in pO2 following blockade of lactate uptake. However, unlike the results in rat hippocampus, ∆[K+]O recovery kinetics and field potential amplitude did not depend on the presence of lactate. Remarkably, inhibition of lactate uptake exerted pH-independent anti-seizure effects both in healthy rat and chronic epileptic tissue and this effect was partly mediated via adenosine 1 receptor activation following decreased oxidative metabolism

Sevoflurane Effects on Neuronal Energy Metabolism Correlate with Activity States While Mitochondrial Function Remains Intact

During general anesthesia, alterations in neuronal metabolism may induce neurotoxicity and/or neuroprotection depending on the dose and type of the applied anesthetic. In this study, we investigate the effects of clinically relevant concentrations of sevoflurane (2% and 4%, i.e., 1 and 2 MAC) on different activity states in hippocampal slices of young Wistar rats. We combine electrophysiological recordings, partial tissue oxygen (ptiO2) measurements, and flavin adenine dinucleotide (FAD) imaging with computational modeling. Sevoflurane minimally decreased the cerebral metabolic rate of oxygen (CMRO2) while decreasing synaptic transmission in naive slices. During pharmacologically induced gamma oscillations, sevoflurane impaired network activity, thereby decreasing CMRO2. During stimulus-induced neuronal activation, sevoflurane decreased CMRO2 and excitability while basal metabolism remained constant. In this line, stimulus-induced FAD transients decreased without changes in basal mitochondrial redox state. Integration of experimental data and computer modeling revealed no evidence for a direct effect of sevoflurane on key enzymes of the citric acid cycle or oxidative phosphorylation. Clinically relevant concentrations of sevoflurane generated a decent decrease in energy metabolism, which was proportional to the present neuronal activity. Mitochondrial function remained intact under sevoflurane, suggesting a better metabolic profile than isoflurane or propofol

Event-Associated Oxygen Consumption Rate Increases ca. Five-Fold When Interictal Activity Transforms into Seizure-Like Events In Vitro

Neuronal injury due to seizures may result from a mismatch of energy demand and adenosine triphosphate (ATP) synthesis. However, ATP demand and oxygen consumption rates have not been accurately determined, yet, for different patterns of epileptic activity, such as interictal and ictal events. We studied interictal-like and seizure-like epileptiform activity induced by the GABAA antagonist bicuculline alone, and with co-application of the M-current blocker XE-991, in rat hippocampal slices. Metabolic changes were investigated based on recording partial oxygen pressure, extracellular potassium concentration, and intracellular flavine adenine dinucleotide (FAD) redox potential. Recorded data were used to calculate oxygen consumption and relative ATP consumption rates, cellular ATP depletion, and changes in FAD/FADH2 ratio by applying a reactive-diffusion and a two compartment metabolic model. Oxygen-consumption rates were ca. five times higher during seizure activity than interictal activity. Additionally, ATP consumption was higher during seizure activity (~94% above control) than interictal activity (~15% above control). Modeling of FAD transients based on partial pressure of oxygen recordings confirmed increased energy demand during both seizure and interictal activity and predicted actual FAD autofluorescence recordings, thereby validating the model. Quantifying metabolic alterations during epileptiform activity has translational relevance as it may help to understand the contribution of energy supply and demand mismatches to seizure-induced injury