Glutathione Protects other Cellular Thiols against Oxidation by CuII-Dp44mT.

Cu-thiosemicarbazones have been intensively investigated for their application in cancer therapy or as antimicrobials. Copper(II)-di-2-pyridylketone-4,4-dimethyl-thiosemicarbazone (CuII-Dp44mT) showed anticancer activity in the submicromolar concentration range in cell culture. The interaction of CuII-Dp44mT with thiols leading to their depletion or inhibition was proposed to be involved in this activity. Indeed, CuII-Dp44mT can catalyze the oxidation of thiols although with slow kinetics. The present work aims to obtain insights into the catalytic activity and selectivity of CuII-Dp44mT toward the oxidation of different biologically relevant thiols. Reduced glutathione (GSH), L-cysteine (Cys), N-acetylcysteine (NAC), D-penicillamine (D-Pen), and the two model proteins glutaredoxin (Grx) and thioredoxin (Trx) were investigated. CuII-Dp44mT catalyzed the oxidation of these thiols with different kinetics, with rates in the following order D-Pen>Cys≫NAC>GSH and Trx>Grx. CuII-Dp44mT was more efficient than CuII chloride for the oxidation of NAC and GSH, but not D-Pen and Cys. In mixtures of biologically relevant concentrations of GSH and either Cys, Trx, or Grx, the oxidation kinetics and spectral properties were similar to that of GSH alone, indicating that the interaction of these thiols with CuII-Dp44mT is dominated by GSH. Hence GSH could protect other thiols against potential deleterious oxidation by CuII-Dp44mT.

Deciphering the mechanism of glutaredoxin-catalyzed roGFP2 redox sensing reveals a ternary complex with glutathione for protein disulfide reduction.

Glutaredoxins catalyze the reduction of disulfides and are key players in redox metabolism and regulation. While important insights were gained regarding the reduction of glutathione disulfide substrates, the mechanism of non-glutathione disulfide reduction remains highly debated. Here we determined the rate constants for the individual redox reactions between PfGrx, a model glutaredoxin from Plasmodium falciparum, and redox-sensitive green fluorescent protein 2 (roGFP2), a model substrate and versatile tool for intracellular redox measurements. We show that the PfGrx-catalyzed oxidation of roGFP2 occurs via a monothiol mechanism and is up to three orders of magnitude faster when roGFP2 and PfGrx are fused. The oxidation kinetics of roGFP2-PfGrx fusion constructs reflect at physiological GSSG concentrations the glutathionylation kinetics of the glutaredoxin moiety, thus allowing intracellular structure-function analysis. Reduction of the roGFP2 disulfide occurs via a monothiol mechanism and involves a ternary complex with GSH and PfGrx. Our study provides the mechanistic basis for understanding roGFP2 redox sensing and challenges previous mechanisms for protein disulfide reduction.

Ultrafast Yb:YAG laser oscillator with gigahertz repetition rate.

We present a SESAM modelocked Yb:YAG solid-state laser providing low-noise narrowband pulses with a pulse duration of 606 fs at a 1.09-GHz repetition rate, delivering up to 2.5 W of average output power. This laser provides access to a new parameter space that could previously not be reached by solid-state lasers and, to the best of our knowledge, is the first modelocked solid-state Yb:YAG laser in the gigahertz regime. This is achieved by introducing a single additional intracavity element, specifically a nonlinear birefringent YVO4 crystal, for soliton formation, polarization selection, and cavity intensity clamping. The isotropic pump absorption in Yb:YAG allows for stable and low-noise operation with multimode fiber pumping. This laser is ideally suited as a seed source for many commercial high-power Yb-doped amplification systems operating at a center wavelength around 1.03 µm. The laser exhibits a high power per comb line of 5.0 mW which also makes it interesting for applications in frequency comb spectroscopy, especially if it is used to pump an optical parametric oscillator. We measure a relative intensity noise (RIN) of 0.03%, integrated from 1 Hz to 10 MHz. Furthermore, we show that the laser timing jitter for noise frequencies >2 kHz is fully explained by a power-dependent shift in the center wavelength of 0.38 nm/W due to the quasi-three-level laser gain material. The narrow gain bandwidth of Yb:YAG reduces this contribution to noise in comparison to other SESAM modelocked Yb-doped lasers.

Stable Isotope Dilution Analysis (SIDA) to Determine Metabolites of Furan and 2-Methylfuran in Human Urine Samples: A Pilot Study.

Furan and 2-methylfuran (2-MF) are food contaminants that are classified as potentially carcinogenic to humans. The main source of exposure for adults via food is coffee consumption. Furan and 2-MF are volatile, which complicates exposure assessment because their content measured in food prior to consumption does not afford a reliable dosimetry. Therefore, other ways of exposure assessment need to be developed, preferably by monitoring exposure biomarkers, e.g., selected metabolites excreted in urine. In this study, cis-2-buten-1,4-dial (BDA)-derived urinary furan metabolites Lys-BDA (l-2-amino-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)hexanoic acid), AcLys-BDA (l-2-(acetylamino)-6-(2,5-dihydro-2-oxo-1H-pyrrol-1-yl)hexanoic acid) and GSH-BDA (N-[4-carboxy-4-(3-mercapto-1H-pyrrol-1-yl)-1-oxobutyl]-l-cysteinyl-glycine cyclic sulfide), as well as acetyl acrolein (AcA, 2-oxo-pent-2-enal)-derived metabolites Lys-AcA (l-2-(acetylamino)-6-(2,5-dihydro-5-methyl-2-oxo-1H-pyrrol-1-yl)-hexanoic acid) and AcLys-AcA (l-2-amino-6-(2,5-dihydro-5-methyl-2-oxo-1H-pyrrol-1-yl)-hexanoic acid) and their stable isotopically labeled analogs, were synthesized and characterized through NMR and MS, and a stable isotope dilution analysis (SIDA) with UPLC-ESI-MS/MS was established. As a proof of concept, urinary samples of a four-day human intervention study were used. In the frame of this study, ten subjects ingested 500 mL of coffee containing 0.648 µmol furan and 1.059 µmol 2-MF. Among the furan metabolites, AcLys-BDA was the most abundant, followed by Lys-BDA and GSH-BDA. Exposure to 2-MF via the coffee brew led to the formation of Lys-AcA and AcLys-AcA. Within 24 h, 89.1% of the ingested amount of furan and 15.4% of the ingested amount of 2-MF were detected in the urine in the form of the investigated metabolites. Therefore, GSH-BDA, Lys-BDA, AcLys-BDA, Lys-AcA and AcLys-AcA may be suitable as short-term-exposure biomarkers of furan and 2-MF exposure.

Glutathione kinetically outcompetes reactions between dimedone and a cyclic sulfenamide or physiological sulfenic acids.

Dimedone and its derivates are used as selective probes for the nucleophilic detection of sulfenic acids in biological samples. Qualitative analyses suggested that dimedone also reacts with cyclic sulfenamides. Furthermore, under physiological conditions, dimedone must compete with the highly concentrated nucleophile glutathione. We therefore quantified the reaction kinetics for a cyclic sulfenamide model peptide and the sulfenic acids of glutathione and a model peroxiredoxin in the presence or absence of dimedone and glutathione. We show that the cyclic sulfenamide is stabilized at lower pH and that it reacts with dimedone. While reactions between dimedone and sulfenic acids or the cyclic sulfenamide have similar rate constants, glutathione kinetically outcompetes dimedone as a nucleophile by several orders of magnitude. Our comparative in vitro and intracellular analyses challenge the selectivity of dimedone. Consequently, the dimedone labeling of cysteinyl residues inside living cells points towards unidentified reaction pathways or unknown, kinetically competitive redox species.

Characterization of the planarian surface electroencephalogram.

BACKGROUND: Despite large morphological differences between the nervous systems of lower animals and humans, striking functional similarities have been reported. However, little is known about how these functional similarities translate to cognitive similarities. As a first step towards studying the cognitive abilities of simple nervous systems, we here characterize the ongoing electrophysiological activity of the planarian Schmidtea mediterranea. One previous report using invasive microelectrodes describes that the ongoing neural activity is characterized by a 1/fx power spectrum with the exponent 'x' of the power spectrum close to 1. To extend these findings, we aimed to establish a recording protocol to measure ongoing neural activity safely and securely from alive and healthy planarians under different lighting conditions using non-invasive surface electrodes. RESULTS: As a replication and extension of the previous results, we show that the ongoing neural activity is characterized by a 1/fx power spectrum, that the exponent 'x' in living planarians is close to 1, and that changes in lighting induce changes in neural activity likely due to the planarian photophobia. CONCLUSIONS: We confirm the existence of continuous EEG activity in planarians and show that it is possible to noninvasively record this activity with surface wire electrodes. This opens up broad possibilities for continuous recordings across longer intervals, and repeated recordings from the same animals to study cognitive processes.

Influence of disk aberrations on high-power thin-disk laser cavities.

We present a systematic study on the influence of thin-disk aberrations on the performance of thin-disk laser oscillators. To evaluate these effects, we have developed a spatially resolved numerical model supporting arbitrary phase profiles on the intracavity components that estimates the intracavity beam shape and the output power of thin-disk laser oscillators. By combining this model with the experimentally determined phase profile of the thin-disk (measured with interferometry), we can predict the operation mode of high-power thin-disk lasers, including mode degradation, higher-order mode coupling, and stability zone shrinking, all of which are in good agreement with experiment. Our results show that one of the main mechanisms limiting the performance is the small deviation of the disk's phase profile from perfect radial symmetry. This result is an important step to scaling modelocked thin-disk oscillators to the kW-level and will be important in the design of future active multi-pass cavity arrangements.

Characterization of the glutathione-dependent reduction of the peroxiredoxin 5 homolog PfAOP from Plasmodium falciparum.

Peroxiredoxins use a variety of thiols to rapidly reduce hydroperoxides and peroxynitrite. While the oxidation kinetics of peroxiredoxins have been studied in great detail, enzyme-specific differences regarding peroxiredoxin reduction and the overall rate-limiting step under physiological conditions often remain to be deciphered. The 1-Cys peroxiredoxin 5 homolog PfAOP from the malaria parasite Plasmodium falciparum is an established model enzyme for glutathione/glutaredoxin-dependent peroxiredoxins. Here, we reconstituted the catalytic cycle of PfAOP in vitro and analyzed the reaction between oxidized PfAOP and reduced glutathione (GSH) using molecular docking and stopped-flow measurements. Molecular docking revealed that oxidized PfAOP has to adopt a locally unfolded conformation to react with GSH. Furthermore, we determined a second-order rate constant of 6 × 105 M-1  s-1 at 25°C and thermodynamic activation parameters ΔH‡ , ΔS‡ , and ΔG‡ of 39.8 kJ/mol, -0.8 J/mol, and 40.0 kJ/mol, respectively. The gain-of-function mutant PfAOPL109M had almost identical reaction parameters. Taking into account physiological hydroperoxide and GSH concentrations, we suggest (a) that the reaction between oxidized PfAOP and GSH might be even faster than the formation of the sulfenic acid in vivo, and (b) that conformational changes are likely rate limiting for PfAOP catalysis. In summary, we characterized and quantified the reaction between GSH and the model enzyme PfAOP, thus providing detailed insights regarding the reactivity of its sulfenic acid and the versatile chemistry of peroxiredoxins.

Optical flow analysis reveals that Kinesin-mediated advection impacts the orientation of microtubules in the Drosophila oocyte.

The orientation of microtubule (MT) networks is exploited by motors to deliver cargoes to specific intracellular destinations and is thus essential for cell polarity and function. Reconstituted in vitro systems have largely contributed to understanding the molecular framework regulating the behavior of MT filaments. In cells, however, MTs are exposed to various biomechanical forces that might impact on their orientation, but little is known about it. Oocytes, which display forceful cytoplasmic streaming, are excellent model systems to study the impact of motion forces on cytoskeletons in vivo. Here we implement variational optical flow analysis as a new approach to analyze the polarity of MTs in the Drosophila oocyte, a cell that displays distinct Kinesin-dependent streaming. After validating the method as robust for describing MT orientation from confocal movies, we find that increasing the speed of flows results in aberrant plus end growth direction. Furthermore, we find that in oocytes where Kinesin is unable to induce cytoplasmic streaming, the growth direction of MT plus ends is also altered. These findings lead us to propose that cytoplasmic streaming - and thus motion by advection - contributes to the correct orientation of MTs in vivo. Finally, we propose a possible mechanism for a specialized cytoplasmic actin network (the actin mesh) to act as a regulator of flow speeds to counteract the recruitment of Kinesin to MTs.

Power-scaling of nonlinear-mirror modelocked thin-disk lasers.

We present a first power-scaled nonlinear-mirror (NLM) modelocked thin-disk laser based on an Yb-doped gain material. The laser oscillator delivers average output powers up to 87 W and peak powers up to 14.7 MW with sub-600-femtosecond pulses at ≈9-MHz repetition rate. We demonstrate a threefold improvement in average output power and sixfold improvement in pulse energy compared to previous NLM-modelocking results. We obtain peak powers in excess of 10 MW for the first time from an NLM-modelocked laser oscillator. In our laser, the NLM is assisted by a semiconductor saturable absorber mirror (SESAM) to reliably initiate pulsed operation. We validate the high-power suitability of the NLM modelocking technique using low-absorption χ(2) crystals and optimized dichroic-mirror coating designs. Furthermore, we discuss stability against Q-switching and study how the tuning of the nonlinear mirror affects the laser performance.

Image-based autofocusing system for nonlinear optical microscopy with broad spectral tuning.

We present an image-based autofocusing system applied in nonlinear microscopy and spectroscopy with a wide range of excitation wavelengths. The core of the developed autofocusing system consists of an adapted two-step procedure maximizing an image score with six different image scorings algorithms implemented to cover different types of focusing scenarios in automated regime for broad wavelength region. The developed approach is combined with an automated multi-axis alignment procedure. We demonstrate the key abilities of the autofocusing procedure on different types of structures: single nanoparticles, nanowires and complex 3D nanostructures. Based on these experiments, we determine the optimal autofocusing algorithms for different types of structures and applications.

Reshaping the Second-Order Polar Response of Hybrid Metal-Dielectric Nanodimers.

We combine the field confinement of plasmonics with the flexibility of multiple Mie resonances by bottom-up assembly of hybrid metal-dielectric nanodimers. We investigate the electromagnetic coupling between nanoparticles in heterodimers consisting of gold and barium titanate (BaTiO3 or BTO) nanoparticles through nonlinear second-harmonic spectroscopy and polarimetry. The overlap of the localized surface plasmon resonant dipole mode of the gold nanoparticle with the dipole and higher-order Mie resonant modes in the BTO nanoparticle lead to the formation of hybridized modes in the visible spectral range. We employ the pick-and-place technique to construct the hybrid nanodimers with controlled diameters by positioning the nanoparticles of different types next to each other under a scanning electron microscope. Through linear scattering spectroscopy, we observe the formation of hybrid modes in the nanodimers. We show that the modes can be directly accessed by measuring the dependence of the second-harmonic generation (SHG) signal on the polarization and wavelength of the pump. We reveal both experimentally and theoretically that the hybridization of plasmonic and Mie-resonant modes leads to a strong reshaping of the SHG polarization dependence in the nanodimers, which depends on the pump wavelength. We compare the SHG signal of each hybrid nanodimer with the SHG signal of single BTO nanoparticles to estimate the enhancement factor due to the resonant mode coupling within the nanodimers. We report up to 2 orders of magnitude for the SHG signal enhancement compared with isolated BTO nanoparticles.

Anapoles in Free-Standing III-V Nanodisks Enhancing Second-Harmonic Generation.

Nonradiating electromagnetic configurations in nanostructures open new horizons for applications due to two essential features: a lack of energy losses and invisibility to the propagating electromagnetic field. Such radiationless configurations form a basis for new types of nanophotonic devices, in which a strong electromagnetic field confinement can be achieved together with lossless interactions between nearby components. In our work, we present a new design of free-standing disk nanoantennas with nonradiating current distributions for the optical near-infrared range. We show a novel approach to creating nanoantennas by slicing III-V nanowires into standing disks using focused ion-beam milling. We experimentally demonstrate the suppression of the far-field radiation and the associated strong enhancement of the second-harmonic generation from the disk nanoantennas. With a theoretical analysis of the electromagnetic field distribution using multipole expansions in both spherical and Cartesian coordinates, we confirm that the demonstrated nonradiating configurations are anapoles. We expect that the presented procedure of designing and producing disk nanoantennas from nanowires becomes one of the standard approaches to fabricating controlled chains of standing nanodisks with different designs and configurations. These chains can be essential building blocks for new types of lasers and sensors with low power consumption.