High average power transform limited picosecond laser with flexible repetition rate and pulse duration.

This Letter presents a pulsed, Fourier transform limited 1030 nm laser with a variable pulse duration between 47 and 733 ps resulting in a spectral bandwidth of roughly 1 to 10 GHz. The laser system is based on ytterbium-doped fiber amplifiers and acousto-optic and electro-optic modulation technology. The repetition rate can be set arbitrarily between 1 and 10 MHz. After three sequential amplifier stages, the average output power reaches a maximum of over 60 W. The particular fiber amplifier geometry allows to prevent the emergence of unwanted nonlinear effects. Due to its unique features, the laser system lends itself to a variety of applications wherever flexibility in terms of pulse duration and corresponding Fourier limited bandwidth are required, such as laser cooling at storage rings, lidar applications, or coherent molecular spectroscopy.

An Optogenetic Tool for Induced Protein Stabilization Based on the Phaeodactylum tricornutum Aureochrome 1a Light-Oxygen-Voltage Domain.

Control of cellular events by optogenetic tools is a powerful approach to manipulate cellular functions in a minimally invasive manner. A common problem posed by the application of optogenetic tools is to tune the activity range to be physiologically relevant. Here, we characterized a photoreceptor of the light-oxygen-voltage (LOV) domain family of Phaeodactylum tricornutum aureochrome 1a (AuLOV) as a tool for increasing protein stability under blue light conditions in budding yeast. Structural studies of AuLOVwt, the variants AuLOVM254, and AuLOVW349 revealed alternative dimer association modes for the dark state, which differ from previously reported AuLOV dark-state structures. Rational design of AuLOV-dimer interface mutations resulted in an optimized optogenetic tool that we fused to the photoactivatable adenylyl cyclase from Beggiatoa sp. This synergistic light-regulation approach using two photoreceptors resulted in an optimized, photoactivatable adenylyl cyclase with a cyclic adenosine monophosphate production activity that matches the physiological range of Saccharomyces cerevisiae. Overall, we enlarged the optogenetic toolbox for yeast and demonstrated the importance of fine-tuning the optogenetic tool activity for successful application in cells.

Optogenetic Downregulation of Protein Levels with an Ultrasensitive Switch.

Optogenetic control of protein activity is a versatile technique to gain control over cellular processes, for example, for biomedical and biotechnological applications. Among other techniques, the regulation of protein abundance by controlling either transcription or protein stability found common use as this controls the activity of any type of target protein. Here, we report modules of an improved variant of the photosensitive degron module and a light-sensitive transcription factor, which we compared to doxycycline-dependent transcriptional control. Given their modularity the combined control of synthesis and stability of a given target protein resulted in the synergistic down regulation of its abundance by light. This combined module exhibits very high switching ratios, profound downregulation of protein abundance at low light-fluxes, and fast protein depletion kinetics. Overall, this synergistic optogenetic multistep control (SOMCo) module is easy to implement and results in a regulation of protein abundance superior to each individual component.

Allosteric communication between DNA-binding and light-responsive domains of diatom class I aureochromes.

The modular architecture of aureochrome blue light receptors, found in several algal groups including diatoms, is unique by having the LOV-type photoreceptor domain fused to the C-terminus of its putative effector, an N-terminal DNA-binding bZIP module. The structural and functional understanding of aureochromes' light-dependent signaling mechanism is limited, despite their promise as an optogenetic tool. We show that class I aureochromes 1a and 1c from the diatom Phaeodactylum tricornutum are regulated in a light-independent circadian rhythm. These aureochromes are capable to form functional homo- and heterodimers, which recognize the ACGT core sequence within the canonical 'aureo box', TGACGT, in a light-independent manner. The bZIP domain holds a more folded and less flexible but extended conformation in the duplex DNA-bound state. FT-IR spectroscopy in the absence and the presence of DNA shows light-dependent helix unfolding in the LOV domain, which leads to conformational changes in the bZIP region. The solution structure of DNA bound to aureochrome points to a tilted orientation that was further validated by molecular dynamics simulations. We propose that aureochrome signaling relies on an allosteric pathway from LOV to bZIP that results in conformational changes near the bZIP-DNA interface without major effects on the binding affinity.

Proscriptive versus prescriptive morality: two faces of moral regulation.

A distinction is made between two forms of morality on the basis of approach-avoidance differences in self-regulation. Prescriptive morality is sensitive to positive outcomes, activation-based, and focused on what we should do. Proscriptive morality is sensitive to negative outcomes, inhibition-based, and focused on what we should not do. Seven studies profile these two faces of morality, support their distinct motivational underpinnings, and provide evidence of moral asymmetry. Both are well-represented in individuals' moral repertoire and equivalent in terms of moral weight, but proscriptive morality is condemnatory and strict, whereas prescriptive morality is commendatory and not strict. More specifically, in these studies proscriptive morality was perceived as concrete, mandatory, and duty-based, whereas prescriptive morality was perceived as more abstract, discretionary, and based in duty or desire; proscriptive immorality resulted in greater blame, whereas prescriptive morality resulted in greater moral credit. Implications for broader social regulation, including cross-cultural differences and political orientation, are discussed.

Mitochondrial inhibition prior to oxygen-withdrawal facilitates the occurrence of hypoxia-induced spreading depression in rat hippocampal slices.

Oxygen withdrawal blocks mitochondrial respiration. In rat hippocampal slices, this triggers a massive depolarization of CA1 neurons and a negative shift of the extracellular DC potential, the characteristic sign of hypoxia-induced spreading depression (HSD). To unveil the contribution of mitochondria to the sensing of hypoxia and the ignition of HSD, we modified mitochondrial function. Mitochondrial uncoupling by carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, 1 microM) prior to hypoxia hastened the onset and shortened the duration of HSD. Blocking mitochondrial ATP synthesis by oligomycin (10 microg/ml) was without effect. Inhibition of mitochondrial respiration by rotenone (20 microM), diphenyleneiodonium (25 microM), or antimycin A (20 microM) also hastened HSD onset and shortened HSD duration. 3-nitropropionic acid (1 mM) increased HSD duration. Cyanide (100 microM) hastened HSD onset and increased HSD duration. At higher concentrations, cyanide (1 mM), azide (2 mM), and FCCP (10 microM) triggered SD episodes on their own. Compared with control HSD, the spatial extent of the intrinsic optical signals of cyanide- and azide-induced SDs was more pronounced. Monitoring NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide) autofluorescence and mitochondrial membrane potential verified the mitochondrial targeting by the drugs used. Except 1 mM cyanide, no treatment reduced cellular ATP levels severely and no correlation was found between ATP, NADH, or FAD levels and the time to HSD onset. Therefore ATP depletion or a cytosolic reducing shift due to NADH/FADH2 accumulation cannot serve as a general explanation for the hastening of HSD onset on mitochondrial inhibition. Additional redox couples (glutathione) or events downstream of the mitochondrial depolarization need to be considered.

Sulfhydryl oxidation reduces hippocampal susceptibility to hypoxia-induced spreading depression by activating BK channels.

The cytosolic redox status modulates ion channels and receptors by oxidizing/reducing their sulfhydryl (SH) groups. We therefore analyzed to what degree SH modulation affects hippocampal susceptibility to hypoxia. In rat hippocampal slices, severe hypoxia caused a massive depolarization of CA1 neurons and a negative shift of the extracellular DC potential, the characteristic sign of hypoxia-induced spreading depression (HSD). Oxidizing SH groups by 5,5'-dithiobis 2-nitrobenzoic acid (DTNB, 2 mM) postponed HSD by 30%, whereas their reduction by 1,4-dithio-dl-threitol (DTT, 2 mM) or alkylation by N-ethylmaleimide (500 microM) hastened HSD onset. The DTNB-induced postponement of HSD was not affected by tolbutamide (200 microM), dl-2-amino-5-phosphonovaleric acid (150 microM), or 6-cyano-7-nitroquinoxaline-2,3-dione (25 microM). It was abolished, however, by Ni2+ (2 mM), withdrawal of extracellular Ca2+, charybdotoxin (25 nM), and iberiotoxin (50 nM). In CA1 neurons DTNB induced a moderate hyperpolarization, blocked spontaneous spike discharges and postponed the massive hypoxic depolarization. DTT induced burst firing, depolarized glial cells, and hastened the onset of the massive hypoxic depolarization. Schaffer-collateral/CA1 synapses were blocked by DTT but not by DTNB; axonal conduction remained intact. Mitochondria did not markedly respond to DTNB or DTT. While the targets of DTT are less clear, the postponement of HSD by DTNB indicates that sulfhydryl oxidation increases the tolerance of hippocampal tissue slices against hypoxia. We identified as the underlying mechanism the activation of BK channels in a Ca(2+)-sensitive manner. Accordingly, ionic disregulation and the loss of membrane potential occur later or might even be prevented during short-term insults. Therefore well-directed oxidation of SH groups could mediate neuroprotection.