A-Si/SiO2 nanolaminates for tuning the complex refractive index and band gap in optical interference coatings.

A-S i/S i O 2 nanolaminates are deposited by magnetron sputtering and show a decreasing absorption when the a-Si single-layer thickness is reduced from 2.4n m to 0.7n m. Moreover, an increase of the Tauc band gap by 0.18e V is measured. Experimental Tauc band gaps are compared to calculated effective band gaps, utilizing a numerical Schrödinger solver. Further, it is demonstrated that the refractive index can be controlled by adjusting the a-Si and S i O 2 single-layer thicknesses in the nanolaminates. The nanolaminates are optically characterized by spectroscopic ellipsometry, transmittance, and reflectance measurements. Additionally, TEM images reveal uniform, well-separated layers, and EDX measurements show the silicon and oxygen distribution in the nanolaminates.

Implications of different membrane compartmentalization models in particle-based in silico studies.

Studying membrane dynamics is important to understand the cellular response to environmental stimuli. A decisive spatial characteristic of the plasma membrane is its compartmental structure created by the actin-based membrane-skeleton (fences) and anchored transmembrane proteins (pickets). Particle-based reaction-diffusion simulation of the membrane offers a suitable temporal and spatial resolution to analyse its spatially heterogeneous and stochastic dynamics. Fences have been modelled via hop probabilities, potentials or explicit picket fences. Our study analyses the different approaches' constraints and their impact on simulation results and performance. Each of the methods comes with its own constraints; the picket fences require small timesteps, potential fences might induce a bias in diffusion in crowded systems, and probabilistic fences, in addition to carefully scaling the probability with the timesteps, induce higher computational costs for each propagation step.

Improving optical thickness monitoring by including systematic and process-influenced transmittance deviations.

During optical monitoring using broadband transmittance measurement, the accuracy depends on how both the substrate and the optical path are aligned. We present a correction procedure to improve the accuracy of the monitoring, even if the substrate has features such as absorption or if there is misalignment of the optical path. The substrate in this case can either be a test glass or a product. The algorithm is proven by experimental coatings which were produced with and without the correction. Additionally, the optical monitoring system was also used to perform an in situ quality check. The system allows a detailed spectral analysis of all substrates with a high position resolution. Both plasma and the temperature effects on the central wavelength of a filter are identified. This knowledge enables the optimization of the following runs.

An auto-inhibited state of protein kinase G and implications for selective activation.

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory (AI) pseudo-substrate sequences to PKG Iα and Iß that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here, we present a crystal structure of PKG Iß in which the AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iß AI sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iß auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.

Dynamical Basis of Allosteric Activation for the Plasmodium falciparum Protein Kinase G.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is required for the progression of the Plasmodium's life cycle and is therefore a promising malaria drug target. PfPKG includes four cGMP-binding domains (CBD-A to CBD-D). CBD-D plays a crucial role in PfPKG regulation as it is the primary determinant for the inhibition and cGMP-dependent activation of the catalytic domain. Hence, it is critical to understand how CBD-D is allosterically regulated by cGMP. Although the apo versus holo conformational changes of CBD-D have been reported, information on the intermediates of the activation pathway is currently lacking. Here, we employed molecular dynamics simulations to model four key states along the thermodynamic cycle for the cGMP-dependent activation of the PfPKG CBD-D domain. The simulations were compared to NMR data, and they revealed that the PfPKG CBD-D activation pathway samples a compact intermediate in which the N- and C-terminal helices approach the central ß-barrel. In addition, by comparing the cGMP-bound active and inactive states, the essential binding interactions that differentiate these states were identified. The identification of structural and dynamical features unique to the cGMP-bound inactive state provides a promising basis to design PfPKG-selective allosteric inhibitors as a viable treatment for malaria.

Regulation of Cardiac PKA Signaling by cAMP and Oxidants.

Pathologies, such as cancer, inflammatory and cardiac diseases are commonly associated with long-term increased production and release of reactive oxygen species referred to as oxidative stress. Thereby, protein oxidation conveys protein dysfunction and contributes to disease progression. Importantly, trials to scavenge oxidants by systemic antioxidant therapy failed. This observation supports the notion that oxidants are indispensable physiological signaling molecules that induce oxidative post-translational modifications in target proteins. In cardiac myocytes, the main driver of cardiac contractility is the activation of the ß-adrenoceptor-signaling cascade leading to increased cellular cAMP production and activation of its main effector, the cAMP-dependent protein kinase (PKA). PKA-mediated phosphorylation of substrate proteins that are involved in excitation-contraction coupling are responsible for the observed positive inotropic and lusitropic effects. PKA-actions are counteracted by cellular protein phosphatases (PP) that dephosphorylate substrate proteins and thus allow the termination of PKA-signaling. Both, kinase and phosphatase are redox-sensitive and susceptible to oxidation on critical cysteine residues. Thereby, oxidation of the regulatory PKA and PP subunits is considered to regulate subcellular kinase and phosphatase localization, while intradisulfide formation of the catalytic subunits negatively impacts on catalytic activity with direct consequences on substrate (de)phosphorylation and cardiac contractile function. This review article attempts to incorporate the current perception of the functionally relevant regulation of cardiac contractility by classical cAMP-dependent signaling with the contribution of oxidant modification.

Mechanism of allosteric inhibition in the Plasmodium falciparum cGMP-dependent protein kinase.

Most malaria deaths are caused by the protozoan parasite Plasmodium falciparum Its life cycle is regulated by a cGMP-dependent protein kinase (PfPKG), whose inhibition is a promising antimalaria strategy. Allosteric kinase inhibitors, such as cGMP analogs, offer enhanced selectivity relative to competitive kinase inhibitors. However, the mechanisms underlying allosteric PfPKG inhibition are incompletely understood. Here, we show that 8-NBD-cGMP is an effective PfPKG antagonist. Using comparative NMR analyses of a key regulatory domain, PfD, in its apo, cGMP-bound, and cGMP analog-bound states, we elucidated its inhibition mechanism of action. Using NMR chemical shift analyses, molecular dynamics simulations, and site-directed mutagenesis, we show that 8-NBD-cGMP inhibits PfPKG not simply by reverting a two-state active versus inactive equilibrium, but by sampling also a distinct inactive "mixed" intermediate. Surface plasmon resonance indicates that the ability to stabilize a mixed intermediate provides a means to effectively inhibit PfPKG, without losing affinity for the cGMP analog. Our proposed model may facilitate the rational design of PfPKG-selective inhibitors for improved management of malaria.

Potential based, spatial simulation of dynamically nested particles.

BACKGROUND: To study cell biological phenomena which depend on diffusion, active transport processes, or the locations of species, modeling and simulation studies need to take space into account. To describe the system as a collection of discrete objects moving and interacting in continuous space, various particle-based reaction diffusion simulators for cell-biological system have been developed. So far the focus has been on particles as solid spheres or points. However, spatial dynamics might happen at different organizational levels, such as proteins, vesicles or cells with interrelated dynamics which requires spatial approaches that take this multi-levelness of cell biological systems into account. RESULTS: Based on the perception of particles forming hollow spheres, ML-Force contributes to the family of particle-based simulation approaches: in addition to excluded volumes and forces, it also supports compartmental dynamics and relating dynamics between different organizational levels explicitly. Thereby, compartmental dynamics, e.g., particles entering and leaving other particles, and bimolecular reactions are modeled using pair-wise potentials (forces) and the Langevin equation. In addition, forces that act independently of other particles can be applied to direct the movement of particles. Attributes and the possibility to define arbitrary functions on particles, their attributes and content, to determine the results and kinetics of reactions add to the expressiveness of ML-Force. Its implementation comprises a rudimentary rule-based embedded domain-specific modeling language for specifying models and a simulator for executing models continuously. Applications inspired by cell biological models from literature, such as vesicle transport or yeast growth, show the value of the realized features. They facilitate capturing more complex spatial dynamics, such as the fission of compartments or the directed movement of particles, and enable the integration of non-spatial intra-compartmental dynamics as stochastic events. CONCLUSIONS: By handling all dynamics based on potentials (forces) and the Langevin equation, compartmental dynamics, such as dynamic nesting, fusion and fission of compartmental structures are handled continuously and are seamlessly integrated with traditional particle-based reaction-diffusion dynamics within the cell. Thereby, attributes and arbitrary functions allow to flexibly describe diverse spatial phenomena, and relate dynamics across organizational levels. Also they prove crucial in modeling intra-cellular or intra-compartmental dynamics in a non-spatial manner, and, thus, to abstract from spatial dynamics, on demand which increases the range of multi-compartmental processes that can be captured.

New cGMP analogues restrain proliferation and migration of melanoma cells.

Melanoma is one of the most aggressive cancers and displays high resistance to conventional chemotherapy underlining the need for new therapeutic strategies. The cGMP/PKG signaling pathway was detected in melanoma cells and shown to reduce migration, proliferation and to increase apoptosis in different cancer types. In this study, we evaluated the effects on cell viability, cell death, proliferation and migration of novel dimeric cGMP analogues in two melanoma cell lines (MNT1 and SkMel28). These new dimeric cGMP analogues, by activating PKG with limited effects on PKA, significantly reduced proliferation, migration and increased cell death. No decrease in cell viability was observed in non-tumor cells suggesting a tumor-specific effect. These effects observed in melanoma are possibly mediated by PKG2 activation based on the decreased toxic effects in tumor cell lines not expressing PKG2. Finally, PKG-associated phosphorylation of vasodilator-stimulated-phosphoprotein (VASP), linked to cell death, proliferation and migration was found increased and with a change of subcellular localization. Increased phosphorylation of RhoA induced by activation of PKG may also contribute to reduced migration ability of the SkMel28 melanoma cell line when treated with cGMP analogues. These findings suggest that the cGMP/PKG pathway can be envisaged as a therapeutic target of novel dimeric cGMP analogues for the treatment of melanoma.

Structural Basis of Analog Specificity in PKG I and II.

Cyclic GMP analogs, 8-Br, 8-pCPT, and PET-cGMP, have been widely used for characterizing cellular functions of cGMP-dependent protein kinase (PKG) I and II isotypes. However, interpreting results obtained using these analogs has been difficult due to their low isotype specificity. Additionally, each isotype has two binding sites with different cGMP affinities and analog selectivities, making understanding the molecular basis for isotype specificity of these compounds even more challenging. To determine isotype specificity of cGMP analogs and their structural basis, we generated the full-length regulatory domains of PKG I and II isotypes with each binding site disabled, determined their affinities for these analogs, and obtained cocrystal structures of both isotypes bound with cGMP analogs. Our affinity and activation measurements show that PET-cGMP is most selective for PKG I, whereas 8-pCPT-cGMP is most selective for PKG II. Our structures of cyclic nucleotide binding (CNB) domains reveal that the B site of PKG I is more open and forms a unique π/π interaction through Arg285 at ß4 with the PET moiety, whereas the A site of PKG II has a larger ß5/ß6 pocket that can better accommodate the bulky 8-pCPT moiety. Our structural and functional results explain the selectivity of these analogs for each PKG isotype and provide a starting point for the rational design of isotype selective activators.