FRCaMP,a Red Fluorescent Genetically Encoded Calcium Indicator Based on Calmodulin from Schizosaccharomyces Pombe Fungus

Red fluorescent genetically encoded calcium indicators (GECIs) have expanded the available pallet of colors used for the visualization of neuronal calcium activity in vivo. However, their calcium-binding domain is restricted by calmodulin from metazoans. In this study, we developed red GECI, called FRCaMP, using calmodulin (CaM) from Schizosaccharomyces pombe fungus as a calcium binding domain. Compared to the R-GECO1 indicator in vitro, the purified protein FRCaMP had similar spectral characteristics, brightness, and pH stability but a 1.3-fold lower ΔF/F calcium response and 2.6-fold tighter calcium affinity with K_d of 441 nM and 2.4–6.6-fold lower photostability. In the cytosol of cultured HeLa cells, FRCaMP visualized calcium transients with a ΔF/F dynamic range of 5.6, which was similar to that of R-GECO1. FRCaMP robustly visualized the spontaneous activity of neuronal cultures and had a similar ΔF/F dynamic range of 1.7 but 2.1-fold faster decay kinetics vs. NCaMP7. On electrically stimulated cultured neurons, FRCaMP demonstrated 1.8-fold faster decay kinetics and 1.7-fold lower ΔF/F values per one action potential of 0.23 compared to the NCaMP7 indicator. The fungus-originating CaM of the FRCaMP indicator version with a deleted M13-like peptide did not interact with the cytosolic environment of the HeLa cells in contrast to the metazoa-originating CaM of the similarly truncated version of the GCaMP6s indicator with a deleted M13-like peptide. Finally, we generated a split version of the FRCaMP indicator, which allowed the simultaneous detection of calcium transients and the heterodimerization of bJun/bFos interacting proteins in the nuclei of HeLa cells with a ΔF/F dynamic range of 9.4 and a contrast of 2.3–3.5, respectively.     

Kinetics of the formation of titanium oxynitride and nitride under the action of VUV, studied by means of ellipsometry

Preparation of thin nanolayers (approximately 10 nm) with the required stoichiometry and minimal grain size is important for the development of nanostructures. The kinetics of the formation of titanium nitride and titanium oxynitride layers through nitridation of the titanium matrix was investigated in the present work. It was shown that the UV radiation causes an increase in nitridation rate, depending on radiation energy, within the studied range 4 to 21 eV. The investigation was carried out with the help of ellipsometry and independent methods: atomic force microscopy, electron microscopy, and X-ray photoelectron spectroscopy. The mechanism of titanium interaction with nitrogen resulting in the formation of nitride and oxynitride layers at the Si/Ti and SiO2/Ti interfaces was investigated, the role of the UV radiation in solid-state reactions was revealed, and the mechanism of the process was proposed on the basis of the experimental data and results of quantum chemical simulation. The use of UV radiation allowed us to obtain the layers at low temperatures (0-5 degrees C) and to achieve a short reaction time due to an increase in reaction rate. The resulting layers have good masking properties (minimal density of pores and defects, high smoothness of the surface). This allows one to use these layers for chemical and electronic passivation and stabilization of the surface of semiconductor nano-objects (quantum dots, quantum wires, nanowhiskers etc.) for electronic and photon nanodevices.     

Planar Graph Coloring Avoiding Monochromatic Subgraphs: Trees and Paths Make It Difficult

We consider the problem of coloring a planar graph with the minimum number of colors so that each color class avoids one or more forbidden graphs as subgraphs. We perform a detailed study of the computational complexity of this problem. We present a complete picture for the case with a single forbidden connected (induced or noninduced) subgraph. The 2-coloring problem is NP-hard if the forbidden subgraph is a tree with at least two edges, and it is polynomially solvable in all other cases. The 3-coloring problem is NP-hard if the forbidden subgraph is a path with at least one edge, and it is polynomially solvable in all other cases. We also derive results for several forbidden sets of cycles. In particular, we prove that it is NP-complete to decide if a planar graph can be 2-colored so that no cycle of length at most 5 is monochromatic.     

Spin Polarization in Single Spin Experiments on Defects in Diamond

Generation of pure spin states is an important step towards coherent control of single spin systems. Especially for nitrogen-vacancy defects in diamond, where readout of single spins using optical detection is available, fast initialization of spin is significant in the context of quantum computing applications. In this system the spin polarization is caused by an intersystem crossing process via the meta-stable singlet state ¹ . The slowest relaxation rate is the intersystem to the triplet ground-state ³ on a timescale of 400 ns. The intersystem crossing process mainly populates the ms = 0 spin sublevels of the paramagnetic ground state. Although no direct nuclear spin polarization is seen for low magnetic fields, frequency selective microwave pulses can be used to transfer electron spin polarization to ¹³C nuclear spin states.     

Automating Mashups for Next-Generation Enterprise Portals

The increasing number of resources available through portals establish a need to tailor information to individual needs and situations. Mashups are tools for dynamically integrating independent applications. For portals, what is needed are means to automatically create personalized mashups that optimally fit a user's information needs in a given situation. At the core of our approach are different ontology-based models that describe the user, the domain, possible information needs in this domain, and personalization rules determining which information to present to which user in which situation.     

Supermartingales in prediction with expert advice

The paper applies the method of defensive forecasting, based on the use of game-theoretic supermartingales, to prediction with expert advice. In the traditional setting of a countable number of experts and a finite number of outcomes, the Defensive Forecasting Algorithm is very close to the well-known Aggregating Algorithm. Not only the performance guarantees but also the predictions are the same for these two methods of fundamentally different nature. The paper also discusses a new setting where the experts can give advice conditional on the learner’s future decision. Both the algorithms can be adapted to the new setting and give the same performance guarantees as in the traditional setting. Finally, an application of defensive forecasting to a setting with several loss functions is outlined.     

Influence of yeast strain on aromatic profile of Gewürztraminer wine

Three selected autochthonous and one commercial Saccharomyces cerevisiae strains used as starters for fermentation of the same Gewürztraminer must were compared in terms of their influence on primary and secondary aroma of wine. At the same testing conditions, each strain S. cerevisiae produced individual metabolites in different concentrations and combinations which significantly influenced resulting wine flavour. Higher concentrations of 2-phenylethanol and cis-rose oxide, associated with S. cerevisiae TC-NAX-A, considerably distinguished this strain from the others. Occurrence of these VOCs has important influence on sweet rose-like flavour typical for Gewürztraminer wine. This paper deals with the characterization of chemical substances that directly affect the primary varietal aroma of Gewürztraminer wine, while looking for a tool to produce an original and attractive product in form of suitable autochthonous strain S. cerevisiae.     

Kaiso Regulates DNA Methylation Homeostasis

Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in H19/Igf2 locus and Oct4 promoter in mouse embryonic fibroblasts. However, nothing is known about how Kaiso influences DNA methylation at the genome level. Here we show that deficiency of Kaiso led to whole-genome hypermethylation, using Kaiso deficient human renal cancer cell line obtained via CRISPR/CAS9 genome editing. However, Kaiso serves to protect genic regions, enhancers, and regions with a low level of histone modifications from demethylation. We detected hypomethylation of binding sites for Oct4 and Nanog in Kaiso deficient cells. Kaiso immunoprecipitated with de novo DNA methyltransferases DNMT3a/3b, but not with maintenance methyltransferase DNMT1. Thus, Kaiso may attract methyltransferases to surrounding regions and modulate genome methylation in renal cancer cells apart from being methyl DNA binding protein.