Reengineering of a flavin-binding fluorescent protein using ProteinMPNN.

Recent advances in machine learning techniques have led to development of a number of protein design and engineering approaches. One of them, ProteinMPNN, predicts an amino acid sequence that would fold and match user-defined backbone structure. Its performance was previously tested for proteins composed of standard amino acids, as well as for peptide- and protein-binding proteins. In this short report, we test whether ProteinMPNN can be used to reengineer a non-proteinaceous ligand-binding protein, flavin-based fluorescent protein CagFbFP. We fixed the native backbone conformation and the identity of 20 amino acids interacting with the chromophore (flavin mononucleotide, FMN) while letting ProteinMPNN predict the rest of the sequence. The software package suggested replacing 36-48 out of the remaining 86 amino acids so that the resulting sequences are 55%-66% identical to the original one. The three designs that we tested experimentally displayed different expression levels, yet all were able to bind FMN and displayed fluorescence, thermal stability, and other properties similar to those of CagFbFP. Our results demonstrate that ProteinMPNN can be used to generate diverging unnatural variants of fluorescent proteins, and, more generally, to reengineer proteins without losing their ligand-binding capabilities.

Refixation behavior in naturalistic viewing: Methods, mechanisms, and neural correlates.

When freely viewing a scene, the eyes often return to previously visited locations. By tracking eye movements and coregistering eye movements and EEG, such refixations are shown to have multiple roles: repairing insufficient encoding from precursor fixations, supporting ongoing viewing by resampling relevant locations prioritized by precursor fixations, and aiding the construction of memory representations. All these functions of refixation behavior are understood to be underpinned by three oculomotor and cognitive systems and their associated brain structures. First, immediate saccade planning prior to refixations involves attentional selection of candidate locations to revisit. This process is likely supported by the dorsal attentional network. Second, visual working memory, involved in maintaining task-related information, is likely supported by the visual cortex. Third, higher-order relevance of scene locations, which depends on general knowledge and understanding of scene meaning, is likely supported by the hippocampal memory system. Working together, these structures bring about viewing behavior that balances exploring previously unvisited areas of a scene with exploiting visited areas through refixations.

Two distinct mechanisms of flavoprotein spectral tuning revealed by low-temperature and time-dependent spectroscopy.

Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins, yet have very similar spectra when in the oxidized state. Recently, we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue (Q148V) or red (I52V A85Q) shifts of fluorescence emission maxima. Here, we use time-resolved and low-temperature spectroscopy to show that whereas the chromophore environment is static in Q148V, an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I52V A85Q variant. Consequently, in Q148V, excitation, emission, and phosphorescence spectra are shifted, whereas in I52V A85Q, excitation and low-temperature phosphorescence spectra are relatively unchanged, while emission spectrum is altered. We also determine the x-ray structures of the two variants to reveal the flavin environment and complement the spectroscopy data. Our findings illustrate two distinct color-tuning mechanisms of flavin-binding proteins and could be helpful for the engineering of new variants with improved optical properties.

Planning to revisit: Neural activity in refixation precursors.

Eye tracking studies suggest that refixations-fixations to locations previously visited-serve to recover information lost or missed during earlier exploration of a visual scene. These studies have largely ignored the role of precursor fixations-previous fixations on locations the eyes return to later. We consider the possibility that preparations to return later are already made during precursor fixations. This process would mark precursor fixations as a special category of fixations, that is, distinct in neural activity from other fixation categories such as refixations and fixations to locations visited only once. To capture the neural signals associated with fixation categories, we analyzed electroencephalograms (EEGs) and eye movements recorded simultaneously in a free-viewing contour search task. We developed a methodological pipeline involving regression-based deconvolution modeling, allowing our analyses to account for overlapping EEG responses owing to the saccade sequence and other oculomotor covariates. We found that precursor fixations were preceded by the largest saccades among the fixation categories. Independent of the effect of saccade length, EEG amplitude was enhanced in precursor fixations compared with the other fixation categories 200 to 400 ms after fixation onsets, most noticeably over the occipital areas. We concluded that precursor fixations play a pivotal role in visual perception, marking the continuous occurrence of transitions between exploratory and exploitative modes of eye movement in natural viewing behavior.

AlGaN HEMT Structures Grown on Miscut Si(111) Wafers.

A complex study was performed on a set of AlGaN/GaN high-electron-mobility transistor structures grown by metalorganic vapor phase epitaxy on miscut Si(111) wafers with a highly resistive epitaxial Si layer to investigate the influence of substrate miscut on their properties. The results showed that wafer misorientation had an influence on the strain evolution during the growth and surface morphology, and could have a strong impact on the mobility of 2D electron gas, with a weak optimum at 0.5° miscut angle. A numerical analysis revealed that the interface roughness was a main parameter responsible for the variation in electron mobility.

Impact of Local Composition on the Emission Spectra of InGaN Quantum-Dot LEDs.

A possible solution for the realization of high-efficiency visible light-emitting diodes (LEDs) exploits InGaN-quantum-dot-based active regions. However, the role of local composition fluctuations inside the quantum dots and their effect of the device characteristics have not yet been examined in sufficient detail. Here, we present numerical simulations of a quantum-dot structure restored from an experimental high-resolution transmission electron microscopy image. A single InGaN island with the size of ten nanometers and nonuniform indium content distribution is analyzed. A number of two- and three-dimensional models of the quantum dot are derived from the experimental image by a special numerical algorithm, which enables electromechanical, continuum k→·p→, and empirical tight-binding calculations, including emission spectra prediction. Effectiveness of continuous and atomistic approaches are compared, and the impact of InGaN composition fluctuations on the ground-state electron and hole wave functions and quantum dot emission spectrum is analyzed in detail. Finally, comparison of the predicted spectrum with the experimental one is performed to assess the applicability of various simulation approaches.

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging.

Flavin-binding fluorescent proteins are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy.

Electromechanically Coupled III-N Quantum Dots.

We exploit the three-dimensional (3D) character of the strain field created around InGaN islands formed within the multilayer structures spaced by a less than 1-nm-thick GaN layer for the creation of spatially correlated electronically coupled quantum dots (QDs). The laterally inhomogeneous vertical out-diffusion of In atoms during growth interruption is the basic mechanism for the formation of InGaN islands within as-deposited 2D layers. An anisotropic 3D strain field created in the first layer is sufficient to justify the vertical correlation of the islands formed in the upper layers spaced by a sufficiently thin GaN layer. When the thickness of a GaN spacer exceeds 1 nm, QDs from different layers under the same growth conditions emit independently and in the same wavelength range. When extremely thin (less than 1 nm), a GaN spacer is formed solely by applying short GI, and a double wavelength emission in the blue and green spectral ranges evidences the electromechanical coupling. With k→·p→ calculations including electromechanical fields, we model the optoelectronic properties of a structure with three InGaN lens-shaped QDs embedded in a GaN matrix, with three different configurations of In content. The profiles of the band structures are strongly dependent on the In content arrangement, and the quantum-confined Stark effect is significantly reduced in a structure with an increasing gradient of In content from the top to the bottom QD. This configuration exhibits carrier tunneling through the QDs, an increase of wave functions overlap, and evidence emerges of three distinct peaks in the spectral range.

Episodic memory formation in unrestricted viewing.

The brain systems of episodic memory and oculomotor control are tightly linked, suggesting a crucial role of eye movements in memory. But little is known about the neural mechanisms of memory formation across eye movements in unrestricted viewing behavior. Here, we leverage simultaneous eye tracking and EEG recording to examine episodic memory formation in free viewing. Participants memorized multi-element events while their EEG and eye movements were concurrently recorded. Each event comprised elements from three categories (face, object, place), with two exemplars from each category, in different locations on the screen. A subsequent associative memory test assessed participants' memory for the between-category associations that specified each event. We used a deconvolution approach to overcome the problem of overlapping EEG responses to sequential saccades in free viewing. Brain activity was time-locked to the fixation onsets, and we examined EEG power in the theta and alpha frequency bands, the putative oscillatory correlates of episodic encoding mechanisms. Three modulations of fixation-related EEG predicted high subsequent memory performance: (1) theta increase at fixations after between-category gaze transitions, (2) theta and alpha increase at fixations after within-element gaze transitions, (3) alpha decrease at fixations after between-exemplar gaze transitions. Thus, event encoding with unrestricted viewing behavior was characterized by three neural mechanisms, manifested in fixation-locked theta and alpha EEG activity that rapidly turned on and off during the unfolding eye movement sequences. These three distinct neural mechanisms may be the essential building blocks that subserve the buildup of coherent episodic memories during unrestricted viewing behavior.

Perceptual bias contextualized in visually ambiguous stimuli.

The visual appearance of an object is a function of stimulus properties as well as perceptual biases imposed by the observer. The context-specific trade-off between both can be measured accurately in a perceptual judgment task, involving grouping by proximity in ambiguous dot lattices. Such grouping depends lawfully on a stimulus parameter of the dot lattices known as their aspect ratio (AR), whose effect is modulated by a perceptual bias representing the preference for a cardinal orientation. In two experiments, we investigated how preceding context can lead to bias modulation, either in a top-down fashion via visual working memory (VWM) or bottom-up via sensory priming. In Experiment 1, we embedded the perceptual judgment task in a change detection paradigm and studied how the factors of VWM load (complexity of the memory array) and content (congruency in orientation to the ensuing dot lattice) affect the prominence of perceptual bias. A robust vertical orientation bias was observed, which was increased by VWM load and modulated by congruent VWM content. In Experiment 2, dot lattices were preceded by oriented primes. Here, primes regardless of orientation elicited a vertical orientation bias in dot lattices compared to a neutral baseline. Taken together, the two experiments demonstrate that top-down context (VWM load and content) effectively controls orientation bias modulation, while bottom-up context (i.e., priming) merely acts as an undifferentiated trigger to perceptual bias. These findings characterize the temporal context sensitivity of Gestalt perception, shed light on the processes responsible for different perceptual outcomes of ambiguous stimuli, and identify some of the mechanisms controlling perceptual bias.

The Influence of Nitrogen Flow on the Stoichiometric Composition, Structure, Mechanical, and Microtribological Properties of TiN Coatings.

Utilizing reactive DC magnetron sputtering method, TiN coatings were deposited on the silicon substrates at different nitrogen flows and powers. A study of the X-ray phase composition of the coatings was carried out. The stoichiometric composition of the coatings was determined using energy dispersive x-ray spectroscopy. The structure of the surface, cross-section, and thickness of the coatings were determined using scanning electron (SEM) and atomic force microscopy (AFM). A significant change in the surface structure of TiN coatings was established with changes in deposition power and nitrogen flow. SEM images of cross-sections of all coated samples showed that the formation of coatings occurs in the form of a columnar structure with a perpendicular orientation relative to the silicon substrate. The mechanical properties (elastic modulus E and microhardness H) of TiN coatings of the first group demonstrate a maximum at a nitrogen flow of 3 sccm and are 184 ± 11 GPa and 15.7 ± 1.3 GPa, respectively. In the second group, the values of E and H increase due to a decrease in the size of the structural elements of the coating (grains and crystallites). In the third group, E and H decrease. Microtribological tests were carried out in 4 stages: at a constant load, multi-cycle for 10 and 100 cycles, and with increasing load. The coefficient of friction (CoF) and specific volumetric wear ω depend on the roughness, topology, and mechanical properties of the resulting coatings. Fracture toughness was determined using nanoscratch and depends on the mechanical properties of TiN coatings. Within each group, coatings with the best mechanical and microtribological properties were described: in the first group-TiN coating at 3 sccm (with (29.6 ± 0.1) at.% N), in the second group-TiN coating at 2 sccm (with (40.8 ± 0.2) at.% N), and in the third group-TiN coating at 1 sccm (c (37.3 ± 0.2) at.% N).

Scattering Analysis of AlGaN/AlN/GaN Heterostructures with Fe-Doped GaN Buffer.

The results of the study of the influence of Fe segregation into the unintentionally doped GaN channel layer in AlGaN/AlN/GaN heterostructures with Fe-doped GaN buffer layer on the electrical properties of two-dimensional electron gas are presented. A set of several samples was grown by metal-organic vapor-phase epitaxy and characterized by the van der Pauw method. The dependence of concentration and mobility of the two-dimensional electron gas on the channel layer thickness was analyzed theoretically by self-consistent solving of 1D Poisson and Schrödinger equations and scattering rate calculations within the momentum relaxation time approximation. It was found that both concentration and mobility decreases were responsible for the increase in the sheet resistance in the structures with a thinner channel layer, with a drop in mobility being not only due to ionized impurity scattering, but also due to a combined effect of weakening of screening, lower carrier energy and change in form-factors on scattering by interface roughness, dislocations and polar optical phonons.

Effects of Temporal Expectations on the Perception of Motion Gestalts.

Gestalt psychology has traditionally ignored the role of attention in perception, leading to the view that autonomous processes create perceptual configurations that are then attended. More recent research, however, has shown that spatial attention influences a form of Gestalt perception: the coherence of random-dot kinematograms (RDKs). Using ERPs, we investigated whether temporal expectations exert analogous attentional effects on the perception of coherence level in RDKs. Participants were presented fixed-length sequences of RDKs and reported the coherence level of a target RDK. The target was indicated immediately after its appearance by a postcue. Target expectancy increased as the sequence progressed until target presentation; afterward, remaining RDKs were perceived without target expectancy. Expectancy influenced the amplitudes of ERP components P1 and N2. Crucially, expectancy interacted with coherence level at N2, but not at P1. Specifically, P1 amplitudes decreased linearly as a function of RDK coherence irrespective of expectancy, whereas N2 exhibited a quadratic dependence on coherence: larger amplitudes for RDKs with intermediate coherence levels, and only when they were expected. These results suggest that expectancy at early processing stages is an unspecific, general readiness for perception. At later stages, expectancy becomes stimulus specific and nonlinearly related to Gestalt coherence.

Non-Isothermal Decomposition as Efficient and Simple Synthesis Method of NiO/C Nanoparticles for Asymmetric Supercapacitors.

A series of NiO/C nanocomposites with NiO concentrations ranging from 10 to 90 wt% was synthesized using a simple and efficient two-step method based on non-isothermal decomposition of Nickel(II) bis(acetylacetonate). X-ray diffraction (XRD) measurements of these NiO/C nanocomposites demonstrate the presence of ß-NiO. NiO/C nanocomposites are composed of spherical particles distributed over the carbon support surface. The average diameter of nickel oxide spheres increases with the NiO content and are estimated as 36, 50 and 205 nm for nanocomposites with 10, 50 and 80 wt% NiO concentrations, respectively. In turn, each NiO sphere contains several nickel oxide nanoparticles, whose average sizes are 7-8 nm. According to the tests performed using a three-electrode cell, specific capacitance (SC) of NiO/C nanocomposites increases from 200 to 400 F/g as the NiO content achieves a maximum of 60 wt% concentration, after which the SC decreases. The study of the NiO/C composite showing the highest SC in three- and two-electrode cells reveals that its SC remains almost unchanged while increasing the current density, and the sample demonstrates excellent cycling stability properties. Finally, NiO/C (60% NiO) composites are shown to be promising materials for charging quartz clocks with a power rating of 1.5 V (30 min).

Comparative Investigations of AlCrN Coatings Formed by Cathodic Arc Evaporation under Different Nitrogen Pressure or Arc Current.

Tools and machine surfaces are subjected to various types of damage caused by many different factors. Due to this, the protecting coatings characterized by the best properties for a given treatment or environment are used. AlCrN coatings with different compositions, synthesized by different methods, are often of interest to scientists. The aim of the presented work was the deposition and investigation of two sets of coatings: (1) formed in nitrogen pressure from 0.8 Pa to 5 Pa and (2) formed at arc current from 50 A to 100 A. We study relationships between the above technological parameters and discuss their properties. Coatings formed at nitrogen pressure (pN2) up to 3 Pa crystallize both in hexagonal AlN structure and the cubic CrN structure. For pN2 > 3 Pa, they crystallize in the CrN cubic structure. Crystallite size increases with nitrogen pressure. The coatings formed at different arc currents have a cubic CrN structure and the crystallite size is independent of the current. The adhesion of the coatings is very good, independent of nitrogen pressure and arc current.

Characterization of Enamel and Dentine about a White Spot Lesion: Mechanical Properties, Mineral Density, Microstructure and Molecular Composition.

The study focuses on in vitro tracing of some fundamental changes that emerge in teeth at the initial stage of caries development using multiple approaches. The research was conducted on a mostly sound maxillary molar tooth but with a clearly visible natural proximal white spot lesion (WSL). Values of mineral density, reduced Young's modulus, indentation hardness and creep as well as the molecular composition and surface microstructure of the WSL and bordering dentine area were studied. The results obtained were compared to those of sound enamel and dentine on the same tooth. A decrease of mechanical properties and mineral density both for the WSL and bordering dentine was detected in comparison to the sound counterparts, as well as increase of creep for the enamel WSL. Differences in molecular composition and surface microstructure (including the indenter impressions) were found and described. WSL induces a serious change in the state of not only the visually affected enamel but also surrounding visually intact enamel and dentine in its vicinity. The results provide the basis for future studies of efficacy of minimal invasive treatments of caries.

Neural correlates of task-related refixation behavior.

Eye movement research has shown that attention shifts from the currently fixated location to the next before a saccade is executed. We investigated whether the cost of the attention shift depends on higher-order processing at the time of fixation, in particular on visual working memory load differences between fixations and refixations on task-relevant items. The attention shift is reflected in EEG activity in the saccade-related potential (SRP). In a free viewing task involving visual search and memorization of multiple targets amongst distractors, we compared the SRP in first fixations versus refixations on targets and distractors. The task-relevance of targets implies that more information will be loaded in memory (e.g. both identity and location) than for distractors (e.g. location only). First fixations will involve greater memory load than refixations, since first fixations involve loading of new items, while refixations involve rehearsal of previously visited items. The SRP in the interval preceding the saccade away from a target or distractor revealed that saccade preparation is affected by task-relevance and refixation behavior. For task-relevant items only, we found longer fixation duration and higher SRP amplitudes for first fixations than for refixations over the occipital region and the opposite effect over the frontal region. Our findings provide first neurophysiological evidence that working memory loading of task-relevant information at fixation affects saccade planning.

Factoring in the spatial effects of symbolic number representation.

Spatial constituents of adult symbolic number representation produce effects of size-value congruity, Spatial Numerical Association of Response Codes (SNARC), and numerical distance. According to behavioral experiments, these effects belong to distinct processing stages. Yet, these effects evoke overlapping responses in both early and late Event Related Potentials (ERPs). To probe whether these overlaps indicate sharing of resources, all relevant stimulus and response conditions were factorially combined in a numerical value comparison task. To secure ERP validity, same numbers were compared against variable reference values. This design resulted in previously unobserved interactions in behavior but inhibited late ERP effects. All effects arose early in the P1 component (around 100 ms) and most showed hemispheric specificity. Independency of congruity and SNARC effects was observed, whereas SNARC and numerical distance were closely intertwined. Differences in hemispheric specificity, rather than stage-wise separation, were key to independence.

High-density scalp EEG data acquired in an inattentional blindness paradigm with background Gestalt stimuli.

Behavioral and electroencephalography (EEG) data were analyzed from 30 participants performing a demanding, attention-capturing task in which they had to detect an occasional decrement of luminance of small discs. Crucially, numerous white segments in the background were presented either in random orientations or configuring a contour shape of a square or a diamond. The participants' awareness of these background configurations was tested with a questionnaire after the first and second sessions. Based on the questionnaire responses, participants were divided into "aware" and "unaware" groups. These data may be of interest to researchers who wish to understand attentional effects on Gestalt integration processes, as well as luminance discrimination.

Refixation patterns reveal memory-encoding strategies in free viewing.

We investigated visual working memory encoding across saccadic eye movements, focusing our analysis on refixation behavior. Over 10-s periods, participants performed a visual search for three, four, or five targets and remembered their orientations for a subsequent change-detection task. In 50% of the trials, one of the targets had its orientation changed. From the visual search period, we scored three types of refixations and applied measures for quantifying eye-fixation recurrence patterns. Repeated fixations on the same regions as well as repeated fixation patterns increased with memory load. Correct change detection was associated with more refixations on targets and less on distractors, with increased frequency of recurrence, and with longer intervals between refixations. The results are in accordance with the view that patterns of eye movement are an integral part of visual working memory representation.