Rational Design of Far-Red Archaerhodopsin-3-Based Fluorescent Genetically Encoded Voltage Indicators: from Elucidation of the Fluorescence Mechanism in Archers to Novel Red-Shifted Variants.

Genetically encoded voltage indicators (GEVIs) have found wide applications as molecular tools for visualization of changes in cell membrane potential. Among others, several classes of archaerhodopsin-3-based GEVIs have been developed and have proved themselves promising in various molecular imaging studies. To expand the application range for this type of GEVIs, new variants with absorption band maxima shifted toward the first biological window and enhanced fluorescence signal are required. Here, we integrate computational and experimental strategies to reveal structural factors that distinguish far-red bright archaerhodopsin-3-based GEVIs, Archers, obtained by directed evolution in a previous study (McIsaac et al., PNAS, 2014) and the wild-type archaerhodopsin-3 with an extremely dim fluorescence signal, aiming to use the obtained information in subsequent rational design. We found that the fluorescence can be enhanced by stabilization of a certain conformation of the protein, which, in turn, can be achieved by tuning the pK a value of two titratable residues. These findings were supported further by introducing mutations into wild-type archeorhodopsin-3 and detecting the enhancement of the fluorescence signal. Finally, we came up with a rational design and proposed previously unknown Archers variants with red-shifted absorption bands (λmax up to 640 nm) and potential-dependent bright fluorescence (quantum yield up to 0.97%).

Fluorescence of the Retinal Chromophore in Microbial and Animal Rhodopsins.

Fluorescence of the vast majority of natural opsin-based photoactive proteins is extremely low, in accordance with their functions that depend on efficient transduction of absorbed light energy. However, several recently proposed classes of engineered rhodopsins with enhanced fluorescence, along with the discovery of a new natural highly fluorescent rhodopsin, NeoR, opened a way to exploit these transmembrane proteins as fluorescent sensors and draw more attention to studies on this untypical rhodopsin property. Here, we review the available data on the fluorescence of the retinal chromophore in microbial and animal rhodopsins and their photocycle intermediates, as well as different isomers of the protonated retinal Schiff base in various solvents and the gas phase.

Simultaneous Catechol and Hydroquinone Detection with Laser Fabricated MOF-Derived Cu-CuO@C Composite Electrochemical Sensor.

The conversion of metal-organic frameworks (MOFs) into advanced functional materials offers a promising route for producing unique nanomaterials. MOF-derived systems have the potential to overcome the drawbacks of MOFs, such as low electrical conductivity and poor structural stability, which have hindered their real-world applications in certain cases. In this study, laser scribing was used for pyrolysis of a Cu-based MOF ([Cu4{1,4-C6H4(COO)2}3(4,4'-bipy)2]n) to synthesize a Cu-CuO@C composite on the surface of a screen-printed electrode (SPE). Scanning electron microscopy, X-ray diffractometry, and Energy-dispersive X-ray spectroscopy were used for the investigation of the morphology and composition of the fabricated electrodes. The electrochemical properties of Cu-CuO@C/SPE were studied by cyclic voltammetry and differential pulse voltammetry. The proposed flexible electrochemical Cu-CuO@C/SPE sensor for the simultaneous detection of hydroquinone and catechol exhibited good sensitivity, broad linear range (1-500 µM), and low limits of detection (0.39 µM for HQ and 0.056 µM for CT).

Effect of Gd3+, La3+, Lu3+ Co-Doping on the Morphology and Luminescent Properties of NaYF4:Sm3+ Phosphors.

The series of luminescent NaYF4:Sm3+ nano- and microcrystalline materials co-doped by La3+, Gd3+, and Lu3+ ions were synthesized by hydrothermal method using rare earth chlorides as the precursors and citric acid as a stabilizing agent. The phase composition of synthesized compounds was studied by PXRD. All synthesized materials except ones with high La3+ content (where LaF3 is formed) have a ß-NaYF4 crystalline phase. SEM images demonstrate that all particles have shape of hexagonal prisms. The type and content of doping REE significantly effect on the particle size. Upon 400 nm excitation, phosphors exhibit distinct emission peaks in visible part of the spectrum attributed to 4G5/2→6HJ transitions (J = 5/2-11/2) of Sm3+ ion. Increasing the samarium (III) content results in concentration quenching by dipole-dipole interactions, the optimum Sm3+concentration is found to be of 2%. Co-doping by non-luminescent La3+, Gd3+ and Lu3+ ions leads to an increase in emission intensity. This effect was explained from the Sm3+ local symmetry point of view.

Fluorescence Imaging of Cell Membrane Potential: From Relative Changes to Absolute Values.

Membrane potential is a fundamental property of biological cells. Changes in membrane potential characterize a vast number of vital biological processes, such as the activity of neurons and cardiomyocytes, tumorogenesis, cell-cycle progression, etc. A common strategy to record membrane potential changes that occur in the process of interest is to utilize organic dyes or genetically-encoded voltage indicators with voltage-dependent fluorescence. Sensors are introduced into target cells, and alterations of fluorescence intensity are recorded with optical methods. Techniques that allow recording relative changes of membrane potential and do not take into account fluorescence alterations due to factors other than membrane voltage are already widely used in modern biological and biomedical studies. Such techniques have been reviewed previously in many works. However, in order to investigate a number of processes, especially long-term processes, the measured signal must be corrected to exclude the contribution from voltage-independent factors or even absolute values of cell membrane potential have to be evaluated. Techniques that enable such measurements are the subject of this review.

Lipid Alteration Signature in the Blood Plasma of Individuals With Schizophrenia, Depression, and Bipolar Disorder.

Importance: No clinically applicable diagnostic test exists for severe mental disorders. Lipids harbor potential as disease markers. Objective: To define a reproducible profile of lipid alterations in the blood plasma of patients with schizophrenia (SCZ) independent of demographic and environmental variables and to investigate its specificity in association with other psychiatric disorders, ie, major depressive disorder (MDD) and bipolar disorder (BPD). Design, Setting, and Participants: This was a multicohort case-control diagnostic analysis involving plasma samples from psychiatric patients and control individuals collected between July 17, 2009, and May 18, 2018. Study participants were recruited as consecutive and volunteer samples at multiple inpatient and outpatient mental health hospitals in Western Europe (Germany and Austria [DE-AT]), China (CN), and Russia (RU). Individuals with DSM-IV or International Statistical Classification of Diseases and Related Health Problems, Tenth Revision diagnoses of SCZ, MDD, BPD, or a first psychotic episode, as well as age- and sex-matched healthy controls without a mental health-related diagnosis were included in the study. Samples and data were analyzed from January 2018 to September 2020. Main Outcomes and Measures: Plasma lipidome composition was assessed using liquid chromatography coupled with untargeted mass spectrometry. Results: Blood lipid levels were assessed in 980 individuals (mean [SD] age, 36 [13] years; 510 male individuals [52%]) diagnosed with SCZ, BPD, MDD, or those with a first psychotic episode and in 572 controls (mean [SD] age, 34 [13] years; 323 male individuals [56%]). A total of 77 lipids were found to be significantly altered between those with SCZ (n = 436) and controls (n = 478) in all 3 sample cohorts. Alterations were consistent between cohorts (CN and RU: [Pearson correlation] r = 0.75; DE-AT and CN: r = 0.78; DE-AT and RU: r = 0.82; P < 10-38). A lipid-based predictive model separated patients with SCZ from controls with high diagnostic ability (area under the receiver operating characteristic curve = 0.86-0.95). Lipidome alterations in BPD and MDD, assessed in 184 and 256 individuals, respectively, were found to be similar to those of SCZ (BPD: r = 0.89; MDD: r = 0.92; P < 10-79). Assessment of detected alterations in individuals with a first psychotic episode, as well as patients with SCZ not receiving medication, demonstrated only limited association with medication restricted to particular lipids. Conclusions and Relevance: In this study, SCZ was accompanied by a reproducible profile of plasma lipidome alterations, not associated with symptom severity, medication, and demographic and environmental variables, and largely shared with BPD and MDD. This lipid alteration signature may represent a trait marker of severe psychiatric disorders, indicating its potential to be transformed into a clinically applicable testing procedure.

Measuring internal inequality in capsule networks for supervised anomaly detection.

In this paper we explore the use of income inequality metrics such as Gini or Palma coefficients as a tool to identify anomalies via capsule networks. We demonstrate how the interplay between primary and class capsules gives rise to differences in behavior regarding anomalous and normal input which can be exploited to detect anomalies. Our setup for anomaly detection requires supervision in a form of known outliers. We derive several criteria for capsule networks and apply them to a number of Computer Vision benchmark datasets (MNIST, Fashion-MNIST, Kuzushiji-MNIST and CIFAR10), as well as to the dataset of skin lesion images (HAM10000) and the dataset of CRISPR-Cas9 off-target pairs. The proposed methods outperform the competitors in the majority of considered cases.

Copper-Ruthenium Composite as Perspective Material for Bioelectrodes: Laser-Assisted Synthesis, Biocompatibility Study, and an Impedance-Based Cellular Biosensor as Proof of Concept.

Copper is an inexpensive material that has found wide application in electronics due to its remarkable electric properties. However, the high toxicity of both copper and copper oxide imposes restrictions on the application of this metal as a material for bioelectronics. One way to increase the biocompatibility of pure copper while keeping its remarkable properties is to use copper-based composites. In the present study, we explored a new copper-ruthenium composite as a potential biocompatible material for bioelectrodes. Sample electrodes were obtained by subsequent laser deposition of copper and ruthenium on glass plates from a solution containing salts of these metals. The fabricated Cu-Ru electrodes exhibit high effective area and their impedance properties can be described by simple R-CPE equivalent circuits that make them perspective for sensing applications. Finally, we designed a simple impedance cell-based biosensor using this material that allows us to distinguish between dead and alive HeLa cells.

Laser-assisted surface activation for fabrication of flexible non-enzymatic Cu-based sensors.

A rapid and effective technique has been develped for the fabrication of sensor-active copper-based materials on the surface of such flexible polymers as terephthalate, polyethylene naphthalate, and polyimide using the method of laser surface modification. For this purpose, we optimized the polymer surface activation parameters using laser sources with a picosecond pulse duration for subsequent selective metallization within the activated region. Furthermore, the fabricated copper structures were modified with gold nanostructures and by electrochemical passivation to produce copper-gold and oxide-containing copper species, respectively. As a result, in comparison with pure copper electrodes, these composite materials exhibit much better electrocatalytic performance concerning the non-enzymatic identification of biologically important disease markers such as glucose, hydrogen peroxide, and dopamine.

4,5-Bis(arylethynyl)-1,2,3-triazoles-A New Class of Fluorescent Labels: Synthesis and Applications.

Cu-catalyzed 1,3-dipolar cycloaddition of ethyl 2-azidoacetate to iodobuta-1,3-diynes and subsequent Sonogashira cross-coupling were used to synthesize a large series of new triazole-based push-pull chromophores: 4,5-bis(arylethynyl)-1H-1,2,3-triazoles. The study of their optical properties revealed that all molecules have fluorescence properties, the Stokes shift values of which exceed 150 nm. The fluorescent properties of triazoles are easily adjustable depending on the nature of the substituents attached to aryl rings of the arylethynyl moieties at the C4 and C5 atoms of the triazole core. The possibility of 4,5-bis(arylethynyl)-1,2,3-triazoles' application for labeling was demonstrated using proteins and the HEK293 cell line. The results of an MTT test on two distinct cell lines, HEK293 and HeLa, revealed the low cytotoxicity of 4,5-bis(arylethynyl)triazoles, which makes them promising fluorescent tags for labeling and tracking biomolecules.

Uncertainty-aware and interpretable evaluation of Cas9-gRNA and Cas12a-gRNA specificity for fully matched and partially mismatched targets with Deep Kernel Learning.

The choice of guide RNA (gRNA) for CRISPR-based gene targeting is an essential step in gene editing applications, but the prediction of gRNA specificity remains challenging. Lack of transparency and focus on point estimates of efficiency disregarding the information on possible error sources in the model limit the power of existing Deep Learning-based methods. To overcome these problems, we present a new approach, a hybrid of Capsule Networks and Gaussian Processes. Our method predicts the cleavage efficiency of a gRNA with a corresponding confidence interval, which allows the user to incorporate information regarding possible model errors into the experimental design. We provide the first utilization of uncertainty estimation in computational gRNA design, which is a critical step toward accurate decision-making for future CRISPR applications. The proposed solution demonstrates acceptable confidence intervals for most test sets and shows regression quality similar to existing models. We introduce a set of criteria for gRNA selection based on off-target cleavage efficiency and its variance and present a collection of pre-computed gRNAs for human chromosome 22. Using Neural Network Interpretation methods, we show that our model rediscovers an established biological factor underlying cleavage efficiency, the importance of the seed region in gRNA.

Azobenzene/Tetraethyl Ammonium Photochromic Potassium Channel Blockers: Scope and Limitations for Design of Para-Substituted Derivatives with Specific Absorption Band Maxima and Thermal Isomerization Rate.

Azobenzene/tetraethyl ammonium photochromic ligands (ATPLs) are photoactive compounds with a large variety of photopharmacological applications such as nociception control or vision restoration. Absorption band maximum and lifetime of the less stable isomer are important characteristics that determine the applicability of ATPLs. Substituents allow to adjust these characteristics in a range limited by the azobenzene/tetraethyl ammonium scaffold. The aim of the current study is to find the scope and limitations for the design of ATPLs with specific spectral and kinetic properties by introducing para substituents with different electronic effects. To perform this task we synthesized ATPLs with various electron acceptor and electron donor functional groups and studied their spectral and kinetic properties using flash photolysis and conventional spectroscopy techniques as well as quantum chemical modeling. As a result, we obtained diagrams that describe correlations between spectral and kinetic properties of ATPLs (absorption maxima of E and Z isomers of ATPLs, the thermal lifetime of their Z form) and both the electronic effect of substituents described by Hammett constants and structural parameters obtained from quantum chemical calculations. The provided results can be used for the design of ATPLs with properties that are optimal for photopharmacological applications.

Why animals swirl and how they group.

We report a possible solution for the long-standing problem of the biological function of swirling motion, when a group of animals orbits a common center of the group. We exploit the hypothesis that learning processes in the nervous system of animals may be modelled by reinforcement learning (RL) and apply it to explain the phenomenon. In contrast to hardly justified models of physical interactions between animals, we propose a small set of rules to be learned by the agents, which results in swirling. The rules are extremely simple and thus applicable to animals with very limited level of information processing. We demonstrate that swirling may be understood in terms of the escort behavior, when an individual animal tries to reside within a certain distance from the swarm center. Moreover, we reveal the biological function of swirling motion: a trained for swirling swarm is by orders of magnitude more resistant to external perturbations, than an untrained one. Using our approach we analyze another class of a coordinated motion of animals-a group locomotion in viscous fluid. On a model example we demonstrate that RL provides an optimal disposition of coherently moving animals with a minimal dissipation of energy.

Ultrasound-Assisted Synthesis of Luminescent Micro- and Nanocrystalline Eu-Based MOFs as Luminescent Probes for Heavy Metal Ions.

The luminescent coarse-, micro- and nanocrystalline europium(III) terephthalate tetrahydrate (Eu2bdc3·4H2O) metal-organic frameworks were synthesized by the ultrasound-assisted wet-chemical method. Electron micrographs show that the europium(III) terephthalate microparticles are 7 µm long leaf-like plates. According to the dynamic light scattering technique, the average size of the Eu2bdc3·4H2O nanoparticles is equal to about 8 ± 2 nm. Thereby, the reported Eu2bdc3·4H2O nanoparticles are the smallest nanosized rare-earth-based MOF crystals, to the best of our knowledge. The synthesized materials demonstrate red emission due to the 5D0-7FJ transitions of Eu3+ upon 250 nm excitation into 1ππ* state of the terephthalate ion. Size reduction results in broadened emission bands, an increase in the non-radiative rate constants and a decrease in both the quantum efficiency of the 5D0 level and Eu3+ and the luminescence quantum yields. Cu2+, Cr3+, and Fe3+ ions efficiently and selectively quench the luminescence of nanocrystalline europium(III) terephthalate, which makes it a prospective material for luminescent probes to monitor these ions in waste and drinking water.

Laser-Assisted Surface Modification of Ni Microstructures with Au and Pt toward Cell Biocompatibility and High Enzyme-Free Glucose Sensing.

We investigated the influence of morphology of Ni microstructures modified with Au and Pt on their cell biocompatibility and electrocatalytic activity toward non-enzymatic glucose detection. Synthesis and modification were carried out using a simple and inexpensive approach based on the method of laser-induced deposition of metal microstructures from a solution on the surface of various dielectrics. Morphological analysis of the fabricated materials demonstrated that the surface of the Ni electrode has a hierarchical structure with large-scale 10 µm pores and small-scale 10 nm irregularities. In turn, the Ni-Pt surface has large-scale cavities, small-scale pores (1-1.5 µm), and a few tens of nanometer particles opposite to Ni-Au that reveals no obvious hierarchical structure. These observations were supported by impedance spectroscopy confirming the hierarchy of the surface topography of Ni and Ni-Pt structures. We tested the biocompatibility of the fabricated Ni-based electrodes with the HeLa cells. It was shown that the Ni-Au electrode has a much better cell adhesion than Ni-Pt with a more complex morphology. On the contrary, porous Ni and Ni-Pt electrodes with a more developed surface area than that of Ni-Au have better catalytic performance toward enzymeless glucose sensing, revealing greater sensitivity, selectivity, and stability. In this regard, modification of Ni with Pt led to the most prominent results providing rather good glucose detection limits (0.14 and 0.19 µA) and linear ranges (10-300 and 300-1500 µA) as well as the highest sensitivities of 18,570 and 2929 µA mM-1 cm-2. We also proposed some ideas to clarify the observed behavior and explain the influence of morphology of the fabricated electrodes on their electrocatalytic activity and biocompatibility.

Ultrafast Excited-State Dynamics of CuBr3- Complex Studied with Sub-20 fs Resolution.

Ultrafast excited-state dynamics of CuBr3- complex was studied in acetonitrile and dichloromethane solutions using femtosecond transient absorption spectroscopy with 18 fs temporal resolution and quantum-chemical DFT calculations. Upon 640 nm excitation, the CuBr3- complex is promoted to the ligand-to-metal charge transfer (LMCT) state, which then shortly undergoes internal conversion into the vibrationally hot ligand field (LF) excited state with time constants of 30 and 40 fs in acetonitrile and dichloromethane, respectively. The LF state nonradiatively relaxes into the ground state in 2.6 and 7.3 ps in acetonitrile and dichloromethane, respectively. Internal conversion of the LF state is accompanied by vibrational relaxation that occurs on the same time scale. Based on the analysis of coherent oscillations and quantum-chemical calculations, the predominant forms of the CuBr3- complex in acetonitrile and dichloromethane solutions were revealed. In acetonitrile, the CuBr3- complex exists as [CuBr3(CH3CN)2]-, whereas three forms of this complex, [CuBr3CH2Cl2]-, [CuBr3(CH2Cl2)2]-, and [CuBr3(CH2Cl2)3]-, are present in equilibrium in dichloromethane.

Copper and Nickel Microsensors Produced by Selective Laser Reductive Sintering for Non-Enzymatic Glucose Detection.

In this work, the method of selective laser reductive sintering was used to fabricate the sensor-active copper and nickel microstructures on the surface of glass-ceramics suitable for non-enzymatic detection of glucose. The calculated sensitivities for these microsensors are 1110 and 2080 µA mM-1·cm-2 for copper and nickel, respectively. Linear regime of enzymeless glucose sensing is provided between 0.003 and 3 mM for copper and between 0.01 and 3 mM for nickel. Limits of glucose detection for these manufactured micropatterns are equal to 0.91 and 2.1 µM for copper and nickel, respectively. In addition, the fabricated materials demonstrate rather good selectivity, long-term stability and reproducibility.

Simple Models to Study Spectral Properties of Microbial and Animal Rhodopsins: Evaluation of the Electrostatic Effect of Charged and Polar Residues on the First Absorption Band Maxima.

A typical feature of proteins from the rhodopsin family is the sensitivity of their absorption band maximum to protein amino acid composition. For this reason, studies of these proteins often require methodologies that determine spectral shift caused by amino acid substitutions. Generally, quantum mechanics/molecular mechanics models allow for the calculation of a substitution-induced spectral shift with high accuracy, but their application is not always easy and requires special knowledge. In the present study, we propose simple models that allow us to estimate the direct effect of a charged or polar residue substitution without extensive calculations using only rhodopsin three-dimensional structure and plots or tables that are provided in this article. The models are based on absorption maximum values calculated at the SORCI+Q level of theory for cis- and trans-forms of retinal protonated Schiff base in an external electrostatic field of charges and dipoles. Each value corresponds to a certain position of a charged or polar residue relative to the retinal chromophore. The proposed approach was evaluated against an example set consisting of twelve bovine rhodopsin and sodium pumping rhodopsin mutants. The limits of the applicability of the models are also discussed. The results of our study can be useful for the interpretation of experimental data and for the rational design of rhodopsins with required spectral properties.

High rate fabrication of copper and copper-gold electrodes by laser-induced selective electroless plating for enzyme-free glucose sensing.

In the current study, the method of Selective Surface Activation Induced by Laser (SSAIL) was used for the fabrication of metallic and bimetallic structures based on copper and gold on the surface of glass and glass-ceramics. It was shown that the fabricated electrodes are suitable for non-enzymatic detection of biologically essential analytes such as glucose. The implemented approach allows performing high-rate metallization of various dielectrics. Voltammetric methods were applied to evaluate the electrocatalytic activity of the obtained structures, which were used as working electrodes. The most promising results were revealed by copper-gold electrode structures manufactured on glass-ceramics. For these structures, sensitivity towards glucose sensing was 3060 µA mM-1 cm-2. The linear range of glucose detection varied between 0.3 and 1000 µM. Besides, the manufactured electrodes exhibited high selectivity and long-term stability.

In Situ Laser-Induced Fabrication of a Ruthenium-Based Microelectrode for Non-Enzymatic Dopamine Sensing.

In this paper, we propose a fast and simple approach for the fabrication of the electrocatalytically active ruthenium-containing microstructures using a laser-induced metal deposition technique. The results of scanning electron microscopy and electrical impedance spectroscopy (EIS) demonstrate that the fabricated ruthenium-based microelectrode had a highly developed surface composed of 10 µm pores and 10 nm zigzag cracks. The fabricated material exhibited excellent electrochemical properties toward non-enzymatic dopamine sensing, including high sensitivity (858.5 and 509.1 µA mM-1 cm-2), a low detection limit (0.13 and 0.15 µM), as well as good selectivity and stability.