Discovering and Targeting Dynamic Drugging Pockets of Oncogenic Proteins: The Role of Magnesium in Conformational Changes of the G12D Mutated Kirsten Rat Sarcoma-Guanosine Diphosphate Complex

KRAS-G12D mutations are the one of most frequent oncogenic drivers in human cancers. Unfortunately, no therapeutic agent directly targeting KRAS-G12D has been clinically approved yet, with such mutated species remaining undrugged. Notably, cofactor Mg2+ is closely related to the function of small GTPases, but no investigation has been conducted yet on Mg2+ when associated with KRAS. Herein, through microsecond scale molecular dynamics simulations, we found that Mg2+ plays a crucial role in the conformational changes of the KRAS-GDP complex. We located two brand new druggable dynamic pockets exclusive to KRAS-G12D. Using the structural characteristics of these two dynamic pockets, we designed in silico the inhibitor DBD15-21-22, which can specifically and tightly target the KRAS-G12D-GDP-Mg2+ ternary complex. Overall, we provide two brand new druggable pockets located on KRAS-G12D and suitable strategies for its inhibition.     

Myofibrillar Lattice Remodeling Is a Structural Cytoskeletal Predictor of Diaphragm Muscle Weakness in a Fibrotic mdx (mdx Cmah−/−) Model

Duchenne muscular dystrophy (DMD) is a degenerative genetic myopathy characterized by complete absence of dystrophin. Although the mdx mouse lacks dystrophin, its phenotype is milder compared to DMD patients. The incorporation of a null mutation in the Cmah gene led to a more DMD-like phenotype (i.e., more fibrosis). Although fibrosis is thought to be the major determinant of ‘structural weakness’, intracellular remodeling of myofibrillar geometry was shown to be a major cellular determinant thereof. To dissect the respective contribution to muscle weakness, we assessed biomechanics and extra- and intracellular architecture of whole muscle and single fibers from extensor digitorum longus (EDL) and diaphragm. Despite increased collagen contents in both muscles, passive stiffness in mdx Cmah−/− diaphragm was similar to wt mice (EDL muscles were twice as stiff). Isometric twitch and tetanic stresses were 50% reduced in mdx Cmah−/− diaphragm (15% in EDL). Myofibrillar architecture was severely compromised in mdx Cmah−/− single fibers of both muscle types, but more pronounced in diaphragm. Our results show that the mdx Cmah−/− genotype reproduces DMD-like fibrosis but is not associated with changes in passive visco-elastic muscle stiffness. Furthermore, detriments in active isometric force are compatible with the pronounced myofibrillar disarray of the dystrophic background.     

Computational Pipeline for Reference-Free Comparative Analysis of RNA 3D Structures Applied to SARS-CoV-2 UTR Models

RNA is a unique biomolecule that is involved in a variety of fundamental biological functions, all of which depend solely on its structure and dynamics. Since the experimental determination of crystal RNA structures is laborious, computational 3D structure prediction methods are experiencing an ongoing and thriving development. Such methods can lead to many models; thus, it is necessary to build comparisons and extract common structural motifs for further medical or biological studies. Here, we introduce a computational pipeline dedicated to reference-free high-throughput comparative analysis of 3D RNA structures. We show its application in the RNA-Puzzles challenge, in which five participating groups attempted to predict the three-dimensional structures of 5′- and 3′-untranslated regions (UTRs) of the SARS-CoV-2 genome. We report the results of this puzzle and discuss the structural motifs obtained from the analysis. All simulated models and tools incorporated into the pipeline are open to scientific and academic use.     

The Strong Enhancement of Electron-Impact Ionization Processes in Dense Plasma by Transient Spatial Localization

Recent experiments have observed much higher electron–ion collisional ionization cross sections and rates in dense plasmas than predicted by the current standard atomic collision theory, including the plasma screening effect. We suggest that the use of (distorted) plane waves for incident and scattered electrons is not adequate to describe the dissipation that occurs during the ionization event. Random collisions with free electrons and ions in plasma cause electron matter waves to lose their phase, which results in the partial decoherence of incident and scattered electrons. Such a plasma-induced transient spatial localization of the continuum electron states significantly modifies the wave functions of continuum electrons, resulting in a strong enhancement of the electron–ion collisional ionization of ions in plasma compared to isolated ions. Here, we develop a theoretical formulation to calculate the differential and integral cross sections by incorporating the effects of plasma screening and transient spatial localization. The approach is then used to investigate the electron-impact ionization of ions in solid-density magnesium plasma, yielding results that are consistent with experiments. In dense plasma, the correlation of continuum electron energies is modified, and the integral cross sections and rates increase considerably. For the ionization of Mg9+e+1s22s2S→1s21S+2e, the ionization cross sections increase several-fold, and the rates increase by one order of magnitude. Our findings provide new insight into collisional ionization and three-body recombination and may aid investigations of the transport properties and nonequilibrium evolution of dense plasma.     

Time-Dependent Image Restoration of Low-SNR Live-Cell Ca2 Fluorescence Microscopy Data

Live-cell Ca2+ fluorescence microscopy is a cornerstone of cellular signaling analysis and imaging. The demand for high spatial and temporal imaging resolution is, however, intrinsically linked to a low signal-to-noise ratio (SNR) of the acquired spatio-temporal image data, which impedes on the subsequent image analysis. Advanced deconvolution and image restoration algorithms can partly mitigate the corresponding problems but are usually defined only for static images. Frame-by-frame application to spatio-temporal image data neglects inter-frame contextual relationships and temporal consistency of the imaged biological processes. Here, we propose a variational approach to time-dependent image restoration built on entropy-based regularization specifically suited to process low- and lowest-SNR fluorescence microscopy data. The advantage of the presented approach is demonstrated by means of four datasets: synthetic data for in-depth evaluation of the algorithm behavior; two datasets acquired for analysis of initial Ca2+ microdomains in T-cells; finally, to illustrate the transferability of the methodical concept to different applications, one dataset depicting spontaneous Ca2+ signaling in jGCaMP7b-expressing astrocytes. To foster re-use and reproducibility, the source code is made publicly available.     

Non-Covalent Forces in Naphthazarin—Cooperativity or Competition in the Light of Theoretical Approaches

Non-covalent interactions responsible for molecular features and self-assembly in Naphthazarin C polymorph were investigated on the basis of diverse theoretical approaches: Density Functional Theory (DFT), Diffusion Quantum Monte Carlo (DQMC), Symmetry-Adapted Perturbation Theory (SAPT) and Car-Parrinello Molecular Dynamics (CPMD). The proton reaction paths in the intramolecular hydrogen bridges were studied. Two potential energy minima were found indicating that the proton transfer phenomena occur in the electronic ground state. Diffusion Quantum Monte Carlo (DQMC) and other levels of theory including Coupled Cluster (CC) employment enabled an accurate inspection of Potential Energy Surface (PES) and revealed the energy barrier for the proton transfer. The structure and reactivity evolution associated with the proton transfer were investigated using Harmonic Oscillator Model of Aromaticity - HOMA index, Fukui functions and Atoms In Molecules (AIM) theory. The energy partitioning in the studied dimers was carried out based on Symmetry-Adapted Perturbation Theory (SAPT) indicating that dispersive forces are dominant in the structure stabilization. The CPMD simulations were performed at 60 K and 300 K in vacuo and in the crystalline phase. The temperature influence on the bridged protons dynamics was studied and showed that the proton transfer phenomena were not observed at 60 K, but the frequent events were noticed at 300 K in both studied phases. The spectroscopic signatures derived from the CPMD were computed using Fourier transformation of autocorrelation function of atomic velocity for the whole molecule and bridged protons. The computed gas-phase IR spectra showed two regions with OH absorption that covers frequencies from 2500 cm−1 to 2800 cm−1 at 60 K and from 2350 cm−1 to 3250 cm−1 at 300 K for both bridged protons. In comparison, the solid state computed IR spectra revealed the environmental influence on the vibrational features. For each of them absorption regions were found between 2700–3100 cm−1 and 2400–2850 cm−1 at 60 K and 2300–3300 cm−1 and 2300–3200 cm−1 at 300 K respectively. Therefore, the CPMD study results indicated that there is a cooperation of intramolecular hydrogen bonds in Naphthazarin molecule.     

Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-Like Membrane

The execution step in apoptosis is the permeabilization of the outer mitochondrial membrane, controlled by Bcl-2 family proteins. The physical interactions between the different proteins in this family and their relative abundance literally determine the fate of the cells. These interactions, however, are difficult to quantify, as they occur in a lipid membrane and involve proteins with multiple conformations and stoichiometries which can exist both in soluble and membrane. Here we focus on the interaction between two core Bcl-2 family members, the executor pore-forming protein Bax and the truncated form of the activator protein Bid (tBid), which we imaged at the single particle level in a mitochondria-like planar supported lipid bilayer. We inferred the conformation of the proteins from their mobility, and detected their transient interactions using a novel single particle cross-correlation analysis. We show that both tBid and Bax have at least two different conformations at the membrane, and that their affinity for one another increases by one order of magnitude (with a 2D-KD decreasing from ≃1.6μm−2 to ≃0.1μm−2) when they pass from their loosely membrane-associated to their transmembrane form. We conclude by proposing an updated molecular model for the activation of Bax by tBid.     

Proteome-Wide Structural Computations Provide Insights into Empirical Amino Acid Substitution Matrices

The relative contribution of mutation and selection to the amino acid substitution rates observed in empirical matrices is unclear. Herein, we present a neutral continuous fitness-stability model, inspired by the Arrhenius law (qij=aije−ΔΔGij). The model postulates that the rate of amino acid substitution (i→j) is determined by the product of a pre-exponential factor, which is influenced by the genetic code structure, and an exponential term reflecting the relative fitness of the amino acid substitutions. To assess the validity of our model, we computed changes in stability of 14,094 proteins, for which 137,073,638 in silico mutants were analyzed. These site-specific data were summarized into a 20 square matrix, whose entries, ΔΔGij, were obtained after averaging through all the sites in all the proteins. We found a significant positive correlation between these energy values and the disease-causing potential of each substitution, suggesting that the exponential term accurately summarizes the fitness effect. A remarkable observation was that amino acids that were highly destabilizing when acting as the source, tended to have little effect when acting as the destination, and vice versa (source → destination). The Arrhenius model accurately reproduced the pattern of substitution rates collected in the empirical matrices, suggesting a relevant role for the genetic code structure and a tuning role for purifying selection exerted via protein stability.     

Shear Stress Enhances the Paracrine-Mediated Immunoregulatory Function of Human Periodontal Ligament Stem Cells via the ERK Signalling Pathway

Relevant immunomodulatory effects have been proposed following allogeneic cell-based therapy with human periodontal ligament stem cells (hPDLSCs). This study aimed to examine the influence of shear stress on the immunosuppressive capacity of hPDLSCs. Cells were subjected to shear stress at different magnitudes (0.5, 5 and 10 dyn/cm2). The expression of immunosuppressive markers was evaluated in shear stress-induced hPDLSCs using qRT-PCR, western blot, enzyme activity and enzyme-linked immunosorbent assays. The effects of a shear stress-derived condition medium (SS-CM) on T cell proliferation were examined using a resazurin assay. Treg differentiation was investigated using qRT-PCR and flow cytometry analysis. Our results revealed that shear stress increased mRNA expression of IDO and COX2 but not TGF-β1 and IFN-γ. IDO activity, kynurenine and active TGF-β1 increased in SS-CM when compared to the non-shear stress-derived conditioned medium (CTL-CM). The amount of kynurenine in SS-CM was reduced in the presence of cycloheximide and ERK inhibitor. Subsequently, T cell proliferation decreased in SS-CM compared to CTL-CM. Treg differentiation was promoted in SS-CM, indicated by FOXP3, IL-10 expression and CD4+CD25hiCD127lo/− subpopulation. In conclusion, shear stress promotes kynurenine production through ERK signalling in hPDLSC, leading to the inhibition of T cell proliferation and the promotion of Treg cell differentiation.     

Synthesis,Properties, and Selected Technical Applications of Magnesium Oxide Nanoparticles: A Review

In the last few decades, there has been a trend involving the use of nanoscale fillers in a variety of applications. Significant improvements have been achieved in the areas of their preparation and further applications (e.g., in industry, agriculture, and medicine). One of these promising materials is magnesium oxide (MgO), the unique properties of which make it a suitable candidate for use in a wide range of applications. Generally, MgO is a white, hygroscopic solid mineral, and its lattice consists of Mg2+ ions and O2− ions. Nanostructured MgO can be prepared through different chemical (bottom-up approach) or physical (top-down approach) routes. The required resultant properties (e.g., bandgap, crystallite size, and shape) can be achieved depending on the reaction conditions, basic starting materials, or their concentrations. In addition to its unique material properties, MgO is also potentially of interest due to its nontoxicity and environmental friendliness, which allow it to be widely used in medicine and biotechnological applications.     

Phase transformation behavior of 3 mol% yttria partially-stabilized ZrO2 (3Y–PSZ) precursor powder by an isothermal method

The phase transformation behavior of freeze-dried 3 mol% yttria–partially-stabilized zirconia (3Y–PSZ) precursor powder has been studied. When the freeze-dried 3Y–PSZ precursor powder was calcined at 773–1073 K for 2 h, the crystalline structure was composed of tetragonal and monoclinic ZrO₂ as primary and secondary phases, respectively. The freeze-dried 3Y–PSZ precursor powder after calcination at 773 K, the monoclinic ZrO₂ content abruptly increased from 8.00% to 31.51% and the tetragonal ZrO₂ content suddenly decreased from 92.00% to 68.49%, with the duration increasing from 0.5 to 1 min. The activation energy of the isothermal transformation from tetragonal to monoclinic was 7.02 kJ/mol. The kinetics equation for the phase transformation from tetragonal to monoclinic in the freeze-dried 3Y–PSZ precursor powder between 773 K and 1273 K for various durations is described as ln(1/1−α)=1/2.61[t^2.61(1.50×10⁻³)^2.61exp(−7.02×10³/RT)]$\ln (1/1-\alpha )=1/2.61[t^{2.61}{(1.50\times {10}^{-3})}^{2.61}\exp (-7.02\times {10}^3/RT)]$; whereas, the HRTEM image shows a typical monoclinic ZrO₂ domain because of the stress-induced tetragonal to monoclinic ZrO₂ martensitic transformation that has occurred.     

Formulation of Magneto-Responsive Hydrogels from Dually Cross-Linked Polysaccharides: Synthesis,Tuning and Evaluation of Rheological Properties

Smart hydrogels based on natural polymers present an opportunity to fabricate responsive scaffolds that provide an immediate and reversible reaction to a given stimulus. Modulation of mechanical characteristics is especially interesting in myocyte cultivation, and can be achieved by magnetically controlled stiffening. Here, hyaluronan hydrogels with carbonyl iron particles as a magnetic filler are prepared in a low-toxicity process. Desired mechanical behaviour is achieved using a combination of two cross-linking routes—dynamic Schiff base linkages and ionic cross-linking. We found that gelation time is greatly affected by polymer chain conformation. This factor can surpass the influence of the number of reactive sites, shortening gelation from 5 h to 20 min. Ionic cross-linking efficiency increased with the number of carboxyl groups and led to the storage modulus reaching 103 Pa compared to 101 Pa–102 Pa for gels cross-linked with only Schiff bases. Furthermore, the ability of magnetic particles to induce significant stiffening of the hydrogel through the magnetorheological effect is confirmed, as a 103-times higher storage modulus is achieved in an external magnetic field of 842 kA·m−1. Finally, cytotoxicity testing confirms the ability to produce hydrogels that provide over 75% relative cell viability. Therefore, dual cross-linked hyaluronan-based magneto-responsive hydrogels present a potential material for on-demand mechanically tunable scaffolds usable in myocyte cultivation.     

Ab Initio Study of Carrier Mobility,Thermodynamic and Thermoelectric Properties of Kesterite Cu2ZnGeS4

The kesterite Cu2ZnGeS4 (CZGS) has recently gained significant interest in the scientific community. In this work, we investigated the thermodynamic and thermoelectric properties of CZGS by employing the first-principals calculation in association with the quasi-harmonic approximation, Boltzmann transport theory, deformation potential theory, and slack model. We obtained a bandgap of 2.05 eV and high carrier mobility. We found that CZGS exhibits adequate thermoelectric properties as a promising material for thermoelectric applications. The calculated Seebeck coefficient at room temperature is 149 µV·K−1. We also determined the thermal and electrical conductivity, the power factor, and the figure of merit. In addition, the thermodynamic properties such as Debye temperature, entropy, and constant volume heat capacity are estimated. According to our results, it is concluded that the Slack model fails to provide correct values for lattice thermal conductivity in this material.