Origin of Enantioselectivity in Engineered Cytochrome c-Catalyzed Carbon-Radical FePP Hydrolysis Revealed Using QM/MM (ABEEM Polarizable Force Field) and MD Simulations.

The origin of highly efficient asymmetric aminohydroxylation of styrene catalyzed by engineered cytochrome c is investigated by the developed Atom-Bond Electronegativity Equalization Method polarizable force field (ABEEM PFF), which is a combined outcome of electronic and steric effects. Model molecules were used to establish the charge parameters of the ABEEM PFF, for which the bond-stretching and angle-bending parameters were obtained by using a combination of modified Seminario and scan methods. The interactions between carbon-radical Fe-porphyrin (FePP) and waters are simulated by molecular dynamics, which shows a clear preference for the pre-R over the pre-S. This preference is attributed to the hydrogen-bond between the mutated 100S and 101P residues as well as van der Waals interactions, enforcing a specific conformation of the carbon-radical FePP complex within the binding pocket. Meanwhile, the hydrogen-bond between water and the nitrogen atom in the active intermediate dictates the stereochemical outcome. Quantum mechanics/molecular mechanics (QM/MM (ABEEM PFF)) and free-energy perturbation calculations elucidate that the 3RTS is characterized by sandwich-like structure among adjacent amino acid residues, which exhibits greater stability than crowed arrangement in 3STS and enables the R enantiomer to form more favorably. Thus, this study provides mechanistic insight into the catalytic reaction of hemoproteins.

Atomic charges in molecules defined by molecular real space partition into atomic subspaces.

Atomic charge (AC), which is the charge distribution of a molecule, is an important property that is closely associated with structures, reactivities, and intra- and inter-molecular interactions among molecules. Several theoretical models or methods can be used to obtain the magnitudes of AC with different characteristics. These models can be classified into fuzzy-atoms models and models partitioning a molecule into individual atoms with sharp boundaries. The first category includes Mulliken, natural population analysis (NPA), Hirshfeld, Merz-Kollman-Singh (MK), CHELPG, the electronegativity equalization method (EEM), the atom-bond electronegativity equalization method (ABEEM), and atomic polar tensor (APT). The second category is derived from quantum chemical topology (QCT) and includes the quantum theory of atoms in molecules (QTAIM) and QCT analysis based on the potential acting on one electron in a molecule (PAEMQCT). Herein, after giving a bird's-eye view of the population methods of the first category, we specifically describe some features of the second category. We only present the basic framework of QCT for obtaining ACs from QTAIM and PAEMQCT and show their important characteristics. QCT establishes the basis of the following chemical concept: a molecule is spatially partitioned into individual atoms with sharp boundaries. The ACs from QTAIM are close to the atomic valence in chemistry, and ACs from PAEMQCT may be practically suitable for modeling intra- and inter-molecular interactions.

Clinical Study on the Efficacy of Bevacizumab in Combination with Pembrolizumab on Cellular Immune Function in the Treatment of Driver Gene-Negative Stage IV Lung Adenocarcinoma.

Purpose: To explore the efficacy of bevacizumab in combination with PD-1 immune drug pembrolizumab on cellular immune function in the treatment of driver gene-negative stage IV lung adenocarcinoma and its short-term survival effect. Methods: From February 2020 to December 2021, 85 patients with driver gene-negative stage IV lung adenocarcinoma were admitted to our hospital and treated with first-line therapy, and their clinical records were reviewed retrospectively. According to the treatments, the patients were separated into two groups the combination group (n = 45) and the control group (n = 40). The treatment regimen of the control group was an AP chemotherapy regimen (pemetrexed combined with cisplatin) + PD-1 immune drug pembrolizumab. The treatment regimen of the combination group was AP chemotherapy regimen + PD-1 immune drug pembrolizumab combined with bevacizumab. We evaluated the pre- and post-treatment cellular immunological function of the two patient groups and discussed the difference between them. Results: There was a substantial difference in the overall effective rate and the disease control rate between the two groups, with the former being 27.50% compared to 48.89% and the latter being 72.50% compared to 93.33% among these 85 patients studied. The KPS for the combination group improved and stayed at 91.11% after treatment, which is considerably better than the KPS for the control group, which was 42.50% (χ 2 = 23.09, P < 0.05). There was no significant difference (P > 0.05) in the numbers of CD3+, CD4+, CD19+, CD8+, or CD4+/CD8+ cells pretreatment between the two groups, but after treatment, the combination group had significantly higher numbers of all these cells. Neither the CD8+ nor the CD19+ level was significantly different between the control and combination groups (P > 0.05). Furthermore, the incidence of common clinical side effects was similar between the two groups (P > 0.05). Proteinuria, tiredness, increased alanine aminotransferase, hypertension, immunological pneumonia, muscle pain, arthralgia, hypothyroidism, etc. were the most common side effects reported among both groups throughout therapy. A grade IV side effect is rare. After follow-up until March 2022, the median PFS for the control group was 9.00 ± 1.65 months (95% CI, 5.76-12.24) and the mean PFS was 11.48 ± 0.91 months (95% CI, 9.69-13.26). Comparison of the median PFS of the combination group (13.00 ± 1.10) months (95% CI: 10.84-15.16) with the average PFS of the group (15.52 ± 0.88) months (95% CI = 13.79-17.25) reveals a statistically significant difference (P < 0.05). Conclusion: Combining bevacizumab with the PD-1 immune medication pembrolizumab to treat patients with stage IV lung adenocarcinoma improves the quality of life, short-term therapeutic effectiveness, immune function, and PFS.

Case Report: Rare Acute Abdomen: Focal Nodular Hyperplasia With Spontaneous Rupture.

Focal nodular hyperplasia (FNH) of the liver is a benign lesion characterized by hypertrophic nodules with central star-shaped fibrous scars. The etiology and pathogenesis of FNH are not completely understood. A 43-year-old man was hospitalized because of acute abdominal pain. Emergency computed tomography(CT) showed hepatic tumor rupture and bleeding. The patient's condition improved following arteriographic embolization to stop bleeding. Laparotomy confirmed spontaneous rupture and hemorrhage of focal hyperplasia and the patient remains asymptomatic after an uneventful recovery. FNH with spontaneous rupture and bleeding is extremely rare. Currently, there is no unified management standard for FNH and most previous studies recommend observation and follow-up. We recommend consideration of surgical treatment of cases with spontaneous rupture and bleeding.

Refined models of coordination between Al3+/Mg2+ and enzyme in molecular dynamics simulation in terms of ABEEM polarizable force field.

MFX (AlF30, AlF4- and MgF3-) as transition state analogues of phosphoryl transfer enzymes (enzyme-MFX-TSAs) is of great significance for study of the catalytic mechanism of phosphoryl transfer enzymes. Bonded model and non-bonded model based on the ABEEM polarizable force field (ABEEM PFF) are developed and applied to study the coordination of enzyme-MFX-TSAs. The bond stretching of the bond containing metal is simulated by Morse potential energy function, because the change of chemical bond is described more accurately in a large range. The charge distribution of the system is distributed to multiple-charge-sites, including atomic site, σ bond site, π bond site and lone pair electron site. Partial charge can fluctuate according to the surrounding environment and molecular conformation. The reasonable charge distribution of 68 model molecules can be obtained, and the energy minimizations are performed in vacuum. Then, with the same parameters the charge distribution and the charge transfer of four complexes are obtained, and the energy minimization and molecular dynamics simulation in NVT ensemble are carried out in vacuum and explicit water solution. The results verify the correctness, rationality and transferability of the new parameters of ABEEM PFF, and the bonded model simulates more reasonable charge distribution and geometry. The parameters determined in this paper make up the blank of the parameters of MFX and phosophoryl transfer enzymes containing Mg2+. The development of ABEEM PFF provides a refined tool for MFX-TSAs to study the catalytic mechanism of phosphoryl transfer enzymes.

Energetics and J-coupling constants for Ala, Gly, and Val peptides demonstrated using ABEEM polarizable force field in vacuo and an aqueous solution.

The development of an atom-bond electronegativity equalisation method at the σπ-level (ABEEM) polarisable force field (PFF) for peptides is presented. ABEEM PFF utilises a fluctuating charge model to explicitly describe the polarisation effects in an extensive environment. The partial charge of any individual site changes in response to changes in its surroundings. The peptide parameters are derived from ab initio methods in vacuum using a consistent and automatic protocol. By including explicit σ- and π-bond sites and lone pair sites, the anisotropy around an atom has been characterised. The fluctuating charge at each site ensures the distinction between the intrinsic behaviour observed among the various conformations of peptides, as corroborated by the agreement between quantum mechanics (QM) and ABEEM PFF concerning the calculated energy order, charge distribution, locations of minima, and potential energy surface (PES) in vacuo. The energy barriers in the PES have been clearly described using ABEEM PFF, in which a good charge distribution plays a vital role. Molecular dynamic simulations have been performed for short peptides in explicit ABEEM 7P-water boxes to examine their conformational properties in solution. The J-coupling constants obtained using ABEEM PFF are consistent with the experimental nuclear magnetic resonance (NMR) spectra and the influence of the chain length and temperature also investigated. The results demonstrate that the ABEEM PFF method is capable of locating conformations and describing the energetics of peptides with high accuracy and efficiency both in vacuo and an aqueous solution.

Water-Mediated Oxidation of Guanine by a Repair Enzyme: Simulation Using the ABEEM Polarizable Force Field.

The recognition mechanism of oxidative damage in organisms has long been a research hotspot. Water is an important medium in the recognition process, but its specific role remains unknown. There is a need to develop a suitable force field that can adequately describe the electrostatic, hydrogen bond, and other interactions among the molecules in the complex system of the repair enzyme and oxidized base. The developing ABEEM polarizable force field (PFF) has been used to simulate the repaired enzyme hOGG1 and oxidized DNA (PDB ID: 1EBM) in a biological environment, and the corresponding results are better than those of the fixed-charge force fields OPLS/AA and AMBER OL15. 8-Oxo-G is recognized by Gln315 of hOGG1 mainly through hydrogen bonds mediated by continuous exchange of 2 water molecules. Phe319 and Cys253 are stacked on both sides of the π planes of bases to form sandwich structures. The charge polarization effect gives an important signal to drive the exchange of water molecules and maintains the recognition of oxidation bases by enzymes. The mediated main water molecule A and mediated auxiliary water molecule B together pull Gln315 to recognize 8-oxo-G by hydrogen bond interactions. Then, the charge polarization signal of solvent water molecule C with a large absolute charge causes the absolute charge of O atoms in water molecule A or B to increase by approximately 0.2 e, and water molecule A or B leaves Gln315 and 8-oxo-G. The other water molecule and water molecule C synergistically recognize 8-oxo-G with Gln315. Even though the water molecules between Gln315 and 8-oxo-G are removed, the MD simulation results show that water molecules appear between Gln315 and 8-oxo-G in a very short time (<2 ps). The dwell time of each water molecule is approximately 60 ps. The radial distribution function and dwell time support the correctness of the above mechanism. These polarization effects and hydrogen bonding interactions cannot be simulated by a fixed-charge force field.

ALYREF Drives Cancer Cell Proliferation Through an ALYREF-MYC Positive Feedback Loop in Glioblastoma.

BACKGROUND: While RNA-binding proteins (RBPs) are known to affect RNA homeostasis during cancer cell initiation and development, their characteristics and biological function in glioblastoma (GBM) remain unclear. METHODS: Differences in RBP expression were explored by differential analysis of The Cancer Genome Atlas-GBM and Genotype-Tissue Expression (GTEx) datasets. Real-time PCR was conducted to verify the expressional levels of Aly/REF export factor (ALYREF) in normal brain and GBM tissues. Proliferative assays were performed to investigate molecular functions of ALYREF in GBM cells in vitro and in vivo. Real-time PCR and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed to analyze the ALYREF downstream signaling pathways. A chromatin immunoprecipitation (ChIP) assay was performed to identify key transcriptional factors that regulate ALYREF expression at RNA level. UV crosslinking, immunoprecipitation (CLIP) and RNA stability assays were conducted to reveal the bound RNAs and their stability regulated by ALYREF. RESULTS: The results showed that ALYREF is frequently increased in GBM tissues, and its mRNA expression is regulated by the MYC proto-oncogene, bHLH transcription factor (MYC). Inhibition of ALYREF expression decreased GBM cell proliferative ability in vitro and tumor formation in vivo. KEGG analysis revealed that high ALYREF expression in GBM tissues was enriched in the upregulation of oncogenic pathways such as the Wnt/ß-catenin signaling pathway. The CLIP assay showed that ALYREF drives GBM carcinogenesis by binding to and stabilizing MYC mRNAs. Overexpression of MYC restored the oncogenic property of ALYREF-deficient GBM cells. CONCLUSION: Our data showed that ALYREF is regulated by MYC at the transcriptional level. ALYREF drives GBM cell proliferation by activating the Wnt/ß-catenin signaling pathway and stabilizing MYC mRNA, suggesting that an ALYREF-MYC positive feedback loop might be a potential therapeutic target for treating GBM patients.

Dividing the Periodic Box into Subdivisions with Their Surroundings to Accelerate Molecular Dynamics Simulation with High Accuracy.

A major field of current research in chemistry and biology is the development of the tools that enable in situ analysis of complex systems. However, the long-time dynamics simulation for an extremely large system in solution is almost impossible by an all-atom force field combined with an explicit solvent model. The results show that the larger the periodic box is, the closer the properties of the system are to the experimental values. Therefore, how can we carry out simulations for systems that are fast, accurate, and large enough? A method of dividing the periodic box into subdivisions with their surroundings (DBSS) is presented here, and it clearly increases the computation speed without losing accuracy and enables the simulation of extremely large systems by strongly decreasing the dimension of the charge matrix. The DBSS method divides a single periodic box or unit in an extremely large system into several subdivisions with a suitable choice according to atomic coordinates. This method ensures that these subdivisions are always changing and allows the atoms to communicate with each other. Intermolecular communication is important for molecular properties and functions but is not possible with other fragment methods. The partial charges are calculated in each subdivision with an overlapping surrounding used to take hydrogen bond interaction between the subdivisions into account. This is an iterative process because the charge population will be recalculated at intervals during the dynamics simulations. Taking a water system as an example, each subdivision is extended by 4 Å as the surrounding. The computation time scales almost linearly with the size of the system, and the slope is small. MD simulations for several properties have been performed by the ABEEM-DBSS method. The results indicate that the ABEEM-DBSS method can accurately simulate the properties of water system, and the accuracy can reach or approach that of the experimental data or of other water potentials. Interestingly, the properties become closer to the experimental data as the sizes of the periodic box increase, further validating the need for the simulation of a large system and demonstrating the value of the DBSS method.

Partitioning a Molecule into the Atomic Basins and the Resultant Atomic Charges from Quantum Chemical Topology Analysis of the Kohn-Sham Potential.

Quantum chemical topology (QCT) solidifies the chemical basic concepts demonstrating how a molecular system is intrinsically partitioned into its components and what the interaction lines between them are. Here, QCT analysis using a Kohn-Sham one-electron potential (KSpot) in KS equation as a scalar function is initiated and explored, showing KSpot and its resultant electron force lines have novel spatial features which reveal that an atom in a molecule is a spatial basin governed by its nucleus as a 3D-attractor that terminates all the electron force lines defined by the negative gradient of KSpot and that a chemical bond line is just a minimum path of KSpot for the electron motion. Particularly, the atomic charges from this KSpot QCT analysis are moderate and good, having much lower dependence on basis sets chosen for computation. This may provide a platform for the study of molecular structures and properties, intra- and intermolecular electrostatic interaction, energy decomposition, and construction of force field.

Polarizable TIP7P Water Model with Perturbation Charges Evaluated from ABEEM.

A polarizable version of the rigid seven-site (TIP7P) water model with the atom-bond electronegativity equalization method (ABEEM) is proposed. The model uses direct polarization, where an isolated water monomer in the equilibrium geometry is assumed as a reference state and the polarization of the monomer arises from interacting with other molecules as a perturbation of the reference state. The charge on each site of the monomer splits into reference charge and perturbation charge. The perturbation charge arises only because of other reference charges. The interaction of the perturbation charge with other perturbation charges is replaced using polarization scaling to enhance the interaction of perturbation charge with the reference charges of the sites from other molecules. The perturbation charges are updated by evaluating explicit expressions once. This direct polarization is time-reversible because the charge update is independent of the charges in previous simulation steps. A Slater-type damping function moderates the short-range electrostatics to treat charge diffusion. The Ewald method corrects the long-range electrostatics both in the nuclei movement and in electronegativity equalization to diminish the size effect. The water model is parameterized by fitting the ab initio results of water clusters and the experimental results of water monomers and thermodynamic properties for liquid water. Owing to polarizability, the model performs better than the TIP7P model in terms of vaporization enthalpy, isothermal compressibility, and shear viscosity of the liquid phase. It performs better at the melting point of ice but slightly worse under critical conditions than the TIP7P model. Direct polarization has a low time complexity of O(N) and is a good choice for ABEEM to improve its computational efficiency.

Seven-Site Effective Pair Potential for Simulating Liquid Water.

Constant pressure simulations were carried out to construct a new rigid nonpolarizable seven-site water model (TIP7P), which is an effective and efficient version of flexible seven-fluctuating-charge water model. In this model, the positive charges are located on three nuclei and the negative charges disperse on two bond sites at the geometric center of each OH bond and two lone-pair sites along the tetrahedral direction away from the oxygen atom. Our new model performs better than other models in properties, such as radial distribution function, liquid density, thermal expansion coefficient, isothermal compressibility, vaporization enthalpy, isobaric heat capacity, static dielectric constant, self-diffusion coefficient, critical temperature, and density. This model reproduces liquid density and static dielectric constant over the temperatures from 253 to 373 K at 1 atm with the standard deviation of 0.0010 g/cm3 and 1.37 to the experimental data, respectively. The maximum density is 1.0006 g/cm3 at 277 K. The calculated isobaric compressibility presents a minimum at about 310 K close to the experimental value of 319 K. The self-diffusion coefficient agrees the experimental data with the standard deviation of 0.55 × 10-5 cm2/s, although it is not the target property for parameterization. Liquid-vapor phase equilibrium was examined in slab simulations. The evaluated critical temperature and density are 633 K and 0.337 g/cm3 close to the experimental values of 647.096 K and 0.322 g/cm3. This model also presents reasonable vaporization enthalpy and isobaric heat capacity. Based upon good performances mentioned, our new model is a good choice for more accurate investigation to large molecular systems.

Reaction mechanism of NO with hydrolysates of NAMI-A: an MD simulation by combining the QM/MM(ABEEM) with the MD-FEP method.

Nitrosylation reaction mechanisms of the hydrolysates of NAMI-A and hydrolysis reactions of ruthenium nitrosyl complexes were investigated in the triplet state and the singlet state. Activation free energies were calculated by combining the QM/MM(ABEEM) method with free energy perturbation theory, and the explicit solvent environment was simulated by an ABEEMσπ polarizable force field. Our results demonstrate that nitrosylation reactions of the hydrolysates of NAMI-A occur in both the triplet and the singlet states. The Ru-N-O angle of the triplet ruthenium nitrosyl complexes is in the range of 132.0°-138.2°. However, all the ruthenium nitrosyl complexes at the singlet state show an almost linear Ru-N-O angle. The nitrosylation reaction happens prior to the hydrolysis reaction for the first-step hydrolysates. The activation free energies of the nitrosylation reactions show that the H2 O-NO exchange reaction of [RuCl4 (Im)(H2 O)] in the singlet spin sate is the most likely one. Comparing with the activation free energies of the hydrolysis reactions of the ruthenium nitrosyl complexes, the results indicate that the rate of the DMSO-H2 O exchange reaction of [RuCl3 (NO)(Im)(DMSO)] is faster than that of [RuCl3 (H2 O)(Im)(DMSO)] in both the triplet spin state and the singlet spin state. © 2018 Wiley Periodicals, Inc.

Evaluation of changes in magnetic resonance diffusion tensor imaging after treatment of delayed encephalopathy due to carbon monoxide poisoning.

Diffusion tensor imaging of the brain tissue microstructure was performed to predict or diagnose the pathophysiological mechanism underlying delayed encephalopathy after carbon monoxide poisoning and the treatment effect was analyzed. The changes in the diffusion parameters (average diffusion coefficient and fractional anisotropy) in adult patients after hyperbaric oxygen therapy of delayed encephalopathy after carbon monoxide poisoning were not significant differences of the two lateral ventricles or anterior or posterior limb of the internal capsule. In the group exposed to hyperbaric oxygen therapy, the fractional anisotropy values of the white matter in the ventricles of the brain and anterior and posterior limbs of the internal capsule were higher than those recorded before therapy, while the average diffusion coefficient values were significantly lower. These finding provide important monitoring indicators for clinicians.

Poly-adenine-mediated fluorescent spherical nucleic acid probes for live-cell imaging of endogenous tumor-related mRNA.

Identification of tumor-related mRNA in living cells hold great promise for early cancer diagnosis and pathological research. Herein, we present poly-adenine (polyA)-mediated fluorescent spherical nucleic acid (FSNA) probes for intracellular mRNA detection with regulable sensitivities by programmably adjusting the loading density of DNA on gold nano-interface. Gold nanoparticles (AuNPs) functionalized with polyA-tailed recognition sequences were hybridized to fluorescent "reporter" strands to fabricate fluorescence-quenched FSNA probes. While exposed to target gene, the "reporter" strands were released from FSNA through strand displacement and fluorescence was recovered. With polyA20 tail as the attaching block, the detection limit of FSNA probes was calculated to be 0.31 nM, which is ~55 fold lower than that of thiolated probes without surface density regulation. Quantitative intracellular mRNA detection and imaging could be achieved with polyA-mediated FSNA probes within 2 hours, indicating their application potential in rapid and sensitive intracellular target imaging.

Label-Free Electrochemical Sensing Platform for MicroRNA-21 Detection Using Thionine and Gold Nanoparticles Co-Functionalized MoS2 Nanosheet.

Herein, we demonstrated a label-free and simple electrochemical sensing platform to detect microRNA-21 (miR-21) with high sensitivity by using MoS2 nanosheet functionalized with thionine and gold nanoparticles (MoS2-Thi-AuNPs). Interestingly, thionine (Thi) was used as a reducing agent to successfully synthesize MoS2-Thi-AuNPs nanohybrid and as a signaling molecule to monitor DNA-RNA hybridization, which provided an ideal platform for label-free miR-21 detection. Upon hybridization with miR-21, the formation of the DNA-RNA duplex on the electrode would greatly hinder the electron transfer, which caused the electrochemical signal decrease of Thi. After optimization of experimental conditions, the signal change of peak currents of Thi has a linear relationship with the logarithm of miR-21 concentration ranging from 1.0 pM to 10.0 nM and the limit of detection (LOD) was 0.26 pM. Moreover, this biosensor could detect miR-21 in biological samples like human serum with satisfactory results.