Efficient and reliable calculation of protein–ligand binding free energy is a grand challenge in computational biology and is of critical importance in drug design and many other molecular ...recognition problems. The main challenge lies in the calculation of entropic contribution to protein–ligand binding or interaction systems. In this report, we present a new interaction entropy method which is theoretically rigorous, computationally efficient, and numerically reliable for calculating entropic contribution to free energy in protein–ligand binding and other interaction processes. Drastically different from the widely employed but extremely expensive normal mode method for calculating entropy change in protein–ligand binding, the new method calculates the entropic component (interaction entropy or −TΔS) of the binding free energy directly from molecular dynamics simulation without any extra computational cost. Extensive study of over a dozen randomly selected protein–ligand binding systems demonstrated that this interaction entropy method is both computationally efficient and numerically reliable and is vastly superior to the standard normal mode approach. This interaction entropy paradigm introduces a novel and intuitive conceptual understanding of the entropic effect in protein–ligand binding and other general interaction systems as well as a practical method for highly efficient calculation of this effect.
In this paper, a padlock probe-based exponential rolling circle amplification (P-ERCA) assay is developed for highly specific and sensitive detection of microRNA (miRNA). The padlock probe is ...composed of a hybridization sequence to miRNA and a nicking site for nicking endonuclease. Using the miRNA as a template, specific ligation to the padlock probe and linear rolling circle reaction (LRCA) are achieved under isothermal conditions. After multiple nicking reactions, many copies of short DNA products are successively produced and then used as triggers in next circle amplification. Thus, a small amount of miRNAs are converted to a large number of triggers to initiate the rolling circle amplification reaction, and circular exponential signal amplification is achieved. This padlock probe-based exponential rolling circle amplification assay exhibits a remarkable sensitivity of 0.24 zmol using optimized sequences of the padlock probe. The target-dependent circularization of the padlock probe and the ligation reaction could improve the specificity effectively, leading to single–nucleotide difference discrimination between miRNA family members. The miRNA analysis in human lung cells was performed with this method. The result indicates this highly sensitive P-ERCA strategy will become a promising miRNA quantification method in early clinical diagnostics.
Entropy effects play an important role in drug-target interactions, but the entropic contribution to ligand-binding affinity is often neglected by end-point binding free energy calculation methods, ...such as MM/GBSA and MM/PBSA, due to the expensive computational cost of normal mode analysis (NMA). Here, we systematically investigated entropy effects on the prediction power of MM/GBSA and MM/PBSA using >1500 protein-ligand systems and six representative AMBER force fields. Two computationally efficient methods, including NMA based on truncated structures and the interaction entropy approach, were used to estimate the entropic contributions to ligand-target binding free energies. In terms of the overall accuracy, we found that, for the minimized structures, in most cases the inclusion of the conformational entropies predicted by truncated NMA (enthalpynmode_min_9Å) compromises the overall accuracy of MM/GBSA and MM/PBSA compared with the enthalpies calculated based on the minimized structures (enthalpymin). However, for the MD trajectories, the binding free energies can be improved by the inclusion of the conformation entropies predicted by either truncated-NMA for a relatively high dielectric constant (εin = 4) or the interaction entropy method for εin = 1-4. In terms of reproducing the absolute binding free energies, the binding free energies estimated by including the truncated-NMA entropies based on the MD trajectories (ΔGnmode_md_9Å) give the lowest average absolute deviations against the experimental data among all the tested strategies for both MM/GBSA and MM/PBSA. Although the inclusion of the truncated NMA based on the MD trajectories (ΔGnmode_md_9Å) for a relatively high dielectric constant gave the overall best result and the lowest average absolute deviations against the experimental data (for the ff03 force field), it needs too much computational time. Alternatively, considering that the interaction entropy method does not incur any additional computational cost and can give comparable (at high dielectric constant, εin = 4) or even better (at low dielectric constant, εin = 1-2) results than the truncated-NMA entropy (ΔGnmode_md_9Å), the interaction entropy approach is recommended to estimate the entropic component for MM/GBSA and MM/PBSA based on MD trajectories, especially for a diverse dataset. Furthermore, we compared the predictions of MM/GBSA with six different AMBER force fields. The results show that the ff03 force field (ff03 for proteins and gaff with AM1-BCC charges for ligands) performs the best, but the predictions given by the tested force fields are comparable, implying that the MM/GBSA predictions are not very sensitive to force fields.
Chemical contamination is an important issue for large-scale soils in China, especially the contamination of chemical heavy metals. Under this background, this paper focuses on the experimental ...method to explore the effects of two soil amendments (lime and phosphate) on reducing soil heavy metal activity and improving soil quality. In this paper, the biological evaluation method was used to test the effects of different soil amendments by taking the soil conductivity, chlorophyll and heavy metal content in the plant as the main evaluation indicators. The research results show that the effect of different soil amendments is greatly related to the concentration of the soil amendment solution, of which 5% of lime water can effectively reduce the activity of heavy metal Zn in soil, but not effective on Pb; the concentration 7.5% of the phosphate solution can effectively reduce the activity of heavy metal Pb in soil but cannot reduce the activity of Zn. This provides an effective solution to the problem of heavy metal pollution in Chinese soil chemistry, especially the pollution of Pb and Zn.
•Studies showed vitamin K2 intake reduced 7% T2DM risk with each 10-μg increment.•Vitamin K2 has a more significant effect than vitamin K1 on T2DM.•Vitamin K2 increased insulin sensitivity via ...osteocalcin metabolism.•Vitamin K2 improved IR via anti-inflammatory property and lipid-lowering effects.•Vitamin K2 suppresses inflammation via inactivating NF-κB signalling pathway.
Type 2 diabetes mellitus (T2DM) continue to be a major public health problem around the world that frequently presents with microvascular and macrovascular complications. Individuals with T2DM are not only suffering from significant emotional and physical misery, but also at increased risk of dying from severe complications. In recent years, evidence from prospective observational studies and clinical trials has shown T2DM risk reduction with vitamin K2 supplementation. We thus did an overview of currently available studies to assess the effect of vitamin K2 supplementation on insulin sensitivity, glycaemic control and reviewed the underlying mechanisms. We proposed that vitamin K2 improved insulin sensitivity through involvement of vitamin K-dependent-protein osteocalcin, anti-inflammatory properties, and lipid-lowering effects. Vitamin K2 had a better effect than vitamin K1 on T2DM. The interpretation of this review will increase comprehension of the development of a therapeutic strategy to prevent and treat T2DM.
MicroRNAs (miRNAs) play significant roles in a diverse range of biological progress and have been regarded as biomarkers and therapeutic targets in cancer treatment. Sensitive and accurate detection ...of miRNAs is crucial for better understanding their roles in cancer cells and further validating their function in clinical diagnosis. Here, we developed a stable, sensitive, and specific miRNAs detection method on the basis of cooperative amplification combining with the graphene oxide (GO) fluorescence switch-based circular exponential amplification and the multimolecules labeling of SYBR Green I (SG). First, the target miRNA is adsorbed on the surface of GO, which can protect the miRNA from enzyme digest. Next, the miRNA hybridizes with a partial hairpin probe and then acts as a primer to initiate a strand displacement reaction to form a complete duplex. Finally, under the action of nicking enzyme, universal DNA fragments are released and used as triggers to initiate next reaction cycle, constituting a new circular exponential amplification. In the proposed strategy, a small amount of target miRNA can be converted to a large number of stable DNA triggers, leading to a remarkable amplification for the target. Moreover, compared with labeling with a 1:1 stoichiometric ratio, multimolecules binding of intercalating dye SG to double-stranded DNA (dsDNA) can induce significant enhancement of fluorescence signal and further improve the detection sensitivity. The extraordinary fluorescence quenching of GO used here guarantees the high signal-to-noise ratio. Due to the protection for target miRNA by GO, the cooperative amplification, and low fluorescence background, sensitive and accurate detection of miRNAs has been achieved. The strategy proposed here will offer a new approach for reliable quantification of miRNAs in medical research and early clinical diagnostics.
Thrombosis is a principle cause of cardiovascular disease, the leading cause of morbidity and mortality worldwide; however, the conventional anti-thrombotic approach often leads to bleeding ...complications despite extensive clinical management and monitoring. In view of the intense crosstalk between inflammation and coagulation, plus the contributing role of ROS to both inflammation and coagulation, it is highly desirable to develop safer anti-thrombotic agent with preserved anti-inflammatory and anti-oxidative stress activities. Nattokinase (NK) possesses many beneficial effects on cardiovascular system due to its strong thrombolytic and anticoagulant activities. Herein, we demonstrated that NK not only effectively prevented xylene-induced ear oedema in mice, but also remarkably protected against LPS-induced acute kidney injury in mice through restraining inflammation and oxidative stress, a central player in the initiation and progression of inflammation. Fascinatingly, in line with our in vivo data, NK elicited prominent anti-inflammatory activity in RAW264.7 macrophages via suppressing the LPS-induced TLR4 and NOX2 activation, thereby repressing the corresponding ROS production, MAPKs activation, and NF-κB translocation from the cytoplasm to the nucleus, where it mediates the expression of pro-inflammatory mediators, such as TNF-α, IL-6, NO, and PAI-1 in activated macrophage cells. In particular, consistent with the macrophage studies, NK markedly inhibited serum PAI-1 levels induced by LPS, thereby blocking the deposition of fibrin in the glomeruli of endotoxin-treated animals. In summary, we extended the anti-thrombus mechanism of NK by demonstrating the anti-inflammatory and anti-oxidative stress effects of NK in ameliorating LPS-activated macrophage signaling and protecting against LPS-stimulated AKI as well as glomeruler thrombus in mice, opening a comprehensive anti-thrombus strategy by breaking the vicious cycle between inflammation, oxidative stress and thrombosis.
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•NK protects against LPS-induced AKI via inhibiting inflammation and oxidative stress.•NK inhibits LPS-induced TRL4 and NOX2 activation in macrophages.•NK inhibits inflammation and oxidative stress both in vitro and in vivo.•NK inhibits LPS-induced PAI-I levels, thereby blocking glomerular thrombus in mice.•NK may break the vicious loop between inflammation, oxidative stress and coagulation.
A new highly efficient interaction entropy (IE) method combined with the polarized protein-specific charge (PPC) force field is employed to investigate the interaction mechanism of CDK2-ligand ...binding and the effect of the bridging water. Our result shows that the computed binding free energies for five CDK2-ligand complexes using the IE method have a significantly linear correlation with the experimentally measured values with a correlation coefficient of 0.98 in consideration of the bridging water under the PPC force field. And the correlation coefficient is found to be slightly weaker with a value of 0.95 using the traditional normal mode (Nmode) method for calculation of entropy change. Importantly, the rank of the predicted binding free energies is significantly consistent with the experimental rank based on the IE method calculated entropy change using the PPC force field. However, without including the bridging water under PPC simulation, the correlation coefficient is below 0.83. For comparison, the result obtained from the simulation using the nonpolarized AMBER force field gives a much weaker correlation with the correlation coefficients of 0.44 and 0.45 using the Nmode method and IE method, due to the lack of electrostatic polarization. Furthermore, hydrogen bond analysis indicates that the bridging water makes a significant contribution to mediating the hydrogen bond network of protein-ligand binding and stabilizing the complex structure. The current study demonstrates that the new IE method is superior to the standard Nmode method in computing the binding free energy. And our results also emphasize the importance of electronic polarization and bridging water in MD simulations and free energy calculations.
B-cell lymphoma/leukemia gene-2(Bcl-2) protein family known for regulating cell cycle arrest and subsequent cell death is highly expressed in a variety of cancers. Among them, the Bcl-xL and Bcl-2 ...are two essential proteins in the Bcl-2 family. In the present work, the differences in binding modes as between the two proteins and two ligands ABT-263/43b were investigated and compared. And the computational alanine scanning combined with the recently developed interaction entropy (AS-IE) method was employed for predicting their binding free energies and finding those amino acids that were more critical during the binding process. The result showed that the binding free energy calculated by the AS-IE method was more in line with experimental values than the molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method. Besides, no significant difference was found between Bcl-xL and ABT-263/43b in the binding free energy, which Bcl-xL showed slightly weaker binding free energy to 43b because of the fewer number of key residues with interactions. Nonetheless, compared with the Bcl-2 and 43b complex, the Bcl-2 and ABT-263 system had greater number of key residues interacting with ABT-263, in particular, contribute favorably, resulting in a stronger binding ability for the Bcl-2 and ABT-263 systems. The van der Waals and hydrogen bond contributions were significant in the four protein–ligand complexes. Overall, Tyr108 was found to be the common key residues in the Bcl-xL–ligand complex, while Tyr105, Glu100, and Glu143 were established as the common key residue in the Bcl-2–ligand systems. We hope that the predicted hot spot residues and their energy distributions can guide the design of peptide and small-molecule drugs targeting Bcl-xL and Bcl-2.
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