NPEX: Never give up protein exploration with deep reinforcement learning.

Elucidating unknown structures of proteins, such as metastable states, is critical in designing therapeutic agents. Protein structure exploration has been performed using advanced computational methods, especially molecular dynamics and Markov chain Monte Carlo simulations, which require untenably long calculation times and prior structural knowledge. Here, we developed an innovative method for protein structure determination called never give up protein exploration (NPEX) with deep reinforcement learning. The NPEX method leverages the soft actor-critic algorithm and the intrinsic reward system, effectively adding a bias potential without the need for prior knowledge. To demonstrate the method's effectiveness, we applied it to four models: a double well, a triple well, the alanine dipeptide, and the tryptophan cage. Compared with Markov chain Monte Carlo simulations, NPEX had markedly greater sampling efficiency. The significantly enhanced computational efficiency and lack of prior domain knowledge requirements of the NPEX method will revolutionize protein structure exploration.

Unusual effects of a nanoporous gold substrate on cell adhesion and differentiation because of independent multi-branch signaling of focal adhesions.

A variety of cell behaviors, such as cell adhesion, motility, and fate, can be controlled by substrate characteristics such as surface topology and chemistry. In particular, the surface topology of substrates strongly affects cell behaviors, and the topological spacing is a critical factor in inducing cell responses. Various works have demonstrated that cell adhesion was enhanced with decreasing topological spacing although differentiation progressed slowly. However, there are exceptions, and thus, correlations between topological spacing and cell responses are still debated. We show that a nanoporous gold substrate affected cell adhesion while it neither affected osteogenic nor adipogenic differentiation. In addition, the cell adhesion was reduced with decreasing pore size. These do not agree with previous findings. A focal adhesion (FA) is an aggregate of modules comprising specific proteins such as FA kinase, talin, and vinculin. Therefore, it is suggested that because various extracellular signals can be independently branched off from the FA modules, the unusual effects of nanoporous gold substrates are related to the multi-branching of FAs.

Bioengineering of High Cell Density Tissues with Hierarchical Vascular Networks for Ex Vivo Whole Organs.

The development of tissue-like structures such as cell sheets, spheroids, and organoids has contributed to progress in regenerative medicine. Simultaneous achievement of scale up and high cell density of these tissues is challenging because sufficient oxygen cannot be supplied to the inside of large, high cell density tissues. Here, in vitro fabrication of vessels to supply oxygen to the inside of millimeter-sized scaffold-free tissues whose cell density is ≈200 million cells mL-1 , corresponding to those of native tissues, is shown. Hierarchical vascular networks by anastomosis of capillaries and a large vessel are essential for oxygen supply, whereas a large vessel or capillary networks alone make negligible contributions to the supply. The hierarchical vascular networks are formed by a top-down approach, which offers a new option for ex vivo whole organs without decellularization and 3D-bioprinting.

Effects of actuation of nanoporous gold on cell orientation in a fibroblast sheet.

Mechanical stimulation such as flood flow often plays a vital role in the growth and maintenance of a living body, and it is important to investigate cell responses to mechanical stimulation. To date, cell responses to mechanical stimulation have been investigated in detail. However, the cell responses have been little known in a cell sheet. In the present study, a small cyclic strain (CS) of ~0.5% generated by a nanoporous gold actuator was loaded on a cell sheet of fibroblasts, and the effects of the CS on cell orientation were investigated. Individual cells were randomly distributed after the CS application, whereas cells were oriented in a specific direction after the CS application for the cell sheet. Thus, the CS had a different effect on the cell sheet from that on the individual cells. It is suggested that the cadherin/p-120 catenin complex played an important role in the cell response to mechanical stimulation in a cell sheet.

Heterogeneous role of integrins in fibroblast response to small cyclic mechanical stimulus generated by a nanoporous gold actuator.

It is important to understand the effects of mechanical stimulation on cell behaviors for homeostasis. Many studies have been performed on cell responses to mechanical stimuli, but the mechanosensing mechanism is still under debate. In the present study, experiments employing molecular dynamics (MD) simulations concerning the effects of cyclic mechanical stimulus on cell proliferation were performed based on the hypothesis that mechanosensing depends on integrin types. We used a nanoporous gold (NPG) actuator to prevent transfer of a mechanical stimulus via molecules other than integrins. Surprisingly, a small cyclic strain of only 0.5% enhanced the proliferation of fibroblasts. α5ß1 and αvß3 integrins showed high sensitivity to the mechanical stimulus, whereas α1ß1 and α2ß1 integrins exhibited low mechanosensitivity. The MD simulations showed that different conformational changes of the integrin headpiece induced by binding to the ECM led to a difference in mechanosensitivity between αI and αI-less integrin types. Thus, the present study provides evidence to support the hypothesis and suggests the mechanism for the heterogeneous roles of integrins in mechanosensing.

A new mechanism for reduced cell adhesion: Adsorption dynamics of collagen on a nanoporous gold surface.

Nanostructured materials such as nanoparticles and nanoporous materials strongly affect cell behaviors such as cell viability. Because cellular uptake of nanoporous materials does not occur, mechanisms for the effects of nanoporous materials on cells are different from those of nanoparticles. The effects of nanoporous materials on cells are thought to result from large conformational changes in the extracellular matrix (ECM) induced by the nanoporous materials, although the mechanotransduction and the critical focal adhesion cluster size also have an effect on the cell response. However, we show that the adhesion of mesenchymal stem cells to a gold surface is reduced for nanoporous gold (NPG), despite the conformational changes in collagen induced by NPG being below the detection limits of the experimental analyses. The adsorption dynamics of collagen on NPG are investigated by molecular dynamics simulations to determine the origin of the reduced cell adhesion to NPG. The adsorption energy of collagen on NPG is lower than that on flat gold (FG) despite there being little difference between the global conformation of collagen segments adsorbed on NPG compared with FG. This finding is related to the surface strain of NPG and the limited movement of collagen amino acids owing to interchain hydrogen bonds. The results obtained in this study provide new insight into the interactions between nanostructured materials and the ECM.

Antibacterial activity of ultrathin platinum islands on flat gold against Escherichia coli.

Nanoporous Au exhibits high antibacterial activity (AA) without releasing reactive oxygen species or metal ions, instead its AA depends on the work function (WF) because cell walls are affected by peculiar electronic states at the surface. Based on this mechanism, a flat surface without nanostructure should show high AA if the WF of the surface is suitably tuned. To verify this, ultrathin Pt islands with high WF was fabricated on flat Au by underpotential deposition (UPD) of copper and subsequent redox replacement with Pt, and the AA of the Pt/Au substrate on Escherichia coli was evaluated. The Pt/Au substrate showed higher AA than Pt and Au surfaces, and a positive relationship between AA and WF was demonstrated. In addition, first principles calculations were performed to investigate the mechanism for the high WF of the Pt/Au substrate. The findings suggest that the high WF of the Pt/Au substrate is at least partly due to charge transfer from Au to Pt.

Electronic origin of antimicrobial activity owing to surface effect.

Nanomaterials have displayed promising potential as antimicrobial materials. However, the antimicrobial mechanism owing to surface effects, where the emission of harmful substances such as metallic ions and reactive oxygen species is not required, is still poorly understood. It is important to figure out relationship between the physical properties and antimicrobial activity based on deep understanding of antimicrobial mechanism for their safe and effective applications. Here, we show that the work function is representative of the surface effect leading to antimicrobial activity, which originates from the electronic states of the surface. We investigated the antimicrobial activity and the work function of nanoporous Au-Pt and Au without the emission of Ag ion, and found that there was a positive correlation between them. In addition, we performed a first-principles calculation and molecular dynamics simulation to analyze the electronic states of the Au surface and the cell wall. These demonstrated that positive correlation was owing to peculiar electronic states at the Au surface, namely, the spilling out phenomenon of electrons. Our finding will contribute to advance the understanding of biological phenomena from a physical view.

Antimicrobial mechanisms due to hyperpolarisation induced by nanoporous Au.

Nanomaterials such as nanoparticles exhibit remarkable antimicrobial activities. Nanoparticles directly disturb the cell membrane or cytoplasmic proteins because they pass through the cell wall. Nanoporous Au (NPG) is another antimicrobial nanomaterial, which cannot pass through the cell wall of bacteria but can still kill bacteria, utilising interactions between the surface of NPG and cell wall of bacteria. The origins of antimicrobial activities without direct interactions are unknown. It is necessary to elucidate these mechanisms to ensure safe usage. Here we show that the antimicrobial mechanism of NPG consists of two interactions: between the surface of NPG and cell wall, and between the cell wall and cell membrane. Fluorescent experiments showed that the cell wall was negatively hyperpolarised by NPG, and molecular dynamics simulations and first-principles calculations suggested that the hyperpolarisation of the cell wall leads to delicate structural changes in the membrane proteins, rendering them bactericidal. Thus, the hyperpolarisation induced by NPG plays a critical role in both interactions. The combination of molecular dynamics simulations and first-principles calculations allows a deeper understanding of the interactions between metallic surfaces and biomolecules, because charge transfer and exchange interactions are calculated exactly.

Softening due to disordered grain boundaries in nanocrystalline Co.

Nanocrystalline Co consisting of fcc and hcp phases was processed by electrodeposition, and its mechanical properties were investigated by hardness tests. In addition, high-resolution transmission electron microscopy observations and molecular dynamics (MD) simulations were performed to investigate the grain boundary structure and dislocation nucleation from the grain boundaries. A large amount of disorders existed at the grain boundaries and stacking faults were formed from the grain boundaries in the as-deposited Co specimen. The as-deposited specimen showed a lower hardness than did the annealed specimen, although the grain size of the former was smaller than that of the latter. The activation volume of the as-deposited specimen (=1.5b(3)) was lower than that of the annealed specimen (=50b(3)), thus indicating that nucleation of dislocations from grain boundaries is more active in the as-deposited specimen than in the annealed specimens. The MD simulations showed that dislocation nucleation was closely related to a change in the defect structures at the boundary. Therefore, it is suggested that a significant amount of defects enhance changes in the defect structures at the boundary, resulting in softening of the as-deposited specimen.

Electrochemical stability of self-assembled monolayers on nanoporous Au.

Desorption of thiolate self-assembled monolayers (SAMs) seriously limits the fabrication of thiol-based devices. Here we demonstrate that nanoporous Au produced by dealloying Au-Ag alloys exhibits high electrochemical stability against thiolate desorption. Nanoporous Au has many defective sites, lattice strain and residual Ag on the ligament surface. First-principles calculations indicate that these surface aspects increase the binding energy between a SAM and the surface of nanoporous Au.

Nanoporous gold prism microassembly through a self-organizing route.

We report a simple and spontaneous synthesis of a nanoporous gold prism microassembly with highly dense skins, which is achieved just by immersing nanoporous gold into concentrated hydrochloric acid. The ligament size was coarsened to several hundred nanometers, but the crystal face orientation was still preserved. The same trends were seen in the case of coarsening by annealing; however, the morphology of the nanoporous gold prism microassembly was significantly different from the annealed nanoporous gold.