Active-Site Conformational Fluctuations Promote the Enzymatic Activity of NDM-1.

ß-Lactam antibiotics are the mainstay for the treatment of bacterial infections. However, elevated resistance to these antibiotics mediated by metallo-ß-lactamases (MBLs) has become a global concern. New Delhi metallo-ß-lactamase-1 (NDM-1), a newly added member of the MBL family that can hydrolyze almost all ß-lactam antibiotics, has rapidly spread all over the world and poses serious clinical threats. Broad-spectrum and mechanism-based inhibitors against all MBLs are highly desired, but the differential mechanisms of MBLs toward different antibiotics pose a great challenge. To facilitate the design of mechanism-based inhibitors, we investigated the active-site conformational changes of NDM-1 through the determination of a series of 15 high-resolution crystal structures in native form and in complex with products and by using biochemical and biophysical studies, site-directed mutagenesis, and molecular dynamics computation. The structural studies reveal the consistency of the active-site conformations in NDM-1/product complexes and the fluctuation in native NDM-1 structures. The enzymatic measurements indicate a correlation between enzymatic activity and the active-site fluctuation, with more fluctuation favoring higher activity. This correlation is further validated by structural and enzymatic studies of the Q123G mutant. Our combinational studies suggest that active-site conformational fluctuation promotes the enzymatic activity of NDM-1, which may guide further mechanism studies and inhibitor design.

3D-printed surfaces of titanium implant: the fibroblasts response.

Ti-6Al-4V (wt%) is the most widely used titanium alloy and its additive manufactured (or 3D printed) parts with near net-shape have provided great advantages for biomedical applications. While the impact of surface roughness on the biocompatibility of 3D-printed Ti-6Al-4V part is recognized, further exploration is needed to fully understand this complex relationship. Hence, this study presents a comprehensive evaluation of as-printed Ti-6Al-4V structures, both with and without surface texturing, with particular focus on the fibroblast response. Alongside a flat surface, or as-printed surface, two different types of surface textures: diamond texture and diamond crystal texture, were meticulously designed and printed through laser powder bed fusion (LPBF). The viability, cell adhesion, and morphology of human and murine fibroblasts seeded on the surface patterns was investigated, as well as the distribution of extracellular matrix (ECM) proteins (collagen I, fibronectin). The results demonstrated that the as-fabricated surface morphologies did not impact fibroblast viability, however, a reduced density of human fibroblasts was observed on the diamond texture surface, likely owing to the upright strut structure preventing cell adhesion. Interestingly, spreading of the human, but not murine, fibroblasts was limited by the remaining partially-sintered powders. The relative intensity of ECM protein signals was unaffected, however, ECM protein distribution across the surfaces was also altered. Thus, the as-printed substrates, particularly with diamond crystal struts, present a promising avenue for the cost-effective and efficient fabrication of Ti-6Al-4V components for medical applications in the future.

Evaluating the Optical Response of Heavily Decorated Black Silicon Based on a Realistic 3D Modeling Methodology.

Combining black silicon (BS), a nanostructured silicon containing highly roughened surface morphology with plasmonic materials, is becoming an attractive approach for greatly enhancing light-matter interactions with promising applications of sensing and light harvesting. However, precisely describing the optical response of a heavily decorated BS structure is still challenging due to the increasing complexity in surface morphology and plasmon hybridization. Here, we propose and fully characterize BS-based multistacked nanostructures with randomly distributed nanoparticles on the highly roughened nonflat surface. We demonstrate a realistic 3D modeling methodology based on parametrized scanning electron microscopy images that provides high-precision morphology details, successfully linking the theoretical analysis with experimental optical response of the complex nanostructures. Far-field calculations very nicely reproduce experimental reflectance spectra, revealing the dependency of light trapping on the thickness of the conformal reflector and the atop nanoparticle size. Near-field analysis clearly identifies three types of stochastic "hotspots". Their contribution to the overall field enhancement is shown to be very much sensitive to the nanoscale surface morphology. The simulated near-field property is then used to examine the measured surface-enhanced Raman scattering (SERS) response on the multistacked structures. The present modeling approach combined with spectroscopic characterizations is expected to offer a powerful tool for the precise description of the optical response of other large-scale highly disordered realistic 3D systems.

Using cryo-electron microscopy maps for X-ray structure determination of homologues.

The combination of cryo-electron microscopy (cryo-EM) and X-ray crystallography reflects an important trend in structural biology. In a previously published study, a hybrid method for the determination of X-ray structures using initial phases provided by the corresponding parts of cryo-EM maps was presented. However, if the target structure of X-ray crystallography is not identical but homologous to the corresponding molecular model of the cryo-EM map, then the decrease in the accuracy of the starting phases makes the whole process more difficult. Here, a modified hybrid method is presented to handle such cases. The whole process includes three steps: cryo-EM map replacement, phase extension by NCS averaging and dual-space iterative model building. When the resolution gap between the cryo-EM and X-ray crystallographic data is large and the sequence identity is low, an intermediate stage of model building is necessary. Six test cases have been studied with sequence identity between the corresponding molecules in the cryo-EM and X-ray structures ranging from 34 to 52% and with sequence similarity ranging from 86 to 91%. This hybrid method consistently produced models with reasonable Rwork and Rfree values which agree well with the previously determined X-ray structures for all test cases, thus indicating the general applicability of the method for X-ray structure determination of homologues using cryo-EM maps as a starting point.

Knockdown EIF3C Suppresses Cell Proliferation and Increases Apoptosis in Pancreatic Cancer Cell.

Increasing evidence shows that eukaryotic initiation factor subunit (EIF3C) plays a crucial role in development of tumors. However, the underlying roles of EIF3Cin the development of pancreatic cancer (PC) remain unknown. In this study, we examined the expression of EIF3C in PC tissues, their adjacent normal tissues and 3 cell lines (SW1990, PANC-1 and AsPC-1). Moreover, the EIF3C-shRNA lentivirus was constructed to suppress EIF3C expression. Following this, the cell colony formation assay was employed to evaluate proliferation ability of PC cells. Meanwhile, the cell cycle and apoptotic assays were also performed by flow cytometry. We found that level of EIF3C in PC tissues was significantly increased compared with that in adjacent normal tissues. Furthermore, the knockdown of EIF3C can significantly reduce cell proliferation, block cell cycle in G2/M and induce apoptosis in both SW1990 and PANC-1 cells. Our findings suggest that EIF3C plays a crucial role in the progression of PC and may be a potential target in the treatment of PC.

THBS2, a microRNA-744-5p target, modulates MMP9 expression through CUX1 in pancreatic neuroendocrine tumors.

The underlying molecular mechanisms of pancreatic neuroendocrine tumor (pNET) development have not yet been clearly identified. The present study revealed that thrombospondin 2 (THBS2) was downregulated in pNET tissues and cells. Forced expression of THBS2 inhibited the proliferation and migration of pNET cells in vitro. MicroRNA(miR)-744-5p was indicated to be a direct regulator of THBS2. Upregulation of miR-744-5p potentially caused THBS2 repression. Furthermore, THBS2 inhibited the production of matrix metalloproteinase (MMP) MMP9 through suppressing the transcriptional activity of CUT-like homeobox 1 (CUX1). CUX1 and MMP9 mediated the effect of THBS2 on pNET proliferation and migration, respectively. The results of the present study revealed a mechanistic role for THBS2 in pNET proliferation and migration, indicating that THBS2 was downregulated by miR-744-5p and further affected the CUX1/MMP9 cascade, which promoted the development of pNET.

Using cryo-electron microscopy maps for X-ray structure determination.

X-ray crystallography and cryo-electron microscopy (cryo-EM) are complementary techniques for structure determination. Crystallography usually reveals more detailed information, while cryo-EM is an extremely useful technique for studying large-sized macromolecules. As the gap between the resolution of crystallography and cryo-EM data narrows, the cryo-EM map of a macromolecule could serve as an initial model to solve the phase problem of crystal diffraction for high-resolution structure determination. FSEARCH is a procedure to utilize the low-resolution molecular shape for crystallographic phasing. The IPCAS (Iterative Protein Crystal structure Automatic Solution) pipeline is an automatic direct-methods-aided dual-space iterative phasing and model-building procedure. When only an electron-density map is available as the starting point, IPCAS is capable of generating a completed model from the phases of the input map automatically, without the requirement of an initial model. In this study, a hybrid method integrating X-ray crystallography with cryo-EM to help with structure determination is presented. With a cryo-EM map as the starting point, the workflow of the method involves three steps. (1) Cryo-EM map replacement: FSEARCH is utilized to find the correct translation and orientation of the cryo-EM map in the crystallographic unit cell and generates the initial low-resolution map. (2) Phase extension: the phases calculated from the correctly placed cryo-EM map are extended to high-resolution X-ray data by non-crystallographic symmetry averaging with phenix.resolve. (3) Model building: IPCAS is used to generate an initial model using the phase-extended map and perform model completion by iteration. Four cases (the lowest cryo-EM map resolution being 6.9 Å) have been tested for the general applicability of the hybrid method, and almost complete models have been generated for all test cases with reasonable Rwork/Rfree. The hybrid method therefore provides an automated tool for X-ray structure determination using a cryo-EM map as the starting point.

Integrating Pharmacophore into Membrane Molecular Dynamics Simulations to Improve Homology Modeling of G Protein-coupled Receptors with Ligand Selectivity: A2A Adenosine Receptor as an Example.

Homology modeling has been applied to fill in the gap in experimental G protein-coupled receptors structure determination. However, achievement of G protein-coupled receptors homology models with ligand selectivity remains challenging due to structural diversity of G protein-coupled receptors. In this work, we propose a novel strategy by integrating pharmacophore and membrane molecular dynamics (MD) simulations to improve homology modeling of G protein-coupled receptors with ligand selectivity. To validate this integrated strategy, the A2A adenosine receptor (A2A AR), whose structures in both active and inactive states have been established, has been chosen as an example. We performed blind predictions of the active-state A2A AR structure based on the inactive-state structure and compared the performance of different refinement strategies. The blind prediction model combined with the integrated strategy identified ligand-receptor interactions and conformational changes of key structural elements related to the activation of A2 A AR, including (i) the movements of intracellular ends of TM3 and TM5/TM6; (ii) the opening of ionic lock; (iii) the movements of binding site residues. The integrated strategy of pharmacophore with molecular dynamics simulations can aid in the optimization in the identification of side chain conformations in receptor models. This strategy can be further investigated in homology modeling and expand its applicability to other G protein-coupled receptor modeling, which should aid in the discovery of more effective and selective G protein-coupled receptor ligands.