Fabrication and characterizations of simvastatin-containing mesoporous bioactive glass and molybdenum disulfide scaffold for bone tissue engineering.

Due to the limitations of the current treatment approaches of allograft and autograft techniques, treating bone disorders is a significant challenge. To address these shortcomings, a novel biomaterial composite is required. This study presents the preparation and fabrication of a novel biomaterial composite scaffold that combines poly (D, L-lactide-co-glycolide) (PLGA), mesoporous bioactive glass (MBG), molybdenum disulfide (MoS2), and simvastatin (Sim) to address the limitations of current bone grafting techniques of autograft and allograft. The fabricated scaffold of PLGA-MBG-MoS2-Sim composites was developed using a low-cost hydraulic press and salt leaching method, and scanning electron microscopy (SEM) analysis confirmed the scaffolds have a pore size between 143 and 240 µm. The protein adsorption for fabricated scaffolds was increased at 24 h. The water adsorption and retention studies showed significant results on the PLGA-MBG-MoS2-Sim composite scaffold. The biodegradation studies of the PLGA-MBG-MoS2-Sim composite scaffold have shown 54% after 28 days. In vitro, bioactivity evaluation utilizing simulated body fluid studies confirmed the development of bone mineral hydroxyapatite on the scaffolds, which was characterized using x-ray diffraction, Fourier transform infrared, and SEM analysis. Furthermore, the PLGA-MBG-MoS2-Sim composite scaffold is biocompatible with C3H10T1/2 cells and expresses more alkaline phosphatase and mineralization activity. Additionally, in vivo research showed that PLGA-MBG-MoS2-Sim stimulates a higher rate of bone regeneration. These findings highlight the fabricated PLGA-MBG-MoS2-Sim composite scaffold presents a promising solution for the limitations of current bone grafting techniques.

Biomimetic chitosan with biocomposite nanomaterials for bone tissue repair and regeneration.

Biomimetic materials for better bone graft substitutes are a thrust area of research among researchers and clinicians. Autografts, allografts, and synthetic grafts are often utilized to repair and regenerate bone defects. Autografts are still considered the gold-standard method/material to treat bone-related issues with satisfactory outcomes. It is important that the material used for bone tissue repair is simultaneously osteoconductive, osteoinductive, and osteogenic. To overcome this problem, researchers have tried several ways to develop different materials using chitosan-based nanocomposites of silver, copper, gold, zinc oxide, titanium oxide, carbon nanotubes, graphene oxide, and biosilica. The combination of materials helps in the expression of ideal bone formation genes of alkaline phosphatase, bone morphogenic protein, runt-related transcription factor-2, bone sialoprotein, and osteocalcin. In vitro and in vivo studies highlight the scientific findings of antibacterial activity, tissue integration, stiffness, mechanical strength, and degradation behaviour of composite materials for tissue engineering applications.

Effects of an HIV-1 maturation inhibitor on the structure and dynamics of CA-SP1 junction helices in virus-like particles.

HIV-1 maturation involves conversion of the immature Gag polyprotein lattice, which lines the inner surface of the viral membrane, to the mature capsid protein (CA) lattice, which encloses the viral RNA. Maturation inhibitors such as bevirimat (BVM) bind within six-helix bundles, formed by a segment that spans the junction between the CA and spacer peptide 1 (SP1) subunits of Gag, and interfere with cleavage between CA and SP1 catalyzed by the HIV-1 protease (PR). We report solid-state NMR (ssNMR) measurements on spherical virus-like particles (VLPs), facilitated by segmental isotopic labeling, that provide information about effects of BVM on the structure and dynamics of CA-SP1 junction helices in the immature lattice. Although BVM strongly blocks PR-catalyzed CA-SP1 cleavage in VLPs and blocks conversion of VLPs to tubular CA assemblies, 15N and 13C ssNMR chemical shifts of segmentally labeled VLPs with and without BVM are very similar, indicating that interaction with BVM does not alter the six-helix bundle structure appreciably. Only the 15N chemical shift of A280 (the first residue of SP1) changes significantly, consistent with BVM binding to an internal ring of hydrophobic side chains of L279 residues. Measurements of transverse 15N spin relaxation rates reveal a reduction in the amplitudes and/or timescales of backbone N-H bond motions, corresponding to a rigidification of the six-helix bundles. Overall, our data show that inhibition of HIV-1 maturation by BVM involves changes in structure and dynamics that are surprisingly subtle, but still sufficient to produce a large effect on CA-SP1 cleavage.

Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR.

Recent studies of noncrystalline HIV-1 capsid protein (CA) assemblies by our laboratory and by Polenova and coworkers (Protein Sci 19:716-730, 2010; J Mol Biol 426:1109-1127, 2014; J Biol Chem 291:13098-13112, 2016; J Am Chem Soc 138:8538-8546, 2016; J Am Chem Soc 138:12029-12032, 2016; J Am Chem Soc 134:6455-6466, 2012; J Am Chem Soc 132:1976-1987, 2010; J Am Chem Soc 135:17793-17803, 2013; Proc Natl Acad Sci USA 112:14617-14622, 2015; J Am Chem Soc 138:14066-14075, 2016) have established the capability of solid state nuclear magnetic resonance (NMR) measurements to provide site-specific structural and dynamical information that is not available from other types of measurements. Nonetheless, the relatively high molecular weight of HIV-1 CA leads to congestion of solid state NMR spectra of fully isotopically labeled assemblies that has been an impediment to further progress. Here we describe an efficient protocol for production of segmentally labeled HIV-1 CA samples in which either the N-terminal domain (NTD) or the C-terminal domain (CTD) is uniformly 15N,13C-labeled. Segmental labeling is achieved by trans-splicing, using the DnaE split intein. Comparisons of two-dimensional solid state NMR spectra of fully labeled and segmentally labeled tubular CA assemblies show substantial improvements in spectral resolution. The molecular structure of HIV-1 assemblies is not significantly perturbed by the single Ser-to-Cys substitution that we introduce between NTD and CTD segments, as required for trans-splicing.

An Alternative Phosphorylation Switch in Integrin ß2 (CD18) Tail for Dok1 Binding.

Integrins are involved in cell migration and adhesion. A large number of proteins interact with the cytoplasmic tails of integrins. Dok1 is a negative regulator of integrin activation and it binds to the phosphorylated membrane proximal NxxY motif in a number of integrin ß tails. The ß tail of the ß2 integrins contains a non-phosphorylatable NxxF motif. Hence it is unclear how Dok1 associates with the ß2 integrins. We showed in this study using NMR and cell based analyses that residues Ser745 and Ser756 in the integrin ß2 tail, which are adjacent to the NxxF motif, are required for Dok1 interaction. NMR analyses detected significant chemical shift changes and higher affinity interactions between Dok1 phospho-tyrosine binding (PTB) domain and integrin ß2 tail peptide containing pSer756 compared to pSer745. The phosphorylated ß2 peptide occupies the canonical ligand binding pocket of Dok1 based on the docked structure of the ß2 tail-Dok1 PTB complex. Taken together, our data suggest an alternate phosphorylation switch in ß2 integrins that regulates Dok1 binding. This could be important for cells of the immune system and their functions.

Characterization of the near native conformational states of the SAM domain of Ste11 protein by NMR spectroscopy.

The sterile alpha motif or SAM domain is one of the most frequently present protein interaction modules with diverse functional attributions. SAM domain of the Ste11 protein of budding yeast plays important roles in mitogen-activated protein kinase cascades. In the current study, urea-induced, at subdenaturing concentrations, structural, and dynamical changes in the Ste11 SAM domain have been investigated by nuclear magnetic resonance spectroscopy. Our study revealed that a number of residues from Helix 1 and Helix 5 of the Ste11 SAM domain display plausible alternate conformational states and largest chemical shift perturbations at low urea concentrations. Amide proton (H/D) exchange experiments indicated that Helix 1, loop, and Helix 5 become more susceptible to solvent exchange with increased concentrations of urea. Notably, Helix 1 and Helix 5 are directly involved in binding interactions of the Ste11 SAM domain. Our data further demonstrate that the existence of alternate conformational states around the regions involved in dimeric interactions in native or near native conditions.

NMR characterization of the near native and unfolded states of the PTB domain of Dok1: alternate conformations and residual clusters.

BACKGROUND: Phosphotyrosine binding (PTB) domains are critically involved in cellular signaling and diseases. PTB domains are categorized into three distinct structural classes namely IRS-like, Shc-like and Dab-like. All PTB domains consist of a core pleckstrin homology (PH) domain with additional structural elements in Shc and Dab groups. The core PH fold of the PTB domain contains a seven stranded ß-sheet and a long C-terminal helix. PRINCIPAL FINDINGS: In this work, the PTB domain of Dok1 protein has been characterized, by use of NMR spectroscopy, in solutions containing sub-denaturing and denaturing concentrations of urea. We find that the Dok1 PTB domain displays, at sub-denaturing concentrations of urea, alternate conformational states for residues located in the C-terminal helix and in the ß5 strand of the ß-sheet region. The ß5 strand of PTB domain has been found to be experiencing significant chemical shift perturbations in the presence of urea. Notably, many of these residues in the helix and in the ß5 strand are also involved in ligand binding. Structural and dynamical analyses at 7 M urea showed that the PTB domain is unfolded with islands of motionally restricted regions in the polypeptide chain. Further, the C-terminal helix appears to be persisted in the unfolded state of the PTB domain. By contrast, residues encompassing ß-sheets, loops, and the short N-terminal helix lack any preferred secondary structures. Moreover, these residues demonstrated an intimate contact with the denaturant. SIGNIFICANCE: This study implicates existence of alternate conformational states around the ligand binding pocket of the PTB domain either in the native or in the near native conditions. Further, the current study demonstrates that the C-terminal helical region of PTB domain may be considered as a potential site for the initiation of folding.