Lige Tonggu

Collagen intrafibrillar mineralization as a result of the balance between osmotic equilibrium and electroneutrality

Mineralisation of fibrillar collagen with biomimetic process-directing agents has enabled scientists to gain insight into the potential mechanisms involved in intrafibrillar mineralisation. Here, by using polycation- and polyanion-directed intrafibrillar mineralisation, we challenge the popular paradigm that electrostatic attraction is solely responsible for polyelectrolyte-directed intrafibrillar mineralisation. Because there is no difference when a polycationic or a polyanionic electrolyte is used to direct collagen mineralisation, we argue that additional types of long-range non-electrostatic interactions are responsible for intrafibrillar mineralisation. Molecular dynamics simulations of collagen structures in the presence of extrafibrillar polyelectrolytes show that the outward movement of ions and intrafibrillar water through the collagen surface occurs irrespective of the charges of polyelectrolytes, resulting in the experimentally verifiable contraction of the collagen structures. The need to balance electroneutrality and osmotic equilibrium simultaneously to establish Gibbs-Donnan equilibrium in a polyelectrolyte-directed mineralisation system establishes a new model for collagen intrafibrillar mineralisation that supplements existing collagen mineralisation mechanisms.

Antimicrobial activity of a quaternary ammonium methacryloxy silicate-containing acrylic resin: a randomised clinical trial

Quaternary ammonium methacryloxy silicate (QAMS)-containing acrylic resin demonstrated contact-killing antimicrobial ability in vitro after three months of water storage. The objective of the present double-blind randomised clinical trial was to determine the in vivo antimicrobial efficacy of QAMS-containing orthodontic acrylic by using custom-made removable retainers that were worn intraorally by 32 human subjects to create 48-hour multi-species plaque biofilms, using a split-mouth study design. Two control QAMS-free acrylic disks were inserted into the wells on one side of an orthodontic retainer, and two experimental QAMS-containing acrylic disks were inserted into the wells on the other side of the same retainer. After 48 hours, the disks were retrieved and examined for microbial vitality using confocal laser scanning microscopy. No harm to the oral mucosa or systemic health occurred. In the absence of carry-across effect and allocation bias (disks inserted in the left or right side of retainer), significant difference was identified between the percentage kill in the biovolume of QAMS-free control disks (3.73 ± 2.11%) and QAMS-containing experimental disks (33.94 ± 23.88%) retrieved from the subjects (P ≤ 0.001). The results validated that the QAMS-containing acrylic exhibits favourable antimicrobial activity against plaque biofilms in vivo. The QAMS-containing acrylic may also be used for fabricating removable acrylic dentures.

Membrane protein reconstitution for functional and structural studies.

Membrane proteins are involved in various critical biological processes, and studying membrane proteins represents a major challenge in protein biochemistry. As shown by both structural and functional studies, the membrane environment plays an essential role for membrane proteins. In vitro studies are reliant on the successful reconstitution of membrane proteins. This review describes the interaction between detergents and lipids that aids the understanding of the reconstitution processes. Then the techniques of detergent removal and a few useful techniques to refine the formed proteoliposomes are reviewed. Finally the applications of reconstitution techniques to study membrane proteins involved in Ca2+ signaling are summarized.

Flow-induced structured phase in nonionic micellar solutions.

In this work, we consider the flow of a nonionic micellar solution (precursor) through an array of microposts, with focus on its microstructural and rheological evolution. The precursor contains polyoxyethylene(20) sorbitan monooleate (Tween-80) and cosurfactant monolaurin (ML). An irreversible flow-induced structured phase (NI-FISP) emerges after the nonionic precursor flows through the hexagonal micropost arrays, when subjected to strain rates ~10(4) s(-1) and strain ~10(3). NI-FISP consists of close-looped micellar bundles and multiconnected micellar networks as evidenced by transmission electron microscopy (TEM) and cryo-electron microscopy (cryo-EM). We also conduct small-angle neutron scattering (SANS) measurements in both precursor and NI-FISP to illustrate the structural transition. We propose a potential mechanism for the NI-FISP formation that relies on the micropost arrays and the flow kinematics in the microdevice to induce entropic fluctuations in the micellar solution. Finally, we show that the rheological variation from a viscous precursor solution to a viscoelastic micellar structured phase is associated with the structural evolution from the precursor to NI-FISP.

Worming their way into shape: toroidal formations in micellar solutions.

We report the formation of nanostructured toroidal micellar bundles (nTMB) from a semidilute wormlike micellar solution, evidenced by both cryogenic-electron microscopy and transmission electron microscopy images. Our strategy for creating nTMB involves a two-step protocol consisting of a simple prestraining process followed by flow through a microfluidic device containing an array of microposts, producing strain rates in the wormlike micelles on the order of 10(5) s(-1). In combination with microfluidic confinement, these unusually large strain rates allow for the formation of stable nTMB. Electron microscopy images reveal a variety of nTMB morphologies and provide the size distribution of the nTMB. Small-angle neutron scattering indicates the underlying microstructural transition from wormlike micelles to nTMB. We also show that other flow-induced approaches such as sonication can induce and control the emergence of onion-like and nTMB structures, which may provide a useful tool for nanotemplating.

Effects of N-glycosylation on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization and conduct an inward cation current, which contributes to rhythmic electrical activity of neural and cardiac pacemaker cells. HCN channels have been shown to undergo N-linked glycosylation, and the N-glycosylation has been shown to be required for membrane trafficking and possibly function. In this study, recombinant wild-type (WT) and glycosylation-defective N380Q HCN2 channels were individually or co-expressed in HEK-293 cells. We demonstrate that glycosylation is required for trafficking to the plasma membrane and for the stability of HCN channels in the cell. Interestingly, the heteromeric HCN2 channels of WT and glycosylation-defective N380Q have been observed on cell membranes, indicating that not all four subunits of a tetrameric HCN2 channel need to be glycosylated for HCN2 channels to traffic to plasma membranes. Subsequently, we investigate the effect of N-glycosylation on the function of HCN2 channels. We developed a fluorescence-based flux assay, which makes it possible to establish a negative potential inside liposomes to open HCN2 channels. Using this flux assay, we demonstrate that glycosylation-defective N380Q HCN2 channels reconstituted into liposomes function similarly to WT HCN2 channels. This suggests that N-glycosylation is not required for HCN2 channels to function.

Substrate Effects on Structural Studies using Cryo-Em

In electron microscopy, glow discharge has been widely used to render the carbon film hydrophilic for better adhesion of aqueous solution. However, the target macromolecules (e.g. proteins) may also interact with the charge on the carbon film, which may introduce preferred orientation and/or some other artifacts. A promising alternative is to use two-dimensional (2D) crystal as a substrate. This method has been successfully employed to study DNA molecules, proteoliposomes, and multiprotein complexes. Here we will use liposomes (i.e. lipid vesicles) as a model system to study the effect of substrate on targeted structures. Four types of substrates will be studied: untreated carbon film, glow-discharged carbon film, holey carbon film, and 2D streptavidin crystal. Liposomes, doped with a few copies of biotinylated lipid and osmotically swollen to ensure a spherical shape, will be allowed to attach to the substrate before blotting and rapid freezing of the specimen. Then cryo-electron microscopy of tilted specimens and cryo-electron tomography will be employed, and the resulted images and tomographs will be compared with spherical liposome models. The deformation of the liposomes will be used to quantify the effect of the substrate on the targeted structure.