Catalytic properties of 26 S and 20 S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities*

20 and 26 S proteasomes were isolated from rat liver. The procedure developed for the 26 S proteasome resulted in greatly improved yields compared with previously published methods. A comparison of the kinetic properties of 20 and 26 S proteasomes showed significant differences in the kinetic characteristics with certain substrates and differences in the effects of a protein substrate on peptidase activity. Observed differences in the kinetics of peptidylglutamyl peptide hydrolase activity suggest that the 26 S complex cannot undergo the conformational changes of 20 S proteasomes at high concentrations of the substrate benzyloxycarbonyl (Z) -Leu-Leu-Glu-b-naphthylamide. Various inhibitors that differentially affect the trypsin-like and chymotrypsin-like activities have been identified. Ala-Ala-Phe-chloromethyl (CH2Cl) inhibits chymotrypsin-like activity assayed with succinyl (Suc) -Leu-Leu-Val-Tyr-AMC, but surprisingly not hydrolysis of Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC). Tyr-Gly-Arg-CH2Cl inhibits Suc-Leu-Leu-Val-Tyr-AMC hydrolysis as well as trypsinlike activity measured with t-butoxycarbonyl (Boc) -Leu-Ser-Thr-Arg-AMC, while Z-Phe-Gly-Tyr-diazomethyl (CHN2) was found to inhibit only the two chymotrypsin- like activities. Radiolabeled forms of peptidyl chloromethane and peptidyl diazomethane inhibitors, [3H]acetyl-Ala-Ala-Phe-CH2Cl, [3H]acetyland radioiodinated Tyr-Gly-Arg-CH2Cl, and Z-Phe-Gly- Tyr-(125I-CHN2), have been used to identify catalytic components associated with each of the three peptidase activities. In each case, incorporation of the label could be blocked by prior treatment of the proteasomes with known active site-directed inhibitors, calpain inhibitor 1 or 3,4-dichloroisocoumarin. Subunits of labeled proteasomes were separated either by reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis or by twodimensional polyacrylamide gel electrophoresis followed by autoradiography/fluorography and immunoblotting with subunit-specific antibodies. In each case, label was found to be incorporated into subunits C7, MB1, and LMP7 but in different relative amounts depending on the inhibitor used, consistent with the observed effects on the different peptidase activities. The results strongly suggest a relationship between trypsin-like activity and chymotrypsin-like activity. They also help to relate the different subunits of the complex to the assayed multicatalytic endopeptidase activities

A fundamental numerical and theoretical study for the vibrational properties of nanowires

Based on the molecular dynamics (MD) simulation and the classical Euler-Bernoulli beam theory, a fundamental study of the vibrational performance of the Ag nanowire (NW) is carried out. A comprehensive analysis of the quality (Q)-factor, natural frequency, beat vibration, as well as high vibration mode is presented. Two excitation approaches, i.e., velocity excitation and displacement excitation, have been successfully implemented to achieve the vibration of NWs. Upon these two kinds of excitations, consistent results are obtained, i.e., the increase of the initial excitation amplitude will lead to a decrease to the Q-factor, and moderate plastic deformation could increase the first natural frequency. Meanwhile, the beat vibration driven by a single relatively large excitation or two uniform excitations in both two lateral directions is observed. It is concluded that the nonlinear changing trend of external energy magnitude does not necessarily mean a nonconstant Q-factor. In particular, the first order natural frequency of the Ag NW is observed to decrease with the increase of temperature. Furthermore, comparing with the predictions by Euler- Bernoulli beam theory, the MD simulation provides a larger and smaller first vibration frequencies for the clamped-clamped and clamped-free thin Ag NWs, respectively. Additionally, for thin NWs, the first order natural frequency exhibits a parabolic relationship with the excitation magnitudes. The frequencies of the higher vibration modes tend to be low in comparison to Euler-Bernoulli beam theory predictions. A combined initial excitation is proposed which is capable to drive the NW under a multi-mode vibration and arrows the coexistence of all the following low vibration modes. This work sheds lights on the better understanding of the mechanical properties of NWs and benefits the increasing utilities of NWs in diverse nano-electronic devices.

Numerical investigation of mechanical properties of nanowires : a review

Nanowires (NWs) have attracted intensive researches owing to the broad applications that arise from their remarkable properties. Over the last decade, immense numerical studies have been conducted for the numerical investigation of mechanical properties of NWs. Among these numerical simulations, the molecular dynamics (MD) plays a key role. Herein we present a brief review on the current state of the MD investigation of nanowires. Emphasis will be placed on the FCC metal NWs, especially the Cu NWs. MD investigations of perfect NWs’ mechanical properties under different deformation conditions including tension, compression, torsion and bending are firstly revisited. Following in succession, the studies for defected NWs including the defects of twin boundaries (TBs) and pre-existing defects are discussed. The different deformation mechanism incurred by the presentation of defects is explored and discussed. This review reveals that the numerical simulation is an important tool to investigate the properties of NWs. However, the substantial gaps between the experimental measurements and MD results suggest the urgent need of multi-scale simulation technique.

Influence of pre-exsiting surface defects on the vibrational properties of Ag nanowires

Large-scale molecular dynamics simulations are performed to characterize the effects of pre-existing surface defects on the vibrational properties of Ag nanowires. It is found that the first order natural frequency of the nanowire appears insensitive to different surface defects, indicating a defect insensitivity property of the nanowire’s Young’s modulus. In the meanwhile, an increase of the quality (Q)-factor is observed due to the presence of defects. Particular, a beat phenomenon is observed for the nanowire with the presence of a surface edge defect, which is driven by a single actuation. It is concluded that different surface defects could act as an effective mean to tune the vibrational properties of nanowires. This study sheds lights on the better understanding of nanowire’s mechanical performance when surface defects are presented, which would benefit the development of nanowire-based devices.

Nagelschmidtite bioceramics with osteostimulation properties : material chemistry activating osteogenic genes and WNT signalling pathway of human bone marrow stromal cells

Bioactive materials with osteostimulation properties are of great importance to promote osteogenic differentiation of human bone marrow stromal cells (hBMSCs) for potential bone regeneration. We have recently synthesized nagelschmidtite (NAGEL, Ca7Si2P2O16) ceramic powders which showed excellent apatite-mineralization ability. The aim of this study was to investigate the interaction of hBMSCs with NAGEL bioceramic bulks and their ionic extracts, and to explore the osteostimulation properties of NAGEL bioceramics and the possible molecular mechanism. The cell attachment, proliferation, bone-related gene expression (ALP, OPN and OCN) and WNT signalling pathways (WNT3a, FZD6, AXIN2 and CTNNB) of hBMSCs cultured on NAGEL bioceramic disks were systematically studied. We further investigated the biological effects of ionic products from NAGEL powders on cell proliferation and osteogenic differentiation of hBMSCs by culturing cells with NAGEL extracts. Furthermore, the effect of NAGEL bioceramics on the osteogenic differentiation in hBMSCs was also investigated with the addition of cardamonin, a WNT inhibitor. The results showed that NAGEL bioceramic disks supported the attachment and proliferation of hBMSCs, and significantly enhanced the bone-related gene expression and WNT signalling pathway of hBMSCs, compared to conventional beta-tricalcium phosphate (β-TCP) bioceramic disks and blank controls. The ionic products from NAGEL powders also significantly promoted the proliferation, bone and WNT-related gene expression of hBMSCs. It was also identified that NAGEL bioceramics could bypass the action of the WNT inhibitor (10 μM) to stimulate the selected osteogenic genes in hBMSCs. Our results suggest that NAGEL bioceramics possess excellent in vitro osteostimulation properties. The possible mechanism for the osteostimulation may be directly related to the released Si, Ca and P-containing ionic products from NAGEL bioceramics which activate bone-related gene expression and WNT signalling pathway of hBMSCs. The present study suggests that NAGEL bioceramics are a potential bone regeneration material with significant osteostimulation capacity.

Mechanical properties of graphene : effects of layer number, temperature and isotope

Herein the mechanical properties of graphene, including Young’s modulus, fracture stress and fracture strain have been investigated by molecular dynamics simulations. The simulation results show that the mechanical properties of graphene are sensitive to the temperature changes but insensitive to the layer numbers in the multilayer graphene. Increasing temperature exerts adverse and significant effects on the mechanical properties of graphene. However, the adverse effect produced by the increasing layer number is marginal. On the other hand, isotope substitutions in graphene play a negligible role in modifying the mechanical properties of graphene.

Influence of grain boundaries on the vibrational properties of silver nanowires

Metal and semiconductor nanowires (NWs) have been widely employed as the building blocks of the nanoelectromechanical systems, which usually acted a resonant beam. Recent researches reported that nanowires are often polycrystalline, which contains grain boundaries (GBs) that transect the whole nanowire into a bamboo like structure. Based on the larger-scale molecular dynamics (MD) simulations, a comprehensive investigation of the influence from grain boundaries on the vibrational properties of doubly clamped Ag NWs is conducted. It is found that, the presence of grain boundary will result in significant energy dissipation during the resonance of polycrystalline NWs, which leads a great deterioration to the quality factor. Further investigation reveals that the energy dissipation is originated from the plastic deformation of polycrystalline NWs in the form of the nucleation of partial dislocations or the generation of micro stacking faults around the GBs and the micro stacking faults is found to keep almost intact during the whole vibration process. Moreover, it is observed that the closer of the grain boundary getting to the regions with the highest strain state, the more energy dissipation will be resulted from the plastic deformation. In addition, either the increase of the number of grain boundaries or the decrease of the distance between the grain boundary and the highest strain state region is observed to induce a lower first resonance frequency. This work sheds lights on the better understanding of the mechanical properties of polycrystalline NWs, which benefits the increasing utilities of NWs in diverse nano-electronic devices.

Continuum mechanics modelling of microfilament networks with different architectures based on molecular investigation of single F-actin

The actin microfilament plays a critical role in many cellular processes including embryonic development, wound healing, immune response, and tissue development. It is commonly organized in the form of networks whose mechanical properties change with changes in their architecture due to cell evolution processes. This paper presents a new nonlinear continuum mechanics model of single filamentous actin (F-actin) that is based on nanoscale molecular simulations. Following this continuum model of the single F-actin, mechanical properties of differently architected lamellipodia are studied. The results provide insight that can contribute to the understanding of the cell edge motions of living cells.

Molecular dynamics simulation of fracture strength and morphology of defective graphene

Different types of defects can be introduced into graphene during material synthesis, and significantly influence the properties of graphene. In this work, we investigated the effects of structural defects, edge functionalisation and reconstruction on the fracture strength and morphology of graphene by molecular dynamics simulations. The minimum energy path analysis was conducted to investigate the formation of Stone-Wales defects. We also employed out-of-plane perturbation and energy minimization principle to study the possi-ble morphology of graphene nanoribbons with edge-termination. Our numerical results show that the fracture strength of graphene is dependent on defects and environmental temperature. However, pre-existing defects may be healed, resulting in strength recovery. Edge functionalization can induce compressive stress and ripples in the edge areas of gra-phene nanoribbons. On the other hand, edge reconstruction contributed to the tensile stress and curved shape in the graphene nanoribbons.

Targeted control of feral pigs in far north Queensland : defining management units using molecular techniques

The feral pig, Sus scrofa, is a widespread and abundant invasive species in Australia. Feral pigs pose a significant threat to the environment, agricultural industry, and human health, and in far north Queensland they endanger World Heritage values of the Wet Tropics. Historical records document the first introduction of domestic pigs into Australia via European settlers in 1788 and subsequent introductions from Asia from 1827 onwards. Since this time, domestic pigs have been accidentally and deliberately released into the wild and significant feral pig populations have become established, resulting in the declaration of this species as a class 2 pest in Queensland. The overall objective of this study was to assess the population genetic structure of feral pigs in far north Queensland, in particular to enable delineation of demographically independent management units. The identification of ecologically meaningful management units using molecular techniques can assist in targeting feral pig control to bring about effective long-term management. Molecular genetic analysis was undertaken on 434 feral pigs from 35 localities between Tully and Innisfail. Seven polymorphic and unlinked microsatellite loci were screened and fixation indices (FST and analogues) and Bayesian clustering methods were used to identify population structure and management units in the study area. Sequencing of the hyper-variable mitochondrial control region (D-loop) of 35 feral pigs was also examined to identify pig ancestry. Three management units were identified in the study at a scale of 25 to 35 km. Even with the strong pattern of genetic structure identified in the study area, some evidence of long distance dispersal and/or translocation was found as a small number of individuals exhibited ancestry from a management unit outside of which they were sampled. Overall, gene flow in the study area was found to be influenced by environmental features such as topography and land use, but no distinct or obvious natural or anthropogenic geographic barriers were identified. Furthermore, strong evidence was found for non-random mating between pigs of European and Asian breeds indicating that feral pig ancestry influences their population genetic structure. Phylogenetic analysis revealed two distinct mitochondrial DNA clades, representing Asian domestic pig breeds and European breeds. A significant finding was that pigs of Asian origin living in Innisfail and south Tully were not mating randomly with European breed pigs populating the nearby Mission Beach area. Feral pig control should be implemented in each of the management units identified in this study. The control should be coordinated across properties within each management unit to prevent re-colonisation from adjacent localities. The adjacent rainforest and National Park Estates, as well as the rainforest-crop boundary should be included in a simultaneous control operation for greater success.

Structural and electronic properties of diazonium functionalized (4, 4) single walled carbon nanotube : an ab initio study

In this work, ab initio density functional theory (DFT) calculations are performed to study the structural and electronic properties of diazonium reagent functionalized (4, 4) single-walled carbon nanotube (SWCNT). We find the aryl group covalently bonds with SWCNT and prefers to be perpendicular to the side wall of nanotube. It has a rotational barrier of 0.35 eV around the formed aryl-tube bond axis and should be thermodynamically stable at room temperature. Additionally, new peaks appeared around the Fermi energy in the density of state (DOS) due to the weak band dispersion. Increasing of the coverage of the functional group will result in significant upshift of the Fermi level.

Molecular dynamics simulation of fracture strength and morphology of defective graphene

Different types of defects can be introduced into graphene during material synthesis, and significantly influence the properties of graphene. In this work, we investigated the effects of structural defects, edge functionalisation and reconstruction on the fracture strength and morphology of graphene by molecular dynamics simulations. The minimum energy path analysis was conducted to investigate the formation of Stone-Wales defects. We also employed out-of-plane perturbation and energy minimization principle to study the possible morphology of graphene nanoribbons with edge-termination. Our numerical results show that the fracture strength of graphene is dependent on defects and environmental temperature. However, pre-existing defects may be healed, resulting in strength recovery. Edge functionalization can induce compressive stress and ripples in the edge areas of graphene nanoribbons. On the other hand, edge reconstruction contributed to the tensile stress and curved shape in the graphene nanoribbons.

Measuring the mechanical properties of ground ophthalmic polymer surfaces using nanoindentation

This work is motivated by the need to efficiently machine the edges of ophthalmic polymer lenses for mounting in spectacle or instrument frames. The polymer materials used are required to have suitable optical characteristics such high refractive index and Abbe number, combined with low density and high scratch and impact resistance. Edge surface finish is an important aesthetic consideration; its quality is governed by the material removal operation and the physical properties of the material being processed. The wear behaviour of polymer materials is not as straightforward as for other materials due to their molecular and structural complexity, not to mention their time-dependent properties. Four commercial ophthalmic polymers have been studied in this work using nanoindentation techniques which are evaluated as tools for probing surface mechanical properties in order to better understand the grinding response of polymer materials.

Mechanical properties of bioinspired bicontinuous nanocomposites

Inspired by the wonderful properties of some biological composites in nature, we performed molecular dynamics simulations to investigate the mechanical behavior of bicontinuous nanocomposites. Three representative types of bicontinuous composites, which have regular network, random network, and nacre inspired microstructures respectively, were studied and the results were compared with those of a honeycomb nanocomposite with only one continuous phase. It was found that the mechanical strength of nanocomposites in a given direction strongly depends on the connectivity of microstructure in that direction. Directional isotropy in mechanical strength and easy manufacturability favor the random network nanocomposites as a potentially great bioinspired composite with balanced performances. In addition, the tensile strength of random network nanocomposites is less sensitive to the interfacial failure, owing to its super high interface-to-volume ratio and random distribution of internal interfaces. The results provide a useful guideline for design and optimization of advanced nanocomposites with superior mechanical properties.