Subcellular localization of proteasomes and their regulatory complexes in mammalian cells

Proteasomes can exist in several different molecular forms in mammalian cells. The core 20S proteasome, containing the proteolytic sites, binds regulatory complexes at the ends of its cylindrical structure. Together with two 19S ATPase regulatory complexes it forms the 26S proteasome, which is involved in ubiquitin-dependent proteolysis. The 20S proteasome can also bind 11S regulatory complexes (REG, PA28) which play a role in antigen processing, as do the three variable c-interferoninducible catalytic b-subunits (e.g. LMP7). In the present study, we have investigated the subcellular distribution of the different forms of proteasomes using subunit speci®c antibodies. Both 20S proteasomes and their 19S regulatory complexes are found in nuclear, cytosolic and microsomal preparations isolated from rat liver. LMP7 was enriched approximately two-fold compared with core a-type proteasome subunits in the microsomal preparations. 20S proteasomes were more abundant than 26S proteasomes, both in liver and cultured cell lines. Interestingly, some signi®cant differences were observed in the distribution of different subunits of the 19S regulatory complexes. S12, and to a lesser extent p45, were found to be relatively enriched in nuclear fractions from rat liver, and immuno¯uorescent labelling of cultured cells with anti-p45 antibodies showed stronger labelling in the nucleus than in the cytoplasm. The REG was found to be localized predominantly in the cytoplasm. Three- to six-fold increases in the level of REG were observed following cinterferon treatment of cultured cells but c-interferon had no obvious effect on its subcellular distribution. These results demonstrate that different regulatory complexes and subpopulations of proteasomes have different distributions within mammalian cells and, therefore, that the distribution is more complex than has been reported for yeast proteasomes.

Low temperature synthesis of carbon nanotubes on indium tin oxide electrodes for organic solar cells

The electrical performance of indium tin oxide (ITO) coated glass was improved by including a controlled layer of carbon nanotubes directly on top of the ITO film. Multi-wall carbon nanotubes (MWCNTs) were synthesized by chemical vapor deposition, using ultra-thin Fe layers as catalyst. The process parameters (temperature, gas flow and duration) were carefully refined to obtain the appropriate size and density of MWCNTs with a minimum decrease of the light harvesting in the cell. When used as anodes for organic solar cells based on poly(3-hexylthiophene) (P3HT) and phenyl-C61-butyric acid methyl ester (PCBM), the MWCNT-enhanced electrodes are found to improve the charge carrier extraction from the photoactive blend, thanks to the additional percolation paths provided by the CNTs. The work function of as-modified ITO surfaces was measured by the Kelvin probe method to be 4.95 eV, resulting in an improved matching to the highest occupied molecular orbital level of the P3HT. This is in turn expected to increase the hole transport and collection at the anode, contributing to the significant increase of current density and open circuit voltage observed in test cells created with such MWCNT-enhanced electrodes.

Reduced electron recombination of dye-sensitized solar cells based on TiO 2 spheres consisting of ultrathin nanosheets with [001] facet exposed

An anatase TiO 2 material with hierarchically structured spheres consisting of ultrathin nanosheets with 100% of the [001] facet exposed was employed to fabricate dye-sensitized solar cells (DSC s). Investigation of the electron transport and back reaction of the DSCs by electrochemical impedance spectroscopy showed that the spheres had a threefold lower electron recombination rate compared to the conventional TiO 2 nanoparticles. In contrast, the effective electron diffusion coefficient, D n, was not sensitive to the variation of the TiO 2 morphology. The TiO 2 spheres showed the same Dn as that of the nanoparticles. The influence of TiCl 4 post-treatment on the conduction band of the TiO 2 spheres and on the kinetics of electron transport and back reactions was also investigated. It was found that the TiCl 4 post-treatment caused a downward shift of the TiO 2 conduction band edge by 30 meV. Meanwhile, a fourfold increase of the effective electron lifetime of the DSC was also observed after TiCl4 treatment. The synergistic effect of the variation of the TiO 2 conduction band and the electron recombination determined the open-circuit voltage of the DSC. © 2012 Wang et al.

Analysis and correction of voltage profile in low voltage distribution networks containing photovoltaic cells and electric vehicles

Voltage drop and rise at network peak and off–peak periods along with voltage unbalance are the major power quality problems in low voltage distribution networks. Usually, the utilities try to use adjusting the transformer tap changers as a solution for the voltage drop. They also try to distribute the loads equally as a solution for network voltage unbalance problem. On the other hand, the ever increasing energy demand, along with the necessity of cost reduction and higher reliability requirements, are driving the modern power systems towards Distributed Generation (DG) units. This can be in the form of small rooftop photovoltaic cells (PV), Plug–in Electric Vehicles (PEVs) or Micro Grids (MGs). Rooftop PVs, typically with power levels ranging from 1–5 kW installed by the householders are gaining popularity due to their financial benefits for the householders. Also PEVs will be soon emerged in residential distribution networks which behave as a huge residential load when they are being charged while in their later generation, they are also expected to support the network as small DG units which transfer the energy stored in their battery into grid. Furthermore, the MG which is a cluster of loads and several DG units such as diesel generators, PVs, fuel cells and batteries are recently introduced to distribution networks. The voltage unbalance in the network can be increased due to the uncertainties in the random connection point of the PVs and PEVs to the network, their nominal capacity and time of operation. Therefore, it is of high interest to investigate the voltage unbalance in these networks as the result of MGs, PVs and PEVs integration to low voltage networks. In addition, the network might experience non–standard voltage drop due to high penetration of PEVs, being charged at night periods, or non–standard voltage rise due to high penetration of PVs and PEVs generating electricity back into the grid in the network off–peak periods. In this thesis, a voltage unbalance sensitivity analysis and stochastic evaluation is carried out for PVs installed by the householders versus their installation point, their nominal capacity and penetration level as different uncertainties. A similar analysis is carried out for PEVs penetration in the network working in two different modes: Grid to vehicle and Vehicle to grid. Furthermore, the conventional methods are discussed for improving the voltage unbalance within these networks. This is later continued by proposing new and efficient improvement methods for voltage profile improvement at network peak and off–peak periods and voltage unbalance reduction. In addition, voltage unbalance reduction is investigated for MGs and new improvement methods are proposed and applied for the MG test bed, planned to be established at Queensland University of Technology (QUT). MATLAB and PSCAD/EMTDC simulation softwares are used for verification of the analyses and the proposals.

The cementogenic differentiation of periodontal ligament cells via the activation of Wnt/β-catenin signalling pathway by Li+ ions released from bioactive scaffolds

Lithium (Li) has been widely used as a long-term mood stabilizer in the treatment of bipolar and depressive disorders. Li+ ions are thought to enhance the remyelination of peripheral nerves and also stimulate the proliferation of neural progenitor cells and retinoblastoma cells via activation of the Wnt/β-catenin signalling pathway. Until now there have been no studies reporting the biological effects of released Li+ in bioactive scaffolds on cemetogenesis in periodontal tissue engineering applications. In this study, we incorporated parts of Li+ ions into the mesoporous bioactive glass (MBG) scaffolds and showed that this approach yielded scaffolds with a favourable composition, microstructure and mesopore properties for cell attachment, proliferation, and cementogenic differentiation of human periodontal ligament-derived cells (hPDLCs). We went on to investigate the biological effects of Li+ ions themselves on cell proliferation and cementogenic differentiation. The results showed that 5% Li+ ions incorporated into MBG scaffolds enhanced the proliferation and cementogenic differentiation of hPDLCs on scaffolds, most likely via activation of Wnt/β-catenin signalling pathway. Further study demonstrated that Li+ ions by themselves significantly enhanced the proliferation, differentiation and cementogenic gene expression of PDLCs. Our results indicate that incorporation of Li+ ions into bioactive scaffolds is a viable means of enhancing the Wnt canonical signalling pathway to stimulate cementogenic differentiation of PDLCs.

Proteomics Approaches in the Identification of Molecular Signatures of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are undifferentiated, multi-potent stem cells with the ability to renew. They can differentiate into many types of terminal cells, such as osteoblasts, chondrocytes, adipocytes, myocytes, and neurons. These cells have been applied in tissue engineering as the main cell type to regenerate new tissues. However, a number of issues remain concerning the use of MSCs, such as cell surface markers, the determining factors responsible for their differentiation to terminal cells, and the mechanisms whereby growth factors stimulate MSCs. In this chapter, we will discuss how proteomic techniques have contributed to our current knowledge and how they can be used to address issues currently facing MSC research. The application of proteomics has led to the identification of a special pattern of cell surface protein expression of MSCs. The technique has also contributed to the study of a regulatory network of MSC differentiation to terminal differentiated cells, including osteocytes, chondrocytes, adipocytes, neurons, cardiomyocytes, hepatocytes, and pancreatic islet cells. It has also helped elucidate mechanisms for growth factor–stimulated differentiation of MSCs. Proteomics can, however, not reveal the accurate role of a special pathway and must therefore be combined with other approaches for this purpose. A new generation of proteomic techniques have recently been developed, which will enable a more comprehensive study of MSCs. Keywords

Biological responses of human bone marrow mesenchymal stem cells to Sr-M-Si (M = Zn, Mg) silicate bioceramics

Strontium (Sr), Zinc (Zn), magnesium (Mg), and silicon (Si) are reported to be essential trace elements for the growth and mineralization of bone. We speculated that the combination of these bioactive elements in bioceramics may be effective to regulate the osteogenic property of boneforming cells. In this study, two Sr-containing silicate bioceramics, Sr2ZnSi2O7 (SZS) and Sr2MgSi2O7 (SMS), were prepared. The biological response of human bone marrow mesenchymal stem cells (BMSCs) to the two bioceramics (in the forms of powders and dense ceramic bulks) was systematically studied. In powder form, the effect of powder extracts on the viability and alkaline phosphatase (ALP) activity of BMSCs was investigated. In ceramic disc form, both direct and indirect coculture of BMSCs with ceramic discs were used to investigate their biological response, including attachment, proliferation, ALP activity, and bone-related genes expression. Beta-tricalcium phosphate (b-TCP) and akermanite (Ca2MgSi2O7, CMS) were used as control materials. The results showed that the Sr, Zn, and Si (or Sr, Mg, and Si)-containing ionic products from SZS and SMS powders enhanced ALP activity of BMSCs, compared to those from b-TCP. Both SZS and SMS ceramic discs supported the growth of BMSCs, and most importantly, significantly enhanced the ALP activity and bone-related genes expression of BMSCs as compared to b-TCP. The results suggest that the specific combination of bioactive ions (Sr, Zn, Si, e.g.) in bioceramics is a viable way to improve the biological performance of biomaterials, and the form of materials and surface properties were nonnegligible factors to influence cell response.

Computational design of cyclic nitroxides as efficient redox mediators for dye-sensitized solar cells

Cyclic nitroxide radicals represent promising alternatives to the iodine-based redox mediator commonly used in dye-sensitized solar cells (DSSCs). To date DSSCs with nitroxide-based redox mediators have achieved energy conversion efficiencies of just over 5 % but efficiencies of over 15 % might be achievable, given an appropriate mediator. The efficacy of the mediator depends upon two main factors: it must reversibly undergo one-electron oxidation and it must possess an oxidation potential in a range of 0.600-0.850 V (vs. a standard hydrogen electrode (SHE) in acetonitrile at 25 °C). Herein, we have examined the effect that structural modifications have on the value of the oxidation potential of cyclic nitroxides as well as the reversibility of the oxidation process. These included alterations to the N-containing skeleton (pyrrolidine, piperidine, isoindoline, azaphenalene, etc.), as well as the introduction of different substituents (alkyl-, methoxy-, amino-, carboxy-, etc.) to the ring. Standard oxidation potentials were calculated using high-level ab initio methodology that was demonstrated to be very accurate (with a mean absolute deviation from experimental values of only 16 mV). An optimal value of 1.45 for the electrostatic scaling factor for UAKS radii in acetonitrile solution was obtained. Established trends in the values of oxidation potentials were used to guide molecular design of stable nitroxides with desired E° ox and a number of compounds were suggested for potential use as enhanced redox mediators in DSSCs. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A screen for modifiers of epigenetic reprogramming

Epigenetic modifiers are the proteins involved in establishing and maintaining the epigenome of an organism. They are particularly important for development. Changes in epigenetic modifiers have been shown be lethal, or cause diseases. Our laboratory has developed an ENU mutagenesis screen to produce mouse mutants displaying altered epigenetic gene silencing. The screen relies on a GFP transgene that is expressed in red blood cells in a variegated manner. In the orginal transgenic FVB mice expression occurs in approximately 55% of red blood cells. During the course of my Masters, I characterised four different Mommes (Modifiers of murine metastable epiallele), MommeD32, MommeD33, MommeD35 and MommeD36. For each Momme, I identified the underlying mutation, and observed the corresponding phenotype. In MommeD32 the causative mutation is in Dnmt1, (DNA methyltransferase 1). This gene was previously identified in the screen, as MommeD2, and the new allele, MommeD32 has a change in the BAH domain of the protein. MommeD33 is the result of a change at the transgene itself. MommeD35 carries a mutation in Suv39h1 (suppressor of variegation 3-9 homolog 1). This gene has not previously been identified in the screen, but it is a known epigenetic modifier. MommeD36 had the same ENU treated sire as MommeD32, and I found that it has the same mutation as MommeD32. These mutant strains provide valuable tools that can be used to further our knowledge of epigenetic reprogramming. An example being the cancer study done with MommeD9 which has a mutation in Trim28. By crossing MommeD9+/- mutant mice with Trp53+/- mice, it can be seen if Trim28 has an effect on the rate of tumour genesis. However no clear effect of Trim28 haploinsufficiency can be observed in Trp53+/- mice.

Strontium-containing mesoporous bioactive glass scaffolds with improved osteogenic/cementogenic differentiation of periodontal ligament cells for periodontal tissue engineering

To achieve the ultimate goal of periodontal tissue engineering, it is of great importance to develop bioactive scaffolds which could stimulate the osteogenic/cementogenic differentiation of periodontal ligament cells (PDLCs) for the favorable regeneration of alveolar bone, root cementum, and periodontal ligament. Strontium (Sr) and Sr-containing biomaterials have been found to induce osteoblast activity. However, there is no systematic report about the interaction between Sr or Sr-containing biomaterials and PDLCs for periodontal tissue engineering. The aims of this study were to prepare Sr-containing mesoporous bioactive glass (Sr-MBG) scaffolds and investigate whether the addition of Sr could stimulate the osteogenic/cementogenic differentiation of PDLCs in tissue engineering scaffold system. The composition, microstructure and mesopore properties (specific surface area, nano-pore volume and nano-pore distribution) of Sr-MBG scaffolds were characterized. The proliferation, alkaline phosphatase (ALP) activity and osteogenesis/cementogenesis-related gene expression (ALP, Runx2, Col I, OPN and CEMP1) of PDLCs on different kinds of Sr-MBG scaffolds were systematically investigated. The results show that Sr plays an important role in influencing the mesoporous structure of MBG scaffolds in which high contents of Sr decreased the well-ordered mesopores as well as their surface area/pore volume. Sr2+ ions could be released from Sr-MBG scaffolds in a controlled way. The incorporation of Sr into MBG scaffolds has significantly stimulated ALP activity and osteogenesis/cementogenesis-related gene expression of PDLCs. Furthermore, Sr-MBG scaffolds in simulated body fluids environment still maintained excellent apatite-mineralization ability. The study suggests that the incorporation of Sr into MBG scaffolds is a viable way to stimulate the biological response of PDLCs. Sr-MBG scaffolds are a promising bioactive material for periodontal tissue engineering application.

ATF5, a possible regulator of osteogenic differentiation in human adipose-derived stem cells

The regulatory pathways involved in maintaining the pluripotency of embryonic stem cells are partially known, whereas the regulatory pathways governing adult stem cells and their "stem-ness" are characterized to an even lesser extent. We, therefore, screened the transcriptome profiles of 20 osteogenically induced adult human adipose-derived stem cell (ADSC) populations and investigated for putative transcription factors that could regulate the osteogenic differentiation of these ADSC. We studied a subgroup of donors' samples that had a disparate osteogenic response transcriptome from that of induced human fetal osteoblasts and the rest of the induced human ADSC samples. From our statistical analysis, we found activating transcription factor 5 (ATF5) to be significantly and consistently down-regulated in a randomized time-course study of osteogenically differentiated adipose-derived stem cells from human donor samples. Knockdown of ATF5 with siRNA showed an increased sensitivity to osteogenic induction. This evidence suggests a role for ATF5 in the regulation of osteogenic differentiation in adipose-derived stem cells. To our knowledge, this is the first report that indicates a novel role of transcription factors in regulating osteogenic differentiation in adult or tissue specific stem cells. © 2012 Wiley Periodicals, Inc.

Development of a gas nanosensor node powered by solar cells

This work focuses on the development of a stand-alone gas nanosensor node, powered by solar energy to track concentration of polluted gases such as NO2, N2O, and NH3. Gas sensor networks have been widely developed over recent years, but the rise of nanotechnology is allowing the creation of a new range of gas sensors [1] with higher performance, smaller size and an inexpensive manufacturing process. This work has created a gas nanosensor node prototype to evaluate future field performance of this new generation of sensors. The sensor node has four main parts: (i) solar cells; (ii) control electronics; (iii) gas sensor and sensor board interface [2-4]; and (iv) data transmission. The station is remotely monitored through wired (ethernet cable) or wireless connection (radio transmitter) [5, 6] in order to evaluate, in real time, the performance of the solar cells and sensor node under different weather conditions. The energy source of the node is a module of polycrystalline silicon solar cells with 410cm2 of active surface. The prototype is equipped with a Resistance-To-Period circuit [2-4] to measure the wide range of resistances (KΩ to GΩ) from the sensor in a simple and accurate way. The system shows high performance on (i) managing the energy from the solar panel, (ii) powering the system load and (iii) recharging the battery. The results show that the prototype is suitable to work with any kind of resistive gas nanosensor and provide useful data for future nanosensor networks.

Purine Nucleoside Phosphorylase mediated molecular chemotherapy and conventional chemotherapy: A tangible union against chemoresistant cancer

Background Late stage Ovarian Cancer is essentially incurable primarily due to late diagnosis and its inherent heterogeneity. Single agent treatments are inadequate and generally lead to severe side effects at therapeutic doses. It is crucial to develop clinically relevant novel combination regimens involving synergistic modalities that target a wider repertoire of cells and lead to lowered individual doses. Stemming from this premise, this is the first report of two- and three-way synergies between Adenovirus-mediated Purine Nucleoside Phosphorylase based gene directed enzyme prodrug therapy (PNP-GDEPT), docetaxel and/or carboplatin in multidrug-resistant ovarian cancer cells. Methods The effects of PNP-GDEPT on different cellular processes were determined using Shotgun Proteomics analyses. The in vitro cell growth inhibition in differentially treated drug resistant human ovarian cancer cell lines was established using a cell-viability assay. The extent of synergy, additivity, or antagonism between treatments was evaluated using CalcuSyn statistical analyses. The involvement of apoptosis and implicated proteins in effects of different treatments was established using flow cytometry based detection of M30 (an early marker of apoptosis), cell cycle analyses and finally western blot based analyses. Results Efficacy of the trimodal treatment was significantly greater than that achieved with bimodal- or individual treatments with potential for 10-50 fold dose reduction compared to that required for individual treatments. Of note was the marked enhancement in apoptosis that specifically accompanied the combinations that included PNP-GDEPT and accordingly correlated with a shift in the expression of anti- and pro-apoptotic proteins. PNP-GDEPT mediated enhancement of apoptosis was reinforced by cell cycle analyses. Proteomic analyses of PNP-GDEPT treated cells indicated a dowregulation of proteins involved in oncogenesis or cancer drug resistance in treated cells with accompanying upregulation of apoptotic- and tumour- suppressor proteins. Conclusion Inclusion of PNP-GDEPT in regular chemotherapy regimens can lead to significant enhancement of the cancer cell susceptibility to the combined treatment. Overall, these data will underpin the development of regimens that can benefit patients with late stage ovarian cancer leading to significantly improved efficacy and increased quality of life.