Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Conjugated Polymer Nanoparticles for Biological Labeling and Delivery

Cancer remains one of the world’s most devastating diseases, with more than 10 million new cases every year. However, traditional treatments have proven insufficient for successful medical management of cancer due to the chemotherapeutics’ difficulty in achieving therapeutic concentrations at the target site, non-specific cytotoxicity to normal tissues, and limited systemic circulation lifetime. Although, a concerted effort has been placed in developing and successfully employing nanoparticle(NP)-based drug delivery vehicles successfully mitigate the physiochemical and pharmacological limitations of chemotherapeutics, work towards controlling the subcellular fate of the carrier, and ultimately its payload, has been limited. Because efficient therapeutic action requires drug delivery to specific organelles, the subcellular barrier remains critical obstacle to maximize the full potential of NP-based delivery vehicles. The aim of my dissertation work is to better understand how NP-delivery vehicles’ structural, chemical, and physical properties affect the internalization method and subcellular localization of the nanocarrier. In this work we explored how side-chain and backbone modifications affect the conjugated polymer nanoparticle (CPN) toxicity and subcellular localization. We discovered how subtle chemical modifications had profound consequences on the polymer’s accumulation inside the cell and cellular retention. We also examined how complexation of CPN with polysaccharides affects uptake efficiency and subcellular localization. This work also presents how changes to CPN backbone biodegradability can significantly affect the subcellular localization of the material. A series of triphenyl phosphonium-containing CPNs were synthesized and the effect of backbone modifications have on the cellular toxicity and intracellular fate of the material. A mitochondrial-specific polymer exhibiting time-dependent release is reported. Finally, we present a novel polymerization technique which allows for the controlled incorporation of electron-accepting benzothiadiazole units onto the polymer chain. This facilitates tuning CPN emission towards red emission. The work presented here, specifically, the effect that side-chain and structure, polysaccharide formulation and CPN degradability have on material’s uptake behavior, can help maximize the full potential of NP-based delivery vehicles for improved chemotherapeutic drug delivery.

Occurrence and Function of Hoogsteen Base Pairs in Nucleic Acids

Nucleic acids (DNA and RNA) play essential roles in the central dogma of biology for the storage and transfer of genetic information. The unique chemical and conformational structures of nucleic acids – the double helix composed of complementary Watson-Crick base pairs, provide the structural basis to carry out their biological functions. DNA double helix can dynamically accommodate Watson-Crick and Hoogsteen base-pairing, in which the purine base is flipped by ~180° degrees to adopt syn rather than anti conformation as in Watson-Crick base pairs. There is growing evidence that Hoogsteen base pairs play important roles in DNA replication, recognition, damage or mispair accommodation and repair. Here, we constructed a database for existing Hoogsteen base pairs in DNA duplexes by a structure-based survey from the Protein Data Bank, and structural analyses based on the resulted Hoogsteen structures revealed that Hoogsteen base pairs occur in a wide variety of biological contexts and can induce DNA kinking towards the major groove. As there were documented difficulties in modeling Hoogsteen or Watson-Crick by crystallography, we collaborated with the Richardsons’ lab and identified potential Hoogsteen base pairs that were mis-modeled as Watson-Crick base pairs which suggested that Hoogsteen can be more prevalent than it was thought to be. We developed solution NMR method combined with the site-specific isotope labeling to characterize the formation of, or conformational exchange with Hoogsteen base pairs in large DNA-protein complexes under solution conditions, in the absence of the crystal packing force. We showed that there are enhanced chemical exchange, potentially between Watson-Crick and Hoogsteen, at a sharp kink site in the complex formed by DNA and the Integration Host Factor protein. In stark contrast to B-form DNA, we found that Hoogsteen base pairs are strongly disfavored in A-form RNA duplex. Chemical modifications N1-methyl adenosine and N1-methyl guanosine that block Watson-Crick base-pairing, can be absorbed as Hoogsteen base pairs in DNA, but rather potently destabilized A-form RNA and caused helix melting. The intrinsic instability of Hoogsteen base pairs in A-form RNA endows the N1-methylation as a functioning post-transcriptional modification that was known to facilitate RNA folding, translation and potentially play roles in the epitranscriptome. On the other hand, the dynamic property of DNA that can accommodate Hoogsteen base pairs could be critical to maintaining the genome stability.

DNA damage induced by low energy electrons (LEEs)

Abstract : The major objective of our study is to investigate DNA damage induced by soft X-rays (1.5 keV) and low-energy electrons (˂ 30 eV) using a novel irradiation system created by Prof. Sanche’s group. Thin films of double-stranded DNA are deposited on either glass and tantalum substrates and irradiated under standard temperature and pressure surrounded by a N[subscript 2] environment. Base release (cytosine, thymine, adenine and guanine) and base modifications (8-oxo-7,8-dihydro -2’-deoxyguanosine, 5-hydroxymethyl-2’-deoxyuridine, 5-formyl-2’-deoxyuridine, 5,6-dihydrothymidine and 5,6-dihydro-2’-deoxy uridine) are analyzed and quantified by LC-MS/MS. Our results reveal larger damage yields in the sample deposited on tantalum than those on glass. This can be explained by an enhancement of damage due to low-energy electrons, which are emitted from the metal substrate. From a comparison of the yield of products, base release is the major type of damage especially for purine bases, which are 3-fold greater than base modifications. A proposed pathway leading to base release involves the formation of a transient negative ion (TNI) followed by dissociative electron attachment (DEA) at the N-g lycosidic bond. On the other hand, base modification products consist of two major types of chemical modifications, which include thymine methyl oxidation products that likely arises from DEA from the methyl group of thymine, and 5,6-dihydropyrimidine that can involve the initial addition of electrons, H atoms, or hydride ions to the 5,6-pyrimidine double bond.

Conjugated polymer nanoparticles for biological labeling and delivery

Cancer remains one of the world’s most devastating diseases, with more than 10 million new cases every year. However, traditional treatments have proven insufficient for successful medical management of cancer due to the chemotherapeutics’ difficulty in achieving therapeutic concentrations at the target site, non-specific cytotoxicity to normal tissues, and limited systemic circulation lifetime. Although, a concerted effort has been placed in developing and successfully employing nanoparticle(NP)-based drug delivery vehicles successfully mitigate the physiochemical and pharmacological limitations of chemotherapeutics, work towards controlling the subcellular fate of the carrier, and ultimately its payload, has been limited. Because efficient therapeutic action requires drug delivery to specific organelles, the subcellular barrier remains critical obstacle to maximize the full potential of NP-based delivery vehicles. The aim of my dissertation work is to better understand how NP-delivery vehicles’ structural, chemical, and physical properties affect the internalization method and subcellular localization of the nanocarrier. ^ In this work we explored how side-chain and backbone modifications affect the conjugated polymer nanoparticle (CPN) toxicity and subcellular localization. We discovered how subtle chemical modifications had profound consequences on the polymer’s accumulation inside the cell and cellular retention. We also examined how complexation of CPN with polysaccharides affects uptake efficiency and subcellular localization. ^ This work also presents how changes to CPN backbone biodegradability can significantly affect the subcellular localization of the material. A series of triphenyl phosphonium-containing CPNs were synthesized and the effect of backbone modifications have on the cellular toxicity and intracellular fate of the material. A mitochondrial-specific polymer exhibiting time-dependent release is reported. Finally, we present a novel polymerization technique which allows for the controlled incorporation of electron-accepting benzothiadiazole units onto the polymer chain. This facilitates tuning CPN emission towards red emission. ^ The work presented here, specifically, the effect that side-chain and structure, polysaccharide formulation and CPN degradability have on material’s uptake behavior, can help maximize the full potential of NP-based delivery vehicles for improved chemotherapeutic drug delivery.^

Integrated Omics for the global mapping of biomolecules in LDL

Circulating low density lipoproteins (LDL) are thought to play a crucial role in the onset and development of atherosclerosis, though the detailed molecular mechanisms responsible for their biological effects remain controversial. The complexity of biomolecules (lipids, glycans and protein) and structural features (isoforms and chemical modifications) found in LDL particles hampers the complete understanding of the mechanism underlying its atherogenicity. For this reason the screening of LDL for features discriminative of a particular pathology in search of biomarkers is of high importance. Three major biomolecule classes (lipids, protein and glycans) in LDL particles were screened using mass spectrometry coupled to liquid chromatography. Dual-polarity screening resulted in good lipidome coverage, identifying over 300 lipid species from 12 lipid sub-classes. Multivariate analysis was used to investigate potential discriminators in the individual lipid sub-classes for different study groups (age, gender, pathology). Additionally, the high protein sequence coverage of ApoB-100 routinely achieved (≥70%) assisted in the search for protein modifications correlating to aging and pathology. The large size and complexity of the datasets required the use of chemometric methods (Partial Least Square-Discriminant Analysis, PLS-DA) for their analysis and for the identification of ions that discriminate between study groups. The peptide profile from enzymatically digested ApoB-100 can be correlated with the high structural complexity of lipids associated with ApoB-100 using exploratory data analysis. In addition, using targeted scanning modes, glycosylation sites within neutral and acidic sugar residues in ApoB-100 are also being explored. Together or individually, knowledge of the profiles and modifications of the major biomolecules in LDL particles will contribute towards an in-depth understanding, will help to map the structural features that contribute to the atherogenicity of LDL, and may allow identification of reliable, pathology-specific biomarkers. This research was supported by a Marie Curie Intra-European Fellowship within the 7th European Community Framework Program (IEF 255076). Work of A. Rudnitskaya was supported by Portuguese Science and Technology Foundation, through the European Social Fund (ESF) and "Programa Operacional Potencial Humano - POPH".

Chemical and microstructural modifications in LiPON thin films exposed to atmospheric humidity

Lithium phosphorus oxynitride (LiPON), the widely used solid electrolyte for thin film microbatteries, is not compatible with the ambient humid temperatures. The reasons for reduction in ionic conductivity of LiPON thin films from 2.8 x 10(-6) Scm(-1) to 9.9 x 10(-10) Scm(-1) when exposed to air are analyzed with the aid of AC impedance measurements, SEM, XPS and stylus profilometry. Initially, particulate-free film surfaces obtained soon after rf sputter deposition in N-2 ambient conditions becomes covered with microstructures, forming pores in the film when exposed to air. LiPON films are deposited on Ti coated silicon in addition to bare silicon, ruling out the possibility of stress-related rupturing from the LiPON/Si interface. The reduction of nitrogen, phosphorus, and increased presence of lithium, oxygen and carbon over the film surface lowers the ionic conductivity of LiPON films when exposed to air. (c) 2011 Elsevier B.V. All rights reserved.

Chemical synthesis of DNA and RNA containing thio-substituted bases and their post-synthetic modifications

Modified oligonucleotides containing sulphur group have been useful tools for studies of carcinogenesis, protein or nucleic acid structures and functions, protein-nucleic acid interactions, and for antisense modulation of gene expression. One successful example has been the synthesis and study of oligodeoxynucleotides containing 6-thio-2'-deoxyguanine. 6-Thio-2-deoxyguanosine was first discovered as metabolic compound of 6- mercaptopurine (6-MP). Later, it was applied as drug to cure leukaemia. During the research of its toxicity, a method was developed to use the sulphur group as a versatile position for post-synthetic modification. The advantage of application of post-synthetic modification lies in its convenience. Synthesis of oligomers with normal sequences has become routine work in most laboratories. However, design and synthesis of a proper phosphoramidite monomer for a new modified nucleoside are always difficult tasks even for a skilful chemist. Thus an alternative method (post-synthetic method) has been invented to overcome the difficulties. This was achieved by incorporation of versatile nucleotides into oligomers which contain a leaving group, that is sufficiently stable to withstand the conditions of synthesis but can be substituted by nucleophiles after synthesis, to produce, a series of oligomers each containing a different modified base. In the current project, a phosphoramidite monomer with 6-thioguanine has been successfully synthesised and incorporated into RNA. A deprotection procedure, which is specific for RNA was designed for oligomers containing 6-thioguanosine. The results were validated by various methods (UV, HPLC, enzymatic digestion). Pioneer work in utilization of the versatile sulphur group for post-synthetic modification was also tested. Post-synthetic modification was also carried out on DNA with 6- deoxythioguanosine. Electrophilic reagents with various functional groups (alphatic, aromatic, fluorescent) and bi-functional groups have been attached with the oligomers.

Synthesis and modifications of metal oxide nanostructures and their applications

Transition metal oxides are functional materials that have advanced applications in many areas, because of their diverse properties (optical, electrical, magnetic, etc.), hardness, thermal stability and chemical resistance. Novel applications of the nanostructures of these oxides are attracting significant interest as new synthesis methods are developed and new structures are reported. Hydrothermal synthesis is an effective process to prepare various delicate structures of metal oxides on the scales from a few to tens of nanometres, specifically, the highly dispersed intermediate structures which are hardly obtained through pyro-synthesis. In this thesis, a range of new metal oxide (stable and metastable titanate, niobate) nanostructures, namely nanotubes and nanofibres, were synthesised via a hydrothermal process. Further structure modifications were conducted and potential applications in catalysis, photocatalysis, adsorption and construction of ceramic membrane were studied. The morphology evolution during the hydrothermal reaction between Nb2O5 particles and concentrated NaOH was monitored. The study demonstrates that by optimising the reaction parameters (temperature, amount of reactants), one can obtain a variety of nanostructured solids, from intermediate phases niobate bars and fibres to the stable phase cubes. Trititanate (Na2Ti3O7) nanofibres and nanotubes were obtained by the hydrothermal reaction between TiO2 powders or a titanium compound (e.g. TiOSO4·xH2O) and concentrated NaOH solution by controlling the reaction temperature and NaOH concentration. The trititanate possesses a layered structure, and the Na ions that exist between the negative charged titanate layers are exchangeable with other metal ions or H+ ions. The ion-exchange has crucial influence on the phase transition of the exchanged products. The exchange of the sodium ions in the titanate with H+ ions yields protonated titanate (H-titanate) and subsequent phase transformation of the H-titanate enable various TiO2 structures with retained morphology. H-titanate, either nanofibres or tubes, can be converted to pure TiO2(B), pure anatase, mixed TiO2(B) and anatase phases by controlled calcination and by a two-step process of acid-treatment and subsequent calcination. While the controlled calcination of the sodium titanate yield new titanate structures (metastable titanate with formula Na1.5H0.5Ti3O7, with retained fibril morphology) that can be used for removal of radioactive ions and heavy metal ions from water. The structures and morphologies of the metal oxides were characterised by advanced techniques. Titania nanofibres of mixed anatase and TiO2(B) phases, pure anatase and pure TiO2(B) were obtained by calcining H-titanate nanofibres at different temperatures between 300 and 700 °C. The fibril morphology was retained after calcination, which is suitable for transmission electron microscopy (TEM) analysis. It has been found by TEM analysis that in mixed-phase structure the interfaces between anatase and TiO2(B) phases are not random contacts between the engaged crystals of the two phases, but form from the well matched lattice planes of the two phases. For instance, (101) planes in anatase and (101) planes of TiO2(B) are similar in d spaces (~0.18 nm), and they join together to form a stable interface. The interfaces between the two phases act as an one-way valve that permit the transfer of photogenerated charge from anatase to TiO2(B). This reduces the recombination of photogenerated electrons and holes in anatase, enhancing the activity for photocatalytic oxidation. Therefore, the mixed-phase nanofibres exhibited higher photocatalytic activity for degradation of sulforhodamine B (SRB) dye under ultraviolet (UV) light than the nanofibres of either pure phase alone, or the mechanical mixtures (which have no interfaces) of the two pure phase nanofibres with a similar phase composition. This verifies the theory that the difference between the conduction band edges of the two phases may result in charge transfer from one phase to the other, which results in effectively the photogenerated charge separation and thus facilitates the redox reaction involving these charges. Such an interface structure facilitates charge transfer crossing the interfaces. The knowledge acquired in this study is important not only for design of efficient TiO2 photocatalysts but also for understanding the photocatalysis process. Moreover, the fibril titania photocatalysts are of great advantage when they are separated from a liquid for reuse by filtration, sedimentation, or centrifugation, compared to nanoparticles of the same scale. The surface structure of TiO2 also plays a significant role in catalysis and photocatalysis. Four types of large surface area TiO2 nanotubes with different phase compositions (labelled as NTA, NTBA, NTMA and NTM) were synthesised from calcination and acid treatment of the H-titanate nanotubes. Using the in situ FTIR emission spectrescopy (IES), desorption and re-adsorption process of surface OH-groups on oxide surface can be trailed. In this work, the surface OH-group regeneration ability of the TiO2 nanotubes was investigated. The ability of the four samples distinctively different, having the order: NTA > NTBA > NTMA > NTM. The same order was observed for the catalytic when the samples served as photocatalysts for the decomposition of synthetic dye SRB under UV light, as the supports of gold (Au) catalysts (where gold particles were loaded by a colloid-based method) for photodecomposition of formaldehyde under visible light and for catalytic oxidation of CO at low temperatures. Therefore, the ability of TiO2 nanotubes to generate surface OH-groups is an indicator of the catalytic activity. The reason behind the correlation is that the oxygen vacancies at bridging O2- sites of TiO2 surface can generate surface OH-groups and these groups facilitate adsorption and activation of O2 molecules, which is the key step of the oxidation reactions. The structure of the oxygen vacancies at bridging O2- sites is proposed. Also a new mechanism for the photocatalytic formaldehyde decomposition with the Au-TiO2 catalysts is proposed: The visible light absorbed by the gold nanoparticles, due to surface plasmon resonance effect, induces transition of the 6sp electrons of gold to high energy levels. These energetic electrons can migrate to the conduction band of TiO2 and are seized by oxygen molecules. Meanwhile, the gold nanoparticles capture electrons from the formaldehyde molecules adsorbed on them because of gold’s high electronegativity. O2 adsorbed on the TiO2 supports surface are the major electron acceptor. The more O2 adsorbed, the higher the oxidation activity of the photocatalyst will exhibit. The last part of this thesis demonstrates two innovative applications of the titanate nanostructures. Firstly, trititanate and metastable titanate (Na1.5H0.5Ti3O7) nanofibres are used as intelligent absorbents for removal of radioactive cations and heavy metal ions, utilizing the properties of the ion exchange ability, deformable layered structure, and fibril morphology. Environmental contamination with radioactive ions and heavy metal ions can cause a serious threat to the health of a large part of the population. Treatment of the wastes is needed to produce a waste product suitable for long-term storage and disposal. The ion-exchange ability of layered titanate structure permitted adsorption of bivalence toxic cations (Sr2+, Ra2+, Pb2+) from aqueous solution. More importantly, the adsorption is irreversible, due to the deformation of the structure induced by the strong interaction between the adsorbed bivalent cations and negatively charged TiO6 octahedra, and results in permanent entrapment of the toxic bivalent cations in the fibres so that the toxic ions can be safely deposited. Compared to conventional clay and zeolite sorbents, the fibril absorbents are of great advantage as they can be readily dispersed into and separated from a liquid. Secondly, new generation membranes were constructed by using large titanate and small ã-alumina nanofibres as intermediate and top layers, respectively, on a porous alumina substrate via a spin-coating process. Compared to conventional ceramic membranes constructed by spherical particles, the ceramic membrane constructed by the fibres permits high flux because of the large porosity of their separation layers. The voids in the separation layer determine the selectivity and flux of a separation membrane. When the sizes of the voids are similar (which means a similar selectivity of the separation layer), the flux passing through the membrane increases with the volume of the voids which are filtration passages. For the ideal and simplest texture, a mesh constructed with the nanofibres 10 nm thick and having a uniform pore size of 60 nm, the porosity is greater than 73.5 %. In contrast, the porosity of the separation layer that possesses the same pore size but is constructed with metal oxide spherical particles, as in conventional ceramic membranes, is 36% or less. The membrane constructed by titanate nanofibres and a layer of randomly oriented alumina nanofibres was able to filter out 96.8% of latex spheres of 60 nm size, while maintaining a high flux rate between 600 and 900 Lm–2 h–1, more than 15 times higher than the conventional membrane reported in the most recent study.

Histone deacetylase inhibitors target diabetes via chromatin remodeling or as chemical chaperones?

Globally, obesity and diabetes (particularly type 2 diabetes) represents a major challenge to world health. Despite decades of intense research efforts, the genetic basis involved in diabetes pathogenesis & conditions associated with obesity are still poorly understood. Recent advances have led to exciting new developments implicating epigenetics as an important mechanism underpinning diabetes and obesity related disease. One epigenetic mechanism known as the "histone code" describes the idea that specific patterns of post-translational modifications to histones act like a molecular "code" recognised and used by non-histone proteins to regulate specific chromatin functions. One modification which has received significant attention is that of histone acetylation. The enzymes which regulate this modification are described as lysine acetyltransferases or KATs and histone deacetylases or HDACs. Due to their conserved catalytic domain HDACs have been actively targeted as a therapeutic target. Some of the known inhibitors of HDACs (HDACi) have also been shown to act as "chemical chaperones" to alleviate diabetic symptoms. In this review, we discuss the available evidence concerning the roles of HDACs in regulating chaperone function and how this may have implications in the management of diabetes. © 2009 Bentham Science Publishers Ltd.

Surface chemical studies of Thiobacillus ferrooxidans with reference to copper tolerance

A strain of Thiobacillus ferrooxidans was adapted to grow at higher concentrations of copper by single step culturing in the presence of 20 g/L (0.314 mol/L) cupric ions added to 9K medium. Exposure to copper results in change in the surface chemistry of the microorganism. The isoelectric point of the adapted strain (pI=4.7) was observed to be at a higher pH than that of the wild unadapted strain(pI=2.0). Compared to the wild strain, the copper adapted strain was found to be more hydrophobic and showed enhanced attachment efficiency to the pyrite mineral. The copper adsorption ability of the adapted strain was also found to be higher than that of the wild strain. Fourier transform infrared spectroscopy of adapted cells suggested that a proteinaceous new cell surface component is synthesized by the adapted strain. Treatment of adapted cells with proteinase-K, resulted in complete loss of tolerance to copper, reduction in copper adsorption and hydrophobicity of the adapted cells. These observations strongly suggest a role played by cell surface modifications of Thiobacillus ferrooxidans in imparting the copper tolerance to the cells and bioleaching of sulphide minerals.

Shear sum rules at finite chemical potential

We derive sum rules which constrain the spectral density corresponding to the retarded propagator of the T-xy component of the stress tensor for three gravitational duals. The shear sum rule is obtained for the gravitational dual of the N = 4 Yang-Mills, theory of the M2-branes and M5-branes all at finite chemical potential. We show that at finite chemical potential there are additional terms in the sum rule which involve the chemical potential. These modifications are shown to be due to the presence of scalars in the operator product expansion of the stress tensor which have non-trivial vacuum expectation values at finite chemical potential.

Modelling induction skull melting design modifications

Induction Skull Melting (ISM) is a technique for heating, melting, mixing and, possibly, evaporating reactive liquid metals at high temperatures with a minimum contact at solid walls. The presented numerical modelling involves the complete time dependent process analysis based on the coupled electromagnetic, temperature and turbulent velocity fields during the melting and liquid shape changes. The simulation model is validated against measurements of liquid metal height, temperature and heat losses in a commercial size ISM furnace. The observed typical limiting temperature plateau for increasing input electrical power is explained by the turbulent convective heat losses. Various methods to increase the superheat within the liquid melt, the process energy efficiency and stability are proposed.