Deoxycytidine glyoxal:Lesion induction and evidence of repair following vitamin C supplementation in vivo

Oxidative DNA damage is postulated to be involved in carcinogenesis, and as a consequence, dietary antioxidants have received much interest. A recent report indicates that vitamin C facilitates the decomposition of hydroperoxides in vitro, generating reactive aldehydes. We present evidence for the in vivo generation of glyoxal, an established product of lipid peroxidation, glucose/ascorbate autoxidation, or free radical attack of deoxyribose, following supplementation of volunteers with 400 mg/d vitamin C. Utilizing a monoclonal antibody to a deoxycytidine-glyoxal adduct (gdC), we measured DNA lesion levels in peripheral blood mononuclear cells. Supplementation resulted in significant (p = .001) increases in gdC levels at weeks 11, 16, and 21, with corresponding increases in plasma malondialdehyde levels and, coupled with previous findings, is strongly suggestive of a pro-oxidative effect. However, continued supplementation revealed a highly significant (p = .0001) reduction in gdC levels. Simultaneous analysis of cyclobutane thymine dimers revealed no increase upon supplementation but, as with gdC, levels decreased. Although no single mechanism is identified, our data demonstrate a pro-oxidant event in the generation of reactive aldehydes following vitamin C supplementation in vivo. These results are also consistent with our hypothesis for a role of vitamin C in an adaptive/repair response and indicate that nucleotide excision repair specifically may be affected. © 2003 Elsevier Science Inc.

Biomorphous SiC ceramics prepared from cork oak as precursor

Porous ceramic materials of SiC were synthesized from carbon matrices obtained via pyrolysis of natural cork as precursor. We propose a method for the fabrication of complex-shaped porous ceramic hardware consisting of separate parts prepared from natural cork. It is demonstrated that the thickness of the carbon-matrix walls can be increased through their impregnation with Bakelite phenolic glue solution followed by pyrolysis. This decreases the material's porosity and can be used as a way to modify its mechanical and thermal characteristics. Both the carbon matrices (resulted from the pyrolysis step) and the resultant SiC ceramics are shown to be pseudomorphous to the structure of initial cork. Depending on the synthesis temperature, 3C-SiC, 6H-SiC, or a mixture of these polytypes, could be obtained. By varying the mass ratio of initial carbon and silicon components, stoichiometric SiC or SiC:C:Si, SiC:C, and SiC:Si ceramics could be produced. The structure, as well as chemical and phase composition of the prepared materials were studied by means of Raman spectroscopy and scanning electron microscopy.

A highly emissive inorganic hexamolybdenum cluster complex as a handy precursor for the preparation of new luminescent materials

The synthesis and characterisation of a new, highly luminescent inorganic cluster complex, (Bu4N)2[Mo6I 8(NO3)6], are described. The complex possesses labile nitrato ligands and is therefore a useful precursor for the design of new luminescent materials. To exemplify this, functionalised polystyrene beads have been utilised as "polymeric ligands" to immobilise the molybdenum cluster complex. This journal is © the Partner Organisations 2014.

Functional neuroanatomy and behavioural correlates of the basal ganglia:evidence from lesion studies

Introduction: The basal ganglia are interconnected with cortical areas involved in behavioural, cognitive and emotional processes, in addition to movement regulation. Little is known about which of these functions are associated with individual basal ganglia substructures. Methods: Pubmed was searched for literature related to behavioural, cognitive and emotional symptoms associated with focal lesions to basal ganglia structures in humans. Results: Six case-control studies and two case reports were identified as relevant. Lesion sites included the caudate nucleus, putamen and globus pallidus. These were associated with a spectrum of behavioural and cognitive symptoms, including abulia, poor working memory and deficits in emotional recognition. Discussion: It is often difficult to precisely map associations between cognitive, emotional or behavioural functions and particular basal ganglia substructures, due to the non-specific nature of the lesions. However, evidence from lesion studies shows that most symptoms correspond with established non-motor frontal-subcortical circuits. © 2013-IOS Press and the authors. All rights reserved.

A proteo-liposome system for the analysis of the intracellular interactome of membrane proteins using amyloid precursor protein as a model

Transmembrane proteins play crucial roles in many important physiological processes. The intracellular domain of membrane proteins is key for their function by interacting with a wide variety of cytosolic proteins. It is therefore important to examine this interaction. A recently developed method to study these interactions, based on the use of liposomes as a model membrane, involves the covalent coupling of the cytoplasmic domains of membrane proteins to the liposome membrane. This allows for the analysis of interaction partners requiring both protein and membrane lipid binding. This thesis further establishes the liposome recruitment system and utilises it to examine the intracellular interactome of the amyloid precursor protein (APP), most well-known for its proteolytic cleavage that results in the production and accumulation of amyloid beta fragments, the main constituent of amyloid plaques in Alzheimer’s disease pathology. Despite this, the physiological function of APP remains largely unclear. Through the use of the proteo-liposome recruitment system two novel interactions of APP’s intracellular domain (AICD) are examined with a view to gaining a greater insight into APP’s physiological function. One of these novel interactions is between AICD and the mTOR complex, a serine/threonine protein kinase that integrates signals from nutrients and growth factors. The kinase domain of mTOR directly binds to AICD and the N-terminal amino acids of AICD are crucial for this interaction. The second novel interaction is between AICD and the endosomal PIKfyve complex, a lipid kinase involved in the production of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol-3-phosphate, which has a role in controlling ensdosome dynamics. The scaffold protein Vac14 of the PIKfyve complex binds directly to AICD and the C-terminus of AICD is important for its interaction with the PIKfyve complex. Using a recently developed intracellular PI(3,5)P2 probe it is shown that APP controls the formation of PI(3,5)P2 positive vesicular structures and that the PIKfyve complex is involved in the trafficking and degradation of APP. Both of these novel APP interactors have important implications of both APP function and Alzheimer’s disease. The proteo-liposome recruitment method is further validated through its use to examine the recruitment and assembly of the AP-2/clathrin coat from purified components to two membrane proteins containing different sorting motifs. Taken together this thesis highlights the proteo-liposome recruitment system as a valuable tool for the study of membrane proteins intracellular interactome. It allows for the mimicking of the protein in its native configuration therefore identifying weaker interactions that are not detected by more conventional methods and also detecting interactions that are mediated by membrane phospholipids.

The amyloid precursor protein controls PIKfyve function

While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease.

The role of amyloid precursor protein in neuronal and non-neuronal cell lines

Models of Alzheimer’s disease (AD) have provided useful insights into the pathogenesis and mechanistic pathways that lead to its development. One emerging idea about AD is that it may be described as a hypometabolic disorder due to the reduction of glucose uptake in AD brains. Inappropriate processing of Amyloid Precursor Protein (APP) is considered central to the initiation and progression of the disease. Although the exact role of APP misprocessing is unclear, it may play a role in neuronal metabolism before the onset of neurodegeneration. To investigate the potential role of APP in neuronal metabolism, the SHSY5Y neuroblastoma cell line was used to generate cell lines that stably overexpress wild type APP695 or express Swedish mutated-APP observed in familial AD (FAD), both under the control of the neuronal promoter, Synapsin I. The effects of APP on glucose uptake, cellular stress and energy homeostasis were studied extensively. It was found that APP-overexpressing cells exhibited decreased glucose uptake with changes in basal oxygen consumption in comparison to control cell lines. Similar studies were also performed in fibroblasts taken from FAD patients compared with control fibroblasts. Previous studies found FAD-derived fibroblasts displayed altered metabolic profiles, calcium homeostasis and oxidative stress when compared to controls. As such, in this study fibroblasts were studied in terms of their ability to metabolise glucose and their mitochondrial function. Results show that FAD-derived fibroblasts demonstrate no differences in mitochondrial function, or response to oxidative stress compared to control fibroblasts. However, control fibroblasts treated with Aβ1-42 demonstrated changes in glucose uptake. This study highlights the importance of APP expression within non-neuronal cell lines, suggesting that whilst AD is considered a brain-associated disorder, peripheral effects in non-neuronal cell types should also be considered when studying the effects of Aβ on metabolism.

The amyloid precursor protein (APP) binds the PIKfyve complex and modulates its function

Phosphoinositides are important components of eukaryotic membranes that are required for multiple forms of membrane dynamics. Phosphoinositides are involved in defining membrane identity, mediate cell signalling and control membrane trafficking events. Due to their pivotal role in membrane dynamics, phosphoinositide de-regulation contributes to various human diseases. In this review, we will focus on the newly emerging regulation of the PIKfyve complex, a phosphoinositide kinase that converts the endosomal phosphatidylinositol-3-phosphate [PI(3)P] to phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2)], a low abundance phosphoinositide of outstanding importance for neuronal integrity and function. Loss of PIKfyve function is well known to result in neurodegeneration in both mousemodels and human patients. Our recent work has surprisingly identified the amyloid precursor protein (APP), the central molecule in Alzheimer s disease aetiology, as a novel interaction partner of a subunit of the PIKfyve complex, Vac14. Furthermore, it has been shown that APP modulates PIKfyve function and PI(3,5)P2 dynamics, suggesting that the APP gene family functions as regulator of PI(3,5)P2 metabolism. The recent advances discussed in this review suggest a novel, unexpected, â-amyloid-independent mechanism for neurodegeneration in Alzheimer s disease.

Self-assembled SnO2 micro- and nanosphere-based gas sensor thick films from an alkoxide-derived high purity aqueous colloid precursor

Tin oxide is considered to be one of the most promising semiconductor oxide materials for use as a gas sensor. However, a simple route for the controllable build-up of nanostructured, sufficiently pure and hierarchical SnO2 structures for gas sensor applications is still a challenge. In the current work, an aqueous SnO2 nanoparticulate precursor sol, which is free of organic contaminants and sorbed ions and is fully stable over time, was prepared in a highly reproducible manner from an alkoxide Sn(OR)4 just by mixing it with a large excess of pure neutral water. The precursor is formed as a separate liquid phase. The structure and purity of the precursor is revealed using XRD, SAXS, EXAFS, HRTEM imaging, FTIR, and XRF analysis. An unconventional approach for the estimation of the particle size based on the quantification of the Sn-Sn contacts in the structure was developed using EXAFS spectroscopy and verified using HRTEM. To construct sensors with a hierarchical 3D structure, we employed an unusual emulsification technique not involving any additives or surfactants, using simply the extraction of the liquid phase, water, with the help of dry butanol under ambient conditions. The originally generated crystalline but yet highly reactive nanoparticles form relatively uniform spheres through self-assembly and solidify instantly. The spheres floating in butanol were left to deposit on the surface of quartz plates bearing sputtered gold electrodes, producing ready-for-use gas sensors in the form of ca. 50 μm thick sphere-based-films. The films were dried for 24 h and calcined at 300°C in air before use. The gas sensitivity of the structures was tested in the temperature range of 150-400°C. The materials showed a very quickly emerging and reversible (20-30 times) increase in electrical conductivity as a response to exposure to air containing 100 ppm of H2 or CO and short (10 s) recovery times when the gas flow was stopped.

Silica promoted self-assembled mesoporous aluminas. Impact of the silica precursor on the structural, textural and acidic properties

This paper investigates the effect of silica addition on the structural, textural and acidic properties of an evaporation induced self-assembled (EISA) mesoporous alumina. Two silica addition protocols were applied while maintaining the EISA synthesis route. The first route is based on the addition of a Na-free colloidal silica suspension (Ludox®), and the second method consists of the co-hydrolysis of tetraethyl orthosilicate (TEOS) with aluminium tri-sec-butoxide, to favour a more intimate mixing of the Al- and Si-hydrolysed species. The properties of the so derived materials were compared to the SiO2-free counterpart. The SiO2 addition was always beneficial from a structural and textural standpoint. TEOS appears to have a truly promoting effect; the ordering, surface area and pore volume are all improved. For Ludox®, the enhancement comes from the formation of smaller pores by a densification of the structure. The crystallization of γ-alumina depends on the interaction between the Al- and Si-species in the mesophase. Ludox®-based materials achieved crystallization at 750 °C but the intimate mixing in the TEOS-based mesophases shows a suppression of the phase transformation by 50-100 °C, with respect to the SiO2-free counterpart. This reduces the textural features substantially. For all SiO2-modified materials, the enhancement in the surface area is not accompanied by a concomitant improvement of total acidity, and the formation of weak Lewis acid sites was promoted. These effects were ascribed to SiO2 migration to the surface that blocks part of the acidity.

Instability of CTG Repeats is Governed by the Position of a DNA Base Lesion through Base Excision Repair

Trinucleotide repeat (TNR) expansions and deletions are associated with human neurodegeneration and cancer. However, their underlying mechanisms remain to be elucidated. Recent studies have demonstrated that CAG repeat expansions can be initiated by oxidative DNA base damage and fulfilled by base excision repair (BER), suggesting active roles for oxidative DNA damage and BER in TNR instability. Here, we provide the first evidence that oxidative DNA damage can induce CTG repeat deletions along with limited expansions in human cells. Biochemical characterization of BER in the context of (CTG)20 repeats further revealed that repeat instability correlated with the position of a base lesion in the repeat tract. A lesion located at the 59-end of CTG repeats resulted in expansion, whereas a lesion located either in the middle or the 39-end of the repeats led to deletions only. The positioning effects appeared to be determined by the formation of hairpins at various locations on the template and the damaged strands that were bypassed by DNA polymerase b and processed by flap endonuclease 1 with different efficiency. Our study indicates that the position of a DNA base lesion governs whether TNR is expanded or deleted through BER.

Electrical properties of platinum interconnects deposited by electron beam induced deposition of the carbon-free precursor, Pt(PF3)4

Comprehensive analysis of the electrical properties, structure and composition of Pt interconnects, developed via mask-less, electron beam induced deposition of the carbon-free Pt precursor, Pt(PF3)4, is presented. The results demonstrate significantly improved electrical performance in comparison with that generated from the standard organometallic precursor, (CH3)3Pt(CpCH3). In particular, the Pt interconnects exhibited perfect ohmic behavior and resistivity that can be diminished to 0.24 × 10−3 Ω cm, which is only one order of magnitude higher than bulk Pt, in comparison to 0.2 Ω cm for the standard carbon-containing interconnects. A maximum current density of 1.87 × 107 A cm−2 was achieved for the carbon-free Pt, compared to 9.44 × 105 A cm−2 for the standard Pt precursor. The enhanced electrical properties of the as-deposited materials can be explained by the absence of large amounts of carbon impurities, and their further improvement by postdeposition annealing in N2. In-situ TEM heating experiments confirmed that the annealing step induces sintering of the Pt nanocrystals and improved crystallinity, which contributes to the enhanced electrical performance. Alternative annealing under reducing conditions resulted in improved performance of the standard Pt interconnects, while the carbon-free deposit suffered electrical and structural breakage due to formation of larger Pt islands