Structural characterization of a cytosine-rich potential quadruplex forming sequence in the EGFR promoter

I-motifs are tetra-helixes that may form in cytosine-rich strands. They are based on cytosine-cytosine(+) base pairs that require the N3 hemi-protonation of the nucleobases, and therefore, the stability of these non-canonical DNA arrangements depends on pH. These structures are promising targets for the development of new cancer therapies since they are enriched in the promoters of oncogenes where they can play a role in the regulation of transcription. The proximal promoter of the EGFR oncogene has multiple regions with a significant potential to form such a tetra-helix arrangement. Here, we present the thermodynamic characterization of a C-rich sequence located 37 nucleotides upstream of the transcription starting site of EGFR. We confirmed the ability of this sequence to fold into an I-motif. By applying a global analysis of calorimetric and spectroscopic data, we derived the dependency of the apparent standard Gibbs free energy change associated with the I-motif folding upon temperature and pH. The results showed that, in contrast to in silico prediction, only 4 CC+ base pairs formed while additional GC and TT base pairings were detected in the I-motif. Noteworthy, a single residue mutation at G14 largely shifts the equilibrium toward the formation of multimeric species

Multispectral satellite imaging improves detection of large individual fossils

Palaeontological field surveys in remote regions are a challenge, because of uncertainty in finding new specimens, high transportation costs, risks for the crew and a long time commitment. The effort can be facilitated by using high-resolution satellite imagery. Here we present a new opportunity to investigate remote fossil localities in detail, mapping the optical signature of individual fossils. We explain a practical workflow for detecting fossils using remote-sensing platforms and cluster algorithms. We tested the method within the Petrified Forest National Park, where fossil logs are sparse in a large area with mixed lithologies. We ran both unsupervised and supervised classifications, obtaining the best estimations for the presence of fossil logs using the likelihood and spectral angle mapper algorithms. We recognized general constraints and described logical and physical pros and cons of each estimated map. We also explained how the outcomes should be critically evaluated with consistent accuracy tests. Instead of searching for fossiliferous outcrops, our method targets single fossil specimens (or highly condensed accumulations), obtaining a significant increase in potential efficiency and effectiveness of field surveys. When repeatedly applied to the same region over time, it could also be useful for monitoring palaeontological heritage localities. Most importantly, the method here described is feasible, easily applicable to both fossil logs and bones, and represents a step towards standard best practices for applying remote sensing in the palaeontological field

Advanced microfabrication technologies for microspacecraft

Advanced microfabrication technologies offer the prospect of reducing the weight and size of spacecraft through the use of lighter and stronger materials in conjunction with new mechanical/structural design concepts and design optimization methods. At the same time, electronic components have been scaled down while increasing functional utility. A two-fold benefit is derived for space applications through the use of less expensive components and the lower launch costs associated with lighter components. GE-CRD is actively pursuing research in these key technologies for a wide range of applications including satellites. These key technologies will be reviewed and an update on GE progress will be given. The need to reduce weight and lower cost, while maintaining product quality and reliability are primary drivers in the design of satellites, in general, and microsatellites in particular. For the structural subsystem, these requirements pose a complex design problem unless new mechanical design concepts and computer-aided design optimization methods are employed. Several new concepts, such as battery packs doubling as panel reinforcements and fuel tanks as integral structural members, need to utilized. In addition, new viscoelastic material damping concepts for spacecraft components provide for lighter weight/lower cost designs, while satisfying the structural dynamics requirements. High density interconnect (HDI) technology permits the use of bare IC's on a ceramic substrate with 90 percent active area utilization. A copper/polyimide multilayer structure is the backbone of the technology, which has demonstrated a size/weight reduction of greater than 10x compared to printed circuit board with performance up to the GHz level. HDI modules have exceptional mechanical robustness as evidenced by survival of 180 kg rapid acceleration tests. Microelectromechanical systems (MEMS) are redefining sensors and actuators by miniaturization through micromachining techniques. Sharing many fabrication steps with HDI, we have developed a new technique for surface micromachining of copper/polyimide structures using computer-aided laser patterning

Na+, K+-ATPase activity in children with autism spectrum disorder: Searching for the reason(s) of its decrease in blood cells

Na+, K+-ATPase (NKA) activity, which establishes the sodium and potassium gradient across the cell membrane and is instrumental in the propagation of the nerve impulses, is altered in a number of neurological and neuropsychiatric disorders, including autism spectrum disorders (ASD). In the present work, we examined a wide range of biochemical and cellular parameters in the attempt to understand the reason(s) for the severe decrease in NKA activity in erythrocytes of ASD children that we reported previously. NKA activity in leukocytes was found to be decreased independently from alteration in plasma membrane fluidity. The different subunits were evaluated for gene expression in leukocytes and for protein expression in erythrocytes: small differences in gene expression between ASD and typically developing children were not apparently paralleled by differences in protein expression. Moreover, no gross difference in erythrocyte plasma membrane oxidative modifications was detectable, although oxidative stress in blood samples from ASD children was confirmed by increased expression of NRF2 mRNA. Interestingly, gene expression of some NKA subunits correlated with clinical features. Excess inhibitory metals or ouabain-like activities, which might account for NKA activity decrease, were ruled out. Plasma membrane cholesterol, but not phosphatidylcholine and phosphatidlserine, was slighty decreased in erythrocytes from ASD children. Although no compelling results were obtained, our data suggest that alteration in the erytrocyte lipid moiety or subtle oxidative modifications in NKA structure are likely candidates for the observed decrease in NKA activity. These findings are discussed in the light of the relevance of NKA in ASD. Autism Research 2018. \ua9 2018 The Authors. Autism Research published by International Society for Autism Research and Wiley Periodicals, Inc. Lay Summary: The activity of the cell membrane enzyme NKA, which is instrumental in the propagation of the nerve impulses, is severely decreased in erythrocytes from ASD children and in other brain disorders, yet no explanation has been provided for this observation. We strived to find a biological/biochemical cause of such alteration, but most queries went unsolved because of the complexity of NKA regulation. As NKA activity is altered in many brain disorders, we stress the relevance of studies aimed at understanding its regulation in ASD

Oxidative stress and erythrocyte membrane alterations in children with autism: correlation with clinical features

It has been suggested that oxidative stress may play a role in the pathogenesis of Autism Spectrum Disorders (ASD), but the literature reports somewhat contradictory results. To further investigate the issue, we evaluated a high number of peripheral oxidative stress parameters, and some related issues such as erythrocyte membrane functional features and lipid composition. Twenty-one autistic children (Au) aged 5 to 12 years, were gender and age-matched with 20 typically developing children (TD). Erythrocyte thiobarbituric acid reactive substances, urinary isoprostane and hexanoyl-lysine adduct levels were elevated in Au, thus confirming the occurrence of an imbalance of the redox status of Au, whilst other oxidative stress markers or associated parameters (urinary 8-oxo-dG, plasma radical absorbance capacity and carbonyl groups, erythrocyte superoxide dismutase and catalase activities) were unchanged. A very significant reduction of Na+/K+-ATPase activity (-66%, p<0.0001), a reduction of erythrocyte membrane fluidity and alteration in erythrocyte fatty acid membrane profile (increase in monounsaturated fatty acids, decrease in EPA and DHA-\u3c93 with a consequent increase in \u3c96/\u3c93 ratio) were found in Au compared to TD, without change in membrane sialic acid content. Some Au clinical features appear to be correlated with these findings; in particular, hyperactivity score appears to be related with some parameters of the lipidomic profile and membrane fluidity. Oxidative stress and erythrocyte membrane alterations may play a role in the pathogenesis of ASD and prompt the development of palliative therapeutic protocols. Moreover, the marked decrease in NKA could be potentially utilized as a peripheral biomarker of ASD

Optimising calcium phosphate cement formulations to widen clinical applications

The demand for reconstructive orthopaedic implants continues expanding at a reasonable pace as the incidence of fracture injuries and infectious diseases rises. There has been an increase in the clinical need for more effective synthetic bone graft materials due to the drawbacks of autogenous grafts. Since the 1980’s calcium phosphate cements (CPC’s) have attracted a great deal of interest due to their chemical similarities to natural bone; chemical, physical and mechanical characteristics have been investigated and manipulated to maximise osteoconductivity and osteointegration of these CPC’s since the start of their commercialisation. Here in this thesis, a series of investigation are complied to demonstrate novel and inventive approaches to expand the application of CPC’s: (1) limiting the liquid phase in the setting reaction of a brushite cement to produce monetite (dehydrated brushite) based cement, with increased solubility to overcome the problems faced by long term stability of hydroxyapatite (HA) cements; (2) manipulating the cement formulation to produce a cement that can set on a change in temperature, upon implantation, increasing handling time during surgeries; (3) incorporating therapeutic molecules to eliminate secondary surgeries following infectious diseases; (4) to enhance osteointegration of CPC’s by synchronising the degradation to natural bone formation. Results exhibit compressive strength appropriate for the application of cranioplasty; long term ageing studies demonstrates that the novel cement formulations do not hydrolyse to HA, eliminating the risk of catastrophic brittle failure that is commonly associated with CPC’s.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Association between SNAP-25 gene polymorphisms and cognition in autism: functional consequences and potential therapeutic strategies.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is involved in different neuropsychiatric disorders, including schizophrenia and attention-deficit/hyperactivity disorder. Consistently, SNAP-25 polymorphisms in humans are associated with hyperactivity and/or with low cognitive scores. We analysed five SNAP-25 gene polymorphisms (rs363050, rs363039, rs363043, rs3746544 and rs1051312) in 46 autistic children trying to correlate them with Childhood Autism Rating Scale and electroencephalogram (EEG) abnormalities. The functional effects of rs363050 single-nucleotide polymorphism (SNP) on the gene transcriptional activity, by means of the luciferase reporter gene, were evaluated. To investigate the functional consequences that SNAP-25 reduction may have in children, the behaviour and EEG of SNAP-25(+/-) adolescent mice (SNAP-25(+/+)) were studied. Significant association of SNAP-25 polymorphism with decreasing cognitive scores was observed. Analysis of transcriptional activity revealed that SNP rs363050 encompasses a regulatory element, leading to protein expression decrease. Reduction of SNAP-25 levels in adolescent mice was associated with hyperactivity, cognitive and social impairment and an abnormal EEG, characterized by the occurrence of frequent spikes. Both EEG abnormalities and behavioural deficits were rescued by repeated exposure for 21 days to sodium salt valproate (VLP). A partial recovery of SNAP-25 expression content in SNAP-25(+/-) hippocampi was also observed by means of western blotting. A reduced expression of SNAP-25 is responsible for the cognitive deficits in children affected by autism spectrum disorders, as presumably occurring in the presence of rs363050(G) allele, and for behavioural and EEG alterations in adolescent mice. VLP treatment could result in novel therapeutic strategies