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Simultaneous SAXS-WAXD measurements are carried out to gain information about the microstructure and the molecular orientation developed by polymeric chains during the electrospinning process. Three semicrystalline polymers were studied, namely polyacrylonitrile, Nylon 6,6, and poly(ethylene oxide), as non-woven mats with either randomly arranged or aligned electrospun fibers. Mat thermal and morphological properties are investigated, together with their structural details in order to derive their hierarchical structure from the macro to the nano-scale. SAXS patterns have an elliptical shape with the main axis along the equator direction. No reflections are noticeable along the meridional direction, suggesting that the investigated electrospun fibers have a fibrillar structure with no trace of lamellar morphology. Combining the values of the unit cell and of the crystal size it can be concluded that in the fibers the ordered domains are organized into a bundle of fibrils due to the lateral aggregation of roughly 10 unit cells and the regular ordering of about either 50 (Nylon 6,6 and polyacrylonitrile) or 25 (poly(ethylene oxide)) cells in the chain direction.

The fragile X premutation (PM) allele contains a CGG expansion of 55-200 repeats in the FMR1 gene's promoter. Male PM carriers have an elevated risk of developing neurological and psychiatric changes, including an approximately 50% risk of the fragile X-associated tremor/ataxia syndrome (FXTAS). The aim of this study was to assess the relationships of regional white matter hyperintensities (wmhs) semi-quantitative scores, clinical status, motor (UPDRS, ICARS, Tremor) scales, and cognitive impairments, with FMR1-specific genetic changes, in a sample of 32 unselected male PM carriers aged 39-81 years. Half of these individuals were affected with FXTAS, while the non-FXTAS group comprised subcategories of non-affected individuals and individuals affected with non-syndromic changes. The dynamics of pathological processes at the cellular level relevant to the clinical status of PM carriers was investigated using the enzyme AMP-activated protein kinase (AMPK), which is a highly sensitive cellular stress-sensing alarm protein. This enzyme, as well as genetic markers - CGG repeat number and the levels of the FMR1 mRNA - were assessed in blood lymphoblasts. The results showed that the repeat distribution for FXTAS individuals peaked at 85-90 CGGs; non-FXTAS carriers were distributed within the lowest end of the PM repeat range, and non-syndromic carriers assumed an intermediate position. The size of the CGG expansion was significantly correlated, across all three categories, with infratentorial and total wmhs and with all motor scores, and the FMR1 mRNA levels with all the wmh scores, whilst AMPK activity showed considerable elevation in the non-FXTAS combined group, decreasing in the FXTAS group, proportionally to increasing severity of the wmhs and tremor/ataxia. We conclude that the size of the CGG expansion relates to the risk for FXTAS, to severity of infratentorial wmhs lesions, and to all three motor scale scores. FMR1 mRNA shows a strong association with the extent of wmhs, which is the most sensitive marker of the pathological process. However, the AMPK activity findings - suggestive of a role of this enzyme in the risk of FXTAS - need to be verified and expanded in future studies using larger samples and longitudinal assessment.

The fragile X premutation (PM) allele contains a CGG expansion of 55–200 repeats in the FMR1 gene’s promoter. Male PM carriers have an elevated risk of developing neurological and psychiatric changes, including an approximately 50% risk of the fragile X-associated tremor/ataxia syndrome (FXTAS). The aim of this study was to assess the relationships of regional white matter hyperintensities (wmhs) semi-quantitative scores, clinical status, motor (UPDRS, ICARS, Tremor) scales, and cognitive impairments, with FMR1-specific genetic changes, in a sample of 32 unselected male PM carriers aged 39–81 years. Half of these individuals were affected with FXTAS, while the non-FXTAS group comprised subcategories of non-affected individuals and individuals affected with non-syndromic changes. The dynamics of pathological processes at the cellular level relevant to the clinical status of PM carriers was investigated using the enzyme AMP-activated protein kinase (AMPK), which is a highly sensitive cellular stress-sensing alarm protein. This enzyme, as well as genetic markers – CGG repeat number and the levels of the FMR1 mRNA – were assessed in blood lymphoblasts. The results showed that the repeat distribution for FXTAS individuals peaked at 85–90 CGGs; non-FXTAS carriers were distributed within the lowest end of the PM repeat range, and non-syndromic carriers assumed an intermediate position. The size of the CGG expansion was significantly correlated, across all three categories, with infratentorial and total wmhs and with all motor scores, and the FMR1 mRNA levels with all the wmh scores, whilst AMPK activity showed considerable elevation in the non-FXTAS combined group, decreasing in the FXTAS group, proportionally to increasing severity of the wmhs and tremor/ataxia. We conclude that the size of the CGG expansion relates to the risk for FXTAS, to severity of infratentorial wmhs lesions, and to all three motor scale scores. FMR1 mRNA shows a strong association with the extent of wmhs, which is the most sensitive marker of the pathological process. However, the AMPK activity findings – suggestive of a role of this enzyme in the risk of FXTAS – need to be verified and expanded in future studies using larger samples and longitudinal assessment.

Summary The human protein arginine methyltransferase NDUFAF7 controls the assembly of the ∼1-MDa mitochondrial complex I (CI; the NADH ubiquinone oxidoreductase) by methylating its subunit NDUFS2. We determined crystal structures of MidA, the Dictyostelium ortholog of NDUFAF7. The MidA catalytic core domain resembles other eukaryotic methyltransferases. However, three large core loops assemble into a regulatory domain that is likely to control ligand selection. Binding of MidA to NDUFS2 is weakened by methylation, suggesting a mechanism for methylation-controlled substrate release. Structural and bioinformatic analyses support that MidA and NDUFAF7 and their role in CI assembly are conserved from bacteria to humans, implying that protein methylation already existed in proteobacteria. In vivo studies confirmed the critical role of the MidA methyltransferase activity for CI assembly, growth, and phototaxis of Dictyostelium . Collectively, our data elucidate the origin of protein arginine methylation and its use by MidA/NDUFAF7 to regulate CI assembly.

A diamond-patterned polyvinylpyrrolidone (PVP) fibre mat manufactured by electrospinning onto a structured target was studied by the combination of electron microscopy and synchrotron small angle X-ray scattering. Multi-scale hierarchical structure analysis revealed the relationship between different morphological levels, from an overall mat layout to individual fibres to polymer fibril orientation within them.

The continuous increase in the demand for highly aesthetic and natural-appearing dental restorations and the development of strong ceramic materials has led to the adoption of sintered ceramics as new load-bearing components used in dental prosthetics. Zirconia is one of the most attractive restorative materials due to its advantageous mechanical properties and biocompatibility. Veneering porcelains are used to coat the surface of zirconia to enhance the aesthetic appearance of prostheses. Nevertheless, porcelain-veneered zirconia restorations are prone to failure primarily by the fracture of the veneering layers. In this paper, the nature of the interfacial bonding and failure modes on samples of broken porcelain-veneered sintered zirconia restorations were studied using Environmental Scanning Electron Microscopy (ESEM) with Energy-Dispersive X-ray (EDX) analysis. Typical fractographic features were observed in broken porcelain-zirconia prosthesis. The chipping mode fractures in the veneering porcelain indicated the dominance of the cohesive fracture mode, in agreement with clinical experience reported in the literature. The crack initiation and propagation within the veneered porcelain layer was also observed and analyzed by a further examination of the fractographic features on both the prosthetic samples and the fractured surface of porcelain zirconia bars. The result indicates that the crack initiated at the location of maximum stress (point of occlusal contact). In addition, it is surmised that the fragility of the prosthesis may result from the high Vickers hardness and the associated low toughness of porcelain.

Yttria Partially Stabilized Zirconia (YPSZ) is one of the most important engineering ceramic materials in that it displays a whole host of outstanding structural and functional properties. Of particular importance for load-bearing applications is the remarkable fracture toughness of YPSZ that arises from its ability to undergo martensitic transformation, a phase transformation that is dependent on stress, temperature, time, humidity, grain size, and the proximity of an interface. The present study was aimed at revealing the influence of the thermal ageing on the tetragonal to monoclinic phase transformation in the near-surface regions of YPSZ. In order to perform qualitative and quantitative characterisation of the phase composition, three principal microscopic techniques were employed: atomic force microscopy, depth resolved Raman micro-spectroscopy, and synchrotron X-ray diffraction. Satisfactory consistency was achieved between the results obtained using different techniques. Moreover, the data obtained in this way displayed complementarity that provided valuable input for the development of thermodynamic modelling of the complex inter-dependence between phase state and processing history of zirconia ceramics.

The single Dictyostelium chaperonin 60 gene, hspA, was cloned, sequenced and characterized. Sequence comparisons and a three-dimensional model for the structure of the encoded protein showed that it exhibits the conserved sequence and structural features expected for its role as the Dictyostelium mitochondrial chaperonin 60. Dictyostelium hspA contains two introns and, unusually for a member of this major heat shock gene family, is not stress-inducible in response to heat, cold or cadmium ions. Although transcription of hspA is down regulated during early Dictyostelium development in response to starvation, the levels of the chaperonin 60 protein remain constant throughout the life cycle. Consistent with the essential role of chaperonin 60 in mitochondrial biogenesis, we were unable to isolate mutants in which the hspA gene had been disrupted. However, transformants were isolated that exhibited differing levels of antisense inhibition of chaperonin 60 expression, depending upon the number of copies of the antisense-expressing plasmid in the genome. Orientation in phototaxis (and thermotaxis) was severely impaired in all antisense transformants, while growth and morphogenesis were markedly defective only in transformants with higher levels of antisense inhibition. This pattern of phenotypes is similar to that reported previously to result from targeted disruption of the mitochondrial large subunit rRNA gene in a subpopulation of mitochondria. This suggests that, regardless of the nature of the underlying genetic defect, mitochondrial deficiency impairs signal transduction more sensitively than other cellular activities.

Background Most neurodegenerative diseases are associated with mitochondrial dysfunction. In humans, mutations in mitochondrial genes result in a range of phenotypic outcomes which do not correlate well with the underlying genetic cause. Other neurodegenerative diseases are caused by mutations that affect the function and trafficking of lysosomes, endosomes and autophagosomes. Many of the complexities of these human diseases can be avoided by studying them in the simple eukaryotic model Dictyostelium discoideum . Scope of review This review describes research using Dictyostelium to study cytopathological pathways underlying a variety of neurodegenerative diseases including mitochondrial, lysosomal and vesicle trafficking disorders. Major conclusions Generalised mitochondrial respiratory deficiencies in Dictyostelium produce a consistent pattern of defective phenotypes that are caused by chronic activation of a cellular energy sensor AMPK (AMP-activated protein kinase) and not ATP deficiency per se . Surprisingly, when individual subunits of Complex I are knocked out, both AMPK-dependent and AMPK-independent, subunit-specific phenotypes are observed. Many nonmitochondrial proteins associated with neurological disorders have homologues in Dictyostelium and are associated with the function and trafficking of lysosomes and endosomes. Conversely, some genes associated with neurodegenerative disorders do not have homologues in Dictyostelium and this provides a unique avenue for studying these mutated proteins in the absence of endogeneous protein. General significance Using the Dictyostelium model we have gained insights into the sublethal cytopathological pathways whose dysregulation contributes to phenotypic outcomes in neurodegenerative disease. This work is beginning to distinguish correlation, cause and effect in the complex network of cross talk between the various organelles involved. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research

To investigate protein import into the mitochondria of Dictyostelium discoideum , green fluorescent protein (GFP) was fused as a reporter protein either to variable lengths of the N-terminal region of chaperonin 60 (the first 23, 40, 80, 97, and 150 amino acids) or to the mitochondrial targeting sequence of DNA topoisomerase II. The fusion proteins were expressed in AX2 cells under the actin-15 promoter. Fluorescence images of GFP transformants confirmed that Dictyostelium chaperonin 60 is a mitochondrial protein. The level of the mitochondrially targeted GFP fusion proteins was unexpectedly much lower than the nontargeted (cytoplasmic) forms. The distinction between targeted and nontargeted protein activities was investigated at both the transcriptional and translational levels in vivo. We found that targeting GFP to the mitochondria results in reduced levels of the fusion protein even though transcription of the fusion gene and the stability of the protein are unaffected. [ 35 S]methionine labeling and GFP immunoprecipitation confirmed that mitochondrially targeted GFP is translated at much slower rates than nontargeted GFP. The results indicate a novel phenomenon, import-associated translational inhibition, whereby protein import into the mitochondria limits the rate of translation. The simplest explanation for this is that import of the GFP fusion proteins occurs cotranslationally, i.e., protein synthesis and import into mitochondria are coupled events. Consistent with cotranslational import, Northern analysis showed that the GFP mRNA is associated with isolated mitochondria. This association occurred regardless of whether the GFP was fused to a mitochondrial leader peptide. However, the presence of an import-competent leader peptide stabilized the mRNA-mitochondria association, rendering it more resistant to extensive EDTA washing. In contrast with GFP, the mRNA of another test protein, aequorin, did not associate with the mitochondria, and its translation was unaffected by import of the encoded polypeptide into the mitochondria.

The single Dictyostelium chaperonin 60 gene, hspA, was cloned, sequenced and characterized. Sequence comparisons and a three-dimensional model for the structure of the encoded protein showed that it exhibits the conserved sequence and structural features expected for its role as the Dictyostelium mitochondrial chaperonin 60. Dictyostelium hspA contains two introns and, unusually for a member of this major heat shock gene family, is not stress-inducible in response to heat, cold or cadmium ions. Although transcription of hspA is down regulated during early Dictyostelium development in response to starvation, the levels of the chaperonin 60 protein remain constant throughout the life cycle. Consistent with the essential role of chaperonin 60 in mitochondrial biogenesis, we were unable to isolate mutants in which the hspA gene had been disrupted. However, transformants were isolated that exhibited differing levels of antisense inhibition of chaperonin 60 expression, depending upon the number of copies of the antisense-expressing plasmid in the genome. Orientation in phototaxis (and thermotaxis) was severely impaired in all antisense transformants, while growth and morphogenesis were markedly defective only in transformants with higher levels of antisense inhibition. This pattern of phenotypes is similar to that reported previously to result from targeted disruption of the mitochondrial large subunit rRNA gene in a subpopulation of mitochondria. This suggests that, regardless of the nature of the underlying genetic defect, mitochondrial deficiency impairs signal transduction more sensitively than other cellular activities.

The dentine-enamel junction (DEJ) is an Important internal interface with strong and durable bonding between the hard outer layer (enamel) and the soft inner tooth core (dentine). This study investigated the microstructure of the DEJ by X-ray imaging method and synchrotron X-ray scattering techniques. Further high resolution elastic modulus mapping indicates that the DEJ is a band with a graded mechanical property rather than a discrete interface. The microstructure-property relationship is also illustrated. The knowledge of the architecture and properties of the natural DEJ will help in the biomimetic development of dental restorations and novel replacement materials and application techniques.

The relationship between the ultrastructure of human enamel and its mechanical behaviour is studied in this paper. Two synchrotron X-ray diffraction techniques, wide and small angle X-ray scattering (WAXS/SAXS) were used in combination to obtain multi-scale quantitative information about the response of human enamel to in situ uniaxial compressive loading. The interpretation of WAXS data gives elastic lattice strains within the hydroxyapatite (HAp) crystals, the stiff reinforcing phase in human enamel. The apparent modulus was determined linking the external load and the internal HAp strain. SAXS interpretation, allows the quantification of the nano-scale HAp crystallite distribution within human enamel. A multi-scale Eshelby equivalent inclusion model of the enamel was proposed that represents the hierarchical mineralized tissue as a two-level composite: micro-level model with rod embedded in the homogenised enamel material, and nano-level model with HAp crystallites embedded in the rod. Satisfactory agreement was achieved between model and experiment, suggesting that the new multi-scale approach accurately reflects the structure and mechanics of human enamel, and may help guide new biomimetic designs.

Engineering microscopy is a term we use to refer to a suite of versatile techniques for spatially resolved characterisation of material structure and properties for the purpose of optimising design, performance and durability of structures and technological systems. The range of tools that can be used for this purpose includes beams of photons (including X-rays), electrons, neutrons, and ions. Different modes of imaging include absorption and emission, spectroscopy, and scattering that can be used in full field or scanning regimes. The approaches that collect information in the form of 2D images can also be extended to 3D characterisation by serial sectioning or reconstruction tomography. An important additional mode of near-surface property evaluation arises through the use of nanoscale contact tip sensors, such as AFM, nanoindentation, electrochemical probes, etc. Crucial underpinning for multi-beam microscopic characterization is provided by multi-scale materials modelling. The lecture will provide an overview of flavours of engineering microscopy and highlight the exciting opportunities presented by the combination of techniques in the form of so-called correlative microscopy. Examples of multi-modal correlative microscopy will include partially stabilized zirconia, biomaterials such as flax fibres and human dental tissues, and also advanced engineering alloys and ceramics″.

Digital Image Correlation (DIC), as a technique based on image processing, has been intensively used for displacement measurement. The setup of DIC analysis starting condition is crucial to the validity of the results. In this paper, the influence of correlation window size (corrsize), one of the parameters that need to be chosen by users for setup, is studied. The results show that DIC is reliable as long as the corrsize is between a lower bound (corssizemin) and an upper bound (corssizemax). The reason why DIC returns high error factor when beyond corssizemax is related to image boundary effect, which is demonstrated by an equation serving as a criterion for corrsize choice. The study in this paper is particularly vital in cases where the Region of Interest (ROI) is close to the image and the real displacement is large.