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The objectives were to characterize the kinetics of acute phase proteins (APPs) α-2 macroglobulin (A2M), α-1 acid glycoprotein (A1AGP), and fibrinogen (FIB), and injection site macroscopic and microscopic findings following intramuscular administration of tetanus toxoid, reduced diphtheria toxoid, and acellular pertussis vaccine (TDaP; Adacel); adjuvants (aluminum phosphate [AlPO4]; aluminum hydroxide, Al[OH]3; CpG/Al[OH]3; or Quillaja saponaria 21 [QS-21]); or saline to female Wistar Han rats. Intravascular lipopolysaccharide (LPS) was a positive control. Injection sites and lymph nodes were evaluated microscopically, using hematoxylin and eosin (H&E) stained sections, 48 hours postdose (HPD) and compared with APP concentrations; A2M and A1AGP were measured using Meso Scale Discovery analyzer. Fibrinogen was measured on STA Compact analyzer. In a time-course study, APP peaked at 24 or 48 HPD. In a subsequent study at 48 HPD, injection site microscopic changes included inflammation and muscle degeneration/necrosis, which was different in severity/nature between groups. The APPs were not increased in rats administered saline, Al(OH)3, or AlPO4. Fibrinogen and A1AGP increased in rats administered CpG/Al(OH)3, QS-21, or TDaP; and A2M increased in rats administered QS-21. Fibrinogen, A2M, and A1AGP increased after LPS administration. Acute phase proteins can be used to monitor inflammatory responses to adjuvants; however, some adjuvants may induce inflammation without higher APPs.

Plastic has rapidly transformed our world, with many aspects of human life now relying on a variety of plastic materials. Biological plastic degradation, which employs microorganisms and their degradative enzymes, has emerged as one way to address the unforeseen consequences of the waste streams that have resulted from mass plastic production. The focus of this review is microbial hydrolase enzymes which have been found to act on polyethylene terephthalate (PET) plastic. The best characterized examples are discussed together with the use of genomic and protein engineering technologies to obtain PET hydrolase enzymes for different applications. In addition, the obstacles which are currently limiting the development of efficient PET bioprocessing are presented. By continuing to study the possible mechanisms and the structural elements of key enzymes involved in microbial PET hydrolysis, and by assessing the ability of PET hydrolase enzymes to work under practical conditions, this research will help inform large-scale waste management operations. Finally, the contribution of microbial PET hydrolases in creating a potential circular PET economy will be explored. This review combines the current knowledge on enzymatic PET processing with proposed strategies for optimization and use, to help clarify the next steps in addressing pollution by PET and other plastics.

Host-guest complexes exhibiting a 1 : 1 binding stoichiometry need not consist of a single host and guest. A series of oligopeptides, which were previously reported to have abnormally high binding enthalpies were investigated to deduce whether they exist as a 2 : 2 quaternary or a 1 : 1 binary complex with cucurbit[8]uril (CB[8]). Through a systematic study of the sequence-specific binding pathways of peptide-CB[8] association, a phenylalanine-leucine dipeptide was found to be capable of switching from a 1 : 1 stoichiometric complex to a 2 : 1 complex. By studying the differences in size-based diffusion properties of these two binding modes, the presence of a 1 : 1 pairwise inclusion complex was verified for the regime where CB[8] is in excess. Findings in this study can be utilised to 'customise' the precise CB[8]-oligopeptide self-assembly pathway, acting as a useful toolbox in the design of supramolecular systems.

Electrodeposition has been widely used for the low-cost formation of large-area thin films of various crystalline materials. In this work, we use thermodynamic calculations to guide electrochemical experiments, in which we conduct a systematic investigation of crystalline iron sulfides formation on electrically poised iron substrates in H2S aqueous solutions. Whereas thermodynamic calculations predict the formation of iron pyrite (FeS2) films at the conditions tested, the principal iron–sulfur mineral phases observed were mixtures of mackinawite (Fe1+xS) and cubic iron sulfide (phase identification and characterization included X-ray diffraction, Raman spectroscopy, scanning electron microscopy, and transmission electron microscopy). These data suggest that the phases formed are determined kinetically, not thermodynamically, across all tested electrodeposition conditions. The results are compared with the formation of iron sulfides produced by the “sour corrosion” of iron and steels, and in addition the e...

A pentapeptide sequence was functionalized with an asymmetric arylated methyl-viologen (AVI3D2) and through controllable β-sheet self-assembly, conductive nanofibers were formed. Using a combination of spectroscopic techniques and conductive atomic force microscopy, we investigated the molecular conformation of the resultant AVI3D2 fibers and how their conductivity is affected by β-sheet self-assembly. These conductive nanofibers have potential for future exploration as molecular wires in optoelectronic applications. ispartof: CHEMICAL COMMUNICATIONS vol:55 issue:51 pages:7354-7357 ispartof: location:England status: published

The endocannabinoid system is dysregulated in schizophrenia. Mice with heterozygous deletion of neuregulin 1 (Nrg1 HET mice) provide a well-characterised animal model of schizophrenia, and display enhanced sensitivity to stress and cannabinoids during adolescence. However, no study has yet determined whether these mice have altered brain endocannabinoid concentrations. Nrg1 application to hippocampal slices decreased 2-arachidonoylglycerol (2-AG) signalling and disrupted long-term depression, a form of synaptic plasticity critical to spatial learning. Therefore we specifically aimed to examine whether Nrg1 HET mice exhibit increased 2-AG concentrations and disruption of spatial learning. As chronic stress influences brain endocannabinoids, we also sought to examine whether Nrg1 deficiency moderates adolescent stress-induced alterations in brain endocannabinoids. Adolescent Nrg1 HET and wild-type (WT) mice were submitted to chronic restraint stress and brain endocannabinoid concentrations were analysed. A separate cohort of WT and Nrg1 HET mice was also assessed for spatial learning performance in the Morris Water Maze. Partial genetic deletion of Nrg1 increased anandamide concentrations in the amygdala and decreased 2-AG concentrations in the hypothalamus. Further, Nrg1 HET mice exhibited increased 2-AG concentrations in the hippocampus and impaired spatial learning performance. Chronic adolescent stress increased anandamide concentrations in the amygdala, however, Nrg1 disruption did not influence this stress-induced change. These results demonstrate for the first time in vivo interplay between Nrg1 and endocannabinoids in the brain. Our results demonstrate that aberrant Nrg1 and endocannabinoid signalling may cooperate in the hippocampus to impair cognition, and that Nrg1 deficiency alters endocannabinoid signalling in brain stress circuitry.

The neural cell adhesion molecule (NCAM) is involved in developmental processes and age-associated cognitive decline; however, little is known concerning the effects of NCAM in the visual system during aging. Using anatomical, electrophysiological, and behavioral assays, we analyzed age-related changes in visual function of NCAM deficient (−/−) and wild-type mice. Anatomical analyses indicated that aging NCAM −/− mice had fewer retinal ganglion cells, thinner retinas, and fewer photoreceptor cell layers than age-matched controls. Electroretinogram testing of retinal function in young adult NCAM −/− mice showed a 2-fold increase in a- and b-wave amplitude compared with wild-type mice, but the retinal activity dropped dramatically to control levels when the animals reached 10 months. In behavioral tasks, NCAM −/− mice had no visual pattern discrimination ability and showed premature loss of vision as they aged. Together, these findings demonstrate that NCAM plays significant roles in the adult visual system in establishing normal retinal anatomy, physiology and function, and in maintaining vision during aging.

The possibilities of identifying new classes of oxides having lower thermal conductivity at high temperatures is approaching the theoretical minimum value. At the same time, the drive for higher engine efficiency requires higher gas temperatures, increasing the radiative heat flux incident on the turbine blades. The significance of radiative heat transfer in the selection of materials is examined. It is concluded that as well as having the lowest thermal conductivity, future TBC oxide coatings must also be optically opaque to minimize radiative heating through a coating. This can be achieved by a combination of doping and scattering.

Our current understanding of epidermal growth factor receptor (EGFR) autoinhibition is based on X-ray structural data of monomer and dimer receptor fragments and does not explain how mutations achieve ligand-independent phosphorylation. Using a repertoire of imaging technologies and simulations we reveal an extracellular head-to-head interaction through which ligand-free receptor polymer chains of various lengths assemble. The architecture of the head-to-head interaction prevents kinase-mediated dimerisation. The latter, afforded by mutation or intracellular treatments, splits the autoinhibited head-to-head polymers to form stalk-to-stalk flexible non-extended dimers structurally coupled across the plasma membrane to active asymmetric tyrosine kinase dimers, and extended dimers coupled to inactive symmetric kinase dimers. Contrary to the previously proposed main autoinhibitory function of the inactive symmetric kinase dimer, our data suggest that only dysregulated species bear populations of symmetric and asymmetric kinase dimers that coexist in equilibrium at the plasma membrane under the modulation of the C-terminal domain., To prevent ligand-independent dimerisation the epidermal growth factor receptor (EGFR) is autoinhibited by an extracellular dimer interaction. Here, the authors use several imaging technologies and simulations to provide structural insights on the inactive species and on how intracellular mutations circumvent the autoinhibition of the basal state.

Super-resolution fluorescence microscopy achieves 20-30 nm resolution by using liquid-immersion objectives to optimize light collection and chemical sample fixation to minimize image blurring. It is known that fluorophore brightness increases substantially under cryogenic conditions and that cryo-fixation is far superior in preserving ultrastructure. However, cryogenic conditions have not been exploited to improve resolution or sample quality because liquid immersion media freezes at the objective, losing its optical properties. Here, simply by replacing the immersion fluid with a low-cost super-hemispherical solid immersion lens (superSIL), we effortlessly achieve uperSIL microscopy delivers a straightforward route to achieve unmatched nanoscale resolution on both bacterial and mammalian cell samples, which any laboratory can effortlessly and inexpensively implement.

There is a limited range of methods available to characterize macromolecular organization in cells on length scales from 5–50 nm. We review methods currently available and show the latest results from a new single-molecule localization-based method, fluorophore localization imaging with photobleaching (FLImP), using the epidermal growth factor (EGF) receptor (EGFR) as an example system. Our measurements show that FLImP is capable of achieving spatial resolution in the order of 6 nm. Abbreviations: BM-I, bisindolylmaleimide-I; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; FLImP, fluorophore localization imaging with photobleaching; PALM, photoactivated localization microscopy; PKC, protein kinase C; SNR, signal-to-noise ratio

Bevacizumab, a recombinant humanized monoclonal antibody targeting vascular endothelial growth factor (VEGF), is approved for treatment of metastatic colorectal cancer, nonsquamous non-small-cell lung cancer, metastatic kidney cancer, and glioblastoma. To support clinical development of the potential bevacizumab biosimilar PF-06439535, nonclinical studies evaluated structural, functional, toxicological, and toxicokinetic similarity to bevacizumab sourced from the European Union (bevacizumab-EU) and United States (bevacizumab-US). Peptide mapping demonstrated the amino acid sequence of PF-06439535 was identical to bevacizumab-EU and bevacizumab-US. Biologic activity, measured via inhibition of VEGF-induced cell proliferation in human umbilical vein endothelial cells and binding to VEGF isoforms, was similar across the three drugs. In vivo similarity was demonstrated in cynomolgus monkeys administered intravenous PF-06439535 or bevacizumab-EU (0 or 10 mg/kg/dose twice weekly for 1 month; total of nine doses). Systemic exposure appeared similar and test article-related effects were limited to physeal dysplasia of the distal femur. The potential for non-target-mediated toxicity of PF-06439535 was evaluated in rats administered intravenous PF-06439535 (15 or 150 mg/kg/dose twice weekly for 15 days; total of five doses). Nonadverse higher liver weights and minimal sinusoidal cell hyperplasia were observed. Collectively, these studies demonstrated similarity of PF-06439535 to bevacizumab, supporting entry into clinical development.

Little is known about the exact genes that confer vulnerability or resilience to environmental stressors during early neurodevelopment. Partial genetic deletion of neuregulin 1 (Nrg1) moderates the neurobehavioural effects of stressors applied in adolescence and adulthood, however, no study has yet examined its impact on prenatal stress. Here we examined whether Nrg1 deficiency in mice modulated the impact of prenatal stress on various behaviours in adulthood. Male heterozygous Nrg1 mice were mated with wild-type female mice who then underwent daily restraint stress from days 13 to 19 of gestation. Surprisingly, prenatal stress had overall beneficial effects by facilitating sensorimotor gating, increasing sociability, decreasing depressive-like behaviour, and improving spatial memory in adulthood. Such benefits were not due to any increase in maternal care, as prenatal stress decreased nurturing of the offspring. Nrg1 deficiency negated the beneficial behavioural effects of prenatal stress on all measures except sociability. However, Nrg1 deficiency interacted with prenatal stress to trigger locomotor hyperactivity. Nrg1 deficiency, prenatal stress or their combination failed to alter acute stress-induced plasma corticosterone concentrations. Collectively these results demonstrate that Nrg1 deficiency moderates the effects of prenatal stress on adult behaviour, but it does so in a complex, domain-specific fashion.

Agonism of GPR119 is viewed as a potential therapeutic approach for the treatment of type II diabetes and other elements of metabolic syndrome. During progression of a previously disclosed candidate 1 through mice toxicity studies, we observed tonic-clonic convulsions in several mice at high doses. An in vitro hippocampal brain slice assay was used to assess the seizure liability of subsequent compounds, leading to the identification of an aryl sulfone as a replacement for the 3-cyano pyridyl group. Subsequent optimization to improve the overall profile, specifically with regard to hERG activity, led to alkyl sulfone 16. This compound did not cause tonic-clonic convulsions in mice, had a good pharmacokinetic profile, and displayed in vivo efficacy in murine models. Importantly, it was shown to be effective in wild-type (WT) but not GPR119 knockout (KO) animals, consistent with the pharmacology observed being due to agonism of GPR119.

Aims Tellurium-based devices, such as photovoltaic (PV) modules and thermoelectric generators, are expected to play an increasing role in renewable energy technologies. Tellurium, however, is one of the scarcest elements in the earth's crust, and current production and recycling methods are inefficient and use toxic chemicals. This study demonstrates an alternative, bacterially mediated tellurium recovery process. Methods and Results We show that the hydrothermal vent microbe Pseudoalteromonas sp. strain EPR3 can convert tellurium from a wide variety of compounds, industrial sources and devices into metallic tellurium and a gaseous tellurium species. These compounds include metallic tellurium (Te0), tellurite (TeO32−), copper autoclave slime, tellurium dioxide (TeO2), tellurium-based PV material (cadmium telluride, CdTe) and tellurium-based thermoelectric material (bismuth telluride, Bi2Te3). Experimentally, this was achieved by incubating these tellurium sources with the EPR3 in both solid and liquid media. Conclusions Despite the fact that many of these tellurium compounds are considered insoluble in aqueous solution, they can nonetheless be transformed by EPR3, suggesting the existence of a steady state soluble tellurium concentration during tellurium transformation. Significance and Impact of the Study These experiments provide insights into the processes of tellurium precipitation and volatilization by bacteria, and their implications on tellurium production and recycling.

P-glycoprotein (P-gp) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. The protein is organized into two halves. The halves contain a transmembrane domain (TMD) with six transmembrane segments and a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the TMD1/TMD2 and NBD1/NBD2 interfaces, respectively. ATP-dependent drug efflux involves changes between the open inward-facing (NBDs apart, extracellular loops (ECLs) close together) and the closed outward-facing (NBDs close together, ECLs apart) conformations. It is controversial, however, whether the open conformation only exists transiently in intact cells because of the presence of high levels of ATP. To test for the presence of an open conformation in intact cells, reporter cysteines were placed in extracellular loops 1 (A80C, N half) and 4 (R741C, C half). The rationale was that cysteines A80C/R741C would only come close enough to form a disulfide bond in an open conformation (6.9 Å apart) because they are separated widely (30.4 Å apart) in the closed conformation. It was observed that the mutant A80C/R741C cross-linked spontaneously (>90%) when expressed in cells. In contrast to previous reports showing that trapping P-gp in a closed conformation highly activated ATPase activity, here we show that A80C/R741C cross-linking inhibited ATPase activity and drug efflux. Both activities were restored when the cross-linked mutant was treated with a thiol-reducing agent. The results show that an open conformation can be readily detected in cells and that cross-linking of cysteines placed in ECLs 1 and 4 inhibits activity.

A safe and facile method for the formation of 3-trifluoromethyl-5-amino-1,2,4-oxadiazoles, via a reversed addition of hydroxylamine to cyanamides, is reported. This two-pot procedure is suitable to scale-up and avoids the hazards associated with trifluoromethyl amidoxime synthesis.

Ghrelin plays a major physiological role in the control of food intake, and inverse agonists of the ghrelin receptor (GHS-R1a) are widely considered to offer utility as antiobesity agents by lowering the set-point for hunger between meals. We identified an acylurea series of ghrelin modulators from high throughput screening and optimized binding affinity through structure-activity relationship studies. Furthermore, we identified specific substructural changes, which switched partial agonist activity to inverse agonist activity, and optimized physicochemical and DMPK properties to afford the non-CNS penetrant inverse agonist 22 (AZ-GHS-22) and the CNS penetrant inverse agonist 38 (AZ-GHS-38). Free feeding efficacy experiments showed that CNS exposure was necessary to obtain reduced food intake in mice, and it was demonstrated using GHS-R1a null and wild-type mice that this effect operates through a mechanism involving GHS-R1a.

Oxide compositions of equimolar YO1.5 and TaO2.5 in the Y–Ta–Zr–O system have attractive properties for high-temperature applications, including as thermal barrier coatings. The effect of zirconia concentration, from 0 to 20 mol.% cation, on the tetragonal-to-monoclinic phase transition has been studied using high-temperature X-ray diffraction, Raman spectroscopy and electron microscopy. The transformation is reversible and the temperature variation of an order parameter based on the spontaneous strain is consistent with the transformation being ferroelastic, a critical feature for toughening at high temperatures. The presence of twin domains further supports this conclusion. Additionally, stabilization of the tetragonal phase with increasing ZrO2 is evident from the amount of partially retained tetragonal phase at room temperature.

P-glycoprotein (P-gp, ABCB1) is an ATP-binding cassette drug pump that protects us from toxic compounds and confers multidrug resistance. Each homologous half contains a transmembrane domain with six transmembrane segments followed by a nucleotide-binding domain (NBD). The drug- and ATP-binding sites reside at the interface between the transmembrane domain and NBDs, respectively. Drug binding activates ATPase activity by an unknown mechanism. There is no high resolution structure of human P-gp, but homology models based on the crystal structures of bacterial, mouse, and Caenorhabditis elegans ATP-binding cassette drug pumps yield both open (NBDs apart) and closed (NBDs together) conformations. Molecular dynamics simulations predict that the NBDs can be separated over a range of distances (over 20 Å). To determine the distance that show high or low ATPase activity, we cross-linked reporter cysteines L175C (N-half) and N820C (C-half) with cross-linkers of various lengths that separated the halves between 6 and 30 Å (α-carbons). We observed that ATPase activity increased over 10-fold when the cysteines were cross-linked at distances between 6 and 19 Å, although cross-linking at distances greater than 20 Å yielded basal levels of activity. The results suggest that the ATPase activation switch appears to be turned on or off when L175C/N820 are clamped at distances less than or greater than 20 Å, respectively. We predict that the high/low ATPase activity switch may occur at a distance where the NBDs are predicted in molecular dynamic simulations to undergo pronounced twisting as they approach each other (Wise, J. G. (2012) Biochemistry 51, 5125-5141).

Tensile deformation was applied to the naturally produced poly-γ-glutamic acid, which can be enzymatically degraded and is, therefore, of interest for biomedical use. However, natural polyamino acids have a similar chemical structure to synthetic polyamides (“nylons”), which are known to feature strong inter-molecular hydrogen bonding that prevents large-scale molecular motion in their solid state. Through esterification, this hydrogen bonding was partially shielded, allowing orientation of the polyamino acid macromolecules through tensile deformation. An increase in Young's modulus and tensile strength was achieved of solution-cast films of the chemically modified poly-γ-glutamic acids, consistent with enhanced uniaxial polymer chain orientation. The latter was confirmed by both wide-angle X-ray scattering and polarized Raman spectroscopy. The films thus produced were found to be non-cytotoxic. These mechanically tailorable, biocompatible polymers may be excellent candidates for use in musculoskeletal tissue engineering applications that have different loading requirements within the body.

A large number of correctors have been identified that can partially repair defects in folding, stability and trafficking of CFTR processing mutants that cause cystic fibrosis (CF). The best corrector, VX-809 (Lumacaftor), has shown some promise when used in combination with a potentiator (Ivacaftor). Understanding the mechanism of VX-809 is essential for development of better correctors. Here, we tested our prediction that VX-809 repairs folding and processing defects of CFTR by promoting interactions between the first cytoplasmic loop (CL1) of transmembrane domain 1 (TMD1) and the first nucleotide-binding domain (NBD1). To investigate whether VX-809 promoted CL1/NBD1 interactions, we performed cysteine mutagenesis and disulfide cross-linking analysis of Cys-less TMD1 (residues 1-436) and ΔTMD1 (residues 437-1480; NBD1-R-TMD2-NBD2) truncation mutants. It was found that VX-809, but not bithiazole correctors, promoted maturation (exited endoplasmic reticulum for addition of complex carbohydrate in the Golgi) of the ΔTMD1 truncation mutant only when it was co-expressed in the presence of TMD1. Expression in the presence of VX-809 also promoted cross-linking between R170C (in CL1 of TMD1 protein) and L475C (in NBD1 of the ΔTMD1 truncation protein). Expression of the ΔTMD1 truncation mutant in the presence of TMD1 and VX-809 also increased the half-life of the mature protein in cells. The results suggest that the mechanism by which VX-809 promotes maturation and stability of CFTR is by promoting CL1/NBD1 interactions.

We demonstrate the preparation of surface-bound cucurbit[8]uril (CB[8]) catenanes on silica nanoparticles (NPs), where CB[8] was employed as a tethered supramolecular "handcuff" to selectively capture target guest molecules. In this catenane, CB[8] was threaded onto a methyl viologen (MV(2+) ) axle and immobilized onto silica NPs. The formation of CB[8] catenanes on NPs were confirmed by UV/Vis titration experiments and lithographic characterization, demonstrating a high density of CB[8] on the silica NPs surface, 0.56 nm(-2) . This CB[8] catenane system exhibits specific molecular recognition towards certain aromatic molecules such as perylene bis(diimide), naphthol and aromatic amino acids, and thus it can act as a nanoscale molecular receptor for target guests. Furthermore, we also demonstrate its use as an efficient and recyclable nano-platform for peptide separation. By embedding magnetic NPs inside silica NPs, separation could be achieved by simply applying an external magnetic field. Moreover, the peptides captured by the catenanes could be released by reversible single-electron reduction of MV(2+) . The entire process demonstrated high recoverability.

G protein coupled receptor 119 (GPR119) is viewed as an attractive target for the treatment of type 2 diabetes and other elements of the metabolic syndrome. During a program toward discovering agonists of GPR119, we herein describe optimization of an initial lead compound, 2, into a development candidate, 42. A key challenge in this program of work was the insolubility of the lead compound. Small-molecule crystallography was utilized to understand the intermolecular interactions in the solid state and resulted in a switch from an aryl sulphone to a 3-cyanopyridyl motif. The compound was shown to be effective in wild-type but not knockout animals, confirming that the biological effects were due to GPR119 agonism.

The microwave condensation of mono-amino functionalised polyhedral oligomeric silsesquioxane (POSS) with a range of mono- and bis-anhydrides yielded the corresponding POSS imide compounds, which were characterized by UV–Vis and fluorescence spectrophotometry. The perylene POSS imide derivative was further characterized by single crystal X-ray crystallography. The naphthyl POSS bis/monoimide exhibited extremely weak fluorescence, whilst the perylene POSS bis-imide displayed particularly strong fluorescence, with a quantum yield approaching unity.

The apatite crystal structure of the gadolinium calcium silicates can accommodate a wide range of point defects, including oxygen and cation vacancies, as well as anti-site defects, depending on the Gd/Ca ratio. Compositions having only cation or oxygen vacancies were identified and the thermal diffusivity and conductivity were measured up to 1000 C. All the compositions, including the stoichiometric composition, exhibit low thermal conductivities from room temperature to high temperature with the defect-containing compositions having even lower thermal conductivities. The high-temperature thermal conductivity, at temperatures below the onset of significant radiative heat transport, decreases with the inverse square root of the cation and anion vacancy concentration, consistent with simple defect scattering models. Based on the data, it is concluded that the oxygen vacancies are slightly more effective in reducing thermal conductivity.

RNA-binding proteins, and in particular, the Musashi genes, function as essential regulators of progenitor functioning in both the developing and adult organism. In this report, we characterize the differential subcellular distribution of Musashi-1 in cells engaged in either proliferating or differentiating contexts in the developing mouse retina, and in cultured Müller glia. During retinal cell differentiation, Musashi-1 immunoreactivity shifts from exclusively cytoplasmic in retinal progenitor cells, to predominantly nuclear localization in differentiating neurons. This nuclear shift is transient, with localization in the adult retina becoming predominantly perinuclear and cytoplasmic in Müller glia and photoreceptors. A correlation between cell cycle progression and subcellular distribution of Musashi-1 is observed in passageable, adult Müller glial cells in vitro. Furthermore, treatment of Müller cultures with neuron-promoting differentiation media induces asymmetric cytoplasmic Musashi-1 immunoreactivity in dividing daughter cells. The observed shifts in subcellular Musashi-1 localization are consistent with contrasting roles for Musashi-1 during cell proliferation and differentiation. These data provide evidence that nuclear, and cytoplasmic sequestering of Musashi-1 in retinal cells is context-specific, and may contribute to downstream functioning of Musashi-1.

Retinal Müller glia have received considerable attention with regard to their potential to function as quiescent retinal precursors. Various activation strategies induce characteristic features of retinal progenitor cells in Müller glia; however, these are often accompanied by hallmark features of reactive gliosis. We investigated the effects of an intravitreal injection of epidermal growth factor (EGF), a known mitogen, and erythropoietin (EPO) on activation and expression of developmental phenotypes within the adult retina. Using thymidine-analogue labeling as well as immunocytochemical and confocal analyses, we assayed the responses of retinal cells exposed to intravitreal administration of either EGF or EPO. We report that adult Müller glia incorporate bromodeoxyuridine (BrdU) and undergo a process of nuclear translocation to ectopic retinal layers following exposure to EGF. These cells survive within the retina for at least 23 days and express the developmental markers Pax6 and Chx10 as well as nestin and glial fibrillary acidic protein. Furthermore, we demonstrate that cotreatment with EGF and EPO suppresses aspects of EGF-induced glial reactivity, alters the retinal distribution of BrdU-positive nuclei, and serves to regulate the expression of developmental phenotypes seen in these cells. These data further our understanding of Müller cell responsiveness to intravitral, combinatorial growth factor treatments.

Thermal barrier coatings typically fail on cooling after prolonged thermal cycling or isothermal exposure. The mechanics of spalling requires that first a critical sized portion of the coating separates from the underlying material, then buckles and finally spalls away. The critical size for buckling depends on the thickness of the coating but is several millimeters for typical zirconia coatings 150 lm thick. As-deposited coatings do not have interface separations but they form on thermal cycling as described in this work based on observations of coating crosssections combined with the stress redistribution in the thermally grown oxide imaged using a piezospectroscopic luminescence method. Analysis of the images reveals that small, isolated regions of damage initially form and then grow, linking up and coalescing to form percolating structures across the coating until the buckling condition is attained, the buckle extends and failure occurs by spallation. The piezospectroscopic imaging of the stresses in the thermally grown oxide formed by oxidation beneath thermal barrier coatings provides a form of ‘‘stress tomography’’ enabling the subcritical separations to be monitored.

In Part I, it was shown that the critical thickness required for mechanical failure of lateral epitaxial overgrown films of ZnO on GaN buffered Al 2 O 3 substrates could be significantly increased by masking and patterning the substrate to produce periodic, hexagonal arrays of circular windows. It was also observed that the critical thickness could be increased by increasing the distance between the circular windows. In Part II, it is shown, using Raman piezo-spectroscopy, that a stress gradient exists within the hexagonal prisms of ZnO that grow from the circular windows. The compressive stress, due to lattice mismatch, was highest in the center of the prisms and lowest near the perimeter where they overgrow the masked region to form ‘wings’. For one orientation of the hexagonal array of circular windows, relative to the substrate, where the hexagonal prism coalesce face to face, the stress gradient persisted after the hexagonal prisms coalesced to form a continuous film because residual voids surround the base of the coalesced, hexagonal prisms. For a second orientation where the hexagonal prisms coalesce corner to corner, the stress gradient was greatly diminished due to a larger amount of coalescence at the base of the prisms. Most important, it was shown that the compressive stress for both arrays decreased with increased spacing between the circular windows. Namely, for window orientation producing face to face coalescence of the prisms, the maximum compressive stress decreases from ∼ 520 MPa for windows separated by twice their diameter to ∼ 350 MPa for windows separated by four times their diameter. These results are consistent with the critical thickness observations reported in Part I.

The absorption and fluorescence spectral properties of several merocyanines with very high first hyperpolarizabilities in fluid solutions are reported. These molecules contain a cyanodicyanomethylidenedihydrofuran electron acceptor group and with either a pyridinylidene or quinolinylidene electron donor. The molecules also contain a bulky substituent group which has been incorporated in order to hinder molecular aggregation. No effect of merocyanine concentration on the absorption spectral distributions of these compounds was observed in any of the solvents studied indicating there was no molecular aggregation in solution. The absorption spectra of these compounds display changes in their vibronic structure in solvents with different polaritiesipolarizabilities while the fluorescence spectra exhibit the standard solvent polarity stabilizing behaviour. The first hyperpolarizabilities decreased with decreasing solvent polarity for merocyanines with a quinolinylidene electron donor and this trend was reversed for compounds with a pyridinylidene electron donor. This is consistent with changes in electron delocalization within the merocyanines induced by changes in the dielectric of the solvent. (C) 2010 Elsevier B.V. All rights reserved.

Polyhedral oligomeric silsesquioxane (POSS) cages containing an iminofullerene species are reported herein. Monosubstituted benzyl chloride POSS was synthesized, and subsequently reacted with sodium azide to form mono benzyl azide POSS. The azide was subsequently reacted with C60 in anhydrous, degassed toluene to yield the desired POSS iminofullerene compound. The prepared compounds were characterized by multinuclear NMR, electrospray mass spectrometry, elemental analysis, UV–vis, fluorescence and optical power limiting measurements. Copyright © 2009 John Wiley & Sons, Ltd.

Deletion of Phe508 in cystic fibrosis transmembrane conductance regulator (DeltaF508 CFTR) causes cystic fibrosis. CFTR consists of two homologous halves with each containing a nucleotide-binding domain (NBD) and a transmembrane domain (TMD). DeltaF508 CFTR appears to be trapped in an incompletely folded state. Small molecules (correctors) promote folding of DeltaF508 CFTR with relatively low efficiency. Understanding the mechanism of repair may lead to the development of more effective correctors. Here we tested the effect of correctors and the DeltaF508 mutation on interactions between the halves of CFTR when expressed as separate polypeptides. Glycosylation of C-half CFTR was defective when expressed alone as a mixture of core and unglycosylated proteins was detected. Coexpression of C-half CFTR with either wild-type N-half or DeltaF508/N-half CFTR, however, increased the amount of core-glycosylated protein, but only coexpression with wild-type N-half promoted maturation of C-half CFTR (Endo H resistant). This suggested that the DeltaF508 mutation inhibited some interactions between N-half and C-half CFTRs. Interaction of A52-tagged wild-type N-half or DeltaF508/N-half CFTR with histidine-tagged C-half CFTR was then followed by nickel-chelate chromatography. Coexpression of A52-tagged wild-type N-half or DeltaF508/N-half CFTR with histidine-tagged C-half CFTR resulted in the wild-type N-half CFTR but not DeltaF508/N-half CFTR protein being retained on the column. Coexpression of DeltaF508/N-half and C-half CFTR in the presence correctors VX-325 and corr-4a, however, restored interactions between the two halves. An interaction that was restored was that between NBD1 and TMD2 as the correctors restored cross-linking of mutant DeltaF508/NBD1(V510C)/TMD2(A1067C). Therefore, correctors promote proper interactions between the two halves of CFTR.

P-glycoprotein (P-gp, ATP-binding cassette B1) is a drug pump that extracts toxic drug substrates from the plasma membrane and catalyzes their ATP-dependent efflux. To map the residues in the drug translocation pathway, we performed arginine-scanning mutagenesis on all transmembrane (TM) segments (total = 237 residues) of a P-gp processing mutant (G251V) defective in folding (15% maturation efficiency) (glycosylation state used to monitor folding). The rationale was that arginines introduced into the drug-binding sites would mimic drug rescue and enhance maturation of wild-type or processing mutants of P-gp. It was found that 38 of the 89 mutants that matured had enhanced maturation. Enhancer mutations were found in 11 of the 12 TM segments with the largest number found in TMs 6 and 12 (seven in each), TMs that are critical for P-gp-drug substrate interactions. Modeling of the TM segments showed that the enhancer arginines were found on the hydrophilic face, whereas inhibitory arginines were located on a hydrophobic face that may be in contact with the lipid bilayer. It was found that many of the enhancer arginines caused large alterations in P-gp-drug interactions in ATPase assays. For example, mutants A302R (TM5), L339R (TM6), G872R (TM10), F942R (TM11), Q946R (TM11), V982R (TM12), and S993R (TM12) reduced the apparent affinity for verapamil by approximately 10-fold, whereas the F336R (TM6) and M986R (TM12) mutations caused at least a 10-fold increase in apparent affinity for rhodamine B. The results suggest that P-gp contains a large aqueous-filled drug translocation pathway with multiple drug-binding sites that can accommodate the bulky arginine side chains to promote folding of the protein.

The measurement of high temperatures in oxides and oxide-based structures in practical applications often presents challenges including steep thermal gradients, the presence of flames or chemically aggressive environments, and the transparency or translucency of most oxides. For turbine engines, oxide coatings are of great commercial importance, and the rapid motion of parts prohibits contact thermometry. Luminescence thermometry offers a number of advantages for measuring temperature in such systems and has been the subject of ongoing study for many years. Recent work on rare-earth-doped thermal barrier coatings, environmental barrier coatings, and related oxides has demonstrated the feasibility of luminescence thermometry to temperatures well in excess of 1000°C. The luminescent properties of these materials and the analytical techniques used to extract reproducible temperature measurements from the measured luminescence are reviewed.

Short cracks, extending to the interdiffusion zone, have been observed in platinum-modified nickel-aluminide diffusion coatings on single-crystal CMSX-4 superalloys after these have been subject to short (10 min) rapid thermal cycles. The cracks were revealed after removing the thermally grown oxide formed during cycling on the coating surfaces. Cracking was observed for coatings subject to thermal cycling between 1150 and 1050 °C, with and without an applied compressive stress, as well as on thermal cycling between 1150 °C and room temperature.

We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl group at the 9-substituent. These compounds were examined for affinity and ATPase stimulation in isolated MDR3 CL P-gp and human P-gp-His(10), for their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant MDCKII-MDR1 cells, and for transport in monolayers of MDCKII-MDR1 cells. Thioamide 31-S gave K(M) of 0.087 microM in human P-gp. Small changes in structure among this set of compounds affected affinity as well as transport rate (or flux) even though all derivatives examined were substrates for P-gp. With isolated protein, tertiary amide groups dictate high affinity and high stimulation while tertiary thioamide groups give high affinity and inhibition of ATPase activity. In MDCKII-MDR1 cells, the tertiary thioamide-containing derivatives promote uptake of calcein AM and have very slow passive, absorptive, and secretory rates of transport relative to transport rates for tertiary amide-containing derivatives. Thioamide 31-S promoted uptake of calcein AM and inhibited efflux of vinblastine with IC(50)'s of approximately 2 microM in MDCKII-MDR1 cells.

Thermal cycling of a platinum modified, nickel aluminide (Ni,Pt)Al coated single crystal superalloy, between 1000 and 1150 °C with 10 min holds at each temperature, and subject to a compressive uniaxial stress is reported. There are two major effects of the superimposed compressive stress not observed in the absence of the stress. One is that the rumpling pattern exhibits an asymmetry with an increase of the bond coat surface roughness perpendicular to the applied loading axis. The other is the formation of cracks in the thermally grown oxide aligned parallel to the stress axis.

The evolution of the surface roughness of an initially flat platinum-modified nickel aluminide (NiPtAl) coating on a single crystal superalloy with cyclic oxidation has been measured by optical profilometry and correlated with the aluminide microstructure. The roughness evolution is dominated by the relative motion of individual grains on thermal cycling, with the larger sized grains moving up and the smaller grains moving down with respect to the average surface. Detailed crystallographic analysis indicates that the grains in the coating are randomly distributed and have no crystallographic relationship with the underlying single crystal superalloy. Furthermore, there is a correlation between the number of sides a grain has and the propensity for its center to move relative to the average surface plane: grains with six or more sides bow up, whereas the surfaces of those grains with fewer than six sides bow down. It is proposed that rumpling initiates due to the vertical displacement of the smaller grains having fewer than six sides. Once initiated, rumpling proceeds driven by the strain energy in the thermally grown oxide.

Silica-bound aldehyde functional silane was used as a versatile bridge to attach porphyrin to the surface of silica and as a surface-bound free radical initiator to yield PMMA brushes. The aldehyde functionality is obtained by a simple ozonolysis of the surface-bound allylsilane, whereby the double bonds are oxidized in high yield to the desired aldehyde. Mono-amino porphyrin was attached to the silica surface under mild conditions via Schiff-base linkages, whilst anchored PMMA was obtained by free radical initiation using an aldehyde/redox free radical polymerization. FTIR and TGA data were used to determine and characterize the attachment to the silica surface.

The evolution in surface morphology of platinum modified nickel aluminide (Ni,Pt)Al oxidation coatings during thermo-mechanical testing has been evaluated. One type of test consisted of cyclic oxidation between an upper temperature of 1150°C and a lower temperature varying from room temperature to 1050°C. The other type of test was cycling between 1000°C/1150°C under an applied compressive stress. Profilometry using optical interferometry was used to quantify the surface “rumpling”. First and second-order statistical parameters including RMS roughness and the auto-correlation function were calculated from the profilometry measurements. The results indicate that the grain structure of the aluminide coating plays a major role in the early stages of rumpling and set its wavelength. Also, the superimposed compressive stress during thermal cycling leads to an asymmetry in the rumpling pattern with respect with the loading axis as well as cracking along the applied stress direction.

An analysis has been conducted that characterizes the susceptibility to delamination of thermal barrier coated (TBC) hot-section aero-turbine components when penetrated by calcium-magnesium-alumino-silicate (CMAS). The assessment has been conducted on stationary components (especially shrouds) with relatively thick TBCs after removal from aero-engines. In those segments that experience the highest temperatures, the CMAS melts, penetrates to a depth about half the coating thickness, and infiltrates all open areas. Therein the TBC develops channel cracks and sub-surface delaminations, as well as spalls. Estimates of the residual stress gradients made on cross-sections (by using the Raman peak shift) indicate tension at the surface, becoming compressive below. By invoking mechanics relevant to the thermo-elastic stresses upon cooling, as well as the propagation of channel cracks and delaminations, a scenario has been presented that rationalizes these experimental findings. Self-consistent estimates of the stress and temperature gradients are presented as well as predictions of channel cracking and delamination upon cooling.

A multi-layered oxide forms on single crystal PWA 1484 alloy when oxidized at high temperatures, consisting of a spinel ((Ni(Cr,Al,Co) 2 O 4 ) at the air/oxide interface, interspersed with tantalum rich oxide particles (CrTaO 4 /NiTa 2 O 6 ), and an inner-most layer of α-alumina (α-Al 2 O 3 ) in contact with the alloy. Observations on rapid cooling after oxidation show that the spallation behavior of this multi-layer oxide depends on time in room temperature laboratory air, the sulfur content of the alloy, and the alloy surface preparation. Time-lapse optical microscopy reveals that spallation of the multi-layer oxide occurs differently on polished surfaces in comparison to ground surfaces. On ground surfaces spallation occurs solely at the metal/alumina interface in both alloys, and follows the grinding scratches’ long axes. The higher S alloy (1.7 ppmw S) almost completely spalls to bare metal (∼95% oxide spalled), whereas very little spallation (∼5%) occurs on the lower S alloy (0.2 ppmw S). On polished surfaces spallation occurs along both the alumina/spinel and alumina/alloy interfaces of the higher sulfur containing alloy but only along the alumina/spinel interface of the oxide on the low-sulfur alloy, leaving the alumina in contact with the metal. The lack of significant difference in measured residual stress in the α-alumina combined with the fact that spallation occurred over periods of several hours at room temperature suggests that failure is a time-dependent process likely associated with the presence of moisture, once a critical oxide thickness has been exceeded. The role of sulfur is likely associated with a reduction in fracture toughness of the oxide and oxide/metal interfaces through formation of voids or reduction in bond energy. Re-heating of the alumina-covered, low-sulfur alloy (0.2 ppmw) indicates that outward grain boundary diffusion of aluminum and tantalum occurs through the α-alumina but not nickel. Also, no new Ni-rich oxides reform confirming that they are indeed transient oxides formed during the initial stages of oxidation of the bare alloy.

The first reported examples of polyhedral oligomeric silsesquioxane (POSS) cages containing a fulleropyrrolidine species are reported herein. Monosubstituted POSS-dioxalane species were synthesized through the hydrosilylation of a silyl-dioxalane with mono-vinyl substituted POSS. Subsequent deprotection yielded the desired aldehyde functionality. An alternative synthetic pathway, involving the nucleophilic substitution of mono-benzyl chloride POSS with 4-hydroxybenzaldehyde yielded the desired aldehyde functionality. Each mono-aldehyde POSS was then reacted with N-methylglycine and C60 to yield the desired POSS fulleropyrrolidines. The prepared compounds were characterized by multinuclear NMR, electrospray mass spectrometry, elemental analysis, UV–vis, fluorescence and optical power limiting measurements. Copyright © 2008 John Wiley & Sons, Ltd.

One of the adverse effects of sand ingestion in gas turbines is that the thermal barrier coatings on the blades and vanes can be infiltrated at high temperatures by molten calcium–magnesium–aluminum–silicate (CMAS) and cause premature failure of the coating. To investigate the effect of CMAS penetration, the optical properties of a synthetic glass representative of CMAS are reported from 500 nm to 2.5 μm. Results are then presented to show that silicate infiltration of an electron beam-deposited TBC can increase radiative transport through the coating. The results are qualitatively consistent with a simple optical scattering model for radiative transport through a porous coating.

The measurement of temperature based on the characteristic lifetime decay in luminescence intensity of dopant ions following excitation is described with illustrations drawn from europium doping in yttria-stabilized zirconia. The method is particularly attractive for making measurements in a temperature gradient, such as those in thermal barrier coatings.

One form of degradation of platinum-modified nickel-aluminide (NiPtAl) coatings on single-crystal superalloys at high temperatures involves a surface instability known as rumpling. In this work, the evolution of rumpling during isothermal and cyclic oxidation at 1150 °C of a NiPtAl aluminide coating deposited on five different modifications of a commercial second generation CMSX-4 superalloy is characterized as a function of oxidation time. The rumpling is sensitive to the Hf and C content of the underlying superalloy, being largest for the alloy containing low hafnium and high carbon concentrations and smallest for the low-carbon, high-hafnium alloy. It is argued that hafnium decreases the propensity for rumpling when it diffuses into the coating and to the growing aluminum oxide increasing their creep resistance. An increase of the carbon content in the superalloy has the opposite effect due to the formation of tantalum-rich mixed carbides, which tie up hafnium, thereby decreasing its amount available to diffuse into the coating and the growing oxide. The effect of yttrium additions on rumpling is minor.

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.

One approach to measuring temperature in oxide coatings is to incorporate, within the crystal structure of the coating material itself, ions (“chromophores”) with temperature-dependent luminescence properties. The attraction of this “crystal chemistry” approach is that the other properties of the coating material, with appropriate choice of chromophore, are unaffected by solid solution doping. The method of luminescence sensing of temperature in both EB-PVD and plasma-sprayed coatings is described as well as the identification of the chromophores for the highest temperature measurements, the optimum doping concentrations and the excitation conditions. In addition, practical aspects in making precise temperature measurements will be presented.

The damping behavior of several materials used in current thermal barrier coating systems are reported from room temperature up to 1200 °C. The effect of long-term aging at 1150 °C on the damping behavior of a PWA-1484 single crystal superalloy is presented as well as the effect of a platinum-modified diffusion nickel aluminide bond-coat and an electron-beam evaporated, yttria-stabilized zirconia (EB-YSZ) thermal barrier coating. The nickel aluminide bond coat is shown to enhance the damping of the superalloys at all temperatures while the EB-YSZ shows a damping peak at ∼ 220 °C. The damping behavior of two top-coat materials, tetragonal-prime YSZ and gadolinium zirconate is compared. The latter exhibits a damping peak at a higher temperature (∼ 350 °C) than zirconia giving it the potential of being used as a damping coating for compressor blade.