Toxicant-Induced Leakage of Germ Cell–Specific Proteins from Seminiferous Tubules in the Rat: Relationship to Blood-Testis Barrier Integrity and Prospects for Biomonitoring

Evaluation of testicular toxicity during drug development is currently based on histopathological evaluation. A sensitive biomarker for testicular toxicology could provide an in-life and “early warning” measurement. Previous studies suggested that disruption of spermatogenesis induced leakage of germ cell proteins from seminiferous tubules (STs) into interstitial fluid (IF); such proteins have potential for use as biomarkers. To investigate this possibility further, adult male rats were treated with three testicular toxicants thought to have differing sites of action; cadmium chloride affects the blood-testis barrier (BTB), methoxyacetic acid (MAA) disrupts pachytene spermatocytes, and 1,3-dinitrobenzene (DNB) targets Sertoli cells. IF proteins were assessed by Coomassie-based dye-stained gels. Immunostaining was used to identify toxicant-induced damage (DAZL) and BTB integrity (ZO-1, occludin, N-cadherin, and β-catenin) and function (biotin). Cadmium chloride induced dose-dependent leakage of proteins from STs into IF coincident with loss of integrity and function of the BTB. Two of the “leaked” proteins were identified on Westerns as being germ cell specific, namely VASA and fatty acid–binding protein 9 (FABP9). In contrast, similar protein leakage was not evident after either MAA-induced or DNB-induced disruption of spermatogenesis and neither of these treatments affected BTB integrity or function. These results suggest that loss of BTB integrity is required for germ cell–specific proteins to leak from STs into IF, implying that use of such biomarkers has very limited potential for noninvasive monitoring of compound-induced disruption to spermatogenesis

Effects of thermally-induced changes of Cu grains on domain structure and electrical performance of CVD-grown graphene

During the chemical vapor deposition (CVD) growth of graphene on Cu foils, evaporation of Cu and changes in the dimensions of Cu grains in directions both parallel and perpendicular to the foils are induced by thermal effects. Such changes in the Cu foil could subsequently change the shape and distribution of individual graphene domains grown on the foil surface, and thus influence the domain structure and electrical properties of the resulting graphene films. Here, a slower cooling rate is used after the CVD process, and the graphene films are found to have an improved electrical performance, which is considered to be associated with the Cu surface evaporation and grain structure changes in the Cu substrate.open

A cookbook for using model diagnostics in integrated stock assessments

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Oxidation resistance of graphene-coated Cu and Cu/Ni alloy

The ability to protect refined metals from reactive environments is vital to many industrial and academic applications. Current solutions, however, typically introduce several negative effects, including increased thickness and changes in the metal physical properties. In this paper, we demonstrate for the first time the ability of graphene films grown by chemical vapor deposition to protect the surface of the metallic growth substrates of Cu and Cu/Ni alloy from air oxidation. SEM, Raman spectroscopy, and XPS studies show that the metal surface is well protected from oxidation even after heating at 200 \degree C in air for up to 4 hours. Our work further shows that graphene provides effective resistance against hydrogen peroxide. This protection method offers significant advantages and can be used on any metal that catalyzes graphene growth

NMR-Based Structural Modeling of Graphite Oxide Using Multidimensional 13C Solid-State NMR and ab Initio Chemical Shift Calculations

Chemically modified graphenes and other graphite-based materials have attracted growing interest for their unique potential as lightweight electronic and structural nanomaterials. It is an important challenge to construct structural models of noncrystalline graphite-based materials on the basis of NMR or other spectroscopic data. To address this challenge, a solid-state NMR (SSNMR)-based structural modeling approach is presented on graphite oxide (GO), which is a prominent precursor and interesting benchmark system of modified graphene. An experimental 2D C-13 double-quantum/single-quantum correlation SSNMR spectrum of C-13-labeled GO was compared with spectra simulated for different structural models using ab initio geometry optimization and chemical shift calculations. The results show that the spectral features of the GO sample are best reproduced by a geometry-optimized structural model that is based on the Lerf-Klinowski model (Lerf, A. et al. Phys. Chem. B 1998, 102, 4477); this model is composed of interconnected sp(2), 1,2-epoxide, and COH carbons. This study also convincingly excludes the possibility of other previously proposed models, including the highly oxidized structures involving 1,3-epoxide carbons (Szabo, I. et al. Chem. Mater. 2006, 18, 2740). C-13 chemical shift anisotropy (CSA) patterns measured by a 2D C-13 CSA/isotropic shift correlation SSNMR were well reproduced by the chemical shift tensor obtained by the ab initio calculation for the former model. The approach presented here is likely to be applicable to other chemically modified graphenes and graphite-based systems

Evaluation of biomarkers for testicular toxicity

Non-clinical safety assessment is essential during the drug development process in the pharmaceutical industry, and involves numerous, detailed in vitro and in vivo toxicology tests (general, reproductive and genetic), and safety pharmacology studies. The testis is a common organ for adverse drug effects leading to attrition of potential compounds. It would, therefore, be useful to detect testicular toxicity as early as possible in the drug development process. Histopathology is the standard method for assessing testis toxicity, but a biomarker for ‘early warning’ detection of testicular toxicity would be far more useful in non-clinical toxicology studies. The aim of this thesis was to evaluate the feasibility of this approach. It is thought that proteins can leak from seminiferous tubules into testicular interstitial fluid following testicular damage, due to either loss of integrity of the blood-testis barrier (BTB) or germ cell damage. A potential biomarker protein could, therefore, leak out of seminiferous tubules into interstitial fluid and then into blood following toxicological insult to the testis. A suitable biomarker protein must be testis specific, abundant, and not normally be present in blood. It may also need to have a low molecular weight. To investigate if proteins do leak out of seminiferous tubules following testicular damage, three known testicular toxicants which affect different aspects of the testis were used; cadmium chloride causes disruption to the blood-testis barrier and spermatogenesis, methoxyacetic acid (MAA) specifically causes a loss of pachytene spermatocytes, and 1,3-dinitrobenzene (DNB) causes Sertoli cell vacuolation and subsequent germ cell disruption. Adult male Wistar rats were treated with various doses of these toxicants to give mild and moderate responses. Samples were collected 24 hours later. Testicular damage was investigated by immunohistochemistry for well-known germ cell markers (DAZL, VASA) and using a general antibody to seminiferous tubule proteins. The integrity of the BTB was evaluated using immunofluorescent co-localisation of occludin, ZO-1, claudin-11, N-cadherin and β-catenin, and a biotin tracer. Protein leakage was investigated using analysis of interstitial fluid samples by 1D gel electrophoresis and staining with Coomassie-based dye or Western blotting for germ cell proteins and with the general antibody to seminiferous tubule proteins. Protein leakage from seminiferous tubules into interstitial fluid was observed with high dose cadmium chloride treatment. This was coincident with a loss of integrity of the BTB. No leakage was observed with MAA treatment which caused a specific loss of pachytene spermatocytes, or DNB which caused Sertoli cell vacuolation. With both treatments the BTB did not appear to be damaged suggesting that protein leakage occurs only following loss of integrity of the BTB. This was further investigated using treatments reported to specifically disrupt the BTB, namely intra-testicular administration of glycerol or transforming growth factor-β3, with samples collected 48 hours later. The damage caused was very localised, although BTB disruption with glycerol treatment caused some protein leakage. The presence of germ cell proteins in interstitial fluid samples before and after the development of the BTB during normal development was also evaluated, although most proteins of interest were not expressed in germ cells of the immature testis before BTB formation. Finally, five potential biomarker candidate proteins (ADAM3, Calpastatin, DAZL, FABP9, VASA) were selected and investigated using samples from the testicular toxicant studies. Smaller molecular weight proteins were thought to be more likely to leak out of seminiferous tubules, however, VASA, a large molecular protein (76kDa) was shown to leak into interstitial fluid following high dose cadmium chloride treatment. However, FABP9 (low molecular weight) was found to be the most promising biomarker for loss of BTB integrity. The results suggest that a biomarker could only be detected if there is a loss of integrity of the BTB and severe disruption of spermatogenesis, thus conferring no real advantage over present histopathology-based toxicity evaluations. Therefore, an automated immunohistochemistry and image analysis method was investigated as a refined method for detection of testicular toxicity at the end of a toxicology study, and shown to have promise.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Synthesis and exfoliation of isocyanate-treated graphene oxide nanoplatelets

A number of functionalized graphite oxides were prepared by treatment of graphite oxide (GO) with organic isocyanates. These isocyanate-treated GOs (iGOs) can then be exfoliated into functionalized graphene oxide nanoplatelets that can form a stable dispersion in polar aprotic solvents. Characterization of iGOs by FT-IR spectroscopy and elemental analysis suggested that the isocyanate treatment results in the functionalization of the carboxyl and hydroxyl groups in GO via formation of amides and carbamate esters, respectively. The degree of GO functionalization can be controlled via either the reactivity of the isocyanate or the reaction time. When used with functionalized isocyanates, the described methodology allows for the elaboration of graphene oxide nanoplatelets with different surface functional groups