Automation and validation of micronucleus detection in the 3D EpiDerm™ human reconstructed skin assay and correlation with 2D dose responses.

Recent restrictions on the testing of cosmetic ingredients in animals have resulted in the need to test the genotoxic potential of chemicals exclusively in vitro prior to licensing. However, as current in vitro tests produce some misleading positive results, sole reliance on such tests could prevent some chemicals with safe or beneficial exposure levels from being marketed. The 3D human reconstructed skin micronucleus (RSMN) assay is a promising new in vitro approach designed to assess genotoxicity of dermally applied compounds. The assay utilises a highly differentiated in vitro model of the human epidermis. For the first time, we have applied automated micronucleus detection to this assay using MetaSystems Metafer Slide Scanning Platform (Metafer), demonstrating concordance with manual scoring. The RSMN assay's fixation protocol was found to be compatible with the Metafer, providing a considerably shorter alternative to the recommended Metafer protocol. Lowest observed genotoxic effect levels (LOGELs) were observed for mitomycin-C at 4.8 µg/ml and methyl methanesulfonate (MMS) at 1750 µg/ml when applied topically to the skin surface. In-medium dosing with MMS produced a LOGEL of 20 µg/ml, which was very similar to the topical LOGEL when considering the total mass of MMS added. Comparisons between 3D medium and 2D LOGELs resulted in a 7-fold difference in total mass of MMS applied to each system, suggesting a protective function of the 3D microarchitecture. Interestingly, hydrogen peroxide (H2O2), a positive clastogen in 2D systems, tested negative in this assay. A non-genotoxic carcinogen, methyl carbamate, produced negative results, as expected. We also demonstrated expression of the DNA repair protein N-methylpurine-DNA glycosylase in EpiDerm™. Our preliminary validation here demonstrates that the RSMN assay may be a valuable follow-up to the current in vitro test battery, and together with its automation, could contribute to minimising unnecessary in vivo tests by reducing in vitro misleading positives

Types of albinism in the black Southern Africa population

Background: Oculocutaneous albinism (OCA) is the most common inherited disorder in Southern African blacks and several types have been described. Molecular techniques, where available, can be used to confirm a clinical diagnosis and the type of OCA, if necessary, and for prenatal diagnosis.Objectives: To investigate and classify the different types of albinism commonly found and to determine the clinical implications for each type.Design: A descriptive survey.Setting: Gauteng province, South Africa, and Lesotho.Subjects: Three groups of subjects with OCA (96 from a genetics clinic, 62 from a dermatology clinic, and 31 from community surveys) from the black African population participated.Main outcome measures: Subjects underwent clinical and/or dermatological examinations and were then classified according to type of OCA.Results: Four forms of OCA were identified: most (82%) subjects had OCA2 (a tyrosinasepositive type) with three sub-types: those without large freckles (ephelides) on exposed areas (named OCA 2a in this study), those with such freckles (named OCA 2b), and those with brown albinism (BOCA); the remainder had red/ rufous albinism, ROCA (OCA 3). The four forms could be distinguished from each other clinically without using molecular genetic testing.Conclusion: The most common types of albinism found in the black population of Southern Africa are OCA 2 and OCA 3. Given the high prevalence of the disorder, together with the high risk of skin cancer, and the recent persecution of affected individuals in certain East African countries, these findings and their clinical implications have significance in terms of both education and awareness for health professionals andlay people caring for those with albinism

In Vitro Primary-Indirect Genotoxicity in Bronchial Epithelial Cells Promoted by Industrially Relevant Few-Layer Graphene

Few-layer graphene (FLG) has garnered much interest owing to applications in hydrogen storage and reinforced nanocomposites. Consequently, these engineered nanomaterials (ENMs) are in high demand, increasing occupational exposure. This investigation seeks to assess the inhalation hazard of industrially relevant FLG engineered with: (i) no surface functional groups (neutral), (ii) amine, and (iii) carboxyl group functionalization. A monoculture of human lung epithelial (16HBE14o-) cells is exposed to each material for 24-h, followed by cytotoxicity and genotoxicity evaluation using relative population doubling (RPD) and the cytokinesis-blocked micronucleus (CBMN) assay, respectively. Neutral-FLG induces the greatest (two-fold) significant increase (p 1 µm diameter). The findings of the present study have demonstrated the capability of neutral-FLG and amine-FLG to induce genotoxicity in 16HBE14o- cells through primary indirect mechanisms, suggesting a possible role for carboxyl groups in scavenging radicals produced via oxidative stress

First direct observation of the Van Hove singularity in the tunneling spectra of cuprates

In two-dimensional lattices the electronic levels are unevenly spaced, and the density of states (DOS) displays a logarithmic divergence known as the Van Hove singularity (VHS). This is the case in particular for the layered cuprate superconductors. The scanning tunneling microscope (STM) probes the DOS, and is therefore the ideal tool to observe the VHS. No STM study of cuprate superconductors has reported such an observation so far giving rise to a debate about the possibility of observing directly the normal state DOS in the tunneling spectra. In this study, we show for the first time that the VHS is unambiguously observed in STM measurements performed on the cuprate Bi-2201. Beside closing the debate, our analysis proves the presence of the pseudogap in the overdoped side of the phase diagram of Bi-2201 and discredits the scenario of the pseudogap phase crossing the superconducting dome.Comment: 4 pages, 4 figure

Triggered optical coherence tomography for capturing rapid periodic motion

Quantitative cross-sectional imaging of vocal folds during phonation is potentially useful for diagnosis and treatments of laryngeal disorders. Optical coherence tomography (OCT) is a powerful technique, but its relatively low frame rates makes it challenging to visualize rapidly vibrating tissues. Here, we demonstrate a novel method based on triggered laser scanning to capture 4-dimensional (4D) images of samples in motu at audio frequencies over 100 Hz. As proof-of-concept experiments, we applied this technique to imaging the oscillations of biopolymer gels on acoustic vibrators and aerodynamically driven vibrations of the vocal fold in an ex vivo calf larynx model. Our results suggest that triggered 4D OCT may be useful in understanding and assessing the function of vocal folds and developing novel treatments in research and clinical settings

A review of clinical decision-making: Models and current research

Aims and objectives: The aim of this paper was to review the current literature with respect to clinical decision-making models and the educational application of models to clinical practice. This was achieved by exploring the function and related research of the three available models of clinical decision making: information processing model, the intuitive-humanist model and the clinical decision making model. Background: Clinical decision-making is a unique process that involves the interplay between knowledge of pre-existing pathological conditions, explicit patient information, nursing care and experiential learning. Historically, two models of clinical decision making are recognised from the literature; the information processing model and the intuitive-humanist model. The usefulness and application of both models has been examined in relation the provision of nursing care and care related outcomes. More recently a third model of clinical decision making has been proposed. This new multidimensional model contains elements of the information processing model but also examines patient specific elements that are necessary for cue and pattern recognition. Design: Literature review Methods: Evaluation of the literature generated from MEDLINE, CINAHL, OVID, PUBMED and EBESCO systems and the Internet from 1980 – November 2005

Few-layer graphene induces both primary and secondary genotoxicity in epithelial barrier models in vitro

Background Toxicological evaluation of engineered nanomaterials (ENMs) is essential for occupational health and safety, particularly where bulk manufactured ENMs such as few-layer graphene (FLG) are concerned. Additionally, there is a necessity to develop advanced in vitro models when testing ENMs to provide a physiologically relevant alternative to invasive animal experimentation. The aim of this study was to determine the genotoxicity of non-functionalised (neutral), amine- and carboxyl-functionalised FLG upon both human-transformed type-I (TT1) alveolar epithelial cell monocultures, as well as co-cultures of TT1 and differentiated THP-1 monocytes (d.THP-1 (macrophages)). Results In monocultures, TT1 and d.THP-1 macrophages showed a statistically significant (p < 0.05) cytotoxic response with each ENM following 24-h exposures. Monoculture genotoxicity measured by the in vitro cytokinesis blocked micronucleus (CBMN) assay revealed significant (p < 0.05) micronuclei induction at 8 µg/ml for amine- and carboxyl-FLG. Transmission electron microscopy (TEM) revealed ENMs were internalised by TT1 cells within membrane-bound vesicles. In the co-cultures, ENMs induced genotoxicity in the absence of cytotoxic effects. Co-cultures pre-exposed to 1.5 mM N-acetylcysteine (NAC), showed baseline levels of micronuclei induction, indicating that the genotoxicity observed was driven by oxidative stress. Conclusions Therefore, FLG genotoxicity when examined in monocultures, results in primary-indirect DNA damage; whereas co-cultured cells reveal secondary mechanisms of DNA damage

Effect of Galactose Ingestion Before and During Exercise on Substrate Oxidation, Postexercise Satiety, and Subsequent Energy Intake in Females.

OBJECTIVE: To examine the effects of consuming a galactose carbohydrate (CHO) drink on substrate oxidation, postexercise satiety, and subsequent energy intake. METHODS: Nine recreationally active eumenorrheic females undertook 3 trials, each consisting of running for 60 minutes at 65% VO2peak followed immediately by a 90-minute rest period. Prior to (300 ml) and at 15-minute intervals during exercise (150 ml), participants consumed either a glucose (GLU: GI 89) or galactose (GAL: GI 20) drink, each of which contained 45 g of CHO, or an artificially sweetened placebo (PLA). Following the rest period, participants were provided with an ad libitum test lunch and asked to record food intake for the remainder of the day. RESULTS: Plasma glucose was significantly greater throughout exercise and rest following the GLU trial compared with the GAL and PLA trials (P < 0.05); however there were no differences in CHO oxidation. Hunger was significantly lower (P < 0.05) throughout the GAL compared to the GLU and PLA trials. There were no significant differences between trials for energy intake during the postexercise meal. Overall net energy balance for the 24 hours was negative in both the GAL (-162 ± 115 kcal; P < 0.05 vs GLU) and PLA trials (-49 ± 160 kcal). CONCLUSIONS: Results demonstrate that ingesting a solution containing GAL before and during exercise can positively impact postexercise satiety and energy balance throughout the day, compared to a more readily available and widely consumed form of CHO. Despite this, there appears to be no apparent benefit in consuming a CHO beverage on fuel utilization for this moderate exercise intensity and duration

Spatial heterogeneity and peptide availability determine CTL killing efficiency in vivo

The rate at which a cytotoxic T lymphocyte (CTL) can survey for infected cells is a key ingredient of models of vertebrate immune responses to intracellular pathogens. Estimates have been obtained using in vivo cytotoxicity assays in which peptide-pulsed splenocytes are killed by CTL in the spleens of immunised mice. However the spleen is a heterogeneous environment and splenocytes comprise multiple cell types. Are some cell types intrinsically more susceptible to lysis than others? Quantitatively, what impacts are made by the spatial distribution of targets and effectors, and the level of peptide-MHC on the target cell surface? To address these questions we revisited the splenocyte killing assay, using CTL specific for an epitope of influenza virus. We found that at the cell population level T cell targets were killed more rapidly than B cells. Using modeling, quantitative imaging and in vitro killing assays we conclude that this difference in vivo likely reflects different migratory patterns of targets within the spleen and a heterogeneous distribution of CTL, with no detectable difference in the intrinsic susceptibilities of the two populations to lysis. Modeling of the stages involved in the detection and killing of peptide-pulsed targets in vitro revealed that peptide dose influenced the ability of CTL to form conjugates with targets but had no detectable effect on the probability that conjugation resulted in lysis, and that T cell targets took longer to lyse than B cells. We also infer that incomplete killing in vivo of cells pulsed with low doses of peptide may be due to a combination of heterogeneity in peptide uptake and the dissociation, but not internalisation, of peptide-MHC complexes. Our analyses demonstrate how population-averaged parameters in models of immune responses can be dissected to account for both spatial and cellular heterogeneity

Anti-Allergic Cromones Inhibit Histamine and Eicosanoid Release from Activated Human and Murine Mast Cells by Releasing Annexin A1

PMCID: PMC3601088This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited