A mechanistic modelling approach for the determination of the mechanisms of inhibition by cyclosporine on the uptake and metabolism of atorvastatin in rat hepatocytes using a high throughput uptake method

(1) Determine the inhibition mechanism through which cyclosporine inhibits the uptake and metabolism of atorvastatin in fresh rat hepatocytes using mechanistic models applied to data generated using a high throughput oil spin method. (2) Atorvastatin was incubated in fresh rat hepatocytes (0.05–150 nmol/ml) with or without 20 min pre-incubation with 10 nmol/ml cyclosporine and sampled over 0.25–60 min using a high throughput oil spin method. Micro-rate constant and macro-rate constant mechanistic models were ranked based on goodness of fit values. (3) The best fitting model to the data was a micro-rate constant mechanistic model including non-competitive inhibition of uptake and competitive inhibition of metabolism by cyclosporine (Model 2). The association rate constant for atorvastatin was 150-fold greater than the dissociation rate constant and 10-fold greater than the translocation into the cell. The association and dissociation rate constants for cyclosporine were 7-fold smaller and 10-fold greater, respectively, than atorvastatin. The simulated atorvastatin-transporter-cyclosporine complex derived using the micro-rate constant parameter estimates increased in line with the incubation concentration of atorvastatin. (4) The increased amount of data generated with the high throughput oil spin method, combined with a micro-rate constant mechanistic model helps to explain the inhibition of uptake by cyclosporine following pre-incubation

Safeguarding children in dentistry: 1. Child protection training, experience and practice of dental professionals with an interest in paediatric dentistry

* Few dental professionals with child protection training have experience of making referrals. * There is a wide gap in practice between recognising signs of child abuse and neglect and responding effectively. * This may indicate missed opportunities to save children from continuing abuse. * There is a need for improved child protection information, support and training for dental professionals. Abstract Following several highly publicised inquiries into the deaths of children from abuse and neglect, there has been much recent interest in the role and responsibility of all health professionals to protect children at risk of maltreatment. The findings of a postal questionnaire, sent in March 2005 to 789 dentists and dental care professionals with an interest in paediatric dentistry working in varied settings in the UK, are presented in a two-part report and discussed in the context of current multi-agency good practice in safeguarding and promoting the welfare of children. This first part explores reported child protection training, experience and practice. There was a significant gap between recognising signs of abuse and responding effectively: 67% of respondents had suspected abuse or neglect of a child patient at some time in their career but only 29% had ever made a child protection referral. The dental profession is alerted to the need to ensure necessary appropriate action to safeguard children is always taken when child abuse or neglect are suspected

Differential phenotypic expression of a novel PDHA1 mutation in a female monozygotic twin pair

Pyruvate dehydrogenase complex (PDC) deficiency caused by mutations in the X-linked PDHA1 gene has a broad clinical presentation, and the pattern of X-chromosome inactivation has been proposed as a major factor contributing to its variable expressivity in heterozygous females. Here, we report the first set of monozygotic twin females with PDC deficiency, caused by a novel, de novo heterozygous missense mutation in exon 11 of PDHA1 (NM_000284.3: c.1100A>T). Both twins presented in infancy with a similar clinical phenotype including developmental delay, episodes of hypotonia or encephalopathy, epilepsy, and slowly progressive motor impairment due to pyramidal, extrapyramidal, and cerebellar involvement. However, they exhibited clear differences in disease severity that correlated well with residual PDC activities (approximately 60% and 20% of mean control values, respectively) and levels of immunoreactive E1α subunit in cultured skin fibroblasts. To address whether the observed clinical and biochemical differences could be explained by the pattern of X-chromosome inactivation, we undertook an androgen receptor assay in peripheral blood. In the less severely affected twin, a significant bias in the relative activity of the two X chromosomes with a ratio of approximately 75:25 was detected, while the ratio was close to 50:50 in the other twin. Although it may be difficult to extrapolate these results to other tissues, our observation provides further support to the hypothesis that the pattern of X-chromosome inactivation may influence the phenotypic expression of the same mutation in heterozygous females and broadens the clinical and genetic spectrum of PDC deficiency

Differential phenotypic expression of a novel PDHA1 mutation in a female monozygotic twin pair.

Pyruvate dehydrogenase complex (PDC) deficiency caused by mutations in the X-linked PDHA1 gene has a broad clinical presentation, and the pattern of X-chromosome inactivation has been proposed as a major factor contributing to its variable expressivity in heterozygous females. Here, we report the first set of monozygotic twin females with PDC deficiency, caused by a novel, de novo heterozygous missense mutation in exon 11 of PDHA1 (NM_000284.3: c.1100A>T). Both twins presented in infancy with a similar clinical phenotype including developmental delay, episodes of hypotonia or encephalopathy, epilepsy, and slowly progressive motor impairment due to pyramidal, extrapyramidal, and cerebellar involvement. However, they exhibited clear differences in disease severity that correlated well with residual PDC activities (approximately 60% and 20% of mean control values, respectively) and levels of immunoreactive E1α subunit in cultured skin fibroblasts. To address whether the observed clinical and biochemical differences could be explained by the pattern of X-chromosome inactivation, we undertook an androgen receptor assay in peripheral blood. In the less severely affected twin, a significant bias in the relative activity of the two X chromosomes with a ratio of approximately 75:25 was detected, while the ratio was close to 50:50 in the other twin. Although it may be difficult to extrapolate these results to other tissues, our observation provides further support to the hypothesis that the pattern of X-chromosome inactivation may influence the phenotypic expression of the same mutation in heterozygous females and broadens the clinical and genetic spectrum of PDC deficiency

Estrogen Receptor Alpha Is Expressed in Mesenteric Mesothelial Cells and Is Internalized in Caveolae upon Freund's Adjuvant Treatment

Transformation of epithelial cells into connective tissue cells (epithelial-mesenchymal transition, EMT) is a complex mechanism involved in tumor metastasis, and in normal embryogenesis, while type II EMT is mainly associated with inflammatory events and tissue regenaration. In this study we examined type II EMT at the ultrastructural and molecular level during the inflammatory process induced by Freund's adjuvant treatment in rat mesenteric mesothelial cells. We found that upon the inflammatory stimulus mesothelial cells lost contact with the basal lamina and with each other, and were transformed into spindle-shaped cells. These morphological changes were accompanied by release of interleukins IL-1alpha, -1beta and IL-6 and by secretion of transforming growth factor beta (TGF-beta) into the peritoneal cavity. Mesothelial cells also expressed estrogen receptor alpha (ER-alpha) as shown by immunolabeling at the light and electron microscopical levels, as well as by quantitative RT-PCR. The mRNA level of ER-alpha showed an inverse correlation with the secretion of TGF-beta. At the cellular and subcellular levels ER-alpha was colocalized with the coat protein caveolin-1 and was found in the plasma membrane of mesothelial cells, in caveolae close to multivesicular bodies (MVBs) or in the membrane of these organelles, suggesting that ER-alpha is internalized via caveola-mediated endocytosis during inflammation. We found asymmetric, thickened, electron dense areas on the limiting membrane of MVBs (MVB plaques) indicating that these sites may serve as platforms for collecting and organizing regulatory proteins. Our morphological observations and biochemical data can contribute to form a potential model whereby ER-alpha and its caveola-mediated endocytosis might play role in TGF-beta induced type II EMT in vivo

Neighbour identity hardly affects litter-mixture effects on decomposition rates of New Zealand forest species.

The mass loss of litter mixtures is often different than expected based on the mass loss of the component species. We investigated if the identity of neighbour species affects these litter-mixing effects. To achieve this, we compared decomposition rates in monoculture and in all possible two-species combinations of eight tree species, widely differing in litter chemistry, set out in two contrasting New Zealand forest types. Litter from the mixed-species litter bags was separated into its component species, which allowed us to quantify the importance of litter-mixing effects and neighbour identity, relative to the effects of species identity, litter chemistry and litter incubation environment. Controlling factors on litter decomposition rate decreased in importance in the order: species identity (litter quality) >> forest type >> neighbour species. Species identity had the strongest influence on decomposition rate. Interspecific differences in initial litter lignin concentration explained a large proportion of the interspecific differences in litter decomposition rate. Litter mass loss was higher and litter-mixture effects were stronger on the younger, more fertile alluvial soils than on the older, less-fertile marine terrace soils. Litter-mixture effects only shifted percentage mass loss within the range of 1.5%. There was no evidence that certain litter mixtures consistently showed interactive effects. Contrary to common theory, adding a relatively fast-decomposing species generally slowed down the decomposition of the slower decomposing species in the mixture. This study shows that: (1) species identity, litter chemistry and forest type are quantitatively the most important drivers of litter decomposition in a New Zealand rain forest; (2) litter-mixture effects—although statistically significant—are far less important and hardly depend on the identity and the chemical characteristics of the neighbour species; (3) additive effects predominate in this ecosystem, so that mass dynamics of the mixtures can be predicted from the monocultures

The influence of mosquito resting behaviour and associated microclimate for malaria risk

<p>Abstract</p> <p>Background</p> <p>The majority of the mosquito and parasite life-history traits that combine to determine malaria transmission intensity are temperature sensitive. In most cases, the process-based models used to estimate malaria risk and inform control and prevention strategies utilize measures of mean outdoor temperature. Evidence suggests, however, that certain malaria vectors can spend large parts of their adult life resting indoors.</p> <p>Presentation of hypothesis</p> <p>If significant proportions of mosquitoes are resting indoors and indoor conditions differ markedly from ambient conditions, simple use of outdoor temperatures will not provide reliable estimates of malaria transmission intensity. To date, few studies have quantified the differential effects of indoor <it>vs </it>outdoor temperatures explicitly, reflecting a lack of proper understanding of mosquito resting behaviour and associated microclimate.</p> <p>Testing the hypothesis</p> <p>Published records from 8 village sites in East Africa revealed temperatures to be warmer indoors than outdoors and to generally show less daily variation. Exploring the effects of these temperatures on malaria parasite development rate suggested indoor-resting mosquitoes could transmit malaria between 0.3 and 22.5 days earlier than outdoor-resting mosquitoes. These differences translate to increases in transmission risk ranging from 5 to approaching 3,000%, relative to predictions based on outdoor temperatures. The pattern appears robust for low- and highland areas, with differences increasing with altitude.</p> <p>Implications of the hypothesis</p> <p>Differences in indoor <it>vs </it>outdoor environments lead to large differences in the limits and the intensity of malaria transmission. This finding highlights a need to better understand mosquito resting behaviour and the associated microclimate, and to broaden assessments of transmission ecology and risk to consider the potentially important role of endophily.</p