Entry pathways of herpes simplex virus type 1 into human keratinocytes are dynamin- and cholesterol-dependent

Herpes simplex virus type 1 (HSV-1) can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s). Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-beta-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol

Surfactant protein A and D polymorphisms and methylprednisolone pharmacogenetics in donor lungs

Objective: Surfactant proteins A and D are important molecules involved in lung allograft innate immunity. Genetic polymorphisms of surfactant proteins A and D are associated with various lung diseases. In this study, surfactant protein A and D expression responses were investigated during pharmacogenetics upon methylprednisolone treatment as observed during lung transplantation. Methods: A human cell line (NCI-H441) and precision-cut lung slices from 16 human donors were incubated with methylprednisolone, and surfactant protein A1, surfactant protein A2, and surfactant protein D messenger RNA and surfactant protein A protein expression were assayed. Surfactant protein A1, A2, and D polymorphisms and surfactant protein A gene and protein expressions were determined. Results: In NCI-H441 cells, methylprednisolone treatment at 10−5 M and 10−6 M reduced surfactant protein A1 and surfactant protein A2 messenger RNA and surfactant protein A protein expression (P <.05). A pharmacogenetic relationship was observed in human donor precision-cut lung slices between the surfactant protein A2 (1Ax) variants: Surfactant protein A1, A2, and D messenger RNA expression were greater for 1A0 versus 1A1 (P <.05); surfactant protein A1/surfactant protein A2 genotype 6A26A2/1A01A0 (n = 5) showed greater surfactant protein A1, A2, and D messenger RNA expression and surfactant protein A protein expression compared with the other surfactant protein A1/surfactant protein A2 genotypes (n = 11) (P <.05). Conclusions: The surfactant protein A genotype and methylprednisolone stimuli influence donor lung surfactant protein A and D expression. Lungs carrying the surfactant protein A2 variant 1A0 have a greater expression of surfactant protein A when treated with methylprednisolone. Surfactant protein A polymorphisms could be used to personalize immunosuppressive regimens

Acute and Chronic Effects of Capsaicin in Perfused Rat Muscle: The Role of Tachykinins and Calcitonin Gene-Related Peptide 1

ABSTRACT In perfused rat skeletal muscle (hindlimb), capsaicin either stimulates (submicromolar concentrations) or inhibits (micromolar concentrations) oxygen consumption (VO 2 ). Both VO 2 effects are associated with vasoconstriction, evident as an increase in perfusion pressure (PP), under constant flow. We have proposed that these effects are mediated by two vanilloid receptor subtypes: VN 1 (stimulation of VO 2 ) and VN 2 (inhibition of VO 2 ) The vanilloid spice principle capsaicin and its structural analogs (dihydrocapsaicin, resiniferatoxin, piperine, gingerols and shogaols) produce concentration-dependent vasoconstriction and a biphasic effect on skeletal muscle VO 2 in the constant-flow perfused rat hindlimb Although our previous findings show that the dual effect

'Surely the most natural scenario in the world’: Representations of ‘Family’ in BBC Pre-school Television

Historically, the majority of work on British children’s television has adopted either an institutional or an audience focus, with the texts themselves often overlooked. This neglect has meant that questions of representation in British children’s television – including issues such as family, gender, class or ethnicity - have been infrequently analysed in the UK context. In this article, we adopt a primarily qualitative methodology and analyse the various textual manifestations of ‘family’, group, or community as represented in a selected number of BBC pre-school programmes. In doing so, we question the (limited amount of) international work that has examined representations of the family in children’s television, and argue that nuclear family structures do not predominate in this sphere

Auto-titrating continuous positive airway pressure treatment for obstructive sleep apnoea after acute quadriplegia (COSAQ): study protocol for a randomized controlled trial

BACKGROUND: Quadriplegia is a severe, catastrophic injury that predominantly affects people early in life, resulting in lifelong physical disability. Obstructive sleep apnoea is a direct consequence of quadriplegia and is associated with neurocognitive deficits, sleepiness and reduced quality of life. The usual treatment for sleep apnoea is nasal continuous positive airway pressure (CPAP); however, this is poorly tolerated in quadriplegia. To encourage patients to use this therapy, we have to demonstrate that the benefits outweigh the inconvenience. We therefore propose a prospective, multinational randomized controlled trial of three months of CPAP for obstructive sleep apnoea after acute quadriplegia. METHODS/DESIGN: Specialist spinal cord injury centres across Australia, New Zealand, the UK and Canada will recruit medically stable individuals who have sustained a (new) traumatic quadriplegia (complete or incomplete second cervical to first thoracic level lesions). Participants will be screened for obstructive sleep apnoea using full, portable sleep studies. Those with an apnoea hypopnoea index greater than 10 per hour will proceed to an initial three-night trial of CPAP. Those who can tolerate CPAP for at least 4 hours on at least one night of the initial trial will be randomized to either usual care or a 3-month period of auto-titrating CPAP. The primary hypothesis is that nocturnal CPAP will improve neuropsychological functioning more than usual care alone. The secondary hypothesis is that the magnitude of improvement of neuropsychological function will be predicted by the severity of baseline sleepiness measures, sleep fragmentation and sleep apnoea. Neuropsychological tests and full polysomnography will be performed at baseline and 3 months with interim measures of sleepiness and symptoms of autonomic dysfunction measured weekly. Spirometry will be performed monthly. Neuropsychological tests will be administered by blinded assessors. Recruitment commenced in July 2009. DISCUSSION: The results of this trial will demonstrate the effect of nocturnal CPAP treatment of obstructive sleep apnoea in acute quadriplegia. If CPAP can improve neurocognitive function after injury, it is likely that rehabilitation and subsequent community participation will be substantially improved for this group of predominantly young and severely physically disabled people. TRIAL REGISTRATION: Australian New Zealand Clinical Trial Registry ACTRN1260500079965

New generalized fuzzy metrics and fixed point theorem in fuzzy metric space

In this paper, in fuzzy metric spaces (in the sense of Kramosil and Michalek (Kibernetika 11:336-344, 1957)) we introduce the concept of a generalized fuzzy metric which is the extension of a fuzzy metric. First, inspired by the ideas of Grabiec (Fuzzy Sets Syst. 125:385-389, 1989), we define a new G-contraction of Banach type with respect to this generalized fuzzy metric, which is a generalization of the contraction of Banach type (introduced by M Grabiec). Next, inspired by the ideas of Gregori and Sapena (Fuzzy Sets Syst. 125:245-252, 2002), we define a new GV-contraction of Banach type with respect to this generalized fuzzy metric, which is a generalization of the contraction of Banach type (introduced by V Gregori and A Sapena). Moreover, we provide the condition guaranteeing the existence of a fixed point for these single-valued contractions. Next, we show that the generalized pseudodistance J:X×X→[0,∞) (introduced by Włodarczyk and Plebaniak (Appl. Math. Lett. 24:325-328, 2011)) may generate some generalized fuzzy metric NJ on X. The paper includes also the comparison of our results with those existing in the literature

11 beta-hydroxysteroid dehydrogenase type 1 regulates glucocorticoid-induced insulin resistance in skeletal muscle

OBJECTIVE: Glucocorticoid excess is characterized by increased adiposity, skeletal myopathy, and insulin resistance, but the precise molecular mechanisms are unknown. Within skeletal muscle, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone (11-dehydrocorticosterone in rodents) to active cortisol (corticosterone in rodents). We aimed to determine the mechanisms underpinning glucocorticoid-induced insulin resistance in skeletal muscle and indentify how 11beta-HSD1 inhibitors improve insulin sensitivity. \ud RESEARCH DESIGN AND METHODS: Rodent and human cell cultures, whole-tissue explants, and animal models were used to determine the impact of glucocorticoids and selective 11beta-HSD1 inhibition upon insulin signaling and action. \ud RESULTS: Dexamethasone decreased insulin-stimulated glucose uptake, decreased IRS1 mRNA and protein expression, and increased inactivating pSer$^{307}$ insulin receptor substrate (IRS)-1. 11beta-HSD1 activity and expression were observed in human and rodent myotubes and muscle explants. Activity was predominantly oxo-reductase, generating active glucocorticoid. A1 (selective 11beta-HSD1 inhibitor) abolished enzyme activity and blocked the increase in pSer$^{307}$ IRS1 and reduction in total IRS1 protein after treatment with 11DHC but not corticosterone. In C57Bl6/J mice, the selective 11beta-HSD1 inhibitor, A2, decreased fasting blood glucose levels and improved insulin sensitivity. In KK mice treated with A2, skeletal muscle pSer$^{307}$ IRS1 decreased and pThr$^{308}$ Akt/PKB increased. In addition, A2 decreased both lipogenic and lipolytic gene expression.\ud CONCLUSIONS: Prereceptor facilitation of glucocorticoid action via 11beta-HSD1 increases pSer$^{307}$ IRS1 and may be crucial in mediating insulin resistance in skeletal muscle. Selective 11beta-HSD1 inhibition decreases pSer$^{307}$ IRS1, increases pThr$^{308}$ Akt/PKB, and decreases lipogenic and lipolytic gene expression that may represent an important mechanism underpinning their insulin-sensitizing action